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John R Dean, Alan M Jones, David Holmes, Rob Reed, Jonathan Weyers and Allan Jones
Practical Skills in
Chemistry
Pearson Education
-
We work with leading authors to develop the strongest educational materials
in chemistry, bringing cutting.rogc thinking and best learning practice to a gloool markel.
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Education lImiled 2002
The righh of John R. Dean. Alan M. JOl'leI. DIMd HolI'l1M, Rob Reed. Jooalt\8n Weyers and AIlBn ..Jones 10 t. ldenIifllld _llUltKln ollhla WOflc hlIVe "-" ~ by !hem in IIOOOtd_ wiItllhe Copyrighl. Desigll8lld PMem, AcI 1988.
All righlS reserved: 110 pIIrt oIlhis publielrtion mev bII
reproduced. IlOred fr1 8flV n,nillvlll SVlI1IIm. Of t"rlImilted Of by IIf"IV mBIIfl8, electrol'lie, mectlenlc,l, pholOcOpylrog. .eootdi!'lll. 01' otherwise wilhoU eil:hef"!he prior
ir'l aov form
wrinen pemIiQlon 01 thlI publither or • licerooe permitt~ rtl$1.ie1ed ~ in the Uriled Kingdom i - * by lhot ~ UoaoIlrog Agerlcy L1d. 90 TOIlenham Court~. London W1T 'lP. ISBN 918413428002-2 BrlthhLbwy~o.ta
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10987.
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Contents Boxes Preface
vii ix x
AcktlOll'ledgemellts Abbreriations
xiii
1 2 3 4 5 6 7
Fundamental laboratory techniques Basic principles Health and safety Working with liquids Basic laboratory procedures I Basic laboratory procedures II Principles of solution chemistry pH and bulTer solutions Resources for fundamental laboratory tochniques
3 6 9 15 26 45 56 62
,
The investigative approach Making and recording measurements
9 10 11
SI units and their use Scientific method and design of experiments
12 13 14 15 16 17
Melting points Recrystallization
Project work Resources fOl" the investigative approach
65 70 75 80 '3
Laboratory techniques
18
Solvent extraction
Distillation Reflux Evaporation Inert atmosphere methods Resources for laboratory lechniques
87
92 102 107 116 121 125 131
Classical techniques
19 20 21 22 23 24 25
26 27 2' 29 30 31 32
Qualitative techniques for inorganic analysis Gravimetry Procedures in volumetric analysis Acid-base titrations Complexometric titrntions
Redox litrations Precipitation titrations Resources for classical tcchniques
Instrumental techniques Basic spectroscopy Atomic spectroscopy Infrared spectroscopy Nuclear magnetic resonance spectrometry Mass spectromelry Chromatogrnphy - introdoction Gas and liquid chromatography
135 139 141 14' 151 155 157 160 163 170 180 190 200 205 211 v
Contents
33
34 35 36
Electrophoresis Ek:cuoonalylical techniques Radioactive isotopes and their uses Thermal analysis Resoun:ai rOf" instrumental techniques
225
229 235 243 246
Analysis and presentation of data
37 38 39 40
41 42 43 44
Using graphs
Presenting data in tahles Hints for solving numerical problems De!icripti\'e statistics Choosing and using statistical tc:su Dra~;ng chemical SlrUClUI'e$ Chemomd.rics
Computational chemistry Resources for ana.!yl;is and presentation of data
45 46 47 48 49 50
51 52 53 54 55 56
Infonnation technology and library resources The Internet and Work! W~ Web Internet resowa:s for chemistry
299
Using sprea~
302 307
Word processors, dalabllscs and other padc:agt$ Fmding and citing published information
317
Communicating information Genenl.l aspects of scientifIC 'Hiring
311
Writing~ys
325 330
Reporting prada! and project work
332
Writing literature surveys and re\iews
339 341 344 348 354
Organizing a po;lcr display Giving an oral presentation Examinations Resources for communicating information References
Index
vi
251 256 259 264 271 280 285 291 294
355 357
Boxes 4.1
27.3
How to make up an aqueous solution of known concentmtion from a solid chemical How to make up an aqueous solution of known concentration from a solid chemical for use in quantitative analysis How to make up a linear dilution series for use in quantitative analysis How to weigh Olll a sample of a solid for use in quantitative analysis How 10 nute a filler paper for gravity filtration Isolation of a solid by suction filtration How 10 dry a solution over magnesilUll sulphate Useful procedures for calculations involving molar concentrations How 10 convert ppm into mass of chemical required An example of complex fonnalion The use of oxidation numbers 10 identify re
FAAS
172
27.4
Sample size and certifted reference materials
173
4.2 4.3 4.4 5.1 5.2 5.3 6.1 6.2 6.3 6.4 6.5 7.1 9.1 9.2 10.1 HI
13.2 13.3 13.4 14.1 14.2 15.1 15.2 15.3 15.4 15.5 16.1 16.2 17.1 18.! 19.1 20.1 21.1 26.1 26.2 27.1 27.2
17 19 21 24
28 30 41 46
47 53 54
55 59 72
73
n
94
95 97 99
105 106 109 109 110 113 114 117
118 122 130 137
139 142 166 169 170 171
vii
Boxes
27.5 27.6 27.7 28.1 28.2 28.3 28.4 29.1 30.1 30.2 32.1
Idealized frdgmemation PI"OCCSSCS for the molCallar iOIl (M+) How 10 prepare a set of live calibration solutions in the concentration rangeo-lOligmL- 1 (mgt-I)
32.2 32.3 35.1 35.2 36.1 37.1 38.1 39.1 40.1 40.2 41.1 41.2 43.1 43.2 45.1 50.1
How 10 make micropipeues for TLC How to run a thin-layer chromatogram How to determine the specifIC activity of an experirnenlHI solution Tips for preparing samples for liqUid scintillation counting How to interpret a thermal analysis trace Checklist for the stages in drawing a graph Checklist for preparing a table Example of using the rules of Table 39.2 Calculation of descriptive statistics for a sample of grouped data Three examples where simple arithmetic means are inappropriate How 10 carr}' out a t-test Worked example of a I-test Example of a two-level factorial design Example of principal component analysis Important guidelines for using computers and networks How to achieve a de-dr. readable style Improve your writing ability by consulting a personal reference librdry The structure of reports of experimental work Writing experimental procetlures Steps in producing a scientific paper Writing under exam conditions
50.2
52.1 52.2 52.3 56.1
viii
Analysis of a sample: dilution faclOr How to operate a flame atomic absorption spectrometer How to acid-digest a I;ampJe using a hot plate How to run an infrared spectrum ora liquid or solid film. mull or KRr disk How to prepare Liquid ami solid films and mulls How to prepare a KBr di<;k How to interpret an IR spectrum How to prepare a sample for NMR spectroscopy How to identify the number of bromine or chlorine atoms in a molecule from the molecular ion
173 177 179
183 185 188 189 193 202 203
212 217 218 238 239 244 253 257 261 265 266 276 277 287 290 301 327 328 333 335 337 352
Preface Practical skills fonn the cornerstone of chemistry. However, the diversity of skills required in the laboratory means that a student's experience may be limited. While some techniques do require specific skills. many of them are transferable, generic skills thai are required throughout the subject area. Limited time constraints of the modern curriculum often preclude or minimize laboratory time. It is the aim of this book 10 provide general guidance for use in aDd outside practical sessions and also to cover a range of techniques from the basic to the morc advanced. The book is primarily aimed at the undergraduate level (principally first and second year), although some information is relevant to final year undergraduates and postgraduate courses. The diversity of the book means that recipe--Jike solutions are not provided for every potential problem. but guidance on a range of topics is provided. As the book focuses on practiall aspects it is important not to apportion 'labels' to the areas covered. However, for the traditionalist the areas covered are primarily focused on synthetic, physical and analytical aspects of chemistry. It is intended that the book will be useful in the laboratory, during practical classes, and during project work. It is not intended to replace rceipelike laboratory scripts but to enable students to become familiar with the practical aspects of the subject both at the time of performing the experiment and during the writing-up process. In addition, lecturers should find that the text provides an effective means of suppk'menting the infoonation given in practical classes, where constraints on time and resources can lead to the under-performance of students. We have selec1ed material for inclusion in Practical Skills in Chemist/")· based on our own teaching experience, highlighting those areas where our students ha~ needed further guidance. As a result of our comprehensive cover of practical skills, some techniques such as microscale methods and specialized 'vacuum techniques ha,,'e been omitted, but specific references arc provided. Instead, we have attempted to provide sufficient detail so that students will have the skills to carry out experiments successfully and not produce poor data as a result of poor technique. Most students will have access to specialist textlx>oks giving in-depth coverage of the theoretical principles and knowledge covered in lectures. This lx>ok aims to suppk'll1ent - rather than replace - such textbooks, and covers the skills required in laboratory classes, together with practical advice, tips, hilUs, worked examples, definitions, key poims. 'how to' boxes and checklists which are useful in the field of chemistry. The text provides an outline on the underlying theoretical principles where necessary, but emphasis throughout is on the practical applications of this information. To students who buy this book, we hope that you will find it useful in the laboratory, during your prnctiall classes and in your project work - this is not a book to be left on the bookshelf. Lecturers should find that the text provides an effective means of supplementing the information gi\"CTI in practical classes. We would like to acknowledge the support of our families and the help provided by colleagues who read early drafts of the material. In particular, special thanks are due to Gary Askwith, Dave Bannister (Manchester
..
Preface
Metropolitan University), Jon Bookham, Susan Carlile, Jim Creighton, $anth Cresswell, Marlin D'lVies. Us Oix, Jackie Eager, Derek Holmes, Ed Ludkin, Dave Osborne, Justin Perry, hne Shaw, Tony Simpson, Dave Wealleans and Ian Winship. Despite this help, the responsibility for any errors rests with us and we would be grateful if readers could alert us to any errors or problems so that we can make amends liS soon as possible. Please write to US at the University of Norlhumbria ill Newc.1stle. or bye-mail at the following addresses:
[email protected] (JRD);
[email protected] (Alan M. Jones); rob.recd@unn.'lc.uk (RHR);
[email protected] (DH); j.d.b.wc)'
[email protected](JDBW);
[email protected] (Allan Jones).
Acknowledgements We are gmteful to the following for pennission to reproduce copyright material: Text extracts from the following databllses: Alit/lyrical Abstracts, Chemical B,LsiJle.fS Nell's/klse, Chemict/I Safety Nell'sIJase, Mas.f Spectrometry Bulletin, Chemical Safety Data Sheets and Chromatography Abstract~' in Chapter 36, 'Internet resources for chemistry', reproduced by kind permission of The Royal Society of Chemistry. Whilst e\'ery effort has been made to trace the o,""crs of copyright material, in a few UlSCS this has proved impossible and we take this opportunity to oOer our apologies to any copyright holders whose: rights we may have unwittingly infringed.
,
For the student This text is designed to help you in your Chemistry studies, before and during laboratory classes, in project work, in tutorials and even in examination'!. Chapters 1-36 CO"('r general and specific: skills for laboratory work.
These are based on the authors' experience of questions that students oOcn u8k and difficulties they ha"e in performing 1:looratory wOrk. They include tips. hints. worked examples. definitions and "how to" boxes that sel out procedures for you step by step. Chapters 37-44 explain data anal}'sis and presentation.
This will be an im(Xlrtant element of your course and you will find thai this section guide.1ii you through the key skills that you will need to develop, from presenting graphs and tables to drawing chemical structures and using statistical tests. Qaapters 45-49 co,'er infonnation tedJoology and librllry rCSQUrces. These chapters will help you get the most from chemistry resources available on I.he World Wide Web. Chapters 50-56 deal with the "jlltl subject of communicating infOf"mation.
Designed to help you report practical and projoct work and give oral presentations effectively, and to succeed in examinations, We hope you will find this book a helpful guide throughout your course. and beyond,
xi
Abbreviations A, AAS
AC ACS AES ANOVA AO
ATP
BTDS b.pl.
CCD CCP
CE CEC CGE COSHH CoY CRm
CZE DAD DCM
DNA DSC
DTA
ECD EDTA EI EIE EMH
""EOF F
FAAS FlO FT Ff-JR
GC GFC OPe h HASAW HCB
BeL Hep HIe HPLC
ICP IEC IEF
relative atomic mass atomic absorption speclromell}' affinity chromatography American Chemical Society atomic emission spectrometry analysis of variant'e atomic orbital adenosine triphosphate Bath Information and Data Services boiling point central oomposit design cubic close packed capillary e1e(trophoresis capillary electTochromalography capillary gel electrophoresis Control of Substances Ha;mrdous to Health coefficient of variance certified reference material capillary zone electrophoresis diode array detection dichloromethane deoxyribonucleic acid differential scanning colorimetry dilTerentialthennal analysis electron capture detector clhylenediaminetetraacetic acid electron impact ionization easily ionizable element electromagnetic radiation ethylenediamine electro-osmotic flow Faraday constant flame atomic absorplion spectrometer flame ionization detector Fourier tmnsfonnation Fourier lransfonn - infrared (spcclroscopy) gas chromatography geJ filtration chromatography gel penneation chromatography Planck constant Health lind Safety at Work hexachloro-l ,3-butadiene hollow-cathode lamp hexagonal dose packed hydrophobic interaction chromatography high-perfonnance liquid chromalography inductively coupled plasma ion-exchange chromatography isoelcctric focusing xiii
Abbreviations
lit ISE IUPAC K,. K.
K. KHP
LGC M, MOL MEKC MEL
MO m.pt. MS
NH NIST
NMR NP-HPLC ODA
OEL PCA PFA PLOT
PMT PTFE R
R, RA
RNA RP-HPLC 'I'm RSC SAX SCOT
sex SDS SE SEM SI
STP TeA TCD TG TLC TMS TRIS URL USEPA
uv WCOT
www ,iv
infmred (radiation) ion selective electrode International Union of Pure and Applied Chemistry acid dissociation constant solubility product ion product of water potassium hydro~n phthulatt: L'l.bomlory of the Government Chemist relative molecular mass minimum detectable level micellar electrokinetic chromatography maximum eXJX)Sure limit molecular orbitnl melting point mass spectrometry null hYPOlhesis National Institute of Standards und Technology nuclear magnetic resonance nomlal phase high·perfonnance liquKl chromatography octadecylsilane occupational exposure slandard principal component analysis perfluoroalkoxyvinylether porous layer open tubular (column) pholomultiplier lube polytetrnnuoroethylene uni\'ersul gas constant relative frontal mobility relative lIbundance ribonucleic acid reverse phase high-perfonnance liquid chromatogmphy revolutions per minute Royal Society of Chemistry strong anion exchange support-caated open tubular (capillary column) strong calion exchange sodium dodecyl sulphate stllndard error (of the sample mean) scanning electron microscopy Sysleme Internationale d'Unites st..1ndard tempemture and pressure trichloracetic acid thennal conductivity detector thermogravimetry thin-layer chromatography tetramethylsilane tris(bydroxymethyl)aminomethane or 2-.amino-2-hydroxymethyl-I ,3-propanediol uniform resource locator United States Environmental Protection Agency ultraviolet wall-coated open tubular (column) World Wide Web
-0 Developing practical skills - these will include.: •
observing and measuring
•
recording data
• designing experiments • •
analysIng and interpreting data reportinwpresenting.
Basic principles All knowledge and theory in science has originated from practical observation and experimentation: this is equally true for disciplines as diverse as nnalysis and synthesis. Labonttory work is an essential part of flll chemistry courses and often accounts for a significunl proportion of the assessment mOlrks. This book aims to provide lin easy·to-use reference source dealing with basic practical techniques and infonnation. The skills developed in pr.lctical classes will continuc to be useful throughout your course nnd beyond, some within science and others in
Being prepared
~ You will get the most out of laboratory work if you prepare well. Do not go into a practical session assuming that everything will be provided, without any input on your part.
TIle main poinls to remember are: •
Using textbook in the lab - take this book along to the refevant CI85S8S, 80 that vou can make full use of the information during the p.-aetical souions.
• • • •
If in doubt over any part of the practical procedure - ASKI There is no such thing as 8 silly Question in the laboratory.
•
• •
Read any handouts in advance: make sure you understand lhe purpose of the practical and the particular skills involvoo. Docs lhc practiCal relate to, or expand upon, a current topic in your leclUrcs'! Is there any additional preparatory reading tbat witl belp? Take along appropriatc textbooks. to explain aspects in the practic<\I. Consider what safety hazards might be involved, and any precautions you might need to take, before you begin (p. 7). Listen carefully to any instructions and note any importanl points: adjust your schedulefhandout, as necessary. During the practical session, organizc your bench space - makc surc your lab book is adjacCt1t to, but not within, your working area. You will ollen find it easiesl 10 keep clean items of glassware etc. on one side of your working space, with used equipment on the other side. All chemical waste (solid or liquid) should be disposed of in the appropriate containers provided (consult thc demonstrator or lecturerin-charge). Write up your work as soon as possible, and submit it on time, or you may lose marks. Catch up on any work you have missed as soon as possible - preferably before the next practical session.
Basic requirements
RecOI'ding pmcticlt/ results Presenting results - while you don't need to be 8 graphic designer 10 produce wort of 8 satisfactory standard, presentation and layout Bre important and you \/ViII lose marks for poorlV presenled work.
An A4 loose-leaf ring binder olTers flexibility, since you can insert labor-dtory handouts. and lined and graph paper, at appropriate points. The danger of losing one or more pages from a loose-leaf system is the main drawback. Bound books avoid this problem. although those containing alternating lined/graph or lined/blank pages tend to be wasteful - it is often better to paste sheets of graph paper into a bound book, as required. Fundamental labof"alory techniques
3
Basic principles
All experimental observations and data should be recorded in a notebook in ink at the time they are made because il is easy to forget when you lire busy. A good-quality HB pencil or propelling pencil is recommended for making diagrdms eiC. as mistakes an: easily correcled with a vinyl eraser. Buy a black, spirit-based (pennanent) marker to label experimental glassware, sample tubes., etc. Fibre-tipped fine line dra\ving,llctlering pens are useful for J)f"eparing final versions of graphs and diagrams for assessment purposes. Use a clear ruler (with an undamaged edge) for gmph drawing, SO that you can see data !X>inL~/infomlation below the ruler a~ you draw.
ClllclIl,,'OI'S These range from basic machines with no pre-programmed functions and only one memory, to sophisticated programmable minicomputers with many memories. The following m;IY be helpful when using a c:llculutor. •
•
Using calculators - take particular care when using the exponential key 'EXP' or 'EE'. PressIng this key produces l()8"""tth,ng. For example if you want to enter 2)( 10 ., the order entry is 2, EXP, -,4 nOt 2, x. 10, EXP, -.4.
Presenting graphs and diagrams - ensure these are large enough to be easily read: a common error is to present graphs Of' diagrams that are too small, with poorly chosen scales.
4
Fundamental laboratory teChniques
•
•
Power sources. Choose a battery-powered machine, rather than a mainsopenlted or solilr-powered type. You will need one with basic m:Hhematical/scientific operations including powers, log:uithms (p. 262), roots and parentheses (brackets), together with statistical functions such as sample means and standard deviations (Chapter 40). Mode of operation. Calculators fall into two distinct groups. The older system used by, for eJmmple. Hewlett Packard calculators is known ,IS the reverse Polish notation: to C'
P"csellliIlC mOl'e IIdJ'tlllced pmcticalll'ol'k In some practical reports and in project work, you m,'ly need to use more sophisticated presentation equipment. Word processing may be essential and computer-based graphics packages can be useful. Choose easily·rcad fonts such as Arial or Times New Roman for project work and posters and consider the la)'out and content carefully (p. 341). Alternatively, you could use fine line drawing pens plus dry-transfer lenering and symbols, such as those made by Lelraset ~, i1lthough this approach is usually more time consuming and less flexible thiln computer-based systems.
Basic principles
Printing on acetates - standfrd overhead transparencie5 are not 6uitable for U89 in laser printenl or photocoP'ers: you need to make 8Uf'8 that you use the-colTEd type.
To prepare overhead t....dnsparencies for oral presentations, you can use spirit-based markers and acetate sheets. An illtemativc approach is to print directly from a computer-based p
Fundament81 laboratory techniques
5
Health and safety HASAW - in the UK, the Hearth and Safety at Work etc. Act 1974 provides the main legal framework for health and safety. The Control of Substances Hazardous to Heatth ICOSHHI Regulations 1994 and 1996 impose specific legal requirements for risk assessment wherever hazardous chemicals or biological agents are used, with Approved Codes of Practice for the control of hazardous substances, carcinogens and biological agents, including pathogenic microbes.
Health and safety law requires institutions to provide a working environment that is safe and withoul risk to health. Where approprialc, training and information on safe working practices must be provided. Students and stafT must take reasonable care to cnsure the health and safety of themselV'CS and of others. and must not misuse any safely equipment.
~ All practical work must be carried out with safety in mind, to minimize the risk of harm to yoursetf and to others - safety is everyone's responsibility by law.
Risk assessment Thc most widespread approach to safc working practice involves the usc of risk asscs.~ment. which aims to establish: J.
Definitions HazMd - the abilitY of 8 substance to cause harm.
Risk - the likelihood that a substance might be harmful under specific circumstances.
Risk - is often associated with the quantitY of chemical to be used, e.g. there is a much greater risk when using a large volume of flammable solvent than 8 few millilitres, even though the hazard is the same.
I-:;~:::=-_ Inhtl~lHm j~ll.stil)/1
j,.JII--
inocul,t"m ~
,1J.so,plil)ll
Fig. 2.1
Major routes of entry of harmful substances into the body.
6
Fundamental laboratory techniques
2.
3. 4.
The inlrinsic chemical and physi~1 hazards. togethcr with any maximum CllPOSUrc limits (MEu) or occuplttionnl ellposurc standards (OESs). where appropriate. All chemical m
The outcome of the risk assessment prOCCS'i mu!>1 be recorded and appropriate safety informalion must be passed on to those at risk. For most practical classes. risk assessmcnts will have been carried out in advance by the person in charge and the information ncttssar}' 10 minimi7.C thc risk.<; to students may be given in the practical schedule. You will be asked to carry out risk assessments to familiarize yourself wilh the process and sources of information. Makc sure you know how your department provides such infonnation and that you have rend the nppropriate material before you begin your practical work. You should also pay dose attention 10 the person in charge at the beginning of Lhe pr"delical session, as they may emphasize the m
Health and safety
LABORATORV RISK ASSESSMENT This stleet must 00 com~ted behlre corrme.u:i,'ll eadl ~
I
Name: Experiment
Synt/lesis 01 NiJ/len~lllanamidll
I
Datil:
1. HlIliJtd and Risk Codes Entet below the coneo Hazard COlle an:! Risk COlle f or IllIdl ,eagcnt. sduenl r-Olkct and trv r-oo..ct tleller an:! runber in the relMlnl boQSI
HAZAfID IHI 3I1d RISK {AI CODES
REAGENT AND
INSTRUMENTS A
2 2 2
W R
M R
,"""" ....
ethilnamde
Elhaooic: acid ldilite aquews soIutionl
.--
__
,.w~ _ _ _ _ , .-
R
, , , , , , ,, , , , , ,,,,,,, ,,,,
Ethar(licll1hydride
A~
F R
"2 R ,", ,, ," ,,R "2 ,, ", ,, ",, ,, " R "
AmirdlenZene laniline)
"
T
C R
2 2
2
2
,-_-
1=_.. --
'_l__
"'-~-
J_...,._
~-o
_
~
~"""
t-$poo;ioI"Ilh_
_
2. f'r«:lIlIIions: cootaiom8llll and pmu,ctian Conu>lnmetlt
St:::t: 01 ExperimlO tine d~ disposall Weillhinll 0llI chenicllk
Penooal Pto1ecOon
,"'d.
_.. --_......__ -----
AddiliOllillltlformation
""'" ............... ,.,..,...,_ ..... ...
1 _ _......... _ _
1_~1.olo
,.....
J~I_~
.~_
~-~
r.. . . - - . - . -....... _ _ """..-_
Protective clothing is worn as a first barrier to spillage of chemicals on to your
body. lab coats are for protection of you and your clothing. Eye protection special spectacles with side pieces to protect you from your own mistakes Bnd those of your colleagues. If you wear spectacles, eye protection with
prescription lenses Bnd side pieces is available from your optician, an expensive but worthwhile investment. Otherwise goggles can be worn over spectacles.
Contact lenses should not be worn in the laboratory. Chemicals can get under the lens and damage the eye before the lens can be removed. It is often very difficult to remove the contact lens from the eye after a chemical splash. Shoes should cover the feet: no open-
toed sandals, for example. long hai, should be tied back and hats le.g. baseball caps) should not be worn.
3. SignaIlII"
Student:
Solpervisllr:
Oae:
Fig. 2.2 An example of a laboratory haUird assessment form.
names and telephone numlx.>J"S of safety personnel, lirst aiders. hospitals, etc. Make sure you read this and abide by any instructions.
Basic rules for laboratory work •
• • •
We-dr appropriate protective clothing at all times - a clean lab coat (butlom.>d up). eye protection, appropriatc rootwear and ensure Y0LU' hair does not constitute a haY..ard. Never smoke, eat or drink in any laboratory, because or the risks of contamination by inhalation or ingestion (Fig. 2.0. Ncvcr work alone in a laboratory. Make sure that you know what to do in case of lire, including exit routes, how to raise the alaon, and where to gather on leaving the building. Remember that the mosl important consideration is human safcty: do not altempt to light a lire unless it is safc to do so. Fundamental laboratory techniques
7
Health and safety
~ ~ e"JIlosive
•
\
/
radioactive
~[K] toKic
oltidiziog
~oo co.-rosrve
he<mflll, Dr Ifrita~1
~[E f1ammahle
biological hazard (biohazardl
Fig. 2.3 Warning labels for specific chemical hazards.
8
Fundamentill laboratory techniques
• •
• • •
• • • •
All laboratories display notices telling you where to find the first aid kit and who to contact in case of accident/emergency. Repon all accidents. even those appearing insignifiamt - your depRrtmcnt will have a reporting procedure 10 comply with safety legislation. Know the warning symbols for specific chemical hazards (Fig. 2.3). Never touch chemicals unless they are known to have minimal hazard: use a spatula to transfer and manipulate solids, and piiXtiCs for liquids sec p. 9. Never mouth pip:::tte any liquid. Use a pipette filler (see p. 10). Take care when handling glassware - see p. 13 for details. Use a fume cupboard for hazardous chemicals. Make sure that it is working and then open the front only as far as necessary: many fume cupboards are marked with a maximum op::ning. Always usc the minimum quantity of any hazardous materials. Work in a logical, tidy manner and minimi7.e risks by thinking ahead. Alway dear up spiJIages, espcci
-----------18
Working with liquids Measuring and dispensing liquids The '-'Quipmcnl you should choose to mc-
volume to be dispensed, the accuracy required and the number of timcs the job must be repeated (Table 3.1). Table 3.1 Criteria for choosing a method for measuring oul a liquid Usefulf"lOlll; for repetitlvo measurement
Best volume ,,,,..
Pasteur pipette Conical f1asklbaaker Measuring cylinder
Volumetric flask Burene Glass pipette Mechanicsl pipettor Syril"lg(l
Microsyringo Weighing
1-5mL 25-5000mL 5-2000mL 5-2000mL l-100mL l-100mL 5-1000/1L
O.5-2iJJ,L O.5-50J,L Any (depends on accuracy
Low Very Jow Medium
High High High HighMedium.... High Very high
Convenient Convenient Conveni6flt
Convenient Convenient Convenient
Convenient Convenient Convenient Inconvenient
01 balance
""If calibrated correctly and used properly (see p. 10). ·--Accuracy depends on width of barnll: large volumes less accurate.
Reading any volumetric scale - make sure your eye is level with the bottom of ttle liquid's meniscus and take the reading from this point
Conical nasks, beakers, measuring cylinders and volumetric nasks measure the volume of liquid contained in them, while buretles, pipettes, pipettors, syringes and microsyringcs mostly measure the volume delivered from them: think about the rcquirentents of the experiment. Certaio liquids may cause problems: • • •
High-viscosity liquids arc difficull to dispense: allow time for the liquid 10 tmnsfcr. Organic solvl.'flls may evapomte rapidly, making mC'dsurcments inaccurate: work quickly; seal containers quickly. Solutions prone to frothing (e.g. surfactant solutions) are difiicult 10 measure and dispense: avoid fonning bubbles; do not tmnsfer quickl}.
Paste",. pipette.\· Hold corn..-ctly during use (Fig. 3.1) - keep the pipette vertical, with Ihe middle finger gripping the barrel to support the pipette while the thumb and index finger provide controlled pressure on the bulb, and squCC'".I.c gently to provide individual drops. To prc\"C'Dtliquid being sucked into the bulb and hence cross-contamination:
--
ill side of Ilillene
bene! $lJJllOf1inQ
ltepipelte
Fig. 3.1
How to hold a Pasteur pipette.
• •
•
Ensure that the capacity of the bulb docs not excetX1 that of the barrel. Do not remove the tip of the pipette from the liquid while drawing up the liquid; the inrush of air may splash the liquid into the bulb. This is panicularly true when you lose patience trying to draw up viscous liquids. Do not lie the pipette on ils side during use.
Com'en;ely, if volatile liquids such as dichJoromethane (DCM), ethanol, propanone (acetone) or diethylether (ether), for example. are to tx: Fundamental laboratory techniques
9
Working with liquids
dispensed, the wamuh of the gills.... pipcuc will cause the liquid 10 squirl
from the pipette witholll any pressure on the bulb. To prevent this. suck up the liquid scvcrdl times inlO the pipette ~o a~ to cool the glass and thcn dispense as normal.
Conical flash and beaken These have llpproximatc gradmllions and should only be used for measuring volumes of solutions{liquids where accuracy is unimporllllli.
Measw'ing cylinders and volllmetJ'ic flu.\'k~' These must be USl-'
Burettes
(.(
('(
(,(
Fig. 3.2 Glass pipettes: Ie) graduated pipette. reading from zero to shouldet"; (b) graduated pipette, reading from maximum to lip, by
gravity; leI bulb Ivolumetric) pipette. showing
Thcsc must be mountL'
volume on bulb.
Pipette!>' Glass pipettes - always check that the pipette is a 'drain-down' type. There may be old 'blow-our pipettes lurking in the back of drawers.
There are various designs, including gradulitL'd and bulb (volumetric) pipettes (Fig. 3.2). Take care to look at the volume scale before use: some graduated pipcucs empty from full volume to 7..cro. others from zero to full volume; some scales refer to the shoulder of the lip, others to the tip by grnvity. Never blowout volumetric (bulb) pipettcs, jLL"t touch the tip against the inside wall of the vessel. Rinse out pipettcs with a little of the solution to be dclivered before commencing the accurate measuremenl. To prevcn! cross-contamination, never draw the solution inlo Ihe pipette !iller. . - . For safety reasons. it is no longer permissible to mouth pipette - various aids (pipette fillersl are available. such as the rubber-bulb and Pi-Pump'l!! lFig. 3.31.
Pipettors (alltopipetton) Using a pipettor - check your technique (precision) by dispensing volumes of distilled water and weighing on a balance. assuming 1mg = 1pL = 1 mm3.
There ,Ire two basic types:
For small volumes, measure several aJiquots together, e.g. 10)( 15pL '"' 150 mg. Aim for accuracy of ± 1%.
2.
10
Fundamental laboratory techniques
I.
Air displacement pipettors. For routine work with dilute aqueous solutions. One of the most widely used is the Gilson Pipetman R (Fig. 3.4). Positive displacement pipenors. For non-standard applications, including dispensing viscous. dense or volatile liquids where an air displacement pipetlor might create aerosols leading to errors.
Working with liquids
Air displacement and positive displacement pipettors may be:
.....
~
• •
_. 0,
• •
1m
E
....
~ipt1te
(.(
Ib(
Fig. 3.3 Pipette fillers: lal rubber-bulb type; (bl
Pi_PumpK.
Fixed volume: capable of delivering a single factory-set volume. Adjustable: where the \'alume delivered is dctcnnincd by the operator across a particular range of values. Pre-set: movable between a limited number of values. Multichannel: able to deliver several replicate volumes at the same time.
Whichever type of these rouline but expensive devices you use. you must ensure Ihal you understand the D!Xrating principles of the volume scale and the method for changing the volume delivered - some pipeltors are easily misread. A pipettor must be fitted with the correct disposable tip before usc and each manufacturer produces different tips to fit particular models. Specialized tips are available for pftrticular applications. If you a«:identally draw liquid into the barrel. seek assistance from your demonstrator/supervisor since the barrel win need to be cleaned before further use (to prevent eross-contamination) and unskilled dismantling of the device will cause irreparable damage. Syringe~'
Using syringes - take great care when handling syringe needles. They are very sharp and may be contaminated by chemicals.
....-
8djustment ring
lwUnell!f
....
Syringes should be used by placing the tip of the needle into the solution and slowly drawing the plunger up to lhe required point on the scale. Check the barrel to make sure no air bubbles have been drawn up. and expel the solution slowly. touching the needle tip on the side of the vessel to remove any adhering solution. If there is air in the barrel. fill past the mark. invert the syringe and push the plunger to the mark so that the air and a little of thc solution are expelled into a waste collection vessel. Then dispense thc solution. The use of syringes for dispensing air-sensitive reagents is described in Chapter 18. Mierosyringcs should always be cleaned before and after use by repeatedly drawing up and expelling pure solvent. 1lle dead space in the needle can o«:upy up to 4% of the nominal syringe volume. Some microsyringes have a fine wire attached to tbe plunger, wbieh fills the dead space. Never pull the plunger out of the barrel.
Balances These can be used to weigh accurately (p. 22) how much liquid you have dispensed. Convert mass to volume using the equation: Mass/density = volume
disposahJe tip -
Fig. 3.4
A pipettor - the Gilson Pipetman \l.
e.g. a liquid (9.0g) of density (1.2gmL~t) = 7.5mL. Densities of common solvents and common chemicals can be found in Lide (2000). You will also need to know the liquid's temperatuTC. since density is temperatuTC dependent.
Holding and storing liquids
Test Illhes Both 'nonna!" and the much smaller 'semi-micro' test tubes arc used for small-scale reactions and tests, e.g. qualitative analysis (p. 135) or solvent selection for recrystallization (p. 93). Fundamelltallaboratory techniques
"
Working with liquids
Beakers Beakers arc used for general purpo~s, e.g. hC'
COlliet,1 (Erlellmeyer) flusks 11lCSC atn be used for gencml purposes. but Ihc) have more specialist applications. The narrow mouth and sloping shoulders reduce losses on stirring/swirling and evaponuion and make them easier 10 seal. The absence of a lip does not fl'l\!our Quantitative transfer: useful in manual titrat;ons (p. 145) and rccryslalli7..at;ons (p. 96). Volume markings arc approximate.
Bottles tlnd .·iuls Storing light-sensitive chemicals - use a brown-glass vessel or wrap aluminium
foil around a clear vessel and ils stopper.
These Oln be used when the solution needs to be scaled for ~fcty, storage or to prevcnl evaporation or oxidation. They usually ha\'c a screwtop. plastic cap or stopper or a ground-glass stopper and comc in various styles and si7..es: from 2.5 L glass bottles used for storing largc volumes of solutions to small plastie
Creating specialized apparatus Glassware systems incorporating ground-glass connections, such as Quickfil!!.. are essential for setting up combinations or standard glass components for reactions. distillation. etc. In project work you may nt."Cd to adapt standard fonns of glassware for a special need. It may be necessary to contaCt a glassblowing service to make special ilems tD order. Table 3.2 Spoctral cutoff values for glass and
plastics ().so = wavelength al which transmission of electromagnetic radiBtion is
reduced to 50%1 Material Routine glassware
Bear in mind the follmving points: ':'50 lnml
Polycarbonate
340 292 396
Acrylic Polyester Quartz
318 220
Pyrex" glassware
12
Choosing between glass and plastic containers
342
Fundamental laboratory techniques
•
Reactivity. Plastic vessels often distort at relalively low temperature, may be inflammable. may dissolve in certain organic solvellts and may be
•
afTected by prolonged exposure tD ultraviolet (UV) light. Opacity. Both glass and plastic absorb light in Ihe UV range of the electromagnetic spectrum (Table 3.2). Quartz should be used where this is important. e.g. in cells ror UV spectrophotometry (see p. 165) or photochemistry.
Working with liquids
•
•
•
Contamination. Some plaStici7Xrs may IC'
Cleaning glass and plastic Special cleaning of glass - for an acid wash use dilute acid, rinse with tap water (three times) to remove the washing solution and then rinse thoroughly at least three times with distilled or deionized water. To remove acidic deposits, wash with KOH}athanol solution followed bV rinsing with dekmizoo water. Glassware, which must be exceptionally clean. should be washed in 8 chromic acid bath, but this must only be made up and used under supervision.
In quantitativc analytiCliI work, beware or contaminat..ion arising rrom prior use or chemicals or inadequate rinsing follo.... ;ng washing. A thorough rinse with distilled or deionized water immediately beforc use will remove dust and other soluble deposits, bUI ensure that the rinsing solution is not lert in the vessel. For analyses on lhe ).lg scale' and below, you should clean glass and plastic containers by immersion in nitric acid (10% vfv) overnight and thcn rinsing with 11 large volumc or distilled or deionized w:llcr. Thc elcan vcssels should be stored either upside down or covered with Oingfilm ~ _ to prevent dust contamination. For general work, 'strong' basic delCr!!Cnts (c.g. Deccln'l or Pyroncg") arc good for solubilizing acidic deposits and an acid wash will rt:1110Vt: remaillingbasic residues. A rinse with ethanol or propanone (aectonc) will remove mallY organic deposits.
Safety with glass Many minor accidents in the laboratory lire due to lack of care with glassware. You should follow these general precaulions: • •
•
•
•
• Fig. 3.5
Handling glass tubing.
Wear eye protection at alltimcs. Don't usc chipped or cracked glassv.are and examine the equipment for 'star' cracks - it may brc-dk under vcry slight strdins and should be disposed of in the broken glassware bin. All laboratories will h~vc a w~stc bin dediolled to broken glass. Never put brokcn glass inlo other bins. Ir heating glassware. use a 'soft' Bunsen flamc (half-opcn air \'Cnt) or 'wavc' the flame around the heating point - this avoids crealing a hot spot where cracks may start. Always use special heat-resistant glovl..'S or rubber 'fingers' (see p. 36) when handling hot glassware. When clamping glassware (see p. 26) ensure that the clamp has a cork. rubber or plastic 'cushion' in the jaws to pfCvent breakages. There must be no metHl-glass contact and you must not over-tighten the clamp. Take care when lluaching rubber or plastic tubing to glass tubes, condensers., etc.• and inserting thcnnomelers and glass tubes into scrcwcap adaptcrs (sec p. 43). Always hold thc tube and the 'holc' close together (Fig. 3.5) and wear thick glovcs where appropriate. Don't rorce bungs too finnly into bollies (see p. 12) they can be difficult to remove. If you need a tight seal. use a screWlop bottle. with a rubber or plastic seal, Parafihn R or ground-glass jointware. such as Quickfit R • fundamental laboratory techniques
13
Working with liquids
• •
14
Fundamental laboratory techniques
Never carry large bottles (> III by their necks - carry them in a bollle basket. Dispose of broken glass thoroughly lmd with great care - usc disposable paper towels, tongs or dust-pan and brush and thick gloves. Always put pil,.-'CCs of broken glass in the correct bin.
--------j8
Basic laboratory procedures I Using chemicals
Safet)' tlspects The Merck Index (Budavari, 1999) and the eRG Handbook of Chemistry and Physics (Lide, 20001 are useful sources on the chemical and physical properties of elements and compounds, including melting and boiling points. solubility and toxicity, etc. Manufacturers' catalogues now include hazard data and disposal
procedures. Table 4.1 Representative risk assessment information for a practical exercise in organic chemistry. the synthesis of N-phenylathanamide
(acetanilidel SUbstance
Hazards/comments
Aminobenzene
Toxic, harmful by skin
absorption. Wear gloves, dispense in fume cupboard Ethanoic anhydride
Corrosive. flammable, toxic,
In pmclical classes, the person in charge has lhe responsibility to infonn you of any hazards associated with the use of chemicals. In project work, your first duty whcn using an unfamiliar chcmical is to find out about its properties, especially those relating to safety. For routine praclical procedures, a risk assessment may have been carried OUI by a member of staff and the relevant safety information may be included in the practical schedule:
reacts with water. Wear gloves, dispense in
•
fume cupboard
• • • • • •
Using chemicals - be considerate [0 others: always return storeroom chemicals promptly to the correct place.
Report when supplies are getting low to the person responsible for looking after the store. If you empty an aspirator or wash bottle. fill it up from the appropriate source. Molar solutions - you will find that chemists talk about '0.1 Molar solutions' or you may see '0.1 M' llS a concentration written on flasks or in books and journals. The term 'Molar" (abbreviated to MI means number of moles per litre. Hence lln aqueous solution of hydrochloric acid (0.1 M) has a concentration of 0.1 mol L-l equivalent to 3.65 9 of hydrogen chloride per litre of solution.
Treat all chemicals as potentially dangerous. Wear a lab cool, wilh the buttons fastened, at all times. Wear eye protection at all times. Make sure you know where safety devices such as eye bath, fire extinguisher, first aid kit arc kept before you start work in the lab. We-.u gloves for toxic, irritant or corrosive chemicals and carry out procedures with Ihem in the fume cupboard. Use appropriate aids such as spatulae, pipette fillers, Pasteur pipettes etc., to minimize risk of contact. label aU solutions, samples, etc., appropriately (see p. 22). Extinguish all naked names when working with nanunable substances. Never drink, eat or smoke where chcmicals are being handled. Report all spillages and clean them up appropriately. Dispose of Chemicals in the correct manner.
SelectiOll Chemicals are supplied in varying degrees of purity and this is always stated on the manufacturer's containers. Suppliers differ in the names given to the grades and there is no confonnity in purity standards. Very pure chemicals cost more, often very much more. and should only be used when the situation demands. If you need to order a chemical, your department will have a defined procedure for doing this.
Preparing solutions Solutions are usuaJly prepared with respect to their molar concentrations (e.g. moll-I, moldm- 3, or molm- 3) or mass concentrations (e.g. gl-l. or kgm- J ): both can be regarded as an amount (usually mass) p;:r unit volume, in accordance with the relationship: . amount concentratIOn = 1 voume
[4.1J
Fundamentatlaboratory te<:hniques
15
Basic laboratory procedures I
Percentage concentration - you may find
that the concentration or a SolUlion is e)(pressed in percentage terms. Thus sodium carbonate (5% wfvl- the symbol w indicates weight of solute and v is the volume of solution. The resulting solution is 8 general-purpose dilute aqueous solution of sodium carbonate prepared from sodium carbonate (59) made up to
100 ml in water and used for neutralizing
The most important aspect of cqn (4.11 i.~ to rccogni7.c clearly the units invoh...-d. and to prql3rc the solution accordingly: for molar concentrations you will need the relative molecular lTlas.~ of the chemical. so that you can calculate the mass of substance required. Further advice on concentrations and intcrconversion of units is given on p. 45. In g....'lCral there are t.....o levels of accuracy rL-quin..d for the preparation of solutions: I.
acid.
(a) solutions used in cxtraction and washing proresscs. e.g. hydrochloric add (0.1 mol L '), sodium hydroxide (2 M), sodium carbonate (5%
The levels of accuracY of sclUllon preparation required are usually indK:ated in the protocol or by the nature of the e)(periment look for phrases such as 'accurately weighed' which means to four decimal places on an anatyhcal balance, together with quantitative transfer.
Volumes Quoted as 250.00 mL 100.00 mL 25.00 ml imply Ihe use of volumetric ftasks and pipettes.
General.purpose solutions - solutions of chemicals used in I.jualitative and preparative procedure; (p. 17) when the col1Ct,."tration of the chemical need not be known to more than one or two decimal places. For example:
wI"); (b) solution~ of chemicals used in preparative experiments where the
2.
techniques of purification - distillation, recrystallization. filtration. etc. - introduce intrinsic losses of substances thai makc accuracy to any grcatt.'f I...." ,d lTleaninglt$.~. Analytical solutions - solutions used in quantitative anal}'tial.l procedures when the concentration needs \0 be known to an accuracy of four decimal places (e.g. O.fXX)1 mol L -I). For example in: (a) volumt:tric procedures (litrations) and gravimetric analysis, where the concentrations of standard solutions of reagents and compound~ to be analysed need to be aceuratt:ly known; (b) spectroscopy, e.g. quantitative UV and visible spt:ctro.scopy. atomic absorption speclroscopy and flame photomelry; (c) electrochemical measurements: pH titraliotls. conductance measurements and polarog"'dphy; (d) chromatographic methods.
The procedures for weighing and the glassw"dl'C USL.od in the preparation of solutions differ according to the level of accur3t.'}' requin.-'d.
Prep(lr(llion ofgenend-pllrpQse .fOllllions Box 4.1 shows the stt:ps involved ill making up general-purpose aqUt.'Ous solutions. The concentration you require is likely to be ddincd by the protocol you arc following and the grade of chemical and supplier may also be spt."Cified. To avoid waste. think carefully about 1m: volume of solution you n..oquire, though it is always advisable to err Oll the high side because you may spill some, make a mistake when dispensing or need to repeat part of the experiment. Otoose one of the standard \'olumes for vessels. as this will make measuring out easier. Use distilled or deionized waler to make up aqueous solutions and stir with a dean Pyrex· glass rod or magnetic stirrer bar ('Ilea") until all the chemK:a1 is dissolved. Magnetic stirrers are a convenient mrnns of stirring solutions but precautions should be taken to prevCl1t losses by splashing. Add the flea to the empty beaker or conical flask. add the chemical and then some water. Place the ves.c;e! centrally on 1m: stirrer plate. switch on the stirrer and gradually increase the speed of stirring. When the crystals or powder have dissolved. switch off the stirrer and remove the Ilea with a magnet. Take care not to contaminate your solution when you do this and rinse the Ilea into the solution with distilled water. In general it is convenient to use glass rods with 16
Fundamental laboratory techniques
Basic laboratory procedures I
Box 4.1
How to make up an aqueous solution of known concentration from a solid chemical
1. And out or decide the concentration of chemical required and the degree of purity necossary,
2. Decide on the volume of solution required. 3. Find out the relative molecular mass of the chemical IM,I. This is the 8um of the atomic {elemental) masses of the component elementls) end can usually be found on the container. If the chemical is hydrated, i.e. has water molecules associated with it, these must be Included when calculating the mass required.
I
4. Work out the mass of chemica' that will give you the concentnrtlon desired in the volume required. bearing in mind the quoted percentage purity of
the chemical. Examplf] 1: Suppose your procedure requires you to prepare 250ml of 0.1 molL-1 sodium chloride solution.
tal Begin by expressing all volumes in the same units. either millilitres or litres (e.g. 250 mL as 0.25 L).
fbi Calculate the number of moles required from eqn (4.11: 0.1 = amount (moll + 0.25. By rearrangement, the required number of moles is thus 0.1 x 0.25 _ 0.025 mol. lei Convert from mol to 9 by multiplying by the relative molecular mass (M, for NBCI = 58.44 9 mol-'). (dJ Therefore, you need to make up 0.025 x 58.44= 1.469 up to 250mL of solution. using distilled water.
Example 2: Suppose you are reqUired to make up l00mL of sodium carbonate (2M). (a) Convert 2M into moIL-'; concentration reQuired "'" 2 mol L-'. (b) Express
all volumes in therefore 100 ml = 0.1 L
the same
units:
(c) Calculate the number of moles required from eqn (4.1}: 2 = amount (mol) -:- 0.1. By rearrangement. the reQuired number of moles is thus 2 x 0.1 _ 0.2 mof. ld) Convert from mol to 9 by multiplying by the M, but note from the container that. the compound is Na2C03.1OHzO. Therefore the
M,. required must include the water of crystallization and M r ;;:: 286.149 mol-'. (0) Therefore. you need to make up 0.2 )( 286.14 :;57.2g up to 100mL of solution using distilled. water. 5. Weigh out the required mass of chemical to en appropriate accuracy. tf the mass is too small to weigh with the desired degree of accuracy, consider the following options: (e) Make up a greeter volume of solution. (b) Make up a more concentrated solution, which can be diluted at a later stege. (c) Weigh the mass first, and calculete whet volume to make the solution up to afterwards using eqn 4.1. 6. Add the chemicaf to a beaker or conical flask and then add a little less water than the finat v<»ume required. If some of tho chemical sticks to the weighing receptacle, use some of the water to wash it off. For accurate solutions. see p.. 19 for accurate weighing Hnd quantitative transfer.
7. Stir and. if necessary. heat the solution to ensure 511 the chemical dissolves. You can determine visually when this has happened by observing the diseppaarance of the crystals or powder. Allow the solution to cool, if heated.
S. Make up the solution to the dasired volume. If the concentration needs to be accurate, use a volumetric flask (see p. 19 for accurate weighing and quantitative transfer); if a high degree of accuracy is not rSQuired, use a measuring cylinder. (a) Pour the solution from the beaker into the measuring vessel using a funnel to avoid spillage, using water to rinse out the vessel. (b) Make up the volume so that the meniscus comos up to the appropriate measurement line. If accuracy is not a major concern,. the graduation marks on the beaker or conical flask may be used to establish the approximate volume.
9. Transfer the solution to a reagent bottle or conical flask and label the vessel clearly, including hazard information. where 8ppt'"Opriate. 00 not use water in this final transfer since you will alter the concentration of the solution by dilution.
beakers - case of access for stirriDg - aDd magnetic neas with conical flasks lower losses through splashing - bUl often il is a matter of your preference and laboratory skills. 'Obstinate' solutions may require heating, bUI only do thjs if you know Ihal the chemical wi]] Dot be damaged at the temperature used. Usc a stirrcrFundementallabotatory techniQUes
17
Basic laboratory procedures I
healer to keep the solution mixed as you heal it. Allow the solution 10 cool 10 room tcmpcralur~ before you finalize its volume.
p,.eptu'ut;on of analytical sollition... The key featufL"S in the prcraration of solutions for analytical purposes are: • •
Make sure that you have the most accurate available knowledge of masses of the chemicals used. Ensure that you have the most accurate available knowledge of the
voluo1t:s of Solulions used. To achieve thdlC features exact techniques of weighing and solution transfcr must be used and the procedure is illuslrall.-d in the following example.
A standard solution is one in which the solute is weighed out to an eceuracy of 4 decimal places and is made up in 8 volumetric flask. A stock solution fs one from which dilutions are made.
'Prepare a standard solution (250.00ml) of ammonium ferrous sulphate (approximately 0.1 M), which is to be used 10 detennine the concentration of a solution of potllssium penmmganale by titration'. You must be aware of the following embedded information: •
• • •
Fig. 4. J Pouring a solution using B glass rod.
Thi.~
is a quantitative experiment therefore requiring an analytical solution to be prepared. You mu~t usc II 250.00ml volumetric flask, which you should nOle is calibrated at 20"C. You must weigh atturately. to four decimal places, the mass of the cht.'fTlical. 11 is
Box 4.2 shows the method for lhe prepanl.lion of the standilrd solution. 11le main practical point is thai you must not lose. by splashing or failure to trdnsft.T by inadt.'
Procedure required with analytical so[,dions prepared from liqlll'Js Many experiments in analytical chemistry, such as chromatography and spectroscopy_ require the preparation of a stilndard solution of a liquid organic compound. 1ncrefore you must know accurately the mas'> of the liquid. The compound can be dispensed by tbe methods described in Chapter 3. provided thaI the pipette. syringe, etc., is aCCUrdte. and thus the mas.." = volume x density, bearing in mind the temperature factor. 18
Fundamental laborlltory techniques
Basic laboratory procedures I
Box 4.2
How to make up an aqueous solution of known concentration from a solid chemical for
use in quantitative analysis Example: Suppose you are to prepare a standard solution (250.00 mll of ammonium ferrous sulphate (approximately 0.1 M), which is to be used to determine the concentration of a solution of potassium permanganate.
is a quant;tative experiment so the ammonium ferrous sulphate must be of the highest purity available to you. Work out the mass of chemical that will give you the conC6ntnmon desired in the volume required. (a) Convert 1.0 M into mol L_1; concentration required = 1.0 mol L-1. (b) Express all volumes in the same units: therefore 250.00 mL = 0.25 L (c) Calculate the number of moles required from eqn [4.1J: 1.0= amount (mol) -;.. 0.25. By rearrangement, the required number of moles is thus 1.0 x 0.25 = 0.025 mol. (d) Convert from mol to g by multiplying by the M.. but note from the container that the compound is (NH t hFeSO•.6H20. Therefore the M r required must include the water of crystallization and M.=392.14gmol l . (e) Therefore, you need to weigh out 0.025 x 392.14 = 9.8035g of (NH t hFeSO•.6H zO. Place a clean. dry weighing boat or appropriately sized sample tube onto a simple two-decimalplace balance (see p. 22) and zero (tare) the balance and weigh about 9.809 of the chemical. Carefully transfer the sample tube plus chemical to a four-decimal-place analytical balance (see p. 231 and record the accurate mass: say 11.9726g. Remove the sample and container from the balam:e and tip the contents into a clean, dry beaker 1400mU. ensuring that there is no spillage outside the beaker. Do not attempt to wash out
1. This
2.
3.
4.
5.
the sample tube with water.
6. Immediately reweigh the sample tube on the analytical balance: say 2.1564g. This is the mass of the container together with a few crystals of the chemical which have remained in the container. However, you now know exactly the mass of the chemical in the beaker: 11.9726 - 2.1564 = 9.8162g of (NH.)2FeS04.6HzO.
7. Add deionized or distilled water (about 100 ml) to the beaker and stir the mixture gently with a dean Pyrex'lJ. glass rod until all the solid has dissolved. Do not splash or spill any of this solution or you cannot calculate its concentration. Remove the glass rod from the solution. rinsing it with a little distilled water into the solution. 8. Clamp a clean volumetric flask for support (see p. 26) and place a clean, dry filter funnel in the top supported by a ring. Carefully pour the solution into the volumetric flask. ensuring no spillage of solution by using the technique illustrated in Fig. 4.1 and pouring slowly so that no air-lock is formed and no solution runs down the side of the beaker. When the addition is complete, do not move the beaker from its position over the funnel.
9. Rinse the inside of the beaker several times with a distilled water wash bottle to transfer all of the solution into the volumetric ftask, paying particular attention to the 'spouf and glass rod. Then place the beaker aside and lift the funnel from the flask while rinsing it with distilled water. You have now achieved a quantitative transfer. Swirl the liquid in the flask to prevent density gradients. 10. Make the solution up to the mark using distilled water. stopper the flask and mix thoroughly by gentle inversion (10 times) of the flask while holding the stopper in place. You now have a solution (250.00 mL), which contains (NH,il2FeS04.6HzO 19.81629). The concentration of this solution is expressed as: 250.00 mL of the solution contains 9.8162 9 of (NH.hFcSO...6H20 Therefore: 10oo.00mL of solution contains
The concentration of the solution is 39.2648gL- 1 =39.2648-;-392.14= 0.1001 moll- 1 = 0.1001 M
Alternatively you can usc a weighing boule as shown in Fig. 4.2. The liquid is placed in [he bottlc, weighed
19
Basic laboratory procedures I
milde up to the mark and stoppcrt:d. You now know the concentration of the standard solution 10 four decimal places.
Preparing dilutions A1aki/lg a single dilution In analytical work, you may m:ed to dilute a standard solution 10 give il particular mils...; concentration or molar concentration. Use the following procedure. I.
2.
3. Fig. 4.2 A weighing bottle.
Making a dilution - use the relationship
IC1IV, = Ic;JV2 to determine volume or concentration
(see
p. 48).
Using the correct volumes - it is important to distinguish between the volumes of the various liquids: a omrin·ten dilution is obtained using one volume of stock solution plus nine ~umes of diluent (1+9-10).
Transfer an appropriate volurnt: of standard solution to a volumetric flask. using appropriale equipment (Table 3.1). Make up 10 the calibration mark with soh't:nt - add the lasl ft:w drops from a Pasteur pipene until the bottom of the meniscus is level with the calibmtion mark. Mix thoroughly, either by repeated inversion (holding the !>1:oppcr firmly) or by prolongt.'C1 stirring, using a magnetic stirrer. Make sure lhat you add the: magnetic flea after the volwnc adjustment slep.
For general-purpose work using dilute aqueous solutions where the hight.... degree of accumcy is not n..'quired, it may be acceptable to sU~lilute conical flasks, beakers or test tubes for volumetric nasks and usc measuring cylinders for volume measurements. In such cases you would calculate: the volumes of 'stock' solutions (usually 'bench' reagents) and dilUt:nt required. with lhe a:..">umption that the final volume is detennined by the individual volumes of stock solulion and diluent uStXi. Thus a two-fold dilution would be pn.'Pan.'C1 by using one volume of slock solution and one volume of diluent. The dilution factor is obtained from the initial concentration of the stock solution and the final concentration of the diluted solUlion. TIle dilution factor can be used 10 calculate the volumes and stock and diluent required in a particular instancc. For example, suppose you wanled 10 prepare lOOmL of a solution of NaOH at 0.1 mol L I. Using the bench rca~nt, commonly containing 2.0mol L- 1 (2.0M), the dilution factor is 0.1 +- 2.0 = 0.05 = 1/20 (a twenty-fold dilution). Therefore the amount of stock solution required is 1{201h of lOOml = 5mL and the amount of diluent needed is 19/20Ih of IOOmL = 95mL.
Preparing a dilution series Dilution series are used in a wide nmge of procedures including Ihe preparation of standard curves for the calibration of analytical instruments (p. 171). A vdriety of different approaches can be used but the most conunon is a lil/Mr di/l//io" series. In a linear dilution series the concentrdlions arc separated by an equal amount, e.g. a series colllflining cadmium at 0, 0.2. 0.4, 0.6, 0.8, 1.0mmol L-' might be used to prepare a calibration curve for atomic absorption spectroscopy (p. 170) when assaying polluted soil samples. Usc ICdV1=LCiIV2 to calculate the volume of !>1:andard solution for each member of the series and pipette or syringe the calculated volume ioto an appropriately sized volumetric flask as described above. Remember to label clearly each diluted SOlution as you prepare it. since it is ~ to get oonfusal. Hie process is oulliDed in Box 4.3.
~
Make all the dilutions from the working stock solution to the required solution. Do not make a solution of lowet'" dilution from one already prepared: if you have made an elTor in the first dilution, it will be r-epeated for the second dilution.
20
Fundamental laboratory techniques
Basic laboratory procedures I
Box 4.3
How to make up a linear dilution series for use in quantitative analysis
The experimental protc:x:cl states: 'Prepare a standard solution {250.oo ml; 0.01 MI of cadmium ions using cadmium nitrste and use this solution to produce 8 linear dilution series of solutions noo.oo mLl of accuratetv' known concentrations of approximately 0.0, 0.2, 0.4, 0.6, 0.8,. 1.0mrnoll-1"
,. Cak:ulate the amount of cadmium nitrate req,,*ed for the standard solution: cadmium nitrate is supplied 8S the tetrehydrate Cd(NOJ~.4H20; M = 308.47 g mol '. Therefore. using eqn [4.1l you should cetculate 250.00 mL of en 0.01 M solution of cadmium ions requires 0.01 x 0.25 = 0.0025 mol C: 0.0025 x 308.47 E 0.077129 of Cd(NO:Jh. 4H20. Remember: this solution will contain 0.0025 moles of 'cadmium nitrate' or 0.0025 moles of cadmium ions and 0.005 moles of nitrate
ions. 2. Make the standard solution by the Quantitative method described in Box 4.2 and calculate its concentration to four decimal places. 3. Calculate the vokJlTNt of soIutkM1 required for the dilution series using lCIJV, = fC:lJV:l, where VI = volume (mL) of standard s06ution required; C, "" concentration of standard solution; V2 = volume of diluted solution (1oo.oomL) and G.! = COl"'lCen-tration of diluted solution. 4. Express the oonc:enbaticMd In the serne units, the most oonvenient in this case being moIL-1. Therefore, Ct = 0.01 M = 1 X 10-2 moll- 1 and for the diluted solution of concentration 0.2 mmoll-'. ~ = 0.2 x 11)3 moll-1. By rearrangement V,
~
5. Transfer th. standard sokItIon ~ mLJ to the volumetric fIMk "00.00 DIU and Il\IIke up to tile mark with distilled water.
'01"
6. RepeIrt: the calculation each of the other diluted solutions _ required, bul note that 8 short cut is possible in Ihis C8/1r. :!linee you require stock solution (2.00mL) for the diluted solution of con'" centration 0.2 mmol L-1, you will need 4..00mL for the 0.4mmoIL-l soIutto"n· etc. Use pure distilled water for the solution of concentration 0.0 mol L_1.
7. Cakt*te the Pact concentrlltlona 01 the diluted solutions using tc.jV, = 1~]V2. since the c0ncentration of the standard solution Is most unlikelv to be exactly 0.0100 mol L-1 (see Box 4.2). For example, if the concentration of the st8ndard soMion IC,I is actually 0.00987 mol L-' =9.87 x 1()3 moa -1, then for the dilute solution of approximate concentration 0.2 mmol L 1, the actual concentr8tion {~) is:
fC2l =
{V, x
ICl l} .... V2
0.2 ml x 9.87 x 10.-.3 moll- l l00.00mL' , j.
,
-= 2 x 9.87 x 10-& moll-l = 0.1974mmoll-.1 . NOle: It Is much simpler to measure out whole-number volumes (2.00 ml. 4.00 mL. etc.) using 8 pipette and produce diluted so/udons of accuretely known concentrelions (but not necessarily whole, numbers) rether then to try to prod~ whole-nun;'lber co~ntTl'ltiOf'lS by measuring out non--whoie-1'lumber v06umes..
.
{Ic,!V,) +fC,]
..;;. 0.2 x ltJ3 moll _1 x 100.00 ml = 2.00 ml 1 x ffil mol L- 1
Sto";ng chemicals and sollilions
Seal[ng flasks - think carefully about the sealing system for a flask 10 be stored at low temperature. Cooling will reduce the volume of vapour (including air) in the flask and create a partial vacuum. Rubber bungs can be i"emovable aftar cooling (another good reason for never using theml and even ground-glass joints can seize up. Plastic stoppers, saewtops or lightly greased glass stoppers, as appropriate, are recommended.
Chemicals wlUch decompose easily (labile cht,'Illicals) may be stored in a fridge or freezer. Take special care when using chemicals which have been stored at low temperalure: lhe container and its contents must be allov.cd to warm up 10 room temperature before use, otherwise 'Nater will condense onto thc chcmical. This may render accurate wt,;ghing impossible and you mllY ruin the chemical. Chemicllis and solutions to be stored al low tcmpe.mturcs musl be in stoppercd or sealed vessels. Do nol store aqueous solutions below OCC since freezing can occur and, with the resulting expansion of the volume, the vessel may crack. Solutions containillg flammable solvents should only be stored in specialized 'spark-proor fridges: consult your laboratory instructor. You must be aware of lhe parlicular problems of storing solutions ill flasks with ground-glass joints. If you are using aqueous SOIUtiOllS you should Fundamental labor1ltory techniques
21
Basic laboratory procedures I
Greasing joints - if you are using solutions of NaOH Of KOH you must grease the ground-glass joint and stopper since the surfaces are anacked by strong alkalis.
lightly grease the joinl and stopper with pctrok:um jelly. since the water will not dissolve the grease as it is poured from the flask. The stopper can be rt.-moved easily and the solution will be uncontaminated. CO",'~I~'. if you are using solutions made up from organic solvents. you should 110' grease the joints since the orgnnic solvent will dissolve the greasc as you pour it from the Oask and contaminate the solution. Moreover, you should not allow the solution to come in contliet with the ungreaSt:d joints, since the solvent will evapor'd.te and leave the solute to 'weld' the stopper to the flask. Fill the flask with solution, using a filter funnel with the stem of the funnel positioned well below the joint. See p. 43 for use of ground-gl'L"5 jointwarc.
~ Always label all stored chemicals dearly with the following information; the name (if a solution. state solute(s) and concentration/sll. plus any relevant hazard warning information. the date made up. and your name,
Balances and weighing Electronic singlt,.... pan balance; with digital readouts lire now favoured over mechanical types and are common in most labor.:ttories, There are es..-.entially two types of ballince: I.
2.
Weighing - neverwcigh anything directly onto a balance's pan: you may contaminate it fOr others. Use an appropriate weighing container such as 8 weighing boat, sample tube, weighing
paper, conical flask, beaker.
'Weighing paper' - It is common practice to put 8 piece of paper onto the pan of general-purpose balances. The mass of
the paper is then 'Iared off before the weighing container is placed on the balance pan. The paper protects the balance pan from corrosion by spillages and also allows you to discard easily any malerial spilt without affecting the
weighing.
Both types are illustrated in Fig. 4.3 and you should familiarize yourself with their operation before use.
Genel'aJ-purpose balances The most useful fealure of this type of balance is the electronic lero facility (self-taring), which means the mass of the weighing container can be subtracted automaticdlly before weighing chemicals. To operate a standard self-taring balance: I.
2. 3.
4.
22
Fundamental laboratory techniques
General purpose balances which v..'Ci.gh to the nelirest 0.01 g with a capacilY of about 300 g. Chemicals may be dispensed for weighing, into a suitable weighing container, directly Ollto these balances. Analytical four-figure balances for quantitative work, which wcigh to the nearest 0.0001 g (0.1 mg) and have a maximum clipacity of libout lOOg. Chemicals must not be transferred onto the ballince at any lime and analytical balances must only be used for weighing by difference.
Check Ihal it is level, u."ing the adjustable feet to centre the bubble in the spirit level (usually at the back of the machine). For relatively accurate work or when using in a fume cupboard, make sure that the drought shield is in place. Ensure that Ihe balance is switched on: the display should be lit. Place an emp(y \\eighing container (see p. 24) centrally on the balance pan and allow the reading to stabili::t--C. If ,lie objet.:t is larger dum Ihe pall. take care ,hal no purl rests OIl the body of the lxlltUlce 0,. 'he draugh, shieltf as this wil/ illl'alidale the rfflding, Press the tare bar 10 bring the reading to zero. Place Ihe chemical or object carefully in the weighing vessel: (a) Solid chemicals should be dispensed with a suitably SiZL'
Basic laboratory procedures I
It is poor elCperimental technique to use large containers to weigh out small m86Se5: you ere attempting to measure small differences between large number.>. For ~mall masses. use a smaM weighir'lg containar (Fig. 4.41.
5.
6.
7.
(b) Non-volalilc liquids should be dispt'fls(''<.! using H PHsteur pipette bUI IHke the weighing comainer off the balance pan before dispensing; lhen re\\t:igh the liquid plus conlainer. Repeal until Ihe dcsir,,-d weighl is obtained. Allow the rcading 10 stabilize and make a note of lhe rcading. If you have added cxcess chemical. lake grcHl care when removing it. Remove the container from Ihe balance, remove the solid (with a spatula) or liquid (with a Pasteur pipctle) and reweigh. If you need 10 clean any deposit acddcntally left on or around the balanre, switch off the balance.
Take Qlre not to exceed the limits for the balance: while most have devices to protcct against overloading, you may damage the mechanism.
Allalytical bala/lce... 1llcse are delicate precision instruments and as such are likely to be found aWlIY from the open laboratory in draught.froc conditions on a vibrationrlampcocd surface. Analytical balances are maintaint:d to the highest specifications and should nL"C(\ no adjustments on your part, such liS kvelling lind zero adjustment. The key points for lIsing an analytical balance are sumlrulrized below:
• • •
No chemicals must be tnmsferred within the weighing compartment of the balance. If il hlls a 'locking' function, the balancc pan must alwlIYS be 'locked' when placing lind removing objccts onlo and from the balance pan. 1lIe doors of the balance must alwlI)'s be dosc.:d when taking mC'
The procedure for weighing a solid chL'Illical for the preparation of an analytical standard solution is shown in Box 4.4.
{.,
Weighing c(Jnfailten These come in various malerials. shapes and sizes: rrom glass weighing boats 10 bclikcrs lind even special glazed paper. The weighing container to be used depends on several factors: • • • •
., Fig. 4.3 Single-pan balance; (a} gen~al purpose. two decimal places: (blanalytica1. four
decimal places.
The amount of chemical to be weighed. The properties of the chemical to be weighed: is it solid, liquid, volatile, corrosive, dcliqucsa:nt. hygroscopic? How and into what type of vessel il is to be transferred. The accuracy to which it is to be weighed.
Somc of the common t)/PeS of weighing container are shown in Fig. 4.4. For analytical procedures. ooly weighing boRts. weighing bottles or glass or plastic sample tubes should be usc..-d. Weighing boats arc used to transfer a solid direclly into 11 volumetric flask via the Deck of the \vcighing boat: this procedul"C is reconuncndcd when the chemical is known 10 be totally soluble in the solvent and allows you to omit the solution preparation stage in a beaker or conical flask (sec Box 4.2). You must ensure thai the neck of the weighing boat will iii wdl inside the ground-glass joint of the neck of the volumetric flask so that all the chemical can be washed down the sidt.'S of the volumetric flask and does not slick Lo the ground-glass joint during the quantitative transfer. Fundament.lll laboratory techniQues
23
Basic laboratory procedures I
Box 4.4
How to weigh out a sample of a solid for use in
quantitative analysis 1. Place the dean. dry weighing boat or sample tube on 8 generalpurpose two-decimal-plllC8 balance and zero the balance. 2. Weigh out the calculated amount of chemical within the accuracy of the balanCe.
\-,
3. Check the zero reading on the anatytical balance by pressing the barlbutton with the balance doors closed.
4. Relock the balance pan bV pressing the bar!button. 5. C.efulty transfer the weighing boat or samp5e tube to the balance pan of the analytical balance (fOt" very accur8te work use tweezers or fine tongs since the S\NCat from your fingers will contribute to the weight recorded) and close the balance door.
6. Reaease the balance pan by pressing the bar/bunon. allow the balance to stabilize and record the weight of the chemical and container. If the last decimal place 'cycles' between two or three numbers, determine the mid-point of the 'cycle' and record this value as the weight.
Ib\
7. Lock the balance pan by pressing the bar/button. remove the sample container and transler the solid to your volumetric flask, beaker or conical flask by pouring, but do not wet the weighing boat or sample tube with solvent. \'\
Fig. 4.4 Weighing containers: (a) plastic weighing dishes; lb) weighing bottles; lei weighing bo8t.
8. Replace the weighing container on the analytical balance pan, close the balance door and weigh the container. Again decide on the mid-point weight if the last decimal place 'cycles' and record this value as the weight of the 'empty' weighing container.
9. Lode the balance pan by pressing the bar/button and remove the weighing container from the balance. 10. Subtract the weight of the 'empty' weighing container from that of the weighing container plus sample and you now know the mass of chemical, to an accuracy of four decimal places, which has been transferred into your volumetric flask. beaker or conical flask.
. -<
. ;~.~ "
"
,,
?
Fig. 4.5 Transferring a solid using glazed peper.
24
Fundllmentallaborlrtory techniQues
For general-purpose work, wcighings can be made directly into pn..weighed or 'tared' conical flasks or beakers, again to avoid a transfer stage. Much more conunon is the use of disposable plastic weighing dishes of the appropriatc sizc. The edges of these dishes can be squeezed together to form a 'funnel' to prevent losses when transferring the solid. Remember that p1a.stic disposable dishes may dissolve in organic solvent.. such as propanone (acetollC), toluene. etc., and should not be used for low-melling organic solids or liquids. Watch- and clock-glasses should be avoided if you wish to transfer the solid into narrow-neckl..od vessels such as conical flasks or sample tubes since it very difficult to dira:t the solid into the narrow opening of the vessel from the large -flar surface of the wateh- or clock-glass. In such cases, or when large amounts of solid are 10 be trdnsferred, it is advisable to tl'iC a wide-necked filter funnel called a 'powder funnel'.
Basic laboratory procedures I
..
....';to.
~(.
/. ...
---
y:.''':::'=:.-- ...
1------ .*:d ~ paper
"
+-- ....
In many prepal'3ti\'e experiments. which art~ carried out on a small scale (involving I g to 109 of solids), the most lL~rul W(.ighing container is special glazr.."<1 paper, providt.'(l Ihal the chemicals do not react with the p
Fig.4.6 Transferring.ll solid 10.11 narrow-
necked flask.
Fundamental laboratory techniques
25
----------18
Basic laboratory procedures II Clamps and support stands When carrying oul experiments il is ,"ital that all aPPanilus is held in phi"'C securely during lhe procedure. It is essential that you know how to assemble supporting i1nd securing equipment to [he highest possible standllrds of safety. The most common types of supporting and securing equipment
Support stU/Ids These are .Illso known as 'retort stands' and comprise ;In aluminium or sleel
---
-- --
==~
-
rod screwed into a heavy metal base. AlwlI}'s check lhal lhe base sits level and thai the rO<.l is tightened fully in place.
Clump holders These are also described as 'clamp bosses' or 'bosses', Make sure lhal the locking screws movc freely and are not distorted. When you atlach the clamp holder 10 lhe supporl stand. tighten the scm.. . firmly and ensure lJUti Ihe open 'slot' 10 be used for lhe damp is poinling upwards (Fig. 5.2).
Clamps General-purpose clamps are used for securing glasswltre - therefore make sure Ihat Ihe inncr surfae~ of thc clamp "jav,'s' and thc 'fingers' are covered with cork or rubber to provide a cushion for the glass: Ihere must be no melal to glass contact in case you overtightcn the clamp and crush the glass. Tighlen the clamp finnly and ensure thai the clamped glassware does not move. Conical flasks should be damped al the neck and ground-glass joinlware should be clamped al the joinl - this usually has lht: greatesl thickn(..~ of glass.
~ Take particular care when using parallel-sided separa-
tory funnels and chromatography columns (Fig. 5.31, where clamping in the middle of the funnel can be the same as squeezing the middle of 8 largtHiiameter glass tube. Clamp at the ground-glass joint using a 'wellcushioned' clamp.
Fig. 5.1
Clamps and supporting equipment.
Using support rings - make sure that the tap on the separating funnel will go throu~h the ring.
26
FundamentallabOfatory techniques
In most clamps. only Oil/;" of the jaws moves when turning the screw. When you use Ihe damp in a horizontal position. make sure that the movable jaw is al the lop (see p. 109). Burette clamps arc specially designed 10 hold the burette vertically. Springs hold lhe burette at two poinls aboul Scm apart - again check for lhe presence of a rubber or plastic 'cushion' at thc poinls of contact - to prevenl slippi.ng and lateral movement. Since the burene damp slides do'Ml the rod of the support stand. then provided lhe support stand is vertical. the burene will be vertical.
Support rings T~ metal rings come in various diameters to support filter funnels and separalory funnels. Often these support ring> are coaled in plastic to provide the cushion between metal and g1a~.
Basic laboratory procedures II
If your support ring is metal. you can make a 'cushion' by finding a piece of thin-walled rubber tubing of the same bore as the metaJ of the ring, cuning it to a length equivalent to the circumference of the ring and then cUlling down the lenglh of the rubber tubing. You can then slide Ihe tubing around the ring to provide the 'cushion',
~ When using damps, support rings and support stands make sure that the clamped/supported apparatus is always in position above the base of the support stand lFig. 5.41 to prevent the stand toppling over.
OlO\llll:llejaw on TOP
~
Cork rings
fixed jaw undemealh 10 suPlJCll'\. e.g. a condenser
WRONG
Right and wrong ways of using suppOI1 stands, clamp holders and clamps. Fig. 5.2
·l1u:se are used 10 hold round-bouom nash on nat surf<1CCS while
manipulalions arc being carried oul. Since they are light in weight Ihey can be used to hold round·bonom nasks on a general-purpose balance.
Filtration
There are a few occasions when these rules do not a~, e.g. when the so.lfd is to be decomposed by heat and the filter paper destroyed, a proceSs known as >ashing·. Some gravimetric analyses, such as the determination of sulphate as barium sulphate, require retention of the precipitate from gravity filtration sinee BaS04 is too fine to be collected on a vacuum filtration system. BaSO. is thermally stable up to 600 C, so the filter paper Can be bumed away,
-
"'' T Fig.
5.3 Clamping a parallel-sided funnel,
Filtmtion is the physical sepamtion of a solid from a liquid and is a process encountered in experimenlal procedures sUl;h as gravimelric analysis (p. 139), recrystallization (p. 92), and solvent drying (p. 41). In principle. the mixture of the solid and liquid is. passed through a porous material, filter paper or simered glass, and the solid is tmpped on the porous material willie the liquid passes through. "l1le type of filtration equipmt"tlt you select for use depends upon which of the Iwo components, Ihe solid or the liquid, you are Irying to isolate. Ln general: • •
If you wish lo isolate the liquid - lise gravi/}'jillra/iotl. If you wish 10 isolate the solid - lise suction (IYlCtlum) jil/ration
Gravity jiJI.-alion In gravity filtration you need to p
27
Basic laboratory procedures II
Box 5.1
How to flute a filter paper for gravity filtration
1. Fold the fitter paper in hatf 1.1, open it out then fold into quarters (bl, open it out and then fold into eighths (e and d), all in the same dieretion.
2. Tum the papet' over and fold each sector in half Ie); you are creating sixteenths but with each fold in the opposite direction to (a), (b), (e). and ld).
3. Finally, fold the paper into. cone Igi ensuring that all the folds are sharp and that the base of the cone comes to a sharp point.
4. The flutes ensure that the filter paper has minimum contact with the filter funnel and the sharp point ensures that the liquid flows rapidly out of the cone and out of the funnel.
Fig. 5.4 Correct use of support stands.
5. If your cone point is blunt it wilt cover the stem of the funnel and so all the liquid must pass through this part of the filter paper, slowing the filtralion.
.,
Fig.5.5 Gravity filtration.
Filtering solids - if you allow the solid to settle you will have difficulty pouring the thick slurI)' of solid and liquid from the bottom of the beaker or conical flask into the filter cone and you will need to use more filtrate to complete the filtration effectively.
To filter the mixture; swirl the suspension of the solid in the liquid so that there is a fairly even distribution of solid in the liquid. and then pour the mixture into the filter cone, making sure that you do not pour any of the mixture outside the filter paper otherwise you wiU need to repeat the filtration, and do not overfill the filter cone. Transfer all the mixture in this way and finally wllsh the last bit of solid and liquid into the filler cone with a small t/llWllnt of fillcred solution and then a /irr/t> pllrt> solVi'nt.
Suction filtration This technique is used for the isolation of a .~o/id from a suspension of a solid in a liquid and relies on prodUl..i ng a partial vacuum in the receiving Oask. The essential comp::ments of a SUClion fillmtion system are: •
28
Fundamental laboratory techniQues
A ceramic funnel containing a flat perforated plate: there are two types based on size and shape called Buchner funnels or Hirsch funnels. When you are filtering. the perforated plate is covered by a filler p
Basic laboratory procedures II
Filtering hot mixtures - never attempt to use suction filtration on 8 hot mixture of 60lid end liquid. As the liquid filters through into the vacuum it may boil under the reduced pressure end be drawn into the pressure tUbing. tf tile liquid is saturated with the solkl, the evaporating liquid will deposit the solid in the holes of the perfOlllted plate and block them.
Filtering charCOIII- never attempt to
remove finely divided charcoal, used in decolouriunion during recrystallization or
•
• •
A receiver flask with a side ann for attachment of the vucuum source. BOChner nasks are conical nasks made from thickened glass, and Hirsch tubes (also known as side-arm boiling lUbes or tcst tubes dt:pending upon size) arc capable ofwilhslanding weak vacuum. e.g. a wt1ler pump. A OCll;ible !ital between the ceramic funnel and the receiving vessel: a BOchner collar or filter seal. A source of vacuum. usually a water pump (water aspirator) which is connected to the receiving flask by thick walled rubber tubing (pressure tubing). Sometimes there will be a trap between the water pump and Ihe receiving flask.
TIle various types of these components are shown in Fig. 5.6: lypical apparatus is shown in Fig. 5.7 and the general procedure for soction filtration is described in Box 5.2.
as a catalySt support, by euction filtration. It is a very fine powder and will always leak into the filtrate. Filler off charcoal by gravity filtration.
~::~~::::::
/
'\
-v
Fig. 5.6
Equipment for suction filtration.
__
,
1lld;-waIed fmfllllsk
Fig. 5.7 Suction filtration
using a Buchner funnel. Fundamental labot"atory techniques
29
Basic laboratory procedures II
Box 5.2
Isolation of a solid bV suction filtration
1. Select the appropriate size of apparatus based on the amount of solid you expect to isolate and the volume of liquid to be coHected in the receiver flask. Consider the following points; A. Thera is no point in using a large Buchner funnel for a small amount of solid since you will collect a layer of solid 'one molecule thick' and be unable to scrape rt from the filter paper cleanly. If there is too much solid for the size of the funnel, you will have to repeat the filtration with a second set of apparatus or the
solid may not suck dry quickly. B. If you use a side-arm boiling tube to try to collect l00mL of liquid, you will overfill the tube and liquid: Ii) will flow into the pressure tubing.
2. 3.
4.
S.
6.
7,
8.
30
contaminating it for your fellow students: Iii) may fill the intermediate trap, if there is one, and you will need to dismantle and clean it; (iii) may be sucked into the weter pump causing corrosion and loss of performance. Clean and dry all the apparatus to be used. C1amp the receiving vessel to a support stand: pressure tubing is heavy and even large Buchner flasks will fall over: do not think that a test tube rack will hold a side-arm boiling tube safely. Place the correct-sized BUchner collar in the neck of the receiving flask: it should sit well into the neck and fit the funnel to form a good seal. Place the funnel into the collar/seal: note that the funnel has a 'point' at the bonom of the stem. Make sure that this 'point" is as far aWBy as possible from the vacuum attachment side arm of the receiver flask, since the fittering liquid runs off this 'poinf and if the point is near the vacuum inlet, the liquid may be drawn into the side arm and then into the trap or water pump (see 18 above). Select a fitter paper, which frts exactly over the perforation in the base of the funne" The filter paper should not fold or crease up the sides of the funnel because the solid will be sucked round the edge of the paper into the receiver flask. If the paper does not fit exactly, trim to size with scissors. PtBce the paper into the funnel and wet it with a few drops of liquid - the same liQuid which is to be used in the filtration. Switch on the tap for the water pump to provide gentle suction. If your system has a trap, don't forget to close the tap on the trap and connect the rubber tubing to the side arm of the receiver. Do not force the rubber tubing too far onto the side arm you may need to pull it off quickly if something goes wrong (see 18 and 5). The fitter paper will be pulled down onto the perforated plate by the vacuum.
Fundamental laboratory techniques
9. Turn on the tap to the water pump to the maximum watliH" (low. If you do not do this, the water pump is not working 8t its maximum efficiency and the vacuum created in your filtration system may cause water to be sucked into a trap, or receiving flask, from the water pump. This is called ·suck-back'. 10. Swirl the mixture to be filtered and then slowty pour it into the Buchnet" or Hirsch funnel at such II rate so that the fittration is rapid. Note that the rate of filtration may slow 8S the 'cake' of solid on the filter becomes thicker. 11. To transfer the last of the solid/liquid from its beaket" or conical flask into the funn~ use a little of the filtrate in the receiving flask. Release the vacuum by opening tho tap on the trap or pull off the vacuum tubing, but do nor rum off rhe rap on the water pump (there is a possibility of 'suckback' (see 9 above)). Dismantle the apparatus, pour a linle of the filtrate into the beaker or conical flask, reassemble the apparatus and continue the filtration. Repeat untit all the material has been filtered. Use the filtrate to wash down any of the solid sticking to the sides of the funnel onto the filter 'cake' - it will not dry quickly on the sides of the funnel. 12. Release the vacuum, by pulling the vacuum tubing from the flask or opening the tap on the trap and tum down the water pressure on the water pump. Transfer the filtrate to 8 clean beaker or conical flask. Add 8 little pure, ice-eold solvent to the filter cake and reconnect the vacuum to provide gentle suction. This will wash the solid. Turn up the vacuum to maximum and suck air through the solid to dry it as much as possible. If 'cracks' appear in the filter 'cake', close them by pressing gently with a clean spatula and repeat until no more filtrate appears to be sucked out. 13. When drying is complete, release the vacuum, tum off the water tap and remove the filter funnel from the apparatus, The solid is best removed as a complete 'cake' by lifting the edge of the filter paper with spatula, inverting the funnel over a watch-glass of clock-glass and the cake should faU out. Peel the filter from the top of the 'cake', break up the 'cake' using a spatula and dry it appropriately. 14. Evaporate the fittrate to haN volume (see p. 1211 and cool to obtain a second crop of crystals. 15. Wash out and clean all the apparatus and dispose of the liquid fittrate safely.
Basic laboratory procedures II
Never use a sintered-glass funnel to remove finely divided charcoal. It is always absorbed into the pores of the
sinter and almost impossible to remove easily.
Instead of a filter paper on a porous plate. sinlcred-glass can be used as the porous malcrial for filtration. Sintercd-gtass funnels come in various lypt.'S and sizes (Fig. 5.8) with different porosity (size of holes) of the sintcred glass. Sintered glass is also used in crucibles (Fig. 5.8) in gravimetric analysis (p. 139). sir1lErBd-~
fikerdisl
sinter r~
gulVimelric lMysis
Slanhlfd sintlll'$lor
taperjoil'll:
fillnllion
Fig.5.8 Tvpical sintered-glass funnels.
Removing charcoal from a cold solution by suction filtration - cover the fiher paper with a 'filter aid" such as Celite.l< . This absorbs the fine particles.
When sintered-gla"s funnels are used instead of Buchner or Hirsch funnels the solid is collected directly on the sintered glass: no filter paper is used. As a result cleaning sintered-glass funnels is a major problem if the solid has been drawn into the pores of the sintered-glass. Therefore if you are to use a sintercd glass funnel. check with your instructor on the appropriate method for cleaning the funnel, before and after usc, so that your product will not be conlaminated. On the other hand, if particle sizes are large enough to prevent this problem, sintered-glass funnels are very effective in suction liltration.
Heating Safety note Never use gas burners to heat flammable chemicals in open containers, in particular solvents, which 8re usually used in relatively large volumes.
In the laboratory you will be required to heat chemicals in dissolution of a solid, promotion of re-d.ction (reflux), distillation of pure compounds and mixtures, extraction. coagulation of precipitatcs, drying solid compounds, etc. Your choice of heat source depends upon several factors: • • • •
First lind foremost. the j7alllmllhilily and rOIl/li1if.l· of the chemical and solvent. The operation 10 be carried out, e.g. simple prepanuion of a solution, reflux or distiUation, The temperature required for the process. -nle amount of chemical or solvent to be heated.
Bumping Before you attempt to he-at any liquid or solution you must take precautions prevent 'bumping'. This is when the liquid suddenly boils without any warning and results in hot liquid and vapour shooting uncontrollably out of the container. 'Bumping' can occur during simple heating in a ll."St lube, conical flask or beaker or in more complex situations such as reflux and distillation. It is necessary 10 provide a point in the liquid or solution where vaporization of the liquid can occur in a controlled manner. Before you ,war! heating a liquid or solution you must a/lra):\' take one of the following 'anti-bumping' precautions: 10
•
Add one or two 'boiling stones' or 'anti-bumping granulcs'; lhese can be filtered off later in the proces..". Fundamental laboratory techniques
31
Basic laboratory procedures II
movablo: collar
,.,
.,
Fig. 5.9 tal Bunsen burner; (bJ micro burner.
•
•
Using a microburner - do not place the micrOburner under the flask and leave it there: you will have no control over the rate of heating.
Heating a test tube - you must 'wave' the test tube in the flame to prevent localized heating, which will cause the liquid to 'bump' out of the tube. Also use an appropriate anti-bumping device and make sure that the tube is not pointing at anyone.
\
\ \
L
0
Fig. 5. 10
Heating an aqueous solution using a Bunsen burner.
32
Fundamental laboratory techniques
• • •
Add a Pyrex. I' glass rod to the beaker or conkal flask. The rod must be longer than the container so thaI it am be removed and rinsed before lhe solution is used further. Add a 'boiling slick'; these are thin pieces of wood sold as 'wooden applicalors', bUI you must be sure lhat nothing will be extracted from the wood into your solution. Stir the liquid or solution with a magnetic 'nea' (see p. 35) which should be removed before furlher processing of the chemicals. Stir the liquid or solution with a mechanical stirrer (sec p. 118). Use an air or inert gas (nitrogen) capiJlary bleed during vacuum distillation (see p. 110).
~
Make sure that you add the anti-bumping device before you start heating since um;:ontrolled boiling may occur immediately. You must allow the liquid to cool back to room temperature and then add the anti-bumping device.
Burners Gas burm:rs come in two common fonus: large burners called Bunsen burners ~md small burners known as microburncrs (Fig. 5.9). Bunsen burners
Basic laboratory procedures II
Tubing for gas burners -this must be of medium wall thickness so that it does not kink or compress easily. With thin-walled tubing, you could accidentally lean Of'Ithe tUbing, cuning off the gas supply, which extinguishes the burner. When you release the constriction, gas will then flow into the laboratory.
Before lighting a gas burner make sure that nobody is using flammable solvents in the laboratory. safety note
Safety note Take partiC\Jlar care when you turn off the gas supply to 8 Bunsen or microburner. Usually the gas tap is situated at the buck of the laboratory bench, so make sure you do not reach over the flame Or knock OV8f" your apparatus.
Do not confuse a waler bath. which is a general-purpose heating device, with a constant temperature bath, which is a precision device used to control the temperature of the liQuid in it (usually water, but not alwaysl to within 0.5 c_
Safety note If you need to increase or decrease the size of the 'hole' by adding or removing rings, do it before you start heating or use tongs to prevent scalding by the steam. Using a "multiple hole' water bath - make sure all the other 'holes' lire covered othelWise the steam will escape through them and heating your flask will be very slow. Safety note Steam lines are very dangerous and you should not anempt to
use them without direct supervision from yoUr laboratory instructor.
rubber tubing, and is not damaged, Close the movable cullar and light the gas with a gas igniter: do not use a splint or a piece of burning puper in case you cause a fire in Ihe waste bin when disposing of the paper. Open the collar to produce a hot bluc flame and adjust the gas now to give the rcquired size of flame. If your work is imerrupted. c10sc thc collar a little to producc a luminous flame. Finally, when the operations are complete, tum off the gas and do not pick up the burner by the barrel. or put il into a cupboard, until it has cooled. The advantages of the burner are that the heat source can be remove
Steam batlu and water bat"s These arc heat sources, which have a maximum tcmperature of 100°C: they arc safe to use even with most flammablc chemicals and solvcnts lind they differ only in the way the slC'.dm is produl"ed. Water baths are the more common of lhe two, and the steam is generated by heating water with an electric element - just like a kettle. The element may have a thennostatic Control. which can control the temperature of the water to some extent, Most water baths have a conSlant level devKe on the side of the bath. which supplies water to the bath 10 a fixed level above the healing element and prevents the water bath from boiling dry. Water baths are 'single hole' or 'multiple hole' types, and the holes are covered by concentric metal or plastic rings. which allows you to vary the size of the hole, When you are to use a water bath. makc sure that water is flowing into and oul of the bath via the conSlant level device - check that water is flowing from the pipe into the drain, Turn on the controller to the le\'el you requirt:the power can be turned down once the bath is boiling. A Sleam bath looks like lile 'single hole' water bath except that there is no heating c!c.:ment and no constant level device. Steam baths require piped steam as their source of heal usually provided by a steam line, which is a pcnnanent supply in the labomtory, Beakers and conical flasks sit firmly on the rings, while round-bottom and pear-shaped nasks should have about half the surface of the nasi< immersed in the steam (Fig. 5.11). The main advanlage of a waler bath is the minimal risk of lire, while the major disadvantages are the maximum tcmpcrature available (..... IOOoq and the special precautions needed if anhydrous reaction conditions are required: remember that steam will condense down the insitlt' of renux condensers.
Hot plate.~ and stirrer "ot platt's Diethyl ether lether) and hydrocarbons (petroleum spiritl must never be heated on a hot plate, Diethyl ether (b.pt. 35 C) should always be heated under reflux using warm water and petroleum hydrocarbons should be heated under reflux on a water bath or oil bath.
These consist of a rIat metal or ceramic plate, which is healed electrically, and varies in size for use by an individual or by several people at thc same time, Thc small versions normally have a built-in magnetK: stirrer, which can be used to stir the liquid with a magnetic 'flea'. Hot plates of both types should only be used ror heating flat-bottom vessels such as beakers or conical flasks and only when the liquid being heated is non-flammable. The vapour of a flammable liquid may 'run' down the outside of the vessd and ignite on the hot metal of me he-.dting surface. Since the contact area betwcen the heating surface and a round-hottom flask Fundamental laboratory techniques
33
Basic laboratory procedures II
,/ walera..1
-
waler out
-+-
water in
,.,
,.,
5. 11 (a) Heating a conical flask on a water bath; (b) heating a reflux set-up on a water bath.
Fig.
Using hot plates - never wrap the power cable round the heating plate. It may be hot and melt the flex, exposing the wires.
is very small, your attempts to heal these flasks effectively will require ellccssi\'e lemperalures of the heating surface, which incre-dses the dangers due to lack of control of the heating process. Therefore round-bottom flasks should only be healed using an oil bath (below) or a mantic (p. 35). The Oat exposed heating plate is extremely dangerous when hot: always check that lhe plate is cool by passing your hand over the plalc without touching it or by placing a drop of water on the plate. If you have to pick up a hOl piaIe, hold il by the sides and do not louch the plale; it may burn. Typical uses of hot plales are illuslnlted in Fig. 5.12a.
Oil baths
Stirring in an oil bath - the stirrer hotplate will stir both the 'flea' in the oil bath and the ·nea'in the reaction vessel.
Using oil baths - oil baths should be used in the fume cupboard because prolonged heating may cause unpleasant and possibly toxic fumes to be released from the oil.
34
Fundamental laboratory techniques
These arc mostly used 10 heal round-boltom flasks at temperatures <1bove 100 C. The oil bath, containing the healing Ouid, is usually a non-ferrous metal or Pyrex" dish and heated on a stirrer hOi plate, and Ihe temperature of the bath is measured with a thennometer. 111e oil bath should never be more than half-full, to allow a margin of safety for thennal expansion of the oil, and stirred wilh a magnelic 'nea' to ensure even heating. TI1C equipment used in a typical oil bath is shown in Fig, 5.12b. 11le nature of the oil used in the bath depends upon the tcmperature range required and a selection of liquids is shown in Table 5.1. You are most likely lo encounter paramn oil baths during your laboratory work and lhe following safety points should be considered: •
•
Paraffin oil discolours rapidly with prolonged heating. If the oil is dark, replace it with fresh oil. Dispose of the old oil in an appropriate manner (check with laboratory st.afl). Check that there is no water in the bottom of the oil bath: look for a separale layer or globules of liquid in lhe bottom of Ihe bath. If you heal
Basic laboratory procedures II
---=:::j
TBbie 5. f
Oil bath liquids
Malerial
Usable rllnge I Cl
Paraffin oil (mineral oil) Ethylene glycol
0-200
Polyethylene glycol 400 Silicone oil Glycerol
• • •
Comments Flammable. cheap, produces acrid smoke above 220 C
0-150 0-250 0-250 0-250
Flammable, cheap, low flash point Water soluble
Expensive Water soluble
the bath above IOOeC, the waler will boil and ma)' spauer hOI oil over you and the apparatus. Dispose of lhe conw.minated oil into the appropriate wasle conLainer, clean the bath with paper towels 10 absorb tJle water and refill with fresh oil once completely dry. If the oil bath is a Pyrex Jt dish, check the dish carefully for cracks and 'star' cracks because you do not do wanl the dish of hoI oil 10 break. Support the stirrer hot plate on a 'Iabjack' so that you cnn quickly remme the heat SQurce if the reaction goes wrong (see m~U1tles, p. 36). When the heating process is finished, altow the Mth to (;001 and raise the nask. let the oil dr-din from il into the bath and Ihen wipe the nask with a doth or pllper towel. Otherwise your hands, gloves, apparatus, com· pounds, etc., may becornc contaminated with oil.
Electric heating mantles
1'1 Fig.5.12 (a) A stirrer hot plate. lbl Using 8 stirrer hot plate with an oil bath 10 heat 8 round-bottom "ask.
Safety note Never connect an electric heating mamie, requiring an elCternal controller, directly to the mains electricity supply.
These are used for heating round-bottom nasks only lind come in various sizes. Always used a healing mantle appropriate 10 the size of Ole flask you are going to heal since you need to COl/fro! the he-J.ting process. The flask should fit snugly into the mantle with the top half of the flask above the case of the mantle. If the mantle is 100 small. heating will be inemciem, whereas if the mantle is too big. the nask ....ill be overheated and decomposition of the contents may occur. Electric heating mantles comprise an electric heating element wr:J.pped in a glass fibre covering, protected by an earth screen and enclosed in an aluminium or, more commonly, a polypropylene case to allow lumdling at moderdle temperatures. Heating control is provided either by a re£ulator built into the heating mantle (Fig. 5.IJa) or by an external conlroller, which is connecled 10 lhe mantle by a plug. Some mantles, 'stirrer mantles', have a built-in magnetic stirrer just like a stirrer hot plate and can be used to mix the liquid using II magnetic flea or bar (Fig. S.J3b). Stirrer mantles have two controls on the side: make sure that you know the function of each, si.nce one controls the extent of heating and the other the stirrer speed. When using mantles make the following safety checks: • •
•
Make sure that the heating element i.s not damaged or worn. If in doubt consult your instructor and get a replacemenL Plug in and test the mantle controls ensuring that both heater and stirrer are working. If fumes are given off from the heating elements someone has spilt chemicals into the mantle: switch off, report the fault to your instructor and obtain a replacement. Mantles are relatively slow to react to changes in the heater control setting and it is easy to 'overshoot' the desired temperature. Therefore always make small incremental changes in the healing control and if a temperature below the boiling point of the liquid is required, make sure lhat a thennomeler is incorporated in the apparatus. Fundamental laboratory techniques
35
Basic laboratory procedures II
•
•
When using a mantic with complex apparatus such as for distillation (p. 107). support the mantlc on a 'labjack' so Ihal it can be removed quickly if overheating occurs (Fig. 5.14). If the mantic is not equipped with a stirrer. remember to add antibumping granules (p. 31) to the liquid.
HoI-ai, guns
.... ""'"" - - SlirCllllln)/---'
.,
Fig. 5. J3 Mantles: (a) heating mantle; (bl stirrer
manlle.
These can be used instead of Bunsen burners or mlCrobumcrs provided thai the temperature required is not 100 high. The main uses of hot-air guns arc for drying glassware and as a heat source for distillation of liquids at relath"Cly moderate temperatures up to about 120°C. HOI-air guns produce heated air, usually at IWO temperatures, and cold air. As with a burner, the heat transferred to the flask being heated can be controlled by 'waving' the hot air Slream around the flask (see p. 32). Remember that the hot-air gUll has a hot-wire heating clement; therefore do nOt use it ill the pf'CSCnce of flammable vapours. The nozzle of the hotair gun \vill become very hot and can cause burns or even ignite somc soIvcnts for some time after it has been switched off. YOll should switch the gun to the cold-air mode for a few minutes after you have finished heating and place it in a 'holster' made from a support ring before finally turning off the gun.
Handling 1Iot glassware The safety precautions necessary for handling hot glassware depend upon: • • • •
---..,.. ,
Normally you do not need any hand protection to handle glassware at temperatures up to 5O"C. For general-purpose work. such as rcmoving glassware from the drying men, assembling hot glassware and manoeuvring hot beakers and conical flasks to and from a burner, steam bath or hot plate, heat-resistant gloves are suitable and should be a\·ailable in the laboratory. Where more intricate lXocesses are required, such as hot filtration, then 'rubber fin~rs' made from medium-wall rubrer tubing (Fig. 5.15) give adequate protection up to about 120"C and are less cumbersome than insulated gloves. "Rubber fingers' are useful for small volumes, up to ISOmL, of liquids whtn the flask or beaker can be easily held by one hand. If larger volumes arc being manipulated then two hands arc required and healresistant gIovcs are essential. The following specific techniques should be noted: •
• Fig. 5.74
36
Heating using a stirrer mantle.
Fundamental laboratory techniques
The temperature of the glassware. The type of glassware: test tube. beaker, conical flask, round-bottom flask, etc. The sUe of the glassware. The manipulation being carried oul.
Tesl "'hes: should be held by a wooden tcst tube holder (Fig. 5.16), which provides adequate insulation and grip. You should nerer usc folded pieces of paper or metal tongs. Conical flasks: are often clamped 10 a support stand during heating and you should 1IJ!\ff" attcrnpt to use the clamp as a device 10 hold the flask when removed from I~ support stand. If you place the flask on the laboratory bench with the clamp attached it will fall over because of the weight of the damp. Furthermore, you will have little control over the
Basic laboratory procedures II
safety note Never attelflpt to lift and milnlpulate a volume of Ilquid/solution that you cannot carlY easily; If in doubt
•
di....ide squally between two flasks.
•
pouring process. Use 'rubber fingers' or an insulated glove and lIerer use folded pieces of paper or metal tongs (Fig. 5.17). 8ellker.~: these have specinc problcms since they ha....e nO narrow neck which can be gripped for lifting. Small bcake~ of volumes up to 400 mL capacity can easily be gripped in one hand protected by an insulated glove or 'rubber fingers'. Rowul-hQltQm flasks: small flasks of capacity up to 250 mL should be held til the neck, gripped in one hand protected by an insulated glove or 'rubber" fingers' and when moving and pouring from larger flasks they should be held by the neck and supported underneath. Do not usc a damp round the ned: of the flask as a support.
Cooling Fig. 5.15 Rubber
'fingers'.
Cooling ra&etions - the heat from exothflfmic reactions is absorbed by the cooling medium. otherwise the reaction rate wi,1I increese rapidly wlth the ridng tempemture and may result il,l ....iolent boiling or even explosion.
Fig. 5.16
Holding a test tube.
Controlling temperature - temperature contrm may be nocfl6sary to achieve the desired reaction. For example, chemK:&fs lTl8y react to give different products at different temperatures.
During laboratory work you will be required to carry out experiments al temperatures below room Icmpcralure. The most common situalions where cooling is required arc: • • • •
Cooling solutions during recrystallization. Completion of precipitation in quantitatative (gravimetric) analysis and preparations. Cooling exothennic reactions. Carrying out reactions at low temperatures.
There are three cooling media commonly used in the laboratory: crushed ice, solid carbon dioxide (Dry Icc''', Drikold'.!i. Cardice
Ice baths
Fig.
5.17 Pouring a hot solution.
A slurry of crushed icc: and water can be used to give a cooling bath in the raDge DOC to 5°C. Pure crushed icc docs not give good contact with the glassware and inefficienl cooling results. If temperatures below D"C arc requi.red, mixtures of crushed icc and various inorganic salts can be used as shown in Table 5.2. Note that these mixtures contain no liquid and therefore cooling is inefficient and the temperatures indicated in the lable are the lowest attainable under ideal conditions. Func!amenlallaboratory
techniques
37
Basic laboratory procedures II
Solid C02 baths Table 5.2 Ice-salt mixtures
s"
Ratio lsalt:ical
CaC1 2·6H20
Ternpel'"afure
1: 2.5
-10 C
NH~CI
1: 4
NaCI CaCh.6H20
1:3 1 :0.8
-15 C -20 C -40 C
Solid ~. when mixed with organic solvenlS. provides cooling media of temperature; ranging from -15"C to ~IOOcC. Some common mixtures together with the minimum achievable temperatures are shown in Table 5.3. The CO! and the organic solvent form a 'slush', which gives excellent contact with nasks and. therefore. efftcicnt cooling. Table 5.3 Solid C~- solvent mixtUr95
Solvent
Temperature
Comments
Ethylene glycol Acetonitrile Chloroform Ethanol Acetone Oiethyl ether
-15 C -40 C
IcWNH~a cheaper Toxic, flammable Toxic flammable Flammable Highly flammable
-60 C
-72 C -78 C
-100 C
Solid CO2 is supplied as large hard blocks or small quanlltlCS can be prepared from cylinders of liquid C~ as a -snow'. Skin contact with solid CO2 will cause frostbite and it must be handled with ;t/slI/tJfed g/OI'es. The cooling bath must be an insulated container, which will need to be topped up with solid C~ at regular intervals to maintain the temperature. or. if prolonged cooling is required, a Dewar flask in which the coolant will maintain ils temperature for 12 houn or so. To prepare a solid C~ cooling bath: • •
Safety note If Ihe cOOling bath is more than half-full of solvent, it will 'boil" out of the bath as you add the first few lumps of solid CO2.
•
• •
Choose the appropriate solvenl and remember to take into account the ha7.ards associated with its use. Break the solid CO:! imo small pieces. The CO:! 'snow' can be broken with a spatula. but the hard blocks should be wrapped in cloth and then broken into pieces with a wooden or polyethylene mallet. (Ialf fill the bath with the solvent and then. using an in~ulatcd glm·e, add small pieces of the solid C02 until the mixture stops 'boiling' and then add a lin1c more solid CO! and stir with a glass rod 10 gi\'C 8 slurry. Use an alcohol or thermocouple thermomeler to check the temperature of the bath. Top up lhe COOling bath with solid CO2 if the tcrnperatUre begins to rise.
~
When using an internal thermometer to measure the temperature of a liquid or solution which is being stirred with a magnetic nea or stirring bar, ensure that the thermometet" bulb does not come into contact with it.
Using cooling baths - remember that a cooling bath will condense water from the atmosphere and therefore lose its effectiveness over a period of time.
To prevent the condensation of water into your nask. you should have an inert gas nowing through it (sec p. 126) and prepare the cold bath around the nask and its contents SO that slow cooling oa:urs. Sudden immersion of a relatively 'hot' flask into the cold bath will cause the bath to 'boir and air (wntaining water vapour) will be sucked into the apparatus despite the inert atmosphere. Alternativcly a 'looseIy packed' CaCb guard tube (sec p. 117) will suffice if cooling is not 100 rapid.
Om/inK p"ohes These arc rigid or nexible metal probes, which are connected to a refrigeration compressor. The probe is placed in the cooling bath and covered with a suitable solvent, which is then cooled to the temperalure desired. 38
Fundamental laboratory techniques
Basic laboratory procedures II
Cooling probes arc commonly found in cOllstam ltmperature baths, when a tcrnperature below ambient temperature is required. and probes can be uscd instead of solid COt to produce temperatures down to -IOO~C. Cooling probes are expensive pieces of equipment; therefore yOll will find them dedicated to a specifie experiment and they arc not usually available for basic laboratory operations.
Drying During your laboratory course it wiU be necessary to dry glassware, analytiall standard compounds (see p. 144), chemicals you have synthesize.'d, crucibles used in gravimetric analysis (sec p. 139) and solvents, Drying solvcnl<; is described on p, 127.
Never place chemicals in the oven on a pieea of fiher paper, If your chemical mehs it will run through the paper and may contaminate the samp&es of fellovv students, If il is on II watch'iJ18SS it may be recovered if it has not decomposed.
DI'j-'ing glussll'uI'e For most general laboratory applications glassware can be dried in an electric oven betwccn 80"C and 9O"C or by rinsing the glassware with a small amount of water-miscible solvent, such as acetone or ethanol. and then evaporating the solvent using a comprcssed+air jet. Remember to remove all plastic components. taps and stoppers from the glassware hefore you put it in the oven. If glassware is required for anhydrous reactions. it must be heated in the oven ab
plastic
"'=--
Fig. 5.18
Desiccators.
=
Heating the chemical. Using a drying agent in a closed container to absorb the soh'Cnt. Reducing the atmospheric pressurc,
Only chemicals which are lhe,ma/~I' stahle should be dried by heating. Most inorganic compounds, which arc salts with relatively high melling points, can be dried in an electric ovcn to remove water, whereas organic compounds, many of which have relatively low mclting points, need to be treated with more care and the OVCll temperaturc should be set betv.'CCn 30"C and so..c beloll' Lhe melting point of the chemical. Chcmicals must be placed in the oven on a clock-glass or watch-glass and be spread as thinly as possible. to increase the rate of solvent evaporation. If you cannol dry your compound in the oven, thcn usc a des;i'mlor. Desiccators are made from glass or plastic and some, vacllum desiccators. arc equipped with a tap to allow evacuation as shown in Fig. 5.18. The bottom of the desiccator is Iilled with a drying agent (desiccant) and the chemical, on a watch·glass or clock-glass, is placed on the mesh shelf abovc and the desiccator closed by sliding the lid onto the desiccator to provide an aiHighl seal. lbe desiccant absorbs the solvent from the 'atmosphere' in the desiccator as it evaporates from the solid, The nature of the desiccant depends upon the solvent to be removed (fable 5.4). TIle most common drying agents for removal of water arc anhydrous CaCl2 and self-indicating silica gel, The eaCh should have the appearance of 'chalky' lumps. Self-indicating silica gel is blue in the 'acti\'e' state and pink FUlldamentallaboratory techniques
39
Basic laboratory procedures II
when its capacity for water absorplion is 'cxhausted', The waler can be removed from the pink silica gcl by heating in an oven above JOOcC and restoration of the blue colour indicates reactivation. Table 5.4 Drying agents for desiccalors
Solvent to be removvd
Drying agent
H,O
Silica gel
CaCI 2
solid KOH Using corrosive acids - P20s and concentrated H2 SO. should be avoided unless there is no suitable alternative. Consult your instructor for their disposal.
P,o.
Cone,
EIOH, MeOH Hydrocarbons
H2SO~
Comments
Corrosive Corrosive Corrosive liquid
CaCI 2
Paraffin wax
Thc ratc of drying can be increased by eV"dcualing thc desiccator and vacuum desiccalOrs are specially designed for this purposc. Thc principal st~ for the usc of a vacuum desiccator are as follows and it is ~scntial that you follow the ordcr of lhe operations: • •
• •
Check that the appropriate desiccant is present and activc. Check that thc desiccator seals perfectly: ensure that rhe ground-glass edges to the lid are greased lightly or, if thc desiccator has a rubber gaskct. carry out a lrial evacuation to cnsurc that the vacuum seals the desiccator by gently pressing the lid onlo the gasket - listen for air being sucked around the seal. Place the samplc onto a walch· or dock-glass or a beaker covered with tissuc paper (secure with an elastic band) and place it on the shelf. Place thc lid on the desicrator and open the tap and cover the desiccator with an appropriately sized safcty cage (Fig. 5.19) [0 prevent injury from flying glass in the case of an implosion.
. ...
•<."'"
Using vacuum desiccators - never switch off the vacuum sup~y before disconnecting the pipe from the desiccator since you will suck water or oil into the supply pipe (see p. 301.
Using vacuum desiccators - never disconnect the vacuum supply with the tap on the desiccator open. Air will rush into the desiccator and blow your dry compound around the inside.
40
Fundamental laboratory techniques
Fig. 5.19 Vacuum desiccator (with mesh safely cage).
•
•
Connect thc lap to an operating source of vacuum: a water pump (p. 30) or vacuum pump (p. IIO) and open the tap slowly to evacuate the desiccator. When drying appears complete, close the tap on the dcssicator and thcn disconnect the vacuum supply.
Basic laboratory procedures II
•
Using weter pumps few pr~nged drying - thlll uses tOO much water: evacuate the deeiocatOf", elose the tap and disconnect and tum off the water pump. Check periodically that the Y8C\lum ts preserved using the fllter paper technique, Re9vacuMa if necessary.
Place a small piece of filter paper on the end of the tap and open the tap slowly. The filter paper will stick to the tap as air is drawn slowly through it. When the air has completely filled the desiccator, the filter paper will fall ofT and il is safe to open the desiccator.
If you ncOO to hcatthe compound under vacuum, then you willllred to usc a vacuum oven (for large quantities of solids). The principles of opcr.ltion or these pieces of equipment arc similar to tho~ of a vacuum desiccator, except that an electric healer is incorporated. AI....'8ys allow the apparatus to cool to room temperature before releasing the vacuum.
Dryillg
, { Drying solvents - the drying &gent used for solutions or pure liquids is not usuallv suitable for clfying solvents for inert atmosphere reactions.
Iiqu;tI~'
This usually means removing water from a liquid chemical or a solution of a chemical in a water·immiscible solvent. You will always need to dry solutions lifter a liquid-liquid extraction (p. 102) and you may need to dry liquid<; aller evaporation (p. 121) or distillation (p. 107). In both cases Ihe liquid is placed in direcl contact with the solid drying agent, i.e. the drying agent is added to the liquid or solulion. Ideally the drying agent should be lotally insoluble in lhe liquid, should not react with it. absorb the water quickly and elliciently. and be easily filtered ofT. A list of the common drying agents is given in Table 5.5. Table 5.5 Drying agents for liquids and solulions
Molecular sieves are synthetic: calcium and sodium aluminosilicates. which have 'holes' of specific siZf!S to allow the absorption of molecules of simitar dimensions. Types 3A and 4A, with 0.3nm and 0.4 nm pores respectivoty, are used tor • drying.
MgSO. Na 2 SO. CaCl2 CaSO. K2C03
Molecular sieve
High High High
Fast Slow Slow low Fast High Fast Moderate Fast
EtflClencv
Comments
Good
Best general use
p~,
Useful
p~,
Reacts with 0 and N compounds Useful Reacts with acidic compounds Must be activated at 3()(J °C
Good
Good Good
Capacity. amounl of waler laken up. Speed; rate of waler absorption. Efficiency; extent of drying after treatment.
Drying solutions and liquids - noto that phrases such as 'drying over magnesium sulphate' mean that the MgSO. is added to the solution.
You must remember lhat the drying agent will absorb some of the liquid or solvent being dried as wcll as lhe water. If you wish to dry a small volume of liquid, it is better to dissolvc it in a low-boiling water-immiscible solvent and dry the solution by the procedure described in Box 5.3.
1, Place the solution to be dried in • eIeen. dry conic:81 ftuIt. The flask should not be more than half-full. 2. Add small quantities of MgSO.. (between '0.1 9 and 1.09 depending upon the ¥OIume of solvent) and swirl the conical flask betweef'l uch addtion. At first the MgSO.. will 'stick' to the sides and bottom of the where it eont8C1:s with the water, but on further additions it will eventually form a free-flowing powder in the liquid and the solution will appear very clear and bright. 3. AIow the mbrtwe to stand for 10 minutes.
"ask
"ant
4.. Gravity filler the Gied layer through a fluted fitter paper into • dean, *v flask. The fluted fiher paper should contain 8 small amount of fresh MgSO... as a safety precaution. in case a few drops of water are floating fn the surface of a denser solYent than water. 5. Rinse the ~ in the conical ftasal: with a few milllitres d pure dry sofvent and filter it, to ensure that you recover all the solute, and then rinse the MgSO.. 00 the fitter· paper. 6. Remove the 50tvent by rotary evaporation (p. 121l or distitlation (p, 1(8).
FUrldamentallaboratory techniques
41
Basic laboratory procedures II
Jointed glassware This type of glassware. commonly known as Quickfit N • comprises a complete
range of
componcnL~
fined with standard-taper ground-glass joims. The
joims arc fully interchangeable with those of the same size and appar-dluS for a whole range of cxperimcnls can be assembled from the simple components without the need to usc rubber bungs, corks, etc. Where there is a mismatch betwccn the sizes of the joints of the pieces of glassware. reduction and expansion adapters can be used. A Iypical range of jointed glassware is illustrated in Fig. 5.20.
66 .............
- .... ........
(ar' hdimrIion aJloml
~ _...
~ "-
~
~
~
-~
-- -~ ~ tr ~ ....... ...... . - - - ......
..-
......".
Fig.5.20 Glass equipment v.ith standard-taper grQund-glass joints.
42
Fundamental laboratory techniques
Basic laboratory procedures II
Do not attempt to use non-standard ground-glass joints in the standard jointware, it will not fit correctly. For example. ground-g18ss stoppers from volumetric flasks do not seal Ouickfie' separatory funnels.
The ground-glass joint on the glassware is classified according to the diameter of the joint at ils widesl poinl (illlemal diameter) and the length of the ground-glass portion of the joint. Thus a 14{23 joinl has a maximum internal diameler of 14 !TIm and a length of 23 mm. Other common joint su..es you will frequently encounter are 19/26, 24/29 and 35/39. The joint size is always elched into glass on the side of or near to the joint. For obvious reasons, joints arc categorized as 'female' and 'male·. Cal"f! ofjointetl glassware Jointed glassware is much more expensive than ordinary glassware because of the precision required in fabricating Ihe joillls. If Ihe joints 'seizc' and cannot be separated the glassware cannot be used again and you may have the problem of a stoppered flask conlaining a volatile organic solvent, which somebody has to open! If this happens, consult your instructor for help and further advice. There are two main causes of 'seizcd' joints: I. Using solutions of potassium hydroxide or sodium hydroxide in water or
other soh'ems, which atlack Lhe glass. 2. Trapping chemicals, including solids and solutions of solids, in Ihe ground-glass joints. Greasing joints - you must grease all the
If you are using jointed glassware with strong alkalis (NaOH. KOH). you
joints when carrying out a vacuum
distilfation.
Separating joints - always separate ground-glass joints as soon as you have finished with the apparatus and wipe the joints clean.
If you break a piece of jointed glassware.
do not throw the item into the brokenglass bin. The glass blower may be able to fe-Use it.
must grease Ihe joints. In most cases a simple hydrocarbon-based grease, such as petroleum jelly, will suffice, since it is easily removed from the joints by wiping with a cloth wet with a hydrocarbon solvent (petroleum spirit, b.pt. 60 80~q. Avoid silicone-based grease, since this is difficult to remove, sohlble in some organic solvents and may contaminate your reaction products. To grease a joint, put a small smear of grease on the upper part of the 'male' joint. push it into the 'female' joint with a twisting movement and the joinl should become 'c1ear' from the top to about half-way down. If more than half the joint has become 'clear', you have used too much grease: separate the joints, clean with a solvenl-soaked c10lh and repeat the process. To avoid trdpping chemicals in the ground-glass joinls, fill flasks elc. using a long-stemmed filter funnel or paper cone, which extends paSI the joint into the flask (sec p. 25).
Sc,'eH'cap
adapter~'
Screwcap or themometer ada piers allow you to place thennometers, glass tubes or air bleeds into jointed glassware flasks. The serewcap adapter works by using the screwcap to compress a rubber ring round the thermometer or glass tube and thus hold it in place. The flexibility of Ihe system allows the height of the thcnnomclCT/glass tube 10 be varied. The adaplers come in difTerent joint sizes and varyillg hole sizes to accommodate difTerenH:liametCT thennomelcrs, lUbes, Pasteur pipencs. etc. TIle component parts of a screwcap adapter are shown in Fig. 5.21. To use the screwcap adapter ",-jth a thermometer: Using screwcap adapters - if the rubber
•
ring is not present in the screwcap adapter, the thermometer will slide out of
the adapter and may smash both the flask and the thermometer.
•
A/lI'ays disassemble the adaptCT to ensure that Ihe rubber ring and the Teflon 11 seal are present. If they are missing, get replacements before use. The Teflon ~ seal is to protect Ihe rubber ring from corrosive and solvent vapours. Ease the rubber ring onto the themometer (see p. 13) and slide the Teflon ll seal on below the ring.
Fundamental laboratory techniques
43
Basic laboratory procedures II
•
•
•
Slide the screwcap OVCf Ihe lOp of the thermometer and then screw the whole assembly onto the base of the adapter and tighten the screw slightly, just enough to hold the thermometer. Trial fit the adapter and thermometer into the apparatus and adjulll Ihe height of the thermometer by disassembling the adapter and moving lhe rubber ring up or down. Reassemble the adapter. Check for final fit and tighten the screwcap firmly, but do not over-tighten.
JQillt
Fig.5.21 The screWCllp adapter.
clips
Plastic joint clips or Keek It dips (Fig. 5.2211) are used for holding groundglass joinls firmly together and may be used 10 replace clamps and support stands at certain points when building apparatus (see p. 109) and are essential when using rotary evaporators (p. 122). The main weakness of these otherwise useful devices is that they soften at about 130°C and this may allow the joints to separate. Therefore they should never be used at the 'hot end' of Ii distillation, for example. The clip should be used to hold a distillation adapter on the end of a water condenser. or the flasks 01110 a fraction collcctor, but nel'f'r on the distilling flask or to hold the condenser onto the still head (I"lg. 5.22b). When usingjoim clips always:
• • • •
Check that you arc ulling the appropriate size of clip for the joints being held. The clips arc often colour coded. Check that the clip is not cracked or splil. CheCk that the wide 'lower jaw' of Ule clip fitll under the rim of the 'female' joint and the 'upper jaw' filS round the male joint. Support the joints wilh your hand as you push the clip imo place. If in doubt use a protective insulated glove.
joint clips t.ll
~ Fig. 5.22
44
Fundamental laboratory techniques
............... I.
Joint clips and their use.
.,
Principles of solution chemistry Preparing solutions - practical advice is given on p. 15.
Definition
a substance that dissociates, either fully or partially, in water to give two or more ions.
Electrofyte -
A solution is a homogeneous liquid, fonned by the addition of solutes to a solvent. The behaviour of solutions is determined by the type of solutes involved and by their proportions. relative to the solvent. Many laboratory exercises involve calculation of concentrations, e.g. when preparing an experimental solution at a particular concentration, or when expressing data in tenns of solute concentration. Make sure that you understand the basic principles sct out in this chapter before you tackle such exercises. Solutes can alleet the properties of solutions in several ways., as follows.
Elet:trolytit: tlissociation This O<.'Curs where a substance dissociates to give charged particles (ions). For a strong electrOlyte, e.g. Na-+·C1-. dissociation is C'>scntiaJly complcte. In contrast. a weak electrolyte. e.g. ethanoic acid, will be only partly dissociated, depending upon the pH and temperature of the solution (p. 57).
Osmotic effects Do not confuse the solubility of a chemical with its strength as an electrolvte. Ethanoic acid is completelv soluble with water In all proportions, yet it is a weak electrolyte because it is only partially dissociated. Barium hydroxide is very insoluble in water, but the small quantity which does dissolve {see Ks below} is dissociated comptetely into Ba 2.,. and QH ions; thus it is a strong electrolyte.
Thesc are the result of solute particles lowering the effective eonL-entration of the solvent (waler). These effccts arc particularly relevant to biological systems since membranes are far more pelmcable to water than to most solutes. Water moves across biological membranes from the solution \vith the higher effective water concentration to that with the lower effective water concentration (osmosis).
Meal/non-Meal behOl'iour This occurs because solutions of real substances do not necessarily conform to the theoretical relationships predicted for dilute solutions of so-called ideal solutes. It is often necessary to take account of the non-ideal behaviour of real solutions. especially at high solute concentrations (sec Lide (2000) for appropriate data).
Concentration Expressing solute concentrations - you should use 51 units wherever possible. However, you are likely to meet non-51 concentrations and you must be able to deal with these units too.
In SI units (p. 71) the concentration of a solute is expressed in mol m-l, which is eS~nlial for calculating specific parameters for substances (e.g. p. 73), but which is ;IICOIII'CII;Cnt when dealing with solutions in the laboratory. A cubic metre (m 3 ) of water weighs approximately ( ton! A common unit of volume used in chemistry is the litre (L): this is a non-SI unit and is converted to the SI unit of volume (m J ) using 1.0 L = 10- 3 m3 • The concentration of a solute is usually symbolized by square brackets. e.g. [Naal Details of how to prepare solutions are given on pp. 17. 19. A number of alternative ways of expressing the relative amounts of solute and solvent are in geneml use. and you may come across these terms in your practical work or in the literature.
Molarity Example A 1.0 molar solution of NaCI would contain 58.44g NaCI {the molecular mass} per litre of solution.
This is the term used to denote molar concentration, lei, expressed as moles of solute per litre volume of solution (mol L -I). This non-SI term continues to find widespread usage. in part !xx:ausc of the familiarity of working scientists with the term. but also because laboratory glassware is ca.librated in Fundamentallaboriltory lechniques
45
Principles of solution chemistry
Box 6.1
Useful procedures for calculations involving molar concentrations
1. Preparing a solution of defined molarity. For a solute of known relative molecular mass 1M.), the
following relationship can be applied:
!CJ -=
mass of solutelM, volume of solution
16.11
So, if you wanted to make up 200 ml (0.2 LI of an aqueous solution of NaCI (M. = 58.44g) at a concentration of 500mmoiL 1 10.Smo1L ,). you could calculate Ihe amounl of NBCI required by inserting these values into eqn [6.1): O5 .
= mass of solutel58.44 0.2
which can be rearranged
10
mass of solute = 0.5 x 0.2 x 58.44 = 5.844 9 The same relationship can be used to calculate the concentration of a solution containing a kno\NO amount of a solule, e.g. if 21.1 9 of NaQ were made up to a volume of 100 mL 10.1 l), this would give: I -, IN a ell -_ 21.1/58.44 _- 3.61moL
o.,
2_
Dilutions and concentrations. The following relationship is very useful if you are diluting (or concentrating) a solulion:
16.21
lNhere lC,) and Ic,l are the initial and final can· centrations, lNhile V1 and V2 are their respective volumes: each pair must be expressed in the same units. Thus, if you wanted to dilute 200 mL of 0.5 moll 1 NaCI to give a final molarity of 0.1 mol L-', then, by substitution into eqn (6.21: 0.5x200=0.1 x V2 Thus V2 "" 1000 mL tin other words, you would have to add water to 200 mL of 0.5 moll 1 NaCI to give a final volume of l000mL to obtain a O.lmoIL-' solution). 3. Intet'COnversion. A simple way of interconverting amounts and volumes of any particular solution is to divide the amount and volume by a factor of 103: thus a molar solution of a substance contains 1 mol l -', which is equivalent to 1 mmol mL " or lJlmollJl 1, or 1 nmolnl-', etc. You may find this technique useful when calculating the amount of substance present in 8 small volume of solution of known concentration, e.g. to calculate the amount of NaCI present in 50 IJL of a solution with a concentration (molarity) of 0.5 mol L-I NaCI: la) this is equivalent to 0.5/lmol/IL-\ lb} therefore SOIIL will contain 50 x O.Spmol =
251JffiOI. The 'unitary method' (p. 263) is an alternative approach to these calculations.
millilitres and liues. making thc preparalion of molar and millimolar solutions relatively straightforward. Ho\\"C\'cr. thc symbols in common use for molar (M) and millimolar (mM) solutions Me al odds with thc SI system and many peopl~ now prefcr to use mol L -I and mmol L- 1 respectivel}', to avoid confusion. Box 6.1 gi\'es details of some useful approllchcs to calculalions involving moillrities.
Molalit)' Example A 0.5 molal solution of NaCI would contain 58.44 x 0.5 ,..- 29.22g NaO per kg of water.
This is used (0 cxpress Ihe concentration ofsolute relative to the mass of soh~nt. i.e. mol kg-I. Molality is l'l tcmpemturo-indepencknt means of cxpressing solute oonccntmtion. mrcly used cxcept when the osmotic properties of a solution are ofinteresl (p. 49).
Example A 5% w/w NaOH solution contains 5 g NaOH and 95 9 water (=95 mL water, assuming a density of 1 gmL -'I to give loog of solution.
Per cellt compoS;t;OII (% wlw) This is the solutc mass (in g) per 100 g solulKln. The advantage of this expression is t~ ease with which a solution can be prepared. since it simply requires each component to be pre-weighxl (for wllter, a volumetric m~Sllrement may be used, e.g. using l'l measuring cylindcr) and then mixed together. Similar t~nns arc parts per thousand (%0), i.e. mgg- l , and pllrts per million (ppm), i.e. Ilgg- 1.
46
Fundamental laboratory techniques
Principles of solution chemistry
Pe,. (:Cllt Example A 5% wlv KQH solution contains 5 9 KOH in 100 mL of solution. A 5% vtv glycerol solution would contain 5 mL glycerol in l00mL of solution. Note that when water is the solvent this is often not specified in the expression,
e.g. a 20 ml v/v ethanol solution contains 20% ethanol made up to l00mL of solulion using water.
NOle that ppm may be a weight/weight (wtw) expression. The origin of the term
ppm derives from a solution whosc concentration is 1 ppm if it contains 1 9 of solute fOr each million (10 61g of solvent.
COllce",,.atioll
(% "'II' and % J'/I'J
For solutes added in solid form, this is the number of grams of solute per IOOmL solution. This is more commonly uscO than per cenl composition, sincc solutions can be aceunHcly prepared by weighing out the required amount of solute and then making this up to a known volume using a volumetric nask. The equivalent expression for liquid solutes is % v/v. The principal usc of mass/mass or mass/volume terms (including gL- I) is for solutcs whose molecular Illass is unkno.....n (e.g. pol}'mers), or for mixtures of certain classes of substance (e.g. total salt in sea water). You should nCI'cr use the per cent term without specifying how the solution was prepared. i.e. by using the qualifier w/w. wlv or v/v. For mass concentrations, it is often simpler to use mass per unit volume, e.g. mgL-I, JIg ilL-I, etc.
Pans per million ('011celltl'arioll (ppm) This is a non-51 weight per volume (W/V) concentmtion teon commonly used in quantitative analysis such as name photometry. atomic absorption spectroscopy and gas chromatogmphy. where low concentr
Box 6.2 How to convert ppm into mass of chemical required Example: Suppose you are asked to prepare an
aqueous solution of sodium ions 1250.00 mU of approximate, but accurately known, concentration of 10 ppm from either sodium chloride or anhydrous sodium carbonate. 1. Convert the ppm concentration into gL-1. Thus 1.0ppm = 1.0 x 10 6 gml-l and hence a solution of 10 ppm = 10 x 10-6 gmL- 1 = 103 x 10x10 6 g l-1 = 10x 1O-3 gl- 1. 2. Convert the concentration from gl-1 to moll-I. The Ar for sodium ion is 23g and for a litre of 10 ppm solution you need 10 x 10- 3 7 23 = 0.435 x 10- 3 mol of sodium ions. 3. Convert the number of moles per litre Into mcHes in the volume required (250.00 mll. Since 1.0 litres of 10 ppm solution of sodium ions requires 0.435 x 10 3 mol of sodium ions, then 250.00ml (O.25U of solution will need 0.25 :<: 0.435 x 10-3mol = 0.1087 x 10-3 mol of sodium ions. 4. Calculate the mass of either sodium chk>ride or sodium carbonate required to make up the smution:
(al Sodium chloride INaCI), Mr _ 58.5: therefore you need to weigh out 58.5 x 0.1087 x 10 3 = 6.359 x 10-3 9 of sodium chloride to be made up to 250.00 ml for a 10 ppm solution of sodium ions. (bJ Sodium carbonate (Na2C03), M, = 106; you must note that cach 'moleculc' of sodium carbonate contains two sodium ions; thus the number of moles of sodium carbonate required for a 10 ppm solution of sodium ions is 0.1087 X 10-3 7 2 = 0.05435 x 10-3 mol. You must therefore weigh out 106 x 0.05435 x 10 3 _ 5.7611 x 10- 3 9 of sodium carbonate to be made up to 250.00ml for a 10 ppm solution of sodium ions. 5. Decide how you are to prepare these solutions using the procedures outlined in Boxes 4.1, 4.2 and 4.3. since the calculation sho1NS Ihat you will need to weigh out small masses of chemicals, which are at the limit of accuracy of an analytical balance.
Fundamental laboratory techniques
47
Principles of solution chemistry
arsenic in water may be 0.05 ppm, but it is more conveniently expressed liS 50 ppb.
Actil'ity (a)
Table 6.' Activity coetfK:ient of NaCI solutions as a functioo of molality. Data from Robin6Oll and Stokes (1970)
Molality
Activity coefficient at 25' C
0.1
0.778 0.681 0.657 0.668 0.783 0.986
0.' 1.0 2.0 4.0 6.0
Example A solution of NaCl with 8 molality of 0.5 mol kg- 1 hil5 an activity coeffICient of 0.iS1 at 25 'C and a molal actIvity of 0.5 x 0.681 _ 0.340 mol kg- 1•
This is a tenn used to describe the effel'l;l"e concentr.ltion of a solute. In dilute solutions, solutes C'
16.31 Equution r6.31 cun be used for SI unils (mol m- 3), molarity (mol L-1) or molality (mol kg-I). In all C'
~ Activity
is often the COrTeet expression for theoretical relationships involving solute concentration (e.g. where 8 property of the solution is dependent on concentration). However, for most practical purposes. it is possible to use the actual concentration of a solute rather than the activity, since the difference between the two terms can be ignored for dilute solutions.
Eqlfh'llient mass (eqllil'alent weigl") Examptes For carbonate ioo& IC~-I, with 8 molecular mass of 60.00 and 8 valency of 2, the equivalent mess is 60.00(2 = 3O.00geq-'.
For slllphurk: acid tHzSO•• molecular
mass 98.081. whem fINO hydrogen ions are available, the equivalent mass is 98.0612 = 191M g eq_1.
Equivllienre lind nonnality an:: outdated tenns, although you may come across them in older texts. TIle magnitude of an equivalCllt mass (equivalent weight) can be simply identified from the balanced equation for the reaction being considered. Remember that the equivalent mass can change, depending on the reaction, as the following reactions illustrate. For: HO + NaOH ...... NnCl + H 20 I mol of HC! rC'
For: H2S04
+ 2NaOH ...... Na2S04 + 2H 20
since I mol of H 2SO4 reacts with 2mol of NaOH, the equivalent mass of H 2S04 is M, .;- 2 = 98..;- 2 = 49, while the equivalent mass of NaOH is still M. = 40. For; 5FeSO. + KMn04 ...... Ft:2(S04h + 2MnS04 since 1 mol of K..Mn04 reucts with 5 mol of FeS04, then the cquivlllcnt l1111SS of KMn04 is /If, -;- 5 = 158 -;- 5 = ]1.6, and lhat of FCS04 is still /If. = 152. BlIt, for: H 2S04 -l Na2C03 ...... Na2S04 + C0:2 + H 20 since the reaction is 1:1, the equivalcnl masses of H2S04 and Na2C03 are their Af, values, 98 and 106 respccth'Cly.
4a
Fundamental laboratory techniques
Principles of solution chemistry
As a result of this possible l:onfusion, the conccpt of equivalent mass (weight) is mrdy used.
NomwlitJ' Example A 0.5 N iOlutlon of sulphuric acid would contsin 0.5 x 49.04 i'" 24.52gl- 1.
A 1 normal solUlion (I f\,') is one that contains OIlC equivalellt maSS of a substance pcr litre of solution. Thc general fonnul~1 is: . mass of substance per litre normahty = equivalent mass
16.41
OSIII(}lm·ity Exa~.
Under ide&! conditions, 1 mol
of NaCI dissolved in water woukt give 1 mol of Na+ 101lii and 1 mol of Cl- tons. 9Quivalent to a theoreticel osmolarity of 201lmoll-1.
This non-SI expression is used to describe the number of moles of osmotically active solute particles per litre of solution (osmol L -I). Tht: need for such a tenn arises because somt: mok:eulcs dissociate to giv<: more than 011t: osmotically active particle in aqueous solution.
OSlIIolality Example A 1.0 mol kg- 1 solution of Natl has an osmotic coefficient of 0.936 at 25'C and an osmolality of 1.0)( 2 )0: 0.936,.,. 1.872 osmol kg-'.
This term describes the number of moles of osmotically :lctive solutc p
Table 6.2 Osmotie coefficients of Nael solutions as a function of molality. Data from Robinson and Stokes (1970)
Molality
Osmotic coeffic;ient at 25 ~C
0.1
0.932 0.921 0.936 0.983 1.116 1.271
0.5 1.0 2.0 '.0 6.0
Exampte A 1.0molkg-1 solution of NaC! at 25 ~C has an osmotalitty of 1.872 osmol kg- J and 8n osmotic pre5Sllre of 1.872 x 2479 ... 4.641 MPa.
II
)0:
1>
(6.5J
If nccessmy, the osmotic coefficients of:l p.articular solutc can be obtained from tables (e.g. Table 6.2): non-ideal behaviour means that 1> may have values> I at high concentrations. Alternativcly. the osmolality of a solution can be measured using an osmometer.
Osmotic pressure This is based on the concept of a mcmbmne pcnncllble to water. but not to solute molecules. For example, if a sucrose solution is plaet:d on one side and pure watc:r on the other, then a passive driving force will be created and water will diffuse across the membranc into the sucrose solution, since thc cffective water con
CoUigative properties and their use in osmometry Several properties vary in direct proportion to the efToctive number of osmotically active solute particles per unit mass of solvent and can be used to detenniTIC the osmolality of a solution. These colfigativc properties include freezing point, boiling point and vapour pressure. Fundamental laboratory techniques
49
Principles of solution chemistry
G
,
~
~
! -----------_.)
0
-
<;>
•, 0
E
•
, , o
Using an osmometer - it is vital that the sample holder and probe ate clean. otherwise small droplets of the previous sample may be carried over. leading to inaccurate measurement.
'~
---- depr"'lllI'l
,
Fig. 6. J Temperature responses of a cryoliCOpfc osmometer. The response can be subdivided into: 1. initial supercooling 2. initiation of crvstalliUltion 3. crystallization/freezing 4. plateau, at the freezing point 5. slow temperature decrease
An osmometer is an instrument which mCllsurt:s the osmolality of u solulion. USLmlly by determining the freezing point depression of the solution in relation to pure water. a technique known as cryoscopic osmomctry. A small amount of sample is cooled rapidly and then brought to th~ freezing point (Fig. 6.1). which is measured by a temperature-sensitive thermistor probe C"dlibmted in mosmol kg-I. An alternative rncthod is used in vllpour pressure osmometry. which mcasures the relative decrease in thc vllpour pressure produ(;ed in the gas phase when a sm111l sl1mple of the solution is equilibraled \\~thin a chamber.
Solubility InsOlubility - no solute cen be shown to be completely insoluble in a given solvent, but for practical purposes, a compound which has less than 0.01% (wI wi solubility in a solvent can be considered to be insoluble in that solvent Variation of solubility - the solubility of a chemical may vary in different sotvents. For example, NaCI is soluble in water but insoluble in DCM whereas for naphthalene the opposite is true. Saturated solutions - theoretically, a saturated solution is one in which the solution is in dynamic equilibrium with the undissolved solute.
The extent to wlUch a solute \~~II dissolve in a solvent is called its solubility. The solubility of a chemical is conventionally expressed as thc maximum number of grams of 11 chemical lrutt will dissolve in loog of solvent but conversion to moiL-lor gL- I is simple and roilY be approprilltc for some applications (see below). Since solubility is temperature dependcnt, is always quoted at a specilic tcmperature. With a wry few cxceptions, incre1LSing the temperrtture of a solvent incrCllSCS the solubility of the solutc.
Saturated solutiotls For prnetical purposes. a saturated solution is one in which no morc solutc will dis.<;Qlve. For cXllmple. the solubility of sodium chloridc in watcr is 35.6g per l00g at 25 cC and J9.1 g per 100g at lOOcC and both solutions arc Sllturnted solutions at their respective temper.lturcs. If the IOO-C solution is cooled to 25 C, then J.5g of NaO crystals will precipitate from the solution, because the solution at 25 ~C requires only 35.6 g of NllCI for saturation. This process is thc bllsis of purification of compounds by rccrystllllizalion (see p. 92).
Solubility product Solubility - remember to use mess;:; volume x density when converting solubilities from grams of solute per 100 g of solvent to 9 L '. when using solvents other than water.
50
Fundamental labOratory techniques
In dilute aqueous solutions. it has been demonstrated experimelltally for poorly soluble ionic salts (solubilities lcss than 0.01 mol L-I) that the mathematical product of the tOlal molar concentrations of the component ions is a constant at constant tcmpt.Tature. This product, K. is called the .foil/bilitr product. Thus for a satumtcd solution of a simple ionic compound AB in water, we haw thc dynamic equilibrimn:
Principles of solution chemistry
AB.oo.J
=== A~l + B~l
where AU rcprcsenl<; the solid which has not dissolved, in equilibrium with ils ions in the aqucous saturated solution. 1lJcn:
K,
~[A'),
[8
J
For example, sih'er chloride is a solid of solubility 0.000 15 g per 100 mL of water in equilibrium with silver C'Ations lind chloride ions. 1lJcn: K, = (Ag t ) ( (CI-I The solubility of AgO is 0.0015gmL- 1 (10 )( solubility per [oog, llssuming lhat the density of water is I.OgmL- 1) and thcrefore the solubility of AgCl is 0.0015 -:- 143.5 1.05 )( 10 S mol L_I. Thus Ihc satunUcd solution (;oulains l.05 x lO-s moIL-1 of Ag' ions and 1.05 x 10-' mol L I of CI- ions and the solubility product K J is
=
KI = (1.05 x IO-s) x (1.05 x IO- s) = I.I
X
10- 10 1001 2 L- 2
If the solid does not have a simple 1:1 r:.ttio of its ionic componcnts, e.g.
PbCiJ:, then the solubility product is given by: K, = fPb 2"1 x [Cn2 In gencralterms, thc solubility proouct for a compound M.•.N... is given by KI = [M'Y x
Example A 0.5 m~al solution of NaCI would contain 58.44)( 0.5 = 29.22g NaCI per kg of water.
[N-Y
The prActical cffects of solubility products arc dcmonstrated ill the detection of anions and cations by precipitlltion (p. 135) Mnd in qUllntitlltive gravimetric analysis (p. 139). For cxample, if dilutc aqueous solutions of silver nitrate (solubility 55.6g per loog of watcr) and sodium chloride (solubility 35.6g per IOOg of water) lire mixed, an immediatc white precipitatc of AgCl is produced because the solubility product of AgCl has been exceeded by the numbers of Ag+ lind CI- ions in thc solution, cven lhough the ions come from different 'molecules'. A satumtcd solution of AboCl is formed and the cxcess AgO precipitlltes out. Thc solubility product of thc other combinAtions of ions is not cxceeded lind thus sodium and nitratc ions remain in solution. Evcn if the concentmtion of Ag-" is cxlrcmely low, the solubility product for AgCl can be cxceeded by Ihe addition of lin excess of a- ions, since it is the multiplication of these two conccntrAtions which defines the solubility product. Thus soluble chlorides can ~ used to detect the presence of Ag+ ions and. convcrsely, soluble silver salts C'An be used to dctect 0- ions, both quantitAtively and qualitativcly.
Reactions of ions in solution Thcre are essentially only four basic reactions of ions in solution: I. 2. 3. 4.
Acid--basc reactions. Precipitation reactions. Complexation reactions Reduction-oxidation (redox) rC'Actions.
Acid-base. precipitation and complcxation reactions are all cxamples of cxchange (metathcsls) reActions in which ions in solution 'cxchange partners', for cxample: A +y-
+ 8"z-
---->
A-'-Z-
+ 8"Y-
Fundamental laboratory techniques
51
Principles of solution chemistry
Met:lthcsL.. rc:letions are rcnlly equilibria between the ionic species, which are dispJnced to the right (to the reuetion product) by a feature which defines the c1llssifiC<\tion of the reaction type.
Acid-base I'eactiolls The most common acid-base reactions arc exempliflCd by the lIeutmli7'Ation reaction between hydrogen ions and bydroxide ions; for example, the reaction between dilute hydrochloric acid and dilute sodium hydroxide: HtClj.caJ
+ Na l OJli~) ..... H.OH(IiqJ + Na' a~)
Since water is essentially a co\'llient compound (sec p. 56) its formation clTecti\'c1y remo\'cs H t and OH- from the equilibrium and drivcs the reaction to completion. Other examples of this ~neml type of reaction include the remO\~
Precipitatiol1 J'eactions In these reactions between ions, one substance is removed from the ionic equilibrium by precipitation (sec solubility product. p. 50) and dri\'es the equilibrium to the right (sec p. 51).
Complex(lJiOIl reactiOlIS A complex ion is formed by the rC4\clion of a metal (;alion, in particular
trnnsition metals, with an electron donor molecule (ligand), which can be llCutnl.1 or have a ncgative charge. The cation can accept an c1oc1ron pair and the ligand donates an electron pair to form a covalent donor (co-ordinalc) bond between the ligand and the melal ion. The ligands arc said to eoordinate wilh the metal ion to give a complex. Many lig:lnds are more powerful electron donors than water :tnd thus the addition of a ligand to an aqueous solution of a melal cation displat"t:S the equilibrium towards the more stable complex ion. (See p. 151 for stability constants and complexes.) The eITects of complex fonnation are illustrated in Box 6.3. The ovemll effect of complex fonnation is to 'remove' a hydrated metal ion from the mixture of ions in solution by displacing the equilibrium in favour of the complex, d. the similar process in the formation of W:lter in acid-rose titrntions and prccipitl\lion reactions.
Redllction-oxidation (redox) reactions The concepts of oxidation and reduction are defined in tcnns of (."omplete electron transfer from one alom. ion or molecule of a chemical to another.
• •
ChemiGlI oxidized - chemicalloscs electron(s). ChemiGlI reduced - chemical gains c!cctron(s).
This approach is gencrally applicablc to most reactions and avoids complications of the older definitions involving hydrogen and oxygen. You should realize that if a chemical is oxidized during a reaction. then another mllst be reduced: oxidation and reduction always occur together. Furthermore: • •
Oxidizing agent - gains elcctron(s) and is therefore reduced. Reducing agent -loses elcctron(s) and is therefore oxidized.
The following reaction between magnesium metal and dilute acid illustrates these concepts:
52
Fundamental laboratory techniques
Principles of solution chemistry
Box 6 3
An example of complex formation
If an aqueous solution of ammonia is added to an aqueous solution of copper 1II) sulphate the following changes are observed.
A. On addition of the ammonie solution to the paleblue cOPPer solution 8 white precipitate forms. B. As addition is continued, the white precipitate dissolves and 8 royal-blue solution is formed. which does not change on further addition of ammonia solution.
3. As the ammonia solution is added. the solubility product of Cu(OHh is exceeded. even by the low concentration of OH- ions and the white solid. Cu(OHh. precipitates from the saturated solution (see p. 51). 4.
As addition of the ammonia solution is continued. the free NH3 molecules displace the water molecules from the pale-blue CUIH20J~+ complex ion to form the roy2l1·b1ue Cu(NH31~1 complex. which is more stabkt than the water complex (larger stability constant).
5.
Since the insoluble CulOHh is in equilibrium with the CU(H20~"'. which is being removed as the Cu(NH3)2+ complex. the Cu(OHh reverts to CU(H20)!~ which then forms the CU(NH31~.f comple)(. Thus the while precipitate dissolves leaving the royal-blue solution of the CU(NH31~+ complex.
These changes can be explained as follows:
1. Ammonia solution is an equilibrium mixture. which lies well to the left. Consequently a dilute solution of ammonia contains a linle OH- and lots of free NHa: NHa
+ H20
i:==:;
NHt
+
OW
2. Copper (Ill sulphate comprises the CU(H20)~'" ion and SO~- ions in solution.
Magnesium melal has 10Sl two eleclrons in forming Mg1+ ions and has thereforc been oxidi::ed. The 1\....0 protons have cl-lch g
So that you rnn work out titrations involving redox reactions, you will find it necessary 10 balance redox equations. and while it is easy for simple reactions such as those abovc. more complcx rt'C!ox reactions. such as the one below, require more Ihoughl :md work. 2KMn04
+ 5H20:z + 3H!S04 ---" 2MnS04 + K 2S04 + 502 + 8H 20
Such problems etln be broken down into se.... cral simplc steps. each wilh its own sct of rules: •
Identify Ihe atoms.. ions or molecules which have been oxidized and reduced.
• •
Identify the ionic half-reactions for the species being oxidized and reduced and combine them. Balance the ionic half-reactions and combine them to give a balanccd equation for the reaction.
The species which are oxidized and reduced can be identificd using the concept of oxidatiofl munbeTs. The rules for
53
Principles of solution chemistry
to blllilDCC redox eqlllHions is shown in Box 6.5. Note that the result or the use or ~rtial ionic equations gives a balanced ionic equation for the redox reaction.
~ In simple acKl-bsse. pt'"ecipitation and complexation reactions. no change of oxidation number occurs at any of the atoms involved.
Box 6.4
The use of oxidation numbers to identify redox systems
The oxidation number is a hypothetical charge assigned to atoms in molecules and ions using a set of specific rules. Since redox reactions involve transfers of electrons, identification of the atoms which change oxidation number will show the atoms. ions or molecules which are specifically involved in the redox process. Rules for oxidation numbers
1. For an atom in its elemental form. the oxidation number is always O. Thus CI in CI2 has an oxidation number O. as does Na metal. and carbon in charcoal, graphite or diamond. 2, For any monatomic ion, the oxidation number is the same as the charge on the ion. Thus Na has an oxidation number of +1, CI- is -1, A13+ is +3. 5 2- is -2, etc. 3. Non-metals usualty have negative values. but there are some exceptions: (a) The oxidation number of fluorine is always -1 in all compounds. (b) The oxidation number of oxygen is always -2, except when bonded to fluorine (OF 2), in peroxides (O~ ), where each oxygen atom is - 1 and superoxides (Oi), where each oxygen atom is -l Ic) The oxidation number of the other halogens is always -1, except when bonded to atoms of greater electronegativity, e.g. CI in CIF3 is +3, Br in BrCl is + 1, etc. (d) The oxidation number of hydrogen is always + 1. except when bonded to electropositive metals. when it is -1, e.g. in HCI it is + 1 in NH 3 it is +1 and in NaH, MgH 2 and AIH 3 it is -1. 4. The sum of the oxidation numbers of all atoms in a neutral compound is zero, e.g. in KCI0 4• K is + 1
54
Fundamental laboratory teChniques
(rule 2), four oxygen atoms, 4)< -2"", -8 (rule 3b); therefore CI must be +7 (rule 3c). 5. The sum of the oxidation numbers of all the atoms in a polyatomic ion is equal to the charge on the ion, e.g. in C~- ion, each oxygen is -2 (rule 3b); thus 3 x -2 = -6. Since the charge on the CO~ ion is 2-, then carbon must be +4. 6. If the oxidation number of an atom becomes mOl"e positive during the reaction, it has lost electrons and has been oxidized. If the oxidation number of an atom becomes more
negative during the reaction, electrons and has been reduced.
it has gained
Example: If you consider the unbalanced equation for
the reaction shown on p. 53; KMn04 +H 202 +H250 4 ~ MnS04+K2S04 + Oz+H20 you can now calculate that the only atoms Which have changed oxidation number are manganese, which has changed from +7 in Mn04 - to +2 as Mn 2-+ in MnS04, and oxygen, which has changed from -1 in H20 2 (rule 3b) to 0 in O2, Thus Mn has gained electrons and been reduced and oxygen has lost electrons and been oxidized. Furthermore. the Mn atom has gained five electrons and the O~ ion has lost tvvo electrons so five molecules of H20 2 should react with two molecules of KMn04. The two partial ionic reactions can now be identified: For oxidation For oxidation
MnOi H20 2
Mnh O2
and the equation balanced as shown in Box 6.5.
Principles of solution chemistry
Box 6.5
How to balance redox equations from partial ionic equations using the ion-electron method
Example: You are to produce
a balanced equation
5e-
-. Mn h + 4H..O HZ0 2 -<' 0 .. +2H' +2e
+ BH' + MnO,
from the partial ionic equations deduced in Box 6.4.
1. Balance the atom which changes oxidation number in each partial ionic equation: MnO, --' Mn2~ H20 .. 2.
-<'
no change necessary in either equation since Mn and are balanced on each side of the equation
°
02
Balance the oxygen atoms on each side of each equation. (a) If the reaction occurs in acid or neutTa' solution. for each 0 atom deficient add one molecule H20 to the side deficient. (b) If the reaction occurs in alkaline solution, for each 0 deficient add two OH to the side deficient and one molecule of H20 to the other. YOur reaction occurs in acid solution, so: MnO; -<' Mn 2+ + 4H 0 2
H20 z
3.
- O2
Balance the hydrogen by addition of H+ to the side deficient BH-'-
+ MnO, ..... HzOz
Mn2~ +4H..0 • 0 .. + 2H+
5. Balance the electrons in each equation, since number of electrons gained by oxidiZing egent must equal number of electrons lost by reducing agent. Therefore multiply top equation by 2 and bottom equation by 5: lOa -+ lSW +-2MnO; 2Mn 2' +8H 20 5H..0 50.. + 10H + 1086. Add the equations together: 10e- + l6W I 5H zC>,; -I 2MnO, - ..... 2Mn2+ + 502 + lOW + BH 2 0+ lOe 1. Cancel t~ on opposite sides of the new equatkm:
6H+
+ 5H 20 2 + 2MnO...... 2Mn2 +- + 50.. +BH..O
This ionic equation is sufficient to work out the mole ratio of the reacting species. i.e. 5 moles of Ht 0 2 will react with 2 moles of MnO;. All the other ions, K~ and S~~. remain in solution and are unchanged by the reaction. If the fully balanced equation is required, the ions can be added to the ionic equation at the end of the process and the numbers adjusted: 3H 2 SO. + 5H 20 2 + 2KMnO....... 2MnSO.
K2S0.
+B~O
+ 502+
4. Balance the charge on each side of the equation by the addition of electrons, each electron having a charge of -1:
Fundamental laboratory techniques
55
-8 Definitions
Acid - a compound that acts as a proton donor in aqueous solution. Base - a compound that acts as a proton acceptor in aqueous solution. Conjugate pair - an acid together with its corresponding base.
pH and buffer solutions pH is a measufC of the amount of hydrogen ions (H+) in a solution. It is usual to think of aqueous solutions as conlaining H+ ions (protons), though protons actually exist in their hydrated form, as hydronium ions (H 30+). The proton concentration of an aqueous solution [H+] is affected by several factors: •
Ionization (dissociation) of water, which liberates protons and hydroxyl ions in equal quantities., according 10 Ihe reversible relationship:
•
Dissociation of acids, according to the equation:
Alkall- a compound that liberates hydroxyl ions when it dissociates. Since hydroxyl ions are strongly basic. this will
reduce the proton concentration. Ampholyte - a compound that can act as both an acid and a base. Water is an ampholyte since it may dissociate to give a proton and a hydroxyl ion (amphoteric behaviour),
H-A
•
<="
H+ +A-
[7.2)
where H-A represents the acid and A-is the corresponding conjugate base. The dissociation of an acid in water will increase the amount of protons, reducing the amount of hydroxyl ions as water molecules arc formed (cqn (7.1)). The addition of a base (usually, as its salt) to water will decrease the amount of H+. owing to the fomlation of the conjugate acid (eqn [7.2)). Dissociation of alkalis, according to the relationship: [7.3)
where X+OH represents the undissociated alkali. Since the dissociation of water is reversible (eqn [7.1]), in an aqueous solution the production of hydroxyl ions will effectively act to 'mop up' protons, lowering the proton concentration. Many compounds act as acids. bases or alkalis: those which are almost completely ionized in solution are usually callt"<1 strong acid<; or bases, while weak acids or bases are only slightly ionized in solution lp. 45). In an aqueous solution. most of the water molecules are not ionized. In facL the extent of ioniwtion of pure water is constant at any given temperature and is usually expressed in tems of the ion product (or ionization constant) of water. K w : K. ~ [H'1I0WI
Example The pH of 0.02 moll- 1 HClcan be calculated as follows: Hel is a strong
acid giving IWl = [CI-I = O.02moIL- 1 • Therefore pH = -log,010.021 = 1.7.
where [H~ J and [OH-J represent the molar concentration (strictly, the activity) of protons and hydroxyl ions in solution, expressed as mol L-I. At 25 c C. the ion product of pure water is 10- 14 mol 2 L-2 (i.e. 10-8 mol 2 m- 6 ). This means that the concentration of protons in solution will be 10-7 mol L-I (10- 4 mol m- J ). with an equivalent concentration of hydroxyl ions (egn (7.1]). Since these values are very low and involve negative powers of 10, it is customary to usc the pH scale, where:
Example The pH of a solution is 6.4. Therefore the IH~J = 10-pH, Le. 3.98 x 10-7 mol L ~1.
[7.5)
and ll-l+l is the proton activity (see p. 48). 56
Fundamental laboratory techniques
[7.4)
pH and buffer solutions
Table 7. 1 Effects of temperature
Of!
the ion
product of water (K'w). H ion contentration and pH 8t neumllily. Values calculated from lide (20001.
lH I al Temp. Imol 2
I'C)
K...
0 4 10 20 25 30
0.11 )( 10-'
37
45
L.-2,
0.17)( 10 • 0.29 )( 10-' 0.68)< 10 • 1.01 x 10-£ 147)(10' 2.39)( 10-' 4,02)(10'
neutrality (nmoll 1)
pH at neutrality
33.9 40.7 53.7 83.2 100.4 120.2 154.9 199.5
7.47 7.'" 7.27 7.08 7.00 6.92 6.81 6.70
~ While pH is strictly the negative logarithm (to the
base 10) of H-+ activity, in practice H~ concentration in moll I (equivalent to kmolm- J in 51 terminology) is most often used in place of activity, since the two are virtually the same, given the limited dissociation of H20. The pH scale is not 51: nevertheless, it continues to be used wldely in chemistry.
The value where an equal amount of H-+ and OH- ions are present is termed neutrality; at 2ST the pH of pure water at neutrality is 7.0. At this temperature, pH values below 7.0 are acidic while values above 7.0 are alkaline. Ho....>tver, the pH of a neutral solution changes with temperature (Table 7.1), owing to the enhanced dissociation of water with increasing tempernturc. This must be taken into account when measuring the pH of any solution and when interpreting your resulls. Always n.-member that the pH scale is a logarithmic one, not a linear one; a solution with a pH of 3.0 is not twice as acidic as a solution of pH 6.0, but a thousand tllTIes as acidic (i.e. contains 1000 times the amount of H ~ ions). TIlerefore, you may need to convert pH values into proton conccntrations before you carry out mathematical manipulations (see Box 40.2). For similar n.'3S0ns. it is important that pH change is expressed in tcrms of the original and final pH values, rather than simply quoting the difference between the values: a pH change of 0.1 has liule meaning unless the initial or final pH is known.
Measuring pH
pH electrodes Table 7.2 Propartics of some pH indiceu>r dyes
Dy.
Useful pH mnge
Thymol blue (acid)
Red-yellow
1.2-6.8
Bromophenol blue
Yellow-bluc
1.2-6.8 2.8-4.0 3.0-5.2
Acid-b&se colour
Methyl orange Congo red
Blue-red
Bromocresol gracn
YellO\o'J-blue
Roo-yellow
Chlorophenol red
Rcd-yellow Red-blue Yellow-red
3.8-5.4 4.3-6.1 4.5-8.3 4.8-6.3
Bromocresol purple
Yellow--purple
5.2-6.8
Methyl red Litmus
Bromothyrnol blue
Yellow-blue
Neutral red
Red-yeIiOlN
Phenol red l-Naphtholphthalein Phenol-
YellOYHed
6.0--7.6 6.8-8.0 6.8-8.2
Yellow-blue
7.2~.6
NoOOHOd
8.3-10.0
phthalein
Accurate pH measurements can be made using a pH electrode, coupled to a pH meter. The pH electrode is usually a combination electrode, comprising two separate systems; an I-'''-sensitive glass ekctrodc and a reference electrode which is unaffecled by H" ion concentration (see Fig. 7.2). Whl"ll this is immersed in a solution. a pH-dependent voltage belwl-cn the two electrodes can be measured using a potentiometer. In mosl cases, the pH electrode assembly (containing the glass and reference electrodes) is connected to a separate pH meter by a cable, although some hand-held instruments (pH probes) have combination electrodes and meter within the same assembly, often using an H+-scnsitive field effect transistor in place of a glass e!cclHx:le, to improve durability and portability. Box 7.1 gi\'CS details of the steps involved in making a pH measurement with a glass pH electrode and meter.
pH indicator dJ't!s These compounds (usually weak acids) change colour in a pH-dependent manner. They may be added in small amounts to a solution. or they can be used in paper strip form. Each indicator dye usually changes colour o\ICr a restricted pH range (Table 7.2): universal indicator dyes/papers make use of a combination of individual dyes to measure a wider pH range. Dyes are not suitable for accurate pH measurement as they are afTectw by other components of the solution including oxidizing and reducing agents and salts. However. they are useful for: •
•
estimating the approximate pH of a solution; determining a change in pi-I, e.g. at the end-point ofa titration. Fundamental
labcwatory techniques
57
pH and buffer solutions
Buffers Definition ~esists
Butter solution - one which
8
change in H'" concentration (pH) on
addition of acid or arkali,
Rather than simply measuring the pH of a solution, you may wish 10 control the pH. during EDTA complexation titrations (see p. 152) or preparative experiments involving carbonyl compounds, and one of the most effective ways to control pH is to usc a buffer solution. A buffer solution is usually a mixture of a weak acid and its conjugate base. Added prOlons will be neutralized by the anionic base while a reduction in protons, e.g. due to the addition of hydroxyl ions, will be counterbalanct.'
eff~tive
,buffering range
Buffer capacity and the effects of pH
.K. r
I
\
I
\
, I
/
II
\
butler capacity
\\ /
alkali edded
\
"
Fig. 7.1 Theoretical pH titration curve for a buffet.- solulion. pH change is lowest and buffer capacity is greatest at the pI(" of the buffer solution.
The extent of resistance to pH change is called the buffer capacity of a solution. The buffer capacity is measured experimentally at a particular pH by titration a~inst a strong acid or alkali: the resultant curve will be strongly sigmoidal, with a plateau where the buffer capacity is greatest (Fig. 7.1). The mid-point of the plateau represents the pH where equal quantities of acid and conjugate base are present, and is given the symbol pKa , which refers to the negative logarithm (to the base 10) of the acid dissociation constant, Ka , where K, ~
(1I+J1A -I
[7.6]
[IIA]
By rearranging eqn [7.6J and taking negative logarithms, we obtain: pH = pKa
W]
+ 10glO [HAl
[7.7)
This relationship is known as the Henderson-Hasselbalch equation and it shows that the pH will be equal to the pKa when the ratio of conjugate base to add is unity, since the linalterm will be zero. Consequently, the pKa of a buffer solution is an important faclOr in determining the buffer capacity at a particular pH. In practical terms, this means that a buffer solution will work most effectively at pH values about one unil either side of the pKa .
Selecting an appropriate buffer Potassium hydrogen phthalate IKHP) is the mono potassium salt of a weak
organic dibasic 8ckl.
58
Fundamental laboratory techniques
When selecting a buffer, you should be aware of certain limitations to its use. Citric acid and phm;phate buffers readily fonn insoluble complexes with diyalent cations, while phosphate can also act as a substrate, activator or inhibitor of certain enzymes. Both of these buffers contain biologically significant quantities of cations, e.g. Na+ or K+. TRIS (Table 7.3) is often toxic to biological systems: owing to its high lipid solubility it can penetrate membranes, uncoupling electron transport reactions in whole cells and isolated organelles. In addition, it is marh.-'dly affected by temperalure, with a lQ-fold increase in H-'. concentration from 4~C to 37°C. A number of zwitterionic molecules (possessing both positive and negative groups) have been inlroduced to overcome some of the disadvantagt.'S of the more traditional buffers. These newer compounds arc often refl'fred 10 as 'Good buffers', to
pH and buffer solutions
Box 7.1
Using a glass pH electrode and meter to measure the pH of a solution
The following procedure should be used whenever you make a pH measurement: consult the manufacturer's handbook for specific information, where necessary_ 00 not be tempted to miss out any of the steps detailed below, particularly those relating to the effects of temperature. or your measurements are
likely to be inaccurate.
pH meter (potentiometerl
I+-+--silver~ilver chloride eletlrodB
Sllt\l'llted---
'0 solution
..,
It+-- calomel reforence
porous
electrode
- - Hi -sensitive IIlan electrode
HCI---
""-
lest solullOl1
Fig. 7.2 Measurement of pH using a combination pH
electrode and meter. The electrical potential difference recorded bv the potentiometer is directly proPOrtional to the pH of the test solution.
1. Stir the test sotution thoroughlV before you make any measurement: it is often best 10 use a magnetic stirrer. Leave the solution for sufficient time to allow equilibration at lab temperature. 2. Record the temperature of every solution you use, including all calibration standards and samples, since this will affect K.... neutrality and pH. 3. Set the temperature compensator on the met..- to the appt'"opriate value. This control makes an allowance fOr the effect of temperature on the electrical potential difference recorded by the meter: it does nOl allow for the other temperaturedependent effects mentioned elsewhere. Basic instruments have no temperature compensator, and should only be used at a specified temperature. either 20 C or 25 C. otherwise they will not give an accurate measurement. More sophisticated systems have automatic temperature compensation.
4. Rinse the electrode assembly wjth distilled water and gently dab off the excess water onto a clean tissue: check for visible damage or contamination of the glass electrode (consult a member of staff if the glass is broken or dirty). Also check that the solution within the glass assembly is covering the metal electrode. 5. Calibt"ate the instrument set the meter to 'pH' mode, if appropriate. and then place the electrode assembly in a standard solution of known pH. usually pH 7.00. This solution may be supplied as a liqUid, or may be prepared by dissolving a measured amount of a calibration standard in water: calibration standards are often provided in tablet form, to be dissolved in water to give a particular volume of solution. Adjust the calibration control to give the correct reading. Remember that your calibration standards will only give the specified pH at a particular temperature, usually either 20 C or 25 -C. If you are working at a different temperature, you must establish the actual pH of your calibration standards, either from the supplier. or from literature information. 6.
Remove the electrode assembty from the calibration solution and rinse again with distilled water: dab off the excess water. Basic instruments have no further calibration steps (single-point calibralion), while the more refined pH meters have additional calibration procedures. If you are using a basic instrument. you should check that your apparatuS is accurate over the appropriate pH range by measuring the pH of another standard whose pH is close to that expected for the test solution. If the standard does not give the expected reading, the instrument is not functioning correctly: consult a member of staff. If you are using an instrument with a slope control function. this will allow you to correct for any deviation in electrical potential from that predicted by the theoretical relationship (at 25 C. a change in pH of 1.00 unit should result in a change in electrical potential of 59.16mV) by performing a two-point calibration. Having calibrated the instrument at pH 7.00, immerse in a second standard at the same temperature as that of the first standard. usually buffered to either pH 4.00 or pH 9.00, depending upon the expected pH of your samples. Adjust the slope control until the exact value of the second standard is achieved (Fig. 7.3). A pH electrode and meter calibrated using the two-point method will give accurate readings over the pH range from 3 to 11: laboratory pH electrodes are not accurate outside this range, since the theoretical
Fundamenlal laboratory techniques
59
pH and buffer solutions
Box 7.1
'continuedl electrode with abrasive material. Allow sufficient time for the pH reading to stabilize in each solution before taking a measurement: for unbuffered solutions, this may take several minutes, so do not take inaccurate pH readings due to impatience!
relationship between electrical potential and pH is no longer valid.
...
...
8.
After use, the electrode assembly must not be allowed to Q
9.
Switch the meter to ZMO (whMe appropriate). but do not tum off the power: pH meters give more stable readings if they are left on during normal working hours. Problems (and solutions) include: inaccurate andl or unstable pH readings caused by crosscontamination (rinse electrode assembly with distilled water and blot dry bet'oNeen measure-mentsl; development of a protein film on the surface of the electrode lsoak in 1% will pepsin in 0.1 mar l-1 HCI for at least an hour); deposition of organic or inorganic contaminants on the glass bulb (use an organic soillent, such as acetone, or a solution of 0.1 moll 1 disodium ethylenediamine-tetraacetic acid, respectively); drying out of the internal reference solutions (drain, flush and refill with fresh solution, then allow to equilibrate in 0.1 moll- 1 HCI for at least an hour); cracks or chips to the surface of the glass bulb (use a replacement electrode).
"00
J
•
S
6
1
I
-u.
~/ I
Fig. 7.3 The relationship between electrical potential and pH. The solid line llhowa the responlie of a calibrated electrode while the other plots are for instruments requiring calibnltion: 1 has the correct slope but incorrect isopotential point (calibration conlroledjUltmCM! is needed); 2 hes the cOrrect isopolentiaJ point but incorrect slope (slope conlrol8djll51ment is needed).
7.
Once the instrument is calibrated, measure the pH of your solutionis). making sure that the electrode assembly is rinsed thoroughly between measurements. You should be particularly aware of this requirement if your solutions contain organic biological material, e.g. soil, tissue fluids, protein
solutions. etc., since these may adhere to the glass electrode and affect the calibration of your instrument. If your electrode becomes contaminated during use, check with a member of staff before cleaning: avoid touching the surface of the glass
acknowledge the C8J"ly work of Dr N.E. Good and co-workers: HEPES is one of the most useful zwitterionic buffers, with a pK~ of 7.5 at 25 cc. These zwitterionic substances arc usually added to water as the free acid: thc solution must then be adjusted to the correct pH with a strong alkali, usually NaOH or KOI-I. Alternatively. they may be uscd as their sodium or potassium salts. adjusted to the correct pH with a strong acid, e.g. HCI. Consequently. you may need to consider what effects such changes in ion concentration may have in a solution where zwittcrions are used as buffers. Fig. 7.4 shows a number of tmditional and zv.'ittcrionic buffcrs and their effective pH ranges. When selecting one of these buffers, aim for a pK~ which is in the direction of the expected pH change (Tables 7.2. 7.3). For example, HEPES buffer would be a hcUer choice of bulTer than PIPES for use at 60
Fundamental laboratory techniques
pH and buffer solutions
Table 7.3 pI(, values at 25
be obtained from the catalogues of most chemical suppliers
Acid or base
pK., value(5)
Acetic acid Carbonic add
'.8 6.1,10.2 3.1, 4.8, 5.4 3.1,8.2
Citric acid G1VCylglycine
2.9,5.5 2.1,7.1,12.3 4.2,5.6 83 9.2
Phthalic acid Phosphoric acid Succinic acid TRIS"
Boric acid
6.1 6.8 7.2 7.5 8.1 8.' 9.3 10.4
MES PIPES MOPS HEPES
TRlC1NE TAPS CHES
CAPS
"Note thai this compound is hygroscopic and
should be stored in a desiccator.
pH 7.2 for experimental systems where a pH increase is anticipated. while PIPES would be a better choice for where acidification is expect(.-'d.
Preparation of huffer solutions Having sclected an appropriate buffer. you will need to make up your solution to give the desired pH. You will need to consider two factors: The nltio of acid and conjuf1ate base required 10 f1ive the corn:.'Cl: pH. The amount of buffering required; buffer capacity depends upOn the absolute quantities of acid and base. as well as their relative proportions. In most instances, buffer solutions are prepared to contain between IOmmol L-I and 200mmol L-I of the conjugate pair. While it is possible to calculate the quantities required from first principles usinf1 the HendersonHasselbalch equation, there are several sources which tabulate the amount of substance required to give a particular vohune of solution wilh a specific pH value for a wide ranf1C of traditional buffers (e.g. Perrin and Dempsey, 1974). For traditional buffers, it is customary to mix stock solutions of acidic and basic components in the correct proportions to give the required pH (Table 7.4). For zwitterionic acids., the usual procedure is to add the compOund to water, and then bring the solution to the required pH by adding a spt:cific amount of strong alkali or acid (obtained from tables). Alternatively, the required pH can be obtained by dropwise addition of alkali or acid, using a meter to check the pH, until the correct value is reached. When preparing solutions of zwittcrionic buffers, the acid may be relatively insoluble. Do not wait for it to dissolve fully before adding alkali to change the pH - the addition of alkali will help bring the acid into solution (but make sure it has all dissolved before the desired pH is reach(.-'d). Finally, when preparing a buffer solution based on tabulated information. always confinn the pH with a pH meter before uSt:. l.
2.
Table 7.4 Preparation of sodium phosphate buffer solutions for use at 25 "C. Prepare separate stock solutions of (al disodium hydrogen phosphate and Ibl sodium dihydrogen phosphate. both at 0.2 mor L-'. Buffer solutions (at 0.1 moll ') are then prepared at the required pH by mixing together the volume of each stock solution shown in the table, and then diluting to a final volume of 100 mL using distilled or deionized water
Aequired pH (at 25 CI
6.0 6.2
6.' 6.6 6.8
7.0 7.2 7.' 7.6 7.8
60
Volume of stock (a) Na2HPO.. (mLi
6.2 9.3
13.3 18.8 24.5 30.5 36.0 40.5 43.5 45.8 47.4
poosphale
TfltCINE
ci1raw
....
tHES
suctinare
,
,
HEPES
acetate
MES
Volume of stock (bl NaH2PO.. (mll
43.8 40.7 367 31.2 25.5 19.5 14.0 9.5 6.5 '.2 2.6
phosphate
2 Fig. 7.4
, •
5
,
TRIS PIPES
1
,
CAe'
9
,H
10
"
Useful pH ranges of some commonly used buffers.
Fundamental laboratory techniques
61
Resources
Resources for fundamental laboratory techniques
Boob Anon. (1989) Saft' Practices in Owmit:al £.,ahor{/fo"ies. Royal Society of Chemistry, London. Anon. (1989) COSI1H in U,!Jvr{/fori{'!J,·. Royal Society of Chemistry, London. Bennetl, S.W. and O'Neale, K. (1999) Progl-e,rsil'C Del'dolmlent of Procti('Q1 SkHls ill Chemistr)', A gllitl{' 10 t'arly-lindergrutilltile experimental Il"Or/.:. Roynl Society of Chemistry. Cambridge. Furniss, B.A., Hannaford, AJ., Smilh, P.\V.G. and Tatchdl. A.R. (1989) Vogel's Textbook of ProctiUl1 Organic Chemisrl")', 51h Edn, Longman, Harlow. Essex. Halpern, A.M. (997) Experimental Phy,rico{ Chemisrry, Prcntit't: Hall. Harlow, Essex. Harwood. LM .. Moody. CJ, and Percy. 1.M. (2000) £'l:perimenlal Organic Chemistr.,'. 2nd Edn. Blackwell Science Ltd, Oxford. Lehman, J.W, (1999) Operario/1al OrgalJic Chemi.rlry. A problem·soMllg apprOOl.'l1 to the laboratory cOllrse, Jrd Edn. PrcUlice Hall, Harlow. Essex. Lenga R.E. (1988) Sigmn-Alt/rich J.Jbmry of Chemical SaJery Data, 2nd Edn, Si!.'ffi3.J\ldrich Ltd_ Gillingham, Lister, T. (1996) Classic Chemisrry DemOlJSrratiollS, Royal Society of Chemistry, Cambridge. Mendham, J .• Denney, R.C.. Barnes, J.D. and Thomas, M.J.K. (2000) Voge/:r Textbook q( Quantiratire Chemical Analysis, 6th Edn, Prentice Hall, Harlow, Essex. Nelson, J.H. (1997) LaboralOry Experimenl!J," for Chemistr}" Prentice Harlow. Essex.
H~lI,
Sharp, J.T., Gosney, l. and Rowley, A.G. (1989) PrOCliml Organic Chemistr.l', Chapman and Hall, London. Suib. S.L. and Tanaka, J. (1999) £"tperimemal Me/hods in lnorgallit.: Chemistry, Prentice Hall. Harlow, ESS(.'X. Urix.'n. P.G. (1999) Brethericks HtDldbook of Reac/il'£' Chemical Ha:ards. Edn. Butterworth-Heinemann, London.
6'"
Zubrick. J.W. (2001) The Organic Chem Lab Slirril"Ul Mmuwl. A ,rful!e/lf'S guitle to leelmil/ues, 5th Edn. John Wiley and Sons Inc" Chichester. Videos Basic labol"".dtory skills, LGC, Royal Society of Chemistry, Cambridge (1998).
Further Laborntory Skills. LOC, Royal Sociely of Chemistry, Cambridge (1998).
Software SoftCOSHH 2000, Royal Society of Chemistry, Cambridge.
62
FlIndamentallaboratory techniques
-8 Definition Variable - any characteristic or property which cen take one of a range of values.
Making and recording measurements TIle tcnn data (singular - datum, or data value or V
Parametet' - a numerical constant Or
mathematical function used to describe 8 particular population (e.g. the mean
height of 'S.year·old femalesl. Statistic - an estimate of a parameter obtained from a semple le.g. the hetght of 18·year-old females based on those in
•
your c1essl. •
Quantitative: where the lIldividual values arc de:)Cribt:d on a numerical SCl.llc which may be either (i) continuous. taking any vlIluc on the measurement scale, or (ii) discontinuous (or discrete), where only intl.:ger values are possible. Many of the variables measured in chemistry are continuous and quantitative. e.g. weight. tt11lpCrnturc. time, amount of product forml.:d in an enzyme reaction. Ranked: where the data valut'S can be listed in order of magnitude. Where such data are given numbered ranks, they are sometimes callL-d 'semi-quantitative data'. Note that such mnks cannot be treated as 'real' numbers and they should not be addl.-d. averaged. etc. Qualitativc: when: individual values arc assigned to a descriptive C
Variables may be independent or dependent. Usually, the variable under the control of the experimenter (e.g.. time, reah'Cnt conccntl"'"dtion, pH. etc.) is the independent variable, while the variable being nll.'asurt.-d is the dependent variable (p. 76). Sometimes, it is inappropriate to describe variables in this way. and they arc often referred 10 as interdependt:nt. Another group of vlllucs. onen termed derived (or computed) data, are calculated from IWO or more individual measurements. and these include ratios, percentages and rates.
Measurement scales Examp~ A nominal scale for temperature is not feasible, since the relevant descriptive terms can be ranked in order of magnitude.
An ordinal scale for temperature measurement might use descriptive terms, ranked in ascending order, e.g.
Variables may be ml'asured on different types of scale; • • •
cold = 1, cool = 2, warm"" 3, hot _ 4. The celsius scale is an interval scale for
temperature measurement, since the arbitrary zero corresponds to the freezing point atwater (O~C). The Kelvin scale is a ratio scale for temperature measurement since 0 K represents a temperature of absolute zero (for information, the freezing point of water is 273.15 K on this scale!.
•
Nominal - where classification is based 011 a descriptive characteristic (e.g. colour). 'Ibis is the only scalc for qualitative data. Ordinal - this classifies by numerical rank, from smallest to greatest, but \\ith no assumption of equal spacing between ranks. Intl....wl - for ccrtain quantitative variables, where numbers on an equal unit scale are related to an arbitrary zero, e.g. temperature in ·C. RatiO - similar 10 the interval scale. except that lhe zero point represents an absence of that character, i.e. it is an absolute ;;,-cro.
You should aim to do quantitative measurements using a ratio scale whenever possible. to allow you to use the broadest range of mathematical opCTtltions and statistical procedures. For example, if you are measuring the surface area of a sample of graphite you could give the area as 200m 2 but. if you know the mass of tile sample (lOg). you should quote Ihe surface area as 20m'!g-1.
Accuracy and precision Accuracy is the closeness of a measured or derived data Wllue 10 its true value, while precision is the closeness of repeated measurements 10 each other (Fig. 8.1). A balance with a fault in it (i.e. a bias. see below) could give precise (i.e. \'~ry repeatable) but inaccurate (i.e. untrue) results. Unless there is bias in a measuring system. precision will lead 10 aecura(.j' and it is The investigative approach
65
Making and recording measurements
......
p'leGe lecunlll
@
...... .....
lCe"''''1
precision lhal is genemlly the most important pmclical considemlion, if there is no reason to suspect bias. You c'tIn investig'tlte the precision of any measuring system by repealed measurements of the same s'tlmple: Ihe nearer the rep/ic:lle "'tIlues 'tire 10 e"
Bias (systematic error)
,,,,
inleCllfll1 in'lplaeiMl
,,,,
pnldse ICtUfll\tl
Fig. 8. 1 'Target' diagrams illustrating precision
and accuracy. Minimizing errors - determine early in your study what the dominant errors are likely to be and concentrate your time and effort on reducing these.
Working with derived data - special effort should be made to reduce measurement errors because their effects can be magnified when differences, ratios, indices 0, rates are calculated.
Bias is a syslematic or non·rdndom inaccuracy and is one of lhe most troublesome dilTtcull.ies in u."ing numerical data. OiaSt:s rnay be associated with incorrectly calibraled instruments. e.g. a faulty electrode or syrinb'C, or with experimental manipulations. e.g. decomposition of chemical compound on stordge. Oias in measurement ean also be subjective. or persona!. e.g. an experimenter's preconceived ideas about an 'expected' result. Oias cHn be minimized by u.sing a carefully standardized procedure, wilh fully calibrated instruments. InvesligHte biHs in 'trial runs' by measuring a variable in several different \....
Measurement lrandom) error All measurements are subject 10 error. but the dangers of misinlerpretation are reduced by recogni7jng and understanding the lil..c1y sources of error and by adopling appropriate protocols and calculation procedures. A common source of random error is carelessness. e.g. reading H SC'dle in the wrong direction or parallax errors.. This can be reduced greatly by Careful recording and m'tly be detccted by rcpe-
Collecting and recording primary data Recording primary data - never be tempted to jot down data on scraps of paper: you arc likely to lose them, or to forget what individual values mean.
66
The investigative approach
When carrying out lab work or research projttts, you will need to master the importanl skills of recording Hod managing data. Individual observations (e.g. laboratory temperature) can be recorded in the text of your notes, but tables are Ihe most convenient way to collect large amounts of information.
Making and recording measurements
~
A good set of lab notes shoutd:
• outline the purpose of your experiment or obset'vation; • set down all the information required to describe your eMperimental section; • record all r&levant information about your results or observations and provide B visual representation of the data; • note your immediate conclusions and suggestions for further eMperiments.
When preparing a table for data collection. you should:
Designing a table for data coUeaioomake sure there Is sufficient space In each column for the values; if mdoYbt. err 00 the generous side.
Recording numerical data - write down only those numbers that can be justified by your measurement technique (significant figures).
I. Use a concise title or a numbered code for cross·refcrencing. 2. Deckle on the number of variables to be measured and their relationship with ~eh other and layout the table appropriately: (a) The first column of your table should show values of the independent (colltrolkd) variable, with subsequent columns for the individual (measured) values for each replicate or sample. (b) If several variables arc measured for the same organism or sample, each should be given a row. (c) In time-coursc studies, put the repliattes as columns grouped according to t.reatment. with the row'S relating to different times. 3. Make sure the arrdllgemenl rcnccts the order in which lhe values will be collecled. Your table should be designed 10 make lhe recording procc!<s as stmighlforwHrd as possible, to minimizc the p~sibilily of mistakes. For final presentation, a different arrangemenl may be best (Chapter 37). 4. Consider whether additional columns are ft'Quircd for subsc::quelll eulculations. Creale a scpamte column for each mathematiC
Recording details of project work The recommended system is one where you m.ake a dual record.
I
Choosing a lab notebook - a spiral-bound notebook is good for making a primary record: it lies conveniently open on the bench and jlf"ovides a simpk! method of dealing with major mistakesl
p,.imm")' j·ecOJ·d The primary record is made at the bench and you must concenlrate on the detail of materials, methods and results. Include infonnalion Ihal would not be used elsewhere, but which might prove useful in error tracing: for example, if you note how a solution W
67
Making and recording measurements
volumes and weights used rather than concentration alone), this could reveal whether a miscalculation had been the cause of a rogue result. Note the origin, type and state of the chemicals used. In the experimental section, the basic rule is to H.-cord enough infomtation to allow a reasonably competent scientist to repeat your work exactly, You must tread a line between the extremes of pedantic, irrelevant detail and the omission of information essential for proper interpretation - belter perhaps to err on the side of extra detail to begin with. An experienced worker C
You should make a secondary ft.·(:ord concurrently or laler in a bound book and it ought to be neater. in both organization and presentation. This book will be used when discussing results with your supervisor, and when writing up a report or thesis. and may be part of your course assessment. Writing a second, neater version forces you to consider again details thai might have been overlooked in the primary record and providL'S a duplicate in case of loss or damage. While the.<;e notes should retain the essential features of the primary record, they should be more concise and the emphasis should move towards analysis of the experiment. Don't repeat the experimental section for a series of similar experiments; use devices such as 'method as for Expt B4', A photocopy may be sufficient if the method is derived from a text or article (check with your supervisor). Outline the aims more carefully at the start and link the experiment to others in a series (e.g. 'Following the results of Expt 024, I decided to test whether .. .'). You should present data in an easily digested form, e.g. as tables of means or as summary graphs. Use appropriate statistical tests (po 271) to support your analysis of the results. Always analyse and think about data immediately after collecting them as this may influence your subsequent activities. Write down any conclusions: sometimes those which seem obvious at the time of doing the work are forgotten when the time comes to write up a report or the.<;is. ukewise. ideas for furlher studies may prove valuable later. Even if your experiment appears to be a failure. suggestions as to the likely causes might prove useful.
Using communal records If working with a research team. you may need to use their communal databases. These avoid duplication of efTon and ensure unifonnity in techniques. They may also form pan of the legal safety requirements for lab work. They might include; 68
The investigative approach
Making and recording measurements
• • • •
• •
a shan.'d notebook of common techniques (e.g. solutions or calibration technique); a set of simplified stcp-by-.step instructions for usc of equipment; an alphabetiC
The investigatjve approach
69
-8 Dimensionless measurements - some
quantities can be expressed as dimensionless ratios or logarithms (e.g. absorbance and pHI. and in these cases you do not need to use 8 qualifying unit.
Table 9.1 Tho base and supplementary 51 units Measured quantity
Name of SI unit Symbol
Base units
Length
metre
M'M
kilogram
Amount of substance
m kg
mO<,
Time Electric currenl
,mol
"""nd
ampere Temperature kelvin luminous intensity candela
Supplementary units Plane angle Solid angle
A
radian stcrodian
81 units and their use When describing a measurement. you normally state both a number and a
unit (e.g. 'the length is 1.85 melrt:s'). The number expresses lhe ralio of the measured Quantity to a fixed standard, while the unit identifies thai standard measure or dimension. Clearly. a liingle unified system of units is essential for efficient communication of such data within the scientific community. The 5ysteme International d'UnitCs (SI) is thc intcrnationally rdtificd form of the metre-kilogram-second system of measurement Rnd represents the accepted scientific convention for measurements of physical quantitIes. Another important fCllloon for adOpting consist(:nt units is 10 simplify complex calculations when: you may be deating with several measured quantities (I« p. 160). Although Ihe rules of the 51 are complex and the SC'
K
•
'"
•
nod
"
•
seven base units and two supplementary units. each having a specified abbrevlation or symbol (Table 9.1); derived units, obtained from combinations of ba~e and supplementary units, which may also be given speci<\1 symbols [fable 9.1); a set of prefixes to denote multiplication factors of 103, used for convenience to express multiples or frdctions of units (Table 9.3).
Table 9.2 Some important derived SI units
Table 9.3
Prefixes used in the SI
Multiple Prefix Symbol Multiple Prefix Symbol
'0 ' 10 • 10-9
10- 12 10- 15 10 18
milli micro nano pica femto
m
,0'
kilo
Il
HI' ,0'
~M
"p f '"0 ,
10 12 10 15 10 18
k
giga tera po"
G
T
,~
E
P
Measored quantity
Name of unit
Symbol
Energy Force Pressure
joule newton pascal
J N
p""",
w,"
Electric charge Electric potential difference Electric resistance Electric conductance Electric capacitance Luminous flux lIlumination Frequency Radioactivity Enzyme aclivity
coulomb
p,
W C
",,10
V
ohm siemens farad lumen I",
n
10"" becquerel katal
S F
1m I,
H, Bq
'"
Alternative in derived units
Definition in base units m1kgs 2 mkgs 2 kgm 1S-2 m2kgs 3
•
A'
Nm Jm' Nm' J, ' JV '
m2kgA-1 s 3 m2kgA 2 S-3 wA2kg-1m 2 S4 A2 kg-1 m 2
JC ' VA' A V-lor 0- 1 CV'
"'" ,-,
1m m- 2
cdsrm- 2
,-,
mol substrate
S-l
Recommendations for describing measurements in SI units Ba.\-ic format Example 10/1g is correct while 10jlg, 10/19. and 10119 are incorrect; 2.6mol is right, but 2.6 mols is wrong.
70
The investigative appfOach
•
Express each measurement as a number separated from its units by a space. If a preflX is required. no space is left between the prefix and the unit it refers to. Symbols for units are only written in their singular form and do not require full stops 10 show that they are abbreviated or that they are being multiplied together.
51 units and their use
Example newtons.
n stands for nano and N for
•
•
Give symbols and preflxes appropriatc upper or lower C'dSC initial letters as this may define their meaning. Upper case symbols are named after persons but when written out in full they arc not given initial capital leHers. Show the decimal sign as a full point on the line. Some metric countries continue to usc the comma for this purpose and you may come acros.<; this in the literature: commas shOUld not therefOre be used to separate groups of thousands. In numbers that contain many significant flgures. you should separate multiples of 103 by spaces rather than commas.
Compolilld express;oll!l' /0/' deril'ed ",,;t~· •
•
•
Take care to scparate symbols in compound expressions by a space 10 avoid the potential for confusion with prefixes. Note, for examplc, that 200ms (metre seconds) is differenl from 200ms (milliseconds). Expres.<; compound units using negative powers rather than a solidus (j): for example. write mol m- 3 rather than mol/m 3 . The solidus is r5ervet:l for separating a descriptive label from its units (see p. 251). Where there is a choicc_ selccI relevant (natural) combinations of derived and bH.se units, e.g. you might choose units of Pam-I to describe a hydrostatic pressure gradienl rather than kgm- 2 s- l • even though these units are equivalent and the measurements are numerically the same.
Use o.t"preJixes Examples 10 jjm is preferred to 0.00001 m or O.010mm. 1 mm 2 = 1 x 10-3 m 2 = 1 x 1O- 6 m 3 lnot one-thousandth of a square metre). 1dm3 (1 litre) is more properly expressed as 1 x{10 1 m ):! = 1 x lO-3 m 3. Avogadro's constant is 6.022174 x 1()Z3 mol- 1 •
•
•
• •
State as MW m-2. rather than W mm- 2.
•
In this book, we use land ml where you would normally find equipment calibrated in that way, but use SI units where this simplifies calculations. In formal scientific writing, constructions such as 1 x lO-6 m 3 1:= 1 ml) and 1 mm:! 1= 1 jll) may be used.
The other common non-51 unit of volume is the cubic centimetre, cm 3 , (10-2. m)3. Even though they are not exactly the same, ml, and em:! arc used interchangeably, as are cubic dccimetrc dm3{10-1 m)3 = 10- 3 m 3 and litre Ill.
Use prefixes to denote multiples of IW (Table 9.3) so that numlx'rs arc kept between 0.\ and 1000. Treat a combination of a prefix and a symbol as a single symbol. 11ms, when a modified unit is raised to a power. this refers to the whole unit including the prefix. Avoid the prefIXes deci (d) for 10- 1 and cellli (e) for 10-2 as they are not stricl!y SI. Express very large or small numbers as a number between I and 10 muhipliet:l by a power of 10 if they are outside the range of prefixes shown in Table 9.3. Do not use prefixes in the middle of derived units.: they should be attachet:l only to a unit in the nwnerator (lhe exception is in the unit for maS';, kg).
~
For the foreseeable future. you will need to make conversions from othet'" units to 51 units, as much of the literature quotes data using imperial, c.g.s. or other systems. You will need to recognize these units and find the conversion factors required. Examples relevant to chemistry are given in Box 9.1. Tabte 9.4 provides values of some important physical constants in 51 units.
Some implications of 51 in chemistry
Volume TIle SI unit of volume is the cubic metre. mJ. which is mther large for pmctical purposes. The litre (L) and the millililre (ml) are IcchnicaUy obsolete. but are widely used and glass......dre is still c:dibmtL'(! using them. The investigative approach
71
SI units and their use
Table 9.4
Some physical constants in SI terms
Physical constant Avogadro's constant Boltzmann's constant Charge of electron Gas constant Faraday's constant Molar volume of ideal gas at STP Speed 01 light in vacuo Planck constant Acceleration due to gravity Atomic mass unit Rydberg conslant Permitivity of vacuum
Box 9.1
Value and units
N,
6.022174 x lQ2J mo l-t 1.380626J K-l 1.602192 x 1O- 19 C B.31443J K-1 mol- 1 9.648675 x 10·Cmol- 1 0.022414m J mol- 1 2.997924 x lQ8ms- 1 6.626205 x 10 3'Js 9.807ms- 1 16605402 x 10-27 kg 1.097 x 107 m- I 8.854 x 10 12Fm-1
k
•R F
,V, h
•
'"
R~
"
Conversion factors between some redundant units and the 51
Quantity
SI unitJsymbol
Area
SQuare metreJmZ
Multiply numbet" in old unit by this factor for equivalent in SI unit-
Multiply number in SI unit by this factor for efluivalent in old unit-
square loot1ftl square incMn2 SQuare yBrdlylP
4.04686 x 10' lOx 1(jl 0.092903 645.16 x 10-' 0.836127
0.247105 x 10- 3 0.1 X 10~3 10.7639 1.55000 x 1()6 1.19599
Old uni1/symbol
.". h.'."_
Angle
radian/rad
17.4532 x 10.a
57.2958
Energy
joulelJ
,,'. kilOYIlItt hourlkWh
0.1 x 10-6 3.6 x 1()6
10xl()6 0.277 n8 x 10-6
Length
metrelm
A~mlA
0.1 x 10-' 0.3048 25.4 x 10 3 1.60934 x 10' 0.9144
10 x 10' 3.28084 39.3701 0.621373 x 10 3 1.09361
28.349 5 x 10-3 0.453592 6.3502. 50.8024 1.01605 >( 1()3
35.2740 2.20462 0.151473 19.6841 x 10- 3 0.984203 x 10 J
101325
f~tIf<
inchl'in mile y.nl/yd
Mass
kilogramlkg
ounce!oz poundltb ~~
hundredweight/cwt: ton IUK) Pressure
pascalJ1"a
atmosphere/atm I'''/b millimetre of mercury/mmHg torrfTorr
133.322 133.322
9.869 23 x 10 6 10x10 6 7.50064)( 10 3 1.50064)( 10~3
'00000
Radioactivity
becquerellBq
curieJCi
37 x lOS
27.0270 x 10 12
Temperature
kelvirV'K
centigrade (Celsius) degreeFC Fahrenheit degree! F
C+213.15 ( F + 459.67) x 5/9
K-273.15 (K )( 9/5) - 459.67
Volume
cUbic metreJm 3
cubic footlf13 cubic inchlin3 cubic yardfydl UK pint/pt US pint/liq pi UK gallon/gal US gallon/gal
0.0283168 163811 x 10-11 0.764555 0.568261 x 10 3 0.473176,. 1Q--3 4.50*609 x 104 3.78541 x 10 3
35.3147 61.0236 x 10' 1.30795 1759.75 2113.38 219.969
-In the case of temperature measurements, use formulae shown.
72
Symbol
The investigative approach
264.172
51 units and their use
Mas!l' The 51 unit for mass is the kiJogmm (kg) rather than the gmm (g): this i<; unusual becftltse the base unit has a prefix applied.
Amount of substance You should use the mole (mol, i.e. Avogadro's con<;tant, see Table 9.4) to express very large munbers. The mole gives the number of atoms in the atomic mass, a convenient constant.
COll(·Cllt,'atiOll The 51 unit of concentmtioll. mol m- 3 , is not convenient for general laboratory work. It is equiValent to the non-51 term 'millimolar" (mM) while 'molar' (M) becomes kmolm- 3 • If the solvent is not specified, then it is assumed to be water (see Chapter 6).
Time In geneml, use the second (s) when reporting physical quantities having a time element. Hours (h), days (d) and years should be used if seconds are clearly absurd (e.g. samples were taken over a 5-year period). Note, however, that you may have to convert these units to seconds when doing calculations.
Tcmperatw'c Definition STP - Standard Temperature and Pressure = 293.15 Kand 101325Pa (or 101.325kPa or 0.101325 M Pal.
Box 9 2
The 51 unit is the kelvin. K. The degrt"C Celsius scale has units of the same magnitude, °C, but starts at 273.15 K, the melting point of ice at STP. Temperature is similar to time in that the Celsius scale is in widespread use, but nOle that conversions to K may be required for calculations. Note also that you must not use the degree sign ( ) with K and that this symbol must be in upper case to avoid confusion with k for kilo; however. you sholiltl retain the degree sign with "C 10 avoid confusion wilh the coulomb, C.
How to interconvert 51 units
Example: You are required to calculate the molecular weight of a polymer by measurements of its osmotic pressure in solution. At infinite dilution. measured graphically from your experiments, the equation below applies:
n c
RT
2. look at the units and decide which are common: since the gas constant is expressed in joules. you should convert the osmotic pressure term into joules. The derived unit for pressure is N m- 2 and, since the derived units for N are Jm~l, the full derived unit of pressure is (J m- 1 ) x m- 2 = J m- 3.
M,
3, Substitute the units into the equation for M
where n = osmotic pressure at infinite dilution (Pal, R= gas constant (JK-lmol~ll. T= temperature (KI. c = concentration of solution (kgm-3j and M, = molecular weight. 1. Reararange the equation for M,: M,= RTc
n
M,=RTq=JK~lmOI-' x ~xkgm~3 =kgmol- 1
n
Jm
4. Substitute the appropriate numerical values into the equation for M,.: you know that the units of the calculation will be correct since the molecular weight is the weight of 1 mole of polymer, expressed in kg.
The investigative approach
73
51 units and their use
lntel"Conversion of SllInits You will find th
as kgm- J or molm- J , elc., and that you may need to use alternatives in derived units (Table 9.2). TIle application of these principles is shown in Box 9.2.
74
The investigative approach
------0 Definition Hypothesis - One possil»e explanation for an observed evenl. A mechanistic hypothesis is one based on some intuition about the mechanism underlying 8 phenomenon.
explel\liljon~ I1yjlllthesis
Bnd allllf1lllliv8llWlDlheces
I I
--
-~. femain
I
teIlabill
IDle remaining ~lhelllS
I
I conlifmMilll'l
I
,,,.,,
Scientific method and design of experiments Science is a systematized body of knowledge derive
Some bmnches of chemistry are designed to provide fundamental infomtution, rather th'in to lest a particular hypothesis: for example, the purirlC'.dtion and chamaerization of a newly discovered natur.dlly occurring molecule, In contr.d5t, an experimellf is a contrived situation de.otignL'd to t~t one or more hypothesis under conditions controlled by the investigator. Any hypothesis that C'.dnnot be rejected from the results of an experiment is provis.ionally accepted. This 'sieve' effcct leaves us with a set of (;urrent explanations for our observations. lllese explanation... are not permanent and may be rejected on the basis of a future investigation. A hypothesis that has withstood many such tests and has been shown to allow predictions to be made is known as a theory, and a theory may generate such confidence through its predictive abilities to be known as a law (Fig. 10.1). Omervations are a prelude 10 experimentation. but they are preconditioned by a framework ofperiphcr.dl knowledge. While there is an elemenl of luck in being at the righl place and time to make important observations, as P".dsteur stated. ·chance: favours only the prepan:<1 mind'. A fault in scicnlific method is that the design of the experiment and choice of method may influence the outcome the decisions involved may not be as objective as some scicntists assume. Another flaw is lhat radical alternative hypotheses may be overlooked in favour of a modification to the original hypothesis. and yet just such leaps in thinking have frequently been required before grei.t1 scientific advances. No hypothesis can evcr be rejected with certainty. Statistics allow us to quantify as vanishingly small the probability of an erroneous conclusion, but we are nevertheless left in the position of never being \00% ccrtain that we havc rejected all relevant alternative hypotheses, nor 100% certain that our decision to reject some alternative hypotheses was conect! However, despite these problems. experimental science has yielded and continues to yield many important findings.
hypolhellill becameH
I IheOfy OOc0lll85
I. .
Fig. 10.1
~
The fallibility of scientific 'facts' is essential to grasp. No eXplanation can eve.- be 100% certain as it is always possible for a new alternative hypothesis to be generated. Our understanding of chemistry changes all the time as new observations and methods force old hypotheses to be retested.
How scientific investigations prooeed.
Quantitati\·e hypotheses in\'ulve a mathematical description of the system. They <:an be fonnulated concisely by mathematical models. Models are often useful because they force det:per thought abant mechanisms and encoumge simpliflcation of the system. A mathematical model:
Definition Mathematical model - an algebraic summary of the relationship between the variables in a system.
• • • •
is inherently testable through experiment: identifies areas where information is lacking or uncertain: encapsulates many observations: allows you to predict the behaviour of the system. The investigative approach
75
Scientific method and design of experiments
Remember, however, that assumptions and simplific'ltions required 10 creale II model may result in it bcillg unrealistic. Further, the results obtained from any mo
Experimentation and variables Definitions
Treatment - a particular set of conditions applied to one or more experimental subjects. Block - 8 group of replicates: a sub-division of the entire set of experimental subjects (the fieldl. This terminology originates from agricuttuml experiments.
In many experimellls, the aim is to provide evidence for causality. If x CllUSes y. we cxpect, repc-dtably. to find thaI a change in x results in a changc in y. Hence. the idCll1 experiment of this kind involves measurement of y. the
dependent (measured) vdriable, al Olle or more values of x, the independent varillble, and subsequent dcmonstmtioll of some relationship between them. Experimcnts therefore involve comparisons of the results of treatments changes in the independent variable as applied to an experimental subject. The change is engineered by the experimenter under controlled conditiOnS, Experirnenl
ConfOimt/ing J'ariables Example In an experiment using a reagent prepared in an organic solvent. the concentration of solvent will V81Y alongside the concentration of reagent. A control, using sol....ent alone (usually called a 'blank'), will allow its effects to be determined,
These increase or decrease systematically ;IS the independent variable inacases or decreases. Their eITcets are known as systematic variation. 'Illis fonn of variation can be disentangled rrom that cau..cd directly by tre:lIments by incorpomting appropriate controls in the experiment. A control is really just another treatment where a potentially confounding Variable is adjusted so lhat its effects, if any. can be lakcn into account. The results from a conlrol may therefore allow an altcmalive hypothesis to be rejected. Thcre are often many potential controls for any experiment. The consequence of sySlt.'Tl1atic variation is that you can nevcr be cert:lin that the tl'"C'dtment. and the treatment alollc. has caused an observed result. By careful design. you call. however. 'minimize the uncertainty' involvcd in }our conclusion. Methods available include: • • • •
A....oiding personal bias - it may be necessary to encode the subjects, so that the in....estigator is 'blind' as to whkh subject is in which treatment regime_
• •
Enwring. through experimental design, that the independent variable is the only major faClor that changes in any treatment. Incorpomting appropriate controls to show that potential confounding variables have little or no effect. Selecting experiment31 subjects mndomly to cancel out systematic variation arising from biased seleclion. Matching or pairing individuals among treatments so tltat differeIX'Cs in response due to their initial status are eliminated. Arranging subjects and treatments randomly so that responses to systematic differences in conditions do not influence the results. Ensuring thai experimental conditions are uniform so that responses to systematic differences in conditions are minimized.
Nuisance l'ariables 11lese are uncontrolled variables which cau~ differences in the value of y inde{Xndently of the Value of x. resulting in random vdriation. Nuisance variables are not common in chemistry except where molecules from natural SOlJrces are used, To reduce and assess the consequences of nuisance variables: • • • 16
The investigative approach
incorpomte replicates to allow random variation to be quantified; choose experimental subjects that are as similar as possible; control random fluctualions in environmental conditions.
Scientific method and design of experiments
Constraints on experimental design
Evaluating design constraints - 8 good way to do this is by processing an individual subject through the experimental procedures - a 'preliminary
run' can help to identify potential difficulties.
Rox IO.1 outlines the important stages in designing an eJ(pcriment. At an early stage. YOu should find out how your resources mn}' constrain the design. For example. limits may be sel by a\~lilability of subjects. COSI of Ircalmen(, availability of a chemical or bench space. Logistics may be il
factor (e.g. lime taken to record or analyse dalal, Of your equipment
Using replicates Using independent replicates - remember that the degree of independence of replicates Is important: sub-samples
cannot act as replicate samples; they tell you about variability in the measurement method but not in the quantity being
meawrcd.
Box 10.1
Rcpliaue results show how variable the response is within trc.atn1t:nts. They allow you 10 compare lhe differences among treatments in the collte)(t of the variability within treatments - you can do this via stmistical tests such as analysis of V'dnance (Olapter 41). Larger sample sizes lend to increllsc the precision of estim
Checklist for designing and executing an experiment
1. Preliminaries
3. Planning laJ List all the materials you wi. need. Order any chemicals and make up solutions; grow, collect or prepare the experimental material you reQuire; check equipment is available.
(81 Read background material and decide on 8 subject area to investigate.
(bl Formulate a simple hypothesis to test. It is preferable to have a clear answer to one Question than to be uncertain about several Questions.
lb) Organize
lc) Decide which dependent variable you are going
lei Account for the t;me taken to appty treatments
to measure and how: is it relevant to the problem? Can you measure it accurately, precisely and without bias?
4.
lal Find out the limitations on your resources.
possible. Idl Keep the number of replicates as high as is feasible. Ie) Ensure that the same number of replicates is present in each treatment with random allocation to individual treatments.
Carrying out the experiment lal Record the resutts and make careful notes of ....erything you do. Make additional observations to those planned if interesting things happen.
2. Designing
lcl Incorporate as many effective controh as
and/or time in which to do the
and record resufts. Make out a timesheet if things will be hectic.
ldl Think about and plan the statistical analysis of your resutts. Will this affect your design?
lb) Choose treatments which alter the mjnjmum of confounding variables.
$p8C8
experiment.
lbl Repeat experiment if time and resources allow. 5.
Analysing
lal Graph data as soon as possible (during the experiment if you can). This will allow you to visualize what has happened and make adjustments to the design (e.g. timing of measurements). lbl CatTY out any planned statistical analysis. lei Jot down conclusions and new hypotheses arising from the experiment.
The investigative approach
77
Scientific method and design of experiments
,
'x,
ox.
C
B
A
C
B
B
,
C
0
B
C
C
B
A
C
0
,
,
,
B
0
B
C
0
Fig. 10.2 Examples of Ultin square arrangements for three and four treatments. letteN; indicate treatments; the number of possible Brrangements increases greatly as the number of treatments increases.
Example If you knew that soil type varied in a graded fashion across a field, you might arrange blocks to be long thin rectangles at right angles to the gradient to ensure conditions within the block were as even as possible.
fllIlUraFw81af
,
2
3
•
I
0
,
0
B
,
B
,
C
A
C
0
B
C
A
A
0
B
" ,, , • ,
C
B
A
C
,
§
0
Fig. 10.3 The experimenter wishes to investigato the effect of pH A-E on the
extraction of phenols from fiW! natural waters. Each water sample is extmeted at a different pH and the influence of pH on the recovery of phenols noted.
If the total number of replicates aVailable for an experiment is limiled by n.-sources, you may nt.'Cd to compromise between the number of treatments and the number of replicates per treatment. Statistics can help here, as it is possible to work out the minimum number of replicates you would need to show a eenain difference between pairs of means (say 10%) :11 a specified le\'el of significance (say P = 0.05). For this. you need to obtain a prior eslimate of vtlriability within treatments (see Miller and Miller, 2000).
Randomization of treatments For relatively simple experiments. you can adopt a completely mndomized design; here. the pOSition and tre.ltment as....igned to any subject is defined randomly. YOlJ can dr
Multifactorial experiments
Definition Interaction - where the effects of trealments given together are greater or less than the sum of their individual effects.
78
The inve!>1.igatiW! appmach
The simplcst experimentS arc those in which one trc."1tment (faclor) is applied al a time to the samples. This approach is likely to give clear-cut answers, but it could be criticized for lacking realb.'D1. In particular, it cannol take account of interactions among two or more condilions that are likely to occur in real life. A multifactorial experiment (Fig. 10.4) is an attempt lo do this; lhe interactions among treatments can be: analysed by specialized rorms of analysis of \'ariance. Multifactorial experiments are economical on resources because of 'hidden replication'. This arises when two or more treatments are given to a subject because the result acts statistically as a replicate for each treatment. Choice or relevanl treatments to combine is important in multiraclOrial experiments; ror instance, an interaction may be present at certain concentrations of a chemical but not al others (perhaps because the response is saturated). It is
Scientific method and design of experiments
-
facto! B
,
,
,
•
8+ b. c
factor
A
•
Fig. 10.4 Design of a simple multifactorial experiment. Factors A and B have effects a and b when applied alone. When both are applied together, the effect is
denoted by 8 -+- b + c.
•
If c == 0, there is no interaction {e.g.2+2+c=41. If c is positive, there is a positive interaction (synergisml between A and B
•
(e.g.2+2+c=51. If c is negative, there is a negative interaction iantagonism) between A and B
•
le·9· 2 + 2 +c=3).
also important thai Ihe measurement scale for the response is consistent. otherwise spurious interactions may occur. Beware when planning a muhifactorial experiment that the numbers of replicates do not gct out of hand: you may havc to restrict the treatments to ·plus· or 'minus' the factor of interest (as in Fig. 10.4).
Repetition of experiments
Reporting results - it is good practice to report how many times your experiments were repeated (in the experimental section); in the results section, you nced
either a statement saying that the illustrated experiment is representative or one explaining the differences between results obtained.
Even if you have taken great care to ensure that your cxperiment is well designed and statistically analysed, you lire limited in the conclusions that can be made. Firstly, what you can say is valid for a particular place and time. with a particular investigator. experimental subject and method of applying treatments. Secondly. if your rcsuhs were significant at the S% level of probability, there is stil1 an llpproximately I in 20 chance that the results did arise by chance. To guard against these possibilities. it is important that experiments are repeated. Ideally, this would be done by an independent scientist with independent materials. However. it makes sense to repeat work yourself so that you am ha\e full confidence in your conclusions. Many scientists recommend that all experiments are carried out three times in total. This may not be possible in undergraduate practical classes or project work!
The investigative approach
19
----------10
Project work Research projects are ;w important component of the fmal-year syllabus for most degree progmmmes in chemistI)', while shorter projects may also be carried out during courses in earlier years. Project work prescnls difficulties at many stages but can be extremely rewarding. The asses...ment of your project is likely to contribute significantly to your degree grade, so all aspects of this work should be approached in a thorough manner.
Deciding on a topic to study The Internet as an information sourcesince many university departments have home pages on the World Wide Web. searches using relevant key words may indicate where research in your area is currently being carried out. Academics usually respond positively to e-mailed questions about their area of expertise.
Assuming you have a choice, this important decision should be researched carefully. Make appointments to visit possible supervisors and ask them for advice on topics lhat you find interesting. Use libmry texts and research p
•
• Asking around - one of the best sources of information about supervisors.
laboratories and projects is past students. Some of the postgraduates in your department may be products of your own system and they could provide an
•
alternative source of advice.
•
Opportunities to leam new skills. Ideally, you should altempt to gain experience and skills that you might be able to 'scU' 10 a potential employer. Easc of obtaining valid results. An ideal project provides a means to obtain 'guaranteed' data for your report, but also the chance to extend knowledge by doing genuinely novel research. Assistance. What help will be aVailable to you during Ihe project? A busy lab with many research students mighl provide a supportive em-ironment should your potential supervisor be too busy to mcct you ollen; on the other hand, a smaller lab may provide the opportunity for more personal interaction with your supervisor. Impact. It is not outside the bounds of JX>SSibilily for undergraduate work to contribute to research papers. Your prospective supervisor can alerl you to such opportunities. Success. You are doing a research project and it may not always provide a positive result. Negative results are just as useful.
Planning your work
Liaising with your supervisor(s) - this is essential if your work is to proceed efficiently. Specific meetings may be
timetabled. e.g. to discuss a term's progress, review your work plan or consider a draft introduction. Most supervisors also have an 'open-door"
policy, allowing you to air current problems. Prepare well for all meetings: have a list of questions ready before the meeting; provide results in an easily digestible form (but take your lab notebook along); be clear about your future plans for work.
80
The investigative
approach
As with any lengthy exercise, planning is required to make the best use of the time allocated. This is true on a daily h
Project work
Getting started
I
Fig. 11.1 is a flowchart illu.~tratjng how a project might proro.:d: llt the starl, don't spend too long reading the litcmture <100 working. Out
I
•
I
•
C~IlI'Sllety
.U_
aspectli of wert
•
irldudmgriu
-,
even a 'failed' experiment will pt"ovidc some useful inrormation which may allow you to create a new or modified hypothesis; pilot expcriment~ may point out deftcicncies in experimentlil technique that will need to be rectified; the experience will help you create a realistic plan of work.
(I" ChapI8f 21
Designing experiments or sampling procedures
I
Son
51l1rt writing:
... .-
o~
.......
.II'.ateriiIb liM
wM:;hlp
detailed
~
_
olll9lfofnew
Inlly&und grBph diltB U
you poceed
Design or experiments is covered in Chapter 10. A void being too ambitious at the stan of your work! It is generally best to work with :1 simple hypothesis and design your experiments or sampling around this. A small pilot experiment or test sample will highlight potential stumbling blocks including TCSOurce limitations. whether in materials or time or both.
dillCIISSIOIl
·referllfll:e bt
Working in a laboratory environment
I Fig. ".1 Flowchan showing a recommended sequence of events in r:arrying out an undergraduate research projer:l.
During your time as a project student. yOll are effectively a guest in your supervisor's laboratory. •
•
•
•
Be considerate - keep your 'area' tidy and ofTer to do your share or lab dutk.'S such as calibrating the pH meier, replenishing stock solutions. distilled water, cleaning used glassware. etc. Use instruments carerully - they could be worth more than you'd think. Careless lise may invalidate calibration settings and ruin other people's work as well as your own. Do your homework on techniques you intend to IISC - there's less chance of making costly mistakes if you have a good background understanding or the methods you will be mingo Always seek ad\~cc if you are unsure or what yOIl are doing.
~ tt is essential that you follow all the safety rules ap·
plying to the laboratory or field site. Make sure you are acquainted with all relevant procedures - normally there will be prominent warnings about these. If in doubt, ask!
Keeping notes and analysing your results Tidy record keeping is often associated with good research, and yOli should follow the advice and hints given in Chapter 8. Try to keep copies or all files relating to your project. As you obtain resUlts. you should always calculate, analyse and graph data as soon as you can (see Fig. 11.1). This can reveal aspects that may not be obvious in numerical or readout form. Don't be worried by negative results - these can sometimes be as useful as positive results if they allow you to climinate hypotheses - and don't be dispiritcd ir things do not work l"irst time. Thomas Edison's maxim 'Genius is one per cent inspimtion and ninety-nine per cent pcrspimtion' certainly applies to research work! The investigative approach
81
Project work
Writing the report The structure of scientiftc reports is deall with in Chapler 52. The following advice concerns methods of accumulating relevant infonnation. Brushing up on IT skills - word processors and spreadsheets Bre extremely useful when producing a thesis. Chapters 48 and 49 detail key features of these programs. You might benefit from attending courses on the relevant programs or studying manuals or texts so that you can use them more efficiently.
Imrodllctioll This is a big piece of writing thai Cdn be very timeconsuming. Therefore. the more work you can do on it carlyon, the better. You should allocate some time at the start for Iibmry work (\.Vithout neglecting benchwork), so that you can build up a dataoo.<;c of references (p. 319). While photocopying can be expensive, you will find it valuable to have copies of key fCviews and reference; handy when wriling away from the library. Discuss proJX>Sals for content and strocture with your supervisor to make sure your effort is relevant Leave SJKICC at the end for a section on aims and objectives. This is important 10 orientate readers (including assessoni), but yOll may prefer to finalize the content after the results have been analysed! Experime"tlll You should note as many details as possible when doing the experiment or making obsCTvations. Don't rely on your memory or hope thai the infonnation will still be aV.lilable when you come to write up. Even if it is, chasing these details mighl waste valuable time.
Using drawings or photographs - these C8n pr(l\lide valuable records of sampling sites or experimental set-ups and could be useful in your report. Plan ahead and do the relevant work at the time of carrying out your research rather than afterwards.
Reslllt~·
Show your supervisor gnlphed and tabulated versions of your d;lIa promptly. These can easily be produced using a spreadsheet (p. 307), but you should seck your supen1sor's advice on whether Ihe design and prinl quality i!> appropriate to be included in }'our lhesis. You rna}' wish to acces.<; a specialisl grhable-quality gmphs and charts: allow wrne time for learning its idiosyncr
DisClIssioll Because this comes at lhe end of}'our tllesi!>, and liOme parts can only be written after you have allihe rcsulL<; in place, the temptlltion l<; to leave the di!>Cussion to last. Thi!> means that it ean be rushed - nOl a good idea beciusc of the weight attached by assessors to your analysis of data and thoughts about future experiments. It will help greatly if yOIl keep notes of aims, conclusions and ideas for fulure work (JS .1'011 go (Jlong (Fig. 11.1). Another useful tip is to make notes of compamble data and conclusion!> from the literature as you read papers and reviews. Acknowledgements Make a !>pedal place in your notcbook for noting all those who havc hc1pt:d you carry out the work, for lise when writing this section of the report. Refel'ences Because of the complex formats involved (p. 319). these can be tricky to type. To save time. process them in batches as you go along.
~ Make sure you are absolutely certain about the dead-
line for 5ubmttting your report and try to submit a few days before it. H you leave things until the last moment, you may find access to printers. photocopier.; and binding machines is difficult.
82
The investigative approach
Resources
Resources for the investigative approach
&oks Adams, MJ. (19IJ5) Cl1l'mol/l{'rric$ in Alit/lyrical Ch{'mi.wry, Royal Society of Chemistry, Cambridge.
Crdwforo, K. and Heaton, A. (1999) Problem Soli'ing in Analytical Che"lI:wr)'. Royal Socicty or Chemistry, Cambridge.
Massart, D.L., Vandcginstc, B.O.M., Buydens, L.M.C., de Jong, S.. l.ewi, Handbook of Chem()lll(>fric~' and Qllo/imet";cs: Port A, Elsevier Science B.V.. Amsterdam.
PJ. Clnd Smeyers-Verbcke, J. (1997)
Meier, P.e. (2000) S/ml.Wical k/l'I/uxl!; in John Wiley and Sons Lld. Chichester.
Ana~I'lica/
ChclIJi.stry, 2nd Cdn,
Miller, J.N. Clnd Miller, J.e. (2000) Slatis/ics and Chell/ome/des for AllolYliml ClIl'mislry, 4th Edn, Prentice Hall. Harlow. Essex.
The investigative approach
83
------8 Purity of solids - mahing points do not vary significantly with changes in atmospheric pressure. wnef6aS the boiling points of pure liquids are not constant: they vary with changes in atmospheric pressure and <:ilonat be used as a measure of purity.
Melting points Melting points arc mC
The melting r,lllgc and upper limit arc an indication of the purity of the sample. Comparison of the melting point with thc lilcntture may indicate the identity of the compound or confimlthal it is not the compound required. If the compound is ncw, olher scicntists will need the information. TIlC compound can be identified with reasonable certainty by laking a mi)((:d nlClting point (I". 90).
Criterion of purity Melting point depression - only imlXlrities, which will dissolve in the compound, will lower its melting point. e.g. reactants. by-products. solvents. Insoluble impurities, e.g. salts, charcoal, filter paper, grit, etc., will not dissolve in the melted compound.
When you synthesize compounds in the
laboratory, you will always be asked to determine their melting points so that your practical expertise can be 8ssessed.
Pure solid covalent organic compounds and many inorganic complcxes incorporating organic ligands have definite melting points. The pure solid will melt l'CProducibly owr a narrow lCll1pcraturc range. usually less than I ~C, lind lhis melting range is known as tllC melting point. If the compound is not pure, the melting range will increase significantly and the upper end of the melLing range will be lowered. Thus the melting point (m.pt.) of a compound is a mcasure of its purity (p. 90). Other metll<xls arc uS(:d routinely to estimate purity of solid compounds. such as NM R (p. 190). and the presence of a single 'spot' or a single pc-,1I.-: on a chromatC1;ram from thin-layer (p. 216). gas-liquid (p. 211) or high-pclformance liquid (p. 218) chromatography, butmclting point remains the standard measure of purity.
~ The term metting point really means the melting
range of a chemical and in your laboratory report you should always quote the measured melting range under the heading 'metting point'.
Metting point apparatus The equipment for measuring the melting point of a solid varies in complexity from 3 simple oil bath heated with a mierobumer to a microscope with a heated stage as shown in Fig. 12.1. The essential components of a melting point apparlltus arc: • Thermometers - make sure that you use
a partial-immersion thermometer not a total-immersion thermometer. The type is written on the back of the thermometer.
•
•
A sample holder: usually a glass capillary lube sealed at one end ill the case of the oil bath and heated block systems, or a pair of microscope slides On an electrically heated plate in lIlC Kofler block. A temperature recording dC\~ce, placed as near to the sample as possible. Usually this is a themtometer but it can be a thennocouplc probe with digital readout. A heat source with fine control to allow a grnclual increase in temperatUfC. These SOlIIttS valY with the sophistication of the (,'quipmCl1t.
In the undergraduate labomtory you arc likely to use only the simpler systems such as the oil bath or heated block apparatus.
Capillw'j' tubes Capillary tubes for n~ltillg point measurement are available commercially and are supplied open at both ends or closc(\ at one end or closed at both Laboratory lechniques
87
Melting points
..... ....
...........
-...
./
'"
I-I
'"
1'1
Fig. 12.1 Melting point apparatus: (a) oil bath; (bl healed block - thermometer readout; (e) heated block - digital readout; (dl Kaner hot-stage microscope.
Breaking capillary tubes - if the capillary lUbe is sealed at both ends, yOu can break it into two half-sized single-endsealed melting point tubes by scoring the mid-point of the tube with a glass file and then snapping the tube at the score mark. If you don't score the glass, the edges of the break will be uneven and the tube will be difficult to fill.
88
Laboratory techniques
ends. To seal one end of an open capillary tube, just touch the end of the cllpilllll)' lube onto the ouler 'layer' of the hot flame of a microbumer (see Fig. 12.2). The end of the lube will rollapSe ill and seal the tube. Make sure IlulI the tube is sealed. i.e. there is nol II fine linc in the sealed end, and that thcre is no large globule or glass on the cnd or the lUbe. otherwise it may not fit into tJ1e hole in the healing block or the melting point app
Melting points
, ~o .......... ...... , .......... ..".,..,
1lbIIi'otld 10 plllVllflt_1lI
ccrrect ie.nll
corclonsatlOl1 insid8
Fig. J2.2 Sealing a melting point capillary
tube.
moved to the scaled end by turning the lube o\'er and lapping il on the bench, or by vibrating it by rubbing it against the thread of the screw on a clamp, or dropping It down a long g111SS tube onto the luboratory bench. Remember: you only nct-d 2-3mm ofS
Oil hallr aPP"J'lIlu,'i The component parts of a typical oil bath melting point appar.ttus are shown in Fig. 12.3. •
• Safety note The 'notch' in the. oortl i. essential: it allows the thermometer to be read over aU its length, it allows the thermometer to be gripped by the corte Bnd most Importantly, h allolNS lhe
•
heated air to escape from the apparatus. Nev« use a cork without the ;ootch'.
•
If the rubber band comes into COfltact with the oil it will expand - the capillary tube VlIiI! drop off into the oil bath and it will discolour the oil so that you will not
•
be able to see when your sample melts.
•
Check that the mineral oil is c1C<1n and contains no water (p. 34) lind Ihat the bulb is only two-thirds full, to allow for expal1sion. Clamp the oil bath to a support slllnd (p. 26). Check that your thcmlOllletcr is of the ;lppropriate ral1ge, thllt the mercury thread is int;let and the glass, in particular the bulb, is not crllcked. AttHch the cnpillHI)' to the thennomelcr with a rubber ling, mllking sure that the compound is next 10 the thenllometC!· bulb and remember to hold the thcnnometcr near the bulb while attaChing the capillary tube (p. 13). Press the Iheml0f11Cter into the 'notched' cork, making sure lhat you can see the thennometer scale, and trial fit Ihe thennometer, sample lind cork into the oil bath making SUI"C that the thermometel' bulb and sample arc ill the centre of the oil bath and that the rubber band is not in the oil. and will not be covered by oil, when the oil expands on heHting. If adjustment is needed, carefully s.lide the cork up or down the thermometer (p. 13). You call attach a melting point capillary to both sides of the thelIDOIlleter bulb to Clmy out two separate melting poillt determillHtions simultaneously.
Eleclric healed block appaI'ullls The hC'odting block usually has three holes, to pennit three simultaneous measurements or melting points. Always ensure that: • • • • • Fig. 12.3 Components of an oil bath melting point apparatus. safety note Mercury vapour is a severe cumulative end chronic hazard and the normal vapour Pfessure of met"cury, at
room temperature, is many times above thlt contrellimit (ell of 0.05 mg m--3 tf you break a thermometer or find or suspect the presence of mercury, inform
your instructor immediately.
The heating block is. at room temperature al the outset. The light in the healing block WOrks. TIle thcnllometer is undamaged (as above) al1d fits snugly into its hole in the heating block. All heating controls arc sct at zero. The capillary tubes slide easily into their holes in the heating block.
TIle usual problems encountered with tltis equipment arc broken capillary tubes or brokCrJ thermometer bulbs in the heating block. Consult your instructor in these cases.
Melting point determination The general procedure is to heat the oil bath with the micro burner using the technique described on p. 32, or 10 usc the heating control to give a temperature rise of about IODC per minute. When the temperature is about 20°C below the melting point, lhe rate of heating must be reduced to about 2 C per minute and continued at this rate unlil fhe compound has melted. The melting point is. the temperature range from where the firsl drop of liquid appears to where the last crystal dissolves into the liquid, Laboratory techniques
B9
Melting points
The: following points of technique shouk! he considered: Heating too fast - if you record 8 melting point which is higher than the literature value, you are implying that your compound has a purity >1000/0. which is impossible. Therefore it must be 8 different compound to the one expected.
•
•
It is normal practice to rn",ke at least two measurements of the melting point, one
apprQKimate and at least one accurate reading.
•
•
TIle most common error is he:lting the sample too tjllkkl}'. Thcre is ortcn a lag between slowing the hc=atin8 rate and the rc=duction in tempentture increase. resulting in a reading of the melting point which is too high. If you do not know what Ihe compound is. you will nOl know its expected melting point. TIlCrcfore. you shoukl dete:rminc :1Il approxim:lte value and then repeat the procedure to give an accurate lllC
Thermometer calibration Calibrated thermometers - if your compound is onty slightly impure and
has a melting point 2' C lower than the literature vall18. a thermometer reading 10 C low will give you an error of 12 C. indicating a low Klval of purity and hence a low grade or false information to fellow scientists.
Table 12.1 A typical sefies of standards for thermometer calibration Compound
Literature m.pt.
Naphthalene Benzoic acid
79-80 C
4-Nitroaniline 4.Toluic acid Anthracene
121 C 147 C 180-182 C 216-21B C
Your thermometer may be a major sour<.'C of e:rror in melting point rne::lsuremcnt. Occasionally thermometers for routine labonttolj' use may not be accurate and may read up to IO~C high or low. To avoid this problem you should always calibrate your thermometer to determine any error and be able to correct for it. To Clllibrate your thermometer you must measure the melting points of a series of vcry pure compounds. ava.ilable commercially. having a range of melting points similar to the range over which you will use the thermOmeter: a general-purpose series is shown in Table 12.1. Having measured the melting points of each of the pure comrlOunds. take the mid-point of each value for lhe thermometer re-ddlng of mehing point for each compound and the midpoillt of the literature melting point of each compoulld and plol them graphiC'dlly usin!] litemture temperature as til: x-axis and lhe thermometer reading as the x-axis. The straight-line plot will obey the equation y = til.\: + c. where y = real temperature (literature m,pt.). x = thermometer reading. m = slope and c = intercept. If you use a computer and suitable program. the values of m and t· will be calculated and you thcn solve the equation for using the thermometer reading (x-axis values) lo find the !'Cal temperature of melting u"axis values) to find the true melting ntngc. Of <."Curse, if you break or lose your thermomcter. you must calibrate another.
Mixed melting point determination You mn eoofinn the identity of a compound by determining a mixed melling point. If you prepare a mixture of your unknown chemical and the one you suspect it may be and measure the melting point of the mixture then there are two possible results: I.
90
Laboratory techniques
lbc melting point of the mixture is the same as the pure compound. which means that the unknown compound and the known compound are the slime.
Melting points
2.
The melting point of the mixture is lower than either of the two pure components and the melting range is large. This is because the two compounds are dirrerent with the result that one is an impurity in the· other.
For example. both benzoic acid and mandelic acid are white crystalline solids which melt al 121°C. However a I: I mixture of the two compounds begins to melt at about 80 ~c. The usefulness of mixed melting points is limited in that you must have some idea of the chemical nature of your unknown compound and a sample of tile suspected compound must be available.
laboratory techniques
91
-----10 Recrystallization involves allowing a hot solution of the required compound to
cool. Crystallization implies allowing the solvent to evaporate from a solution of the compound. Crystallization will not remove solvent-soluble impurities since they will be deposited as the solvent
evaporates.
Recrystallization The prodlu..1S from many synthctic preparations are seldom pure and the technique of recrystalli7-
2. 3.
limits of purification - crude solids containing only up to 10-15% impurities can be purified by recrystallization. Otherwise chemical purification or chromatography (p. 217) will be required to produce a compound. which can then be further purified by recrystallization.
Practice in the technique of
recryStallization is important. since the aim of the procedure is to produce the
Insoluble material: anti-bumping gmnules, pieces of filter paper, traces of drying agents. grit, hair and other materials which may huvc been present in the starting chemicals. Small quantities of unreaeted starting chemicals and/or by-products from side reactions or other isomers. Very smull amounts of coloured by-products resulting from oxidation or polymerization of the chemicals used.
Recrystallizalion is designed to remove all these types of impurity and provide a pure product suitable for melting point measurement. Purification by recrystallization is based on the theory of saturated solutions (p. 50) and a suitable recrystallization solvent is olle in which the chemical to be purified is insoluble in the cold solrem and soluble in the hOi soll·enf. When the crudc reaction product is dissolved in the hot solvent the insoluble impUlitics (type I above) can be removed by hot filtration (p. 98). When the hot solution is allowed to cool. the solUlion becomes saturated with the desired compound uod it precipitates from thc cold solution. The cold solution does not becomc saturated with the lower concentration of the contaminants of type 2, which therefore remain in solution. Coloured impUlilies (type 3) can be remO\'oo by absorption using charcoal as described on p. 96. The recrystallization process can be divided into three st.jJaruIC steps:
maximum quantity of the higheh't quality
product. Poor technique ohen results in low recovery of high-quality product or high recovery of fow-quality product.
I. 2. 3.
Selection of a suitable solvent. Recrystallization of the crude compound. Drying the pluified solid.
Sob'ents for reuysttllliztl(;olt You can decide on a suitable solvent for fa"yst
•
The experimental prOiocol may tell you which solvent to usc. If you know the identity of the compolmd you havc made, refcrence texts may indicate a suitable solvent (e.g. Lide, 2000: Buckingham and Macdonald, 1995). If you arc not sure of the identity of the compound you have prepared or if it is a new compound, you must carl)' out a 'solvent selection' to find out which solvent is the most appropliate (p. 93).
When selecting a suitable solvent for recrystallization. chemists work to the general rule 'like dissolves like' whcn considering the polarity of the chemical to be recrystallizt.x1 and the polarity of the sol\'Cnt. In general solvents for recrystallization are classified in terms of polarity and miscibility. Solvent polarity depL"nds upon the overall dislonion of the electron clouds in the I,:ovalent bonds within the solvent molecules, resulting in a dipole. The greater 92
Laboratory techniques
Recrystallization
the dipole, the greater the polarity of the solvent, e.g. Irichloromcthllnc has three polarized C-C1 bond.e;, giving an ovcmll dipole to the molecule :tnd producing 11 good solvent for many molecules. but in ICLrachloromctlwnc all of the G CI dipoles cancel out, giving a non-polar solvent with different solvent properties in comparison. An additional fcatUl'C that adds solvating power is hydrogen /xJl/r/illg. Thus water, a polari7.Cd molecule, can foml hydrogen bonds with oxygen and nitrogen atoms in SOlutes, dissolving them efficiently.
Whcn you arc choosing a solvcnt for recrystalliZJtion, look for lhe following general charneteristics: Yields from recrystalliUltion - some of
•
the compound that you require will
remein di&soMld in the solvent because il has to form a saturated solution. TherefOrp you can flfIVfJI'recover ltlCl% of
your compound by recrystallizatkm.
Dofi_n Solvents which mix in all proponions,
such as water and ethanol. are said to be miscibM, whereas 60lvents which do not mix, e.g. water and petrol, are • immiscible.
• •
A high dissolving power for the solule at high tempemture lind a low dissolving power at room temperature or below. so that a high recovery of purified compound can be achieved. A high or negJigiblc dissolving power for the impurities, so that they will eithcr be filtered off or remain in solution. A relatively low boiling pOint to facilitate drying the purified compound.
The miscibility of soh-ents with each othcr must be taken into account when attempting mixed-soll'em recrystalli=lltiom (see p. 95); the properties of some commQfl rccrystalli,,-ation solvcnts, which you arc likely 10 encountcr in your labonltory work, are shown in Tablc 13.1. R~mcmbcr Ihat this is only a gcneml list of solvents; fU.iher infOrmation on solvent I)ropcrties can be found in standard textbooks such liS Harwood et (II. (2000. p. 133), Loewenthal (1990, p. 146) and Furniss er fll. (1989. p. 137). Table 13.1 Setected solvent properties Solvent
b.pt.
Polar\ty
Miscibitity Commems: dass of organic with water compounds recry8tilllized
rCI
Water
'00
V. high
V"
Ethanol
7.
High
V"
Propanone
56
High
V"
Ethyl cthanoate Dichloromethane 41
Medium Medium
No No
Toluene Light petroleum
L~
V.low
No No
7.
"'
40-60
Always use when suitable: sohs. aromatic acids Flammable: alcohols, acids, amides Flammable: carbonyl compounds Flammable: eSlers Toxic: halogen compounds. ethers Flammable: hydrocarbons Flammable: hydrocarbons
Sob'ent select;oll To fUld a suitable sol\'CJlt for ra::ryslallization you must carry OUI a scnes of tests measuring thc solubility of your crude compound in a series of solvents (cold and hot) of varying solvCJlt polarity. Tlus series of tests is called 'solvent selection' and is carried out on a test tube scale but, as your technique improves, you can carry out the tesls using semi-micro scale since you will usc less of your compound during the process. 111e procedure for solvent sclection at we test tube scalc is desctibed in Box 13.1 and mooificalion of the procedure 10 semi-micro scak requires only a corresponding reduction of the quanlilies of solvcnt and solute used.
~
When carrying out the solvent selection experiments you must always cool the solution after heating. Do not assume that if the compound is insoluble in the cold solvent and dissolves when heated, it will always precipitate on cooling.
Laboratory techniques
93
Recrystallization
Box 13.1
How to carry out a solvent selection for recrystallization of an unknown compound (b) The solid cfissolves and thus the solvent may be useful for recrystallization. {cl Most of the compound dissolves but leaves a small amount of insoluble residue. This means that there Bre solvent-insoluble impurities present in your compound and the solvent may be suitable for use in recrystallization. (d) The solid melts and floats on the meniscus at the top of the solvent giving the appearBnce of having dissofved. This is a common feature of low-melting-point hydrocarbons such as naphthalene and other non-polar aromatic compounds: shake the test tube to see if oily globules are released from the meniscus. If this occurs, the solvent is unsuitable far recrystallization.
A suitable range of solvents for general use. in order of decreasing polarity, is: water, ethanol, propanone, ethyl ethanoate, dichloromethane, toluene and light petroleum lb.pt. 40-60' CI. 1. Clean and dry six Pyrex R test tubes to ensure that you will have no problems with contaminated solvents when carrying aUf the solubility lests. 2.
Add a small sample of the compound under test to each tube, using just enough compound to cover the bottom of the test tube.
3.
Add about 2.0 ml of a pure solvent to the first tube and observe the effect. You should look for the following features, which may help in the next recrystallization stage: (a) Tho solid does not dissolve, but it ts 'wetted' by the solvent. This implies that the solvent may be suitable for use in recrystallization. (bl The compound dissolves easify. Therefore the solvent is unsuirable for recrystallization and there is no need to continue tests with this solvent. (e) Most of the compound dissotves but leaves a small amount of insoluble residue. This means that there are solvent·insoluble impurities pre· sent in your compound, but the solvent itself is unsuitable for use in recrystallization. (dl Sarno cmour is released into the sotvent and the compound becomes lighter in cofour. This means that there could be a coloured impurity present in your compound and it will be necessary to de· colOl'ize the prodUct in the recrystalllzation experiment.
4.
94
Heat the mixture in the test tube by an appropriate means, bearing in mind the flammability and toxicity of the solvent to see if the solid dissolves in the hot solvent For non-f1ammeble solvents, remember to hold the test tube with a holder (see p. 37) and to 'wave' the test tube over the heat source to prevent 'bumping' (p. 31). Flammable solvents should be heated using a steam bath in a fume cupboard: do not lower the test tube into the steam bath and leave it there. Instead you must 'wave' the test tube in the steam escaping from the bath to achieve controlled heating. This is partiCUlarly important wilh lowboiling-point flammable or loxic solvents such as propanone, dichloromethane and light petroleum (b.pt. 4O-6O~C). You should look for the following results: (a) The solid does not dissolve in the hot solvent. which is therefore unsuitable for recrystallization and there is no need to continue tests with this solvent.
Laboratory techniQues
5.
Cool the test tube in an ice-water bath or under a cold stream afwater ensuring that no water gets into the test tube. There are sevtlral possible results: Ie) The compound recrystallizes in high yietd on coding: often there will appear to be mare solid than you started with because of the tine crystals produced. This solvent is suitable. jb) The compound recrystallizes in low yield on cooling: the solvent is unsuitable unless no better alternative can be found. (e) The compound remains in solution after cooling: the solvent is unsuitable or a supersaturated solution has been formed. To check if the formation of a supersaturated solution has occurred. gently scratch the inside of the test tube at the surface of the solution with a Pyrex" rod. The scratches provide points of nucleation for crystal growth and crystals should form rapidly if the solution is super· saturated. Take care when scratching the test tube with the glass rod (see p. 100). If a super· saturated solution is formed, then the solvent is suitable for recrystallization.
6.
Repeat the test using the othet' solvents and record your results in tabular form to include your experimental observations and conclusions.
7.
If you have found sevet'"al su~e solvents, then select one after considering fectors such as flammability, toxicity and boiling point since you will need to use much larger volumes of solvent in the recrystallization process, with consequent complications in the equipment to be used (see Table 13.21-
8.
If you have not found a suitable solvent. then you will need to carry out a mixed·solvent recrystalli· zation and you must find out which combination of solvents will be suilabJe (see Box 13.2).
Recrystallization
Mixed solvel1ts When no single solvent is found to be suitable for recrystallization, then a mixed solvent system must be used. There arc three essential propeltics required for a pair of solvents to be used in a mixC(holvent system: l. Using mixed solvents for recrystallization - never assume that a mixed solvent system is a mixture of equal vofumes ot the two solvents. The ratio of the two solvents is established practically during
the recrystallization experiment.
2. 3.
The two solvellts must be miscible in all proportions over the temperature range to be lIscd. The solute must be insoluble in one of the solvents. The solute must be soluble in the other salven!.
II is an advanta!!c if the two solvents have similar boiling points within 2030 C. Suitable common soh<enl pairs from the solvenlS given in Table 13.1 are watcr/ethanol and dichloromcthane/light petroleum (b.pt. 40-60 C), but many other combinmions arc possible. One of the most frequently cncountered mixed solvent systcms i:s 'aqueous ethanol' (water/ethanol) in which the compound to be fl'Cl)'stallizcd is insoluble in W'dter and vcry soluble in ethanol. You can identify the solubility characteristics of your compound using the solvent selection procedure shown in Box 13.1 and modifications for mixed solvent selection are given in Box 13.2.
The recrystallization process Having chosen a suitable solvent system, the process to be used to purify the bulk of your impure compound can be separated into several distinct steps:
Box 13.2 How to carry out a mixed-solvent selection fOr recrystallization of an unknown compound From your solvent selection tests (Box 3.1) you will have discovered tha individual solvents in which your compound is soluble (good solvents) and in which it is insoluble (poor solvents), when tha solvents are cold. Proceed as follows:
6. Repeat the heating and solvent addition process until the point is reached when the precipitate just dissolves when the solution is heated. You now have a hot solution of your compound with the correct ratio of the two solvents.
1. Choose a miscib~ solvent pair, in which the solubility of your compound is appropriate.
7. If you have added too much poor solvent and the precipitate will not redissolve on heating, add enough good solvent to dissolve the precipitate and then continue to add poor solvent and heat as before.
2. Place a small amount labout 0.2 g} of the compound in a clean dry Pyrex ~ test tube and add a good solvent {about 2.0mll in which it is soluble. 3. Heat the test tube by an appropriate method, dependent on the solvent used. until the solid has dissolved. Remember that your original solvent selection may have shown that solvent-insoluble impurities are present. 4. Add a few drops of the poor solvent from a Pasteur pipette. A slight 'cloudiness' or precipitate should form, since the solubilizing power of the good solvent has been decreased by the poor sotvent and the solution will have been cooled slightly,
5. Reheat the solution until the cloudiness or precipitate disappears and then add a few mare drops of the poor solvent to produce a precipitate.
8. Cool the test tube in an that crystals are formed.
iee-wat~ bath,
to ensure
9. " an oil is formed on cooling, which may happen if the melting point of the solid is below the boiling points of tha solvents used, you should use slightly mare of the good solvent (or less of the poor solvent) or try a different combination of solvents. 10. If crystals do not form on cooling, you may have formed a supersaturated solution and you should 'scratch' with a Pyrex" glass rod, as described far single solvent setection (Box 13.1),
Laboratory techniques
95
Recrystallization
• •
• • • •
Dissolution of the solid using a single-solvent or a mixed-!;olvcnt ~ystem. Decolorization using charcoal: even if your compou[]d is while, decolorization will improve its appearance significantly. Hal filtration (p. 98) to remove solvent-insoluble impurities and charcoal. Cooling, to produce the crystals. Collection of the crystals by suction filtration (p. 28). Drying (p. 39).
It is important to remember lhat for a successful recrystallization, you need to use equipment of a size appropriate to the amount of solid and the volume of solvent you arc likely to use. You can estimate the volume of solvent to be used by extrapolation of the dala from your solvent selection tests. In general terms, conical nash, beakers and round-boltom flasks should never be more than half-full of solution but, on thc othcr hand, using small volumes of solutions in largc tlasks will result in losses of the compound on the sides of the vessels.
Dissolution To carry out a single-solvent recrystallization (Box 13.3) you must g<..'1 the compound into solution and this is achieved by suspending it in the appropriate cold solvent. found in the solvent selection proccss, and then heating the mixture to dissolve the solid. The equipmcnt used will depend on the boiling point of the solvcnt, its flammability and toxicity. Some general systcms arc shown in Table 13.2. Table 13.2 Solvents for recrystallization Solvent
b.pt. 1 CI
Glassware
Heat source
Containment
Water
'00
Burner, hot plate
None
Ethanol Propanone Ethvl ethanoate Dichloromethane Toluene Light petroleum
78 56 78
Conical flasks, beakers Conical flasks Conical flasks Conical flasks Reflux Reflux Reflux
Water bath Water bath Water bath Water bath Hot plate, mantle Water bath
Fume cupboord Fume cupboard Fume cupboard Fume cupboard Fume cupboord Fume cupboard
41 110 40-60
When heating wlvents in conical flasks and beakers you should cover the top of the flask with a clock-glass or watcb-glass to prevent excessive evaporation of the solvent, resulting in the fonuation of crysl 500mL).
DecoJouriZQtion Small amounts of coloured impuriti<..'S can be removed from your product by absorption on finely divided charcoal, which is then f<..'111oved in the hot filtration process (p. 98). In general, you should use charcoal in ew?ry 96
Laboratory techniques
Recrystallization
Box 13.3 How to carry out a single-solvent recrystallization Examples; Suppose that you have prepared 8 crude sample (about 4.0g) of N-phenylethanamide (acetanilide)
and you are to purify if by recrystallization from water. The melting point of pure N-phenylethanamide is 114 C (lide, 2000).
1. Weigh the crude sample and retain a few crystals in case seeding is required. 2. Transfer the solid, using glazed paper or a solids funnel (p. 25), into a clean, dry conical flask (250mLl. 3. Add cold water (about 100 mL) and a glass rod as
an anti-bumping device to the flask and then heat the mixture on a hot plate until the compound has dissolved completely. Then add more water (about 10ml), to ensure that precipitation of the solute does not occur during the following stages.
4. Remove the flask from the heat and allow it to cool for 2 minutes. 5. Add a small amount of decolorizing charcoal labout 0.01 gl to the solution, place a watch-glass on top of the conical flask and heat the mixture gently for 5 minutes. 6. Prepare another clean, dry conical flask 1250mL) and add water (about 20ml) and a glass rod and then heat to boiling on the hot plate with a stemless funnel and fluted filter paper just above the neck (Fig. 13.1), so that the steam heats the funnel and filter paper. 7. Fiher the recrystallization solution through the fiher paper using hand protection (rubber fingers or an insulated glove) and keep the filter topped up with solution to prevent cooling. At the same time,
keep the recrystallization solution hot during the filtration by putting it back onto the hot plate. 8. When filtration is complete, remove the collection flask from the heat, take out the glass rod and clamp the flask in an ice-water bath, covering the top of the flask with a watch-glass. 9. When the solution is cold (about 5' Cl, collect the solid by suction fihration (Box 52), using the filtrate, not fresh water, to transfer the solid completely from the collection flask. If crystals do not form, either add a f8\r\l crystals of the crude solid to 'seed' the supersaturated solution or 'scratch' with a Pyrex'l<, glass rod to induce recrystallization. 10. Rinse the compound on the fiher using a little (about 5 mL) ice·cold water and continue suction, to make the crystals as dry as possible. 11. Transfer the crystals to a clock-glass using a spatula and spread them in a thin layer. 12. Dry the pure compound in the oven at 70 ~C for about 30 minutes and test them with a spatula to check their dryness: they should not stick together. 13. Allow the crystals to cool, covering them with another clock-glass to prevent contamination. 14. Transfer the purified crystals to a 'tared' watchglass on a balance to determine their weight and then transfer them to a labelled sample tube using folded glazed paper (p. 24). 15. Calculate the efficiency of the process:
% recovery =
weight of pure compound . x weight of crude compound
'00
rl"Crystalli7.ation since the colour of white and coloured compounds is improved by ·decolorization'. The amount of charcoal used should be about 2% by weight of the sample to be recrystallized. Note the following important points; Coloured compounds - you should know something about the colour of the compound you are trying to purify. There is no point in trying to remove the colour from a coloured compound. However, charcoal will improve significantly the appearance of a coloured compound.
•
•
•
Add the charcoal only when the solution has been fonned. [f you add charcoal to the cold solution, you will not be able to Sl'C when all of the compound has dissolved. Do not add charcoal to a boiling or a very hot solution otherwise [he solution will boil extremely vigorously, usually boiling out of the flask or reflux apparatus. To add charcoal to a hot solution. ,.en/ore the flask from the heat source and then let it cool a little (oot enough to cause prt."Cipitation). Add the charcoal either from a spatula (into a conical flask or beaker). a paper funnel (p. 25) or powder funnel (into a round·bottom flask with a ground-glass joint in reflux apparatus), and then rcheat the solution. Laboratory techniques
97
Recrystallization
flot filtration Solvent-insoluble impurities - remember that if you have found solvent-insoluble impurities, not a/l your 'compound' will dissolve in thO hot solvent. You can confirm the presence of these by edding en excess of hot 'selected' solvent to e small sample of the crude compound. If it
does not dissolve completely, solventinsoluble impurities ere present
This is a modification of gravity filtration (p. 28), designed to removc solvcnt-insoluble impurities, charcoal, anti-bumping granules or mllgnctic 'fleas' from the hot solution beforc cooling the solution to form the crystals of purified product.
~ Hot filtration is used to prevent cooling of the solu-
tion during the fittration process, which would result in the formation of crystals in the fitter paper.
For a succcs.<;ful hot filtration the solUlion must pass through the liller paper and filter funnel into the collection nask as quickly as possible so that cooling and crystallization do notlX."Cur. The following points should be noted: • • •
•
-
•
lor_bBdll
Fig. 13.1
AJlI'uys usc a nuted filter paper (p. 28). AlwfI.I's usc a 'stemless' glass filter funncf because cooling and thus
crystallization may occur in the funnel stem causing a blockage. AlwQ)'.\" heat the filter funnel and filter paper, either by pre-heating them in an ov(.'I\ or by using boiling solvent in the collecting nask (Fig. 13.1). If filtration is rapid. keep the filter cone 'topped up' with the hot solution being filtered. since lhis keeps the filter cone and filter funnel hot. If you allow the filter cone to empty of liquid. cooling and crystalli7..ation may occur. When attempting a hot filtration with a mixed-solvent system, always ensure that the 'ideal' solvent ratio has not been reached, i.e. therc is not enough of the poor solvent to cause immediate precipitatioll as a result of slight cooling during the hot filtration. The solvent ratio can be adjustt:d to the 'ideal' ratio for maximum rceovel)', after filtration and before cooling (Box 13.4).
Filtration of e hot solution,
Cooling Rapid cooling in an ice-water bath ('crash--cl)'stallizalion') usually produces small crystals ocduded with molher-liquor, whereas slow cooling by allowing the collection Ilask to stand on the laboratory bench often produces large well-defined crystals. Remember to: •
• •
the top of the collection flask with a wateh- or clock-glass to prevent solvent evaporation and entry of dust into the nask: (10 /101 lise rubber bungs or corks since they may be pulled into the nask as it cools (p.21), elamp the Ilask in place. if you usc an ice-water bath_ otherwise it may fall over as the ice melts and the volume of water increaSL'S; make sure that the solution is cold, even after slow cooling, so that maximum precipitation of the solid occurs. CO\'cr
If no crystals appear on cooling, you will have formed a supersaturated solution and, to induce precipitation of the solute, you must provide sites for nucleation and crystal growth. This can be achieved by either seeding the solution by adding a few crystals rdust') of the crude compound or .~l'TlItl'hing the inside of the nask at the surface of the liquid. using a Pyrcx ii glass rod (Fig. 13.2).
Collection of the crystals You should collect the puriftcd crystals by suction filtration by the procedure described in Box 5.2 (p. 30). Remember to transfer the crystals from the 98
Laboratory techniques
Recrystallization
Box 13.4
How to carry out a mixed-solvent recrystallization
Examples: Suppose that you have prepared a crude sample (e.g. "" 4.0 g) of N-phenylbenzamide (benzant.lide). Your solvent selection tests have shown that it is insoluble In water end fairly soIul:Me in cold ethanol. The melting point of pUre N-phenylbenzamide is 158"C lUcie, 2000). Proceed 8S follows: 1. Weigh the crude semple and retain a few crystals In case seeding is required. 2. Transfer the so'id, using glazed paper or a solids funnel (p. 25), into a eleen, dry conical nask
(250ml). 3. Add cold ethanol (about 25 ml) and a glass rod as an anti-bumping device to the flask and then heat the mixture on a water bath until the compound has dissolved completely.
4. Add cold water (about 5 ml) to the hot ethanol solution and a slight precipitate should form, which then redissolves - this is often seen as a 'nash' of white in the solution. Reheat the solution and add more water 15 ml). The precipitate will remain for a little longer and take more time to disappear on reheating. 5. Repeat the water addition and reheating Pf1)C8S$. but note that you will noed to reduce the volume of water added at each consecutive addition, since the precipitate will take longer to redissolve 8S the solvating power of the ethanol is reduced by the water. Eventually, you will reach the situation where the precipitate just redissolves at the boiling point of the solvent milCture. You now have the ideal solvent mixture for recrystallization of Nphenylbenzamide and sny slight cooling of the solution will result in its precipitation.
the neck (Fig. 13.11 so that the ethanol vapour heats the funnel and filter paper. 8, Fitter the recrystalization sotutlon throu.gh the flttet- paper using hand protection (rubber fingers or an insulated glove) and keep the fitter topped up with solution to prevent cooling. At the same time, keep the recrystalli2ation solution hot during the filtration by putting it back onto the hot plate. 9. When filtration is complete, remove the filter funnel and restart the water addition and reheating process until the ideal solvent ratio is reached once again.
10. Remove the coI&ectjng flask from the heat, take out the glass rod and clamp the flask in an ice-water bath, covering the top of the flask with a watchglass. 11. When the solution is cokt (about 5 C), collect the solid by suction filtration (Box 5.2), using the fittrate, to transfer the solid completely from the collection flask. If cryst:lls do not form, either add 8 few crystals of the crude solJd to 'seed' the supersaturated solution or ·scratch' with a Pyrex Jll glass rod to induce recrystallization.
12, Rinse the compound on the fitter using a little (about 5 ml) ice-cold water and continue suction, to make crystals as dry as possible. 13. Transfer the crystals to a cloc:k-glass using a spatula and spread them out in a thin layer. 14. Dry the pure compound in the oven at 70 ~C for about 30 minutes and test them with a spatula to check their dryness: they should not sUck together. 15.
A~w the avstels to coo4, covering them with another c1ock-glass to prevent contamination.
6. Remove the solution from the heet, add ethanol {5 mll, to ensure that the N-phenylbenzamide remains in solution if it cools a little, add charcoal (about 0.01 g), place a watch-glass on top of the flask and reheat the solution on the water bath for about 5 minutes.
16. Transfer the purified crystals to a 'tared' watchglass on a balance to determine their weight and then transfer them to a labelled sample tube using folded glazed paper (p. 241.
7. Prepare another clean. dry conical flask 1250 mW
17. Calculate the efficiency of the process:
and add ethanollabout 10ml) and a glass rod and then heat to boiling on the water bath with a stemless funnel and fluted filter paper just above
'"
/U
recovery =
weight of pure compound 00 . )( 1 weight of crude compound
collecting flask to the filter using a little of the filtrate: do flOt lise fresh solrellt. The filtrate is a saturated solution of the compound being recrystallized and cannot dissoh'C any more solute, but fresh solvent will dissoh'e some of your product resulting in an inefficient recrystallization process. Laboratory techniques
99
Recrystallization
Safety note Take great earn when "scratching' 0 supcrsaturatod solution wlth 8 glass rod. Hold the flask or test tube at the neck. not at the bottom, end make short scratching movements on the side of the flask just above and below the surface of the liquid (Fig. 13.2), If you accidentally break the flask or test tube you will cut your hend if you hold the vessel at the bottom.
I
,-)
Fig. J3.2
.,
'Scratching': (e) right (b) right; (el wrong.
Drying The purified compound should be dried by the llppropriate method as described on p. 39. If you do not know the melting point of your compound, you should always carry out a tcst by placing II small amount on a watch-glass in the ovcn, beforc committing thc bulk of your chemical. Detailed procedures for single-solvent and mixed-solvcnt ret-Tystallizlltions are shown in Box 13.3 and Box 13.4 respectively and the modifications necessary for the usc of other solvent systems can be worked OUI from the infonnation in Tablc 13.2.
Crystallization in the filter paper: prevention - keep everything hot and use a good fluted filter paper;
Problems in recrystallization Thcre arc three common problems encountered during rL'Crystallization: I.
cure - wash through with hOf solvent,
evaporate mosl of the excess and repeat the hot filtration.
Evaporation of excess solvent - remove by rotary evaporation (p. 121) or N z btO\Ndovvn (p. 1231. This is the ooe occasiOn
when water is the less than ideel solvent. since its evaporation is time consuming.
100
laboratory techniQues
2.
The compound crystallizes in the filtcr funnel during hot filtration. This is because the solubility of the solutc decreases rapidly with temperature and the slight cooling during hot filtration causes precipitation of the solid, even though you are heating the funnel. The answer is to usc morc than the minimul11 amount of solvent and then cvaporate off the excess before cooling. The compound does not recrystallize. There arc Iwo reasons: you havc used too much solvent and you must evaporatc off some solvent before cooling, or you have fonned a supersaturated solution and you must 'seed' or 'scratch' the solution (see p. 98).
Recrystallization
3.
The compound precipitates as an oil. This is because compounds with low mdting poinL" often come out of a concentrated solution above their melting point. In such cases more solvent should be added and the compound redissolved and cooled so that prt-cipitation is retarded to the temperature at which the crystalline solid comcs out of solution. Often ·scr.uching· the hot solution as it cools can prevent 'oiling oul'.
laboratory techniques
101
---8 Extraction - making a cup of coffee involves extraction of the flavour chemicals and caffeine from tho insoluble vegetable matter using hot water and is an example of liquid-solid extraction.
Solvent extraction This tcchnique separates the components of chemical mixtures by using the dissimilar solubility properties of the components of the mixture in different solvents. Extraction is USt.'d mainly 10 purify a reaction product partially before final purification by recrystallization (p. 92) or distillation (p. 107). The two common types of extraction process uS<.>d in the laboralory arc: I.
2.
Liquid-liquid extraction: this uses two immiscible solvents; the desired compound in solution or suspension in one solvent is extracted into the other solvent. For example. covalent organic compounds arc exlr
liquid-liquid extraction Several experimental processes in practical chemi"try arc basc<1 on liquidliquid extraction: •
• •
'Extraction': where a solid or liquid suspended or dissolved in one solvent is cxtracted into another. This technique can be used lO separate covalent molecules from ionic compounds in an aqueous solution or suspension. 'Washing': where ionic species are removed from a non-polar solvent by extraction into water. 'Acid-basc extraction': where covalent molecules are converted into their salts and thus rcmov<.'d from a non-polar solvent into water, while neutral covalent species will remain in Ihe non-polar solvent, as shown in Table 14.1.
Table 14.1 Examples of acid-base extraction chemiSlry
NoOH
ArCOOH
RH
Acid insoluble in H20 soluble ;n CH2Cl2
Neutral insoluble in H20 soluble in CH2Cl2
+ Amine insoluble in H20 soluble in CH 2 CI 2 ArCOOH
CH2C~'
CH 2 C12
Neutral insoluble in H 20 soluble in CH2Cl2
+
Acid insoluble in H20 soluble in CH2CI2
Na'
+
SaIl soluble in H2 0
Hel
RH
ArCOO
ArNHj Cl-
+
Salt soluble in H20
A«>H
ArCOO Na
Weak acid Insoluble in H20 soluble in CH 2Cl 2
Salt soluble in H 20
RH Neutral insoluble in H 20 soluble CH 2 Cl 2
RH Neutral insoluble in H20 soluble CH 2 CI 2
+
MlH Weak acid insoluble in H20 soluble CH 2Cl 2
All of these processes involvc mixing the two immiscible solvcnts, one of which contains the mixture, in a separalory funnel (p. 103) and shaking the funnel to promole the extraction process. The immiscible layers are allowed to refonn and arc then separated. 102
Laboratory techniques
Solvent extraction
Safety note Because of its flammability and tendency to form explosive peroxides, the use of diethyl ether for extractions in many undergraduate laboratory has effectively been discontinued. Use dichloromethane or ethyl ethanoate instead. Safety note Dichloromelhane is effectively non-ffammabJe and is often the solvent of choice, but it is a liver toxin and an irritant, and must be handled in the fume cupboard if volumes greater than 50 ml are to be used.
For liquid-liquid extraction. water is usually the polar solvent. Since most extractions involve getting the required compound inlo the organic soh'enl (or removing unwanled ionic chemicals from it). it should have good solvenl power for the desirt.'
Partition coefficients The theory of liquid-liquid extraction is based on the equilibrium between the concentrations of dissolved component in Ihe two immiscible liquids, when they are in contact. The equilibrium constant for this process is called (be partition coefficient or distribution coefficient and is given by: concentration of solule in liquid I ~olute in liquid 2
114.1]
K = concentralion of
You only m..'Cd to calculate such quantities if: •
•
Extraction calculations - it is necessary to calculate volumes of solvents and number of extractions when attempting to maximize tho economics of an industrial-scale extraction process.
Separatory funnels - the coned-shaped funnels are specifically designed for extractions. Only use parallel-sided funnels when no alternative is available. Safety note Extractions involving large volumes of solvents I> 100 mll should always be carried out in cone-shaped separatory funnels supported by a ring, because there is then no danger of the funnel slipping from a clamp at its neck.
you arc carrying out specific experiments to determjne partition coefltcients. when you wilt be given specific instructions or references to lhe appropriate literalure: the solute has appreciable solubility in both solvents.
The reason calculalion is not necessary is that. in the overwhelming majority of extractions you will carry out, the conditions used are designed to ensure that the components will be aJmost totally soluble in one of the liquids and almosl insoluble in the olher, since complete separation is rcquin."d. The number of extraclions needed 10 extract a water-soluble solute into an immiscible organic phase can be calculaled from the following relalionship: 11'"
=
11'0
- -K ' - ' - )" ( Kv+s
[142)
wh(,...e K = partilion coefficient of the solute. I' = volume (mL) of aqueous solution of the solute. s = volume (ml) of immiscible organic solvent. 11'0 = w(,'ight of solule initially in the aqueous layer, II'" = weight of solute remaining in the aqueous layer after 1/ extractions. and n = number of extractions. Evaluation of this expression shows that, for a fixed volume of solvent. il is more effick"J1t to carry out many small extractions than one big one.
SeparatOl'y fiuJIlels These come in a range of !>1ZCS from 5mL to 5000ml and there are two general types: parallel sided and cone shaped (Fig. 14.1). Cone-shaped separatory funnel.. are made of thin glass and should be supported in a ring (p. 104). Small-volume cone-shaped funnels « IOOmL capacity) and parallel-sided separatory funnels should be damped at the ground-glass joint at the neck (p. 27). Separatory funnels will have glass or Tenon!.' taps wilh a rublx.-r ring and clip or screw cap on the end to prevent the tap slipping from the oorrel. or a Laboratory techniques
103
Solvent extraction
Rotaflo Ii tap. You must ensure that the tap assembly is in good condition by making the following ChL'Cks before starting work: •
For glass taps: disassemble the tap by first removing the dip and ring or cap from the lap (note lhe order of the comlX>nent parts for reassembling). Dry lhe tap and barrel with tissue, add a light smL-ar of grease to the tap (making sure you do not clog the hole in the tap) and reassemble lhe tap and fitlings. turning the tap to ensure free movement. Support the st.'paratory funnel in position and add some of the organic solvent to be used (2-3 mL) to the funnel, with the tap closed, to check that the tap does not leak. If the tap k-aks, disassemble and rcgrease. For Teflon \! taps: disassemble the tap, wipe the tap and barrel with dean tissue. reassemble without grease, check for free movement of the tap and for leakage as described above. When you have finished using the funnel, loosen the clip/cap on the tap since Teflon'li, will flow under pressure and the tap may 'scize' in the barrel. For a Rotaflo A tap: unscrew the tap from the funnel and ensure that the plastic locking thread is in place (Fig. 14.2). If it is not present, consult your instructor and obtain a replacement. Dry the barrel of the tap and the tap with a tissue and reassemble. Do not grease the Rotaf1o~[ tap.
•
•
The general proa.'<1ure for using a separatory funnel for extraction is described in Box 14.1 and there are five additional practical tips to aid your
success: Label all flasks to avoid confusion. Nel'er Ihro\l' (limy allY of Ihe separated /iqll;lls until )'ou are absolulely slire of Iheir idemit)'. Alwll.)'s transfer solvents into the scparatory funnel using a stemmed
I. 2. J.
lilter funnel so that solids and liquids will not stick to the inside of the joint and prevent a good sL'a1 when you insert the stopper and then invert the funneL Always place a '!i(ifeIY' beake/" under the separatory funnel to collect liquid just in case the tap leaks (Fig. 14.3a).
Fif). 14.1 Separatory funnels.
4 Drying separatory funnels in an oven always disassemble the tap and do not place the tap and its plastic components in the oven. Dry them with tissue.
0-----
\
o
...
...
-
.
, ~
L ROlallo@tap
Fif). 14.2 A Rotaflo" tap.
104
Laboratory techniques
,
l~
U'
~
1=1 ____
---
....
Y~7
"'
Fig. 14.3 A separatory funnellal ready
0' to use and lbl in use.
Solvent extraction
Box 14.1
How to separate a carboxylic acid and a hydrocarbon using solvent extraction
This is an example of an acid-base extraction. The solid mixture (e.g. 4.0g for the solvent volumes used below) of benzoic acid and naphthalene is soluble in dichloromethane but benzoic acid will dissolve in dilute aqueous sodium hydroxide (2 Ml by forming the sodium salt (sodium benzoate). Naphthalene is insoluble in water. 1. Dissolve the mixture in a clean. dry beaker in dichloromethane ISO mll.
2. Clean and dry the tap of a separatory funnel (250mL) and set up as shown in Fig. 14.3a. 3. Make sure that the tap is closed and then add the solution containing the mixture, using a stemmed funnel to prevent contamination of the joint, and rinse out the beaker with dichloromethane ('" 5 mL). 4. Add sodium hydroxide solution no mll to the separatory funnel, place the stopper in the separatory funnel and gently Invert it and hold it as shown in Fig. 14.4. Do not shake the separatory funnel since you do not know how much heat will be produced in the reaction, which will pressurize the separatory funnel. 5. Open the tap. to release any pressure caused by the heat of reaction. 6. Close the tap, shake the minure once and open the tap to release any pressure. 7. Close the tap, shake the minure twice and open the tap to release any pressure. 8. Repeat until no more vapour is expelled via the tap. 9. Close the tap, and replace the separatory funnel in the ring or clamp.
5.
Fig. 14.4
Holding a separatory funnel.
Batch liquid-liquid extraction - the process described is inefficient if the material being extracted has appreciable solubility in both of the solvents used. In these situations a continuous extraction system is necessary and you should consult the specialist textbooks. e.g. Furniss er a/. (1989, p. 160).
10. Take out the stoPI*'". place it upside down in the top of the separatory funnel and allow the solvent layers to separate. The upper layer is the aqueous layer (lOmL compared with 50mL of dichloromethane). Sometimes a few globUles of dichloromethane will 'cling' to the surtace of the water layer: these can be released by gently swirling the contents of the separafory funnel. 11. When the liquKts have stopped swirling. open the tap gently and slowly run the dichloromethane lower layer into a clean conical flask and label it 'dichloromethane layer". Avoid fast emptying of the funnel because a vortex may be fanned which will cause the upper layer to run out with the lower layer. 12. Run the remaining aqueous layer into a clean. dry conical flask and label it 'sodium hydroxide layer". 13. Return the cichloromethane layer to the separatory funnel and extract it with another portion (lOmL) of sodium hydroxide. Repeat the extraction process for a total of 40 mL (i.e. 4 x 10 mL) of the alkali, collecting all the sodium hydroxide extracts in the same flask. 14. Finally. extract the dichloromethane with water 120ml), to remove any traces of sodium hydroxide and add these 'washings' to the sodium hydroxide layer' flask. 15. You now have a solution of naphthalene in dichloromethane and a solution of sodium benzoate in sodium hydroxide ready for further processing. 16. If an emulsion forms - the layers do not have a well defined boundary: add a few drops of methanol to the upper layer down the inside wall of the funnet. This often 'breaks· the emulsion.
Always take thc stopper from thc separatory funnel before you attempt to allow liquid to run from the funnel. If you do not remove the stoppcr from the top of the funnel, a vacuum is fomled in the funnel after a little of the liquid has run out. Air will be sucked into the funnel through the outlct stem causing bubbles. which will remix your separated layers. If your funnel is equipped with a Quickfit >(0 stopper. it is good practice to take the stopper out of the top and put it back upside down (Fig. l4.3b). This ensures that no vacuum is formt:d and that organic vapours do not escapc easily from the nask.
Solid-liquid extraction In this process the componcnts of a solid mixture arc extracted into a solvent. The 'batch process', analogous to liquid-liquid extraction, involves grinding the solid to a nne powder, mixing it with the appropriate solvent. and filtcring oIT the solid by gravity (p. 27) or under vacuum (p. 30) and then evaporating the solvent (p. 121) from the extract solution. Howl,.,\,cr, a morc Laboratory techniques
lOS
Solvent extraction
Box 14.2 How to set up a Soxhlet extraction system 1. Select apparatus of the appropriate size for the amount of solid to be extr8cted. Specifically, the Soxhlet thimble should fit below the siphon outlet and the volume of the solvent reservoir should be such that it is never more than half-full when all the solvent has siphoned from the elCtractor. 2. Assemble the apparatus as shown in Fig. 14.5, clamping at the joints at the flask and the top of the Soxhlet extractor. The best heat source to use for continuous operation over a long period is a mantle.
3. Disassemble the apparatus, leaving the clamps in position. 4. Fill the flask to about one-third of Its volume whh solvent, add some anti·bumping granules (or a meg netic 'f1ea', if a stirrer-mantle is being used) and clamp it into position in the mantle.
5. Lightly grease the "male" joint of the SolChlet extractor and attach it to the reservoir flask. Clamp the top of the extractor. 6. Add solvent to the extractor until it siphons. This ensures that the reservoir will never be dry. Check that the reservoir is now no more than half-full; if it is, replace with a larger flask.
d... ---~!LQ
.
--
InOlt--- :::::..
...,., ---t.
$did ID be
""'"
.... -----<
Fig. 14.5 A Soxhlet extraction system.
106
laboratory techniques
7. HaN-fill the ewtraetion thimble with the solid to be extracted and plug the top of the thimble with white cotton wool to prevent any solid being carried over Into the solvent. 8. P1ace the thimble in the extractor.
9. Attach a water supply to the reflux condenser, lightly grease the male joint and attach the condenser to the top of the extractor. Turn on the water, ensuring a steady flow. 10. Switch on the heater and turn up the power $0
that the solvent reftuxes and drips into the extractor. 11. Confirm that everything is running smoothly by watching at least two siphoning cycles of the extraction and check the apparatus frequently. 12. When the extraction is complete, allow the apparatus
to cool and dismantle it. Place the extraction thimble in the fume cupboard to allow the solvent to evaporate and the dispose of it appropriately. Gravity filter or decant the solvent in the reservoir flask to remove the anti-bumping granules of magnetic 'flea' and remove the solvent by distillation (p. 107) or rotary evaporation (p. 122).
elegant 'continuous extraction process', called Soxhlet extraction, is available when The most appropriate solvent is known. The apparatus for Soxhlct extraction is shown in Fig. 14.5 and comprises a fla5k containing the solvent. i1 Soxhlet extractor and a reflux condenser (p. 116). The solid to be extracted is placed in a porous thimble. made from hardened filter paper. and Ihe sohoenl is heated so that its vapour flows jl
------<8 Working with azeotropes - not all liquid mixtures can be separated by distillation. In some cases an azeotrOf)tt. a mixture of the liquids of definite composition, which boils at a constant temperature, is formed. For eKarnple, an azeotrope
containing 95.5% ethanol and 4.5% water boils at 78.15 C. which is below the boiling point of pure ethanol (78.3 C). Therefore 100% ethanol cannot be
obtained by distillation of ethanol-water mixtures, evel'\ though their boiling
points are about 22 ~c apart.
Distillation Distillation is u.~ to separ.tle the components of a liquid mixture by vaporizing the liquids. condensing the vapol.lrs and collt'Cting the liquid t'Ondensate. Separation is the result of the differing boiling poims of the individual constituents of the mixture and the efficiency of the distillation column. You m..'y be required to pl/rif)' a liquid by tli.ytillatirm. which involves the rt:moval of small quantities of impurities, or to separate a lllixture of liquids by jmc!iolllll tfiwil/arjOlI, each of which can then te purilied by distillation. You will meet several types of distillation process each applicable to different situations depending on the chemicals to be purilied or separated. •
•
•
Using steam distillation - in practice, the decision to use this approach to separate
•
the components of a mixture rs based on previous experience. Consult your
instructor for advice.
Simple dis/illation: used for sepi.uating liquids, boiling beloll' 200"C at atmospheric pressure, from other compounds. For effective separation there should be a difference in the boiling points of the components of at least BOcC. FmctiOlll11 di:nil/atiOll: used for separating components of liquid mixtures, which have boiling points differing by more than 25 ce, at temperatures beloll' 200'C. VaCII1Ull or redllced-pre.ul/re (Iiflillation: used for separating liquids boiling abo~Y? 200-C, when decomposition may occur at the high temperature. The effect of distilling at reduced pressure is to lower the boiling point of a liquid. This tt'Chnique can be applied to both simple distillation and fractional distillation. Steam distillatiol1: used for separating mixlUrcs of chemicals such as oils. resins, hydrocarbons. etc., which are essentially insoluble in water and muy dt'COmpose at their boiling poinLS. Heating lhe compounds with steam makes them distil bl'lo'l'l' ](X)~c.
Distillation eqllipment Apparatus used for the various types of distillation has several general fealUres: • • Setting up distillation apparatus - do not allow the support stands 10 move during the distillation. since this will allow the
ground-glass joints connecting the still· head and condenser to separate. The hot \lapours will then escape into the laboratory instead of going down the condenser.
Securing distillation cnmponents - do not use plastic joint clips on the 'hot end- of a
distillation apparatus sil"lCe they may melt and the joints may separate.
• •
•
Distillation flask: usually round bollom or pear shaped with one or two necks (to allow a vacuum bleed to be fitted). Still-head: to hokl the lhennometer and to channel the vapour into the condenser. For fraction<'ll distillation. the fr.letionating column is fitted betwccn the distillation nask and the still-head. Condenser: usually with circulating cold water. Take-off adapter: to allow the distillale to run into lhe collecting vessel. For vacuum distillation. the adapler will have a vacuum inlet tube and could ha\e three 'anns' to allow lhree frActions 10 be collccted without breaking the vacuum. Receiving (collection) vessel: this c-.m be a tcst lube, a measuring cylinder, a conical flask. or a round-bottom flask with a ground-glass joint.
No mailer what type of distillation you are attempting, it is essentiallhat you assemble the apparAtus correctly, since you are dealing with hot, often Oammable. liquids and vapours. A typical simple distillation apparatus IS shown in Fig. 15.1 and the method of assembly is described in Box 15.1. Laboratory techniques
107
Distillation ~ You must ensure that all the ground-glass joints are
seated property and the apparatus is damped firmly SO that no move· ment of the joints will allow vapours to escape.
Simple distillation jcint clips
The procedure for doing a simple distillation is described in Box 15.2 and you should note the following jX)inlS: •
•
•
Fig. 15.1 Apparatus for simple distillation.
Do not distil to dryness, i.e. no liquid remaining in the distillalion Oask, since this causes overheating and charring (decomposition) of the residue. A/lrays leave 1 or 2 mL of liquid in the distillation Oask. Inilially. some liquid lmIy distil while the temperature rises rapidly. This is tcrmed 'forerun', for examplc often a small amount of solvent from an extraClion process (po 102): collect and (hen, whl."tl the temperature sl.'1bilizes, collect the desired compound in a clean receiving vessel. Towards the end of the distillation. the temperature may begin to rise again: this will be the higher boiling impurity. Stop heating. quickly remove the receiving flask containing your pure compound and collect these last few drops, termed 'tailings', in a fresh receiving ves:;el.
Fractional distillation Heating a distillation flask - unlike when using a microbumer, it is diHicult to stop the heating process instantly when using an oil bath or mantle on a ·Iabjack'. Therefore have a clean receiver flask ready to collect the 'tailings' as soon as the temperature begins to rise.
Collecting fractions - as the temperature begins to rise 'between fractions' you will have 2 or 3 seconds to change receiving vessels before the liquid runs from the top to the bottom of the condenser. Have three or four pre-weighed receivers ready to hand.
Simple distillation is not very eITI.-'Ctive in separating liquids unless their boiling points are at leaSl80~C apart and a better separation can be achieved if a fractionating column is used. There are many types available (Fig. 15.2) and the device brings the ascending vapours into contact with the condensing (in the fractionating column) liquid and amounlS to a succession of many simple distillations in which the dl.'SCending liquid strips the high·boiling component from the ascending vapour. Overall, the lower boiling component distils first and cleanly. The column packing, usually glass beads, helices or 'fingers', gives a large surface area for contact of the ascending vapours and descending liquid. After the first fraction has distilled. the temperature will rise and the rate of distillation will slow. This is an inlennediate fraction containing a little of both components of thc mixture. Finally, the temperature will become constant and the pure higher boiling compound will distil. The proct'dure for fmetlonal distillation is given in Box 15.3.
Distillation in fume cupboards - it may be necessary to insulate the fractionating
column from the -Wind' by wrapping it in
aluminium foil, since the draught prevonts equilibration in the column.
Don't forget to leave a 'window' in the foil so that you can see the vapour condensing near the top of the column.
108
Laboratory techniques
""'" Fig. """'" 15.2 Common fractionating columns. Vigrew: COIURll
~ A successful fractional distillation relies on thermal equilibration of the components in the fractionating column. You must allow the column to be heated by the vapoufS of the mixture.
Distillation
Box 15.1
How to assemble the apparatus for a simple distillation
In general laboratory work you will usually be distilling small volumes of liquids ('" 25ml). Standard glass joint-ware apparatus size 14/23 (p. 43) is appropriate. If you are to carry out larger scale distillations, you must consider the weight of the components, specifically the receiver flask, which should be supported on a 'labjack', since a joint clip may not be strong enough to hold a large flask 1250ml or larger).
1. Clamp the distilling flask firmly to a support stand ensuring that it is at the correct height to allow the heat source (microburner, oil bath or mantle on a 'Iabjack'l to be removed if necessary.
2. Insert the still-head in the top of the flask.
3. Carefullv line up the condenser on a damp (attached to the same support stand as the distilling flask (Fig. 15.1J, if possible) and adjust the height and angle of the clamp so that the condenser slides onto the still-head joint. Remember to have the nonmoving jaw of the clamp at the bottom (p. 27) otherwise you will lift the condenser when you tighten the clamp and break the joint on the stillhead or condenser or both. Carefully tighten the clamp and ensure that the joints are ·seated'.
4. Attach the take-off adapter to the bottom 01 the condenser with a joint clip. If you are using a Quickfit R flask as a receiving flask, you must use a vacuum-type take-off adapter (p. 42) otherwise you will be distilling in a closed system, which may explode when you begin to heat the distilling flask.
5. Attach the Ouickflt R receiver flask to the take-off adapter with 8 joint clip or place the receiver flask underneath the take-off adapter, supported on a 'Iabjack' or clamped position. The outlet of the take-off adapter must be just inside the receiving vessel so that no spillage of distillate will occur. If
Box 15.2
you clamp the receiving vessel in position, use a separate support stand. 6. Disassemble the apparatus in the reverse order to assembfy by opening the clamps - do not move the clamps and stands.
7. Add the liquid to be distilled to the distillation Dask using a stemmed funnel, together with antibumping granules or boiling stick or a magnetic 'flea'. The flask should not be more than 50% full. Reclamp the flask in position.
8. lightly grease the 'male' joint on the stiD-head and replace it in the flask.
9. Insert a thermometer into a screwcap adapter (p. 43) and adjust so that the bulb is just below the outflow from the still-head. lightly grease the joint on the screwcap adapter and put it into the still-head_ 10. Fit the rubber tubing onto the condenser (p. 117), lightly grease the joint on the still-head and refit the condenser, clamping it firmly into place.
11. Connect the lower tube to the water tap so that the water will flow up the condenser, and upper tube should be routed into the sink. Make sure that there are no kinks in the rubber tubes and, for safety, feed them over the protruding arms at the back of the clamps. Turn on the water tap gently and check that the water flows freely.
12. lightly grease the bottom joint of the condenser and attach the take-off adapter using a joint clip.
13. Attach the Quickfit" receiving flask to the take-ofl adapter with a joint clip {after lightly greasingl or place the receiving vessel underneath the take-off adapter, supported on a 'Iabjaek' or clamped into position. Turn up the water ftow to give a steady stream from the condenser.
How to carry out a simple distillation
1. Set up the apparatus as described in Box 15.1 and make sure that all the joints in the distillation system are secured properly.
2. Slowly appfy the heat source to the distillation until the liquid is boiling gently. 3. Increase the heat slowly until the liquid starts to drip Into the rece;ving vessel at a rate of about one drop every 2 seconds. If the temperature is 'constant' (i.e. it does not vary more than 2-3 CI,
collect the liquid and record the temperature range of the distillate.
4. Remove the heat source, allowing the apparatus to cool down and the remaining drops of distillate to run out of the condenser into the receiving flask.
5. Seal and label the recanting flaskls). 6. When cool, d"lSmantle the apparatus, wash it out with an appropriate solvent and dispose of the washings in the correct solvent residues bonle.
Laboratory techniques
109
Distillation
Box 15.3 How to carry out a fractional distillation 1. Set up the apparatus as described Box 15.1 but insert the fractionating cmumn between the
distilling flask and the stlll.-head. For extra stability, clamp the fractionating column at its top joint to support the stand carrying the distilling flask.
2. Apply the heat source $lowly until the liquid begins to boll gentty. Then slowly increase the heat until the column is warmed up and liquid is condensing from the thermometer bulb, but not distilling over. 3. tf the temperature is constant. increa58 the heat slightty until the first component distils over at 8
rate of about one drop every 2 seconds. Collect this distillate as the first fraction. as long as the temperature is 'constant' (i.e. it does not vary more
than 2-3 C). Record the temperature range of the distillation.
5. lnuease the heat s10wty and collect the intermediate fraction until the temperature stabilizes again. Record the temperature range of the distillation.
6. Change the receiving flask and collect the new fraction (one drop every 2 seconds) for as long as the temperature remains 'constant' (i.e. it does not vary by more than 2-3 C). Record the temperature of the distillation. 7. Remove the heat source, allowing the apparatus to
cool down and the remaining drops of distillate to run out of the condenser into the receiving flask. 8, Seal and label the receiving lIaskls). 9, When cool, !:ismande the apparatus. wash it out with an appropriate solvent and dispose of the washings in the correct solvent residues bQttle.
4. Change the receiving flask when the temperature begins to rise and the rate of distillation slows.
Vacuum or reduced-pressure distillation Safety note You must check all flasks and glassware for 'star' cracks and chips. /( in doubt replace.
Working with reduced pressuresdistillation flasks and receiving fl3Sks must always be round bonom or pear shaped. Do not use flat-bottom or conical flasks under vacuum. Measuring reduced pressures - note that despite 51 nomenclature for pressure, practical work usually involves pressure measurement in mm of Hg. Atmospheric pressure is about 760 mm Hg. Pressures quoted in ·torr" (seen on some old vacuum equipment! are equivalent to mmHg. Moving a merctlry manometer - always carry mercury manometers in a vertical position or tho mercury may spill out. Safety note Do not allow air to rush into an AnSChutz manometer, otherwise the mercury in the left-hand limb of the manometer will shoot up the tube and may break the glass at the top.
'10
Laboratory techniques
Distillation at reduced pressure is used to distil liquidS with few impurities or to fractionate the components of liquid mixtures with high boiling points, which would decompose if distiUed at atmospheric pressure. The modifications to the distillation app.1.ratus (Fig. 15.1) required for rcducedpressure distillation are: •
•
•
•
A vaCuum source: this can be a water pump (sec p. 29) with an 'anti--suckback' trap producing a vacuum of 15--25 mm Hg, aI best~ or a rotary vacuum pump (consult your instructor about its use since it is an expensive piece of equipment and easily contaminatcd or damaged), which will evacuate down to 0.1 mm Hg. Two-slage dry vacuum pumps prodUce a vacuum of 1~5 mm I-Ig. are resistant to organic and acid vapours and are easy to use. A manometer to measure the pressure (vacuum) in the apparatus, since the boiling point of a liquid varies with pressure. Two types are in common use ()-Ig. 15.3): the Anschutz. manometer, which gives a constant reilding of the vacuum throughout the distillation. and the Vacustal" , which is used to take a 'sample' of the vacuum at a given instant. Vacustats It arc very accurdte and are more oflen used in combination with a rDlary oil pump. A take-off adapter, which permits the coltt:ction of several fractions without stopping the distillation to change the receiving nasks. A receiving adapter. termed a 'pig' (Fig. 15.4), can be rotated on the cnd of the condenser to collect three fractions. Appropriate 'anti-bumping' measures: reduced-pressure distillations are notoriously prone to 'bumping' (p. 31) and anti-bumping granules are ineffective. You C'"d.n usc a magnetic 'flea' in conjunction with a hot platestirrer and oil bath; an air bleed, made by pulling out a Pasteur pipette into a fine C3pillary using a microburner (consull your instmctor). inserting it into a screwcap adapter (p. 43) and placing it in the second neck of the distilling flask. The vacuum pulls a fine stream of air into the
Distillation
. ..,
tollSpiMOf
Fig. 15.3 Manometers for vacuum distillation.
'pig' type " ' ,- - ' \ - - - - receivirrg adaplorr
Fig. 15.4 °Pig"_type receiving flask adapter.
Safety note If, during the course of your vacuum distillation using a microbumer and a boiling stick, the liquid stops boiling and appears 'quiescent', it is
flask and forms small bubbles which agitate the liquid during distillation;
or a wooden boiling stick for small-scale distillations of shari duration. since the vacuum will pull air from the stick and agitate (he liquid.
about to 'bump' vigorously. Stop heating, allow;1 to cool, 'break' the vacuum
(Box 15.4). and add a new boiling stick.
•
1\ lhree-way tap inserted between the vacuum source and the manometer, so that you can control the vacuum applied to the distillation system.
A typical systl.."T11 for reduced-pressure distillation is shown in Fig. 15.5 and the procedure, using a water pump, is described in Box 15.4.
"'......lh clip
Ending a distillation - complete distillation of a liquid is not possible because of the
need to leave a few millilitres in the distilling flask, to prevent overheating and due to the 'hold-up' volume of the flask and fractionating column.
Fig. 15.5
Apparatus tor vacuum distillation.
Kugekollr distillatio" 111is is also known as short-path distillation or bulb-to-bulb distillation. It is a procedure for reduced-pressure distillation, which almost eliminates losses owing to the size of the app.·uatus. and is particularly useful for small volumes of liquids. The liquid sample is placed in a small round-bollom flask, connected to a series of bulbs (Fig. 15.6) and then heated and rotated (to prevent bumping) laboratory tcchniques
111
Distillation
Estimating the boiling point of a liquid at reduced pressure - a useful guide is; if
fhe pressure is halved, the boiling point is reduced by ~ 10"(. For example, if the b.pt is 300 'C at 760 mm Hg, it will be
approximately 290 C at 380 mm Hg and 190 'C at _1 mm Hg. Alternatively a nomograph (Fig. 15.8) can be used for a more accurate estimation.
under vacuum in an electric oven. The liquid distils from the Oask inside the oven to a cold bulb outside the oven. Since the distillation path is very short the length of a ground-glass joint losses are minimized. The use of several bulbs as r(:ccivcr nasks allows a small volume of mixture to be fractionated by varying the temperature in the OVI,.>t1. The distillates can be removed from the bulbs by either washing into another nask with a low-boiling-point solvent or using a 'bent' Pasteur pipette (Fig, 15.7) since the bulbs can only be held horizontally.
Fig. 15.6
Kugelrohr distillation apparatus.
Steam di!'>"tiJlation This process can be used to separate water-insoluble covalent compounds
Fig. 15.7 A "bent' Pasteur pipette.
from crude reaction mixtures or to isolate volatile natural product'), e.g. terpencs from plant tissue. The crude mixture is heated wilh water and steam and the steam-volatile m.·uerial co-distils with the steam and is then condensed with the water and collected. You willthcn need to carry out an extraction (p. 102) to separate Ihe waler and the insoluble component.
700"0
Fig. 15.8 Nomograph for estimating boiling point at reduced pressure, To use it, draw a line between the recorded pressure and the boiling
100::
b.p!. corrected
10 160 rro-n Hg
poinl at 760 mm Hg. and then extend the line to the observed boiling point scale. This point is Ihe boiling point at the redlXed pressure. In
0-
this example. a liquid b.pt. of 250 C at 760 mm Hg will boil 81 118'C at 10 mm Hg.
112
Laboratory teChniques
100-
observed b.p(.
Jl'essure in rom Hg
Distillation
Box 15.4
How to carry out a reduced-pressure distillation using a water pump
1. Set up the distillation apparatus 85 described in Box 15.1, but use a two-necked distilling flask and include a short fractionating column between the lop of the distilling flask and the still-head or use 8 Vigreux flask (Fig. 15.2 I or use a Claisen still-head (Fig. 15.21. 2. Connect three Ouickflt,w round-bottom receiving ftasks to the receiving adapter Of' 'pig' using plastic joint clips and attach the 'pig' to the bottom of the condenser. Support the receiving flasks on a 'Iabjack" if they are 100ml size or larger. Rotation of the 'pig' allolNS three fractions to be collected without stopping the distillation - this is a major task when working under reduced pressure. 3. Insert the air bleed into the 'spare' rMK:k of the distilling flask Or into the lovver ioint on the Vigreux flask or Claisen head and make sure that the tip of the air bleed reaches the bottom of the distilling flask. If you are using boiling sticks as an ·anti·bumping' precaution, add the sticks, making sure that they reach to the bottom of the flask and do not float, and put stoppers in the unused joints. 4. Insert a Quickfit" thermometer into the remaining 'male' joint in the apparatus. Do not use an ordinary thermometer in a screwcap adapter: it may be sucked into the apparatus and break or crack the flask.
5. Using thick-walled rubber pressure tubing. connect the water pump (via the anti-suck-back trap) to the three-way tap and then to the Anschutl manometer. Connect another piece of pressure tubing to the manometer but do not connect it to the 'pig' for the moment.
6. To check the available vacuum, turn on the water pump fully, kink or seal the open pressure tubing on the manometer and turn the three-way tap so that a vacuum is created in the manometer. Slowly open the tap on the manometer and the mercury level should rise in one of the manometer 'arms'. When the mercury has stabilized, move the SC8~ so that the zero is level with the lowest mercury level. and read off the upper mercury level. The pressure (vacuum) is the difference in levels in mm. The reading should be between 10-20 mm. Now close the tap on the manometer, unkink or unseal the pressure tubing, and turn the three-way tap so that air is admitted into the system.
7. Connect the pressure tubing from the manometer to the ·pig'. Pressure tubing is heavy and it should be sUPpOrted with a clamp and stand, close to the 'pig', to prevent strain on the glassware.
8. Slowly turn the three-way tap to evacuate the apparatus. As the pressure decreases, bubbles will issue from the air bleed or the boiling stick and low-boiling solvents such as dichloromethane or diethyl ether will begin to boil. causing frothing in the flask. If this happens, stop applying the vacuum via the three-way tap and wait until the frothing has died down and then continue to lower the pressure until the three-way tap is fully open. 9. Check whether the air bleed is too coarse, producing large bubbles and vigorous $plashing in the distillation flask.. If so, place a piece of pressure tubing (about 2 cm long) on the end of the air bleed and constrict the tubing with a screw clip to reduce the air flow through the air bleed to an acceptable rate.
10. Slowly open the tap on the manometer, allow the mercury levels to stabilize and read the vacuum by moving the zero on the scale to the 1000000r mercury level: the reading should be 15-25 mm or better. If this pressure is not obtained, there must be leaks in the system, which usually occur at the groundglass joints or rubber-to-glass joints. Close the tap on the manometer, check. the sealing of the joints by rotating each one in turn and also check the rubber-to..glass joints by carefully pushing the rubber pressure tubing a linle further onto the glass. Open the tap on the manometer and recheck the pressure.
11. Gently heat the dlst.IIlng flask and carry out a fractional distillation as described in Box 15.3, ooUecting the fractions by rotating the ·pig'. Remember to record the temperature and pressure at which the fractions distil. If the liquid in the distilling flask bumps over into the oonden8e1'. you will need to clean out the apparatus with a solvent, evaporate off the solvent to recover the chemicals and restart the whole process.
12. When the distillation is complete, dose the tap on the manometer and allow the apparatus to cool. Support the 'pig' and receiving flasks (with your hand or a 'Iabjack'j and slowly open the three-way tap to allow air into the system. Disconnect the pressure tubing from the 'pig' and place the 'pig' and receiving flasks in a safe place. Disconnect all other tubing and apparatus for washing or storage, turn off the waler pump and finally. very gently, open the tap on the manometer to allow the mercury levels to equalize and then close the IBp.
Laboratory techniques
113
Distillation
_lor rtipIerosting
dimlaliDn Plll
-----I
--
---->{
Fig. 15.9 Apparatus for steam distiJIatiOll.
The steam n..'quired for a steam distillation can be provided by all external source, such as piped S1eam in the laboratory or steam generated by hctlling water in a flask. which is then piped inlo Ihe distilling flask. Steam is vcry dangerous and the safest way 10 generate steam is to heat Ihe compound wilh a vast excess of water in the distilling flask: steam is generated in silll. The equipment for steam distillation is illustrated in Fig. 15.9 and Ihe procedure is described in Box 15.5.
~
You must never attempt a distillation in 8 eompletllly
closed system. Always cheek that the expanding vapours can escape.
114
laboratory techniques
Distillation
Box 15.5
How to carry out a steam distillation
1. Set up the apparatus for distillation as described in Box 15. 1 with the following modifications: (s) Use a Bunsen burner with tripod and gauze as the heat source. fb) Use a large three-necked flask as the distilling
flask. usually 250 ml or 500 ml capacity for most laboratory procedures or a single necked
flask with 8 Claisen head (Fig. 15.9). (e) Use a splash-head instead of a still-head since there is no point in recording the distillation temperature and it is more important not to cOntaminate the distillate bv splashing from the distillation flask. Idl Insert an addition funnel (8 separating funnel with a ground-glass joint on the stem) into the distilling flask and fit a stopper in the remaining neck of the flask. (e) Use a single-surface condenser. it is easier to (f)
unblock than a double-surface condenser. Use a simple take-off adapter leading into a large conical flask since you will be collecting a large volume of water.
water, add a large portion of 'anti·bumping' granules and fill the addition funnel with water. 3.
Heat the flask until steady boiling commences and ds'tillatkm of an oily emulsion begins. Then continue the distillation adding water from the addition funnel
to maintain the level of water in the distillation flask. 4. K the distillate is a solid. it may clog and block the condenser. If this occurs. turn off the condenser water, take the condenser tube off the tap to let the water drain out of the condenser and allow the steam to heat up the condenser and melt the solid, which will then flow into the receiving flask. Then carefully restore the water flow. 5. The distillation is complete when no more oily emulsion is condensing (check by collecting a few drops of distillate in a clean, dry test tubel. Turn off the heat and allow the apparatus to cool before dismantling.
6. Separate the product from the water by extraction into a suitable organic solvent (p. 1021.
Z. Add the compound to be distilled to the flask via the spare neck, half-fill the distillation flask with
Laboratory techniques
115
-------10 When carrying out reactions using volatile or dangerous chemicals or solvents - use a reflux system to keep the vapours in the apparatus even though the
mixture is not heated to its boiling point.
Refluxing overnight - you mus! have the approval of your instructor for these operations and complete the necessary
documentation for the night-staff. A special laboratory may be available for 'overnight' reactions.
Since the water pressure may change during the night, you must fix the coolant tubes in place, on the condanser and the
tap, using copper wire twisted round the rubber tUbing or plastic cable ties.
Reflux Reflux is one of the most common techniques you will encounter in your chemistry laboratory c1as..<>es. Since many reactions between covalent compounds are slow processes rather than instantaneous reactions. prolonged heating forces the equilibrium to give an acceptable amount of product. In the reflux process, the reactants are dissolved or suspended in a suitable solvent, the solvent is boiled and then condensed so that it returns to the reaction flask. Onre set up. a reaction carried out under reflux can be run for minutes. hours or even days to promote the required rea<..1ion. The basic components for a renux apparatus are: • • • •
a reaction flask: a renux condenser, a heat sourre (see p. 32); a coolant soun:e, usually water, for the condenser.
The procedure for setting up a simple reflux apparatus (Fig. 16.1) is given in Box 16.1.
Reaction fla~k'j
Stirring in pear-shaped flasks - special triangular-shaped 'fleas' are now available for the conical-base tubes used for evaporation and microscale reactions,
but they are not yet common in the undergraduate laboratory.
Round-bollom or pear-shaped rea<.1ion flasks are preferred, but note that stirring with the usual type of magnetic 'flea' is not jXlssible in pear~shaped nasks. The flasks can have multiple necks so that the apparatus can be configured for tcmpemture measurement, addition of solids or liquids, mechaniC-eli stirring and inert atmosphere work (p. 125). No matter which arrangement of componenL<; is used, alwelYs clamp the reaction flask at the neck and keep the heaviest components (such as an addition funnel containing another chemical) vertically aocl\'e the flask. A condenser will still function at 30 from vertical and it is not very heavy. 0
Condensers For general-purpose work, these are usually single·surface or double-surface types (Fig. 16.2): the double-surface condenser is used for low-boiling JXlinl solvents such as dichloromethanc. diethyl ether or light petroleum (b.pl. 40We). WlIter oot
therll1(Jl'l1!l\lI'_
iJi'i.--_
Fig. 16.1
116
Apparatus for simple reflux.
laboratory techniques
Fig. 16.2
Condensars.
Reflux
Box 16.1 1.
How to set up a simple reflux apparatus
Choose. round-bottom or pear-shaped Ouickfit'Jl flask of appropriate size so tI1at it will be no more than half-full and clamp it to 8 support stand.
2. Sfiect the appropriate heat source and antibumping protection, and adjust the height of the flask so that the heat source can be removed easily jf something goes wrong.
3. Choose 8 condenser of appropriate size and type so that it will condense the volume of vapour formed in the reflux process. For example, do not use a smalt condenser with a 14/23 ioint on a 250 ml flask, since it will not cope with the
S.
6. UghttV grease the joint on the condenser and place it in the flask, rotating it to ensure a good seal. Do not clamp the condenser - gravity will keep it in place - but make sure that it is vertical. You may need to move the set-up quickly away from the heat source and it is easier to manipulate if only one clamp, on the neck of the flask, is present.
7, Tidy the coolant pipes behind the damp ensuring
volume of vapour to be condensed, or a large
that there are no kinks, the pipes are not touching the heat source and the outlet pipe is positioned in the drain or sink. Turn on the water gently to check for leaks and, if all is correct, turn up the water to give a steady flow.
condenser on 8 small flask, where all the solvent may be converted into vapour before conden.
salion occurs. 4. Add the Itquids/solids and the solvent to the flask. using the solids funnel, paper funnel Of stemmed fitter funnel (p. 24) so that you do not get chemicms on the inside of the ground-glass joint. If you do comaminate the joint. it may not allow the condenser to 'sear properly and hot solvent vapour will escape into the atmosphere. Wipe the joint clean with tissue.
---""""'~
ro
""
--
Fig. 16.3 Plastic connectors for condensers.
Aefluxing anhydrous reactions - if it is necessary to exclude atmospheric water or oxygen or carbon dioxide, guard tubes are Ineffective and the reaction must be carried out under an inert atmosphere (p. 1251. Safety note Always use a fresh guard tube because the action of water on the drying agent may form a saM 'cake' and seal the-guard tube. This will act as a ·stopper' and pressurize the reflux apparatus whan you begin heating and may cause an explosion. Do not pack the tube too tightly with drying agent.
8. Apply enough heat to bring the liquid to the boil, check that the solvent is refluxing at a steady rate and that the vapour is condensing no higher than one-third of the length of the condenser. The apparatus can then be left, with regular monitoring, forthe reaction to proceed.
Rubber tubing for coolant waler is allached to the condenser in two ways: I.
2.
imer ntilef lliIsM:t
Fit the r~r tubing to the condenser; the lower tube is connected to the water tap so that the water flows up the condenser for the most efficient cooling.
If the condenser has glass inlet and outlet pipes with a 'knuckle'. wet the ins.ide of the rubber tube with a little W
~
You should always attach rubber tubing to a condenser before fitting it to the apparatus.
Drying tubes Water can get into your reaction by condensation from the atmosphere or by condensation of the steam produced in a water bath. To exclude water, )'ou should fit a guard tube comaining a solid drying agent such as anhydrous calcium chloride or calcium sulphate 10 the top of the condenser. A typical guard tube is shown in Fig. 16.4; use a coarse-sized drying agent rather than a fine powder, which would 'cake' very quickly as it absorbs moisture. Laboratory techniques
111
Reflux
Reflux wit" addition of chemicals Instead of slopping the reaction and opening the upp.initus, you can put in the new cllemicals using an addition or 'dropping' funnel. Addition funnels nre separatory funnels (see p. 119) with a ground-glass joint on the stem, and there are Iwo Iypes (Fig. 16.5): I.
Fig. 16.4 A CaCI, guard tube.
Safety note Never anempt to use 8 pressure-equalizing dropping funnel as a separatory funnel because, when you invert the funnel. the solvent will flow dovm the side arm, past the tap and onto you or Ihe laboratory benchl
Box 16.2
2.
Addition funnels; when you add a liquid or solution from the funnel 10 the reaction flask, you must take out the stopper. otherwise a vacuum is formed and the liquid doc'S not now out (sec p. 105). This is a disad\'unluge when using compounds with irritating or toxic vapouOi or which are air sensitive. The simples! solulion to Ihis problem is to fit a guard lube. instead of a stopper. to the addition funnel and then the liquid or solution will flow easily into Ihe reaction flask. Pressure--cqwllizing dropping funnels: these ha\'c II side arm linking thc reservoir of Ihe funnel 10 the inlet slcm below the tap. The pr~ures in lhe reservoir and the reaction nask are equal, and liquid will now into Ihe rcaclion nask even when the stopper is in place in the funnel. Pressure-equali7jng dropping funnels are \'cry expensi\'e and are nonnally only used for inert atmosphcre reactions (p. 119).
How to set up the apparatus for reflux with mechanical stirring
1. Clamp a muttineck flask to a heavy support stand
at a height where you can put the heating source (mantle or oil bath) underneath, on a 'labjack·. 2. Slide the stirring rod through the stirTet' gland, add
the stirrer paddle and fit into the clamped joint of the flask. Lift the stirrer rod so that the paddle is not touching the bottom of the "ask.
7. tfths glass stirring rod 'slips' in the rubber tubing, tighten the tubing round the glass rod with twisted copper wire or a plastic cable tie.
3. Slide 8 piece of rubber pressure tubing (about 40 mm long) half·way onto the dri....e shah of the stirrer motor and fix the stirrer motor onto the support stand aboul 3mm above the top end of the stirrer rod.
8. Add the chemicals to the flask lstemmed funnel) through one of the olher necks, grease and fit a
4. Very carefullV line up the centre of the pressure tubing on the motor with Ihe centre of the stirring rod and ensure that the drive shaft, stirring rod, stirrer gland and flask are aligned and vertical: look at the set-up from several different angles and adjust the clamps as appropriate until you are sure.
9. Raise the heat source under the flask and adjust
5. Lift the stirring rod and slide it into the rubber tubing, abOut 10 mm, and Ihen raise the flask so that the stirrer paddle is about2mm above the bottom of the flask. Finally tighten Ril the clamps firmly.
6. Make sure that the stirrer motor speed control is set to the minimum speed or zero. Switch on the power and turn the speed control to the lowest setting. If the stirrer turns smoothly and slowly, all is well. You can increase the speed to check for
118
vibration and 'Whip' and then turn the speed down to zero. If the stirrer is reluctant to stir or seems 'stiff, switch off the power and readjust the apparatus.
Laboratory techniques
reflux condenser and additon funnel (if appropriate). Start the motor and increase the speed to give a steady stirring rate. the power to give steady boiling. 10. When the reaction is complete, remove the heat source and allow the apparatus to cool to room temperature. Switch off the stirrer and disconnect from the mains and remove the condenser and addition funnel. Carefully lOllVer the reaction flask about 40 mm (the stirrer paddle will be lifted from the bottom of the flask by 40 mm), cut the copper wire or plastic tie holding the top of the stirrer rod in the rubber tube and carefuflyease the stirrer rod out of the rubber connection. Unclamp the stirrer motor and put it aside. You can now remove the stirrer and stirrer gland from the reaction flask, rinsing the paddle into the flask with a litHe fresh solvent.
Reflux
Adding a chemical to a refluxing reaction - many reactions are exothermic, so you
The addition of dry solids to refluxing reactions requires sp.:cial equipment
and you shouJd refer to the appropriate texis (Errington. 1997, p. 125; Harwood, ef aI., 2000. p. 88; Furniss, ('1 (II.. 1989. p. 82). The simplest
should add the new chemical to the reaction at such a rate that the height of
approach is to dissolve Ihe solid in a small amount of the solvent being used
the refluxing vapour in the condenser
and add it as a solutioo. using an addition funnel.
does not change much.
Reflux with mechal/ical stirring When a magnetic stirrer is unsuitable, e.g. in reactions involving
vL~OOUS
liquids or mixlures. or solids and liquids, a mechanical slirrer musl be used. A mechanical or ovcrhe'.ld stirrer syslern comprises.: I. 2.
An e1ectnc variable speed motor connected to the mains supply. A flexible connector, usually a short length or rubber tubing.
...,
lOddilion
..-. ...... pressure
_--'oli
--wale." in
-"
Claisen
"'."'''
/
wale, in
,.,
--~C==J
''I
Fig. 16.5 Adding chemicals to a reflux apparatus: (aj addir'on funnel; fbI pressureequalizing funnel.
,.,
,.,
lXIndense,_
,<, Fig. 16.6 Apparatus arrangements for reflux with addition: (a) heating a reaction mixture to reflux during the addition of a reagent; (b) heating a stirred reaction mixture during the addition of a reagent; (c) healing and overhead stirring of a reaction mixture during the addition of a reagent.
Laboratory techniques
119
Reflux
3.
4.
A stirrer gland. which fils inlo the lop ground-glass joint of the nask acting as a seal for Ihe renuxing vapour and a guide for the stirrer shaft. There are sevcrallypes of gland available, but the best is now made from Teflon" and needs no lubrication. If this is not available, II screwcap adapter in which the rubber scaling ring has been lubricated with a louch of silicone oil can be used. Note that the stirrer shan should rotate in the adapter, not the 1lrlapter in the join! of the flask. so do not tighten the SCrcwC.1p 100 much. A Tenon paddle which swivels on the end of the stirrer shan so that il can pass through the sround·glas.~ joint on the lop of the flask.
Setting up the app.mllus for reflux with mechanical stirring is II precision ta.<;,k and is described in Box 16.2. The major problems encountered
•
•
The weight of the stirrer maJOr high up on the support stand: uSC II Illrgc support stand wilh a hrovy base or use a support framework. which is screwed 10 the Iabor-dtory bench. .Besides the motor's weight, the torque of the motor can C"dUse 'whipping' in the suppon sland. The motor, stirrer gland, slirrer shaft and relu..1ion nask mlL~t be absolutely vertical and concentric. otherwise the gl
A selection of oonfigurdlions, Suilllble for most renux procetlun$ is shown in Fig. 16.6.
120
laboratory tecl1niques
--------10) Evaporation - the purpose of evaporation is to remove the solvent from a solution. Purification or separation of the components of the solute is by other means. such as recrystallization or distillation.
Evaporation Evaporation is the process in which the solvent of a solution is converted into a \~dpour to leave a solid or liquid solute. There are many applications of cV'dpomlion, nmging from the slow evaporation of water from a solution of an ionic compound to Ie-ave hydrated LTystals, e.g. crystallization of CuS04.5H l O, to the evaporation of large volumes of low-boiling-point solvent under reduced pressure in the extmction of an organic compound. Since crystallization by evapomtion is a specific technique for a relatively small range of compounds, the tenn evaporation is generally interpreted as the removal of the solvent from a solution.
Evaporation of solvents There are three commonly used techniques for solvent evaporation: I. 2. 3.
Evaporation from open Oasks on a steam bath. Rotary film evaporation. Gas ·blow-<.lown'.
All these techniques have advantages and disadvamagcs. Where your expcrimemal protocol may simply state 'the solvent is evaporatoo orr, you should select the most appropriate procedure based on: • • • •
the volume of solvent to be removed: the amount of solute in solution; the relative boiling points of the solvent and solute; the next step in the experimental procedure,
EI'aporation from open flasks Safety note Clamp the conical flask in position on the steam bath. There is particular risk of the flask falling over when using small conical flasks
Ie:; 100 mLI and a glass rod.
This is useful for evaporating small volumes (~25mL) of low-boiling-point solvents « 70°C) from solutions conwining a solute which has a boiling point above 110°C. Place the solution in a beaker or conical flask, containing a glass rOO or boiling stick, omo a ste'am bath (maximum temperature achievable is lOO'C) in a fume cupboard and evaporate the sol\'em until boiling ceases. The obvious advamage is simplicity; the disadvantages include environmental concerns of release of solvents in the atmosphere, contamination of the solvent by condeI1S<.'
Rota,}' film cl'aporation Using 'rovaps' - these are communal so make sure that the 'rovap' is clean before you use it and clean it up aher use. Empty the solvent collection fia$!<. into the
appropriate waste solvent bottles.
This method, which is also known as rotary evaporation or 'rovap', is the tcchnique of choice for the removal of large volumes of volatile solvents from solutions, e.g. from extractions and column chromatography (p. 217). Rotary evaporators arc now standard communal piecL'S of equipment in the laooratory and the operating principle is that of a reducedwpressure distillation except that the evaporation Oask can be rOlated. This rotation reduces the risk of 'bumping', inherent in all reduced-pressure distillations. and sprl'ads the solution in a thin film on the walls of the nask. This l'ffcetively increases the surface area of the solution and increases the rate of evaporation, which is further enhanced by the use of a vacuum. Laboratory techniques
121
Evaporation
Box 17.1 1.
How to
use
a rotary film evaporator
Check that the apparatus is ready for use by ensuring that: (a) The receiving flask is clippcd in place and is empty_ (b) You have available the correct-size groundglass joint adapters to connect your flask to the rotating 'barrel' protruding from the motor of the evaporator. Many rotary film evaporators have an 'odd' joint size, usually 29/32, which is not common to the routine ground-glass flasks used in the laboratory. Alternatively, special bulb-shaped flasks with 29/32 joints may be available. (c) The rotating barrel is 'clipped' in place in the motor, by pulling it gently. Someone may have had to clean out and reassemble the 'rovap' and, if the barrel is not 'clfpped' in place. it will slide out when you attach your flask. If the barrel slides out of the motor when you pull it, consult your instructor. (d) The rotating barrel is clean and dry. (e) Water is flowing steadily through the condenser. If it is not, adjust the water tap. (f) The temperature of the water in the water bath is about 20 'C below the boiling point of the solvent to be removed.
2. Open the vacuum inlet adapter at the top of the condenser. and turn the watcr pump to maximum
(p.301.
3. Fill the rotating flask half-full or less, using a stemmed filter funnel. You must not contaminate the joint of the rotating flask with solute. which will be deposited there during evaporation, since you may not be able to remove the flask after evaporation. If the flask is too full it may 'bump'. sending solution up into the condcnser and receiver. You will then have to dismantle and clean out the equipment with an appropriate solvent to recover your compound.
system is an electric motor controlled by an 'updown' pressure switch, but on older apparatus the lifting device is either (i) a manual handle with a trigger, which is pulled to lift and released to lock it in place, or (ii) a lever with a twist-grip on the end. To Operate the lattcr mechanism, twist the grip anticlockwise to release the 'lock', pull the lever down to raise the apparatus and twist the grip clockwise to lock it in place. 5. Attach the flask to the barrel and put plastic joint dips on all the joints, while supporting the flask with YOllr hand. If the weight of the flask and contents 'springs' any of the joints, it is too heavy: replacc it with a smaller flask or remove some of the solution. You must not rely on the power of the vacuum to hold your flask in place.
6. Turn on the motor, slowly increasing the speed to the maximum. 7. Close the vacuum inlet adapter slowty untit it is fully shut and observe the flask for a few seconds. If boiling occurs (liquid is condensed to the receiver flask) continue until boiling stops and then lower the flask so that it just touches the surface of the water, lock it in place and boiling should recommence. As the volume of solution decreases, you may need to lower the flask further into thc water bath until all the solvent has been cvaporated. If a white coating of frost forms on the flask evaporation may stop, because the flask is too cold: lower the flask into the water bath to warm it and evaporation should begin again.
8. When evaporation is complete, raise the flask from the bath. switch the motor off. open the vacuum inlet tap to allow air into the system and allow the flask to cool. Support the flask with your hand, take off the plastic joint clips, put the flask on one side and only then turn off the water pump_ Turn off the water supply to the condenser.
9. Unclip the receiving flask, dispose of the solvent
4. Raise the apparatus using the l;Jting mechanism so that, when the flask is attached, it is not touching the water in the bath. On modern equipment the lifting
into a waste solvent bottle and reattach the receiving flask to the apparatus.
~
When using 8 'rovap' you must check that the reduced-pressure boiling point of the solute you are trying to isolate is below the temperature of the water bath. There arc many variations in the details or the form of rotary film evaponuors and a typical assembly is ilIuslmtcd in Fig. 17.1. A geneml guide to the use of a rolary evaporator is given in Box 17.1.
122
laboratory techniques
Evaporation
When using rOfary film evaporatou you should "ake notc of lhc following safety advice: • • •
• •
•
., Fig. 17.1 la) Typical examples of a rotary evaporator; (bl exploded view 01 glassware.
Transferring viscous liquids - it is ohan difficult to transfer small amounts of viscous liquids from a 'rO\lap' flask to a small sample tUbe. Dissolve the liquid in a small amount of dry solvent, transfer a lillie of this solution 11 or 2 mll to a suitable small tube and 'blow off' the solvent with nitrogen.
Safety note
Make sure that you do not put the tip of the Pasteur pipette too dose to the liquid surface or you will blow the liquid out of the tube I Test the flow first
Newr use flat-bottom flasks or conical flasks under rcduttd pressure (p. 110). Alwuys dteek your nask for'star'.-erclcks. AlWays make sure thaI your solution has cooled to room temperature before you bc~in, othcnyisc it may boil vigorously and 'bump' when you apply the Vllcuum, before it is lowered inlo thc water b
If it is necessary to evaporate volumes of solvent greater than the capacity of the rotating flask. you can carry out the process batch-wise involving se,'eral sepamte evaporations or the rotary evaporator can be modified for continuous evalXlrdlion (Fig. 17.1). A thin Teflon W tube is attached to the vacuum inlet adapter so that it feeds down the condenser into the 'barrel' and another glass tube, dipping into the solution to be C\'aporated, is connected to thc air inlet on the vacuum adapter. Once lhc rotary evaporlltor is operating. the tap on the vacuum adaplcr is opened a lillIe. Solution is drawn up by the vacuum. runs into the rotating nask and is evaporated. Careful control of the tap allows a conSlant volume of solution to be sucked into the rolating nask and evaporated without o,'erfilling it.
Gas 'hlow-dOlI'11' This procedure is useful for removing very small volumes of solvenls (about 2ml) from solutes by blowing a stream of nitrogen over the surface of the solution, while wamung lhe Solulion gently. The main application of the gas blow-down is in the isolation of small amounts of solute from rotary evaporation or small-scale liquid-liquid exlmction, for further analysis by instrumentnl techniques, where the sample size mny be 20mg or less: for example, infrared spectroscopy (p. 180), I\.'MR spectroscopy (p. 190). gas chromatography (p. 211) or liquid chromatography (po 218). lne simplest system for eVdporalion by gas blow-down is shown in Fig. 17.3. A Pasteur pipelte is connected by a flexible tube to a cylinder of nitrogen. which has a gas blow-orr safety system (p. 125). The sample is placed in a speciallUbc with n conical base, such as a Rea(.tiVial 'L . Hold the Pasteur pipelle and dirccl a gentle stream of nitrogen towards the side of the tube so that it nows over the surface of the liquid. As the solvent evaporates. the liquid and tube will cool and may condense atmospheric water into the tube. To prevent condensation. clamp the tube in a warm sand bath or above a closed steam bath or in the hole of n purpoge..dcsigned aluminium heating Laboratory techniques
123
Evaporation
Fig. 17.2 The procedure for continuous solvent removal using a rotary evapOfator.
Fig.
17.3 Gas 'blow-down' QVapOration,
block, The following points should be noted when using a system: • • •
124
laboratory techniques
gJ.IS
blow-down
Always carry out the opcr-d!ion in a fume (,l..lpboard. The solute should have negligible vapour pressure at room ternpentture. otherwise il may co-evapOr
------j0 Inert atmosphere reactions - these should be done in the fume cupboard, since most of the solvents and reagents used are flammable and/or toxic.
Nitrogen atmospheres - note that lithium metal reacts slowly with nitrogen.
Inert atmosphere methods During your laboratory work you may need to carry OUI fC.'lClions using chemicals which are described as air sensili~'e or moisture sensith'e, These compounds may react with waler, oxygen, carbon dioxide and even nitrogen (e.g. lithium metal). The most sensitive chemicals may require special apparalus such as glove boxes or vacuum line equipment and you should consult the appropriate speciaJist litemture (Errington, 1997, p. 56). On the other hand. many reaetion~ involving some air-sensitive reagents (e.g. organolithium compounds or hydride reducing agent) can be done on a small scale using standard glassware with appropriate modifications. For simple apparatus for inert atmosphere rcar.-1ions, the basic requirements arc: •
Use of argon - reactions carried out under argon can be opened to the atmosphere briefly (~5 sl. for the addition of other chemicals, without degradation of the inert atmosphere.
•
...........
• •
,,","
Inert atmosphere, usually nitrogen or argon, piped into the apparatus. Nitrogen is the most commonly used inert gas, whereas argon is more expensive but does have the advantage Ihat it is denser Ihan nilrogen and is not lost from the apparaltl~ as quickly. The inert almosphere is mllintaincd in Ihe appamtus by the use of a 'bubbler' (see p. 126) on one of the outlets from the glassware - all other outlets must be stoppered or C"dpped with septa. Appropriate glassware for the experiment (see Chapler 16) which must be dry. Dry solvents and chemicals (p. 127). Syringe lechniques for dispensing llnd transferring chemicals to the apparatus (p. 127).
tOgh-pressure
gauge levtir'O!r1
, 7'C:"'- ...."'" legiElOr control
tlweaded bUlel aclaplllf!(IIi! ;u., (:yli)der
= El
~easeoullel
"""'-
cylinder va/ve
gas cyIiocler
Fig. 78.1
The diaphragm pressure regulator.
satety note Gas cylinders must be supported safely either by clamping to the bench using a special cylinder- clamp
or in a cylinder trolley.
Inert gas flow rate - only low flow rates
are required to provide an inert atmosphere, once the apparatus has been 'swept' with the gas.
Inert atmosphere The source of the inert atmosphere is usually a cylinder of nitrogen or argon gas under pressure. which should be placed as close to the apparatus as possible to avoid long 'runs' of connecting rubber tubing.
Cw,' cylinders The gas flow nite from the cylinder is controllc<.l by the cylinder head regulating valve (Fig. J8.1). Before you start make sure that the regulator outlet tap is off (tum anti-clockwise umil it feels 'free') and then open the valve to the cylinder with the cylinder spanner (tum ami-clockwise) and the cylinder pressure should be indicated on the right-hand pressure dial. Switch on the gas at the regulator (tum slowly clockwise) until there is a reading on the left-hand dial. Use the minimum pressure required to provide a steady now of gas. The gas now nHe from the regulator can be controlled further by a needle valve on the regulator outlet, if one is fitted. To switch ofT, reverse the instructions above.
Connection to the apparatus Use clean, dry. thin-walled rubber tubing and special adaplers with groundglass joints to connect the tubing to your reaction flask or to the inlet pipe of a 'bubbler'. Where a single cylinder supplies seveml outlets, e.g. in a fume cupboard, the gas flow mte may change markedly when someone turns off one of the oudets, resuiling in an incrC'
Laboratory teChniques
125
Inert atmosphere methods
the apparatus. To do this, fit a gla~ lube 'T-piece' in the gas line 10 your apparalus and COflllect it to a Dreschel boille containing mineral oil to a depth a lillIe more than thai of the mineral oil in the 'bubbler', If there is a sudden upward change in gas prc~ure, the gas will be vcnted through the Dreschel bOllle, as well as through your apparatus.
Gas 'bubblers' Using a gas 'bubbler' - this ensures that your apparatus is not a sealed system, which will pressurize as you introduce the inert gas.
The exit of the inert gas from the apparatus must be protected by a gas "bubbler'. The 'bubbler' allows you to monitor the now of incrt gas through the apparalus and prevents the entry of air into the apparatus. Several designs of gas 'bubbler' are available (Fig. 18.2) and it is usual to connect the 'bubbler' to the apparatus at the top of the condenser. You should make sure that the 'bubbler' conlain<; enough mineral oil to create a seal from the atmosphere and that it has a bulb above the mineral oil to collect any mineral oil, which could be sucked back into your apparatus if there is a sudden contraction in the volume of inert ga.. in the apparatus. Such changes in volume will occur if you suddenly cool the apparatus without increasing the ga., flow to compensate for the resulting reduction in inert atmosphere volume.
Apparatus Guard tubes - these absorb relatively little atmospheric water and do not absorb oxygen and carbon dioxide. Always use a gas 'bubble( to protect the exit from your a!>ltaratus,
",.
Depending upon the type of reaction to be carried out. one of the a~mblics shown on p. 119 may be used. with additional modifications for inert gas inlet and outlet. You should consider very carefully what is required: heating or cooling. magnetic or mechanical stirring, temperature measurement, etc. The gas inlet can be either directly into the reaction nask or into the inlet aml of the gas 'bubbler' - it depends on the number of 'necks' available on the reaction flask.
seal or
""~='Jr=~
DrJ'inJ.: glassware All equipment to be used should be dried (e.!!. in an oven overnight at 125~C do not forgl1 to remove aU plastic or Tenon fl components before placing the glassware in the men). After drying, the apparatus should be greas<;d, assembled hot, using heat-resistant gloves as protection. and allowed to cool with the inert gas flowing rapidly through it. Once cool, the water connections for the condenser should be fitted - screw-on water connectors (p. 118) are most useful in this context.
Addition ofc!lcmicals
t
iner1 gas in
Fig. 18.2 Typical 'bubblers'.
126
Laboratory techniQues
Chemicals should be added to the reaction flask using a pressureequalizing dropping funnel (p, 119). Liquids and solid compounds arc best addl'
Inert atmosphere methods
Solvents and chemicals Dry solvents - the term also impliCitly means carbon dioxide-free and oxygenfree,
All chemicals and solvents used in inert atmospherc reactions must be dry. Most of these materials provided by suppliers are nol dry enough, evCn solvents .... hich you consider 10 be immiscible with water. and may contain enough moisture to hinder the reaction or rt:duce the yield of your product. Therefore you mlL~t ensure that Hit chemicals 10 be used in lhc process have been dried to the appropriate b'tls. as described below.
Solid chemical:r These should be dried by the methods outlined on p. 39. TIle most commoll approach is to dry the chemical in an oven and then allow it to cool in a vacuum desiccator (p. 40). Technique<; for extremely air-sensitive solids can be found in lhe specialist literature.
Liquid chemicals All liquids should be dried by a mcthod appropriate 10 Ihe amounl of water they may contain (p. 41). Generally, the liquid should be dried with a solid drying ahoenl (p. 41) which does not reacI with the chemical (consult the appropriate literature or your instructor). filtered, distilled (p. 107). then stored over rnolL'(:ular siev(:s (p. 41) in a bottle capped by a septwn and rL'(/istilled before use. Altem3tivcly. the liquid can be dissolved in a solvent. the solution dried (p. 41). the solvent removed byeV3JXlratio/l (p. 121) and the liquid distilled and stored as described above.
Sob'e"ts Grign8rd reactions arc relatively tolerant and dry solvent can be added to the apparatus using an oven-dried measuring cylinder.
The solvent wi" have the greatest volume in your reaction and it
mllSI be tlly. Most labol"'dtory-grade solvents. as supplied by manufacturers, contain varying amounts of water and therefore must be dried by the appropriate method before use. If you are required to dry the solvent. you should consult the literalUre (Errington. 1997; Harwood. et til.. 2000: Furniss. et til., 1989). Some manufacturers supply dry solvents in 2.5 L quantities for inert atmosphere reactions and I-!PLC (p. 218). These solvents arc relatively expensive but may be economic in term.s of time aoo expense if one-ofT reactions are required. Howc\'CT. such solvents should lx: treated with suspicion if the containers are Ies."i than half-full. since air and moisture may have been allowed into the container by previous users. If you have any doubts. dry the sohenL
Syringe techniques Many air-sensitive chemicals are supplied as solutions in nitrogen-filled which are sealed by a seplUm, and small volumes (up 10 25mL) of these solutions are best transferred 10 the apparatus using glass syringes. Similarly, air-sensitive liquids can be added to the reaction using a syringe. bollle£,
~ When removing air-sensitive reagents from nitrogen-
filled botttes. you must replace the volume of liquid removed with inert gas (nitrogenl from a gas cylinder or balloon, via a needle, otherwise air (water, oxygen and carbon dioxide) will be pulled into the bottle as a rasuh of the vacuum you have created. Laboratory techniques
127
Inert atmosphere methods
Syringe.'I Using syringes - always ensure that the syringe piston is the correct one fOr your syringe. since the components of rhe syringe are atways separated for drying.
.qlllilli1111l1i1111i 1
p.blnlll
~I
_.. :::tJ ~
-- --
q'ili1liii111lilllii l
~O
Tefton{!ltip
~
Fig. 18.3 Syringes 1M inert atmosphere work.
Fig. 18.4 Needle-te-tubing connector.
Glass. gas-tight syringes with II Luer lock fining are the most versatile type of syringe and they come in a rdnge of sizes. 111e Luef lock enables the stainles.o; Sleel needle to be locked in place on the end uf the syrilll.>e so that there is no danger of the needle dropping off the syringe during the tnlOsfer process (Fig. 18.3). Variations in syrinb'C types include those with TeOonil'-tipped pistons (plungers), which are somewhat more expensive. Before using II syringe, always check Ihal it is working by sucking up a lillIe of the solvent to be LLo;cd, ensuring that air is not sucked into the syringe either via the Luer lock or down a gap between the syringe and piston. If all is correct. disassemble the syringe and needle, dry in an oven at 120'C (not ifTeflon k tipped) and allow to cool in a desiccator. Once you have transferred the airsensitive reagent. you mu.'1 clean out the syringe and needle immediately. by the appropriate method, as air will gel into the needle and syringe and decompose the reagent cau.<;ing the syringe lojam or the needle to block.
NceJIe-to-tuhing connectors These adapters allow a Luer lock syringe needle 10 be connected to rubber tubing (Fig. 18.4). An inert gas supply can then be piped, via the needle, into a boule to allow the trnnsfer of lurge volumes of solvellt or air-scnsitive reab'Cnts to the apparatus. Alternatively a balloon can be taped to a short pi~ of thick·wallcd rubber pressure tubing 10 provide a supply of nitrogen when \\ithdrMvin,g air-sensitive reagents using a syringe (Fig. 18.5).
,.,
--<~F='T~f"r---
..,.......
firoG SlCJPOrl to hi*!
1---_..,
=--,,.,,
.,
Fig. 18.5 loon almosphere transfers: fa) balloon and needle; (bl preserving the inen atmosphere While removing reagenL
128
Laboratory techniques
Inert atmosphere methods
Syringe needles and mnnulae Working with viscous liquids - attempting to dlllw the solution up a fine needle creates a strong vacuum in the syringe, which may result in air being pulled into the syringe via the small gap between the syringe barrel and the piston. Use a larger diameter needle.
Stainless steel Luer lock syringe needles come in various length" and diameters. The length of needle you will need depends Oil the size of the vessel from which you wish to withdmw the liquid; the diameter required depends Oil the size of the syringe - you should not use a large--diameter needle with a small-volume syringe - and the viscosity of the solution or liquid. Needle diameters are expressed in 'gauge': the higher the gauge, the narrower the nl.-edle diameter. For most inert atmosphere work you should use a needle with a 'nonoCoring' or 'deflecting' tip (Fig. 18.6), which ensures that a piece of the septum is not trapped in the needle when you push it through. Cannulae are long, flexible. double-ended needles made from stainless steel or inert plastics, which are used to transfer large volumes of reagents or solvents from one vessel to another under inert gas pressure (Fig. 18.7). A generic method for tmnsferring an air-sensitive reagent, by syringe, from a boUle to a pressure-equalizing dropping funnel is described in Box 18.1.
inmQllS
/
Fig. 18.6 Needle with non-coring tip for
piercing septa.
Fig. 18.7 Transfer of air-sensitive reagents using the double-ended needte technique.
!-----pressure-equaliZlOlil
_----'i
addition funnal
,...... ------1~~. \l~!l;:tr----'--~~.i-------lt'o'ee.OOCk
",,,"
-@-
,""'-@-
flask
1---- .....
Fig. 18.8 Transferring an air-sensitive reagent to a pressure-equalizing dropping
funnel. Laboratory techniques
129
Inert atmosphere methods
Box 18 1 How to transfer an air-sensitive reagent using a syringe 1. Assemble the apparatus lFig. 18.8} while hot and allow to cool while nitrogen is flowing through it and then add all the chemicals and solvents as required. 2. Assemble the syringe and needle, making sure that the needle is locked in place having first checked that it Is air-tight. 3. Draw a few millilitres of solvent into the syringe, invert it and squirt the solvent and any air into the waste solvent bonIe. Repeat this step three times to ensure that syringe and needle contain only solvent vapour, 4. Using the syringe, inject a few millilitres of the dry sotvent into the pressure-equatizing dropping funnel, either via the septum or by quickly removing the stopper while diverting the gas flow through funnel by putting your finger tip over the 'bubbler' outlet. This ensures that there is an inert atmosphere in the funnel. Replace the stopper and release the gas flow through the ·bubbler. 5. Clamp the boUle of air-sensitive reagent to a support stand so that it cannot fall over while you manipulate the syringe needles. 6. Remove the needle from the end of a needle-totubing connectoc" canying a balloon. Connect the inert gas supply using clean, dry rubber tUbing and inflate the balloon. Turn off the gas supply, twist the neck of the balloon to stop the gas escaping, reanach the needle, ensuring it is locked in place, and release the neck of the balloon. Dip the end of the needle into a little dry solvent to confirm that nitrogen is being released from the needle - check for bubbles. 7. Hold the needle near the tip and pierce the septum on the bottle. Make sure that the needle tip is in the space above the liquid. Support the needle connector and balloon by a clamp On the support stand. 8. Holding the syw-inge and the needle near the tip, pierce the septum on the bottle. Still holding the
130
Laboratory techniques
needle, ease the syringe needle into the space above the liquid. 9. Draw up some gas into the syringe and expel it back into the bottle. Repeat three times and then ease the syringe needle into the liquid. 10. Draw up the required volume of solution into syringe and carefully ease the needle out of bottle. Make sure that you do not press syringe piston and squirt the reagent out of syringe!
the the the the
11. Inject the solution into the pressure-equalizing dropping funnel, erther by: (a) holding the syringe needle near the lip, piercing the septum on the pressure-equalizing dropping funnel and injecting the reagent; or by (b) puning your finger over the 'bubbler' outlet and removing the stopper from the pressureequalizing dropping funnel. You can now inject the solution into the funnel. protected by the nitrogen and solvent vapour coming out of the funnel neck. Replace the stopper and release the gas flow through the ·bubbler. 12. Draw a few millilitres of dry solvent into the syringe and inject them into the pressure-equalizing dropping funneL This rinses any residual reagent from the syringe. 13. Draw some methanol or another solvent which reacts gently with the reagent to destroy the airsensrtive reagent and squirt it into an eJeC9SS of water. To clean the syringe assembly. draw water into the syringe several times and then disassemble It, wash well with propanone (acetone) and water, and then dry in the oven. 14. Remove the needle-balloon assembly from the reagent bonle, cover the holes in the septum with a little hydrocarbon grease and screw the bottle cap in place. Place the needle in the oven after washing with propanone (acetone) and then water.
Resources
Resources for laboratory techniques
Books Bennell, S.W. and O·Neale. K. (1999) Pmgressiw! Dt'I'i'fopmelll of Prtlcticlll Skil/.f ill Cl1i!misllY. A guide to earIY-lJlIdergwdlllJtt! eXfH!rimcllwlll'ork. Royal Society ofOlemistry. Cambridge.
Errington. RJ. (1997) Adl'(1Jlced PrtlClical Inorgtmic lind Mew/organic Cheml~'ry.
Blackie Academic and Profes.<;iollal. London.
Furniss. B.A., HanMford. A.J.. Smith. PW.G. and Tatchell. A.R. (1989) Vogel's Textbook of Prtlct;c(ll Organic Chemi.flry. 5th Edn, Longman, Harlow, c·,sex. Halpern. A.M. (1997) £.v:perimemal Physical Chemi.ury, Prentice I-Iall, Harlow, Essex. Harwood. L.M., Moody. C.J. and Percy. 1.M. (2000) £y.perimellllll Organic Chemiflry. 2nd Edn, Blackwell Scienl..'t Lid, Oxford.
Lehman, J.W. (1999) Operatiollal Olgol/ic Chemistry. A prohlem·soMng approach 10 the labora/or)' course, 3rd Edn. Prentice Hall. Harlow, E-.sex.
Lister, T. (1996) C/assir Chemisl/}' DemOlI$lrtlfionf. Royal Society or ChemL'itry, Cambridb'e, Loewenthal. HJ.E. (1990) A Gllitkfor Ihe Perplt!.\"l'd Organic Experimenwli.
Mendham. J .• Denney, R.C.. Barnes. .I,D. and Thomas. M.J.K. (2000) Vogel's Tex/book of Q,wn/ilmil'e Chemical Ana/)"sis. 6th ELIn. Prentice Hall. Harlow, Essex. Nelson, J.I.I. (1997) LllboratOl'Y £\·periments for Chemist/yo Prentice Hall. Harlow, E.'iSex. ShaTP. J.T., Gosney, I. and Rowley, A.G. (1989) Prtlcticfll Orgwlic Chemisll)', Chapman and I-Iall. London. Suib, S.L. and Tanaka. J. (1999) £'l:perimenlll/ Methollr ill hrorganir Chemist,}'. Prentice Hall, Harlow. Essex. WiUiamson, K.L. (1999) Macroscu/e lmd Mi,'roseil/e Organic £\"perimems, Jrd Edn, D.C. Heath and Company, Lexington.
Videos Basic Laboratory Skills. Lac. Royal Society or Chemistry, Cambridge (1998).
Further Laboratory Skills, LGC. Royal Sociely or Chemistry, Cambridge (1998).
Laboratory techniques
131
--~0
Qualitative techniques for inorganic analysis Qualitative techniques are used to identify cations and anions in aqueous solution by simple reactions, mosUy involving the production of a precipitate, evolution of gas or a visual colour change. It is important to make observations accurately and 10 interpret them in a step-wise manner. The following basic equipment is required to carry oul qualitative analysis:
• • •
Test tubes - in which the reactions arc perfonned. Cork or rubber stoppers - for the protection of the contents of test lUbes from contamination and for safe storage. Test tube rack - this allows test tubes lo be stored upright when not in usc.
• • • Wash bottle - always keep a wash bottle
of distilled watBr handy.
• • • • • • • •
Test lube holder- this allows individual test lubes to be hcated safely. A glass rod - Ihis has several functions induding the stirring, tmnsfer of solutions, and the break-up of precipitates. Watch-glasses these have several functions induding the covering of beakers 10 prevent contamination and as a receptacle for solutions. A wash bottle containing distilled water. Spatula - for Imnsferring small quantities of solids. Pasteur pipettes - for transferring liquids. Micro-Bunsen burner - for heating solutions to boiling and for evaporating solutions. Evaporation crucible - this is used as a container for solutions when complete evaporation of liquid is required. leaving a solid product. Crucible tongs - for removillg the crucible from the heal source. CenlJifugc - for separating precipitates from solution. Heated water bath.
Table 19.1 Typical reagents for Qualitative analysis 2M NH.OH
Cone. Hel
2M NaOH 2M AgN03 2M CH 3COOH
0.1 M HN03 2M HN03
2M B"CI 2 O.lM Hel 2M Hel
Cone. HN~ 2M H2S04 Cone. H2S0 4
Reagents At the stan of your experimental work always check lhat the appropriate reagents are readily available (a list of commonly used reagents is gi\'cn in Table 19.1). Note that it is essential to use distilled water in all qualitative analysis. Tap water contains ions such as Ca 2+. Mg2~. Fe2 ', Fe)', soiand Cl- and its use could lead 10 'false-positive' fCSUItS.
Testin9 for anions and cations Specific litemture containing tests for the determination of anions and cations can be found in the resources section (p. 160). In general. however. the following tips are useful when carrying out qualitative analysis: Spatula - never place the spatula in the
• •
test solution, it may lead to false-positive tests for iron and chromlum.
•
Always work tidily to prevent cros.'i-contamination of samples. Ensure that all glassware has been cleaned thoroughly in detergent and then rinsed twke with distilled water. Invert the test tubes to drain; never dry the inner surface with t()\velling or tissue. Lahcl test tubes at the start - it may prove difficult to remember what you have done later on. Classical techniques
135
Qualitative techniques for inorganic analysis
OuaIiIBlMlIOSIS kJr canKlS lKld uniI:wlS
-
_..
•
An unknown solution ""3S restOO as f
r.. 2dn:ip$of~
IfCI, ooil. then add 1
drop Ba~ sOOtion
lMlite preciplMe
""",,"00
(S~'l present
•
• Test performed (Ill
..,.,.,"""""
Report oIlhe oll&ervaboos madll
Concwion drawn from Iha ooo_tion
Fig. 19.1 Recording your observations in qualitative analysis.
•
•
A 'clear' solution is transparent; a 'colourless' solution has no colour. •
•
Always t~st solutions with a known composilion before you attempt 10 anaJysc solutions with an unknown content. This allO\vs you to gain the necessary experience in solution manipulation, observation skills and the interpretation of results, The colour of solutions and/or precipitates has to be interpreted from wriuen or oral information, Interpretation of colour can be subjective, so you will fIC(.'$t tube. Never put Pasteur pipettes inside test tubes as this can lead to contamination of the reagents. Effective mixing of the test solution and added reagents is essential. This can be aehieVl..-c! by holding the lest tube between the thumb and index linger of one hand and 'nicking' the tube with the index finger of your other hand. Allernatively, solutions can be mixed by bubbling air from a Pasteur pipette held at the bottom of Ihe ll..'st tube. Evolved gas CUll be drawn up inlo a Pasleur pipelle and then bubbkd throu1!-h a test solutioll, e.g. CO2 can be drawn into a Pasteur pipette and then 'blown' out through lime waler (Ca(OHh solution) giving a milkywhite solution. Solutions can be tested for pH using litmus paper. Never place Iilmus paper directly into the ll..'st solution. Instead, dip a glass rod into the solution, remove, touch the wet glass rod onto the litmus paper and note the colour. Acidic solutions change blue litmus papcr to rl.'d; alkaline solutions change rl.-'d litmus paper to blue. Alternatively, universal indicator paper can be used. In this case, the orange paper turns 'reddish' with acidic solutions and 'bluish' wilh alkaline solutions. By comparing any change in colour with a chart (supp!it'd with Ihe universal indicalor paper), the pH of the solution can be l.'Stimatcd.
Centrifugation of solutions Centrifugation causes particulate material to accumulate al the bouom of the test tube. The procedure for centrifuging your sample is described in Box 19.1. The speed and time of the run will depend on the c(.'otrifuge available, but will typically be in the mnge 5000-10000 rpm for 5-lOmin, respectively. Always allow the cenlrifuge to stop in ilS own time, as abruptly halting the centrifuge will disturb light precipitates. After centrifugalion, hold the tl..'St tube at an angle so Ihat il is easy 10 remove Ihe liquid component (or centrifugate) with a Pasteur pipette (Fig. 19.2). You will find thai it is difficult to remove all the cclllrifugale from the precipitate, and to maximize the transfer of centrifugate it is necessary to wash the precipitate, Ths is carried out as follows: • • • • ","'
19.2 Separation of liquid trom a residue using a pipette,
Fig.
136
Classical techniques
•
Add a small quantity of distilled water to the precipitate. Use a glass rod to break up the precipitate and mix thoroughly. Rccentrifuge the mixture. Transfer the liquid obtained to the original centrifugate and store this solution for further analysis. Repeat the washing process. but this time discard the eenlrifugate, and retain the precipitate for further tests.
Qualitative techniques for inorganic analysis
Box 19.1
How to use a low-speed bench centrifuge
1. Choose the appropriate test ttbe size. lNith stoppers where necessary. Most Iovv-speed machines have four-place or six-place rotors. Use the correct number of samples to fill the rotor assembly whenever possible. 2. Fill the tubes to the appropriate level: do not overfill, or the sample may spill during centrifugation. 3. Ensure that the rotor is balanced during use. To
achieve this prepare identical test tubes and place these diametrically opposite each other in the rotor assembly. However, for low-speed work. where you are using small amounts of particulate matter in aqueous solution it is sufficient to counterbalance a sample with a second test tube filled with water. 4. H you are using centrifuges with swing-out rotors, check that each holderlbucket is correctly positioned in its locating slots on the rotor and that it is able to swing freely. All buckets must be in position on a swing-out rotor, even if they do not
contain sam~e tubes buckets are an integral part of the rotor assembly. 5.
Load the sample test tubes into the centrifuge. Make sure that the outside of the centrifuge tubes, the sample holders and sample chambers are dry: any liquid present will cause an imbalance during centrifugation fas well as potentially causing corr~ sive damage to the rotor). Balanced tubes must be placed opposite each other, use a simple code if necessary. to prevent errors.
6. Bring the centrifuge up to operating speed by genUe acceleration. Do not exceed the maximum speed for the rotor and tubes used. If the centrifuge vibrates at any time during use, switch it off and find the source of the problem. 7. On completion of the run, allow the rotor to stop spinning, release the lid, and remove all test tubes. If any sample has spilled, make sure you etean it up thoroughly. 8. Finaltv. close the lid (to prevent the entry of dust) and return all controls to zero.
Heating test tubes and other containers Safety note
Take care when heating unknown solutions. As well as the risk of burns, some reactions can be violent.
Safety note Never point a test tube towerds yourself or. for that matter, towards anyone else while eV8poration is being carried out.
It is orten necessary to heat a solution in a test tube. eithcr to cause precipitation or to dissolve a precipitate. You can carry out this heating effectively and safely by partially immersing the test tube containing the mixture in a sinunering boiling,wdter bath (remember to usc a test tube holder!). II is possible to reduce the volume of the solution in the test tube, i.e. to pre-concclltrate the sample, by evaporation. Two different methodS can be employt.'d. l.
safety note Never look down into a test tube (even with safety glasses on - there is still a risk of hot solution suddenly being ejected into your face). Always view coloured products through the wall of the test tube.
-
8afety note Noxious fum9$ can be given off in some instances. Be careful not to breathe them in. Always work in a welt-ventilated room or a fume cupboard.
Beware - hot glass looks exactly like cold glass.
2.
Transfer the solution to a small evaporaling dish. Place the e\~su!t in hardening of any residue, making it unusable. Alternatively, evaporate the solution directly in a test lUbe by gentle heating over a micro-Bunsen burner. Remember to use a test tube holder. l)osition the test tube at an angle with the tip of the Bunsen burner name positioned at the upper surface of the liquid. Place a glass rod inside the test tube and rotate constantly. This acts to disperse bubbles of steam that are given off. Extreme caution is required with this method of evaporation. as the steam bubbles can cause the solution to 'bump' out of the test tube. 'Bumping' can result in hot (and maybe toxic) substances being ejt.'Cled over a suprisingly large distancc. To prevent this it is normal to add antibumping granules (p. 31). Classical techniques
131
Qualitative techniques for inorganic analysis
Table 79.2 Flame tests for c8tions CatI~
Barium Calcium Copper Potassium Sodium Strontium load. arsenic. antimony and bilimuth
R.mecofour Apple-green Brick-red·· G""," Lilac· Intense V9I1ow Crimson·· CUI"'"
• The colour is often obliterated by trace impurites of sodium present lsodium gives an intense yellow roIourl. You can overcome this by viewing the colo"," through cobalHwue
glass which aJIolNs the lilac coIouration from potassium to be seen. •• VIeWing through cob&h-b1ue glass l!Ilso allows calcium and strontium to be distinguished. In this case, calcium is light green in colour while strontium appears purploe.
US
Classical techniques
Fig. 19.3 Holding a nichrome wire in a !lome test.
Aametests Simple flalM tC'its can be carrii:d oul on soM samf*$. Place a little of Ihe solid on a watch-glass and moisten with a drop of concentratOO hydrochloric acid 1k purposco of the hydrodllonc acid is to prodoc:c metal chlorides which arc volatile at the temper-llure ofthc Bunsen bumer. Pre-dcan a platinum or nichrome wire by holding it in the houest pan of the Bunsen flame (just above the central b1uc cone) until there is no coloured flame from the "ire. Cool.. then dip the cleaned wire into the moistened solid sample. Plaa: the ",ire at the edge ofthc Bunsen flame (Fig. 19.3) and record the colour of the flame from the sample (sec Table 19.2).
--------0
Gravimetry Gmvimc:tric analySis is the process of convcrting an clement imo a definitive compound, isolating this compound from other con.'.tilucnfs in a swnplc and then weighing the compound (Box 20.1). The weight of the dement eM thell be caJculau..-d from lhc formula of the compound and Ihe relative atomic masses ofthc clements involved. You rJ(:ed to be able to weigh accurately, by difference. a substance 10 four decimal places (sec p. 23).
~ The essential component of gravimetric analysis is
the transformation of the element of interest into a pure stable solid
compound. surtable for weighing.
'Inc most common approadl for isolating the clement is by pn.'Cipitalion from 11 solution where it is prt.'$enl in lonK: form (SL'C p. SO). Ideally, the consliIUl..'tl1 under investigation is precipitated out of solution as a wmerinsoluble compound. so Ihal 110 IOS&-'S ()(:cur when the precipitate is sepamtoo by filtmlion. wasll':d rree or soluble impurities and then wLighed.
Box 20.1
How to carry out gravimetric analysis
Suppose you wanted to analyse the amount of metal in an alloy. For example, you might want to determine the nickel content,. as a w/w percentage (p. 471, in a particular sample of steel. 1.
Select an appropriate solvent for your sample: in this example, you could dissolve the steel in aqua regia. a combination of concentrated nitric acid and concentrated hydrochloric acid in the volume ratio of 1: 3 respectively.
2. Choose an appropriate precipitant and carry out the precipitation reaction Here, an alcoholic solution of dimethylglyoxime could be used to precipitate nickel from a hot solution of aqua regia. by adding a slight excess of aqueous ammonia solution, forming a red precipitate of nickel dimethylglyoximate (Fig. 2O.3J. 3.
4.
5. 6.
Alter the precipitate through a pre-weighed Gooch crucibte. This should have been previously dried in an oven at 120 C and stored in a desiccator until required. Wash the precipitate: in this example, the nickel dimethylglyoximate can be washed with cold water until qualitative testing shows that the wash solution is free of chlOride ions.
Dry the precipitate in an oven, for example, at 120 C, and allow to cool in a desiccator. Determine the weight of the precipitated compound. Suppose in this example that the nickel had
been precipitated from steel (2.0980 g) using dimethylglyoxime (H 2 DMGI. giving a precipitate of nickel dimethylglyeximate tNi(HDMG)2) weighing
0.23709· 7.
Write out the equation for the reaction and perform the calculation. In this example. you need to check the relative atomic masses of the elements involved. The relative molecular mass of nickel dimethyl. glyoximate is 288.91 9 mol 1 (for structure see Fig. 20.3; A,.. H = 1.01; 0 "'" 16.00; C = 12.01; N = 14.01; Ni 3' 58.69). The equation for the reaction can be summarized as: Ni2 -t- + 2H 2DMG '" Ni(HDMGlz + 2W According to the above equation. for each mole of Ni in the steel sample, 1 mote of precipitate will be formed. Therefore. 0.2370 9 of precipitate carre· spends to: 0.2370g NilHDMGh + 288.95g mol- T Ni(HDMGlz = 8.20 x 10-· mol Ni(HDMGh The amount of nickel in the steel sample must therefore be: 8.2 x 10- 4 mol NilHDMGlz x 58.69 9 mol- T Ni = 0.0481gNi
The percentage weight of nickel in the steel sample is therefore:
0.0481 g Ni -:- 2.0980 9 sample x 100 = 2.29% w/w
aassical techniques
139
Gravimetry
Precipitation Table 20.1
Common precipitants IonlsJof
Interwt
.~
Interl_ms Pd 2.,
Dimethylglyoxime
~',
Bi:J.o
and Au'J+ Cu 2 • and Pb2 +
Cupferron a-Hydroxyquinoline (oxine)
Many metals
CH,-C-OOH I CH 3-C-NOH £.Wne!h';tolllyomle
Inorganic ions C'dn be sepamtoo from mixtures using organic reagents (precipitants), with which they form sparingly soluble, often coloured, compounds. The precipitants usually have high molecular weights, so a small quantity of the ion will producc a large amount of precipitate. IdeaU)', the precipitant should be specific for a particular ion. though this is rarely so. Examples of conunon precipitants and their target ions are shown in Table 20.1 and their structures are shown in Fig. 20.1. DimethyIglyoxime is only slightly soluble in water (0.40 g L-I) and it is therefore used as a 1"'/", w/v solution in ethanol. Cupferron, the anunonium salt of N-nitroso-N-phenylhydroxylamine, is used as a 5-10% wlv solution in hydrochloric add or sulphuric acid. Oxine (8-hydroxyquinoline) is almost insoluble in water and is USL'd as either a 2% or a 5% wlv solution in 2mol L-[ acetic acid. When precipitating a compound:
,NO @""N'O-NH;
•
"<'-
•
CQICJ ""
•
S.Hydre»rflO"Jindine
Fig. 20.1
Structures of common precipitants.
_~--li-- Gooch crucible
•
•
•
Mix your reagents slowly, with continuous stirring, to encourage the growth of large crystals of the compound. Improve the precipitation proct.'SS by heating your solutions: ideally, one or boLh solutions should be heatl.'
Filtration Fig. 20.2 Experimental arrangement for filtration of precipitate.
Filtering your precipitate - remember that the particle size of your precipitate must be such that it is not lost during the filtering process.
Fig. 20.3 Structure of nickel-dimethylglyoxime complex.
140
Classical techniques
To carry out this procedure. you will nl.'ed to assemble a Gooch crucible and funnel on a BUchner flask. clamped for stability using a retort stand (Fig. 20.2). The glass Gooch crucible has a porous disk of sinterl.>d ground glass, typically of pore diamelcr 2o-JOprn, which is satisfactory for moderately si7.t.'
-------0
Procedures in volumetric analysis Volumetric analysis, also known as titrimctric analysis, is a quantitative technique used to dctennine the amount of a particular substance in a solution of unknown composition. This requires: •
• •
A standard solution, which is a solution of a compound of accuraLCly known concentration, that reacts with the substance to be analysed. The lest solution, containing an unknown conct:nlraiion of the substance to be analySt."'. Some means of detecting the end-point of the reaction belwt--ell the
standard and tcst solutions, e.g. a chemical indicator or, in the case of potentiometric titralions. a pH electrode (see Chapler 34). Some reactions exhibit a colour change at the t:nd-point without the addition of an indicator. Deflnition
A stoichiometric titration is one with a known reaction path, for which a chemical reaction can be written, and having no alternative or side reactions.
The volume of standard solution that reacts with the substance in the test solution is accurately measured. This volume, together with a knowledge of concentmtion of the standard solution and the stoichiometric relationship between the reactants, is used to calculate the amount of substance present in the wst solution. Specific examples of the different tytX:s of calculations invoh'l.-d are shown in Chapters 22 to 25.
Classification of reactions in volumetric analysis There are four main types of reaction l.
2.
3. 4.
Acid-basc or neutralization reactions, where frcc bases arc reacted with a standard acid (or vice versa). These reactions involve the combination of hydrogen and hydroxide ions to fonn water. Complex fonnation reactions. in which the reactants are combined to form a soluble ion or compound. The most important reagent for fonnation of such complexes is ethylent.>diamine tetra-acetic acid. EDTA (as the disodium salt). Precipitation reactions, involving the combination of reactants to fonn a precipitate. Oxidation-reduction reactions, i.e. reactions involving a gain (reduction) or loss (oxidation) of electrons. The standard solutions used here are either oxidizing agents (e.g. potassium pcrmanganate) or reducing agents (e.g. iron (11) compounds).
What can be measured by trtration7 • • • • •
The concentration of an unknown substance e.g. 0.900 mol L -I. Percenlage purity. e.g. 56%. Water of crystrtllization, e.g. (NH4hS04.nH20. Percentage of a metal in a salt, e.g. 12% Fe in a salt. Water hardness, e.g. determination of the concentrmion of calcium and magne5ium ions.
Examples of the types of calculations uS<.'
141
Procedures in volumetric analysis
Box 21.1
Types of calculations used in volumetric analysis - titrations
In titrations you react a solution of a known concentration with a solution of an unknown concentration. If you know the mole ratio of the two reacting chemicals in solution, you can calculate the amount (the number of moles and thus the number of grams) of the solute in the solution of unknown concentration. let's look at the reaction between NaOH and HCI: HCI
+ NaOH
= NaCI
+ H2 0
Since the equation is balanced we know that 1 mol (36.59) of Hel will react with 1 mol (409) of NaOH. We know that a 1.0 M solution of HCI contains 1 mol of HCI in 1000 ml (1 litre) of water. Then: 1000ml of 1.0M HCI solution
is equivalent to is equivalent to is equivalent to is equivalent to is equivalent to
1.0mol of NaOH l000ml of 1.0M NaOH solution 409 of NaOH 239 of Na+ ions 17 9 of OH- ions
Similarly for the reaction between potassium hydroxide and sulphuric acid:
Since 1 mol of H2S04 reacts with 2 mol of KOH, then: 1000 ml of 1.0 M H2 S0 4 solution
is equivalent to is equivalent to is equivalent to is equivalent to is equivalent to
2.0 mol of KOH 2 x 1000 ml of 1.0 M KOH solution 2 x 569 of KOH 2 x 39 9 of K~ ions 2 x 17 g of OH ions
To work out the results of titrations you must always: • •
Work out the balanced equation to find out the ratio of moles reacting. Decide what you are trying to calculate.
Example: 25.00ml of sodium hydroxide solution were titrated by 24.00ml of 0.1 M HCI solution. Calculate the concentration of the sodium hydroxide solution. HCI + NaOH = NaCI + H2 0 Concentration of NaOH, i.e. moles of NaOH in 1000ml,. since concentration is moll-I.
• •
Now: 1000mlof 1.0M Hel solution is eqUivalent to lOmol of NaOH but the concentration of HCI is only 0.1 M: 1000ml of 0.1 MHCI solution is equivalent to 0.1 x 1.0 mol of NaOH but only 24.00 ml of HCI solution were used:
x 1.0 mL of 0.1 M HCI solution is equivalent to ~.o xl : 1.0 mol of NaOH and 24mL of 0.1 M HCI solution is equivalent to 24 x
l'~;O~_l x
1.0 mol
of NaOH = 2.4
X
10-3 mol of NaOH
but 25.00 mL of NaOH solution were used: 25.00ml of NaOH solution contains 2.4 x 10--3 mol of NaOH
142
Classical techniques
Procedures in volumetric analysis
Box 21.1
(continued)
Then
.
1.0ml of NaG H solution contams
,nd 1000 m l
0
3 2.4x 10I IN OH 25 -mo 0 a
3 fN a OsolutIon H· . 1000 x2.4x 10- rna I a fN aOH contains 25
= 0.096 mol of NaOH Therefore concentration of NaOH solution is O.096molL- 1• Using this set of equations you can calculate directly the mass of NaOH per litre, the mass or moles of sodium ions and the mass or moles of hydroxide ions.
Note: The expression [C,JV, = 1G.!)V2 was not used, even though it is applicable in this case. Problems arise when [Cl l V, = (G.!) V2 is used for reactions whicll are not 1: " e.g.:
H2S04 + 2KOH = K2S04 + 2H20 2MnO;
+ 16H-'- + 5C20~-
= 2Mn2+ + 10C02 -t 8H 20
10; +51 +6W = 312 +3H 2 0
Titrations
Definition A primary standard should be easy to obtain in a pure form. It should be unaffected in air during \IVOighing, be capable of being tested for impurities and be readily soluble under the conditions used. Finally, the reaction with the standard solution should be stoichiometric and instantaneous.
The pr()(X'SS of adding the standard soJUlion to the test solution is called ~ titratioll. and is carried out using a bureUe (sec below). The point at which the rcaClion betwct.>Jl the standard solution and the tcst substance is just complete is calk'd the t-"quivalencc point OT the tht-"OTctical (or stoichiometric) end-point. This is nonnally detected by a visible change. dther of the standMd solution itself or. more commonly, by the addition of an indicator.
SWntlm'tl !J·oluliolts A stand~rd solution can be prepared by weighing out the appropriate amount of a pure Tctlgenl and m~king up the solutioll to a particular volume, as descrilx.'CI on p. 24, The concentration of a st:mdard solulion is expressed in terms of molarity (p. 19). A substance used in a primary standard should fulfil the following criteria: • • • •
• •
It should be obtainable in high purity (>99.9%). II should rt-main unaltert->d in air during weighing (i.e. it should 1I0t be hygroscopic), It must not dt-'Compose when dried by heating or vacuum. It shouJd be capable of !x'ing tested for impuTitit'S. It should be readily soluble in an appropriate solvent. It must re-dct with the test substance stoichiometrically and rdpidJy.
P,'eparillg II Slltnlltw,1 soll/Iiolt Thc molarity of a solution is the concentration of (he solution exprcsS(:d as mol L-I. If x g of a substance of mok'Cular weight M r is dissolvl.'d in y mL of distilled watcr. the moles of substance dissolved = ~ = m. Therefore, Mf IJ'IxlOOO molarity (molL-I) = (21.11 )'
Classical techniques
143
Procedures in volumetric analysis
Primary standards prepared from solid compound.. should be weighed out using the 'weighing by dilTerence' method itS dt-'SCribed in Chapter 4, and accurately made up to volume using a volumetric flask. Complete transfer of the substance from the weighing vt--ssel to the volumetric flask is best achieved by inserting a funnel into the neck of the flask (Fig. 21.1). As much of the solid as possible should be tmnsferred I'fa the funnel. The funnel should be wdshcd with distilled water prior to removal. Distilled water is then addl-d to the flask, with occasional swirling to help to dissolve the solid. lllis is continued until the meniscus is about I em below the volume mark. At this point a stopper is inscrted and the flask is invcrted several timt:s to ensure the solid is completely dissolved. Finally, using a Pasteur pipette. distilled water is added lip to the volume mark. The solution should be thoroughly mixed before use. If the solid is not readily soluble in cold water it may be possible Lo dissolve it by stirring in warm water in a beaker. After allowing the solution to cool to room temperature. it can be transferred to the volumetric flask using a glass rod and filter funnel (Fig. 21.1) followed by several rinses of the glass rod/ftiter funnel. Finally, the solution is made up to the mark with distilk-d water.
Filling a burette For accurate readings - always remember to positton your burette vertically.
•
Clamp a ck'an 50.00mL burette (p. 10) in a laboratol)' stand. Place a beaker on a white tile immediately below the outlet of the burette (Fig.
21.2). •
• •
Place a small filtt-... funnel on top of the burette and, with the tap open. carefully pour in the standard solution (or titrant) until it starts to drain into the beaker. Gose the burette tap, and fill the burette with the standard solution until the meniscus is about 1-2cm above the zero mark. Remove the funnel. Open the tap and allow the solution to drain until the mcniscus falls to the zero mark. The burettc is then rC'ddy for the titration.
Note that to avoid contamination the solution in the beaker should be discarded, rather than rt-'Cyck'
TItrand - this is the soJution of unknown composition in the conical flask. The titrant is the stendard solution in the burette. Safety note Never mouth pipene.
Using a pipette A dean 25.00mL pipette (po JO) is required togcther with a suitable pipette filler. Various designs of pipette Iiller are available. The most common type is based on a rubber-bulb suction device. It is best to evaluate a range of pipeue mlers. if aVdilable in the laboratory, for ease of use and pt-...formance. The pipetting procedure is as follows: •
•
144
Classical techniques
Pour the solution of unknown composition (the titmnd) into a beaker. Never place the pipette in the volumetric nask containing the solution as this can lead to contamination of the solution from the external surface of the pipette. Using your pipette filler, drdw the titrand to just beyond the gntduation mark (Fig. 21.3a). Remove the pipelle filler. and invert the pipette to allow the solution to drain out. This ensures that the titmnd used subsl.>(juently will be undiluted and uncontaminated by any residue or liquids in the pipette.
Procedures in volumetric analysis
"", ""
,...,
,otIJ1 stand
-
~"-,-
if
~....:
rinse_
\
"\ f
a
~
~ ~
Fig.21.1 Quantitative transfer of a solid 10 a volumetric flask.
•
Fig. 21.2 Apparatus for 8 titration.
Refill the pipette until the meniscus of the titrand is above the graduation mark (Fig. 2I.3a). Remove the pipette filler and block the hole at the top of the pipette with the index finger of your right hand (if right-handed; Fig. 21.3b). Carefully raise the pipette to eye level, and allow the tilnmd to drain olll inlo a beaker by lifting your finger slightly from the top of the pipette. Continue until the bottom of the meniscus is on the graduation mark. Classical techniques
t45
Procedures in volumetric analysis
•
Wip: the outside of the pipette with a tissue (Fig. 2I.3c). Be careful not 10 touch the point of the pipette with the tissue otherwise solution will be
by capillary aClion. Allow the pipetle's contents to drain into a conical nask. Finally, touch the end of the pipette on the wall of the flask (Fig. 2I.Jd). :lnd rinse the inside of the neck of the flask with distilled water. This will ensure that exnctly 25.00mL of the test solution has been delivered by the pipette. 1051
• •
Note Ihat it is nonnal for n small (IUantity of solution to remain in the pipette tip. This volume is tnken into account when pipcltes are c:l!ibmted. so do not attempt to ;blow ouC this Liquid into the conical n:lsk.
Performing a titration Add one or two drops of indicator to the titmnd contained in the conic:!1 nask. For a right-handed person the process is as follows: 1.1
1'1
•
•
•
•
"1 Fig.21.3
I.
Using a pipene.
Washing the test solution - any distilled water used for washing the test solution from the walls of the conical flask has no effect on the titration or the calculation.
Volumetric analysis - calculating the concentration of a test substance The calculation should be carried out in a togical order as follows: I. 2.
Reading a burette - your eye-line should be level with the bottom of the meniscus. Then. record the volume used to one decimal place. Rough titration - always carry oul an initial rough titration to determine the approximate volume of litrant required for the end-point. This allows you to anlicipate the end-point in subsequent titrations to determine the accurate volume.
146
Classical techniques
Hold Ihe conical flask containing the litf'cwd and the indicator in your right hand, and control the tap of the burelle 'flith your lef! hand. The bureue should be arranged so Ihat the tap is on the opposite side of the burette to your palm. In this way, your len hand also suppom the body of the burette (Fig. 21.4). Add the titrant by opening Ihe tap. and simultaneously swirl the contents of the conical Oask. This may take a bit of pT'dCtice, so do not worry if you cannot do it stmight llway. The ,itraOl can be added quickly al first, but as the end-point approaches. additions should be made drop-wise. The end-point is indicated when the :lppropriate colour change takes place. When the end-point is reached, one drop of titr:mt should be sufficient to cause the colour change. You should note the volume used for the titration to the nenrest O.I-o.05mL. This is done by reading orr the volume of titrant used (Fig. 21.5). Refill the burette 10 the zero mark using a funnd ready for the next titralion.
Write the balanced equation for the reaction bet\vecn the standard and the test substance. From the stoichiometry of the reaction, detennine how many moles of the test substance react with I mole of the standard substance. For example, in the reaction behwen an H2SO4 standard solution and nn NaOH lest solution:
12I.2J 3.
Therefore 2 moles of NaOH react with I mole of H2S04 . Calculate the nwnbcr of moles of standllrd substance used to reach the end-point of the reaction. This can be detemtined from knowledge ofille concentration of the standard solulion (mol L-I) and the volume of tilrant used (mL). Rem('''ll1ber to take cnre with units - in this instance division by a factor of 1000 is required to convert mL to L:
Procedures in volumetric analysis
•
Ii
Fig.2J.5 Reading a burette. Place 8 white card b610w the level of the meniscus. This alloYJS an accurate reading to be made.
Fig.21.4 Performing a tltration.
For consistency of resuhs - alw8Ys perform two or three titrations (or until consistent resuhs are obtained, Le. titre values within 0.1-0.5 mL).
N 4.
S.
um
be
f Ics concentration (mol L -I) x volume (mL) ro mo = 1000
The number of moles of lest substancc present in the litnl.nd is then obtained from knowledge of the equivalences. In the example given above (point 2) the number of moles of test substance is twice the number of moles of standard substance. Therefore, if X moles of H 2 S04 are used (as calculated in point 3). 2X moles of NaOH were present in the initial volume of test solution. Finally, the concenlnllion of the lest solution C:ln be cdlculated using the fomlula: lOOOxamount of test substance (mol) Concentration of test solution (mol L -I) = initial volume of rCSl solution (mL) Again, the factor of 1000 is used to converr mL to L.
Classical techniques
147
-------0
Acid-base titrations The titrcHion of an acid solulion with a slandard solution of alkali will detennine the amount of alkali which is equivalent to the amount of acid presenl (or vice versa). The point al which this occurs is called the equivalence point or end-point For example, the titration of hydrochloric acid with sodium hydroxide can be expressed as follows: [22.11
safetv note Never pipene by mouth.
_...
Ifbolh the acid and alkali arc strong electrolytes, the rcsullam solulion will be nculml (pH 7). If on lhe other hand either (he acid or alkali is a weak electrolyte the resultant solution will be slightly alkaline or acidic, respectively. In either case, detection of the end-poinl requires accur:lte measureml>J11 of pH. This can be achieved L'ither by using an indicator dye, or by measuring the pH with a glass eleclrode (described in Chapler 7).
Acid-base indicators Typical acid~base indicators are organic dyes that change colour at or near Ihe equivalence or end-point. 'Iltcy have Ihe following characteristics: • •
,.
•
.....
l
(pH 1.1-8.91
They show pH-dependent colour changes. The colour change occurs whhin a fairly narrow pH range (approximately 2pH units). The pH at which a colour change occurs varies from one indicator to :mother, and it is possible 10 select an indicator which exhibits a distinct colour change at a pH close to the equivalence or end-poin!.
Selected common indicators together with their pH ranges and colour changes are shown in Table 22.1. Examples for thymol blue and phenolphthalein arc shown in Fig. 22.1. Table 22.1
Colour changes and pH range of selected indicators
Indicator
""
(pHB.3-101
Figure 22.1 Examples of indicators used in acid-base lilralians: thvmol blue and phenolphthalein
Thymol blue Methyl orange Methyl red Phenol red Phenolphthalein
pH "nge
Colour in acid solution
sokJtion
1.2-6.8 2.8-4.0 4.3-6.1 6.8-8.2 8.3-10.0
Rod R,d Roo
Yellow Yellow Yellow
Yellow Colourless
Colour in alkaline
Red
Pink/red
15,------------, -
"
",
-
_1·
•
• • •••
•
Neutralization curves .-
-
o"!-~_;:_-__c::____c~-c:! o 50 100 150 200 vtJlome of NeOtl added lmtl
Figure 22.2 A typical neutralization curve: 0.1
M Hel with 0.1 M NaOH.
148
Classical te<:hniques
A plot of pH againsl rhe volume of alkali addl-'d (mL) is known as a neutralizarion or titration curve (Fig. 22.2). The curve is generated by a 'polentiomelric titration' in which pH is measured after each addition of alkali (or acid). The significant feature of the curve is lhe vcry sharp and sudden change in pH near to the equivalence point of the titration. For a strong acid and alkali this will occur :It pH 7. If either the acid or base concentration is unknown, :l preliminary litr:ttion is necessary to find the approximate equivalence point followed by a more accurate titration as described on p. 146. The ideal pH range for an indicator is 4.5--9.5.
Acid-base titrations
14 , - - - - - - - - - - - - - - ,
Determination of the equivalence point From the neulrctlization curve (Fig. 22,2). the initial and final slopes are drawn (Fig. 22.3) and a parallel line is drawn such Ihat the mid-point is on the curve. This is the equivalence POilll, producing a titration value of xmL.
Example calculations
.--""j..... ~ J 2
-
o ~O---..----',OO~--"..---200~ \IDImle of NaOH aa:ted (mL)
Standardization of a sodium hydroxide ~iolution What is the molarity of a solution of sodium hydroxide, 25.0ml of which requires 21.0ml of a standard solution of hydrochloric acid of concentration 0.100 mol l-l for neutralization? FolloWing the sequence in Box 21.1: 1. Writc the equation:
Figure 22.3 Determination of the equivalence point.
NaOH(aq) 2.
Subscripts - 'sq' is used to represent aqueous, '1' the liquid state.
3.
+
HO(aq) --t NaC\lIQ)
+
H 20(l)
(22.2)
Determinc the cquiV'
Number of moles ofHCI
lOW
0.100 moiL-I x 21.0ml ~
1000
= 2.1 x 10-3 mol
4.
Calculate thc number of moles of NaOH in the initial volume of test solution. As indicnted in point 2 above. I mole of NaOH is equivalent to I mole of HCI. Therefore: no. of moles of NaOH in initial volume = no. of moles of HO used =
5.
2.1 x IO-)mol
Determine the concentr
woo x amount of test substance initial volume of tcst solution (mL) 1000 x 2.1 x IO-J(mol) 25 (mL) =O.084moIL- 1
Standardization ofa sodium hydroxide solution using potassium hytlrogen phthalate as a primarJ' standard (p. 143) An accumtely weighed amount (5.IIOOg) of potassium hydrogen phthalate (KHCI\H 40 4) was dissolved in distilled wafer (250.00mL). This solution (25.00ml) required sodium hydroxide solution (23.50mL) to reach equivalence. What is the molarity of the sodium hydroxide solution? (Note that, in this case. 25.00mL of Ihe standard solution was U$(:d as the tilr
149
Acid-base titrations
Following the sequt:IlCC in Box 21.1:
J.
Write the equation.
NaOH c....., + 2.
3.
KHC8H404(~q)
-.
NaKC8H40.$/~ql +
H2 0(I)
f22.3J
Determine the equi"Hlences.
Equlltion (22.3J shows fha! I mok of NaOH is equivalent to I mole of KHCHH.10... Calculate the num1x:r of moles of standard used. Firstly. the concentration of the standard solution of polllssium hydrogen phlhalnle must be cllculatcd. The molecular weight of KHCs H4 0 4 is 204.22gmo!-I. Therefore 5.1100g of KHCll H..0 4 is equivalent to 5.1100 (g)/204.22 (g mol-I) = 0.025 mol. We am now cnlculllte the concentmtion of the st:md:trd solution:
Concenlmtion of sland:ud
1000 x amount of =
solution (mol L 1)
KHC~H404 (mol)
volume of standard solution (mt) 1000 x 0.025 (mol)
250(m.L)
= O.IOOmolL- 1 The number of moles of Slandard used in the titration is as follows. Number of moles of KH4H404
conccnlration (mol L ~l) x volumc{mL) ~
1000 1
~
O.IOOmoIL- x 25.0mL I ()()()
= 2.5 4.
IO-J mol
Calculate the number of moles of NaOH used 10 reach equivalence. As indicated in point 2 :lbove. I mole of NaOH is equivalent to mole of KHCI\H 4 0 4 • Therefore:
No. of moles
or N aOH used
. = no. of moles of KHCRH 4 0 4 in the Iltnmd
= 2.5 5.
X
X
10- 1 mol
Dctennine the concentration of the NaOH solution: Concentmtion of NaOH solution (mol L -I)
1000 x amount of NaOH (mol) volume of NaOH solution used(mL) 1000 x 2.5 x 1O-1 (mo[) 23.50 (mL)
=O.I06moIL- 1
150
Classical techniques
---------10
Complexometric titrations Complexomclric litrations :lrc m:linly used 10 determine the concentration of cations in solution. The method is based on the competition between a metal ion (for example) and two ligands, one of which acts as an indicator and the other is a component of a standard solution. Some knowledge of the principles of metal-ligand binding is fL"quired in order to under-nand this method.
Types of ligand Ligands are chemical species that co-ordinate with metal ions to form a complex. They arc classified on the basis of the number of points of attachment to the central ion. • • Fig. 23.1 Structure of [Co(enhl3-l. II is a six-
co-ordinate octahedral complex of ethylenediamine (en) with cobalt till). The complex has three five-membered rings.
/C~-COOH
I-llOC-CHz, HOOC- ~ -"
N-CH;:-C~-N
"Clic eOOH
'0'
•
Monodentate ligand - here the ligand is bound to the central ion at only one point. e.g. H 20. NH). Bidenlalc ligand - this has two poinls of attachment to the central ion, e.g. ethylenediamine (en) (Fig. 23.1). Multidenlalc ligand - these have several points of nllachmcnt. e.g. ethylenediaminetetrct-acetic acid (EDTA), which is a hexadentate ligand (six points of attachment) (Fig. 23.2).
The basis of a complexometric titration involving fOTA The metal ion under investigation is bound to an indicator in solution (under strict pH control). This solution is then titrated against a standard solution of Em·1\. This c:m be l.'Xpressed in the form of an equation: Melal-indiC'
----t
metal-EDTA + indicator
[23.1]
For example. if the indicator being used was solochromc black. the metalindicator solulion would be red while the colour of the free indicator would be blue (in the pH r'
StabilitJ' of(·omplexe...
Fig.23.2 Structure of EDTA. (al EDTA contains two donor N atoms and four donor 0 atoms. It can therefore form a hexadentale complex (bj with a metal ion, e.g. Pt>l' .
The lhermodynamic stability of a species is an indiC'
+ L= ML
K,
~
[MLJ/[M][LJ
[23.2J
ML+ L = MLz
K,
~
[ML,]f[MLJ[LJ
[23.3J
M
Or, in general tcnns:
MLc"..I) + L
=
[23.4J
ML"
where K" K2 , .•• Kif are step-wise stability constants. Classical techniQues
151
Complexometric titrations
An alternative approach for expressing the equilibria mighl be as rollows; M + L= ML M+L2=M~
p, = IMLV[MULj p, = [ML,jJIMILj'
123.61
P. -IML"V1MIL1,
123.71
[23.5]
Or, in generaltenns; M+ L"=ML,,
where 11., Ill•...• 11" lire the O\'erall stability constants and are related step-wise stability constanfS as rollows:
10
the
123.81 Factors influencing the stability ofcomplexes The stability of a complex is rdatt.'d (0 the abilily or (he metal ion 10 complex with a given ligand. and to the characteristics or the ligand. End.points can be determinl"d more t:3sily when a single complex is rormed rather than when the complex is rormcd in a step-wise rashion. This can be achieved by using the IIminopolycarboxylic acid, EDTA (Fig. 23.2). In equations, EDTA can be expressed as H 4 Y. The disodium salt Na2H2 Y is rrequently u...oo a~ a source of the complex-forming ion, H2y 2-. Thus the Iypical reaction of EDTA with a metal ion enn be writtell in the following rorm: M 2+ Teble 23.1 Stability constants of selected metal-EDTA complelles (expressed as log K)'00 Mg~'
Ca~'
Fe~' Co~'
A1 3 • Cd~'
'og k
8.7 10.7
'Nion2,
'og k
Cu~'
18.8 21.9
18.6
~
14.3 16.3 16.3 16.6
23.1
Cr'
24.0 24.9
In3 •
-Ionic strength of solution was 0.1 at 20 C. Adapted from Vogel"s TexrbookofQuanlitBrive Inor(}8nic Analysis, 4th Edn, J. Basse". RC. Denney, G.H. Jeffery and J. Mendham, Longman
Scientific and TechniCal, Har1ow. (19781 p. 264. OH
OH
...""-t'-.-oo '0,
-~
Classical techniques
+ 2H'"
[23.9J
lbc reaction of a melal ion with EDTA is always in the n\tio I; I. The stability constants or selected metaI-EDTA complexes are given in Table 23.1. 1llc detection or lhe coo-point in tltrations involving EDTA is most cOllllnonly achieved using a metal-ion indicator, i.e. a compound that changes its colour when it complexes with a particular metal ion. The structures or seloooo metal-ion indicators are shown in Fig. 23.3 and the properties or a variety of metal-ion indicators are given in Table 23.2.
Types of complexometric titration
Direct titration In this case, the metal ion is titrated with a slandard solulion of EDTA. The solution containing the melal ion is buffered to an appropriate pH at which the stability constant or the mctal-EDTA complex is large. The rrcc indicator has:l different colour from that of the metal-indica lor complex.
Back titration
• • • •
152
_ My2-
In certain circumstances a partK:ular metal ion cannot be titrated directly. This includes situations where:
!eriodTtmllAct:; TI
Fig.23.3 Examples of metal-ion solochrome black and calmagil8.
+ H 2yl-
indicators:
The metal ion precipitates in the abscure of EDTA. The metal ion reacts too slowly with EDTA. "l1tc metal ion fonns an inert complex. No suitable indicator is available.
In these cases a back titration is required. This involves addition or a known excess of EDTA to the mt.'1:al ion (buffered to an :lppropriale pH). Then, the excess EDTA is titrated with a standard solution of a different metal ion. The choice or a second metal ion is important as il must not displace the analyle metal ion from ils EDTA complex.
Complexometric titrations
Table 23.2 Properties of selected indicators
Indicator Murexide < pH9 (H.ln-j pH9-11 (H;:Jnz-J > pH 11 (HvnJ-1 Solochrome ~ < pH5 (Hzln-) pH7-11 (Hln Z ) >pH11.5\1n J-j
Colour of free indicator
Colour of metal·ion complex
Red-violet Violet Blue
orange (Cu 2 'I, yellow (Ni 2 ' and Co 2 ') and red (Cahj
Red
In pH range 7-11 colour change is
Blue Orange
blue-red (Mg, Mn, In, Cd, Hg, Pb, Cu,
Calmagite pH 11.4 (tn J-)
Blue Red-orange
Pyrocatechol viok1t < pH 1.5 (H.lnj pH 2-6 (HJln I pH7 (H zln2-j >pH 10 fln' j
Yellow Violet Blue
Red
Red
AI. Fe,
n. Co, Ni and Pt metalsl
Same colour change as solochrome blad< bu! clearer and sharper
In pH range 2-6, yellow to blue (Bi and Thl; pH 7 violet to blue (Cu 2.., ln 2 ', Cd h , Ni z , and Coh )
Practical considerations • •
• •
For a metal-ion indicator to be useful it must be less stable than the corresponding metal-EDT A compklx.
•
pH adjus!ment is crilical in EDTA lilr:nions. The pH is monilorcd with a pH meter or pH test paper. The metal ion under investigation should ideally be approxima~ly 0.25 roM in a volume of 5tJ.-.-150mL of solulion. Dilution of Ihe melal ion may be m..'Cessary 10 avoid end-point deu.:ction problems. Do not add excess indicator. as too intense a colour can lead to problems. e.g. masking of !he colour change. It is somelimcs difflCull to delcel Ihe end-point because Ihe colour change can be slow to develop. Slirring is t\.'Commended 10 assisl colour ll"dnsformation. The use of meTal-ion indicators 10 indicale lhe end-poinl of complexomclric titralions is based on a specific colour ch:mge. Some individuals may find it difficult to delt.'ct a particular colour change (e.g. Ihose with colour blindnes.<;). Alternalive approaches for end-poin! detection arc aVdilable based on a colorimelcrjspeclropholomctcr (devices ror measuring colour, see Chapler 26) or ekctrochemical detection (sec Chapter 34).
Example calculation A solution or Ni 2+ (25.00mL) was titmted wilh 0.1036moIL- 1 EDTA al pH 5 and r'-"<juircd 20.25mL for the melal-indicalor 10 change colour. What is !he concentmtion (gL -I) of Ni2~? The atomic weight or nickel is 58.71 glllol-i. I.
Write the balancl-d equation ror the reaclion belwecn the standard and the test substance
[23.IOJ 2.
Dcleonine the equivalences of the reacting species: I moleorEDTA iSe
153
Complexometric titrations
3.
Calculate the number of moles of stllndard substance (EDTA) used to reach the end-point of the reaction: moles of EDTA
volume (L) x molarity (mol L-I) = 20.25 x lO-3 L x 0.1036mol L- 1
=
= 2.098 )( 10- 3 mol
4.
Calculate the corresponding number of moles of NiH prescnt in the 25mL of nickel solution. Since I mole of EDTA is equivcllent to 1 mole of Ni 2 +; moles of Ni 2+ = 2.098 x lO-3 mol
5.
Calculate the concentration of the Ni 2 + solution: concentnuion of Ni 2 + solution (mol L -I)
1000 x 2.098
X
10- 3 (mol)
25 (mL) = 83.92 )( 10-3 mol L- 1
In this instance, the Ni 2 -f- concentration is fe(Juired in gL -I. The molecular weight of nickel is 58.71 gmol- l : concentration of mok.'CUlar weight (gmol- l ) 2 Ni + solution (g L-I) = x molarity (mol L-I) = 58.71 x 83.92 x 10- 3 = 4.927gL- 1
154
Classical techniques
----------18
Redox titrations All redUClion-oxidation reactions involve a tmnsrer of electrons. The oxidizing agenl accepts etecuons. and the reducing agent donates electrons. To establish the reaction for a redox tilmlion it is n~cessary 10 determine lhe 'half-equ3tion' for both Ihe oxidizing IIgenl and the reducing agent. By adding the two 'half-cquations' it is possibk 10 determine the ovcmlJ equation for lhe lilmtion. The basic theory of electrochemistry is dt..'SCribcd in Chapter 34. One of the mosl common oxidams is pOiassium pennanganalc wruch in acidic solulion can undergo the following reaction:
[24·11 UnforlUnatcly. potassium pennanganatc is not obtainable in high enough purily and can undergo decomposition by eXpOsure 10 sunlight. Therefore it cannot be used as a primary standard (p. 143). However, it can be used in redox tit rations provided it is standardized with sodium oxalate (which is available in high purity). The redox reaction involving oxalate is as follows:
124.2] The ovcrall reaction between pc:rmang-dnale and oxalatc can be obtained by
balancing the electrons on each side of the equation. This can be achieved by multiplying eqn [24.1] by 2 and eqn {24.21 by 5, and then combining them as follows:
124.3] Another common method for the stand:lrdization of potassium pcnnanganate is to use iron (Il): Fe21 = FeJ+
+ e-
[24.4]
The combined equation is obtained by multiplying eqn 124.41 by 5 and adding to eqn (24.1):
124.5] Potassium pennanganatc has a major ad\~dntage when used for titrations in that it can act as its own indic:ltor. A list of other common oxidizing agcnls and reducing agents is given in Table 24.1. Table 24. f
Common oxidizing and reducing agents used in redox titrations
Oxidizing agents
Reducing agents
"'OlFe"
Arsenite Ferrous
Iodate
NHzOH Sn 21
Hydro"Vtamine Stannous
Permanganate
s,Oj
Thiosulphate
Cerie Dichromate Hydrogen peroltide
Ctassical techniques
155
Redox titrations
Example calculation Standardization ofpotU!i.\lum pe"manganate with a primary standard, sodium oxalate An aa:urately weighed portion of sodium oxalate (O.1550g) was dissolved in dilute sulphuric acid (250 ml). WhiL<;t maintaining the temperalure of the solution above 70°C. it was titrated to equivalence with JXltassium permanganate solution (18.5ml). What is the molarity of JXltassium permanganate? I.
Write the balanced equation for the reactioll between the standard and the test substance (using the two half-equations (24.1) and 124.2J): 2MnOi + 16H+ + 5Cl~- = 2Mn 2 ~ + IOC0 2 + 8H 10 (24.3}
2.
Determine the equivalences of tile reacting species: 2 molcs of MnO; arc equivalent to 5 moles ofC2~-.
3.
Calculate the number of moles of standard SUbslRl1Ce (sodium oxalate) used to reach the end-JXlint of the reaction. The molecular weight of sodium oxalate is l34gmol- 1. Therefore 0.1550 g of sodium oxalate is equivalent to:
~O."15,,5-,O-,,(g,=),, = 1.157 x 10- 3 mol 134 (gmol I)
4.
Calculate the corresJXlnding number of moles of JXltassium perman· ganate present in the volume of titrant added. From the equation: 5 moles of Na 2C 20 4
..,
2 moles of KMn04
Therefore, moles of KMn04 used in the tit....dtion = ~ x 1.157 x 10- 3mol = 4.628 x 10-4 mol 5.
Calculate the concentration of Ihe KMn04 solution: Concentration of KMn04 solution (moiL-I)
1000 x amount of KMn04 (mol) Volume of KMn04 solution used(mL) ~
1000 x 4.628 X 10--4 (mol) 18.5(ml)
= 0.025 mol L- 1
156
Classical techniques
--------10 Definition Argentimetric is derived from the Latin argentum, which means silver.
DefinitJon
Precipitation titrations Precipitation is the lem u<;e
To be exact.. the pfunctlon should be
defined in terms Of l'K:tivities instead of concentrations.
Titration curves - plots of concentration of titrand against volume of titrant used.
pX = -laglO
[Xl
(25.1 J
For example. in the titr.uion of IOOmL of 0.1 mol L-I NaG with 0.1 mol L- 1 AgNO) the initial concentration of ICI-) is 0.1 mol L-I, so by using eqn 125.1) the p function is 1 or pCl- = I. When 25mL of 0.1 moiL-I AgNO) has been added, 75ml of Naa remain.<; in a tOlal volume of 125 mL Therefore. the t;oncentration of the chloride ion is gi\'eIl by [CI ]=75mLxO.lM=6xI0 125mL
Table 25. J Titration of 100 ml of 0.' M NaCl with 0.1 M AgN~ (Note that 1<", for AgCI,..1.1xlO 10)
pCl-
pAg
0 25 50 90 95 98 99 99.6 99.8 99.9 100 100.1 100.2 100.5 101 102 105 110 120
0.1 '.2 1.5 2.3 2.6 3.0 3.3 3.6 '.0 '.3 5.0 6.7 6.0 6.' 6.7 7.0 7.' 7.7 8.0 8.' 8.2 8.3
0.0 8.7 8.5 7.7 7.' 7.0 6.7 6.' 6.0 5.7 6.0 '.3 '.0 3.6 3.3 3.0 2.6 2.3 2.0 1.9 1.8 1.7
140 150
and pCl- = 1.22. (Note that the solubility product. 1.1 x 10 10, see p. 50.) Therefore:
K~.
of Ab<:1 is
0'
Adapted from Vogel's Tex/book of Quantitative Inorganic Analysis, 4th Edn, J. Bassen. R.e. Dennev. G.H. Jeffery and J. Mendham, Longman Scientific and Technical,
Harlow 11978J. p. 200.
(25.2J
(25.3J
0.' M AgN~ tml)
'30
2 mo ll-1
pAg+
+ pCr =
(25.4J
9.96 = pAgCl
It was found above that pCl- = 1.22, hence pAg+ = 9.96 - 1.22 = 8.74. In a similar manner, the pAg+ values can be c.1.lculated. At the cqui,'alence point [Ag+J = [0-]. Therefore:
IAg+]=[Cn=,,[K;=VI.lxIO 10_I.05xlO-5
(25.5]
pAg+ = -)0g(1.05 x 10-5 ) = 4.98
[25.6J
Beyond the equivdlence point the solution:
~ituation
changes. For IOO.lml AgNOl
O.lmlxO.lM_ 0-' 1-' (A g'I_ 200.lmL - 5 xl mol
(25.7J
or pAg+ = 4.3. Therefore, pCI-
=
pAgCl - pAg+ - 9.96 - 4.3 = 5.66
Values calculated in this way up to the addition of 150mL of 0.1 MAgNO) are given in Table 25.1 and the titration curve in fig. 25.1. Classical techniques
157
Precipitation titrations
~poirl,pAg+ ..
10.0
~!
'.0
•:t '.0 • '.0
r
,
0
.,
'00
End-point determination Three techniques art commonly used !o detennine lhe end-point in precipitation tilTations. They are:
-
2.0
.,
!i
-- ,.,
o..lM~{nt)
Fig. 25. 1 Precipit~tion titration curve. Initial and final slopes are drawn (see Fig. 22.31 and a paratlelline Is drawn such that the mid·point ts on the curve. This is the equivalence point.
I.
2. 3.
potentiometric methods; chemical indicator methods; light-scatterring methods. exemplified by turbidimelry or nephelomelry.
Only indicator methods will be discussed rurther. Three types or indicator methods can be applied to determine the end-point or an Ag+ and a CItitration. These are: I.
In acid media (pH < 6), the concentration of C~- is lowered by the following reaction: Cr~- + 2H+ 2HCrOi Cr2~- ... H20. In alkaline media (pH >10), Ag(OHll~ may precipitate.
Mohr titrdtion. which involves the formation or a coloured precipitate by rCl:lction with the indicator. For example. in the determination or chloride concentration with silver nitrate 11 small amount or polassium chromate !>olution is added as an indicator. This results in the ronnation or a red silver chromate (Ag 2Cr04 ) precipitate at the end-point;
2Ag" f- C~ ..... Ag2 Cr04(,)
[25.8)
(red)
2.
In all argentimetric titrations strong light (including daylight) should be avoided as it can lead to decomposition of the silver salts.
In this c~. the precipitate may rorm slightly aner the end-point. but this error can usually be neglected. Also. the tilration should be done in neutral or slighly alkaline solution (pH 6.5-9) otherwise silver chromate might not be rormed. Volhard titration. which involves the formalion or a soluble coloured compound. lllis approach is exemplified by lhe quantitative analysis or chlorides. bromides and iodidt:s by hack titration. In this case. the halide is titrated wilh silver:
125.91 Excess silver ions are then titrdted with standard potas."ium thiocyanate solution in the presence of an iron (III) salt Ag+
+ SCN-
..... AgSCN«,
When all the Ag+- has been reacted. the SCN a red complex. indiC'"dting the end-point: Fe'''
+scw ..... FeSCNH
125.10] rencts with Fe"" to rorm
[25.11]
(red)
:,
:~o o Fig. 25.2
158
CO;
Structure of dichlorofluorescein.
Classical techniques
3.
A problem with the detcrmination of chloride by this approach is that the end-point coloration slowly rades. as AgCl is more soluble than AgSCN. As a consequence the AgCI slowly dissolves to be rep11.lced by the FeSC~+. Two approaches are possible to prevent this secondary reaction rrom taking p11.lce. The most common method is to filter off the AgCl and tilrdte only the Ag+ left in solution. Alternatively. add a rew millililrcs or an imrnisc.ible liquid (e.g. nitrobenzene) to the titrand prior to the back titration. The nitrobenzene acts to 'coat' the AgCI precipitate. thereby isolating it from the SCN-. Fajans titrntion. which involves the adsorption of a coloured indicator onto the precipitate at the end-point, resulting in a colour change. During this adsorption process a change occurs in Ihe indicator resuhing in a change of colour. TIle indicators used for this are often anionic dyes.
Precipitation titrations
Table 25.2 Selected applications of precipitation titralions
Analyt_
Commonts
CI • Br
Mohr method: Ag~r04 used as end-JXIint Volhard method: precipitate remOVal is unnecessary Vol hard method: precipitate removal is required Fajans methOd: titration with Ag'. Detection lNith fluorescein. dichloroftuorescein and eosin Titration with ThIN~4 to prOduce ThF 4 • End-point detection with alizarin red 5
CI-.CN •
COj
CI-. Br-. 1-. SCN F
e.g. fluorescein or eosin. The most common indic:a!Or rOf AgO is dichlorofluorcsccin (Fig. 25.2) (this is greenish yellow in solution but changes colour to pink when it is adsorbed on AgCl). Selected exumples or precipitation titmtions are shown in Table 25.2.
.Adapted from; Quanlitafive chemica/analysis. 4th Edn, D.C. Harris. W.H. Freeman, New York 11995), p. 176.
Classical techniques
159
Resources
Resources for classical techniques Geneml hoob Main supplementary text:
Mendham, J.• Denney, R.C., B
Betmell, S,W. and O'Neale, K. (1999) Progress;\'/! del'dopmenr of practical skills in r.hemiflry. A guide to early wulergraduott! experimental Y1'{)rk. Royal
Society of Chemi.<;try, Camhridge. Crawford, K. and Healon, A. (1999) Problem So/l'ing in Aru,lytical Chemistry, Royal Society afChemistl)', Cambridge. Day R.A. and Underwood, A.L (1991) Q,wntitatil'c Analysis. 6th Edn, Prentice Hall, Harlow, Essex.
Rubinson. J.F. and Rubinson, K.A. (1998) Comcmporary Chemical Analysis, Prentice Hall, Harlow, Essex.
Skoog, D.A.. West. n.M. and Holler, FJ. (19%) FWldomlmtals of Analytical Chemistry. 7th Edn. Saunders College Publishing, Orlando. Ronda.
Specific boob on qltalitatb'e analysis Main supple'llentary text: Svehla, G. (1989) Vogel's Qualiw/il"l' Inorganic Alloly,vis. Longman. Harlow. Essex. Other m;erul sources (chronoiogical order): Whitten. K.W., Davis, R.E. and Peck. M,L (2000) General CllPmistry with Qualiw/irl' Analysi.\', 6th Edn. Saunders College Publishing. Orlando. Florida. Hardcastle. W.A. QUlJlitatil'e Analysis. A guide to best praetit:e, Royal Society or Chemistry, Cambridge.
Videos Basic Labol"dtory Skills. LGC. Royal Society or Chemislry, Qlmbridge (1998), Further Laboratory Skills LGC. Royal Society of Chemistry, Cambridge (1998).
CD-ROMs Chemistry Video Consortium, Practical Laboratory Chemistry. Educalional Media Film and Video LId, Harrow, Essex, UK - Inorganic analysis (gravimetric analysis). Chemistry Video Consonium, Praclical Laboralory Chemistry, Educational Media Film and Video Ltd. Harrow, Essex. UK - Volumetric techniques (using a balance, using a pipette, using a burette and making-up solutions). Chemistry Video Consortium, Practical Laboratory Chemistry, Educational Media Film and Video Ltd. Harrow, Essex. UK - Volumetric analyses methods (doing a litration, some common end-points and potentiometric titralions).
160
Classical techniques
---0 Spectroscopic techniques can be used to: •
•
Identify compounds tentstivety. by determining ttieir absOfption or emission spectra; quantify substances. either singlV or in the presence of other compounds. by moasurlllQ. the 9ignal strength at en appropriate w8Wt1ength;
• determine molocular structure; •
follow reactions, by measuring the disapPearance of B substance. Of" the
appearance of a product as
8
function
qf time.
,0'
.. ",.., , '00
--
,"
--.....
,Ii'
l -1 ..... "'
.... .-... ..",-
,0'
.. •
, f
~
..
.....
,0' ,
0"
"" ,0·>1
"""""" ,D" Fig. 26.1 The electromagnetic spectrum.
Light is most strK.1Jy defined as that pan of the spectrum of electromtlgnctic radialion detected by the human eye. However, the term is al~o Hpplied to rddialion just outside that visible range, e.g. ultraviolet (UV) and infrmed (lR) 'light', Electromagnetic radiation is cmined by the sun and by other sources (e.g. an incandescent lamp) and the electromagnetic spectrum is a broad band of radiation, ranging from cosmic rays to radio waves (Fig. 26.1). Most chemical experimenlS involve measun:menlS within the UV, visible and IR regions (generally, within the wavclt.:ngth ranJ,'e 200-IOOOnm). Rl:ldiation has the char.:lClcristics of a particle and of a vibrating W(lve, travelling in discrete particulate ulliL~. or 'packets'. termed photons. A quantum is the amount of energy contained within a single photon (it is important not to confuse these two tenns., although they ~lre sometimes used interchangeably in I.hc literature). In some circumstances. it is appropriate 10 measure light in tenns of the /lumber of photons. usually expressed directly in moles (6.02 x Ion photons = I mol). Alternatively, the energy content (power) may be measured (e.g. in Wm- 2). Radiation also behaves as a vibrating electrical and magnetic field moving in a particular direction, with the: magnetic and ekctrical compont:l1ls vibrdting perpendicular to one anotht.T and perp(:ndicull:lr to the direction of trd\'el. The wave nature of radiation gi\'es rise to the concepts of wavelength (J.. usually measured in nm), frequency (l', measured in S-I, but often recorded in hert,.. Hz). speed (c, the speed of electromagnetic radiation. which is 3 x l()llms- I in a vacuum), and direction. In other words., radiation i~ a vector quantity, where:
[26.IJ
c=J."
, l
.--
3
,0'
Basic spectroscopy
Sometimes, it is necessary to fe'
[26.21
1'=-
J.
Introduction to spectroscopy The absorption and emission of electromagnelic radialion of specific energy (wavelength) is a characterislic feature of many molecules, involving the movement of electrons between different energy states, in accordance with the laws of quantum mechanics. Electrons in atoms or molecules are distributed at various energy levels, but arc mainly at the lowest energy level, usually tenned the ground state. When exposed to energy (e.g. from electromagnetic radiation). electrons may be excited to higher energy levels (cxt.ited slates). with the associatro absorption of energy at specific wavelengths giving risc to an absorption spectrum. One quantum of energy is absorbed for a single electron tmnsition from the ground state to an excited state. On the other hand, wlx:n an electron returns to its ground state, one quantum of energy is relcaSl..'d: this may be dissipated to the surrounding molecules (as heat) or may give rise to an emission spectrum. The energy change (6.£) ror an electron moving between two energy states, £. and £2, is given by the equation:
[26.3J where 11 is the Planck constant (p. n) and ~ is the frequency of the electromagnetic mdiation (expressed in Hz or S-I). By substituting for Instrumental techniques
163
Basic spectroscopy
frequency in 126.3] it is possible expression;
(0
rearmnge this equation to give the
t::.E = he
A
126.4]
UV Ivisible spectrophotometry This is a widely used technique for measuring the absorption of radiation in the visible and UV regions of the spectrum. A spectrophotometer is an instrument designed to allow precise measurement at a particular wavelength, while a colorimeter is a simpler instrument, using filters to measure broader wavebands (e.g. light in the green, red or blue regions of the visible spectrum).
Principles of light absorption Two fundamental principlcs govern thc absorption of light passing through a solution: I. The absorption of light is exponcntially related to the number of molecules of the absorbing solute that are encountered, i.e. the solute concclltration [C]. 2. The absorption of light is exponentially related to the length of the light path through the absorbing solution, I. These t'""O principles are combined in the Beer-lambert relationship, which is uswdly expressed in tenns of the intensity of the incident light (10) and the emergent light (I):
10glO(~) =£[(11 Definition
where £ is a constant for the absorbing substance at the wavelength the measurement is made and is termed the absorption coefficient or absorptivity, [(1 is expressed as either mol L-lor g L-I (see p. 45) and I is given ill em. This relationship is extremely m;eful, since most spectrophotometers are constructed to give a direct measurement of 10glO(lo/l), tenned the absorbance (A), or extinction (E), of a solution (older texts may m;e the outdated term optical density). Note that for substances obeying the Beer-Lambert relationship. A is linearly related to [C]. Absorbance at a particular wavelength is often shown as a subscript. e.g. A550 represents the absorbance al 550nm. The proportion of light passing through the solution is known as the transmittance (T), and is calculated as the ratio of the emergent and incident light intensities. Some instruments have two scales:
Absorbance (A) - this is given by: A = log10(~/1)
Definition Transmittance (T) - this is usually expressed as a percentage. where: T = (l/~J )( 100(%)
I. 2.
"""" '"
!~D'tU-:[1~) liOurce
em
test slit sokfion
Fig. 26.2 Components of a UV/visible spectrophotometer.
164
Instrumental techniques
[26.5)
leadwt
An eXlxmential scale from zero to infinity. measuring absorbance. A linear scale from 0 to 100. measuring (per cent) tmnsmiuance.
For most practical purposes, the Beer-Lambert relationship will apply and you should use thc absorbance scale.
VII/visible spectrophotometer The principal componcnts of a UVjvlsible spectrophotometer are shown III Fig. 26.2, High-intensity tungsten bulbs are used as the light source in basic instruments, capable of operaling in the visible region (i.e. 400-700nm). Deuterium lamps are used for UV spet.trophotometry (200-400 run); these
Basic spectroscopy
All spectroscopic equipment is costly and equipment must be used under guidance from a demonstrator or technician. All equipment in this section has an inherent hazard due to its use of mains electricity.
Using p'astic disposable cells - these 8(8 adequate far WOfk In the ne8r~UV region.
e.g. fO( Job's plot stucfies, as welt as the vi8ible range. Handling cella - Never handle the ceUs by non-poIish~ sides.
the
Ex.-nples The molsr absorptivity of phenol is 6.20 x 1@lmol- 1 em- 1 at 210nm. For a test solution giving an absorbance 610.21 in 8 cell withe light peth of 5mm, ysing IlQn 126.5) this is equal to II roncentrntlon of:
0.21 _ 6.20 x 'OS >( 0.5 x ICI ICJ = 0.0000677 mol L-J lor f51.7/lmol L-I)
Safety note Never balance full cells on the instrument. Measuring absorbances in colorimetril;: analysis - if any final solution has an absorbance that is too high to be read with accuracy on your spcctTophotomet& (i.e. A > 2), it is bad practice.to dilute the solution so that It can be measu(8d. This dilutes both the sample molecuSes and the cok>ur reagents ttl an equal extent. Instead, you should dikrte lhe ori9i~ sample and re-assav.
lamps lire fitted with quam~ envelopes, since g1l'ls,'l docs not transmit UV mdiation. TIle spectrophotometer is a mlijor improvl,:ment over the simple <.'Olorimctcr since it US(:S a difTrHetion grdting to produce a pamllcl beam of monochromatic light from the (polychromatic) light sauro::. In practice the light emerging from such a monochromator is not of a single wavelength. but is a narrow band of wavelengths. This bandwidth is an important <,·hant<.tcristic, since it determines the wavelengths used in absorption measurements - the bandwidth of basic spectrophotometers is around 5-lOnm while research instruments have bandwidths of less trum I nm. &ndwidth is aITe<.tcd by the width of the exit slit (Ihe slit width), since the bandwidth will be reduced by d~Teasing the git \l,~dth. To obwin accurate data at a parlicular wavelength sclling, the narrowcst possible slit width should be used. However, decrt'asing the slit width also reduccs the amount of light readling the dcte<.1.or. decreasing the si~JTUil-to-noise rMio. The CxlenL to which Ihe slit width can be reduced depends upon the sensitivity and stabilily of the detection/ltlTIplification system and the presen<.'C of str.ty light. Most UVjvisible spectrophotometers are designed 10 take ceUs (cuvettes) with an oplical path length of IOmm. Disposable plastic cells are suitable for rouline work in tlte visible range using aqueous and alcohol-based solvcnts, while glass cells must be USt"d for most other organic solvents. Glass cells are manufactured to more exacting sl3ndards, SO you should usc optically matched glass cells for accurate work. espa::ially at low absorbances « 0.1), where any differences in the optical properties of cells for reference and test samples will be pronounred. Glass and plastic absorb UV light, so quartz cells musl be used al wavelengths below 300 nm. Before taking a rnc-.tsuremenl, make sure that cells arc dean, unscrlHched, dry on the outside, filled to the correct level anti in the correel position in their sample holders. Unwanted material can accumulate on the inside faces of glass/quartz cells, so remove any deposits using acetone on a colton bud, Of" soak overnight in I mol L -I nitric acid. Corrosive Hnd ha7..ardous solution... must be used in cells with tightly fining lids or Tcflon,ji, stoppers, to prevent damage to the instrument and to reduce the risk of accidental spillage. Basic instruments use photocells similar to those used in simple colorimeters or phOlodiode detectors. In many C'.tSCS. a dinerent photocell must be used at wavelengths abovc and below 55O--600nm, owing to differences in the sensitivity of such detectors over the visible waveband. TIle detectors used in more sophisticated instruments, give increased sensitivity and stability when compared with photocells. Digital displays are increasingly usa! in preference to needle-type meters, as they arc not prone to parallax errors anti misreading of the absorbance scale. Some digital instruments can be C'.tlibrated to give a direct readout of the concentration of the test substance.
Types of UYjJ'isihle spectrophotometer Basic instruments are single-beam specuophotometers in which there is only one light path. The instrument is set to zero absorbance using a blank solution, which is then replaced by the tesl solution. to obtain an absorbance reading. An allernative approach is used in double-beam spectrophotometers, where the light beam from the monochromator is split into two separate beams. one beam passing through the tesl solution and the other through a reference blank. Absorbance is then me.tsured by an electronic circuit which compares the outputs from the reference (blank) and sample cells. DoubleInstrumental techniques
165
Basic spectroscopy
Plotting calibration curves in quantitative analysis - do not force your calibration line to pass through lero if clearly it does not. There is no reason to assume that the zero value is any more accurate than any other reading you have made.
beam spectrophotomctry reduces measuremcnt crrors caused by fluctuations in output from the light source or changes in the sensitivity of the detection system. since reference and lest solutions are measured at the same timc (Box 26.1). Recording spc<:trophotometcrs are doublc-beam instruments, designed for usc with a chart recorder or computer. either by recording the dilTt.'ft.'I1ce in absorbrmtt bctwCt.'I1 reft.'fCnce and tcst solutions across a predetermined wavcband to give an absorption spectrum. or by recording thc change in absorbance at a particular wavclcnb>th as a function of time (e.g. in a kinctic determination.
Qllunt;tatil'e spec:trophotometrt'c: anu/J'~'i~' A single (purified) substance in solution can be quantified using the Becrlambert relationship (eqn [26.5]). provided its absorptivity is known at a particular wavelength (usually at the absorption maximum for the substance. since this will give the greatest sensitivity). The molar absorptivity is the absorbance given by a solution with a concentration of I mol L- 1
Box 26.1
Using a UVIvisible spectrophotometer
1. Switch on and select the correct lamp for your measurements (e.g. deuterium for UV, tungsten for visible right). 2. Allow up to 15min for the lamp to waml up and for the instrument to stabilize before use. 3. Select the appropriate wavelength: on older instruments a dial is used to adjust the monochromator, while newer machines have microprocessor-eontrolled wavelength selection. 4. Select the appropriate detector. some instruments choose the correct detector automatically (on the basis of the specified wavelength), while others have manual selection. 5. Choose the correct slit width (if available): this may be specified in the protocol you are following, or may be chosen On the manufacturer's recommendations. 6. Insert appropriate reference blankls): single-beam instruments use a single cell, while double-beam instruments use two cells (a matched pair for accurate work), The reference blank should match the test solution in all respects apart from the substance under test, i.e. they should contain all reagents apart from this substance. Make sure that the cells are positioned correctly, with their polished (transparent) faces in the light path, and that they are accurately located in the cell holder(s). 7. Check/adjust the 0% transmittance: most instruments have a control which allows you to zero the detector output in the absence of any light (termed dark current correction). Some micro-processorcontrolled instruments carry out this slep automatically.
166
Instrumental techniques
8. Set the absorbance reading to zero: usually via a dial, or digital readout. 9. Analyse your samples: replace the appropriate reference blank with a test sample, allow the absorbance reading to stabilize (5-10s) and read the absorbance value from the meter/readout device. For absorbance readings greater than one (i.e. < 10% transmission), the signal-to-noise ratio is too low for accurate results. Your analysis may require a calibration curve or you may be able to use the Beer-lambert relationship (eqn 126.5]) to determine the concentration of test substance in your samples. 10. Ctleck the scale zero at regUlar intervals using a reference blank, e.g. after every 10 samples. 1'. Check the reproducibility of the instrument: measure the absorbance of a single solution several times during your analysis. It should give the same value.
Problems (and solutions): Inaccurate/unstable readings are most often due to incorrect use of cells, e.g. dirt, fingerprints or test solution on outside of cell (wipe the polished faces using a soft tissue before insertion into the cell holder), condensation (if cold solutions are not allowed 10 reach room temperature before use), air bubbles (which scatter light and increase the absorbance; tap gently to remove), insufficient solution (causing refraction of light at the meniscus), particulate material in the solution (check for 'c1oudiness' in Ihe solution and centrifuge before use, where necessary) or incorrect positioning in light path (locate in correct position).
Basic spectroscopy
(= I kmolm- 3) of the compound in 11 liglu path of I <'1n. The approprime value may Ix: available from tabulaled 5pcctrnl data (e.g. A110n., 1%3), or if am be dClcnnincd experimentally by measuring the absorb:mce of known cOIlCl.'fIlmlions of the sub5tance (Box 26.1) and plotting a standard curve.
This should confirm thut the relationship is linear O\'cr the desired OOIlccnlmlion range and the slope of the line will give the molar absorptivity.
Fluorescence
piBi
I ~
f 1 B • •• ~
l
i
•• ....,..,
(
£
~ ~
. ()
s
ii
~
.I ) )
Fluorescence spectrophotometry
Fig. 26.3 Energy levels and energy transitions in fluorescence.
O .... 1Y'\
With most molecules. aftcr electrons arc raised 10 II higher energy Ic"e1 by absorption of cJccuomagnctic radiation, they soon fall back to thc ground state by radiationless transfer of cncrgy (hem) to lhc solvclIl. Howcver, with somc molcculC!\, the C\'cnts shown in Fig.. 26.3 may occur, i.c. electrons mny lose only plm of their energy by non-mdillnt roules llnd the rcst may be emitted as electromagnetic radi,uioll, a phenomcnon known as fluorcsocncc. Since not all of the encrgy that WIIS absorbed is cmilled (due 10 non-radiant loss), thc wlwelcngth of the fluorescent light is longer limn Ihc absorbed light (longer wavelcngth = lo\\cr energy). Thus, a fluon::sccnt molecule hns both an absorption spectrum and an cmission spectrum.
~
....
exatalion rnooochOlTlillor
\) ........
!test iOklIiDnl
}:f
The principal componcnts of a fluorescence spectrophotometcr (fluorimctcr) are shown in Fig. 26.4. Thc inslrumcnt cOnlains two monochromators. onc to select the cxcitation wavelcngth and lhe olhcr 10 monilor thc light cmitlcd, uSWllly at 90" to the incidcnt beam (though lighl is uctually cmillcd in all directions). As an cxamplc. thc wavclcngths used to melsurc thc highly fluorescent compound naphthalenc arc 270nm (cxcitation) and 340nm (emission). Somc examples of molecules with intrinsic fluorescence arc given in Tllblc 26.1. Table 26.1 Examples of compounds with intrinsic fluorescence
Vrtamins
Aspirin, morphine, barbiturates, propanalol, ampicillin, telracyclines Riboflavin, vilamins A. 66 and E, nicotinamide
Pollutants
Naphthalene, anthracene,
Drugs
Comp.'lred with UVJ"jsiblc spectrophotometry, fluorescence speclroscopy has certain lldvantages. including: •
d.~
•
@]ill]
.-,
Fig.26.4 Components of a fluorimeter (fluorescence spectrophctometer). Note that sample cells for fluorimetry must have clear
ben~opyrene
Enhanced sensitivity (up to IOOO-foldl, since thc cmitted light is dctccted llgainst a background of zcro, ill conlmsl to spcctrophotornctry whcre small changes in signal are lTlC'otsured againsl a larl!:c 'backgmund' (sec cqn 126.5]). Increased specificity, because nol onc. bUl two. specific wavelcngths are required for a particular compound.
Howcver, there arc also certain dmwbac\.:s: •
sides all round.
•
Not aU compounds show intrinsic nuorcsa:nce, limiting ils application. Howevcr, somc non-fluorescent compounds may be coupled to f1uorcscent dyes, or fluorophorcs (c.g. alcohol clhoxylalcs may be coupled to naphthoyl chloride). Thc tight emitted can be less than CXpL"Cled owing 10 qucnching, i.e. when substances in the sample (e.g. oxygen) either intcrfere with encrgy Iffinsfcr, or absorb the emitted light (in some instances. the samplc molecules may sclf-quench if they are present at high concentration). Instrumental techniques
167
Basic spectroscopy
TIle sensitivity of fluorescence has made it invaluable in techniques in which specific chemicals, e.g. polycyclic aromatic hydrOClrbons and akohol ethoxylates, are linked to a Ouoresa:nt dye for detection in high-performance liquid chromatography (p, 218).
Phosphorescence and luminescence A phenomenon rdaled to fluorescence is phosphorescence. which is thc emission of light following intcf!>)'stcm crossing between electron orbita.ls (e.g, between excited singlet and triplet slates). Light emission in phosphorescence usually continues after the exciting energy is no longer applied and, since morc energy is lost in intersystem crossing, the emission wavclenb'lhs arc b'Cnerally 10nb'Cr than with fluorescem;e. Phosphorescence has limited applications in chemical sciences, Luminescence (or chl.'ITlilumincscence) is another phenomenon in which light is emitted, but here the energy for Ihe initial excilation of electrons is providcd by a chemical reaction rather than by electromagnetic radiation. An example is the action of the enzyme luciferasc. extracK'd from fireOies. which calalyses the following reaction: luciferin + ATP + 0] ..... oxyluciferin + AMP + PP,
+ CO] + light
(26.6]
The light proouccd is either yellow-green (560nm) or red (620nm). This system cun be used in biomolccular analysis of ATP, e.g. to dctennine ATP concentration in a biological sample. Measurement can be performed using thc photomultiplicr tubes of a scintillation counter (p. 237) to detect the l."ITliued light. with calibration of the output using. a series of standards of known ATP content.
n..""
",...".,
-+-
.---+-D--+-H dlitectlJ'
filter or
~Fig.26,5
Atomic spectroscopy
[QEB] ~ell
I
"""'"
Components of a flame photometer.
Safety note In atomic spectroscopy, the use of high pressure gas sources, e.g. cylinders, can be particularly hazardous. Always consult a demonstrator or technician before use.
Atoms of certain metals will absorb and emit radialion of specific wavelengths when heated in a flame, in direct proportion to the number of atoms present. Atomic spectrophotometric techniques measure the absorption or emission of particular wavelengths of UV and visible light, to identify and quantify such metals.
Flame atomic embH'ion spectrophofometl'y (01' flame photometry) The principal componcnts of a flame photometer arc shown in Fig. 26.5. A liquid sample is convcrted into an aerosol in a ncbulizer (atomizer) before being introouced into the name, where 11 small proportion (typically less than I in 10000) of the atoms will be raised to a higher energy level. releasing this energy as light of a specific wH\'clength, which is passed through a filter to a photocell delcctor. Flame pholometry can be used to measure the alkali metal ions K-t, Na-l- and Ca 2+ in, for example, biological fluids and water samples (Box 26.2).
Atomic absorption specII'oscopJ' This technique is applicable to a broad range of metal ions, including those of Pb, Cu, Zn, etc. It relics on the absorption of light of a splX.;fic wavelength by atoms dispersed in a llame. The appropriate wavelength is provided by a hollow cathode lamp, coated with the element to be analySt.'d. focused through the flame and onto the detector. When the sample is introduced into Ihe flame, it will decrease the light detected in 168
lnstrumcntal tcchniques
Basic spectroscopy
t. AItow time for the lnstrument to stablftz.e. Switch on the instrument. light the flame and wait: at least 5 min befote anefysing your solutions. 2. Check for impurities In VOW' reagents. For example. jf you are measuring
N~+
in an acid digest of sorne material. e.g. soil.
check the Na1 content of a reagent blank, containing everything except the soil, procesged in exactly the same way 88 the sampes. Subtract this value from your sample'values to obtain the true Nat- content 3. Ouentffy your samples using a ~ curve (p.2511. Calibration standards should cover the expected concentration range for the teM solutions - your calibration curve may be
non-linear (especially at concentrations above 1 mmol L-I, i,e. fmolm-3 ). 4. Analyse all IOlutions In duplicate. so that repeatability can be 85Se$S8d. 5. CtIec1l your caHbnltion. Make repeated measurements of a standard solution of known concentration after every six or
seven samples. to confirm that the instrument calibration is still valid. 6. Con&Jder the posdJiIity of ktterference. Other metal atoms may emit light which is detected by the photocell, since the filters cover a wider \'I(8veband than the emission line of a particular &Iement. This cen be a serious problem if you are trying to measure low concentrations of a particular metal In the presence of high concentrations of othel" metals (e.g. Na+ in sea water), or other substances whtch form complexes with the teat metal, suppressing tt18 signal le.g. phosphate).
direct proponion to the amount of mClal prescnt. Practic1.1 advantages over flame pholometry include:
Cautjon is needed when using 6t,rong (concentnltEld) acids: always wort:: in 8 fume cupboard, wear gloves ttl proIect your haAds from 'acid and elw8ys rinee lIffect8d arees with Ierge amounts of water.
Safety note
bums"
• • •
improved sensitivity; increased precision; decreased inlerference.
The tochnitjue can be used with Or without a flame. In Ihe nameless
technique several variations arc possible, including 1J graphite furnace or cold vapour, all of which are more sensitivc than t1
Instrumental techniques
169
----10) All spectroscopic equipment is costly. Tho use of such equipment must alwavs be done under guidance from 8 demonstrator or technician. All equipment in this section has an Inherent risk due to its use of mains electricity.
Safety note In atomic spectroscopy, the use of high-pressure gas sources, e.g. cylinders, can be particularly hazardous. Always consult a demonstrator or technician before use.
Atomic spectroscopy Atomic spcclroscopy is a quantitative technique used for the d\.'tcrmination of metals in S
Atomic Absorption Spectroscopy The components of an atomic absorption spectrometer arc a radiation source. an atomization cell. a sample introduction system. a method of wavelength selection and a detector (Fig. 27.2). Sample/standard dilutions - all dilutioos should be done using appropriate glassware or plastic ware. TypicaliV. ttlis involves the use of grade A pipettes for the transfer of known volumes of liquids and grade A volumetric flaskS for subsequent dilutions.
Box 27.1
RlIdia#on souI'ce The main mdiatioll source for AAS is the hollow-cnlh<xle lamp (HCL). The HeL (Fig. 27.3) emits radiation characteristic of a particular clement. The choice of HCL for AAS is simple. For example. if you are analysing for lead, you will nl'Cd a lead-coatcd HeL It is normlll to pre-warm the HCL for about IOmin prior to use. This c.1.n be done either by using a scPilratc pre-
How to prepare a 1000 f.l9 mL 1 stock solution of a metal ion from a metal salt
Stock solutions can be prepared directly from reagentgrade chemicals. It is important to use only reagentgrade chemicals of the highest purity e.g. AnalaR" . This includes the water to be used - distilled and deionizedMilliQ water. (Note: many reagents (solids and liquids) contain metallic impurities in trace amounts. While you can minimize this risk of contamination by using the highest-purity reagents, it is essential to run 'reagent blanks', especially for elemental determinations trace levels.) 1. Determine the M, of the metal salt. For example, the M, of Pb(N03 h = 331.20 9 moll. 2.
Determine the 207.19gmol-1.
A. of the metal. The A,- for Pb is
3.
Ratio the M, to
A:
170
Instrumental techniques
:;:~~ = 1.59859 of Pb(N03h -t.
in 1 litre
Accuratety weigh out (p. 191 the metal saft. In this case. weigh 1.5985 9 of Pb(N03h.
5. Quantitatively transfer the metal salt to a precleaned 100ml beaket" lind dissolve in 1% IItv HNO:J (AnalaR" or equivalenl). 6. Quantitatively transfer the dissotved metal salt to a 1 L voklmetric flask and make up to the graduation mark with 1% vtv HN03 (AnalaA"lI or equivalent). Often. a certified stock standard with a single or multielement composition can be purchased. usually at a concentration (per element) of 1000 mg l-l (1000 ~gmL-l).
Atomic spectroscopy
Box 27.2
How to prepare a set of five calibration solutions in the concentration range
o-10 ....gml
1
{mgl '1
Assuming that we are starting with a lO00pgmL....' stock solu~on of a particular metal. e.g. 'ead. then you wilt need the following: she 100.00ml grade A volumetric flasks; two 100 mL beakers; and, 8 graduated pipette (0-10.00 mU. 1. Ensure thft aI the g&assw. . is deen (see p. 13). 2. Tr.n..... ,l>;:'16mL ofthi' stock the pre-deaned bea~
~n
into one of
3. Ouantit:lrliwlly bMISfer 10.00mL of thestoek solution Into a 100.00 mL volumetric flask. Then, dilute' to 100.00ml Vllith 1 % v/v HNO:J (high purityl.
10000pg 100ml You now have a 100~gml-' 'working' stock solution of Pb.
S. Tren. . . s::::.15 ml of the working Itodl solution
Into the other pre-dNned b8IIker~
6. Then. quentitdvety tJensler 2.00 mL of the Mlfudon into a 100.00 ml vaIumettic .... and dilute to 100.00 ml with 1 % v!v HN~ (high purity). label the ftesk as the 2J1g mL ' Pb calibration soltltion.
7. Similarly bwlsfw O....00. 0.00. 8.00 and 10.00 mL
4. What .. the c:oncentnItion at this new IIOIution1 Remember that we started with 8n initial 1000~ mL-, Pb stock solution.
8. Talc. the 0, 2, 4, &. 8 end 10 JIQ ml~' Pb caHbratioo solutions for FAAS analysis.
,:~g X 10ml= 10000JIgPb 10000jlgPb
~as
~ into ~ yohanetric flasks end dilute to 1oo.00ml vvith the nitrk acid and label as O. 4, 6, 8 end 10 p.g ml-1 Ph calibration solutions.
p'aced in 8 100.00mL volumetric
flask. so:
heater unit, capable of wanning up sc\'eml HCLs simultaneously. or by inserting the HCl in the AAS instrument .md switching on the current. The lamp is typically operated at an electric currenl between 2 and 3OmA.
A tonlizUl;On cell Safety note Gaution is needed when using strong (concentrated) 9cids. When using concentrated acids always wort in a fumo cupboard. Wear gloves to protect your hands from 'acid bums'. Always rinse affected areas with copious amounts of water.
Sc\!cml lypcs of atomi7...lIion cell arc available: flame. graphite furnace. hydride generation and cold vapour. Flame is the most common. In the premixed laminar flame. the fuel and oxidanl g."lses arc mixed before they enter the burner (Ihe ignition site) in an expansion chamber. The more commonly used flame in FAAS is the air-llcct),lcnc flame (temperature, 2500K), while the nitrous oxide-acetylene name (temperature. 31SOKJ is used for refractory c1eml.Tlts. e.g. AI. Both arc fanned in a slot burner positioned in the light path of the HCl (Fig. 27.4). In the grnphite furnacc atomizer, a small volume of sample (5-I00pL) is introduced onlo the inner surface of a graphite tube (or onto a platform placed within the tube) through a small opening (Fig. 27.5). 'nle graphite tube is armnb'Cd so that light from the HCl passes din.-ctly through the centre. Passing an electric current through the lube allows the operator to program a heating cycle. with scveral stages (Fig. 27.6) including the elimination of water from the sample (drying), removal of the sample matrix (ashing),
171
Atomic spectroscopy
Box 27 3 How to analyse a sample using the method of standard additions in FAAS The method of standard additions is used when the sample matrix may cause difficulties, e.g. chemical interferences, in sam~e concentration determination. Standard additions allO\lVS any adverse effects to be overcome by incorporating a known amount of the sample in the calibration solutions. 1. Prepare a l000JlgmL 1 stock solution (see Box 27.11. 2. Then, prepare a 100 119 ml 1 working stock solution (see Box 27.21. 3. You will also need to have prepared the sample. If the sample is a solid you will need to digest the sample (see Box 27.71. 4. An estimetll of tht metal concentration in the sampae i. required prior to canying out standard addjtions SO that the linear relationship between signal (absorbance) and concentration is maintained. 5. You can then prepare the standard addition solutions. This is most easily done as in Table 27.1. 6. Analyse the samples using FAAS. 7. Plot the graph. The graphical output should appear as shown in Fig. 27.1. 8. The graph should contain several features: it must have a linesr response (signal against concen· tration); it does not pass through the origin; and extrapolation of the graph is required until it intersects the x-axis, e.g. 3.2/19 ml- t .
9. Oet.-mine the concentnrtton of the metal in the original sarrf)le. This can be done by taking into account the dilulfons involved In the standard additions method and any dilutions used to prepare the sample (see dilution f8Ctor, Box 27.51. Table 27. J Standard addilions
solutions
v_ _
-
Volume of 100 .... Volume of mI. ' wartdng ....-au-
1100.00""
1
mx:kMludaft
..............
ImLI
lmLI
o
10 10 10 10 10
,,
,
5
7
2
1
5
o.
I" 1I"
3.2 jlU ml:'
~------.
0.2
o +--I-'O:;::"'~-+--~~~~~~-I -4
-3
-2
-I
0
1
2
3
4
5
6
1
IlOIICllItl'lItim/llU mi.-I
Fig. 27.1
Standard additions graph.
an atomization cell using a carrier g.'1s. Thc atomization cell is normally an electrically heatoo or name-hcllted qum1z tube. Using arsenic as an examplc it is possible to write the following equation for the generation ofarsinc (AsH)):
(27.11 Cold vapour generation is the term cxclusively rcservcd for mercury. Mercury in a samplc is reduced to elemental mercury by tin (11) chloride (eqn 27.2):
In2J and the mercury vapour produced is trnnsponcd to an atomil.ation cell by a carril.T gas. The atomization cell consists of a long-path gl..1.SS absorption cell located in the path of the HCL Mcrcury is monitored at a wavelength of 253.7nm.
Sample ;tltl'Od"ct;on into the flame Samples are almost exclusiVely imroduccd inlo names as liquids. Solid samples need to be convCIlOO to aqueous solutions using mcthods such as decomposition (see p. 177). Once in thc aqueous form the sample is introduced into the flame using a nebulizer/expansion chamber. The pneumatic concenuic nebulizer (sec also p. 176) consists of a stainless steel tube through which a Ptflr capillary tube is located. The aqucous 172
Instrumental techniques
Atomic spectroscopy
Box 27.4
Sample size and certified reference materials
If a lk1ear celtbration graph (plot of conoontrMK>n against
absorbance) has been prepared for klad in FAAS with the concentrations 0, 2. 4, 6, 8 and 10JIQml-'. the absorbance for the digested sample needs to fall
within the linear portion of the graph. This is controUed bv two (noo-instrumentall factors: the weight of the semple digested and the final volume that the digested sample is made up to. As the ~ume of the digested sample is limited by the availability of votumetric
f1a6ks (10.00, 25.00, SO.OO, 100.00 or 250.00 ml are most commonly used. with the 50.00 and 100.00 ml volumetric flasks the most common) it is often easier to alter the sample sileo In order to heve 8 representative sample., a minimum sample size is often recommended. For example. if using a certified reference materiel (CRM) the supplier will recommend a minimum sample size to ensure homogeneity, e.g. not < 0.5g far a powdered solid steel or aUoy samp"'l or not < 1.0g for a powdered biological sampl", such as citrus leaves. Often the maximum sampte size Is limited by the cost of the CAM. If using 8 'resl' ~mple then it is best to take 8 larger sample size, since a CRM has usually been tested and prepared to a high specification with respect to drying, milling and shelflife time. A typical minimum sample size for a 5011
Box 27 5
Analysis of
•
• •
•
SampUng - how it is to be done1 ~w will e representative sample be arrived al? Storage of sample - what containers wi'll be used fOl" storage of sample? Be aWBn'I of contamination for the storage container and 'from the Implements used to sample and transfer the samp1e. lifetime of slored sample - how long will the semple remain stabie? S& preservHtion of the sample necesury?
NO(e: CRMs cen be obtained from appropriate 8uppliefs, e.g. laboratory of the Government Chemist in the UK or the N8ttonallnstitute for Science and Technoklgy in th~ USA. In addition to the CAM. Ii certificate is provided that' contains infOlTTlaiion on the concentr8lion of various metals within the sample as \N8U as their variation (nollTlsUy quoted as one standard deviation either side of the mean vafue), e.g. 2,5:i:0.3/19g-1 Pb. CRMs are used to test the accuracy of a new method or to enable e Quality conb"~ scheme to be operated by a commercial laboratory. In practical wort they are useful to. assess student perfOfTTlSnce in terms of hislher ability to prepare and anelyse a sempm.
a sample: dilution factor
A sample was wolghod (0.4998 9) and digested in concentnitecl nllrK: acid (20 ml). After coofing. the digested sample was Quantitativcly tr.ansferred into 8 100.00 mL volumetric flask and made up with uttrapure water and then analysed for lead by FAAS. Let us suppose that the absorbance obtained corresponds to a concentration of 3.4/lgmL-1. What is the concentration of lead in the ortginal sample? The method of calculation is most epproprtately done as follows: •
might be 5.00 g. It IS Important tf lJ6lng 'real' semples to consider the following additional factOtS.'
Calculate the dilution factor. This can be done if the finel volume of the sample and its original weight are known. In this case 100mL and O.4998g. You then multiply the concentration from the graphwith lhe dilution factor.
3.4JtQ . loomL _ L><:In -1 mL X O.4998g - oou/199
Note: • The volume of acid used Is irrel~vant in the calculation - onty the final volume in the volumetric flask matters. • The units cancel (mL on top line canoels with mL on the bottom line) leaving you whh units of ItQg-l (~g). • A1tematively, the units can be expressed in mgkg- I (mg/kg). i.e. 680mgkg- 1 or % wtw. i.e. 0.068% wlw (see p. 46). (10000llg0"-1."" 1% or for aqueous Mmp6es 10000pg mL_1 =0-1% wlv).
w/W
sample is drawn up through the capillary tube by Ihe action of the oxidant gas (air) escaping Ihrough the exit orifICe that exists between the outside of the capillary lube and the inside of the slainless steel tube. The action of rhe escaping air and aqueous samplc is sufficient to form a co..1.rsc aerosol in a process termed the Venluri effect. The typical uptake rale or Ihe nebulizer is between 3 and 6mLmin- l . Instrumental techniques
173
Atomic spectroscopy
The cxp<msion chamber (Fig. 27.7) has twO [unctions. The firsl is conccmcd wilh aerosol gcncrdtion the objectives of which arc:
• • •
to convcn the aqueous sample solution inlo a coarse aerosol lL<;ing the oxidant gas; to disperse the col.\l~ aerosol further into a fine acrosol. by interaction with lxlffies located within the chamber; to condl,:nse any rcsiduallierosol pal'ticles. which thell go to waste.
The second function involvcs the safe prc~mixing of the oxidant and fuel gases before they are introduced into the laminar Ilow burner.
Fig. 27.2 Components of an atomic absOrption
spectrometcr.
t
f I
I
IRld8
Ne or Ar
Fig. 27.3 Components of 8 hol'ow-eathode lamp IHCLJ.
... ...... ....-..
Fig.
27.4 Components of a slot burner for
FAAP. ~qectlonpm
f:;~-()
I .--
0
l_~O3--1
~
eloctric:aIlllri'cI
Fig. 27.5 Schematic diagram of a graphite furnace atomizer.
174
Instrumental lechniques
'Val'elengtll select;on I1IUI deteet;flIl As AA$ is used to monitor one metal at a time. the spectrometer \Iscd is termed a monochromlltor. Two optiad Ml1Ingcmenls arc possible; single and double beam. The latter is preferred as it corrects for nuctualions in the HCL caused by wann-up, drill and sour<.-e noise, thus leading to improved precision in the absorbmlCe measurement. A schematic diagnlln of the optical llmlT'lgcmcnt is shown in Fig. 27.8. The attenuation of the HCl radiation by the atomic vapour is detected by a photomultiplier tube (PM'I). a device for proportionally convcrting photons of light 10 dcctri(; (;urrenl. Btlckgl'OlInd COl'rection metllod... One of the main practical probk.ms with the usc of AAS is the occurrence of molccuhlr spec.:ies that coincide wilh the atomic signal. One approach to remo\le this molecular abiorbance is by the usc of background correction methods. Several approaches are possible, but lhe most common is based on the usc of a continuum source, D2 • In the atomiztltion cell (e.g. l11.me) absorption is possible from both atomic species and from molecular species (unwanted illlerferencc). By measuring the aOOorption that occurs from the radiation source (HCL) and comparing it with the absorlxmcc that occurs from the continuum source (D1.I a corrected absorption signal can be obtained. This is because the atomic species of interest absorb the specific radiation associated with the HCL source, whereas the absorption of radiation by the continuwn soorce for the same atomic species will be negligible. Intel!erem:es ;11 the Jillme Interferences in the Ilame Cl:m be classified into four categories: dlemical, ioni7.1ltion, physical and spectral. Chemical interferences ou.".r when thc anal}1e forms a thennal1y stllble compound with a molecular or ionic species present in the sample solulion. Examples include the suppression of alkalinc earth metals due to the presence of phosphate, silicate or aluminate in Ihe sample solution in the air-acetylene name. Thc most wcll-known example of this is the absorption signal suppression that occurs for Ca at 422.7nm owing to increasing amounts of phosphate. This signal suppression is due to the fonnalion of calcium pyrophosphate, a thermally slilble compound in the name. Ionization interferences occur most commonly for alkali and alkaline earth metals. The low ionization potential of these metals can lead 10 their ionu..ation in the rclath'Cly hot environment of the l11.mc. If this occurs, no absorption signal is detected, since FAAS is a technique for measuring atoms not ions. This process can be prevented by the addition of an ionization suppressor or 'bulTer', e.g. an alk.1.li metal such as Cs. Addition of excess Cs
Atomic spectroscopy
, •
1
I
2
...
,
Fig. 27.6 Heating cycle for a graphito furnace atomizer. 1. drying; 2. ashing; 3. analysis; 4. cleaning and 5. cooling
..... ~~M~r~~ ........ ...... rine Il!rOId
-'"
Fig.27.7 Schematic diagram of a nebutizerexpansion chamber for FAAS.
-'-~--'--),., i t
;( -
, :
/ItDOlCeI..:
= -+~;""'I"'-+ heJ·u.ered lrirJor
Fig. 27.8 Schematic diagram of the optical arrangement for AAS.
Make sure djstitled water is available to aspirate into the flame once it is ignited.
Contamination risk - wipe the outside of the 8spiretor tube with a clean tissue in between samples/standards to prevent contamination. Safety note Once the flame is ignrted it should 'not be left unattended.
leads to its ioni7.ation in the namc in preferencc to Ihe mewl of interest, e.g. Nu. This process is termed the 'ml.lSs aelion· effect. Physical interferences arc due to the effects of the sample solution on aerosol forlTlation within the spmy chamber. 'Illt: fonnalion of an ftCfosol is dependent upon the surface tension, density and viscosilY of the sample solution. 111is type of interference can be controlled by the matrix matching of sample and standard solutions, i.e. add the same s.ample components to the standard S<.Ilution, but without the metal of interest. If this is not possible• it is then ncccssmy 10 usc the method of standard addilions (Box 27.3). Spectml interferences arc uncommon in AAS owing 10 the selectivity of Ihe lechnique. HowC\'er, some interferences may occur, e.g. the resonance line of Cu occurs at 324.754nm and has a line coincidence from Eu at 324.753nm. Unless thc Eu is 1000 times ill cxcess, howC\'cr, it is unlikely to causc
Atomic Emission Spectroscopy The main components of an atomic emission Spct.'tromeler arc an atomi7;1tion and ioni.1.lltion cell, a method of sample introduction, the spectrometer and detector. In contrasl to AAS, no rndiation source is required. Flame pholOmelry (see also p. 168) is almost exclusively used for the determination of alkali metals because of their low excitation potential (e.g. sodium 5.14eV and potassium 4.34cV). 'Ibis simplifies the instrumentation RXluired and allows a cooler name (air-propane, air-butane or air-natural gas) to 1x: used in conjunction with a simpler spectrometer (interferencc filter). The use of an interference filter allows a huge excess of light to be viewed by the detector. Thus, the expensive photomultiplier tube is not required and a chc.'1pcr detector can be used, e.g. a photodiode or pllOtocmissive detector. 'lllC sample is introduced using a pneumatic nebulizer liS described for FAAS (p. 172). Flame photot11Ctry is therefore a simple, robust and ineX~lSi\'e technique for the determination of potassium (766.5nm) or sodium (589.0nm) in clinical or environmental samples. The technique suffers from the same type of interferences as in FAAS. '1l1e operation of a name photometer is described ill Box 26.2.
Inductively coupled plasma A radio frequency inductively coupled plasma (ICP) is fonned within the confines of three concentric glass tubes or plasma torch (Fig. 27.9). £.1.ch concentric glass tube has a tan~ntially arranged entry point through which argon gas enters the intenncdiate (plasma) and external (coolant) tubes. 'll1e inner tube consists of a cnpillary lube through which the nerosol is introduced from the sample introduction system. Located around the pk'lsma torch is a coil of water-cooled copper tubing. Power input to the fep is achieved lhrough this copper, load or induction coil. typically in the range OS-I.5 kW at a frequency of27 or 40MH7_ Instrumenlallechniqucs
175
Atomic spectroscopy
Be prepared - ensure all standards. blanks and samples are readily and eMily accessible prior to ignition. Slllety note Check that the drain is full of water prior to use.
Initiation of the plasma is achievcd as follows. The carrier gas flow is fi~l switched off and a spark added momentarily from a Tesla coil (attached 10 the ollier edge of the plasma torch). The spark, a sourcc of 'seed' electrons, causes ionization of the argon gas. The co-cxi~lellce of argon, argon ions and electrons conslitutes a plasma located wilhin I he confines of the plasma torch but protruding from the lOp in th~ shape of ~I bright whitc luminous bulle!. In order to introduce the sample aerosol into thc ICP (7000 10 000 K) Ih~ carrier gas is s\\ildlcd on and punches a hole inlo Ihe centre of the plasma crealing Ihe chamctcri<;tic doughnut or toroidal shape. The emiucd radialion L<; viewed laterally (side-on) through !.he luminoull plallma.
Sample introduction
o o
a IoId ooiI o
The most common method of liquid sample introduction in ICP-AES is the nebulizer. The ncbulizer operate.<; in the sam~ manIlCr as that used for FAAS but there are differences in ils con<;truclion malerial and manufactured tolcral1tt (the nebulizer for ICP-A ES gellt:ralCli a finer aerosol. but is morc inefficient). The pneumatic nebulizer consists of a concentric glass tube through which a capillary tube passes (Fig. 27.10). The sample is drawn up through I.he capillary by the action of th~ argOn carrier gas ~scaping through the exit orifice that exists between Ihe oulside of the capillary lube and the inside of the glass concenlric tube. The typical uplake rate of the nebuli....er is between 0.5 and 4mLmin I. In common with FAAS, a meanll to reduce the coaI'SC aerosol generated to a line aerosol is required. In ICP-AES lerminology this device is called a spray chamber (Fig. 27.11).
Spec/I'ome/eI's The nature ofthc ICP is such lhat all e1emenlal information from the sample is
Fig. 27.9 SChematic diagram of an inductively coupled plasma.
...... Fig. 27. 10 SChematic diagram of a pneumatic nebulizer.
contained ",ilhin il. The only limiullion is whether it is pmsiblc to Ob1icTVC all the elemental infonnation at the same time or one element at once. This limitation i~ as.o;;ociated not with the ICP but with the Iype of spectrometer used to view the emilie
De/ectol's The mosl common deloctor for AES ill thc photomultiplier lUbe (sec p. 174). An alternative approach for the detection of multielement (rnulliwavelcngth) infonnation ill the charged-couplcd device (CCO). A CCD ill Cliscnlially an array of closely spaced melahnsulator--scmiconduclor diodes formed on a wafer of semiconductor maLCrial. Incident light lltriking the CCD is converted into an e10ctrical signal.
InterfcI'cnces in ICP-AES Fig. 27.1 1 Schematic diagram of a spray
chamber.
176
Instrumental techniques
Interferences for AES can be c1as.<;ilied into IwO main catcgOriCli, llpe<:tral and matrix interferences. Spectral interference can occur as a rCliult of an inlerfering emission line from either another element or the argon source gall, impurities within or entrained into the source. e.g. molecular species such as N 1 . Such interferences can be eliminaled or reduced either by increasing the resolution of the spectrometer or by selecting an alternative llpectral emission line.
Atomic spectroscopy
Box 27.6
How to operate a flame atomic absorption spectrometer
You should only operate an FAAS svstem under direct supervision. The instrument should be located under a fume extraction hood. ihe spec;:trometer requires approxr· mately 20 min to VoI8fTTl up before SY
Adiust the opM'ating wavelength and slit width of the monochromator. This is done by consulting st8ndard operating conditions, e.g. for lead see Table 27.2.
2. Decide what wevelength " to be used for the ......... For lead the rrwcimum sensitivity is achieved by selecting 217.0 om. 3, Adjust the wawelength selector to the approprNrte WIIVe6ength.
4. Adjustthe 98" control until theenergv meter reading roaches & maximum.
5. Adjust the wavelength 'elec:tcll' for maximum signal reading. You are now ready to ignite the airacet0ene flame. &. Turn on 'It. fume extreetion hood. Thifl aHows toxic gases to be safely removed from t~ laboratory environment.
7. Tum on the air suppty soch that the oxidant Row meter Is 8t'the desired setting. 8. Tum on the ec:etyIene supply such that the fuel flow meter is at the desired setting.
..
Table 27. 2 Standard operating conditions for Ieed
283.3 217.0 205.3 202.~
261.4 368.3 364.0
0.7 0.7 0.7 0.7 0.7 0.7 0.7
c:. III bMion ~1,..~1, , , 0.45 0.19
6.'
7.' 11,0 27.0 87.0
Nato: recommended tIame: alr-acetytene, Ol&iling (1e80, bluel.
9. Press the
9Wte button lor fteme button).
flame should light instantaneously with
8
The
'pop'.
10. Aftsr establishing the flame, insert the aspirator tube into distilled water. Allow the flame to stabilize for up to 1 minute by aspirating distilled water, prior to analysis. 1 1. AIt.... completing your allIItysIs. shut off the acetYlene first Iby closing the cylinder valvel.and vent the acetylene gas line while the air is still on. Then. shut off the air compressor and allow the air line to
vene 12. FUUtlly, switeh off the fume extriKtion hood.
Matrix interferences arc oOen associated with the sample introduction process. For example, pneumatic nebulization ean be affected by the dissolved-solido; content of the aqueous sample, which affccts the uptake rate of the nebulizer and hence the sensitivity of the assay. Matrix effects in the plasma source typically involve the presence of easily ionizable clements (EIEs), c.g. alkali metals, within the pla~na source.
Decomposition techniques for solid inorganic samples Conversion of a solid matrix into a liquid matrix involves the decomposition of the sample. One of the major problems in preparing solid samples for trace clement analysis is the potential risk of contamination. Contamination can arise fmlll several sources: the grade of reagents used: the vcsscls used for digestion and the subsequent dilution of the sample: and human involvement. In order to minimize the risk of contamination you should take the following measures: • •
Use the highest purity of reagents and acids. including the water used for sample dilution. Use sample blanks in the analytical procedure, to identify the base level of impurity in the reagents. Instrumental techniques
177
Atomic spectroscopy
•
• •
Soak sample vessels in an acid leaching bath (e.g. 10% v/v nitric acid) for at least 24 hours., followed by rin.'iing in copious amoulllS of ultrapure water. Store e1eaned volumetric nasks with thcir stopper.; inserted: cover beakers with Qingfilm R or store upside down to protect from dust. In addition to the wcaring of a labor-dlory coat and safety gLasses. it may be ooccss.ary to wear 'oontamimmt'-frec gloves and
Decomposition involves the liberation of the analytc (metal) of inlcfQiit from an intcrfering matrix using a reagcnt (mincral/oxidiling acids or fU'iioll flux) and/or heat. An important aspect in the decomposition of an unknown SlI.mp1c: is the sample size (llox 27.4). You need to consider two aspect... Firstly, the dilution factor required to convert the solid samplc to all aqueou<; solution (Box 27.5), and. secondly. the sensitivity of the analytical instrumcnt. e.g. FAAS.
Acid digestion This involves Ule use of mineral or oxidizing adds and an external heat source to decompose the sample manix. TIle choice of an individual acid or combination of acids depends upon thc nature of thc matrix 10 be decomposed. For examplc. the digestion of a matrix containing silica. SiCh. (e.g. a geological sample), requires the use of hydrofluoric acid (Hf-l. A summary of the mosl common acids used for digestion and their application is shown in Table 27.3. Once ),ou havc chosen an appropriate acid. place your sample into all appropriatc vcsseI for the decomposition stage. Typical vessels include an open glass beaker or boiling tube for conventional heming or for microwave heating, a PTFE or Tcflon II PFA (pcrnuoroalkoxyvinylcthcr) vcsscl. A typical microwave system Operdtes at 2.45GHz with up to 14 sample vessels arranged on a rotaling carousel; commercial systems have additional features such as:
Table 27. 3 Common acids· used for digestion
......,
Boiling point I C1
Hydrochloric acid (HCn
110
Useful far satts of ~rbonates, some oxides and some sulphides. A weak reducing agent; not generally used to dissolve Ofganic marter
112
for digeslion of silica-based materials only. Cannot be used with glass containers luse plastiewareJ. In addition to labOratory coot and safety glasses, extra safety precautions are needed, e.g. gloves. In case of spillages, calcium gluconate gel Is required for treatment of skin contact siles and should be available during use; evacuate to hospital immediately if skin is exposed to liquid HF
122
Useful for the digestion of metals, alloys and biological samples. Oxidizing attack on many samples not dissolved by HCI; liberates trace metals as the soluble nitrate salt
Hydrofluoric acid
(HFI
Nitric acid (HN03 l
Useful tor releasing a volatile product; good oxidizing propenies for ores, metals, alloys, oxides and hydroxides. Often used in combination with HN03 . Note: Sulphuric acid must never be used in PTFE vessels {melting point 327 Cl Hydrochloric/nitric acids IHClJHNO:!)
A 3: 1 VN mixture of HCI and HNo, is called aqua regia. tt forms a reactive intermediate. NOCl. Useful for digesting metals, alloys, sulphides and other ores
"All concentrated acids should be used only in a fume cupboard.
178
Instrumental techniques
Atomic spectroscopy
Box 27.7
How to acid-digest a sample using a hot plate
1. Accuratety weigh yOlK ll8f11p1e into a beaker 1100 mLI. For digestion of a powdered metal sample 0.50009 is appropriate (for details on how to weigh accurately see p. 24).
2. Add 20mL of concentrated acid(s) (see Table 27.3). 3. Cover the beaker with a watch glass. This is done to prevent the loss of sample and to minimize the risk of contamination.
4. PIKe the beaker on a pre-heated hot plate. 5. Reflux the sample for approx. 30 mins to 1 hour; depending on the nature of the sample a coloured,
7. Wash the watch-glass cover into the beaker to 'capture' any splashes of solution.
8. Dilute the digested sample with deionized, distilled water. 9. Quantitatively transfer the diluted. digested sample to a 100.00 mL volumetric flask (see p. 18). Make up to the graduation mark with de-ionized, distilled water.
10. Prepare a sample blank using the same procedure, i.e. perform all of the above tasks, but without adding the actual sample. 11. Prepare samples in at least d'4)licate. For statistical
clear solution should resutt.
6. Remove the beaker from the heat and allow to cool This may take several minutes. Retain the watchglass cover during this stage to reduce airborne contamination.
work on the results, at least seven sample digests and tvvo sample blanks are recommended.
Otl,e,. method!)' ofsample decomposition The usc of aeid(s) and heat is probably the most common approach to the decomposition of samples. However. severnl alternatives exist including dry ashing and fusion. Dry ashing involves heating the sample in air in a muffle furnace at 400800~C to destroy the sample matrix, e.g. soil. After decomposition. the sample residue is dissolved in acid and quantitatively transferred to a volumetric nask prior 10 analysis. The method may lead to the loss of volatile elements. e.g. Hg, As. Some substances. such as silicates and oxides, are not always destroyed by the direct action of acid and heal. In these situation'i an allcrnalive approach is required. FU'iiOil involves the addition of a Io-fold excess of a suitable reagent (e.g. lithium metabornte or letrabornte) to a finely ground sample. TIle mixture is placed in a metal crucible, e.g. Pl. and then healed in a muffle furnace at 900-I000OC. After heating (from several minutes to several hours) a clear 'mel!' should result, indicating completeness of the decomposition. Arter cooling, the mell is dissolved in HF (Table 27.3). This process can lead to a higher risk of contamination.
Instrumental techniques
179
---0
Infrared spectroscopy In addition 10 ultmvioJcI-visiblc (UV-vis) spectroscopy (p. 164). there arc three other C$.o;ential techniques that you will encounter during your laboratory coursc. They are:
Identifying compounds - the combination of techniques described in this and the following chapters can often provide sufficient information to identify a compOund with a tow probability of error.
I. 2.
Definitions Spectroscopy - any technique involving the producticm and subsequent recording of a spectrum of electromagnetic radiation, usually in terms of wavelenglh or energy_
3.
Spectrometry - any technique involving the measurement of a spectrum. e.g. of
Il/fronY! (lR) Spt!ClrO$€Opy: this is concerned with the energy changes involved in the stretching and bending of covalent honds in molecules. NllclMr /lUlgl/elic reS()I/tUIC€ (NM R) speClroscopy. this involVe<; the absorplion of energy by specifIC atomic nuclei ill magnetic fields and is probably the most powerful 1001 availablc for the slructural delcmtination of molecule<; (Chapter 29). Mass SIN'CIl'omelry (MS): this is based on the fragmentation of compounds into smaller units. The resulting positive iOlls are then separated according to their mass..to-charge ratio (m/=) (Chapter 30).
As with UV \'is spectroscopy, IR and NMR spectroscopy are b:lsed on the interaction of electromagnetic radiation wilh molecules, whereas MS is different in that it relies on hig.h--cncrgy particles (electrons or ions) 10 break up the molecules. The relationship between the various types of spectroscopy and the electromagnetic spectrum is shown in Table 28.1.
electromagnetic radiation. molecular
masses, etc. Interpreting spectra - the spectrum produced in UV-vis. IA and NMR spectroscopy is a plot of wavelength or frequency or energy (x-axisl against absorption of energy ly-axis). Convention
Infrared spectroscopy A covalenl bond between two atoms can be crudely modelled as a spring connecting two masses and the frequency of vibration of the spring is defined by Hooke's law (eqn (28.ID, which relatcs the frequency of the vibration (v) to the strength of the spring, expressed as the force constant (k), alld to lhe masses (ml and m2) on the ends of the spring (delined as the reduced mass Jl = (m[ x "'2) -=- ( m] -t- IIIz».
puts high frequency (high energy. short wavelengthl at the leh-hand side of the
spectrum.
,. ~ -'- fE 2ft
[28.11
V"P
In simple terms, this means thaI: •
Table 28.1
the stretching vibration of a bond between two atoms will increase in frequency (energy) if on changing from a single bond to a double bond and then to a triple bond between the same two atoms (masses), i.e. the spring gets stronger. For example,
The elecrromagneric spectrum and types of spectroscopy
Type of radiation
Wavektngth
Or;gin ~
j'-rays X-rays
~
Atomic nuclei Inner shell electrons
7-ray spectroscopy X-ray Iluorescence (XRF)
UltravioletlUV)
loni~ation
UV/Visible
Valency electrons
2.o-200nm 200-800nm
Vacuum UV spectroscopy UVivisible spectroscopy
Infrared
Molecular vibrations
O.8-300Jlm
lR and Raman spectroscopy
Microwaves
Molecular rotations Electron spin
1 mm toJOcm
Radio waves
Nuclear spin
O.6-10m
Microwave spectroscopy Electron spin resonance (ESRI Nuclear magnetic resonance (NMR)
180
Instrumental techniques
Infrared spectroscopy
v for C""C > v for C=C >
•
I'
for C-C
as the massc<; of the aloms on a bond increases, the frequency of the vibration decreases, i.e the errcct of reducing the magnitude of j./; for example, \' for C-H > v for C-e; I' for C-H > v for C-D: v for 0 H > \. for S-H
IR absorption bands - since the frequency of vibration of a bond is a specific value you would expect to see line spectra on the chart. However, eact1 vibration is associated with several rotational motions and bands (peaks) are seen in the spectrum.
Bonds can also bend. but Ihis movement requires less energy than stretching and thus the bending frequency of a bond is always JOJ1'er than the oorresponding stretching frequency. When IR radiation of the same frequency as the bond interacts with Ihe bond it is absorbed and increases the amplitude of vibration of the bond. This absorption is detected by the IR spectrometer and results in a peak in the spectrum. For a vibration to be detected in the IR region the bond must undergo a change in dipole moment when the vibration occurs. Bonds with the greatest change in dipole moment during vibration show the most intense absorption. e.g. C=O and C--o. Since bonds betwecn specific atoms have particular frequencies of vibralion. IR spectroscopy provides a means of identifying the type of bonds in a molecule, e.g. all alcohols will have an O--H stretching frequency and all compounds containing a carbonyl group will have a c=o slretching frequency. This propeny. which does not rely on chemiml tests. is extremely useful in diagnosing the functional groups within a covalenl molt."Cule.
IR spectra A typical IR spectrum is shown
III
Fig. 28.1 and you should note the
following JX)ints:
"
100.00 , - - - - - - - - - - - - - - - - - - - - ,
\I ,
~
,
,
~
M
g , ~
nooL-~-
The use of wavenumber - this is an old
established convention, since high wavenumber"" high frequency", high energy = short wavelength. Expression of the IR range, 4OOOcm-' to 650cm- 1 , is in 'easy' numbers and the high energy is found on the left-hand side of the spectrum. Note that IR spectroscopists often refer to wavenumbers as 'frequencie5', e.g. 'the peak of the C=O stretching 'frequency' is at 1720 em-I •.
4000
Fig. 28.1
•
IR
_ _- ~ ~ - - - _ - - _ - _ _ _ J
3500
3lXXl
2500
2lXXJ
1500
IlXXJ
6OOcm"
spectrum of ethyl ethanoate CH 3 COOCH zCH 3 as a liquid film.
The x-axis, the wavelength of the radiation. is gi\'en in waveflumbers (v) and expressed in reciprocal centimelres (em-I). You may still see some spectra from old instruments using microns (p, equivalent to the SI unit 'micromelres·. j./m, at I x 10-6 m) for wavelength: (he conversion is given by eqn [28.2): wavenumber (em-I) = I/wavelength (em) = IOOOO/wavelength (pm) [28.2] Instrumental techniques
181
Infrared spectroscopy
•
•
The y-axis. expressing the amounl of rndialion absorbed by the molecule. is usually shown as "I" trnnsmittance (p. 164). When no radialion is absorbed (all is trnnsmiued through Ihe sample) we have 100% transmittance and 0% tmnsmiuunce implies all radiation is absorbed at a particular wavenumber. Since the y-axis scale goes from 0 to 100% IrnnsmiUallce. the absorption peaks are displayed (fOlln from Ihe 100% line; this is opposite 10 mosl other common speclra. The cells holding the sample usually display imperfeclions and arc not completely transparent to IR ntdiation. even when emply. Therefore Ihe base line of the spectrum is rarely set on 100% trdilsmiUance and quantitative applications of IR spectroscopy arc more complex than for UV-vis (p. 166).
JR spectl'Ometen· Therc arc two general types: monochromelOr detet:\QI'
(t~~~~-----,
~W ....
chlppel"
grating
slits
't ---
~
1.
SlUre
lWTl
Fig. 28.2 Schematic diagram of a doublebeam IR spectromeler.
(a) scan speed: this is lhe rate at which the chart moves - slower for greater accuracy and sharp peaks: (b) wavelength range: thc full s!XCtrum or a part of the IR range may be selected; (c) 100% conlrol: this is used to set the pen at the 100% transmiuance line when no sample is present Ihe base line. It is usual practice to sel Ihe pen 1.11 90% transmittancc at 4000 em-I when the sample is present. 10 give peaks of the maximum deneclion.
Using double-beam instruments - you can identify the sample beam by quickly placing your hand in the beam. If the pen records a peak, this is the sample beam, but if the pen moves uP. then this is the reference beam.
Using the 1000A, control- if you use this contralto set the base line for the sample, you must turn down the 100% control when you remove the sample, otherwise the pen-drive mechanism may be damaged in trying to drive off the top of the chart.
Double-beam or dispersive instrumenls in which the lR radiation from a single source is split into two identical beams. One beam passes through the sample and the other is used as a reference SOlve the sample. The differencc in intensily of the two beums is detected and recorded as a peak; the principal components of this Iype of instrument are shown in Fig. 28.2. The important controls on the spectrometer are:
2.
You should remember thai this is an electromechanical inslrument and you should always make sure that you align lhe chan .\gainst lhe calibration marks on the chart holder. In the more advanced instrument.s an on-board computer slores a Iibntry of standard spectra. which can be compared with your experimt.1l1.l.l1 spectrum. Fourier transform IR (Vr-JR) spectrometer: the value of IR spectroscopy is greatly enhanced by Fourier tnlllsformation, named after the mathematician J.B. Fourier. The Ff is a procedure for inlercom·erling frequency functions and time or distance functions. The IR beam, composed of all the frequencies in the IR range, is passed through the sample and generales interference p
182
Instrumental techniques
Infrared spectroscopy
Box 28.1
How to run an infrared spectrum of a liquid or solid film. mull or KBr disk
A. Oouble-beam spectrometer
2.
1. Ensure that the instrument is switched on and that it has had a few minutes to warm up. 2, Make sure that the chart is aligned with the
calibration mafi<s on the chart bed or chart drum. Most spectrometers scan from 4000 cm- 1 to 650cm- 1 and the pen should be at the 4000cm- 1 mark. 3. Adjust the 100% transmittance control to about 90%, if necessary.
4. Place the sample cell in the sample beam and adjust the 100% transmittance control to 90%, or the highest value possible. 6. Select the scan speed. You must balance the definition required in the spectrum with the time available for the experiment. For most qualitative applications the fastest setting is satisfactory. 7. Press the 'scan' or 'start' button to run the spectrum. The spectrum will be recorded and the spectrometer will automatically align itself at the end of the run. Do not press any other buttons while the spectrum is running or the instrument may not realign itself at the end of the run. 8. Adjust the 100% transmittance control to about 50%, remove the sample cell from the spectrometer and turn the 100% transmittance control to about 90%. 9. Enter all of the following data on the spectrum: name, date, compound and phase (liquid film, Nujol'l" mull, KBr disk, etc.). B. FT-lR spectrometer 1. Make sure that the sample compartment Is empty and close the lid.
S~ect
the number of scans; usually four is adequate for routine work.
3. Select 'background' on the on·sa-een menu, and scan the baCkground. Do not press any other buttons or icons while the spectrum is running. 4.
Place tho sample coli in tile beam, close the lid, select 'sample' and scan the sample. Do not press any other bunons or icons while the spectrum is running.
5.
Select 'customlze', or a similar function. and enter all the data - name, date, compound, phase (liquid film, Nujoll1 mull. KBr disk, etc.) - on the spectrum.
6. Select 'print', to produce the spectrum from the printer. Problems with IR spectra These are usually caused by poOr sample preparation and the mOre common faults are; 1. The large peaks have tips below the bottom of the chart or the large peaks have 'squared tips' near the bottom of the chart: the sample is too thick; remove some sample from the cell and rerun the spectrum. 2. The spectrum is 'weak', I.e. few peaks: the sample is too thin - add more sample or remake the KBr disk. 3. The base line cannot be adjusted to 90% transmittance: the NaCI plates or KBr disk are 'fogged', scratched or dirty - replace or remake the KBr disk. 4. The pen tries to 'go off' the top of the spectrum: obviously due to some absorption at 4000 cm 1 when you were setting the base line. Repeat baseline set-up but at 80% transmittance and bear in mind that dirty plates. above, can be the cause.
(d) the integral computer system enables the usc of libraries of spectra and simplifies spct:trum manipulation, such as the subtraction of contaminant or solvent spt."Ctra. Thc procedures for running IR spectra on double-beam and spectrometcrs are described in Box 28.1.
FT
Sample handling IR spectra of aqueous solutions - special sample cells made from CaF 2 are available for aqueous solutions, but they are expensive and only used in specific applications.
You can obtain IR spectra of solids, liquids and gafCS by use of the appropriate sample cell (sample holder). The sample holder must be completely transparent to lR rndiation; consequently glass and plastic cells cannot be used. The most common samplc cells you will encounter are made from sodium chloride or potassium bromide and you cannot use aqueous Instrumental techniques
183
Infrared spectroscopy
'" '"
,.,
solutions or very wei sampks, otherwise the s.ample cells will dis,~lve. A typical mngc of sample cells is shown in Fig. 28.3 and for roUline qualitative work you will regularly use NaCI platcs and KBr disks to obtain spectrn of solids and liquids. Solution cells and gas cells are utilized in mOre specialized lIpplications and require specifIC instructions and training.
Liquid ."iumples The most eOllvenient way 10 obtain the IR spectrum of a pure. dry liquid is to make a Ihin liquid film between two NaCI disks (plau,:s). Since the mm thickness is unknown, this procedure: is nOI applicable to quantitative work.
Solid sample."i
'0'
If you were to place a fine powder between two NaCl platcs. a usable spectrum would not be obtained because Ihe IR radialion would be scattered by diffmction at the edges of the particles and would not pass through 10 the detector. There are three solutions to this problem: I.
,., Fig. 28.3 Cells for IR spectroscopy: (a) demountable cell fOr liquid and solid films
and mulls; (b) solution cell; lei gas cell.
2. Storing IR sample cells and KBr powder-
cells are always stored in desiccators to prevent "fogging' by absorption of
moistul'tl. KBr powder must be dried in the oven, cooled and kept in a desiccator.
3.
Mulls:. in which the firely ground solid is mixed with a liquid, usually Nujoill. (liquid paraffin) or, Jess frequently, HCn (hexaehloro-l,3butadiene). 'Inis mulling liquid docs not dissolve the chemical but fills the gapS round the edges of the crystals preventing diffraction and scattering of the IR radiation. Remember that these mulling liquids have their own IR spectrum, which is relatively simple. and can be subtmetcd either 'mentally' or by the eompuler. The choice of mulling liquid dcpends upon the region of the I R spectrum of interest: Nujol'~ is a simple hydrocarbon containing only C-H and e-c bonds, whereas Hcn has no C-H bonds. but has C-C1. C=C and C-C bonds. Examination of the sepamte spectra of your unknown compound in each of these mulling agents enables thc full spectrum to be analysed. KB,. disks: here the finely ground solid compound is mixed with anhydrous KDr and squeezed under pressure. The Kllr becomes nuid and forms a disk containing the solid compound dispersed evenly within it and suitable for oblaining a spectrum. The advantage of the KBr disk technique is the absence of the spectrum from the mulling liquid. but Ihe disadvantages are the equipment required (Fig. 28.4) and thc practice required to obtain suitable transparent disks. which arc very delicate and rapidly absorb atmospheric moisture. Thillsolidfilms: here a dilute solution of the compound in a 10w-boiJingpoint solvent such as dichloromethane or ether is allowed 10 evaporate on a Nael plate producing a thin transparent film. This method gives excellent results but ;s slightly Limited by solubility factors.
Handling NaC! plates and KBr disksNaCI plates are delicate and easily damaged by scratching, dropping or squeezing. Hold them onlv by the edges
When you afC recording speclr.t of mulls. KDr disks and thin solid films air is
and place them on filter paper Of tissue
used as the refcl'CJlCe and they are suitable for qualitative analysis only. The
when adding chemicals. KBr disks should be handled using tweezers.
procedure for the preparation of liquid and solid films and muns is described in Box 28.2 and that for KBr disks in Box 28.3.
I"terpretation of lR spectra To identify compounds from their IR spectrum you should know at which frequencies the stretching and bending vibrations occur. A delailcd analysis can be achieved using thc correlation tables found in specialist textbooks, For interpretation, thc spectrum is divided intO lhree regions. 184
lnslrumenlallectmiques
Infrared spectroscopy
Box 28 2
How to prepare liquid and solid films and mulls
A. Preparing a liquid film
formed. If the mull is too thick. add another drop of mulling agent. or if it is too thin, add a little more solid. Only experience will give you the correct consistency of the mull and the key to a good spectrum is a mull of the correct fluidity.
1. Select a pair of dean NaCI pUtes from a desiccator, clean them by wiping with a soft tissue soaked in dichloromethane and place them on the bench on 8 piece of fitter paper or tissue paper to prevent scratching by the bench surface. 2. Using a glass rod or boiling stick. pMce a small drop of liquid in the centre of one of the plates. Do not use a Pasteur pipette. which may scratch the surface of the plate. 3.
Carefultv. hoIcing it by the edge. place the other plate on top and see if a thin film spreads between the plates. covering the centres. 00 not press to force the plates together. If there is not enough liquid, carefully separate the plates by lifting at the edge and add aoother drop of liquid. If there is 100 much liquid, separate the plates and wipe the liquid from one of them using a soft tissue.
B. Preparing a thm solid film 1.
Dissotve the sample labout 5mgl in a suitable low-boiling-point solvent (about 025 mL), such as DCM or ether.
2. Place two drops of the solution onto the centre of a NaCI ptate and allow the solvent to evaporate. Use a Pasteur pipette. but do not touch the surface of the plate. 3.
at the resulting thin film of solid does not cover the centre of the plate. add a little more solution.
4. Mount the single NaCI plate in the spectrometer and run the spectrum. Note that the NaCl plate can rest on the ·V··shaped wedge on the sample holder in the spectrometer. C. Preparing a mull 1. Grind a small sample of your compound (about 5 mgt using a small agate mortar and pestle for at least 2 minutes. The powder should be as fine as possible.
2. Add one drop of muOing agent INujol fl; or HeB) and continue grinding until a smooth paste is
3. Transfer the mull to the centre or along the diameter of an NaCI plate. on a piece of filter paper or tissue paper to prevent scratching by the bench surface. using a small plastic spatula or a boiling stick. 4.
Carefully. holding it by the edge. place the other plate on top and very gently press to ensure that the mull spreads as a thin film between the plates. If there is not enough liquid, carefully separate the plates by lifting at the edge and add another drop of mull. If there is too much liquid (poor spectrum). separate the plates and wipe the mull from one of them using a tissue.
C. Setting up the cell hold8t" for liquid films and mulls 1. Place the back-plate of the cell holder on the bench. position the rubbet- gasket. place the NBCI plates on the gasket and then put the second gasket on top of the plates. These gaskets are essential to prevent fracture of the plates when you tighten the locking nuts. 2. Carefully place the cell holder top-plate on the top gasket. drop the locking nuts into place and carefully tighten each in rotation. These are safety nuts and if you over-tighten them or if the back- and top-plates are not parallel. they will spring loose 10 prevent the NaCl plates being crushed.
3. Transfer the cell holder assembly to the spectrometer and make sure it is secure4y mounted in the cen compartment. 4.
Clean the plates in the fume cupboard by wiping them with a tissue soaked in OCM, stand them on filter paper or tissue paper to allow the solvent to evaporate and put them in the desiccator. Allow the DCM to evaporate from the tissue swab and dispose of it in the chemical waste.
Instrumental techniques
185
Infrared spectroscopy
Fig.28.4
---,.e- ,.,•. Le--
base die
Equipment for preparation of a KBr disk.
Region J (4000-2000cm- I ): this region contains the high fTL"Quency vibrations such as C-H, N-H and o-H stretching, together with C=C and C==N stretching vibrations. Region2 (2000-1500cm- J ): lhis is known as the -functional group region'
and includes the stretching frequencies for C=C, C=O, C=N. N=O and N-H bending vibrations. Region 3 (J 500--650 ene' ): this region contains stretching bands for C-o, eN' C-Hal and the C-H bending vibrations. It is known as the 'fingerprint region' lx:causc it also contains complex [ow-cnergy vibrations resulting from the overall molecular structure and these arc unique to each different molecule. Fig. 28.5 shows the spL"Ctra of I-propanol and I-butanol, both of which show almost idcnliall peaks for the O-H, C-H and C-o stretching frequencies and the C-H bending frequencies, but the spectra are different in the number and intensity of the peaks between 1500 and 650 em-I. resulting from the prest:ncc of the additional CH 1 in I-butanol. Conversely, these highly specific bands in the 'fingerprint' region are useful for identification ofmolecules by comparison with authentic spectrn via a databasc.
"
'moo , - - - - - - - - - - - - - - - ; - - - - - - - - . , . , Y,--~ l
4000
3500
3lXXl
2500
2llOO
>500
Fig.28.5 IR spectra of 1-propanol 0') and l-butanol (2).
186
Instrumental techniques
"XX)
Infrared spectroscopy
Box 28.3
How to prepare a KBr disk
1. Take spectroscopic.grade KBr powder from the oven and allow it to cool in a desiccator. Z. Grind your compound 11-2mg! in an agate mortar for 2 minutes. then add the KBr (02 gl and continue grinding to 8 fine powder. Put the KBr powder back into the oven.
3. Obtain 8 'disk kit' and make sure that: (a) it is complete - comprising a plunger. two dies (base and top), a die holder and an anvil, as
shown in Fig. 28.4; (b) the components are for the same device - they
are not interchangeable with another disk kit and should be numbered.
4. Press the die holder onto the anvil ensuring 8 proper fit. 5. lower the base die. numbef'ed-side down, into the die holder and make sure it slides into a depth of
9. Place the assembled disk kit in the hydraulic press and tighten the top screw so that it touches the top of the plunger. 10. Connect the anvil to a source of vacuum, e.g. a rotary vacuum pump. 11. Close the hydraulic r~use valve on the side of the press and gently pump the handle until the pressure gauge reads between 8 and 10 tons and leave for 30 seconds. 12. Open the hydraulic refease valve gently and. when the pressure has fallen to zero, disconnect the vacuum from the anvil. 13. loosen the top screw and remove the disk kit from the press. 14. Turn the disk kit upside down and carefully pull off the anvil. Make sure that the plunger does not slide out by supporting it in the palm of your hand.
about 50mm.
6. Pour the compound/KBr powdM mixture, about one-third to one-half of the amount pntpantd.. into the die hoidel' and tap gently to produce an even layer on the base die. 7. lower the top die. numbet'ed-side up. on top of the KBr mixture and make sure it slides down onto the powder. 8. Slide the pJunger. with the beveUed edge at the top, into the die holdel" ensuring that it is touching the top die and press down gently so that the dies slide to the bottom. ensuring that you do not then push off the anvil.
15. Gently push the plunger and the base die wiD emerge from the die holder. Take off the base die leaving the KBr disk exposed. 16. Carefully slide the KBr disk into the special disk holder using a microspatula. 17. Run the IR $p8CfTum immediately, because the disk will begin to cloud over as it absorbs atmospheric moisture. 18. Clean the disk kit components with a tissue and check that all parts are present. 19. tf the crlllS or the plunger stick in the die holder, tell your instructor.
A simple correlation chart indicating the three regions or the spectrum and their associaIL-d bond vibrations is shown in Fig. 28.6. You can obtain most diagnostic information from spectral regions I and 2, since these are the simples. regions containing thc peaks related to specific functional groups, while region 3 iJ; normally used ror confirmation or your findings. Anothcr important aspect or thc IR spectrum is the relalive intensities of the commonly fouod peaks and you should become familiar with peak sizes. A chart indicating the positions, general shapes and relative intensities or commonly found peaks is shown in Fig. 28.7. When you are attcmpting to interpret an IR spectrum you should usc the approach described in Box 28.4. Ir you are studying complcxes rormed from metals and organic ligands, the mctal-ligand stretching vibration wilt occur below 6(X)an- 1 and special JR spectrometers are used to observe this region. However. changes in thc IR spectrum of the organic ligand on complexation can be det~ted in the normal4()()().--650cm- 1 rdngc.. Instrumental techniques
187
Infrared spectroscopy
- .- -
"C-fl =C-fl
~
- --
e-c
-fl
""
••
-<," "0
--
:t-fl =C-fl
-t<
-
..... -
_ - ....
--.""- ----
-
-t<
OOOH
C-<J
""-
C-<J
.1 elcOOoI .2" alcohol Q
_J''llIcd101
atillcl1toride _ _N-H
- -'"
N-H
-"'"
,, ,, ",,:
- -,
C~
-
cc-<>
...
--
..
,
35'"
.500
25'"
-0
"S"'{)l
_
'000
,£
,\', ' .,, :£"1 , \,z, '. . , \
u
i
,,
/
o
,•
"
u
i
/
\,' \, 11\1
. ..
•
"" "
:• :
,
,:
\•, \,,
,
,
"
/
'
,,
:•
"
..\~/ g "
...
3500
..
,
'500
..
,
Fig.28.7 Idealized intensities of some IR bands of common functional groups.
188
Instrumental techniques
'500
o
•M" 5
o"
Fig. 28.6 Simplified correlation chart of functional group absorptions.
__,
_
'000
600(:111"1
Infrared spectroscopy
Box 28,4
How to interpret an IR spectrum will find the Cw-H stretching frequencies for CH3, CH2 and CH in saturated systems. Oth8f" bands for Q-H, N--H, C=:C and C:=N are obvious.
1. Note the conditions under which the SpeCtrum was obtained, which should be written on the spectrum as 'phase', If it is a solution or a mull. you will need to identify and ·subtract· the spectrum of the mull'ng agent or soIvenL
2. Consider carefuRy the reaction you h8ve caned out. You should know, from the correlation table, the functional groups and peaks in the starting materials and those expected in the product. 3. Remember that the absence of peaks may be as useful in intttf'pratlltion as the presence 01 peaks.
7. EQmine region 2 (2OOO-1500cm '1. Here you will find C=O stretch, usually the most intenee band in the spectrum; C""C and C=N stretches, less intense and sharper; N=D stretch (from ND2) intense end sharp and with a twin band in region 3; N-H bending vibrations - do not confuse with C""D.
5. Many sharp peak. of medium to strong intensity throughout the spectrum generally indicate an aromatic compound.
Examine region 3 l1500-650cm-'I. The large bands here are C-O, C-N, C-CI, 5=D, P=O, N=O (twin from region 21 stretches and C-H 'breathing' bands 1900-700cm-'I, whtch indtcate the number and position of subsCituenls on a benzene ring. Medium-intensity peaks of importance include the CH3 and CH 2 bands 8t 1460cm- 1 and 1370cm-' from the carbon skeleton which are also found in Nujol'll:.
6. Examine region 1 14OOO-2OOOc:m-'I. It is useful to
9. Tabulate your result. end meke the 8PPI"opri8te
draw a line on the chart at 3000cm ': just above the line (3DOO-3100cm 1) you will find the stretching frequencies for C",,-H and C"",-H indicating unsaturation, whilst just below {2980-2BOOcm- 1 j VOU
deduetions. aher consulting the detailed correlation table. Remember to correlate the spectroscopic data with the chemical data.
4.
Do not attempt to identify all the "..s, just those which are relevant to your interpretation. Go for the large peaks first
a
Instrumental techniques
189
---8 Nuclear spin quantum numbers and NMR - other common nuclei with non-zero quantum numbers are uN, "0 (1 = 1) and 118 and 35C! (I = ~l. Their NMR spectra
Nuclear magnetic resonance spectroscopy Eloctromagnetic mdialion (typically at radio frequencies of 60-600 MHz) is used La idemify compounds in
l=!.
l=!.
are not used on a routine basis.
""18... field
110 =
.-
I.'
For a given value of the applied field (80), nuclei of different elements hallc different values of the magnctogyric ratio (;') and will give rise to resonance at various radio fl\.'<juencics. The plincipal componenl" of an NMR spectromcter are shown in Fig. 29.2.
htHHHHH+
'.d
}
"'" tttttPttttttt~
t>E~
[29.11
"'IBo/2rr
pole of magrEt
hv
field COO\JoIer
Ibi
-.
.,
Fig. 29.1 Effect of an applied magnetic field. 80. on magnetic nuclei. (a) Nuclei in magnetic
field have one of two orientations - either with the field or against the field (in the absence of an applied field, the nuclei would have random orientation!. (bl Energv diagram for magnetic nuclei in applied magnetic field.
Example
For an external magnetic field
of 2.5 T (lesla), !::.E for 'H is 6.6 x 10 2fl J and since l:lE = hi', the corres~nding
frequency M is l00MHz; for same field, b.E is 1.7
X
25 MHz.
190
1
C in the
10-2ti J, and v is
Instrumental techniques
I~'"
Fig.29.2 Components of an NMR spectrometer.
For maBnetic nuclei in a given molecule, an NMR spectrum is b
B=
~J(l-a)
[29.'J
where (J (the shielding constant) expreSSL"S the contribution of the small secondary field Benerated by the nearby electrons. The magnitude of a
Nuclear magnetic resonance spectroscopy
depends on the electronic environment of a nucleus, so nuclei of the same isotope give rise to small different resonance frequencies according to lhe equation: 1'0 -
129.3J
a)!2n
)'80(1
~
The variation of resonance frequencies with surrounding electron density is crucial to the usefulness of the NMR technique. tf it did not occur. nuclei of a single isotope would come into resonance at the same combination of magnetic field and radio frequency and only one peak would be observed in the spectrum.
a"
Chemical shift Measuring chemical shifts - ppm is not a COncentration term in NMA but is used to reflect the smalt frequeney changes that occur relative to the reference standard, measured in proportional terms.
The scpanllion of resonance frequencies resulting from the different electronic elwironments of the nucleus of the isotope is callt.od the ('hl'lIJiCli/ .I"hili. It is expressed in dimcnsionlt:ss tenns, as parts per million (ppm), against an internal standard, usually Ictramcthyhilane (TMS). By convention, the chemical shift is positive if the sample nuclt:us is less shielded (lower c1CCl.fon densit), in the surrounding bonds) than the lJucleus in the reference and negati\e if it is more shielded (greater electron density in the surrowlding bonds). The chemical shirt scale (b) for a nucleus is defined as: (29.4J
This means that the chemical shift of a specific nucleus in II mokcule is at the same 15 value, no mailer \\hat the operating frequency of the NMR spectrometer. An NM R spectrum is a plot of chemical shirt (b) as lhe x-axis against absorption of energy (resonance) as the .r-axis. On the right-hand side of the spectrum at () = Oppm there may be a small peak. which is the reference (TMSl. A typicalIH-NMR spectrum is shown in Fig. 29.3.
,
•
,
,
I cit b ~J
"
,., .., ..,
Fig. 29.3 'H-NMR spectrum
3.'
2.5
,.,
2.'
"
"
TMS
,.,lllwn)
of l-methoXYPf"opanone.
NMR spectrometers These can operate at different radio frequencies and magllCtic fields and arc llS\lltUy referred to in terms of radio frequency, e.g. 60 MHz, 270MHz and 500MHz spectrometers. Spectrometers openHing above IOOMJ-Iz require expensivc supcrconducting magnets to generatc the high magnetic fields. In routine laboratory work 60MHz and 90MHz instruments arc common but 270 MHz machines are becoming more aITordable. Increasing the operating Instrumental techniques
191
Nuclear magnetic resonance spectroscopy
frequency of the spectrometer effectively increases the resolution of the chemical shifts of the nuclei under examination. For example, We difference in frequency bew,een 0 and lb is 60 Hz in a 60MHz spectrometer but 270 Hz for a 270 MHz instrument. Spectrometers can be divj(led imo two types:
I.
2.
Continuous-wave fCW) speetrometers, which usc a pennanenl magnet or an ckctromagnet, usually operating at 60 or 90 MH7_ In practice the radio frequency is held constant and small electromagnets on the faces of the main magnet (sweep coils) vary the magnet}c field over the chemical shift range. The spectrometer sweeps through the spectroscopic region plotting resonances (absorption peaks) on a chart recorder (cr. dispersion lR spectrometers). CW spectrometers are usually dedicated to observation of a spe"ific nucleus such as 'H. Fourier transform (FT) spectrometers, using supcrconducting magnets containing liquid nitrogen and liquid helium for cooling. Here the magnetic field is held constant and the sample is irntdiated with a radio frequency pulse containing all the radio frequencies over the chemical shift region of the nucleus being examined, cf. FT-IR (p. IS2). Computer control allows rapid repeat scans to accumulate spectnl, presenting the data as a standard CW·typc spectrum via FT processing. Simple variation of the radio frequencies permits ObSCWdtioll of difTerent nuclei (multinuclear NM R sp.-ctromcters). Thus an Ff-NMR spectrometer can be used for obtaining lH, DC, 19F, 15N and lip NMR spectra.
Sample hundling Using deuterated solvents - these are expensive and should not be wasted. CDCI 3 is 100 times more expensive than speCiroscopic-grade CHCI 3 and the others are at least 10-15 times more expensive than CDCI 3 •
Using CDC!;, - when using this solvent 8 peak in the spectrum at 0 = 1.26 ppm is seen. This is due to the presence of CH0 3 as an impurity in the COCI 3.
The majority of NMR spectra arc obtain<.-'d from samples in solution and therefore the solvent should prefembly not contain atoms of the nuclei being observed (except in the case of IJC-NM R). The most common solvents are those in which the hydrogen atoms have been replaced by deutcrium, which is not observed under the conditions under which the sp.'Ctrum is obtained. COCll (deutcciochlorofonn, chlorofonn.J.l is often the solvent of choice, but others such as dimethylsulphoxide--d 6 , «C03hSOj, propanolle--d 6 (CD 3hCOL methanol-l{4 (CD30D) and deuterium oxide (D 20) are in common usc. As it is unlikely that you will be allov.'Cd 'hands-on' usc of an NMR spectrometer, the best approach you can take to obtain a good spectrum is to ensure good sample preparation. The quality of an NMR spectrum is degraded by: • • • • • •
inappropriate solvent; inappropriate concentration of solute; inappropriate solvent volume; solid particles in the solution; water in thc sample (inefficient drying): paramagnetic compounds.
Sample preparation for NMR spectroscopy is described in Box 29.1.
Interpreting NMR !)peclru As a matter of routine in your laboratory work you will be required to interpret IH_NMR spectra (also known as proton spectra). 13C_NMR spectra are becoming more common, while 19F and 31p spectra may be obtained in specialized experiments. Therefore you should concentrate on the interpretation of IH and 13C spectra in the fin;t instance. 192
tnstrument
Nuclear magnetic resonance spectroscopy
Box 29.1
How to prepare a sample for NMR spectroscopy
do not use alumina. You must wear .gloves when handling glasswool.
1. Make sure that your compound is free from water and soJvent (p. 391. 2. Test the solubiUty of your compound in cold C~Cb.. If it is soluble you can use CDCI3 as the solvent for the NMA experiment. If it is insoluble, consult your instructor for the availability of other deuterated solvents. 3. Dissolve your compound CDCI, (about 2 mll in a clean, dry sample tube. Use about 10 mg of sample for CW-NMR or 5mg of sample for FT-NMR. Check to see if the solvent contains TMS; if it does not, consult your instructor.
4. Make a simple fifter in 8 new Pasteur pipette to remove insoluble material and water (Fig. 29.4). Check that your compound does not react with cotton wool and neutral alumina (alcohols and acids are strongly adsorbed on neutral alumina). If it does, replace the cotton wool with glasswool and
5.
Put the filter into a suitable clean, dry NMR tube and, using 8 clean, dry Pasteur pipette, filter the solution into the NMR tube.
6. Fill the NIWt tube to the appropriate level: between 30 and 50 mm in height is sufficient. 7. Cap the NMR tube with the COITect-size tube cap, making sure that it is correctty fitted to prevent oscillation when the tube is spinning in the spectrometer. Make sure that the cap is fitted correctly so that it will not fall off when the tube Is in the spectrometer. 8. Wipe the outside of the tube with a clean, dry tissue to make sure that the spectrometer will not be contaminated. Cleaning the spectrometer probe is a very difficult task.
'H-NMR spectra 'H-NMA spectra - most of the spectra shown in this chapter do not extend over the normal spectral range 0 = 0-10 ppm. They are expanded to show the details of couplinU patterns.
Till'se normally cover the range between 0 = 0 and 10 ppm but the range is increased to 0 = IS ppm when acidic protons are present in the molecule. The ll-1-NMR spectrum of a molecule gives three key piCCL-'S of infonnation about the structure of a molecule: I.
2. 3.
Chemical shin (0): the peak positions indicate the chemical (magnetic) environment of the protons. i.e. difTerent protons in the molecule have difTerent chemical shifts. Integration: the relative size of peak area indicatcs how many protons have the 0 value shown. Coupling: the fine structure on each peak (coupling) indiC
These three features make lH·NMR a powerfUl tool in structure determination and there arc Iwo extreme approaches to it: I. pipene ~eutral
A1,.o)
oononwool
NMA lOOiB
2.
Fig. 29.4 Filtration of solutions for NMR.
Prediction of the spectrum of the expected compound from theoretical knOWledge and then comparison with the spectrum obtained. You should recognize 'patterns' (e.g. triplct and quartct for an ethyl grO\lp: a singlct of peak area six for two identical methyl groups), which were present in the starting materials, but the OH values may have changed in the 'new' molecule. There are computer programs, such as g-NMR R, which will simulate the NMR sJX.'Ctrum from a structural fommla. Interpretation of thc spectrum from correlation tables, but this is very difficult for the inexperient.'ed.
In practice a combination of the two approaches is used with crossreferencing and checking the proposed structure with tabulated OH values and reference spectra until a satisfactory answer is found. Instrumental techniques
193
Nuclear magnetic resonance spectroscopy
. - . Always make sure that your predicted structure is consistent with the spectrum. Factor~· affectilll(
Proton chemical shifts - only hydrogen atoms bonded to carbon will be considered in this simplified treatment.
1.
2. Tllble 29. 1 Chemical shifts of methyl protOfls
Chemical IIhBt Ippm) 0.00 0.90
CH31
2.16
CH3Br
2.65 3.10 3.30 4.26
CH 3CI CH30R CH3F
The hybridization of the Ctrbon atom to which the hydrogen atom is attached: (a) sp) hybridinxl carbon: peaks occur between b = 0.9 and 1.5 ppm in simple hydrocarbon S}'::itl..'ms. "lIe peaks move downficld with change ofstrUC\urc from CH) to CH~ 10 Cli. (b) sp hybridi7.ed carbon: peaks oo::ur at aoout fJ = 1.5-3.5 pplll in alkyncs. (c) Sp2 hybridized carbon: in alkencs the resonances occur around () =" 4-R ppm lind the C-H peaks of aromatic rings are found between 11 = 6 and 9 ppm. The large downlic1d shifts of these C'fl2-H nuclei rcsult rrom deshiclding or the protons by fields set up by circulation or the 1H:lectrons in the magnetic field. The proton or the uldchyde group (CHO) is particularly dcshielded by this eITect and is found at lJ = 9-10 ppm.
Interpreting NMA spectra: changes of fJthe terms used to indicate the movement of a particular peak with change in its chemical (magnetic) environment are: opfie/d - towards" = 0 ppm; downfteldtowards 6 = 10 ppm; slJielded- increased electron density near the proton; deshielded - decreased electron density near the proton.
(CH,I.Si CH,R
chemical shift (lJ u )
The {) \/slues of protons can be predicted to a general approximation rrom knowledge of the effocts which produce \/ariations in chemical shirl.
3.
4.
Elcctron attraction or dectron rekasc by substituent atoms altachct.lIO the carbon alom. Electron auracting atoms. such as N, 0, Hal attached to the carbon, attract electron density from the C-H bonds and thus deshicld the proton. This n:suJts in movement of the chemical s.hirt to higher fJ \/alues (Table 29.1). Conversely. electron-releasing groups produce additional shielding of the C-H bonds resulling in upfidd shill:s of fJ valucs. All the protons in ben~ne arc identical and oa.:ur at (j = 7.27 ppm. In substituted aromatic compounds.. the overall electron-atll'dcling or relcasing effect or the substituent(s) alters the (j values of the remaining ring protons making Ihem non-equiver1 increase with increasing acidity of the 0-1-1 group: thus fJ = 1~6 ppm for alcohols, 4--12 ppm for phenols and 1(}....14 ppm rorcarboxylic acids. Hydro~cns bound to nitrogen (10 and 2"' amines) are round al fJ = 3-8 ppm. The approximate chemical shin regions are shOwn in Fig. 29.5.
I"teg,.atio" ofpeak m·eas Integration of coupled peaks - the area under the singlet, doublet,. triplet,. quartet, etc., is still thai of the type of hydrogen being considered. For example, if the peak for the three protons of a methyl group is split into a triplet by an adjacent methylene group, the Brea of the triplet is
The area of each peak gives the relative number of protons and is produced directly on the spectrum (Fig. 29.6). On CW-NMR spectrometers the height or the peak area integration line must be measured using a ruler, whereas on FT-l'.'MR machines the area is calculated and displayed as a number. You must remember that: •
three.
•
154
Instrumental techniques
The areas are ratios, not absolute vaJ~, and you must find a peak attributable to a specific group to obtain a reference area. e.g. a single peak at (; = 1.0 ppm is likely to be a CH) group and thus the area displayed or measured is equal to three protons. You must ensure thaI you include integrations from all the fine-structure (coupling) peaks in the peak area.
Nuclear magnetic resonance spectroscopy
13 12 11 10 9
B 1
6 5
4 J
2 1 0
Fig. 29.5 Approximate chemical shift positions in the 'H-NMR spectrum.
~
• b
,
CH,CtylCH3
, •
-
TMS
b
•• Fig. 29.6
•
3.0
2.0
1.0
'H-NMR spectrum of melhoxyethane.
Do not expect the pt:ak area integrations to be exact whole numbers, e.g. an area of 2.8 is probably three protons (CH 3). 5.1 is probably five protons (e.g. a Ct;H s group), but 1.5 is probably
Coupling (!<Jpin-spi1l !<Jpliui1lg) Many IH_NMR siglUlls do not eonsisl of a single line but are usuall}' associated with several lines (splitting patterns). Protons gi"ing multiline signals arc said to be cot/pled. This coupling arises from the magnetic influence of protons on one alom with those on an adjacent alom(s). Thus information about the nature of adjacent protons can be detennined and fed into the structural elucidation problem. To a simple first approximation the following three general points are useful in Ihe interpretation of coupling patterns: I.
Aliphatic systems: if adJacel1l carbon atoms have different types of protons (a and b). then the protons will couple. If a prOlon is coupled to 11 (II = I. 2. 3. 4, 5, elc.) other protons on an adjacent carbon atom. the number of lines observed is 11 + I. as shown in the examples below. Protons a arc coupled 10 Iwo protons b: 1/ = 2; CH J CH 20CH 3 abc therefore the peak for protons a is split into three lines (a triplet). Protons b arc coupled to three protons a; 11 = 3; therefore Ihe peak for protons b is split into four lines (a quanet). Protons c have no adjacent prOlons and therefore arc not coupled and give a single line (singlet) (Fig. 29.6). Instrumentat techniques
195
Nuclear magnetic resonance spectroscopy
CH)CHBrCH 2 Br Protons a are coupled to one proton b: a
b
f/ = I; therefore the peak for protons a is split into two lines (doublet) Protons b are coupled to three protons a and two prolOns c: n = 5; therefore the peak for protons b is split inlO six lines (sexlct). Protons c are coupled 10 olle proton b: I; therefore the peak for protons c is split into two lines (doubk:t). ProtonS a and protons c are not adjacenl and do not couple (Fig. 29.7).
"
,,=
.
f I' ..,
,, CI<,'""""" •
,
3D
,.,
0.,
no
li(ppml
Fig.29.7
The intensity of each peak in the resul!ing singlet, doublet, triplct, quartet, etc., is c-.lIculated from Pascal's triangle (Fig. 29.8). The separalion between the coupled lines is called the coupling constant, J, and. fOf" aliphatic protons CH. CH 2 and CH), it is usually '" 8 H:t_
----------. meQts-VelIJ'S,J I - - - - - - - __ IWO lines (dol.tiel or dl
2 :I
1 - - - - - - lIv.. li1es lriPe! Q' tj :I
1 • - - fu.r lines r~el or Ql
•• Fig. 29.8 Intensities of coupled peaks from
Pascal's triangle.
~ The (n + 1) rule only applies in systems where the coupling constant IJ) between the protons is the same. Fortunately this is common in aliphatic systems. 2.
3.
196
Instrumental techniques
lH_NMR spectrum of 1.2-dibmmopropane.
Alkene hydro~ns: hydrogen atoms on double bonds have diITerenl coupling constants depending upon the stereochemistry of the alkene. Alkene hydrogens in the Z (cis) configuration have J = 5-14 Hz, whereas those in the E (trans) configuration have J= 11~19Hz (Figs 29.9a and b). Aromatic hydrogens: coupling of hydrogens. which are non-adjacent, is readily observed in aromatic compounds. Different protons onllO to each other couple with 1 = 7~ IOH7_ while those in a meta relationship have J = 2-3 H7_ Para coupling (1 = o~ I Hz) is nOl usually seen on the spectrum. The types of aromatic compound you are likely to meet most often are:
Nuclear magnetic resonance spectroscopy
(a) Monosubstituted aromatic compounds, in which Ihn:c basic paltcrns are found in the aromatic region of the spectrum. If the substituent exerlS a \\-'eak electronic effec1 on the ring, the Ii values of the ring protOns are similar and the protons appear as a single peak of area five (Fig. 29.IOa). If the group is strongly electron releasing (01-1. NH2. OCH), etc.). the protons appear as complex multiplelS (o,./ho and mela coupled), below b = 7.27 ppm of relative areas two 10 lhrre (Fig. 29. lOb). If the group is electron attracting (e.g. N~, COOH, etc.), then the complex multiplels have fJ > 7.27 ppm (Fig. 29.IOc);
•••
(b) para disubslitutcd aromatic compounds, which are of two types. If the SubslilUcnls are lhe same. then all the rill!! protons arc identical and a singlet. of relative area four. is ~n (Fig. 29.IOd). If the substiluents are different, Ihen the paiN; of hydrogells orlhf) to each substituent are different and ol'/ho-<:ouple to give what appears to be pair of doublels, each of relative area two (Fig. 29.loe). (c) locreasing numbers of substituents, which decrease Lhe number of aromatic hydrogens and the spectrum becomes simpler. Thus the common 1.2.4-trisubstituled pattern lFig. 29. IOf} is recogni7£d easily as an orlho-coupled doublet, a metQ-COuplcd doublel and a doublet of doublets (coupled orll/O and mew).
,.,
.,
The chemical shifts of aromatic protons can be cakulated from detailed correlation tables.
••
""""'
Fig.29.9 lH-NMR spectra of: tal (Z}-Jbromopropanonitrile; (b) (E}-3-bromopropallGnitrile.
13C_NMR spectra The DC nucleus has I=!, like 'H, and the l)e-NMR spectrum of a compound can be observed using a different radio frequency range (in the same magnetic field) to that for 'H. 'I1Je DC spectrum will give peaks for each different type of C'
The natural abundance of DC is only 1.1% compared with 98.9% for 12C ~ in any molecule no two adjacent atoms are likely to be DC and therefore coupling between I)C nuclei will nol be seen. giving a very
•
•
•
•
simple spectrum. In a sample of a compound. which contains many molecules, the l3C isotope is randomly distributed and all the different carbon atoms in a sample of a compound will be seen in the IlC_NMR spectrum. The sensitivity of the I)C nudeus is low and this. togelt~'( with its tow natural abundance, means that FT-NMR is the only practical system to produce a spectrum by accumulation of spectra by repetitions. Larger sample size in bigger NMR lUbes also assist in solving the sensitivityj abundance problem. The chemical shin range for llC is greater (he = 0-250 ppm) lhan for IH (hH = 0-15 ppm) gi\
197
Nuclear magnetic resonance spectroscopy
•
,
•
"b~""
8.0
"" 1.0
"
'.0
>D
'.0
'0
,.,
II
no
'.0
Jif
.,
~"
J.2 1.0
,--..,.....-.-
"
6lppnt
Ibi
•
•
• 'OCl<, ;©r. ,
,
'.0
Illppml
•
,
•
•.:©( " "" "",
'~
,0.
TMS
"
8.0
1.5
1.0
I
,. .,
ao
"
"""'"
'"
'.0
0.0
'.0
'0
litJllYllI
'"
,
•
•
,
&:@;' 0,
[. [. u
~
U
1~
U
~
U
~
U
u
U
~
"""'"
I"
u
u
~
u
~
U
''''
.
1.2
~
U
"
u lilPfll'TlJ
'"
lH_NMR spectra of tal mcthylbenzene; (bl melhoxybenzene; lcl nitrobenzene; ld) 1,4-dimethylbenzene; lei 4methoxynitrobenzene; 4·amino-3-bromonitrobenzene (NH l protons not shown).
Fig.29.10
m
•
The peak areas of the difTerenl carbon atoms are not related to the number of carbon atoms having the same chemical shift, as in the case for IH_NMR sr:ectra.
Inte,·pretinK 1JC_NNIR spectra Interpretation of l:lC-NMA spectra - the spectrum is that of aU the carbOn atoms in the molecule. It is easy to forget that the peaks for carbon atoms carrying no hydrogen atoms are present
198
Instrumental technkjues
Nonnally you will be given two 13C::-NMR spectra (Fig. 29.11). The upper spectrum. which is more complex (more lines) is called the off-resonance decoupled spec1rum and shows the lJC_1H coupling 10 enable you 10 detennine which carbon signals are CH), CH 2 • CH and C. Then overlapping of peaks may make the identification of different carbon aloms difficull. The
Nuclear magnetic resonance spectroscopy
,
d
•
b
'---0
,
"
~~~~t~
d
•
b
Iii) 21~
1~
IU
I~
I~
11~
~
roo
~
~
lM~
ll(pprnl Fig.
29.17 13C-NMR speclra of 1-methoxypropanone: (il off-resonance decoupled;
(ii) broadband decoupled.
I~c-~o-I
lower spectrum is a broadbantl decoupled spectrum in which the moll-'Cule is irradiated with a second radio frequency range for the protons in the molecule and effectively removes all the l3C_1H couplings from the spectrum. The resulting simplicily of the spectrum makes identification of the different types of carbon in the molecule relatively easy. The chemical shifts of l3C alorns (be) vary in the same manner as those of protons (Fig. 29.12):
IAr-C I Sll-c I I_I
I. rT'rl160 120 Z2ll 180 140 100
20
Fig. 29.12 Approximate chemical shih positions in 13C-NMR.
2. 3. 4.
be moves downfield as the hybridization of the carbon atom changes
from Sp3 (0-50 ppm) to sp (75-105 ppm) to Sp2 (100-140 ppm); for Sp3 hybridized carbon: oe moves further downficld with the change from CH 3 to CH 2 to CH 10 C; for Sp2 hybridi
'V
Instrumental tochniques
199
Mass spectrometry Understanding mass spectrometrysince this technique does not involve the production and measurement of electromagnetic spectra and is not based on quantum principles. it should not really be referred to as a spectroscopic technique. Mass-to-charge ratios - In the overwhelming majority of simple cases the ion detected is a monopositive cation; thus :c = 1 and the peaks seen on a lowresolution spectrometer equate to the mass of the ion. Determination of exact molecular masshigh-resolution instruments enable the molecular formula of a compound to be determined by summation of the masses of the individual isotopes of atoms. e.g. both ethane and methanal have integral mass values of 30, but the accurate values are 30.046950 and 30.010565 respectively.
..
o·
CHaCCH 2CHa + le'
M'
Mass spectrometry (MS) involves the bombardment of molecules, in the gas phase. with electrons. An electron is lost from the molecule 10 give a cation, the molecular ion (M~), which then breaks down in characteristic ways to give smaller fragments. which are cations. neutral molecules and unchargt.'
Sample handling For low-resolution spectra obtainable from a 'bench-top' MS. samples should be pn..o<>ented in the same form and quantity as demanded for gas chromatographic analysis (p. 211). For high-resOlution spectra contamination of any sort must be avoided and samples (typically less than 500 fig) should be submiUed in glass sample tubes with scre....'Caps containing an aluminiumfoil insert. MS is so sensitive that the plasticizers from plastic lUbes or plastic push-on caps will be detected, as will contaminating grease from ground-glass joints and taps.
'0
"
CCH~I~
Mass !'pectra
Fig. 30.1 Formation and fragmentation of a molecular ion 1M; I.
The standard low-resolution mass spectrum (Fig. 30.3) is computer generated. which allows easy t.'Omparison with known spectra in a computer database for identification. The peak at Ihe highest mass number is the molecular ion (Mi.), the mass of the molecule minus an electron. The peak at RA = 100%, the base peak, is the mOSI abundant fragment in the spectrum and the computer automatically scales the spectrum to give lhe most abundant ion as 100%. The mas.~ spectrum of a compound gives the following information about its chemical structure: •
• •
200
Instrumental techniques
molecular ion mass, which includes information on the number of nitrogen atoms and the presence of chlorine and bromine atoms (see below), - which is not easily obtained from IR and NMR spectra; the mosl stable major fragmem (base peak), which can be correlated to the structure of Ihe molecule; other important fragmenl ions, which may give information on the structure;
Mass spectrometry
--
•
'"""
ir1eclflll
.f~~ 1 ---,-....
"'ct~
N
.-
"'-- ...... ,,,,,
Fig. 30.2 Components of an electron-impact mass spectrometer.
., i1: •
i ~
the detailed fragmenlation patlern, which can be use
Mo{ee,,{QI' iOn!; The mIt value of the molecular ion is the summation of all the alomic masses in Ihe molecule, illchltJillg Ihe f10tlually ()(,(llrrillg l:fOIOpe~. For organic molecules you will find a small peak (M + I) above the apparent molecular ion mass (M:) value due 10 the pre5enL'e of llc. TIle imporlarn..'e of isolope peaks is the delection of chlorine and bromine in molecules since these IWO elements have large natunl.1 abundanres of isotopes. e.g. 3~CI: 370 = 3: 1 and 19 Br : ~l Br = I : I. The mass spectra produced by molecules containing these alams are very distinctive with peaks at M + 2 and even M + 4 and M f.. 6 depellding on how many chlorine or bromine atoms are pr~scnl. The idenlification of fhe number and type of hal~en atom.'l is illustrated in Box 30.1. Since the low·rewlution mass spectrum produces inleger values for fIl!:!, lhe mass or M" indicatcs the number of nitrogen atoms in the molecule. If III!: for M; is an (HIli imeKer, there is an otItlllllmber of N atoms in the molecule and, iflhe value is an cl'en number. then there is an e"eII number of N aloms.
Base peak
70 50
.f
•"
~
r"
,:1'
10
1114 11
10
'"
mI'
"
"
Fig. 30.3 Mass spectrum for methanol; rrizmass-to-<:harge ratio.
IdentiflC8tion of isotope peakS - the natural abundance of ,3C is 1.1%. For 8 molecule containing n carbon atoms the probability is that 1.1 x n% of these atoms will be 1~. Thus the mass
spectrum of hcxane (six carbons) gives 8 molecular ion tM-l-} at mlz.:. 86 snd a peak st rrjz=87 (M+ 1) which is 6.6% thc intensity of the M~ pesk.
--
The molecular ion M l fmgmcnts into cations, radicals, radical cations and neufral molecules of which only the positively charged species are dCfecled. There are several possible fragmentations for each M I but Ihe base peak represents the most energetically!al'ourel{ process with the ml:! value of thc base peak representing the mass of the most abwulam (and therefore mOSI stable) positively charged species. The fragmentation of M i info the base peak follows fhe simplified rules outlined in Box 30.2, and for a more detailed intCf"pretation you should consult the correlation lables 10 be found in the specialist texts referred 10 at the end of the section. FI'aKl1Ientat;on patte"m;
The mass spectrum of a molecule is unique and can be sfored in a computer. A match of the spectrum with those in the computer library is made in terms of molecular weight and the 10 most abundant peaks and a selection of possibilities will be presenled. At this point you need 10 correlate all thc information obtained from the spectroscopic techniques described in Chapters 26. 2i1. 29 and 30 together with the chemistry of the molecule 10 a!lempt to identify the slructure of the molecule. When you allempt 10 interpret the mass spectrum remember that:
•
Only the base peak is almost rertain 10 be derived from the molecular
•
Some lesser peaks may result from allernative fragmentation palhways, but these may be useful in assigning structural fealures. MS is often used to confirm information from IR and NMR spectra; interpretation of the mass speclrum alone is very difficult. excepl for the simplest molecules.
IOn.
•
Interfacing mass spectrometry Bench-top mass spectrometcrs are to be found interfaced mfh other analytical instruments such as gas chromatographs (p. 215) and highInstrumental techniQUes
21)1
Mass spectrometry
Box 30.1
How to identify the number of bromine or chlorine atoms in a molecule from the
molecular ion 1. Since Cl and Sr have isotopes two mass numbers apart, their presence in a molecule will produce peaks at mJz values above M~ , which are two mass
numbers apart. i.e. M + 2, M + 4, etc.
2. The expression for the number and intensities of these peaks is given by the expansion of the formula:
(8+bl
. OJ
I: i"30 ro
': L.c~~'~'--,'C'":--~"~"-,--,..wJll-~-.-J
n
5
where a and b are the ratio of the two atom isotopes, and n is the number of atoms. Example 1: If the molecule contains one chlorine 8tom then: (a
t-bl =(3+1)' =3+1
•
~:
•
i'"
~:
"
ro 10
CH2CI2 would show M -I- 2 at mlz = 86 mlz = 88 (CH231CI2) peaks will be the
30.4b).
Example 3: If the molecule contains one bromine atom then: (a-l-bl n =(1 -I- 1)1 = 1 + 1
2S 30 35 40 45
(a + b)3 = a3 + 3a1b + 3atJ + Ji3 (11-11 3 = 1 +3+3+ 1
=
Thus the mass spectrum of CHBr3 would show M~ at mlz = 250 (CH79Br3), M t-2 at mlz = 252 (CH19B~lBr). M -I- 4 at m1z = 254 fCH19BrS1Br2) and M + 6 at mJz = 256 (CH81Br3) and the heights of the peaks will be in the approximate ratio 1 :3:3: 1 (Fig. 3O.4d).
202
Instrumental techniques
5(1
I.
55 60 6S 10 15 80 Il5 90 95
mi.
90
"ro
I: i" ., ro 10
81
I
o L,-Jl,-~~~~-'I--$L.c-"'I.J 20 30 40
Thus the mass spectrum of CH3CH2Br would show M~ at mjz = 108 (CH3CH219Br) and M -I- 2 at m/z -= 110 (CH3CH z81Br) and the heights of these peaks will be in the approximate ratio 1: 1 (Fig. 3O.4cl.
EX8mpfe 4: If the molecule contains three bromine atoms then:
•
:l5314,~
o
Example 2: If the molecule contains two chlorine atoms then: (a -I- bin = a" -I- 2ab + tJ2 = (3 + 1)2 = 9 t- 6 + 1 Thus the mass spectrum of M- at mJz"'"" 84 (CH 235 CI 21. (CH235CI37CIl and M t- 4 at and the heights of these approximate ratio 9: 6: 1 (Fig.
mi.
90 OJ
n
Thus the mass spectrum of CH 3CI would show M-I at niz = 50 ICH3 35 CI and M + 2 at m/z = 52 ICH:l7 CIl and the heights of these two peaks will be in the approximate ratio 3: 1 (Fig. 30.4a),
..
101520253035404550~60
50
. "'"
60 10 80 90 100 110
OJ
~
""
.5O 60
111 115
~ 40 30
~~
48
ii
m~21
:j;J
ol,"'~I-l.,~L.--.-'~~--':::ll:,::J 40 60 00 100 120 140 100 11lll2OD 220 24C Z60
101 Fig. 30.4 Mass spectra of: (a) CH~H2Br;
ld) CHBr3'
"'"
CH~I;
lb) CHzCb; fe)
Mass spectrometry
Box 30 2
Idealized fragmentation processes for the molecular ion 1M )
1. Il..cleavage: this involves breaking the ·next but one rondo to a hetero-atom (N, 0, Hal, etc.) in the functional group of a molecule. The following examples illustrate the general principles:
•
R-CIl2-"i~:
(,,,
R-('li-o;
•
H
(
R,
R- + CH-O(i)
•
I
H
I (M-R)
Ani)
R-C--ot
R-e-o;
•
I
R,
RO + e-Olil
•
I R,
M
I
H
R,
(M:,
(M)
2.
R- + Olrl[llllil IM-R)
()ni)
(
R,
•
1M;'
R-(.li-ot
I
()n0
R-CII2-fioJ '
I
R,
(M')
(M-It)
a-Bonds in alkanes: C-e bonds break in preference to C-H bonds and the most stable carbocation will be formed as the base peak. For example, 2,2-dimethylpentane will give the stable (CH 3 hC+ ~tion as the base peak instead of the less stable propyl cation CHJCHzCHi. ?ll CH)-f-CIlIDtplj
~"H)
ell J
ell)
---~•• ~'-f· CH,<.'Hf=Hl ---~•• CH)-r· i."H~Hpt)
<"'H J (M)
3.
Aromatic compounds: simple aromatics cleave to give a phenyl cation, mlz = 77, as the base peak which then loses ethyne to give m1Z:K. 51. Aromatics with CHz next to the ring give the stable tropylium cation m/z = 91, and then lose ethyne to m1z = 65.
rgrX
• @'X
•
rgrCHrX
•
rgr"""
•
©t
•
C4 H,m nrh; -.:II
$
CH ,
•
1fO/?'- 91
1M;,
(M)
-oc_ell
,./:._ n
(M:')
(M)
@$
0:
-IlC_<.l-l
•
0 mlt_M
4. ,B..caeavage or McL.afferty rearrangement: this is applied to molecules with a carbonyl group. If there is a hydrogen atom on the carbon atom four away from the carbonyl oxygen I)' carbon atom), a rearrangement of J the molecular ion occurs and a neutral alkene is lost from M • This process occurs concurrently with the a:;cleavage:
";eH0 c, ,
~
I
•
•
•
R
(M)
Instrumental techniques
203
Mass spectrometry
performance liquid chromatographs (p. 222) giving rise to the 'hyphenated techniques', GC-MS and L MS. respectively. In both techniques the separated compounds from the chromalogrdph arc sampled automalically and the mass spectrum of each compound is ret:orded. Comparison of the mass spcctm with a library in an 'on-board' database pennits idelllification of the components of the mixlure.
2{)4
Instrumental techniques
---0 Chromatograptly ilroften II three-wa;y compromise between: 1. sepal'8don of anatytes: 2. time of analysis;" 3.
volume of eluent.
Chromatography - introduction Chromatography is used to separate the individual components of a mixture on the basis of differences in their physic-al characteristics, e.g. molecular Si7-C, shape, charge, volatility, solubility and/or adsorption properties. The esscllIial oomponent~ of a chromatographic system are:
•
A stationary phase, where a solid, a gel or ,m immobilized liquid is held by a support malrix. A chromatographic bed: the stationary phase may be packf.'
•
bed. A detection system 10 visualize the test substances.
• •
•
~ecti('lg 8 separation method - rr: is often best 10 select a technique that involves direct interaction between the
substanceHil and the stlttionary phase
(e.g. lon.exchange Of affinfty chtom8t~), owing
to their
increased cepeeity 8tId resolution compared with other mettlods (e.g.. partition or gel permeetion chrpmatography) wtJere the anafytes are
not bound to the stationary ph&so.
The individual substances in the mixture interact with the stationary phase 10 different extents. as thcy are carried through the system, enabling separation (0 be achicved.
~ In a chromatographic system. those substances which intet"act strongly with the stationary phase will be retarded to the greatest extent. while those which show little interaction will pass through with minimal delay, leading to differences in distances travelled or elution times.
Chromatography is sub-divided according to the mechanism of interaction of the solute with the stationary phase.
Adso,pt;on c/".omatog,'aplty This is a form of solid-liquid chromatography. The stationary phase is a porous, finely divided solid which adsorbs molecules of the mixlure on its surface by dipole-dipole interactions, hydrogcn bonding and/or \'an der Waals' in(f.Taclions (I-.g. 31.1). The range of adsorbents is limited 10 polystyrene-based resins for non-polar molecules and silica, aluminium oxide and calcium phosphate for- polar molecules. Most adsorbenlS nUL'll be activated by heating to I 1(}'-120°C before use, since thcir adsorptive capacity is significantly decreased if water is adsorbed on the surface. Adsorption chromatography can be carried out in column (p. 217) or thin-layer (p. 216) form, using a wide range of organic solvents. Fig. 31.1 Adsorption chromatography (polar stationary phase).
Par/ition ch,'omalOgl'apltJ' This is based on the partitioning of a substance between two liquid phases, in this instance the stationary and mobile phases. Substances which are more soluble in the mobile phase will pass rapidly through thc system while those which favour the stationary phase will be retarded (Fig. 31.2). ]n nonnaJ phase partition chromatography the stationary phase is a polar solvent, usually water, supported by a solid matrix (e.g. cellulose fibres in paper chromatography) and the mobile phase is an immiscible, non-polar organic solvent, For reversed-phase partition chromatography the stationary phase is Instrumental techniques
205
Chromatography - introduction
....
-
~=~'J- ,~ "o
--
lheewrm
I flow.
Fig.31.2 Uquid-liQuid partilion chromatographV, e.g. reversed-phase HPLC.
Maximizing resolution in IEC - keep your columns as short as pOSSible. Once the sample components have been separated, they should be eluted 8S quickly as possible from the column in order to avoid band bf"Oodening resulting from diffusion of sample ions in the mobile phase.
sellllle cations oisplIal ndlie lXUllOOons Ie.g. H' IllI'CIlIre retllinBd twllfllI cd.ml stlllil;ray p/lasIl is B eatiorr~
,,,;,
Fig. 31.3 IOfHIXI:hange chromatography lcation exchanger).
a non*polar solvent (e.g. a elM hydrOC'.ubon, such as octadcx:ylsilanc) which ill chemically banded 10 a porous support matrix (c.g. silic'd), while the mobile phase can be chosen from a wkle r<1O£e of polar solwnts. IL~ualJy water or an aqueous buffered solution containing one or morc organic solvenls. e.g. acelonilrile. Solutcs interact with the stationary phase through non-polar inleractions and so the lcast polar solutcs clule la.st from the column. Solute retention and sepal""dtion arc controlled by changing the composition of lhe mobile phase (e.g. % v'v 1Icctonitrile). Reverse-phase high-performance liquid chrOmatography (RPIIPlC. p. 218) is used 10 sepaf"dle a broad nlllgc of nonpolar. polar and ionic moloculcs, including environmental (,'(Impounds (e.g. phenols) and pharmaceUlk:al compounds (e.g. .stt:rokls).
r
Jon-4!Xdllnge chronllltog"aphj' IECj Here, sepal""dtions arc carried oul using a (,'(Ilumn packed with a porous matrix which has a l1lrgc number of ioni:red groups on ilS surfaces. i.e. lhe .stationary phase is an ion-exchange resin. The groups may be calion or anion exchangers, depending upon their affinity for positivc or neglllive ions. The net charge on a pUl'licular resin depends on the pKA of the ioni",.able groups and Ihe pH of the solution, in accordana: wilh the Henderson-H.lssclbll1ch equation (p. 58). For most prliCliC"dl appliC"dtion5. you should ~1e<.1 the ion-exdlllnb'e resin and bulTer pH so thai the test substlUX:CS are strongly bound by c1e<:trostatie atlr"detion to the ion--exchange resin on passage through the system, while the other components of the sample arc mpidly eluted (Fig. 31.3). You can then clute the bound componellts by raising the salt concentration of the mobile phase. eilher stepwise or as a continuous gradient, so that exchange of ions of lhe same charge occurs at oppositely charged sites on the stationary phase. Weakl)' bound samplc molecules will elute first. while more strongly bound molecules will e1ule at a higher concenlration. Compuler-controllcd gradient formers arc a\~dilable; if two or more componenls canllot be resolved using a linear salt gradient. an adapled gradient can be used in which the rate of change in salt concentmtioll is decreased over the range where these components are expected to elute. IEC can be used to separate mixtures of a wide range of anionic and cationic compounds. Electrophoresis (Chapter 33) is an altemative means of separdting cruugcd molecules.
Gel permeation c:hl'omatography rGPCj or gel filtratio"
,-.......
lBrgB Il'lOIe<:lAe:s ilfB
from tte pores, pMngrBJidv
Fig.3J.4
206
Gel permeatiOfl chromatography.
Instrumental techniques
Here. the stationary phase is in the fonn of beads of a cross-linked gel containing pores of a discrete size (I-ig. 31.4). lbe size of the pores is controlled so lhal at the molecular level. the pores aet as 'gates' that will exclude large molecules and admit smaller ones (Table 3l.l). However. this gating errect is not an all-or-nothing phenomCllon: molecules of intennediatc size partly enter the pores. A column packed with such beads will have within it two effective volumes that are potentially available 10 sample molecules in lhe mobile phase. i.e. V" lhe volume surrounding the be"dds and Vii. the volume within the pores. If a sample is placed at the top of such a <.'Olumn, the mobile phase will cany lhe sample components down the column. but al dilTerent rates according to their molecular si7..c. A very large molecule w:ill have access to all of V, bUI to none of Vii- and will therefore elutc in the minimum possible volume (the 'void volumc'. or 1'0. equivalent to I'll. A very small molecule will have aQ:CSS to all of Vi and all of V". and therefore it has to pass through the total liquid volume of thc column (1'\. equivalent to
Chromatography - introduction
Using a gel permeation system - keep
your sample volume as small as possible.
in order to minimize band broadening due to dilution of the sample during passage through the column.
Table 31.1
Fractionation ranges of selected
GPC media M,
Medum
50-1000 1000-5000 1500-30000 4000-150 000
Sephadex G15, Biogel P-2 Sephadex G-25 Sephadex G-50, Biogel polO
5000-250000
20 000-1 500 000 60000-20000000
Sephadex G-,OO Sephadex G-200 Sephacryl S 300
Vi + Vii) before it emerges. Molecules of intermediate size have access to all of Vi but only part of Vii, and will elute at a volume betwccn 1'0 and VI> in order of decreasing size depending on their llCt:e:s.<; to Vjj. Cross-linked dextmns (e.g. Sephadex H), agarosc (e.g. Scpharose'~') and polyaerylamide (e.g. Bio-gel R') can be used to separate mixlurcs of macromolecules, particularly enzymes, antibodies and other globular proteins. Selectivity in GPC is solely dependent on the stationary phase, with the mobile phase being used solely to transporl the sample components through the column. Thus. it is possible to estimate the molecular mass of a sample componenl by calibrating a given column using molecules of known molecular mass and similar shape. A plot of e1ulion voluIllC (Ve) against 10£10 molecular mass is approximately linear. A further application of GPC is the gcnero:tl sepamtion of components of low molecular mass and high molecular mass, e.g. 'desalting' a protein exlmct by passage through a Scphadexl/l G-25 column is fa<;ter and morc efficient than dialysis.
Sepharose 4B
Affil1ity chromatograph)' EX8m~es
Biospecific molecules used in affinity chromatography include:
•
enzymes and inhibilorslcofactors/
• • •
hormones and receptor proteins; antibodies and antigens; complementary base sequences in DNA and ANA.
substrates;
Elution of substances from an affinity
system - make sure that your elulioll condilions do not affect the interoction
between the ligand and the stationary phase, or you may elute the ligand from the column. Remember that you cannot quantify 8 particular analyte without first identifying it the presence of a single peak on a ehromalOgram does not prove that a single type of analyte is present.
liglifld
""""~""
OlCJIeclJa bound by
"'<-----,~./ specific interaction with ligend
" \:» V; " ~ fit 0II1cr $OIWlS
6 6
now cI
are etJWd
lIKtIiIe p/Iilsc
Fig.31.5
Affinity chromatography.
Affinity chromatogmphy allows biomolecules to be purified on the basis of lheir biological specificity rather than by differences in physico-chemical properties. and a high degree of purification (more than WOO-fold) can be expected. It is especially ll<;cful for isolating small qU
The chromatogram A plot of the detector response present
lime is called a chromatogram (Fig. 31.6). The time from injcction of the sample until the peak elutes from the column is called the rctention time. I r • The amount of compound present for a given peak can be quantified by measuring the peak height or area (most useful) and comparing it with the response for a known amount of the smne compound. The aim of any chromatographic system is to resolve a number of components in a sample mixture, i.e. to cllSure that individual peaks do not overl
207
Chromatography - introduction
••
unretaincd component. The capacity factor for other components C'dn then be cakulatcd according to the following equation:
k'='r-to
131.1 J
to
'.
'-_J.C
"
0.5'
, ---'..'
- __ r>._-'--L.
......
Fig.31.6 Peak characteristics in a chromatographic separlltion. i.e. a chromatogram. For svmbols, see eqns 131.11 and 131.3).
Sq,llratioll fltelor, « 1bc sCjl.'\rdtioll factor, or selectivity. identifies when the peaks clute relative to each other. It is defined for two pC'dks as the rdtio of thc capacity factors (14 > ki):
14 ki
0:=-=
1',2 - /0
I"t
(31.2)
10
where fr.l and ',.2 arc the rctention times of peak and J'C'dk 2, respectively. If two peaks arc present the separation factor must be greater Ihan one to achievc an effective separation. Column emciel'ley (plate lIumblY), N An additional parametcr used to characterize a scparJ.tion systcrn is the plate number, N. It represents. in generdl term..<;, the narrowness of the peak and is often C'dleulaled as follows: N = 5.54(-"-)2 Il'lU
learning If'om eJ(J)erience - It you . , unable to sep8fl8te your moIecute ustng a par1iciJler nwthod, do not regard this as • fellure. 'but hwtead think about what this tells voo about e~ the sutwtaneeM or the sample.
[31.3J
where I, is the fCIelllion time of the peak and "'0.$ is its width at one-half of its height (Fig. 31.6). For a compound emerging from a column of length L, the number of theoretical plait'S, N, can be expressed a.~: L
N=1i
(31.4)
where H is the plate heighl (or height equivalent to a theoretical plate). In genCl"dl, chromatogI"'dphic columns with larger values of /Ii give the narrowest peaks and generally bettcr sep
-
M)'mmclry factor, A. The plate !lumber. /Ii, assumes that the peak shape is Gaussian, but in practice this is rare. It is more likely that the peak is asymmet.Jiall, i.e. it 'tails'. 'Iltis is quantified using the asymmetry factor, A., calculated a.<; shown in Fig. 31.7. A \'Crttcal line is drawn Ix:tween the pc'
Fig.3J.7 Peak asymmetry.
(31.5]
In general, A. values between 0.9 and 1.2 are acceptable. If A. > I peak tailing is in evidence; if A. < I peak fronting is evident. The pmctical impact ofpcak tailing or fronting is that adjacent peaks arc not as well sepamted as they would be if they were symmetrical, lcuding to difficulties in peak quantitation.
Resolution Jt is often important to be able to separate a large number of compounds. A visual inspection of the chromatogram (Fig. 31.8) will usually indicate whether the separation is appropriate. It is dcsimble that the valley between 208
Instrumental techniques
Chromatography - introduction
adjoining peaks rcrums to the base linc and resolmion is a qUllnlilalivc measure of the scpamtion. The influence of k', a and Non rcsolulion. R, is shown ill the following expression:
,fN
II k+1
a-I
R=-x--x--
4
[31.6]
a
Three conditions must be satisfied in order to achieve some degree of resolution: I.
--
2. 3.
Peaks have to be retained on the column (II > 0). Peaks have 10 be separated from C"dch OIher (a > I). The column must develop some minimum value of N.
48121620242832
Fig.31.8 A mullicomponenl chromatogram. Separation of many compounds. some ~11 resolved, e.g. peaks at 12-13 mins. and others that are 1'01, e.g. peeks at 24--25 mil'S.
These different effeclS and their influence 011 resolution arc ~howll in Fig. 31.9, using high-performance liquid chromatogrdphy (HPLC) liS an example (see Chapter 32). I.
2.
I I. initial
2 1 . WIY "
I J. irc'lIISl! N
I 4. incf9ase 11
A/\
3.
v
Fig.31.9 Influence of Ir'. II and Non resolution.
4.
Initial conditions result in inadeqwlle separation of the two components. Effect of varying the C"dpacity factor, IC: from an initial mobile phllse of 50% methanol: 50% water (v/v) two scenarios arc possibile. Firstly. on the left·hand side. the innuence of increasing the percentage of organic solvent (70% methanol: 30% water) allows a faster throughput, but the peaks arc unresolved. Secondly, on thc right-hllnd side, the percentage organic solvent is reduced (30% methanol: 70% watcr) allowing the components to remain in the system for a longer time. giving some separafion. but causing peak broadening. This is the easiest ehange to m:lke and will affect resolution. As a guide. a twO"" to three-fold change in IC will result for e-dch 10% changc in mobile phase composition. Effect of increasing the plate number, N: reducing the particle size of the HPLC packing from 5~m 10 3~m allows a more cfficicnt scpamtion. It should be noted that the retention times of the peaks arc not altered (from the initial chromatogram) provided the stationary phase is not altered. Alternatively N can be increased by placing t:olumns in series with onc another. Ho\.\-"CVcr, you should nole from cqn [31.61 that R has a square-rOOt dependence on N, i.e. a four-fold increase in N is requirctl to double R. Incrca.sc separation faCIO!", a: resolved peaks can be obtainctl by changing the mobile phase (e.g. methanol to acetonitrile) or the column stationary phase (e.g. C u, to Cd. Unfortunately this is thc lC'dst predictable approach.
Detectors After separating the components of the mixture it is necessary to detect them. A dt:9Cfiplion of a rangc of detectors suitable for chromatography is described in Chaptcr 32. As chromatography is often used as a quantitative technique it is essential to be familiar with the following tcm\s:
• •
Ulliw::rsal defector: this I'CSpOnds to all compounds eluting from lhe column. irrespective of their composition. Selectil't!/specific detector: this responds to certain clements or functional groups. This is a useful approach if the components of the mixture are known.
Instrumental techniques
209
Chromatography - introduction
•
• •
210
Instrumental techniques
Sensitivity: the ratio of detector signal to s
-------0
Gas and liquid chromatography Gas chromatography
,..
t8lTict
Fig. 32.1 Components of a GC system.
Types of capil&ary columns
• Well·coated open tubular {Wean column: liquid stationary phase on inside wall of column.
• Suppon-coalcd open tu~18r (SCan column: liquid stationary phase Coaled on solid Support attached to inside
In gas ehromatogrdphy (GC), a ga~us solute (or the vapour from a volatile liquid) is carried by the gaseous mobile phase. In gas-liquid partition chromatography, the stationary phase is a 1I01l-volatile liquid coated on the inside of the column or on a fine support. In gas-solid adsorption ehromatogr.lphy, solid particles that adsorb the solute a<..1 as the stationary phase. The typical components of a gas chromatograph are shown in Fig. 32.1. A volatile liquid is injected through a septum into a heated port. which volatilizes the sample. A gaseous mobile phase carries the sample through the heated column, and the separdled components arc detccted Mnd recorded. Two types of columns are available: packed and C'.lpillary. Open tubular capillary columl\.<; olTer higher resolution. shoner anal}'sis time and gl'C'dter sensitivity than packet1 columns.. but have lower capacity for the sample.
Sample injection Samples al'C injected onto the 'top' of the column. through a sample injection port containing a gas·tight septum. The two common sample injection methods for capillary GC are: I.
wall of column.
•
Porous layer open tubular (PLOn: solid stationary phase on inside walt of column.
Applications of gas-liquid chromatography - GLC is used 10
separate volatile. non-polar compoonds; substances with polsr groups must be converted to less polar derivatives prior to analysis. in order to prevent adsOfplion on the column. resulting in poor resolution and peak tailing.
2.
Spllt/splitlcs..<; injector: in the split mod<: only a portion of the injccted Sl.lmple (typically, I part in 50) reaches the column. The fCSl is vented to wasle. A splil injector is used for con<;entrated samples (> 0.1 mgmL- 1 for FlO; sec p. 215). In the splitlcss mode all the sample volume injected passes through to Ihe column. It is used. in Ihis mode, for trace samples « 0.1 mgmL- 1 for FlO). Cold on-column injector: all lhe sample is injected onlo the column. It is used for thermally unstable compounds and high~boiling solvenlS.
I n both C'dSCS a syringe (I ~L) is used to inject the sample. Examples of cach Iype of injection systCtn arc shown in Fig. 32.2. The procedure for injection of a sample is shown in Fig. 32.3. In Fig. ]2.3a the syringe (see p. 213) is filled \\~th the S
The co{,mm
Analysing compounds by GC in the split mode - make sure no hazardous
materials enter the laboratory atmosphere through the split vent. A charcoal split-vent trap may be required to eliminate potential hazards.
Modern GC uses capillary columns (internal diameter O.l-Q.5mm) up to 60m in length. The stationary phase is generally a cross-linked sili<..'One pol}<mer, coaled as a thin film on the inner wMIl of the flL<;OO silica (SiOll capillary: al normal operating temperdl ures, this behaves in a similar manner to a liquid film, but is far more robust. Common stationary phases for GC are shown in Fig. 324. The mobile phase ('carrier gas') is usually nitrogen or helium. Sclcclivc separation is achieved as a result of Ihe difTerential partitioning of individual compounds between the C'.llTier gas and silicone jX)lymer phases. lbe separation of most organic molecules is influenced by Instrumental techniques
211
Gas and liquid chromatography
Box 32.1
How to prepare a set of five calibration solutions in the concentration range 0-10 JIg ml
1
ImgL 'I Assuming that we are starting with a l000,ugml- 1
1000,ug 3 ml x20x 10- mL=20,...g2-chlorophenol
stock solution of a particular organic compound, e.g.
2-ehlorophenol. then you will need the following: 6 x 10.00mL grade A volumetric flasks and a syringe {o-loo.DO JILl.
132.11 so 20,...g 2-chlorophenol was placed in the 10.00ml volumetric flask. So,
1. Ensure that all the glassware is clean (see p. 13). 2. Add ~ 9 ml of «gante solvent e.g. dichloromethane. to al0.00mL with dichloromethane.
3. Quamitativetv transfer 20.00 pl of the stock solution into the 10.00ml volumetric flask. Inject the solution from the syringe below the surface of
the dichloromethane. Then, dilute to 10.00mL with dichloromethane.
4. What is the concentration of this new solution? Remember that we started with an initial 1000 fig ml- 1 2·chlorophenol stock solution.
;~~~ =
5. Similarly transfer 0, 40.00, 60.00, 80.00 and 100.00 Jll volumes into separate volumetric flasks and dilute to 10_00mL with dichloromethane and label as 0,4.6,8 and 10 JIg mL _1 2-chlorophenol calibration solutions. 6. Take the 0, 2, 4, 6, 8 and 10jlO ml-1 2-chlorophenol calibration solutions to the chromatograph for analysis.
-
""~
.....
1\
to plJ"g&
1M'll
I
I
\
IIII~,-
~
."
I)
(
syringe needle
132.2)
You now have a 2,ug mL -1 calibration solution of 2chlorophenol.
PTfE\k
IIII
2/19 mL -1 2-chlorophenol
g1assme.
.
.:1
,.rio
-" ..., =-,.
--"""'.
"'~
,,,.., 1 carrier
=
r
tospl~
inside tt.: 0\W1
Ll
err ........-- symge IlOedIe
w
~
Fig.32.2 Sample introduction in GC: (a) splitlsplitless injector; (bl on-column injector.
the tCtnperature of the column. which may be constanl during the analysis ('isothcnnal' - usually SD-2S0"C) or, more commonly, may increase in a preprogrammed manner (c.g. from SOT Lo 2S0~C at lOoe pcr minute).
212
lnstrumernallechniques
Gas and liquid chromatography
")
lal30 m >( O.Z5 rom Ld.
• ,
, •
, 5
1
II
" "
2
'I
,<)
Fig. 32.3 sample injection in GC. la) Fill the syringe. (bl wipe clean the outside of the syringe needle. (el place the syringe noodle into lhe injector and ldl depress the plunger on the syringe 10 inject the s.omple.
:1
• • " " '" "
10llll.~
~
!he IlIasl poiIII' bcnIed phase. USOO kr boiIPJI polO! SllJ&lIIiDnIlu.ents. jllltrDll:uft pmcb;ts. etc.l. ~
_ : DB-I, HP-I,l!tlI-l
lbt 30 m" 0.53 mmld.
,
• •
"
•
, ,
5
"
'"
'"
Fig. 32.4 Common stationary phases fOf" capillary GC.
• • "
" '"
Fig. 32.5 Influence of GC column internal diameter on separation: 1. phenol; 2. 2-
chlorophenol; 3. 2-nitrophenol; 4. 2,4dimethylphenol; 5. 2,4-dichlorophenol; 6. 4chloro-3-methylphenol: 7. 2,4,6-tnchlorophenol; 8. 2,4-dinitrophenol; 9. 4-nitrophenol; 10. 2methyl-4,6-dinitrophenol; 11. pentachlorophenol.
Selecting an appropriate column for capillary GC is a difficult task and one which is u~a1ly left to the technician. Howc\'cr, it is imporlant to be aware of some general issues and what influence they can have on the separation. The column inlemal diameter can affect both resolution and speed of analysis. Smaller internal diamelers columns (0.25 mm i.d.) can provide good resolution of early eluting peaks (Fig. 32.5a). However. the problem is that the analysis limes of the eluting componcl}(s may be longer and thai the linear dynamic range (see p. 210) may be restricted. In contmst. Instrumental techniques
213
Gas and liquid chromatography
60 m xO.25lTfTl i,d.
30 m ,,0.25 rml i,d,
1
, 1
,
• , •
, 8 11 12
'It •
,
,.,
" "
8
"
•
8
" "
, ,8
" "
20
28
"
" "
-
Fig.32.6 Influence of column length on analvsis time. Analysis of phenols under isothermal conditions: ,. phenol; 2. o-cresol; 3. 2,6-
xylenol; 4. /Kresol; 5. n-cresol; 6. o-ethylphenol; 7. 2,4-xylenol; 8. 2,5-xylenol; 9. 2,3·xylenol; 10. p-ethylphenol; 11. m-ethylphenol; 12. 3.5-xylenol; 13. 3.4-xylenoL 30 m" 0.53 rrm i.d.
60 ml< 0.53 Im'i.d.
J
I
.I.
•
8
"
20
28 min
•
8
" "
20
"
28
Fig. 32.7 Influence of column length on analysis time. Analysis of bacterial acid methyl esters under temperature-programmed
conditions.
Solvent selection in GC - choose a solvent with a boiling point at least 20 "C below that of the first eluting compound. Thicker stationary-phase films provide longer retention and higher peak
capacity.
larger internal diameter columns (0.53 mm i.d.) provide less resolution for carly duting compounds (Fig. 32.5b). but this is reflccted in shoner analysis limes and a greater linear dynamic mngc (see p. 210). This type of colmnn may provide sufficient resolution for the analysis of complex mixtures. Fig. 32.5 illustrates the effects of column internal diameter. Another important colmnn effect is the length of lhe column and the influence this can have on the resolution of eluting components. It was previously shown (p. 108) tbat resolution was influenced by II, a and N. Substituting eqn [31.4] into eqn [31.6] produces the lollowing equation:
[32.3J
214
Instrumental techniques
Gas and liquid chromatography
... . air
-+FlD~
The importance of this equation can be 5ho"-11 by considering the influence on resolution. R, of column length. L. Under isothermal analysis condilions, i.e. the same column temperature, the retention of eluting compounds is more dependent upon column length. For example, doubling the column length doubles the analysis limes and increases the resolution by 41%. This is shown in Fig. 32.6 for the analysis of phenols. In contrast. undcr tempen'lturepro1!1ammcd analysis. e.g. 130"C to 250°C at 4~Cmin-l, the retention time of eluting componcnts is more dependent on temperature. For example, doubling the column length has minimal elTect on analysis times. This is shown in Fig. 32.7 for the analysis of bacterial acid methyl csters.
GC detectors
gesflow
"'" '""'"
Fig. 32.8 Components of a flame ionization
delector (FIOI.
Detectors - the most appropriate detector depends on the type of chromatography
and the application: ideally, the detector should show high sensitivity, a low detection limit and minimal noise or drift. These terms are defined in Chapter 31.
coJcctor electrode (anode)
_ _ ECDbodv
t Fig. 32.9 Components of an electron capture detector (ECD).
The output from the GC column is monitored by a detector. The most communly used detcctors for GC analysis of organic molecules arc as follows. The flame ionization detector (FlO) is particularly useful for the analysis of a brood nmge of organic molecules. " involves passing the ex.it gas stream from the column through a hydrogen flame that has a potential of more than 100V applied across it (Fig. 32.8). Most organic compounds, on passage through this flame. produce ions and electrons that create a small current across the electrode", and this is amplified for measurement purposes. The FID is very sensitive (typically down to ~ 0.1 pg), with a linear responsc over a wide concentration range. One dnlwback is that the sample is de"troyed during analysis. The thermal conductivity detector (TCD) is based on changes in the thennal conductivity of the b'
215
Gas and liquid chromatography
_dO
ioo source
~
0
- ).../
0
...
.J11
'.:... ...:..
""""0
;;r"';r;
II'
/
Fig. 32. 10 Schematic diagram of 8 GeMS instrument.
Compounds eluting rrom the column are bombarded by electrons (electron impact. EL mooc) causing fragmentation and production of charged species. These charged species are separated by the mass spectrometer on lhe basis of their mass-to-c::harge ratio. Ions passing throUgh the mass spectrometer arc detected by an electron mulliplier tube. The mass spectrometer can be used in two modes: total ion lind selected ion monitoring. In the fonner mode. the complete mass spectrum of each of the components of the mixture eluting from the column is recorded. In the taller mode, only ions of specified massto-charge ratios are detected. Selected ion monitoring offers increased sensitivity and selectivity.
liquid chromatography Using TLC plates - do not louch the surface of the TLC plate with your fingers. Hold them by the edges to prevent
contamination. Using lLC plates - a plate 12 em x 5 em) will hold three sample 'spots',
The basic chromatographic system comprises a stationary phllse (adsorba.m), usually alumina, silica gel or cellulose, through which a mobile phase travels (elutes). Sepllration of a mixture of compounds is achieved by a combination of the differing 'adsorption' and solubility characteristics of the components on the stationary phase and in the mobile phase respectively. Liquid chrom:nogmphy is used both as an analytical method to determine the complexity of mixtures and the purity of compounds, and as a preparntive system for the separation of mixtures. liquid chromatography is divided into two general types: I.
\1IIl1(;~SS (CM'l"
nc ... lOOml. beaker
2.
two halves 01 filtef papel'
-
0
3nTn 01 ehenl
Fig. 32.11 The developing tank for TLC.
Solutions of the mixture - the mixture must be applied to the plate as a solution. If the solvent for the solution is not the same as the eluent to be used. you must evaporate the solvent from the plate,
Thin-Jape" chromatography The essential components of a TLC system are: •
before placing it in the eluent.
•
•
• Fig. 32.12 How to make micropipettes.
216
Instrumental techniques
Thin-Illyer chromatogrclphy (TLC): in which II glass or plastic pilite is coated with II thin lllyer of the stationary phase and the mobile phase ascClldl' the plate by capillary aL1ion. TlC is essentially an analytical tool and prepamtive TLC has been largely superseded by nash chromalOgraphy. Column chromatography: in which the stationary phase is packed imo a glass column and the mobile phase is passed down the column, either by gravity (gravity chromatogrnphy) or under low pressure from a pump or nitrogen cylinder (flash chromatography). These are the preparcltive systems.
The stationary phase comprising the layer of adsorbant on a solid backing the chromatoplate. Aluminium or plastic-backed chromatoplalcs are now the nonn having replaced glass plates, which needed to be prepared 'inhouse', The chromatoplates (20cm x 2Ocm) can be cut down to the more useful size (2cm x 5em) for analytical work, using a guillotine. The adsorbant often contains a nuorescent compound (ZnS) to en.. .tble visualization of the compounds after elution. The development tank: for plmes of (2cm x 5cm) a dean, dry beaker (IOOmL) covered with a watch-glass is idelil. The eluting solvenl should be about 3 mm deep and filter paper should be placed in the tank to saturale the lank atmosphere with solvent vapour (Fig. 32.11). The application system: a micropipette or a microsyringe to place Ihe solution of the mixture 011 the chromatoplate. Micropipellcs (Fig. 32.12) are the more common and Box 32.2 gives the instruclions for their preparation. The eluent: finding the eluent. which will give the best separation of the components of the mixture. is by experiment - you may need to try
Gas and liquid chromatography
Box 32.2
How to make micropipettes for TLC
1. Heat the middle of an open-ended mefting point tube at the tip of the hot name of a microbumer until it begins to sag. If the melting point tube is sealed at one or both ends, carefully break off the sealed end(s) wearing gloves for protection.
2. Quicklv remove the tube from the flame and pull gentty. forming a short capillary. Do not pull the tube while it is in the flame.
Finding an eluent - l:l medium-polarity solvent such as dichloromethane (DCM) is generally a good starting poim.
Table 32. 1 The erUiropic series of solvents for
chromatography Non-polar
Ught petroleum (b.pt. 4Q--60 C) Cyclohexane
•
3. AHow the tube to cool and then tweak in the centre of the capinary. You now have two micropipettes. tf the capillary is too long, break it near to each end and immediately dispose of the fine waste glass into the broken-glass bin. Do not leave the waste glass on the laboratory bench.
4. Make at least 10 mlcropipettes and store them in a plastic-capped sample tube for future use.
several solvents of differing polarity (Table 32.1) or mixtures of solvents to find the best eluent. A visualjzation system to be able to see colourless separated componenls on Ihe chromatogrclln. If the plate contains a nuorescer. it can be viewed under UV light ()_ = 154 nm) in a special box or cabinet. The ZnS in the stationary phase Iluorcsces green. whereas the 'spots' of separated compounds appear dark. Alternatively, the plate can be placed in a seak"d jar containing a few iodine crystals. The iodine vapour stains the plate light brown and the 'spots' dark brown.
Toluene
You can express the movement of an individual compound up the TLC plate
Oichloromethane Oiethvlether (etherl
Ethyl ethanoote (ethyl acetate) Propanone (acetone) EUlanOH:; acid lacetic acid)
Polar
Methanol
~
Safety note Great care must be taken when using UV light. Do not look directly at the UV source and wear gloves if you put your hands into the UV cabinet.
in terms of its Rr (relative frontal mobility) value. where:
Rr = distance moved by compound/distance moved by solvent
[31.4]
The Rr value is a constant for a particular substance and eluent system on a specific stationary phase. but variations in chromalOgraphic conditions adsorbant, eluent (in particular solvent mixtures), tempef'tlture and atmosphere make the application of Rr values to absolute identification rather problematical. Usually an authentic sample is run alongside the unknowns in the mixture (Fig. 32.13) or on top of the mixture - 'double spotting' - as shown in Fig. 32.14, 10 enable identification. The general procedure for running a TLC plate is described in Box 32.3.
Column chromatography
..rt+- cOIlllOnent spot "H+-lXIITIpOIIl!nt spot
This is used for the preparative scale sepamtion of mixtures of compounds. There are many variations in delail of equipment and technique such as column type, column packing, sample application and fraction collection. many of Which are a mailer of personal choice and apparatus available. Typical arrangements are shown in Fig. 32.15 and for a detailed description of all these variations you should consult the specialist texts such as ErTington (1997. p. 163), Harwood el al. (2000_ p. 175) and Furniss er al. (1989, p. 2(9).
"H+-
mi>
alfl'II)lnm
spot
--I=::::::::::J- base ~ne
Fig. 32.13 A thin-18yer chromatogram.
Gmvity chromatography is used to scpamte the components of a mixture which have a difference in R r value of at least 0.3. Rash chromatogmphy, because of the smaller size: of the adsorbant particles, is more effective separating mixlUres componenls of l:::.Rr = 0.15 and is also faster.
~ Before attempting a preparative mixture separation by column chromatography, you must always analyse the mixture by TlC to establish the stationary phase and solvent parameters for effective separation and to determine the R, values of the components. Instrumental techniques
217
Gas and liquid chromatography
Box 32.3
How to run a thin-layer chromatogram
1. Prepar. the TLC development tank using 8 dean, dry beaker. as shown in Fig. 32.11, and allow it to equilibriate for 10 minutes.
9. Put the nd on the tank and anow the eluent to r;se up the plate to about 1 em from the top of the plate.
2. Prepare the plastic-backed nc plate by drawing a
10. Remove the chromatopbrte from the tank and
fine line (tha base tinel in pencil about 1 em above
quickly mark the height reached by the eluent, using a pencil.
the bottom. Take car. not to scrape oH any of the stationary phase. Put three pencil dots on the base
line - one in the centre and the others equidistant on either side, but no etoser than 3 mm 10 the
edges of the plale.
3. Ois50tve the mixture 11-3mgl in two or three drops of 8 suitable volatile sotvent (dichloromethane or ether are the most common). in 8 small sample tube.
4. Dip the tip of 8 mk:ropipette into the SDtution and capillary action will draw the solution into the pipette. 5. Touch the tip of the mK:r'opipette onto one of the pencil spots on the base line. Capillary action will draw the solution from the micropipette onto the plate as a spot. Do not allow the spot be more than 2 mm in diameter. Put the micropipene back into the sample tube containing the mixture.
6. Spot other samples (e.g. reference compounds) onto the plate as appropriate. using a new micropipett8 in each casco 7. Evaporate the solvent from the plate by waving it in the air, holding the plate by the edges.
8. lower the plate into the developing tank. holding it by the edges and make sure that the eluent docs nat cover the base line. If it does. discard the plate. prepare another and empty some eluent from the tank.
11. Wave the chromatoplate in the air to evaporate
the eluent from the plate. 12. Visualize the chromatogram by either placing the dry chromatoplate in the UV cabinet and using a
pencil to draw round the spots (remember to wear gloves when your hands are in the UV cabinetj or putting the plate in the Iodine Jar until the dark spots develop.
Problems with thin-u.yer chromatogt'aphy Overloading the chromatoplate with sample. nc is an extremely sensitive technique and it is easy to put too much sample on the plate. The sample solution must not be too concentrated and you must not repeat applications of solutions on the same spot. The result is non-separation of the mixture and a 'smear" up the plate. Dilute the solution or don't put so much on the plate. Putting the spots of sample too close together. The separated spots '~eed' into each othet' and you can't tell from which sample they originate. Putting the spots too close to the edge of the plate. This results in in8CCUr8te II, values (sPOts travel Jastet' up the edge of the platel and ·~eeding'. Contamination, which produces unexpected spots. Make sure all apparatus ts clean, solvents are clean and use a fresh micropipette for each application.
High-performance liquid chromatography Safety note Iodine vapour is toxic and the iodine tank should be stored in the fume cupboard.
High-performance liquid chromatognlphy (HPlq uses high pressure to force the mobile phase throUgh a closed column packed with micrometresized particles. 'l1lis allows rgpid separation of complex mixtures. Several opemling modes of HPLC are possible (see also p. 205). These are: •
UV visualization - if you use an eluent such as toluene, which absorbs in the UV region. you must allow all the eluent to evaporate or you will see only a dark. plate.
218
InSTrumental techniQues
•
Normal phase (f'.rpHPlC): the sample should be soluble in a hydrophobic solvent. e.g. hexane. and should be non-ionic. The mobile phase is non-polar while the stationary phase is polar, e.g. silica, cyano, amino. Reversed phase (RPHPLC): the sample should be soluble in water or a polar organic solvent, e.g. methanol, and should be non-ionic. The mobile phase is polar while the stationary phase is non-polar, e.g. CI8 (ODS), Cs (octyl), phenyl.
Gas and liquid chromatography
•
• R,oIA=O.452 R, of B '" 0.476
•
1--+-blHlQA
,.,
B
• • •
• • •
"A+B B
A A+B B
,,'
'"
Fig. 32.14
•
Oouble-sponing technique: (a) compounds A and B with close R, values; lbl figure'S' of double spot shows that A and 8 are different; (e) single spot for double spot shows that A and B could be the same.
Size exclusion chromoloJ:!.rdphy (SEC): this is used when the ITutjOT dilTerence between compounds in a mixture is their molecular weight. It is normally used for compounds with molecular weights grealer than 2000. The mobile phase should be a stomg solvent for the sample. Aqueous SEC is called gel filtr
sex
The essential components of an HPlC system are a solvent delivery system. a method of sample introduction. a column.. a detector and an associaled reAdout devK:e (Fig. 32.16).
So!J'/?nt c1elh'er)' !jj'stem
,"1-_ .... ':
,
,
,,
This should fulfil certain requirements:
--
-
saro
"""""
[al
""'.
lhl
Fig. 32. 15 Column chromatography: lal gravity chromatography; (bl flash chrorrultography.
HPlC ito a versatile form of chromatography, used with a wide variety of stationary and mobile phases, to separate indi....idual compounds of II particular dass of molecules on the basis of size, polarity, solubility or adsorption
• •
It should be chemically inert. It should be capable of delivering a wide now-rate mnge. It should be able to Withstand high pressUI"CS. It should be able to deliver high now-rale precision. It should have a low internal volume.
•
It should provide minimum flow pulsation.
• •
•
Although several systems are available that meet these requiremellls. the most common is the reciprocating or piston pump. The choice of solvent delivery system depends on the type of separ
lsocratic sep.'lffition: a single solvent (or solvent mixture) is used throughout the analysis. Gradient elution separation: the composition of the mobile phase is altered using a microprocessor-eontrolled gradient programmer. which mixes appropriate amounts of two difTerent solvents to produce the required gradient.
The main advalllages of gr
characteristics.
Sample introdllction
Preparing samples for HPlC - fitter aU samples through either II a.2Jlm or II
The most common method of sample introduction in HPLC is via a rotary valve, e.g. a RheOOyne~ valve. A schematic diagram of a rotary valve is shown in Fig. 3217. In the lood position, the sample is introduced via a syringe tQ fill an extcmalloop Qf volume 5. 10 or 20IlL. While this occurs. the mobile phase passes through the valve to the column. In the inje<.'1 position, the valve is rotated so that the mobile phase is diverted thrOUgh the
O.451'm filter prior to injection.
Instrumental techniques
219
Gas and liquid chromatography
..
"'-
collector
,
""'""
..g~~~;A fillR
'"
Fig. 32.16 Components of an HPlC system.
Fig. 32.18
san1Jle inject Fig.32.17 Schematic diagram of 8 rotary valve.
Always use the highest purity solvents.
Safety note Always dispose of organic solvent waste in accordance with laboratory procedure (never down the sink).
r '"'
.- O-Sj- (CH zll1 %
ODS bonded 1Jr0l4l
I
CH,
I
-- 0 - Si -
Cli:l
enj.cil!lPOO silarols
I
0', .- OH
l.flIr9aIed silanols
Fig. 32.19 A C18 station8ry phase.
220
Instrumental techniques
Ibl
'"
Sample injection in HPLC,
sample loop. thereby introducing a reproducible volume of the sample into the mobile phase. The procedure for injection of a sample is shown in Fig. 32.18. In Fig. 32.18a the syringe (see p. 11) is filled with the sample/standard solution (typically I mL). -nlcn the outside of the syringe is wiped dean wilh a tissue (Fig. 32.18b). The syringe is placed into the Rheodynel! injC(;tor of the chromatogmph while ill the 'load' position (Fig. 32.18c) and the plunger on the syringe is depressed to fill the sample loop. Finally, the position of the Rheodyne rR valve is switched to the 'inject' position to introduce the sample into the chromatograph (Fig. 32.J8d) and then the syringe is removed from the injection valve. The procedure for the preparntion of a series of calibrntion solutions is shown ill Box 32.1.
Tile column This is usually made of stainless steel, and all components. valves. etc., are manufactured from materials which c
f1PLC detel"tors Most HPLC systems are linked to a continuous monitoring detector of high sensitivity. e.g. phenols may be detected spectrophotometrically by
Gas and liquid chromatography
Using silica-based HPlC columns - these are limited to a pH range of 2-8 (preferably 3-71. Allow pH the bonded phase may be removed; at high pH the
sitica particles may be dissolved.
UV cutoff for organic solvents: 195nm 190nm 205nm 190nm
Hexane
Acetonitrile Methanol Water
-.. C;
P
I
~
/
i"""" typical/~10
/
/ / i
C :J path lengll1 lYJ)icaly lOmm
Fig. 32.20 UV detector cell for HPLC.
time lminl
Fig.32.21 Diode array detector absorption spectra of the eluent from an HPLC separation of a mixture of four steroids, taken every 15
seconds.
monitoring I he absorbance of the eluent at 280 nm as it passes through a flow cell. Other detectors can be used to measure changes in iluorescence, current or potelUial, as described below. Most detection systems are non-deslru<.1ive, which merills that you can collect eluent with an automatic fmelion colloclOr for further study. UV/visible detectors are widely used and have the advclntages of versatility. sensilivity and stability. Such detectors are of two types: fixed wavelength and variable wavelength. Fixed-wavelength detectors are simple to use, with low operating costs. They usually contain a mercury lamp as a light source. emitting at several wavelengths betv.ecn 254 nm and 578 run; a particular wavelength is selected using suitable cutoff filters. The most frequently used wavelengths for analysis of organic molecules are 254 nm and 280 nm. Variable wavelength detectors use a deuterium lamp and a continuously adjustable monochromator for wavelengths of 190-600nm. For both types of detector. sensitivity is in the absorbance range 0.001-1.0 (down to <':::: I ng), with noise levels as low as 4 )( 10-5. Note that sensitivity is partly influenced by the path length of the flow cell (typically 10rom). see Fig. 32.20. Monitoring at shortwavelength UV (e.g. below 24Onm) may give increased sensitivity but decreased specificity. since many organic molecules absorb in Ihis range. Additional problems with short-wavelength UV detection include instrument instability. giving a vdriable base line. and absorption by components of the mobile phase (e.g. organic solvents. which often absorb at < 210 nm). An important development in chromatographic monitoring is diode arrdY detection (DAD). The incidenl light comprises the whole spectrum of light from Ihe source. which is passed through a diffraction grating and thc diffracted light detected by an array of photodiodes. Typical DAD can measure the absorbance of each sample component at 1-lOnm interv
221
Gas and liquid chromatography
Overcoming interference with
fluorescence detectors - use
D
dual flow
~Il
to offset background fluorescence due to components of the mobile phase.
Maximizing sensitivity with fluorescence detectors - the concentration of other
sample components, e.g. pigments, must not be so high that they cause quenching of fluorescence.
Optimizing electrochemical detection the mobile phase must be free of any compounds that might give a response; all constituents must be of the highest
Mass spectrometry (p. 204) used in conjunction with chromatographic methods can provide a powerful tool for identifying the components of complex mixtures. e.g. phannaceuLicals. One drawback is the limiled cap.;lcilY of the mass spectrometer - due to its vacuum requirements - comjXlred with the volume of material leaving the chromalOgrnphy column. Similarly, in HPLC, devices have been developed for solving the problem of large solvent volumes, e.g. by splitting the eluent from the column so only a small fmction reaches the mass spectrometer. The computer-generated outputs from Ihe mass spectrometer are similar to chromalograms oblained from other methods, and show peaks corresp::l1lding to the elution of parLicular components. However. it is then possible to select an individual peak and obtain a mass spectrum for the component in thai peak to aid in its identification (p. 2(0). This has helped to identify hundreds of componenls present in a single sample. including flavour molecules in food, drug metabolites and water pollutants.
purity.
Recording and intel'pJ'eting chromatogranu Interpreting chromatograms - never assume that a single peak is a guurantee of purity: there may be more than one
compound with the same chromatographic characteristics.
Problems with peaks - non-symmetrical peaks may result from column overloading, co-elution of solutes, poor
packing of the stationary phase. or
interactions between the substance and the support material.
For analytical purposes., the detector output is usually connected 10 a computer-based data acquisition and analysis system. This consists of a personal computer (PC) with data acquisition hardware 10 convert an analogue detector signal 10 digital format, plus software to control the data acquisition process, store the signal information and display the resulting chromatogram. The software will also delect peaks and calculate their retention times and sizes (areas) for quamitalive analysis. The software often incorpomtes functions to control the chromatographic equipment. enabling automatic opcwtion. In sophisticated systems, the detector output may be compared with that from a 'Iibmry' of chromatograms for known compounds. to suggest possible identities of unknown sample peaks. In simpler chromatographic systems, you may need to usc a chart recorder for detector output. Two important scltings must be con<;idered before using a chart recorder: I.
2.
The base-line reading - this should be set only after a suitable quantity of mobile phase has passed through the column (prior to injection of the sample) and stability is established. The chart recorder is usually set a little above the edge of the chart paper grid. to allow for base-line drift. The detector range - this must be set to enSure that the largest peaks do not go off the top of the chart. Adjustment may be based on the expecled quantity of analyte. or by a trial-and-error process. Use the maximum sensitivity Ihat gives intact peaks. If peaks are still too large on the minimum sensitivity, you may need to reduce the amount of sample used, or prepare and analyse a diluted sample.
Interpreting chromatograms Make sure you know the direction of the horiwntal axis of the chromatogram (usually, either volume or time) - it may run from right to left or vice versa - and make a note of the detector sensitivity on the vertical axis. Ideally, the base line should be 'flat' between peaks. but it may drift up or down owing to a number of factors inclUding: • • 222
Inslrumental techniques
changes in the composition of the mobile phase (e.g. in gradient elution, p.219); tailing of material from previous peaks;
Gas and liquid chromatography
•
cany-over of material from previous samples; this can be avoided by efficient cleaning of columns between runs - allow surticient time for the previous sample 10 pass through the column before you introduce the
•
loss of the stationary phase from the column (column 'bleed'), caused by
next sample: extreme elution conditions; Avoiding problems with air bubbles in liquid chromatography - always ensure that buffers are effoctively degassed by vacuum treatment before usc. and regularly clean the flow cell of the
•
detector. Degassing yoor mobile phase solvent - this is an impOrtant step end the best approach is to prepare the solvent composition (e.g. 50:50v/v mettlllnol :water, forisocratk: RPHPLC) and then filter through a O.22prn porosity filter using 8 Buchnerflask arrangement (p. 281.
air bubbles (in liquid chromalogr.lphy); if the buffers used in the mobile phase are not effectively degassed, air bubbles may build up in the flow cell of the detector, leading 10 a gradual upward drin of the ba~ line, followed by a sharp fllll when the accumulilted air is released. Small air bubbles that do not becOme tnlpped may give spurious small peaks as they PllSS through the detector,
A peak dose to the origin mlly be due to non-retained sample molecules., flowing at the same r,He liS the mobile phase, or to artefacts, e.g. air (Ge) OJ" solvent (HPLC) in the sample. Wh..'uever its origin, this peak can be used to mel1sure the void volume and dead time of the column (po 207). No peaks from genuine sample components should appear before this type of pellk. l:>eaks can be denoted on the basis of their elution volume (used mainly in liquid chromatogn~phy) or their retention times (m'linly in Gq. If the peaks are not narrow and symmetrical. they may contain more than one component. Where peaks are more curved on the trdilinll side compared with the leading side (peak tailing. p. 2(8). this may indicate too great an association between the component and the stationary phase, or overloading of the column.
Optimizing chromatographic separations In an ideal chromatographic analysis the sample molecules will be completely separated, and detection of components will result in a series of discrete individunl peaks corresponding to each type of molecule. However. 10 minimize the possibility of overlapping peaks, or of pe
•
the selectivity, as measured by the relative retention times of the two components (p. 2(8), or by the volume of the mobile phase between the peak maxima of the two components afier they have passed through the column; this depends on the ability of the chromatographic method 10 separdtc two I.:Omponents with similar properties: the band-broadening properties of the chromatographic system, which influence the width of the peaks; these are mainly due to the effects of diffusion.
TIle resolution of two adjacent components can be defined in terms of 1<'. a and N. using eqn (31.6). In practical terms. good resolution is achieved when there is a large "distance' (either time or volume) between peak maxima. and the peaks are as narrow as possible. The resolution of components is also affected by the relative amount of each substance: for systems showing low resolution. it can be difficult to resolve small amounts of a pRrticular component in the presence of larger amounts of a second component. If you cannot obtain the desired results from a poorly resolved chromatogram, other chromatographic conditions, or evtn different methods. should be tried in an attempt to improve resolution. For liquid chromatography, changes in the following factors may improve resolution: Instrumental techniques
223
Gas and liquid chromatography
•
• •
•
Stationllry-phase particle size - the smaller the particle, the greater the area available for partitioning between the mobile phase and the stationary phase. This partly accounts for the high resolution observed with HPLC compared with low-pressure methods. The slope of the salt gradient in eluting IEC columns, e.g. using computer-controlled adapted gradients. In low-pressure liquid chromatography, the flow rate of the mobile phase must be optimized because this influences two band-broadening effects which are dependent on diffusion of sample molecules: (i) the flow rate must be slow enough to allow effective partitioning between the mobile phase and the stationary phase: and (ii) it must be fast enough to ensure that there is minimal diffusion along the column once the molecules hllve been separated. To allow for these opposing influences, a compromise flow rate must be used. If you prepare your own columns, they must be packed correctly, with no channels present that might resull in uneven flow lind eddy diffusion.
Quantitative analysis Most detectors and chemical assay systems give a linear response with increasing amounts of the test substance over a given 'working range'. Alternative ways of converting the measured response to an amount of substance arc: Quantifying molecules - note that quantitative analysis often requires assumptions about the identity of separated components and that further techniques may be required to provide information about the nature of the
molecules present, e.g. mass spectrometry (see Chapter 30).
When using external standardization. samples and standards should be
analysed more than once, to confirm the
•
•
External standardization: this is applicable where the sample volume is suffICiently precise 10 give reproducible results (e.g. HPLC). You me-
reproducibility of the technique.
When using an internal standard, you should add an internal standard to the
sample at the first stage in the extraction procedure. so that any loss or degradation of tcst substance during purification is accompanied by an equivalent change in the internal standard, as long as the extraction characteristics of the internat standard
and the test substance arc very similar.
224
InSlrumenlal techniques
,~
peak area (or height) of test substance peak area (or height) of reference subsmnce
132.5]
Use this response factor to quantify the amount of test substance (Ql) in a sample containing a known amount of the reference substance (Ql)' from the relationship: Ql =
[peak area (or height) of test substanceJ x Qr [peak area (or height) of reference substance) /.
132.6)
Internal standardi..-.ation should be the method of choice wherever possible, since it is unaffccled by small variations in sample volume (e.g. for GC microsyringe injection). The internal standard should be chemically similar to the test substancc(s) and must give a peak that is distinct from all other substances in the sample. An additional advantage of an internal standard which is chemically related to the test substance is that it IT41.y show up problems due to changes in detector response. incomplete derivati7..ation. etc. A disadvantage is that it may be difficult to fit an internal standard peak into a complex chromatogT'"
----------10
Electrophoresis Electrophoresis is a sepamlion technique based on the movemelll of charged molecules in an electric field. Dissimilar molecules move at different rates and the components of a mjxture will be separated when an electric field is applied. It is a widely used technique, particularly for the analysis of complex mixtures or for the verification of purity (homogeneity) of isoiftted molecules. While electrophoresis is mostly used for the separation o[ charged macromolecules. techniques are available for high-resolution separations, e.g. capillary electrophoresis. o[ small molecules such as amino adds. anions and catecholamines. TIle electrophoretic mobility of a charged molecule depends on: •
• •
•
Net charge - negatively charged molecules (anions) migrate towards the anode (+). while positively charged molecules (cations) migrate tmvards the cathode (-); highly charged molecules move faster towards the electrode of opposite charge than those with lesser charge. Size - frictional resistance exerted on molecules moving in a solution means that smaller molecules migrate faster than large molecules. Shape - the effect of friction also means that the shape of the molecule will affect mobility, e.g. globular proteins compared with fibrous proteins, linear surfacwnts compared with micellular surfaetants. Electrical field strength - mobility increases with increasing field strength (voltage), but there are pr.l.ctical limitations 10 using high voltages, especially due to heating effects.
The combined influence of net charge and size means that mobility is detennined by the charge-ta-density or the charge-to-mass ratio, according to tl1e fonnula: qE
,
[33.11
p.=-
where q is Ihe net charge on the molecule, r is the molecular radius and E is the field strength. Most types of electrophoresis using supporting media arc simple to c<,rry out and the apparatus can be easily constructed. although inexpensive equipment is conunercially available. High-resolution techniques such as twodimensional electrophoresis and capillary electrophoresis require more sophisticated equipment. both for separation and analysis (see later). Simple e1t:ctrophoretic separations can be performed either vertically (fig. 33.1) or horizontally (Fig. 33.2). The electrodes are nonnally made of platinum wire, each in its own buffer compartment. In vertical electrophoresis, the buffer solution forms the electrical contact between the electrodes and the supporting medium in which the sample separation takes place. In horizontal ekctrophoresis e1ectric-dl conlact can be made by bulTersoaked paper 'wicks' dipping in the bulTer reservoir and laid upon the supporting medium. The buffer reservoir normally contains a divider acting as a barrier to diffusion (but not to electrical current), so that localized pH changes which occur in the region of the electrodes (as a result of electrolysis, p. 232) arc not transmitted to the supporting medium or the sample. Individual samples are spotted onto a solid supporting medium containing Instrumental techniques
22S
Electrophoresis
0gel cast betwllel1 ~ss plal:es. Wells 8"9
.""'
formed in
".""
..
Fig. 33.1 Apparatus for vertical slab eleclrophoresis lcomponents move downwards from wells. lhrough the gel matrix).
lark wiIlllid
lliroctionm 8nSyt8
SJDde
rrwgralioo
(£ll::::::,;o::::::,::::::,r~~
o""""
Fig. 33.2 Agarose gel electrophoresis.
buffer or are applied to 'wells' formed in the supporting medium. The power pack used for most types of electrophoresis should be capable of delivering approltimalely 500 V and approximately 100mA.
The supporting medium The cffects of convection currents (n.'sulting from the healing effect of the applied electric field) and the diffusion of molecules within the buffer solution can be minimized by carrying out the electrophoresis in a porous supporting medium. This contains buffer electrolytes and the sample is added in a discrete location or zone. When the electric field is lIpplicd, individual sample molecules remain in sharp zones as they migrate at different rates. After separation. post-eieclrophoretic diffusion of selected molecules (e.g. proleins) C"dn be avoided by "fixing' them in position on Ihe supporting medium, e.g. using trichloracelic acid (TeA). The heat generdK'd during electrophoresis is proportional to the square of Ihe applied current and 10 the electrical resist<mce of the medium: even when a supporting medium is used, heat production will lead to zone broadening
226
Instrumental techniques
Electrophoresis
Definition Ohm's law: V=:= IR where V = voltage, I = current and R = resistance.
by increasing the rate of diffusion of sample componenlS and buffer ions. Heal denaturation of certain sample types may also o<x:ur, e.g. proteins. Another problem is that heat will reduce bulTer viscosity, leading to a decre'
Inert media - these provide physkal support and minimize convection; separation is based on charge density only (e.!!. cellulose acetate). Porous mc'dia - these imroduce molecular sievin!! as an additional effect: their pore size is of the same order as the size of molecules being separated. restricting the movement of larger molecules relative to smaller ones. Thus, separation depends on both the charge density and the size of the molecule.
in essence, an incomplete form of
With some supporting media, e.g. cellulose acetate, a phenomenon called electro-endosmosis or electro-osmolic flow (EOI') occurs. This is due to the presence of negatively charged groups on the surface of the supporting medium, attracting calions in the electrophoresis buffer solution and creating an electrical double layer. The cations are hydrated (surrounded by water molecules) and when the electric field is applied, they arc auracted towards the cathode, creating a flow of solvent that opposes the direction of migration of anionic molecules towards the anode. The EOI' can be so great that weakly anionic molecules may be carried towards the cathode. Where ncccSSJITY, EOF can be avoided by using supporting media such as a£.arose or polyacrylamide, but it is not always a nindrance to electrophoretic separation. Indeed. the phenomenon of EOF is used in the high·resolution technique of capillary electrophoresis.
electrolysis. since the applied electric field is switched off well before sample molecules reach the electrodes.
Capillary electrophoresis
Definition
Electro-osmotic flow - the osmotically driven mass flow of water resulting from the movement of ions in an
electrophoretic svstem.
Definitions
Electrophoretic mobility - the rate of migration of a particular type of molecule in response to en applied electrical field.
Understancing electrophoresis - this is.
The technique of capillHry electrophoresis (CE) combines thc high resolving power of electrophoresis with the speed and versatility of HPLC (p. 218). The technique largely overcomes the major problem of carrying out electrophoresis without a supportin!! medium, i.e. poor resolution due 10 convection currents and diffusion. A capillary tube has a high surface-area-to-volume ratio, and consequently the heal generated as a result of the applied electric current is rapidly dissipated. A further advdntage is that very small sample volumes (5IOnL) can be analysed. The versatility ofCE is demonstrated by its use in the separation of a range of molecules, e.g. lImino acids, proteins, nucleic adds. dru£,S. vitamins, organic acids and inorganic ions: CE can even separate neutral species, e.g. steroids. aromatic hydrocarbons. The components of a typical CE apparatus are sho\\1) in Fig. 33.3. The capillary is made of fused silica and externally coated with a polymer for mechanical strength. The internal diameter is usually 25-50 pm. a compromise between efficient heat dissipation and the need for a light path that is not too short for detection using VVIvisible spectrophotomelry. A gap in the polymer coaling provides a window for detection purposes. Samples Inslrumental techniques
2Z1
Electrophoresis
are injected into the capillary by a variety of mC'dOS, e.g. electrophoretic loadinl; or displacement. In the former, rhe inlet end or the capillary is immersed in the s,,1.mple and a pulse of high volta&'C is applied. The displacement method invoh'es forcing the sample into the capillary. either by
applying pressure in lh~ sample vial using an inert gas. or by introducing a vacuum at the outlet. The detectors used in CE arc similar 10 those used in
..
.....,
chromatography (p. 220), e.g. UV/visiblc spectrophotomeuic systems. Fluorescell<;C detection is more sensitive. but lhis may require sample
o
_...
""-
dlll'~
Fig. 33.3 Components of a capillary elcctrophorosis system.
denvitization. Electrochemical and conductivity detection arc also used in some applications, e.g. conductivity detection of inorganic cations such as Na+. K+.
EOF, described above. is essential to the mOSt commonly used types of CEo The existence of EOF in the c..'lpillary is the result of the net negative charge on the fused silica surface al pH values over 3.0. The resulting solvent now towards the cathode is greater than the attraction of anions towards the anode. so they will now towards the calhode (note that the detector is situated at the cathodic end of the capillary). The gre-Mer the net negative charge on an anion. the greater is its resislance to lhe EOF and the lower its mobility. Separated components migrate lowards the cathode in the order. (I) cations. (2) neutral species, (3) anions.
Capillary zone electrophoresis (CZE) This is the most widely used fonn of CE, and is based on electrophoresis in free solution and EOF, as discussed alxwe. Separations are due 10 the charge-to-mass ralio of the sample oomponems, and the techniqlle can be used for almost any type of charged molecule, and is especially useful for phannaceutical compound separation and confirmation of purity.
Micellar electrokinetic chromatography IMEKC) This technique involves the principles of both electrophoresis and chromatography. Its main strength is that it CM be used for the separation of neutral molecules as well as char~ ones. This is achieved by including surfactants (e.g. SDS. Triton X· I00) in lhe electrophoresis buffer at concentrations that promote the formation of spherical micelles, with a hydrophobic interior and a charged. hydrophihc surface. When an electric field is applied. these micelles will tend to migrate with or against the EOF depending on their surface charge. Anionic surfaetants like SDS are attracted by the anode. but if the pH of the buO'er is high enough to ensure thal the EOF is faster than the migration velocity of the micelles. tile net migration is in the direction of the EOF. i.e. towards the cathode. During this migration, sample components partition between the buffer and the micelles (acting as a pseudo-stationary phase); this may involve both hydrophobic and e1ectrosmlic interactions. For neutral species it is only the partitioning effect that is involved in separation; lhe more hydrophobic a sample molecule. the more it will inter"<1ct with lhe micelle. and the longer will be its migration time. since the mia:lle resists the EOF. The versatility of MEKC enables it lO be used for separations of molecules as diverse as amino acids and polycydk hydrocarbons.
228
Instrumental techniques
-------0
Electroanalytical techniques Electrochemical methods are used to quantify II broad range of different molecules, including ions, gases. metabolites and drugs.
~ The basis of all electrochemtcal analvsis is the tranS'Definitions
Oxidation - loss of electrons by an atom or molecule (or gain of 0 atoms, loss of H atoms, increase in positive charge, or decrease In negative charge). Reduction - gain of electrons by an atom Or molecule (or loss of 0 atoms, gain of H
fer of electrons from one atom 0' molecule to another atom or molecule in an obligately coupled oxidation-reduction reaction la redox reaction). H is conveniem 10 separate such redox reaclions inlo by convention. each is wriUen as:
IWO
half-reactions and.
reduclioll
atoms, decrease in positive charge or
oxidi7..ed fonn+e1eclron(s) (ne-)
increase in negative charge),
4
reduced form
(34.1]
oxidaliOfl
You should note thai the half-reaction is reversible: by applying suitable conditions, reduction or oxidation can take pillce. As an example, a simple redox reaction occurs when metallic zinc (Zn) is placed in a solution containing copper ions (Cu~+), as follows: Cu~+ + Zn --. Cu + Zn 2+
..-
direclion 01(; fl~
rifleG
anode
KCI SIIIt bridge
r-..
copper
D Clllhooe
Fig. 34.1 A simple galvanic electrochemical cell. The KCI salt bridge allO\lllS migration of ions between the two compartments but prevems mixing of the two solutions.
Definition Galvanic cell- an electrochemical cell in
which reactions occur spontaneously at the electrodes when they are connected eX1ernally bv a conductor, producing
electrical energy.
[34.2J
The half-reactions are (i) Cu l + + 2e- ----0 Cu and (ii) Zn 2+ + 2e- ----0 Zn. The oxidizing power of (i) is greater than that of (ii). so in a coupled system, the latter half-reaction proceeds in the opposite direction to that shown above• i.e. as Zn - 2e- ...... Zn 2t . When Zn llnd Cu electrodes are placed in separate solutions containing their ions, and connected electrically (Fig. 34.1). electrons will Oow from the Zn electrode to Cu 2+ via the Cu electrode owing to the difference in oxidizing power of the two half-reactions. By convention. the electrode potential ofany half-reaction is expressed relative to that of a standard hydrogen electrode (half-reaction 2H-I- + 2e- ...... H2) and is called the standard electrode potential, E". Table 34.1 shows the values of E" for selected half-n:actions. With any pair of half-reactions from this series, electrons will now from that having the lowest electrode potential to that of the highest. E" is detennined at pH = O. It is often more appropriate to express standard electrode pOlcntials at pH 7 for biological systems, and the symbol E'" is used: in all circumstances. it is important that the pH is clearly stated. The arrangement shown in Fig. 34.1 represents n simple galvanic cell where two electrodes serve liS the interfaces between a chemical system and an electrical system. For analytical purposes. the magnitude of the potential (voltage) or the current produced by an electrochemical cell is related to the concentration (strictly the activity. o. p. 48) of a particular chemical species. Electrochemical methods offer the following advantages: • • •
excellent detection limits, and wide operating range (10- 1 to 10-11 mol L-I); measurements may be made on very small volumes (ul) allowing small amounts (pmol) of sample to be measured in some cases; miniature electrochemical sensors can be used for certain in vil'o measurements, e.g. pH. glucose, oxygen content.
Instrumental techniques
229
Eleclroanalylicallechniques
Table 34.1 Standard eloclrode potentials· (EO) for selected half-reactions
Potentiometry and
ion~selective electrodes
Operating principle!; EO 8t 25 C
Half-reaction
IV!
ell -+- 2e- :=' 2 c/O, + 4HO + 4e ;:0 2H,O Br~ r2e ... 2Br
-1-1.36 +1.23 +1.09
Ag' 1-6 ;=!Ag
+0.80
Fe'" + 0-
;:0
Fe"
-1-0,n
Cu'-
+ 29
Hg~Ct2
='" Cu
+ 29 ",. 2Hg + 20
AgCI+e- =Ag+CI Ag(S2031~- of
e- "'" Ag
2H' -I 28
H2
:='
I- 2S10~
Agl+e-=Ag+1 PbSO, + 2e :=' Pb + ScY.
Cd" +2e- 0"" Cd Zn 2! +2e- "'Zn
I.
a ·scnsing' electrode, the half·cell potential of which responds
+{).01
10 changes in the activity (concentration) of the substarx.'c to be mcasured; the most common type of indicator electrodes are ion-selective electrodes (ISEs); a 'referencc' electrode. Ihc potential of which does nol change. forming the second half of the cell.
+0.00 -0.15 -0.35 -(1.40
To assay a particUlar analyte. the potential difference between these electrodes is mmsured by an mV metcr (e.g. a standard pH meter). Reference electrodes for polet1tiometry arc of three main types:
-0.76
I.
+0.5-4 of 0.34 +-0.27 +0.22
IJ+2e ;=:031
These syslems involve galvanic cells (p. 229) and are based on measurement of the potential (voltage) difTcrL'nce between two elet.1rodes in solution when no net current nows between them: no nct clectrochemical reaction occurs and measurcments are made under equilibrium conditions. These systems include methods for measuring pH, iOlls, and gases such as CO} and NH 3. A typical polentiomctric cell is shown in Fig. 34.2. It cOlllains two electrodes:
2.
"From Milano Df al. (1978).
Using a calomel electrode - always ensure that the KCt solution is saturated bV checking that KCI crystals are present.
2.
3. pH/mVmeler
The standard hydrogen electrode. which is the reference half-ccll electrode, defined as 0.0 Vat all temperatures, agolinsl which values of are cxpreslied. H 1 gas at I atmosphere pressure is hubbled over a platinum clectrode immersed in an acid solUlion with all aClivilY of unity. This electrode is rarely used for analytical work, since it is U1lSlable and othcr reference electrodcs are easier to construct and use. The calomel electrode (Fig. 34.3), which consists of a pl.lste of mercury covered by a coat of calomel (HS2C1~), immersed in a saturated solution of KG. The half-reaClion Hg~Ch --l 2e- -- 211g + 20- gives a stable standard electrode potential of +0.24 v. The silver/silver chloride electrode. This is a silver wire coated wilh AgCl and immcrsed in a solution of constanl chloride conccl1lration. The halfreaction AgCl + e- ...... Ag + CI gives a stable, standard electrode potential of --l 0.20 V.
e
~ lon-selective electrodes IISEs) are based on measurement of 8 potential across a membrane which is selective for analyte.
IHI sdtlliOl'l
seOlSlng eIet:lrode
"J--
_______=.J
~,,,,,,
~tIl'rer
Fig. 34.2 Components 01 a potentiometric cell.
Using ISEs. including pH electrodesstandards and samples must be measured at the same temperature, as the Nernst equation shows that the measured potential is temperature dependent.
230
Instrumental techniques
8
particular
An ISE consists of a membrane. an internal reference cketrode. and an intemal refercnce electrolyte of fixed activity. The ISE is immcl1'.Cd in a sample solution that COJllains the analyte of interest. along wilh a reference electrode. The membrane is chosen to Imve a specific affinity for a p.1rticular ion_ and if activity of Ihis ion in lhe sample differs from lhat in the refeI"Cnce electrolyte. a potential develops across the membrane that is dependent on the ratio of these aClivities. Since the potentials of the two reference electrodes (internal and external) are fixed, and the internal e1ectrolytc is of conslant activity. the measured potcntial, E, is dependent on the membrane potential and is given by the Nernst equation: E= K
RT
+ 2.303 =F
log raj
134.3J
where K represents 3 constant potential which is dependent on the reference electrode, z represents the net charge on the analyte, [aJ the activity of analyte in lhe sample and all olher symbols and constants have their usual meaning (p. 70). For a series of standards of known activity, a plot of E against log [aJ should be linear over the working range of lhe elcctrode. with a slope of 2.303RT/=F (0.059 V at 25~C). Although 15Es strictly measure actil'ity, the
Electroanalytical techniques
el.etriclll 1..6
fill" oole and .ubbllf bun~
_
$mel lIoIe in
potential differences can be approximated 10 concentration as long as (i) the analyte is in dilute solution (p. 48), (ii) the ionic strength of the calibration standards matches th..u of the sample. e.g. by adding appropriate amounl$ of a high ionic strength solution to the standards, and (iii) the elTCCI of binding to sample macromolecules (e.g. proteins, nucleic acids) is minimal. Potentiometric measun:ments on undilulOO biological nuids, e.g. K ~ and Na+ le''els in plasma, tissue Ouids or urine, 3re likely to give lower values than n.1.me emission spectrophotometry, since the lauer procedure measurcs tOlal ion levels. rather than juS! those in aqueous solution. All of the various types of membrane used in ISEs operate by ineorpornting the ion to be analysed into the membrane. with the tlccom'''1.nying csttlblish· ment of a membrane potl.:nlial. The scope of electrochemic.1.1 tlnalysis has been extendcd to measuring gases and non-iorlic compounds by combining ISEs with gas-permeable membranes. enzymes. and even immobili7Xd bacteria or tissues.
ime.lclle ~l1'lIJ;J-- I(Cl clYStals - - sinl..ed gllln dill<
Fig. 3-4.3 A calomet refenmce elOClrode.
Glass membra/Ie electrodes The most widely used ISE is the glass membrane electrode for pH measurement (p. 57). The membrane is thin glass (.50Jlm wall thickness) made of silica wbK:h contains some NtI+. When thc membnlOe is soaked in water. a thin hydrated layer is romled on the surrace in which negative oxide groups (Si-O-) in the glass act as ion-exchange SilCS. If the elcctrode is placed in an tlcid solution, H+ exchanges with Nat in Ihe hydrated layer, producing an external surface potential: in alkaline solution, H+ moves out of the mcmbrane in exchange for Na+. Since the inner surface potential is kept constant by exposure 10 a fixed aClivity of H!, a consiSICIll,
Gus-sellsillg glass electrodes
Fig. 34.-4 Underlying principles of a gassensing electrode.
Here, an ISE in contact with a thin external layer of aqueous electrolytc (the 'filling solution') is kept close to the glass mcmbrane by an tldditional, outer membrane thai is selectively penneable to the gas of interest. The arrangement ror a CO2 electrode is shown in Fig. 34.4: in this ease Ihe ollter membrane is made of CO2-permeable silicone rubber. When CO2 gtls in the sample selcc1ively diffuses across lhe membrane and dissolves in the filling solution (in this case an aqucous NaHCOl{NaO mixture), a change in pH occurs owing to the shift in the equilibrium:
134.41 Using CO 2 electrodes - applications include measurement of blood PC0 2 and in enzyme studies .....tune CO2 is utilized or released: calibration of the electrode is accompiished using 5%vIV and 10%vIV mixtures of CO: in an inert gas ; equilibrated against the measuring solution.
The pH change is 'sensed' by the internal ion-selective pH electrode. tlnd its response is proportional to the partial pressure of CO! of the solution (PC02). A similar principle operates in the NH J electrode. where a Teflon I~ membmne is used. and the filling solution is NH4 0.
Liquid and polymer memhmne electrodes In these types of ISEs, the liquid is a water-insoluble viscous solvent containing tI soluble ionophore. i.e. an oTgtlnic ion exchanger, or a neutral carrier molecule, that is specific for the analytc of interest. When this liquid is Instrumental techniques
231
Electroanalyticaltechniques
inWff\il1 solution
external (samplel solution Fig. 34.5 Underlying principles of a liquid
membrane ion-selective electrode. A+ _ analyte; C = neutral carrier ionophore; Em = surface potential; membrane potential = Em(inlernall - Em(externllil.
soaked into a thin membrane such as cellulose acetate, it becomes effectively immobilized. The arrangement of analyte (A~) and ionophore in relation to lllis membrane is shown in Fig. 34.5. The potential on the inner surface of the membrane is kept constant by maintaining a constant activity of At in the internal solution, so the potential change measured is that which results from At in the sample interacting with the ionophore in the outer swface of lhe membrane. A relevant example of a suitable ionophore is the antibiotic valinomycin. which specifically binds K t. Other ionophorcs have been developed for measurement of. for example. NH;. Ca1+. CJ-. 1n ilddition. electrodes have been developed for organic species by using specific ion-pairing reagents in the membrane that inleract with ionic fonns of the organic compound. C.g. with drugs such as 5,5-diphenylhydantoin.
Solid-state mcmhmnc elcctrodcs These contilin membranes made from single cf)'Stills or pressed pellets of salts of the allillyte. The membrane material must show some penneability to ions and must be virtually insoluble in water. Examples include: Definition Jonophore - a compound that enhances membrane permeability to a specific ion: an ionophore may be incorporated into an ISE to detect that ion.
•
•
the fluoride electrode, wllich uses LaF3 impregnated with Eu2+ (the latter to increase penneability to r). A membrane potential is set up when Fin the sample solution enters spaces in the crystal lattice; the chloride electrode, which uses a pressed pellet membrane of A~S and Ab>C1.
Vottammetric methods
Definitions ElectrolytiC cetl- an electrochemiC
application of an external vottage grealer than the SpOntBneous potential of the cell. Ehlctro'ysts - a non-spontaneous chemical reaction resulting from the application of a potential to an electrode.
Voltammetric methods are based on measurements made using an electrochemical cell in which electrolysis is occurring. Voltmnmelry, sometimes also called amJX:rornetry, involves the usc of a potcntiill applied betw{."Cn Iwo electrodes (the working electrode and the reference electrode) to cause oxidation or reduction of an electroactive analyte. The loss or gain of electrons at an electrode surface causes current to flow, and the size of the current (usually measured in rnA or pAl is directly proportional to the concentration of the elcctroactive analyte. The materials used for the working electrode must be good conductors and electrochemically inert. so that Ihey simply transfer electrons 10 and from species in solution. Suitable materials include Pt, Au. I-Ig and glassy carbon. Two widely used devices that operate on the voltammetric principle are fhe oxygen electrode and the glucose electrode. These are sometimes referred to as ampcrometric sensors.
Oxygen electrodes The Clad, (Rank) oxygen electrode These instruments measure oxygen in solution using the polarographic principle, i.e. by monitoring the current Oowing between two electrodes when a voltage is applied. The most widespread electrode is the Clark type (Fig. 34.6), manufactured by Rank Bros. Cambridge. UK. which is suitable for measuring O 2 concenlrations in cell. organelle and enzyme suspensions. Pt and Au electrodes are in contact with a solution or electrolyte (nomtal1y saturated KG). The electrodes are separated from the medium by a Teflon!! membrane. permeable to O 2 • When a potential is applied across the electrodes, 232
Instrumental techniques
Electroanalytical techniques
n.~-SI
&l!jtlsterl
,ubber '0' ring inc;ublIlion
chamber
~
nrrer ba. ~§~fmagneliC rubbar'U
ring
[~~~ll~!~'lr~~~!:'} to c8ftnll - r~ lellon .,""- circulilr salurated ......lJr... e ,silWr, KQ IUlltfron
..... ...
Fig.34.6 Transverse sectioolhrough a Clark (Rank) oxygen electrode.
Ihis generates a current proporlional to the 0.: concentration. The reaclions can be summed up as: 4Ag - 0 41\ g1 +4e
saw'alel! I
.,
~
silver ~
II_~_ GilliS elec... ocla
+ 2e- + 2H+ ~ H 20 2
(al silver anode) (in elCClrolyte solution; O2 replenished by diffusion from test solution) (al platinum cathode)
bodv
Oxygelt probes rubbeor'O' rinG
Teflon "",mbranl!
p1Sbnum
csthode
Fig, 34.7 A Clark-type oxygen probe,
C1ark-IYPC oxygen elea.rodes are also available in probe form for immersion in lhe lesl solution (Fig, 34.7) e.g. for field studies, allowing direct measuremelll of oxygen stalus in .~itll, in contrast to chemical assays. The main poinl 10 nole is Ihal Ihe solulion must be stirred during measuremenl, to replenish Ihe oxygen consumed by the electrode ('boundary layer' effect).
The glucose electrolle BIosensor - 8 device for measuring a substance. combining the seHlctivity of 8 bioiogical reection with that of it sensing
_ode.
The glucose eleclrode is a simple lytle of biosensor, whose basic design is shown in Fig. 34.8. It consists of a Pt electrode, overlaid by two membranes. Sandwiched between these membranes is a layer of the immobilized enzyme glucose oxidase, The outer membrane is glucose pemleable and allows glucose in the sample to diffuse through to the glucose oxidase la)-'er, where it is converted to gluconic acid and H 20 2 _ llle inner membrane is selectively pemlcable to H 2 0 2 , which is oxidized to O2 at the surface of Ihe Pt electrode. The current arising from this release of electrons is proportional 10 the glucose concentration in the sample within the range 10- 7 to 10-) mol L-1. Electrochemical detectors used in chromatography operate by vol tammetric principles and currenls are produced as the mobile phase flows over elCClrodes set at a fixed potential: to achiel'e maximum sensitivity, this potential must be sct at a 1el'e1 that allo\\-'5 elcctrochemical reactions to occur in all analytes of interesl. Instrumental techniques
233
Eleclroanalyticallechniques
Coulometric methods Here. the charge required 10 elecrfolysc a sample completely is mC
Cyclic voltammetry The ttthnique provides qualitative infonnalion about e1eclrochemical reac1ions, e.g. the redox behaviour of compounds and the kinetics of electron Fig. 34.8 Underlying prInciples of a glucose electrode.
potllf'lial M
Fig. 34.9 A typical cyclic voltaml"llOgram for a
reversible 0
234
+ ne- ..... R redox process.
Instrumental techniques
transfer reactions. In practice, a triangular p()Icmial waveform is applied linearly to Ihe working electrode in a unstirred solution. After a few seconds, the ramp is reversed and the potemial is returned to iLS initial value. The process may be repeated several times. The resulting plol of current versus potential is tenned a cyclic voltammogram. Fig. 34.9 shows a typical cyclic voltammogram for a reversible redox couple after a single potential cycle. It is assumed lhal the oxidized fonn. 0, is the only species preSCtll at [he start. Therefore, the first scan is towards the (more) negative direction, commencing at a value ,""ere no reduction occurs. As the applied potential approaches the characteristic t.'" fOl" the redox process, a C<1.tholic currenl starts to increase. up to a maximum. After excet:ding the potemial at which the reduction process takes place. the direction of the potential current is reversed. During trus stage, reduced molc<:ules R. generated during the initial process, are reoxidized back to 0, resulting in an anodic peak.
Radioactive isotopes and their uses Examples 1~C. ~c and I:C are three of the isotopes of carbon. About 98.9% of naturatlv occurring carbon is in the stable tie form. t~c is also a stable IsotoPe but it onlv OOC\Jrs at 1.1% nl!lturalabundance. Trace amounts of radioacttve l:C ara found naturally; this is 8 negatron-emitting radiotllotope (see Table 35.21.
The iSOtopes of a particular clement have the same number of protons in the nucleus but a different number of neutrons, giving them the same proton number (atomic number) but a different nucleon number (mass number, i.e. number of protons + number of neutrons). Isotopes may be stable or radioactive. Radioactive isotopes (radioisotopes) disintesrate spontHneo~ly al random to yield radiation and a decay product.
Radioactive decay There are three forms of radioactivity (Table 35.1) arising from thrcc main types of nuclear decay: Table
35.1 Types of radioactivity and their properties
Radiation Alpha (ct) Beta (PI GammaM
Suitable shielding
Range of maximum energies (MeV·)
Penctrmion range In air (m)
material
4-8
0.025-0.060 0.150--16
Unnecessary Plastic (e.g. PerspeX")
0.01-3 0.03-3
Le,.
1.3-13~~
~Note that 1 MeV _ 1.6)( 10-1~ J. • ~Distance at which radiatioo intensity is reduced to half.
E"""""eo
•
mRe decays to wRn bV loss of an atpha
particl&, as follows; 2::Ra ..... ~ Rn + ~He2i
•
I·e shows beta decaV. as follows:: t:C_'; N+r 'l2Ne decays by positron emission. as follows:
nNa ..... ~Ne -t- p+ !i6Fe decays bV efectron capture and the
produetiocl of an )Hav. as follows; ~Fe -4 ~Mn + Jc
The dec8v of 22 N8 by positron emission (1J~lleads to the production of l!I)""rav when the positron til annihitated on cotljaion with an electron.
•
Alpha decay involves !.he loss of a particle equiv<\lent to a helium nucleus. Alpha (a) particles, being large and positively charged, do not penctratc far in living tissue., but they do cause ionization damage and this makes them generally unsuitable for tracer studies. Beta decay involves the loss or gain of an electron or its positive counterpart, the po6itron. There are three sub-types: (a) Nt¥alron (P-) emission: loss of an electron from the nuclcus wbcn a neutron transforms into a proton. Examples of negatron-cmiuing isotopes are: 3H, 14C, 32p, 45Ca and wCo. (b) Positron (Pi) enlission: loss of a positron when a proton transforms into a neutron. This only occurs when sufficient energy is available from tbe transition and mny involve the production of gnmma rays when the positron is later annihilated by collision with an electron. (c) Electron capture (Ee): when a proton 'captures' an electron and transfonns into a neutron. This may involve the production of x-rays as electrons 'shuffie' about in the atom (as with 125 1) and it frequemly involves electron emission. Internal transition invohoes the emission of electromagnetic radiation in the fonn of gamma b') rays from a nucleus in a metastable state and always follows initial alpha or beta decay. Emission of gamma radiation leads to no further change in atomic number or mass.
Note from the above thai more than one type of radiation may be emitted when a radioisotope decays. The main radioisotopes used in chemistry and their properties are listed in Table 35.2. Each radioacti\'e particle or ray carries energy, usually measured in electron volts (eV). The particles or mrs emitted by a particular radioisotope Instrumental techniques
235
Radioactive isotopes and their uses
exhibil a range of energies, tcntx:d an energy spectrum, chanlcterizcd by lhe maximum energy of lhe radiation produced, E,rw. (Table 35.2). Table 35.2
Properties of selected isolOpeS
""-
Emie8ion(sl
Mutnun I"*VY eMIlY)
'H
IT IT
0.0186 0.156 1.71 0.25 0.571 0.657 0.511 3.72 0.0465
"c
~p ~p
~Cu
PP IT P'
y
'wpb
•y
_....
12.3 years 5730 years 14.262 days 25.34 days 12.701 hours 22.3 years
The energy spectrum of a paT1icular radioisotope is rdevanl to the following: • •
, •,,
•
,
I
OlS,
--+, , ,, ,, ------~--~----i f '--.
-,.
Fig.35.1 DecaV of a radiooetiw isotope with time. The time taken for the radioactivrlV to decline from /( to 0.5/( is the same as the lime taken for the radioactivity to decline from 0.5/( to 0.25x, and so on. This time is tho hatf~rife (t1 /1) of the isotope.
Safcty: isolOpc8 with the highest maximum energies will have the greatest pcm..' tmling power, requiring appropriate shielding (Table 35.1). Delection: different instruments vary in their 1tbilily to detect isotopes with diffl.'ft.'t1t cnergies. Discrimination: somc instruments can distinguish bety.'Cen iSOIOpes, baSt,.'<.i on the energy spectrum of the radiation pl'(xluced (I'. 237).
The decay of an individual alom (a 'disinll.·gnltion') occurs at Illndom, but thai of a population of atoms occurs in a predictable manncr. 111C radioactivity decays exponenlially, having a charactcnstic half-life (lIp), This is the lime taken for the radioactivity to fall from a given value 10 half that value (Fig. 35.1). TIle '1/2 values of different radioisotope'> range from fructions of a second to more than 10 19 yC'ars (sec also Table 35.2). If tl/2 is very short, as with 150 (t1/2 ::::;l 2 min), then it is gt.'nerally impractical to usc the isotope in experiments bt.'C'ausc you would need to l'lccount for the d<:cay during the experiment and counting period. To cakulate the fraction (f) of the origin..'ll radioactivity left after l'l particular time (t). use the following relationship:
f
= e.... when: x = -0.69311'112
[35.1J
Note that the same units must be u.scd for t and 11/2 in Ihe above cquation. Table 35.3
Relationships between units of
radioactivilV
Measuring radioactivity 1 BQ "" 1 d.p.s. 1 BQ = 60d.p.m. 18Q=27pCi 1 d.p.s. _ 1 Sq 1 d.p.m. = 0.0167 Bq 1 Ci = 37G8Q 1 mCi = 37MSq 11,Ci _ 37 kBQ 1 Sv = lOOrem
lGy=100rad 1 GV <>:: 100 roemgen 1 rem = 0.01 Sv 1 rad = 0.01 GV
1 roentgen"", 0.01 Gy
236
Instrumental techniques
Thc SI unit of mdioactivity is the bccqucrel (Bq), equivalent to one disintegration per second (d.p.s.), but disintegmtions per minutc (d.p.m.) arc also used. Thc curie (Ci) is a non..SI wlit equivalent to Ihe number of disintegrations produced by I g of radium (37GBq). Table 35.3 shows the relationships bety.ecn these units. In practice, most instruments are nOI l'lble to deted all of the disinlegrations from a panicular sample, i.e. their efficiency is less than 100% and the rale of decay may be presented as counlS min-I (c.p.m.) or counts S-I (c.p.s.). Mosl modern instruments correct for background radiation and ineffICiencies in counting, converting count data to d.p.lll. Alternatively, the results may be presented as thc measured count rate, although this is only valid where lhe efficiency of counting docs not vary greatly among samples.
Radioactive isotopes and their uses
. . - . The specifIC aetivrty is a measure of the quantity of radioactivity present in a known amount of the substance: .r: ' . ,"~"d~;~O~",='=iv:;,;I~y~(~Bq::!.:..~Ci=·.~d=.~p:;..m~.~.~,,=c*.) SpeclitC acllvlly""amount of substance (mol. g. etc,)
[35.2J
This is an importam cancepl in pmctical wOf"k involving radioisotopes, since it allo.....s intcroonvcrsioll of disintegrettions (activity) and amount of substance (sec Box 35.1). Two 51 units refer to doses of radioactivity and these are u.~d when C'ellculating exposure levels for a particular source. The sievert (5v) is the amount of radioactivity giving a dose in man equivalent to I gray (Gy) of x-rays: I Gy = an energy absorption of I J kg-I. The dose recdvl..'tI in Illost biological experiments is a nt..'gligible fraction of the maximum permitted exposure limit, Conversion factors from older units are givcn ill Tablc 35.3. The most impOf"tant methods of measuring mdioactivity for chemical pUrposc$ are dl..'SCritx.-d below.
Tlte Geiger-Mt7l1er (G-M) II/be This opemtes by detecting radiation when it iOlli7.cs gas between a p'
Tile scilt/illaliolt cOl/nler
Correcting f'N quenching - find out how your instrument corrects for quenching and check the quendl iootC8tion par.,,-,eter (QIP) on the prinrout,. which measures the extent of quenching of , e8Ctl sample, Large differences in the OIP would indk:8tB that quenching is variable among SBmples and might give you cause for Concern,
This operates by detecting the scintillations (fluorescence 'flashes') produced .....hen radiation interacts with certain chemicals called fluors. In solid (or external) scintillation counters (often rererrcd to as 'gamma counters') the radioactivity causes scintillations in a crystal of fluorescent material held close to the sample. This method is only suitable for radioisotopes producing penctmting radiation. Liquid scintillation counters are mainly used for detecting beta decay. The sample is dissoh'cd in a suitable solvent containing the fluor(s) - Ihe 'scintillation cocktail'. The radiation first interacts with the solvent, and the energy from this interaction is passed to the fluors .....hich produce detectable light, The scintillations are measured by photomultiplier tubes which tum the light pulses into electronic pulses, the magnitude of which is directly related to the energy of the original radioactive event. The spectrum of electronic pulses is thus related to the energy spectrum of the radioisotope. Modem liquid scintillation counters use a series of electronic 'windows' to split the pulse spectrum into two or three components. This may allow more than one isotope to be detected in a single sample, provided their energy spectra are sufficiently different (Fig, 35.2). A complication of this approach is that the energy spectrum can be altered by pigments and chemiC-ells in the sample, which absorb scintillations or interrere with the transfer or energy to the fluor; this is known as quenching (Fig, 35.2). Most instruments have computer-operated quench correction facilities (based 011 measurements of standards of known activity and energy spectrum) which correct ror such changes in counting elTK:icncy. Instrumental techniques
237
Radioactive isotopes and their uses
Box 35.1
How to determine the specific activity of an experimental solution
Suppose you need to make up a certain volume of an experimental solution. to contain a particular amount of radioactivity. For example. SOmL of a mannitol solution at a concentration of 25mmoll- 1• to contain 5 Bqjll 1 - using a manufacturer's stock solution of "C·labelled mannitol (specific activity = 0.' Ci mmol- 1 l. 1. Calculate the total amount of radioactivity in the experimental solution, in this example 5 x 1 000 Ito convert III to mL) x 50 (50 mL requiredl s "=" 2.5 )( lO Bq (i.e. 250 kBQ).
2. Establrsh the volume of stock radK>lsotope solution required: for example, a manufacturl'!r's stock solution of I·e_labelled mannitol contains SO/ICi of radioisotope in 1 ml of 90% vlv ethanol; water. Using Table 35.3. this is equivalent to an activity of 50 x 37 = 18SOkBq. So, the volume of solution required is 250/1850 of the stOCk vOlume, i.e. 0.1351 ml f1351Ill. 3. Calculate the amount of non-radioactive substance required as for any calculation involving concentration (see pp. 17, 146), e.9. 50 mL (0.05 L) of a 25 mmol L 1 (0.025 moll 1) mannitof (relative molecular mass 182.17) will contain 0.05 x 0.025 x 182.17 = 0.2277 g. 4. Check the amount of radioactive isotope to be added. In most cases, this represents a negligible amount of substance, e.g. in this instance, 250kBq of stock solution at a specific activity of 14.8)( 1()6kBqmmol- 1 (converted from 0.4Ci mmol 1 using Table 35.3) is equal to 250/14800000 = 16.89 nmol, which is equivalent to approximately 3119 mannitol. This can be ignored in calculating the mannitol concentration of the experimental solution. 5. Make up the experimental solution by adding the appropriate amount of non-radioactive substance and the correct volume of stock solution.
Liquid scintillation counting of highenergy beta emitters - beta particles with energies greater than 1 MeV can be counted in water lCerencov radiationl, with no requirement for additional fluon; fe.g. 32 PI.
238
Instrumentat t~hniques
6. Measure the radioactivity in a known volume of the experimental solution. If you are using an instrument with automatic correction to Sq, your sample should contain the predicted amount of radioactivity, e.g. an accurately dispensed volume of l00,ll of the mannitol solution should give a corrected count of 100 x 5 = SOOBq (Or 500 x 6030 000 d.p.m.). 7.
Note the specific activity of the experimental solution: in this case. l00/ll (1 x 10-4 11 of the mannitol solution at a concentration of 0.025 moll 1 will contain 25 x 10-7 mol (2.5pmoll mannitol. Dividing the radioactivity in this volume 130000d.p.m.) by the amount of substance (eqn 135.21) gives a specifIC activity of 30000/2.5_ 12000d.p.m.llmol 1, or 12 d.p.m. nmol I. This value can be used: lal To assess the accuracy of your protocol for preparing the experimental solution: if the measured activity is sUbstantially different from the predicted value, you may have made an error in making up the solution. lb) To determine the counting efficiency of an instrument; by comparing the measured count rate with the value predicted by your calculations.
Ie) To interoonvert activity and amount of substance: the most important practical application of specific activity is the conversion of experimental data from counts (activityl into amounts of substance. This is only possible where the substance has not been metabolized or otherwise converted into another form; for example, a tissue sample incubated in the experimental solution described above with a measured activity of 245 d.p.m. can be converted to nmol mannitol by dividing by the specific activity, expressed in the correct form. Thus 245/12 = 20.417 nmol mannitoL
Many liquid scintillation counters treat the first sample as a ·background', subtracling whatever value is oblained from the subsequent measurements as part of the procedure for converting to d.p.m. If not you will na-d to subiraci Ihe background count from all olhcr samples. Makc sure thai you use an appropriate background samplc, identical in all respects to your radioactive samplc but with no added radioisotope, in the con-CCI position within the machine. Check that the- background reading is reasonable (1530c.p.m.). Tips ror preparing samples for liquid scintillation counting are givcn in Box 35.2.
Radioactive isotopes and their uses
80x 35.2
Tips for preparing samples for liquid scintillation counting
Modern scintillation counters arc very simple to operate; problems are more likely to be due to inadequate sample preparation than to incorrect operation of the machine. Common pitfalls are the following: • Incomplete dispersal of the radioactive compOund in the scintillation cocktail. This may tead to underestimation of the true amount of radioactivity present: lal Water-based samples may not mix with the scintillation cocktail - change to an emulsifierbased cocktail. Take care to observe the recommended limits. upper and lower, for amounts of water to be added or the cocktail may not emulsify properly.
based cocktails by adding a specific amount of water. • Chemiluminescence. This is where a chemical reacts with the f1uors in the scintillation cocktail causing spurious scintillations, a particular risk with solutions containing strong bases or oxidizing agents. Symptoms include very high initial counts which decrease through time. Possible remedies are:
lb} Solid specimens may absorb disintegrations or scintillations: extract radiochemicals using an intermediate solvent like ethanol (ideally within the scintillation vial) and then add the cocktail.
lei Particulate samples may sediment to the bottom of the scintillation vial- suspend them by forming a gel. This can be done with cenain emulsifier-
la} Leave the vials at room temperature for a time before counting. Check with a suitable blank that counts have dropped to an acceptable level. lb) Neutralize basic samples with acid (e.g. acetic acid or Hel). lcl Use a scintillation cocktail that resists chemiluminescence such as HiconicfluorJ:'. ldl Raise the energy of the lower counts detected to about 8keV - most chemiluminescence pulses are weak (0-7 keY). This approach is not suitable for 3H.
rra)' spect"ometry
This is a method by which a mixture of j·-my-cmitting radionuclides can be resolved quantitatively by pulse-height analysis. It is based on the fact that pulse heights (voltages) produced by a photomultiplier tube are proportional 10 the amOlUllS of I'-ray energy arriving at the scintillant or a lilhium-drirted gennanium detector. The lithium-drirted gelmanium detector, which is abbreviated to GL-(Li) - pronounced 'jelly' provides higher resolution (narrov.-er peaks), essential in the analysis of complcx mixtures.
Anroralliography This is a method where photographic film is exposed to the isotope. It is used mainly to locate mdioactivc tracers in thin sections of all organism or on chromatography papers and gels. but quantitative work is possiblc. The radiation interacts with the film in a similar way to light. silver grains heing fonned in the developed film where the particles or rays have passt.'<1 through. The radiation must have enough cnergy to penetrate into the film. but if ir has too mLlCh energy the grdin ronnation may be too disram from the point whcre the isotope was located to identify precisely the point of origin (e.g. high-energy beta-emillt.-'Ts). Autoradiogmphy is a relatively sIX---cialized method and individual lab protocols should be followed for particular isotopes/applications.
Chemical applications for radioactive isotopes The main advantages of using radioactive isotopes in chemical experiment" are: •
Radioactivity is readily detected. Methods of detection are sufficiently sensitive to measure extremely small amounts of radioactive substances. Instrumental techniQUes
239
Radioactive isotopes and their uses
Fig_ 35.2 Energy spectra for three radioactive samples. detected using a scintillation counter. Sample a is a high-energy beta emitter while b contains a low-energy bela eminer, giving a lower spectral range. Sample ti contains the same amount of low-energy beta eminar, but with quenching. shifting the spectre! distribution to a 10000r energy band. The counter can be sot up to record disintegrations within a selected range (a 'window'), Here, 'window a' could be used to count isotope a while window b' could give a value of isotope b, by applying a correction for tho counts due to isotope a. based on the results from 'window a', Dual counting allows experiments to be carried out using two isotopes (double labelling).
·wtldowa'
'wlOdowb' J I b'
6fl&'llY
• •
Studies can be carritxl out in synthctic chemistry using radiolabelkxl compounds, e.g. 3H or He. Protocols are relatively simple compared with cquiV'dlent methods ror instrumental chemical analysis.
The main disadvantages are: •
Degradation of radiolabelled compounds - you may need to separate individual metabolites before counting, e.g. using chromatography (p. 211), or electro-phoresis (p. 225).
•
The 'isotope effect'. Molecules containing different isotopes of the same atom may react at slightly different ratto'S and behave in slightly different ways to the natural isotope. The isotope effect is more extreme the smaller the atom and is most important for 3H.labelled compounds of low molecular mass. TIle possiblility of mistaken identity. The presence of radioactivity does not tell you anything about the compound in which radioactivity is present: it could be different from the one in which it was applied, owing to chemical breakdown of a 14C-contail\lng organic compound.
The main types of experiments are: •
Example Carbon dating - living organisms have essentially the same ratio of "c to 12.C as the atmosphere; however. when an organism dies, its uCfl 2e falls because the radioactive 14e isotope decays, Since we know the halflife of 14e (5715 years), a sample's 1"C112e ratio will allow us to estimate its age; for example, if the ratio were exactly one-eighth of that in the atmosphere, the sample would be three half-lives old and was formed 17145 years before present. Such estimates carry an error of the order of 10% and are unreliable for samples older than 50000 years, for whtch longer lived isotopes can be used.
240
Instrumental techniques
•
•
•
Radiolabelled compounds: the usc of radiolabcllcd compounds in synthetic and tracer studies is importanl as it allows the scientist to locate the labelled alom, i.e. 14C. lH, in, for example, chemical synthesis and laboratory environmental fate (degradation) studies. If using radiolabelled compounds several issues arise and these include deciding upon the radionuclide itself. its position in the molecule, the specific activity. the solvent and cost, Radio-dating: fhe age of plant or mineral samples can be detennined by measuring the amount of a mdioisotopc in the sample. The age of the specimen can be found using tl/2 by assuming how much was originally incorporated. Ml'dical uses: in radiotherapy the use of gamma radiation from 6OCo to destroy cancerous cells; 24Na can be illlroduccd into the blood stream 10 follow the now of blood and identify obstructions; heart disease can be assessed using WIT! and 99Tc where the ronner concentrates in healthy hem1 tissue and the laller concentrafes in abnormal heart tissuc. Assays: radioisotopes are used in several quantilafive detection methods of value to chemists. Radioimmunoassay is a quantitative method for measurement of a substance (the analyte) using antibodito"S which bind specifically to that analyte. Isotope dilution analysis works on the
Radioactive isotopes and their uses
assumption that introduced radiolabelled molecules will equilibrate with unlabelled mok:<:ules prcselll in the sample. The amount of SU~lanee initially pn:sem can be worked oul from the change in specifIC activity of the radioisotope when it is dilutt.x1 by the 'cold' lTIHterial. A method is required whereby the subsumce can be purirK.'d from the sample and sufficielll substance rnll
Working practices when using radioactive isotopes
In the UK, institutions must be registered for work with specific radioisolopes under the Radioactive Substances Ad (19601.
By law, undergraduate work with radioactive isotopes nm"t be very closely supervised. In practical classes, lhe protocols will be clearly OUllined, hut in project work you may have the opportunity 10 plan and carry out youI' own expcrimenls, albeit under supervision. Some of the factors thaI you should lake into a<.'Counl, based on the assumption that your department and laboratory are registered for radioisotope use. are discussed below: •
In the UK. the Ionizing Radiations Act (19851 provides delalls of local
•
arrangements for the supervision of
radioisotope work. •
•
Must you use radioactivity? If nol it may be a legal requirement that you use the alternalive method. Have you registered for radioactive work? Normal practice is for all users 10 register wilh a IDOlI Radiation Protcclion Supervisor. Details of the project may have to be approved by the appropriate administrator{s). You may have to have a short medical examination before you can start work. What labella:! Compound will you use? Radioactive isotopes must he on!t..rec! "'ell in advance through your department's Radiation Protection Supervisor. Aspects that need to be consKlered include: (a) The radionuclide. With many organic compounds this will be contina:! to JH and 14C (but sec Table 35.2). The risk of a signincant 'isotope effect' may innuence this decision (sec above). (b) The labelling position. This may be a crucial part of a melabolic study. Specifically labelled compounds arc nonnally more expensive than those that are unifonnly ('generally') labelled. (c) The specific activity. The upper limit for this is defint.'<.I by the isotope's half-life. but below this the IUgher the spcciflC activity, the more expensive the compound. Are suitable facilities available? You will nttd a suitable work area, preferably out of the way of general lab trafflc and within a fume cupboard for those cases where volatile radioactive substances arc used or may be produced.
Each new experimem should be planned carefully and expclimental protocols laid down in advance so you work as safely as possible and do not waste ex.pensive radioactively labelled compounds. In conjunction with your supervisor, decide whether your method of application will introduce enough radioactivity into lhe system, how you will account for any loss of radioactivity during recovery of tbe isotope and whether there will be enough activity 10 counl at the end. You should be able to predict approximately the
Jnstrumeotal techniques
241
Radioactive isotopes and their uses
Carrying out a 'dry run' - considor doing this before working with radioactive compounds. perhaps using a dye 10 show the mowmetlt or dilution of introduced liquids, as this will lessen the risks of accident and improve your technique.
amount of radioaclivily in your SAmples. based on the specific activity of the isotope used. the expected rate of uptakc/exchange and Ihe amount of sample to be counted. Use the iooto~'s specifIC activity to l'Stimate whether the nonradioactive ('cold') compound introduced with the radiolabelled ('hal') compound may k-ad 10 excessive concentrations being administered. Advice for handling data is given in Box 35.1.
Safety and p"ocedllral a~peCls Using Benchkole ~ - the correct way [0 use Benchkote ~ and similar prOductS is with the waxed surface down (to protect the bench or tray surface) and the absorbent surface up (to absorb any spillage). Write the date in the comer when you put down a new piece. Monitor using a G-M tube and replace regularly under normal circumstances. If you are aware of spillage, replace immediately and dispose of correctly.
Make sure the bench surface is olle that call be t:'
CAlITlON RAOIOACTtVE MATERIAL
Tape showing the international symbol for radioactivitY. Fig.35.3
• • •
• • •
242
Inslrumentallechniques
RC'"
-------10
Thermal analysis Thl.'rmal methods arc techniques in which changes In physical lind/or chemical properties of a substance are mcasull..d as a function of I(,.·mpcmturc. Scvcrdl methods of analysis Hrc used: •
•
ThermogT"dvimclry (TG) is a technique in which a change in thc weight of the substance under investigalion is monitored wilh rt."Specl 10 It.1nperdlure or lime. Differential thermal analysis (DTA) is a technique for measuring the difference in tl...TIlpcralure between lhe substance under invcsligaLion and
an inert rcft.'TCJlce material with rcspect to It.'JllpcrcllUrc or time. •
DifTt.YCnlial scanning colorimetry (Osq is a technique in which the energy nec::essary 10 establish a .....ero temperature difference between the substance under investigation and a reference material is monitored wilh respect to temperature or time.
When canying Qut a thermal analysis procedure it is important and record the following details: •
•
•
Tabla 36.1
Common sample atmospheres
•
Gesst1 atm Air Carbon dioxide
Helium Nitrogen
33.' 25.1 190.6 32.3
•
10
con.<;id<.'t"
Sample: a c:h<.'tl1K:al description of the sample. plus its source and any pre· treatment. Also. the purity, chemical composition and formula, if known. Other important items to note are: the partick: size, whether the sample has been mixed with a 'binder' (and, if so, what it has been mix<.'(\ with and in what (,"
Thermogravimetry The apparatus required for TG analysis is shown in Fig. 36.1. TG is normally carried oUi on solid samples. Example operating conditions are as rollows: Sample: Crucible: Rate: Atmosphere: Mass:
calcium oxaJate monobydrate platinum pan 10 K min-I nitro!,'CIl,20mLmin- 1 10.5mg
Instrumentattochniques
243
Thermal analysis
Mlrllbellllra
-
""""" """"
I
",·1 ~
-~ '-
0
--j ........ I
I",,,,,
"'-
Fig. 36. f SChematic diagram of a system for thermogravimetry.
Box 36.1
How to interpret a thermal analysis trace
1. Identify the start position of the trace (see Fig. 36.2); this is usually indicated by the scale on the trace. 2. Identity any regions of decomposition: Ihese are where there is a rapid change in the vertical axis. Three distinct regions of decomposilion can be identified in Fig. 36.2: (8) between the start and the first plateau there is a loss of 12.5% (stage 1); (bl between the first and second plateau a loss of 18.75% occurs (stage 2); and (cl between the second plateau and the final residue there is a loss of 29.75% (stage 3).
3. Determine the M, of the starting material. The M,. of CaC,0•. H20 is 146.1. 4.
Using the M, detennine the decomposltion loss associated with each region. Stage 1: 146.1 x 12.51100 = 18.3 (18 corresponds to the loss of water) CaC,0•.H20'.1 ...... Cac,O~.J
+ H20!",
Stage 2: 146.1 )( 18.75/100 = 27.4 (28.01 corresponds to loss of
COl CaC,O.\11 ..... CaC~'1
+ CO/g ,
Stage 3: 146.1 x 29.751100 -, 43.5 144.01 corresponds to loss of
co,l CaCo,l"l ... Ca0 ls) t C02Cgl 5. Determination of the final product. In this case for CaC 20 •. H20 the final residue is CaO (M,. "" 56.08).
6. Check the M,. of the original compound: original sample (M,) -= [residue IM.)1% residuel x 100 Residue from Fig. 36.2 is 39%. Therefore, original sample (Mr) -= {56.08I39%1 )( 100 = 143.8 Thus the calculated (M,) of CaC 20 •.H2 0 is 143.8, which is similar to the known M- of CaC20•.H20 of 146.1 g moll. 7. Assess the purity of the original material. The percentage purity of CaC20•.H20 is calCUlated as follows: 143.8 x 100 )( 1/146.1 = 98.43%
The result can be eltpressed as either a TG cun'e. a plot of changing weight with respect 10 tcm(X'rlllUrc or time, or a derivalive of the curve, i.e. DTG, where the lirst derivative of the TG cun'e is plolled with respect to temperature or lime. As well as providing information on Ihe Ihennal decomposition of inorganic compounds, additional information can be deduced. e.g. sample purity and M .. The oltulale hydrates of the alkaline eaoh metals, e.g. calcium. strontium and barium. are all insoluble. If a calcium salt made acidic with elhanoic acid is treated wilh sodium oltalatc solution. a white precipitate of calcium oxalate 244
Instrumental techniques
Thermal analysis
o
H' .......... resOJeCeO
"
1"-----.
monohydrate.
• • • •
HeMing Illle: 20'1: nino' SwnpIe toolda: pIet;n"m A!fI1lISP/Iere: ritrogen Aowme: IOn min-I
'00
is shown in Fig. 36.2. Box 36.1 shows how to
,."
Applications
I"" START ~
o
Fig. 36.2 A Iypicallhermal analysis trace for
CaC-!O•.H20.
Ca~04.1-I~.
interpret a thennal analysis trace for calciwn oxalate monohydrate.
2>" '-.......... ~ Iltin; CllIllIitions
monohydrdte is fonned quantitatively. After washing the precipitate with ethanol it can be anaIYSL-d. A typical TG curve, for calcium oxalate
As well as inorganic compicxl.'S, thermal .lltullysis is applicable to a wide mngc of substance". e.g. pol)mers. drugs, soils and coals. It can also be applit.'(] to mixture<; of, for example, polymer blends. Degradation of poI)"nlCts The effecl of heat 011 polymers varil.'g according 10 the type of polymer under investigation. In an inert atmosphere. polymeric matl.-nals react in two distinct ways: lhey eithcr dt.'PQlymcrizc or r.:arbonizc. For example, poly(mcthyl methacrylate) may degrdde back to the monomcr.
Soil Thc composition of soil is complex and varies wilh localion ~md geology. Three gcncml stages of soil decomposition on ht:'
2. 3.
Loss of moisture and simple organic compounds (bctw(.>en room tempcmture and 150~C). Ignition of soil organic matler (bctw(.>en 250 and 550~C). Presence of minerals e.g. carbonates. The process can be complicated by the presence of hydmted minerals e.g. aluminium and iron oxides, and micas (above 550
Drugs The prcscnt.'( of water in both 'free' and 'bound' slates III phannaceuticals can be identified.
Instrumental techniques
245
Resources
Resources for instrumental techniques
General books Christilln, G, (1994) Analytical Chemistry, 5th Edn, John Wilcy and Sons Ltd, ChichCSlcr. Crawford, K. and Heaton. A. (1999) Problem Solring in Analytical Chemistry, Royal Society ofChemislry. Cambridge, Currell, G. (2000) Analyticallnstrummtation. Performance and chamCferistics ofquolitJ'. John Wiley and Sons Lid., ChichCSler. Day, R.A. and Underv.'ood, A.L. (1991) QIIOJ1fiwtire Anal.vsis. 61h Edn. Prentice Hall. I-Iarlow. Essex. Harris. D.C. (1995) Quantitative Chemical Analysis, 41h Edn. Freemlln and Co., Inc.• New York. Kellner. R. (1998) Analytical Chemi~try. John Wiley and Sons Ltd, Chichcslcr. Mcndh..'1m. J., Denney. R.C., Barnes. J.D. and Thomas. MJ.K. (2000) Vogel's Textbook of QlIOntitatil'e Chemical Analysis, 61h Edn. Prentice Hall, I-Iarlow, Essex. RouC:SSac. F. and Roucssac. A. (2000) Chemical Analysis. Modem instrumentation methods (ilul techniques. John Wiley and Sons Lid, Chichcster. Rubinson. K.A. and Rubinson, J.F. (2000) COll1empomry Instrumental AlUdysis. Prentice Hall. Harlow. Essex. Schwedt, G. (1997) The Essential Guide to Analytical Chemistry. John Wiley and Sons Lid. Chichester. Skoog, D.A., West, D.M. and I-I oller, F.J. (1996) FIIIllJamell111ls of Analytical Chemistry, 7th Edn. Saundcrs Collcgc Publishing. Orlando. Florida. Skoog, D.A. and Leary. J.L. (1992) Principles of In.f1rumen1l11 Ana(l'sis. 4th. Edn. Saunders College Publishing. Orlando. Florida.
Willard, H.H .. Meriu. Jr.. L.L.. Dean, J.A. and Scule. Jr.. FA (1988) Instrwllental Methods of Anal.l·sis. 7th Edn. Wadsworth Publishing Co., Belmont. CA.
Specific books on atomic spectl'O..,copy Cresser. M.S. (1995) Fkmle Spectromelly ill Em'irol1mental Chemical Analysis: A Pmctical ApPTOllclt, The Royal Society of Chemistry. Cambridge.
Dcan. J.R. (1997) AlOmic Absorption and Plasma Spectroscopy, ACOL, John Wiley and Sons Ltd, Chichcster, EWon, L., Evans, 1-1 .• Fisher, A. and HiU. S. (1998) An l"trOllllction to Atomic AbsOlption Spectrnmelly. John Wiley and Sons Ltd. Chichester. Howard, A.G. and Statham. PJ. (1993) Inorganic TUK:e Analysis. PltilOSOP/I)' and Practice, John Wiley and Sons Ltd, Chichester. LlIjunen, L.I-U. (1992) SpeCl/"Ochemical Analysis by Atomic Ahsorption and Emission, Royal Society of Chemistry. Cambridge. Vandealsteelc, C. and Block. cn (1993) Modem Methods of Trace Element Determination, John Wiley and Sons. Chichester.
Specific books on molecular !Jpectroscopy Braun. S., Kalillov.'Ski. H.G. and Berger, S. (1998) 150 alul more hasic NMR e.t.:perillu?Ilts. A pmctical course, 2nd Edn, John Wilcy and Sons lid, New York. 2-46
Instrumental techniques
Resources
Kemp, W. (1991) Organic Speclroscop)', 3rd Edn. Macmill
Specific hooks on chromatography Braithwaite. A. and Smith. FJ. (1990) ChromalOgraphic Cbapman
Metholl~.
4th Edn.
Fowlis. I.A. (1995) Gas ChrolJllllography. 2nd Edn, Analytical Chemistry by Opcn Learning, John Wiley and Sons LId. Chichester. Fritz, J.S. and Gjerde, D.T. (2000) 1011 Chromatograph..·• 3rd Ed., John Wiley and Sons Ltd., Chichester (2000). Grab, K. (200 I) Splil alia Spfiliess Injection in Capillary Gas Chromatography, 4th Edn, John Wiley and Sons Ltd. Chichester. I-Iahn-Deinstrop. E. (2000) Appliell Thin La.I'eI' Chromatography. John Wiley and Sons Ltd, Chichester. I-lanai. T. (1999) /lPLC: A Practical Guide. Royal Society of Chemistry, Cambridge. Hubschmann. H,J. (2000) /landbook ofGC/MS, John Wiley and Sons Ltd, Chichester. Kromidas. S. (2000) Pra(·tical Problem Solving in J-JPLC, John Wiley and Sons Ltd. Chichester. Schocnmakers, PJ. (1986) OptinliZalioll of Chromatographic Seleclivity. A Guille to Method Derelopmenr. Elsevier Science Publishers B.V.• Amsterdam. Subramanian. G. (2000) ClriraJ Separation Tedmufues. 2nd Edn. John Wiley and Sons Ltd. Chichester. Synder, L.R. and Kirkland, JJ. (1979) ImrodllctiO/1 to Moe/ern Liquill ChromolOgraphy. 2nd Edn. John Wiley and Sons Ltd, New York. Synder. L.R.. Kirkland. U. and Glajch, J.L. (1997) Praclical HPLC Method Del'elopment, 2nd Edn. John Wiley and Sons Ltd, New York.
Instrumental techniques
247
Resources
Specific book
Oil
elew'Oantllyt;cal teclm;qllf.!!i
Wang. J. (2000) Anal)'tical ElectrOChemistry. 2nd Edn, John Wiley and Sons Ltd, Chichcster.
Specific book on rad;ocllem;:;try Newton, G.W.A. (1999) El1l'irQtmumtal Radiochemical Analysis. Royal Society of Chemistry, Cambridgc.
Spuific book on thermal method!i Hatakcyama T.llnd Zhcnhai, L. (1998) Handbook a/Thermal An(dy.I'i.\·. John Wiky and Sons Ltd. Neo.y York.
CD-ROMs Chemistry Video Consortium, Pmctical Laboratory Chemistry, Educational Media Film and Video Ltd. Harrow, Essex. UK - Chromatographic tcchniques (TLC, column, ion crchange and gas chromatography). Chemistry Video Consortium, Practical Laboratory Chemistry, Educational Media Film and Video Ltd. Harrow, Essex, UK - Electrochemical tcchniques {using galV'dnic cells, using conductometrie (;ells., determining standard electrode potentials. dClermining solubilit), products, thennodynamic characteristics of cells. COOOU(:tometric titrations and lIsing an automatic titrator). Chemistry Video Consortium, Prdctical Laboratory Chemistry, Educational Media Film and Video Lid, Harrow, Essex, UK - Flame photometry, AA and TGA measurements (using a flame photometer. using an a(Omic absorption spectrometer and thennogravimctric analysis). Chemistry Video Consortium, Practical Laborntory Chemistry. Educational Media Film and Video Ltd, Harrow, Essex, UK - Microscale chromatography (TLC, column chromatography, gas chromatography and preparation of a Grignard reagent). Chemistry Video Consortium, Practical Labor,lIory Chemistry, Educational Media Film and Video Ltd, Harrow. Essex, UK - PolarimCIIY, refractometry and radiochcmislJ)' (using :I polarimeter. determining the refmcti"e indices of liquids. measuring the rates of radioactive processes and measuring gas phase emission spectra).
Video Grcevcs. N .. Hodson, L. and Kcedall. G. (1998) Imermediate NMR Spec/rasropy, Royal Socicty ofChcmistry.
Sotware AAS softbook, Royal Society ofChcmistry. Cambridge. TIle advanced ICP softbook, Royal Society ofChcmistry, Cambridge. GC application and method de"e1opment sofibook, Royal Socicty of Chemistry, Cambridge. GC softbook, Royal Society of Chemistry, Cambridge. HPLC softbook, Royal Sodety of Chemistry, Cambridge. MS softbook, Royal Society of Chemistry, Cambridge.
248
Instrumental techniques
Using graphs 0.' 0.5
-
••.,., •
0.'
~
•
0'
0 0
Fig. 37.1 Calibration curve for the determination of lead in soil using FAAS. Vertical bars show standard errors (n = 3).
o
100
200
300
400
500
600
100
;.~. -~-
...~ ~:E- ------:.- - - -I • -5 - - - - - - - timel$)
Fig. 37.2 Decomposition of N 20s- The first-
order mte conslant can be determined from the slope of the line.
Selecting a title - it is a common fault to use tides that are grammatically incorrect: a widelv applicable format is to state the relationship b~n the independent and dependent variables within the title. e.g. 'The relationship between absorbance and concentration'.
Graphs can be used to !ihow dctailoo resuils in an abbreviated fom1, displ3ying the maximum amount of inform3tion in the minimum space. Graphs and tables present findings in different ways. A graph (figure) gives a visual impression of the content and meaning of your results, while a table provides an accurate numerical record of data \!Mlues. You must decide whether a graph should be used, e.g. to illustrate a pronounced trend or relationship, or whether a table (Chapter 38) is more appropriate. A well-oonstructcd graph will combine simplicity, accuracy and clarity. Planning of graphs is needed at the C
Practical aspects of graph drawing The follo""1ng comments apply to graphs drawn for laboratory reports. Figures for publication, or similar f0I111<11 prcscntation. arc usually prepared according to specific guidelines. provided by the publisher/organizer. Two examples of graphs are shown (Fig. 37.1 and 37.2). The firsl is typical in quantitative analytical chemistry (Fig. 37.1), while lhe second is typical in physical chemistry (Fig. 37.2).
~
Graphs should be setf-contained - they should include all material necessary to convey the apprOfJf'iate message without reference to the text. Every graph must have a concise exptanatory tide to establish the content. tf several graphs are used, they should be numbered, so they can be quoted in the text.
•
• Remembering which axis is which - a wav of remembering the orientation of the x-axis is that x is a 'cross'. and it runs
'across' the page (horizontal axis).
•
•
Consider the layout and scale of lhe axes carefully. Most graphs are used to illustrate the rclalionship between two \~
251
Using graphs
,,~
•
n
,,}
Handling I'ery /m'ge or ..€ry small IIIl1nbe,'S
-
, '~~
~
o LL.LJ....LJCJ...L.L.LJ-LLJ:=-
o
10
12
A figure legend should be used to provide explanatory detail, including the symbols used for each dala set.
1416182022
weJgnt(gl
Fig. 37.3 Frequency distribution of weights for a range of different soil types (sample size
24085); the size class interval is 2g.
To simplify presentation when your cxpcrimcnllil datn consist of either very large or very small numbers, the plotted values may be the measured numbers multiplied by a po"cr of 10: this multiplying power should be writtcn immediatcly before the descriptive label on thc appropriate axis (as in Fig. 37.3). However. it is ortcn beHcr to modify the primal)' unit with an appropriatc prefix (p. 70) to avoid any confusion rcgllrding negative powcrs of 10.
Size Rcmcmber that the purpose of your graph is to communicatc information. It must not be too Small. so usc al least half all A4 page and design your axes and labels to fill thc available space without overcrowding any adjacent tcxt. If using graph papey-. remember that the white space around the grid is usually too small for effective labelling. The shape of a graph is detcrmined by your choice of scale for the x· and y-axes which. in turn. is governed by your experimental data. It mll.Y be inappropriale to start the axes al zero. In such instances, it is particularly important 10 show the scale clearly. with scale breaks whcre necessary. SO the gmph docs not mislead.
C"aplt paper In addition to conventional linear (squared) graph paper. you may following: • Exampkt For a data set where the smallest number on the log axis is 12 and the largest number is 9000. three-cycle log-linear paper would be used, covering the range 10-10000.
•
•
" -
10
•
~AB~--'-,O""'-
•
0'-'-.;-'---'-, n,;-,-_-,-:: blood grOl:p
Fig. 37.4 Bar chart. showing the number of studenls belonging to each ABO blood group
252
Probability gmph paper. This is useful when one axis is a probability salle. Log-linear graph paper. This is appropriate when one of thc srnJes shows a logarithmic progression, e.g. in c11cmical kinetics. A plot of In k against liT is used to determine the activation energy (E.). where k is the rate constant and T is the temperature. Leg-linear paper is defined by the number of logarithmic divisions covered (usually termed 'cycles') so make sure you usc a paper wilh the appropriate number of cycles for your data. An alternative approach is Ie plot the log-transformed values on 'normal' graph paper. Log.-.-Iog graph paper. This is appropriate when both scales show a logarithmic progression. e.g. the large linear ranges achievable with some analyti(.-al instruments.
Diffcrent graphical forms may be used for different purposes. including:
5
(n =
the
Types of graph
•~
I
llL-OO
29).
Analysis and presentation of data
• •
Ploncd cun'CS - used for data where the relationship between two variables can be represented as a continuum. Scatter diagrams - used to visualize the relationship between individual data \'alucs for two interdepcndem variables (e.g. Fig. 41.6) often as a preliminary part of a correlation analysis (p. 278). Three-dimensional graphs show the inter-relationships of three variables. (e.g. Fig. 32.21). Hislogmms for frequency distributions of continuous variables (e.g. Fig. 37.3).
Using graphs
•
• A
Frequenc)' polygons emphasize Ihe fonn of a frequency dislribUlion by joining the co-ordinalCS with straight lillCS, in contrast to u histogram. This is particularly useful when plotting two or mon:: SCIS of
•
Bar charts represent frequency distributions of a discrete qunlitative or
•
quantiUltivc variable (e.g. Fig. 37.4). Pic churlS illustrate portions of a whole (e.g. Fig. 37.5).
The plotted curve
o Fig. 37.5 Pie chan: relative abundance 01 ABO blood groupli in man.
This is the commonest ronn of graphicsl rcprcscnllillon used in chemistry. The key features arc outlined below and in checklist foml (Box 37.1).
Dum points Each datu point must be shown accurately, so that any reader call determine the exact V'
readily identifiable. A useful technique is to usc a dot for each daHl point, surrounded by a hollow symbol for each treatment. An alternative is 10 usc symbols only, though the co-ordinatcs of each point are defined less accurately. Usc the same symbol for the same entity if it ()(;eurs in several graphs and provide a key to all symbols.
Statistical measures If you are plolting average values for scvcral replicates and if you have the necessary statistical knowledge, you can calculate the standard error (p. 268), or thc 95% confidence limits (p. 278) for each mean value and show these on Box 37.1
Checklist for the stages in drawing a graph
Tho following sequence can be used whenever you need to 8 plotted curve: it will need to be modified for other types of graph.
construct
Colect aM of the data vatues and statistical va....es (In tabular form. where appropriate). 2. Decide on the most sortable form of presentation: this may include transformation, to convert the data to linear form. 3. Choose a conciBe descriptive title, together with a reference 1.
(figure) number and date, where necessary. 4-. Determine whk:tl ~ 8 is to be plotted on the lfoaxis and which on the y-aJris. 6. Select appoprMde seales for both axes and make sure that the numbers and their location (seate marks) are clearly shown, together with .lIly scale breaks. 6. Decide on appropriate desctiptive labels for both axes, with Sl
7.
units of measurement, where appropriace. Choose the symbols for each set 01 data points and decide on the best means of representation for statistical values.
Plot the points to show the co-ordinates of eacb value whh appropriate symbols. 9. Draw a trend line for each set of pointa. 10. Write a figure legend,. to include 8 key whtch identifies all liymbols and statistical values 8nd any deticriptlve footnotes, 8.
as required.
Analysis and presentation of data
253
Using graphs
your gmph as a series of vertical burs lscc Fig. 37.1). Make it dellr in the legend whether the bars refer to standard errors or 95% confidence limits and quote the value of n (the number of replicatcs per dala point). Another
approach is to add a least significant difference bar (p. 277) to the grnph.
Conveying the correct message - the golden rule is: "alwaVs draw the simplest line that fits the data reasonably well and is consistent with the underlying chemical principles'.
Extrapolating plotted curves - try to avoid Ihe need to extrapolate by better
experimental design.
IlIterpolat;OIt Once you havc plolletl each point. you mllst deddc whcther to link them by straight lines or 1I smoothed CIIrve. Each of these techniques conveys a different Illcssage to your reader. Joining the points by straight lines may seem the simplcst option. but may give thc impression that crrors arc vcry low or non-existent and that the relationship between thc variables is complex. Joining points by straight lines is appropriate only in certain graphs, e.g. reeording a patient's tcmperature ill a hospital, to emphasize any variation from onc time point to thc ncxt. Howevcr, in most plOlted curvcs the best str-dight line or curved line should be drawn (according to appropriatc mathematical or statistical models, or by cye). to highlight the relationShip between the variables - after all, your choicc of a plolted curve implies that such a relationship cxists! Don't worry if some of your points do not lie on the line: this is caused by crrors of measurement and/or sample variation. Most cunlcs drawn by eyc should havc an equal number of points lying on eithcr side of the line. You may be guidcd by 95'10 confidence limits, in which case your curvc should pass within these limilS wherevcr possible. Curved lines can be drawn using a nexiblc curvc. a set of French curves. or freehand. In the last casc, turn your paper SO that you can draw the eurve in a single. sweeping stroke by a pivoting movement 11.1 the elbow (for larger curves) or wrist (for smaller ones). Do not try to force your hand to make complex. unnatural mO\'ClllCnts, as the resulting line will not be smooth. Extrapolat;on Ilc wary of cxtrapolation beyond the upper or lower limit of your measured values. 1l1is is rarely justifiable and may lead to serious ClTors. Whenever extmpolation is used. a dotted line ensures that the reader is aware of the uncertainty involved. Any assumptions behind an extrapolated curve should also be stated clearly in your text.
The histogram
In a histogram, each datum is represented by a co'umn with an area proportional to the magnitude of y. in most cases, you should use columns of equal width, so that the height of each
column is then directty proportional ro y. Shading or stippling may be used to identify individual columns. according to your needs.
254
Analysis and presentation of data
While a plotted curve assumes a continuous relationship between the variables by interpolating between individual data points, a histogram involves no such assumptions and is the most appropriate representation if the number of data points is too few to allow a trend line to be drawn. Histograms are also used to represent frequency distributions (p. 265). where the y-axis shows the number of times a partkular value of x was obtained (e.g. Fig. 37.3). As in a plotted curve. the x-axis represents a continuous variable which am take any value within a givcn "mge, so the scale must be broken down into discrete classes and the scalc marks on the x-axis should show eithcr the mid-points (mid-values) of each class (Fig. 37.3), or the boundaries between the classes. The columns are adjaccnt to each other in a histOgram, in contrast to a bar chart (Fig. 37.4), where the columns are separate beanlSe thc x-axis of a bar chart represents discrete valucs.
Using graphs
Interpreting graphs Using computers to produce graphs never allow a computer program to dictate size, shape and other aspects of a
graph: find out how to alter scales, labels. axes, etc., and make appropriate selections. Draw curves freehand if the program only has the capacity to join the individual points by straight lines.
Whenever yOll look 81 gmphs drawn by olher people, make sure }'OU understand the axes before you look at the relationship. It is all 100 easy to take in the shape of a graph without first considering the scale of the axes, a fact that some advertisers and politicians exploit when curves arc used 10 misrepresent information. Such graphs are often used in nL"Wspapers and on television. Examine them critically - many would nol pass the stringent requirements of scientific communication and conclusions clmwn from Ihem may be flawed.
AnalVsis and presenration of data
255
--------10
Presenting data in tables A table is orrcn the most appropriate way to prcscm numerical dllUI in H concise, accumtc and structured form. L
~
Always remember that the primary purpose of your
table is to communicate information and allow appropriate comparison. not simpty to put down the results on paper!
Preparation of tables Title Constructing titles - take care over titles it is a common mistake in student practical reports to present 1B~S without tides, or to misconstruct the title.
85
EveI}' table must have a brief descriptive lille. If several tables are Uf,(:d, number them consecutively so they can be qUoted in your text. The tilles \\'ithin a report s.hould be eompared ""ith one another, making sure they arc logical and consistent and thai they describe accurately the numeri<.
Structure
tablos - you may be able to omit a column of control data if your resutts can be expressed as percenlages of the corr96pOnding contrcl values. Saving space in
Table 38.1
Selected properties of elements in the Periodic Table
Element
Aluminium Cobalt Iron Manganese Zinc '" Determined at
Symbol
AI Co
Relative
~mbe,
atomll;
Atomic radius
m...-
'pm)
26.98 58.93 55.84 54.94 65.38
143
+3
12S 12. 12. 138
+2.+3 +2, +3 +2, +3. +4, +7
72
+2
7.1
13 27
f.
2.
M"
25
'"
.....
Compounds in ores
Atomic
30
28~C.
** Relative to the atomic mass of 12C 1= 121. 256
Display the componenls of each table in a way thaI will help lhe reader understand your data and grasp the signifICance of your results. Organi;,r..e the columns so that C""dch category of like numbers or properties is list<-'
Analysis and presentation of data
Important oxidatioo ,
. 2.7 8.7 7.B
AJ,o, CoAsS, CoAs 2 , CoS
F82O:J, Fe30., FeCO:J, FeS2 MnO:z. Mn 20 a, Mfl30., mixed oxides ZnS, ZnO, ZnCO a
Presenting data in tables
Hem/inKS and sIlb-IIeudings These should identify each sct of data and show the unilS of mcasurement, where necessary. Make sure Ihnt each column is wide cnough for the headings and for the lon~l dala value.
Nmlledelll data Examples Quote the radius of an Mom "O.126nm or 126 pm, rather than 0.OOOOOOOOO126m or 0.126)( 10-' m lor 126)( 10-'2 m), However. some texts still use the angstrom. A. An angstrom is equivalent to 10-'(1 m; hence the radius of an atom would be 1.26A. in this example.
Within the table, do not quote values to more signilicnnt figures limn necessary, as tbis will imply spurious accul1lcy. By careful choice of flppro· priate units for eadl column you should aim to prcsclll numerical dal.."1 within the range 0 10 1000. As with grnphs. it is less ambiguous to use derivt.'d 51 units. with the appropriate prefixes, in the hC~ldings of columnS nnd rows, ruther than quoting Illultiplying factors as J'O,",,'Crs of 10. Alternatively, include exponents in the main body of thc table (see Table 7.1), to avoid filly pos.'~ible confusion regarding the usc of neg
Otltel' notations
Saving further space - in some lnstanceG a footnote can be used to replace a whole column of repetitive data.
Avoid using dashcs in numerical tables. as their meaning is unclear; enter a 7.cro reading as '0' and usc 'NT for not lesled or 'ND' if no datil value was obtained, "1lll a footnote to explain each abbreviation. Olher footnotes, idcntincd by asterisks. superscripts or other symbols in thc table, may be used to prov)dc relevanl cxpcrimcntul delail (if nol givcn in the text) and an cxplanation of column headings and individual resulls. where appropriate. Footnotes should be as cond:nscd as possible. Table 38.1 provides an example.
Stutisties In tables where the disrersion of each data set is shown by an appropriate statistical parameter, you must state whether this is the (s.ample) standmd deviation, the standard error (of the mean) or thc 95% confidence limits and you must givc the valuc of" (the number of replicates). Other descriptivc statistics should be quoted \.I:ith similar detail, and hypothesis-testing statistics should be quoted along with the value of P (the probability). Details of any tcst ust'd should be given in the legend, or in a footnole.
Text Sometimcs a table can be a useful way of prcscl1ling texlUal information in a condensed form (sec cxample on p. 256).
Box 38.1
Checklist for preparing a table
Ev8fY table shouk! have the following components: 1. A title, plus a reference number and date where necessary. 2. Headings few each co4umn and row, with appropriate units of measurement. 3. Data v. . . ., quoted to the nearest signifICAnt figure ahd with statistical parameters, according to your requirements. .t. Footnotes to explain abbreviations. modifications and individual details. 5. RuIinga to emphasize groupings and distinguish items from each other.
Analysis and presentation of data
257
Presenting data in tables
Using microcomputers and word processing packages - these can be used to prepare high-Quality versions of tables for project work (p. 313).
258
An
When you have finished compiling your tabulated dala. c
-------jG
Hints for solving numerical problems Olcmistry. and in particular physical and analytic-<\! chemistI)', oflen requires a numerical or slatjstical approach. Not only is malhcmalie<1I modelling all imporlant aid to uoocnilanding, bUI computations arc oflen needed to IUrn faw dala info meaningful informntion or 10 compare them wilh olher data sets. Moreover, calculations arc p.."ln of k'1bonuory rouline, perhaps required for making up solulions of known concentnlliOIl (see p. 170 and below) or for the C"
Table 39.1
Sets of numbers and operations
Sets of numbers Whole numbers: Natural numbers: Integers: Real numbers: Prime numbers: Rational numbers: Fractions: Irrational numbers: Infinity:
0, 1,2,3, ... 1,2,3, ,., ... -3, -2, -1, 0, 1,2,3, ... integers and anything between le.g. -5, 4.376, 3116, ft, v'S1 SUbsel 01 natural numbers divisible by 1 and themselves only (i.e. 2, 3,5,7, 11, 13, ... 1 plq where p (integer) and q [natural} have no common factor (e.g. 3I4) pIq where p is an integer and q is naturalle.g. -6/8) real numbers with no exact value (e.g. Itl (svmbol ocl is larger lhan any number (technically not a numbaf" as it does not obey the laws of algebra)
Operations and symbols Basic operators: +, -, x and .;- win not need explanation; however,l may substitute for +, • may substitute for x or this operator may beomined Powers: a", i.e. '8 to the pc')Wef" n', means 8 multiplied by itself n limes le.g. til "" 8 X 8 = '8 squared', tiJ - 8:X 8 x 8 '" '8 cubed'). n is said to be the index or exponenl. Note sO = 1 and 8' -= 8 log.'lrithms: the common logarithm (log) of any number x is the power 10 which 10 would have to be raised to give xli.e, the log of 100 is 2; lQ2 "" 1001; the antilog of x is 10". Note that there is no log for 0, so take this into account when drawing log axes bV breaking the axis. Natural or Napierian logarithms (In) use the base e (= 2.11828, .. ) instead of 10 Reciprocals: the reciprocal of a real number 8 is 1/8 (8 0/- 0) Relational 8> b means '8 is greater (more positive) than b', < means operators: less than, ..: means less than ()( equal to, and ." means grealer than or equal to Proportionality: 8 X b means '8 is proportional to b' (i.e. 8 = kb, where kis a constanO. If 8 0( lIb, a is inversely Pftlportional to b (8= klb) Sums: L Xi is shorthand for the sum of all x values from j = 0 to ; - n (more COfrectly the range of the sum is specified under the symbol) Moduli: Ixl signifies modulus of x, i,e. its absolute value fe,g. 141 = 1-41 = 4) Factorials: xl signifies factorial x, the product of all integers from 1 to x (e.g. 3~ = 61. Note O! = 11 = 1
Analysis and presenlation of data
259
Hints for solving numerical problems ~ at you have
8 'block' about numerical work, practice at problem solving is especially important.
PrdCtising at problem solving: •
• •
dcmystifics the prcl(.'c you 10 gain confidence so that you dan', become confused whcn confronted with an unfamiliar or apparenlly complex form of problem; helps you recognize the various fOflns a problcm can take liS, for cx.mlplc, in lhe different fonns of 'itrations (Chapters 21-25 inclusive).
Steps in tackling a numerical problem The step-by-step approuch outlined below may nOI be the fastest method of arriving at an answer, but mosl mistakes occur where steps arc missing, combined or not made obvious., so a logical approach is often beller. Error tracing is distinctly easier when all sli.\&'CS in a calculation arc laid OUI.
Hal'e ,lte r;gllt tools ready A computer spreadsheet may be very useful in repetitive calculations or for 'what in' case studies (see Chapter 47).
Scientilic calculators (p. 4) greatly simplify the numerical part of problem solving, Howel'er, the seeming infallibility of the calculator may lead you to accept an absurd rcsult which could have ariscn because of faulty key-pfL"Ssing or faulty logic. Make sure you know how to use all the fea!Un:s on your calculator, especially how the memory works; how 10 introduce a constant multiplier or divider, and how to obtain an exponent (note that the 'cxp' button on most calculators ~i y;il(}<, not Par y"; so I x 106 would be entered aslIJ~[!], '101 I.!..QJ ex [I]).
Approaclt tlte problem thought/lilly If the indi,~dual slcps have been laid out on a worksheet, the 'tactics' will already have been decided. It is more difficult when you havc to l1dopt a strategy on your own, especially if the problem is presented in a descriptive style and it isn't obvious which equations or rules need to be appli<:d. •
Read Ute problem carefully as the text may give dues as
10
how it should
be tackled. Be certain of what is required as an answer before slarting. Table 39.2 Simple algebfa - rules for manipulating equatiOfls
•
Ifa= b+ c, then b= 8 - C and c= a- b If a = b x c, then b,., ale and e., alb If II" b C , then b = al/c and c = logs/log b
a'/" = V'8 ir" = '/11'
a b x a" = a(b.<-cl and ab/B" = lal>f = a'1>~"1 a x b = antilogUog s + log bl
alb-c)
•
•
260
Analysis and presentation of data
Analyse what kind of problem it is, which eITeclively means dt.'Ciding which cquation(s) or approach will be applicable. If this is not obvious, consider tlte dimensions/units of the information available and think how lhcy could be filled to a relevanl formula. In cxaminations, a favourite ploy of examiners is to present a problem such that thc familiar form of an CQuation mllSt be rearranged (sec Table 39.2 and Box 39.1). Another is to make you usc two or more equations in series. If yOll are unsure whether a recalled fonnula is correct. a dimensional analysis C<1.n help: write in all the units for the variables and make sure that lhey cancel out 10 give lhe expected answer. Check lhat you have, or can derive, all of the information required 10 usc your chosen cquation(s). It is unusual but not unknown for examiners to supply redundant information. So, if you decide nOI to usc some of the informalioll given. be sure why you do nol require it, Decide on what format and (mits the answer should be presented in. This is sometimes suggested to you. If lhe problem requires many changes in
Hints for solving numerical problems
Box 39.1
Example of using the rules of Table 39.2
Problem: if. = (b - cl/(d+ 6"1. find e 1. Multiply both sides by ld + en); formula becomes: a(d+e n ) = (b- c)
2. Divide both sides by a; formula becomes:
b-c
d+e"=-a
3. Subtract d from both sides; formula becomes:
e,,=b-C_ d
a
4. Raise each side to the power lIn; formula becomes:
e={b~C_d}'/n
•
the preftxes to units. it is a good idea to convert all data to base SI units (multiplied by a power of 10) at the outsel. If a problem appears complex, break it down illlo component parts.
Present YOllr anslt'el' clew'ly Show the steps in your calculationsmost markers will only penalize B mistake once and part marks will be given if the remaining operations are performed correctly. This can only be done jf those operations are visible!
The way you present your answer obviously needs to fit the individual problem. Guidelines for presenting an answer include: •
•
• Units - never write any answer without its unit(s) unless it is truly dimensionless.
Rounding off - do not round off numbers until you arrive at the final answer.
•
• •
Make your assumptions explicit. Most mathematical models of chemical phenomena require that certain criteria arc met before they can be legitimately applied (e.g. 'assuming the sample is homogeneous .. :), while some approaches involve approximations which should be clearly stated (e.g. 'to estimate the volume of a tube, it was approximated to a cylinder with radius x lind length y .. .'). Explain your strategy for answering, perhaps giving the applicable formula or definitions which suit the approach to be taken. Give details of what the symbols mean (and their units) at this point. Rearrange the formula to the required form with the desired unknown on the left-hand side (see Table 39.2). Substitute the relev31l1 values into the right-hand side of the formula. using the units and prefixes as given (it may be convenient to convert values to 51 beforehand). Convert prefixes to appropriatc po\vers of 10 as soon as possible. Convert to the desired units step by step, i.e. taking each variable in tum. Whcn you have thc 3nSv.-cr in the desired units, rewrite thc left-hand sidc and underline the answcr. Make surc that the result is prcscnK'd with an appropriatc number of significant figures (see p. 67).
Check ,rOIl/' answe,' Having written out your answer, you should check it mcthodically, answering the following questions; •
Is the answer ofa realistic magnitude':' You should be alerted to an error if an answcr is absurdly large or small. In repeated calculations, a result standing out from others in the same series should be doublc-cheeke
261
Hints for solving numerical problems
•
•
00 the units make sense and match up with the allswer required? DOli'" for example, present a volume in units of ml , Do you get the same answer if you recalculate ;1\ a dirrcrcnl way'! If you have time, recalculate the ans....er using a different 'rOllle', entering (he numbers into yOUf calculmor ill a diftcrelll form and/or cdrrying Qui the
opemtions in a difTerent order.
Some reminders of basic mathematics Errors in calculations sometimes appear because of faults in ffiHlhclIlatics rather than computational errors. For referel"lCe purposes, Tables 39.1 and 39.2 give some basic mathematical principles that may be useful.
Exponent!j' Example
23 =2:.:2>:;2::::8
Exponential nol.'> than J. See Table 39.2 for other mathematic
&ielltijic IIoltltion Example Avogadro's number, :::0602352000000 000 000 000000, is more conveniently expressed 8S 602352:.: 1()23.
In scientifIC notation, also known as 'stanwud form'. the base is 10 and the exponent a whole number. To express numbers that arc 110t whole powers of 10. the form c x 10" is used. where the coefficient c is normally between I and 10. Scientific notation is valuable when you are using vef1' large numbers and wish to avoid suggesting spurious accuracy. Thus if you write 123000, this suggestS that you kllow the number to ±0.5, whereas 1.23 x lOS might give a truer indication of measurement accuracy (i.e. implied to be ..1:500 in this case). Engineering notation is simil.u. bul t.reats numbers as powers of 10 in groups of three. i.e. c x IffJ. 10J. 1()6, 109, etc. llli~ corresponds to the SI system of prefixes (p. 70). A useful property of powers when expressed to the sarnc base is that when mullipl}1ng two numbers together. }OU simply add the powers. while if dividing. you subtract the powers. Thus. suppose you countoo eight molecules in a 10- 7 dilution. there would be 8 x 101 in the same volume of undiluted solution; if you now dilllte this 500-fold (5 x l()l), then the number present in the same volume would be 8/5 x 1()C7~1J = 1.6 x lOS = 160000.
Logarithms Exami»e luse to check the correct use of vour own calculator}
10296385 a log = 5.012681 lto six decimal places) 1OS-0 12681 = 102962.96 (Note loss of accuracy due to loss of
decimal places.l
262
Analysis and presentation of data
When a number is expressed as a logarilhm, lhis refers 10 Ihe pml,'Cr n that the base number (J must be raised to give that number, e.g. 10glO(lOOO) = 3. since loJ = 1000. Any base could be used, but the two most common are 10. when the power is referred to as !oglO or simply log. and the constant e (2.718282). used for mathematical convenience in certain situations, when the power is refcrred to as log. or In. Where a coefficient would be used in scientific notation. then the log is not a whole number. To obtain logs, you will need to usc thc log key 011 your calculator, or special log tables (now largely redundant). To convert back, use: •
the ~ key, "'1th x = log value;
•
the inverse then the log key; or
•
thelL:]kcy. withy = 10 and x = log valuc.
I
I
Hints for solving numerical problems
Wilh log fables, you will find complemelltary antilogarithm tables to do this. There are many uses of logarithms in chemistry, and in paTlicular physical
chemistry. including pH ( = -log[H+]), where [H+] is exprcsS(;,,,l in mol L- 1 (p. 56), and chemical kinetics, e.g. ratc constants, where a plot of In(reactant) against time produces a straighl line if the reaction is first order.
Hints for some typical problems CalclJlations im'o/t'ing proportions OJ' ratios Example A lab schedule states that 59 of a compound of M, 220gmol- 1 are dissolved In 400 ml of solvent. For writlng up your Experimental section, you
wish to express this as moll 1. 1. If there are 5g in 4OOml, then there are 5/400 9 in 1 ml. 2. Hence. l000mL will contain 5/400 x 1000L "" 12.59. 3. 12.5g=12.5j220mol=O.0568mol.so [solution] = 56.8mmoIL- 1
(=
56.8molm- 3 J.
The 'unitary method' is a useful way of approaching calculations involving proponions or ratios, such as those required when making up solutions from stocks (see also Chapter 6) or as a subsidiary part of longer calculations. • •
if given a value for a multiple. work out the eor~sponding value for a single item or 'unit'. Use this 'unitary value' to c
Calclllations im'o/t'ing
se"ie~'
Series (e.g. used in dilutions, sec also p. 10) can be of two main forms:
I.
2.
arithmetic. where the diffi.,,·cna' bet\.\'CC11 two successive numbers in the series is a constanl. e.g. 2, 4. 6. 8, 10, ... ; geometric, where the ratiQ bctwt-'Cn two successive numbers in the series is a constant. e.g. I, 10. 100. 1000, 10000....
Note that the logs of the numbers in a geometric series will foml an arithmetic series (e.g. 0, I, 2. 3. 4, ... in the above case). Thus. if a quantity y varies with a quantity x such that the rate of change in )' is proportional to the value of)! (i.e. it varies in an exponential mannt-'1"), a semi-log plot of such data will form a straight line. This form of relationship is rclev.lnl for chemical kinetics and mdioactivc decay (p. 236). Stati~'tical
calclliations
The need for long, complex cakulations in statistics has largely been removed because of the widespread use of spreadsheets with statistical functions (Chapter 47) and specialized programs such as Minitabii' (p.315). It is, however, important to understand the principles behind what you are trying to do (see Chapters 40 and 41) and interpret the program's output correctly, either using the 'help' function or a reference manual.
Analysis and presentation of data
263
-------8
Descriptive statistics The purpose of most practic-.t1 work is to observe and measure a p:trlicular characteristic of a chemical system. However, it would be cxtremely rare if the same value was obtained every lime the chnraClcristic was measured, or with every experimelltal subject. More commonly, such measurementS will show variability, due 10 measurement errOr and sampling variation. Such variabilily can be displayed as a frequency distribution (e.g. Fig. 37.3), where the)' axis shows the number of limes (frequency. I) each particular value of Ihe measured variable (Y) has been obtained. OcsCTiptive (or summary) Slatistics quantify aspects of Ihe frequency distributiOJl of a S<1.mplc (Box 40.1). You e,lI1 use them 10 condense a large data sci, for pr~nlalion in ligures or rabies. An additional application of descriptive stalistics is to provide eslimates of the true values of Ihe underlying frequency distribUlion of the population being sampled. allowing the sig.nificance and precision of the experimental observations to be assessed (p. 272).
•
A
··." .. ·;"· ...~ .. .,
j ;"
· ···
.....
~
:. .. .
deperdem (meas\;fedl variable Fig. 40. 1 Two distributions with different locations but the same dispersion. The data set labelled B could have been obtained by adding 8 constant to each datum in the dala set labelled A.
~
The appropriate descriptive statistics to choose will depend on both the type of data, i.e. whether quantitative, ranked or qualitative Isee p. 651 and the nature of the underlying frequency distribution_
In many illl,tances, the normal (Gaussian) distribution best describes lhe observed patient. giving a S}'mIT1clrical, bell-shaped frequency distribUlion (p. 274) for example; replicate measurements of a particular chameteristic (e.g. rt:peated mcasLlrements of tile end-point in a titration). Three important features of a frequcncy distribution that C
the sample's location. i.c. its position along a given dimen!>ioll representing lhe dependent (measured) variable (Fig. 40.1); the dispersion of the data, i.e. how spread out the valucs are (Fig. 40.2); the shape of the distribution, i.e. whelher symmetrical, ske....u1, Ushaped, ete. (Fig. 40.3).
A
""-.,.,
depender.t (rr.euurIldJ lIilriaIlle
Frg. 40.2 Two distributions with different dispersions but the same location. The data set labelled A COVers e relativelv narrow range of values of the dependent (measuredl variable while that labelled B covers 11 wider fallge.
264
Analysis and presentation of data
dependenllmuStlfedl variable
F/fJ. 40.3 Symmetrical and skewed frequency distributions, Showing positions of mean, median and mode.
relative
Descriptive statistics
Box 40.1
Calculation of descriptive statistics for a sample of grouped data
' ' ' ' "' 'Y
Vslue
Cumutative
fY'
'YJ
(f'
I 2
0
0
I
,
0 2
3
2 3
3 6
6
4
•
5 6
5 2 0
7
•
fY
"""'en
4
"
14
48
40 30
"
14
21 21
0 l04_I:fY
21"", Ef(= n)
Totels
I. 0
In thls example, for simplicity and ease of calculation, integer val1Je6 of Y ere used. In many practical exercises, where continoous variables arc measured to several sioniflC8nt figures and where the number of data values is small, giving frequencies of 1 for most of the values of Y, h may be simpler to omit the column dealing with Velue-
SIB1isric
200 180 98 0 548_
L fY1
frequency and list all the individual values of Y and y2 in the appropriate columns. To gauge the underlying frequency distribution of such data sets, you would need to group individual data into broader classes le.g. all values between 1.0 and 1.9, all velues between 2.0 and 2.9, ete.) and then drew a histogram (p. 2521.
How calcuhrted
Mean
4.95
E fYln, i.e. 104/21
Median
5
Value of the In + 1112 variate, i.e. the vatue renkl;ld (obtained from the cumulative frequency column)
Modo
5
The m(lll;t common value {Y velue whh highest frequency)
Upper extreme
7
Highest Y value in dlltll sel
lOW'8r extreme
2
Lowest Y value In data set
Range
5
Difference between UpPer and tower extremes
Variance (~)
1.65
s'"""bfY2iri(~fYt2/~ "" 548-~4~/21
Standard devi8l:ion (511
1.28
.;"
Standard errot (SE)
0.280
Coefficient of variation ICoV) "R~nded
25.9%
(21 + 11/2 ,., 11th
5/';11
100s/Y
to appropriate lilignifical1t figur•.
Measuring location Use of symbols - Y is used in Chapters 40 and 41 to llignify the dependent variable in statistical calculetions (fpllCMiing the eKample of Miher and Miller-. 2000. Note, however. thet some authors use X or x in analogous fonnulee lind many cak:uI81Of'S refet to, for example, x, L x2, etc., for their Slatistical functions.
Here, Ihe obja,1ivc is 10 pinpoinl Ihc 'centre' of the frequency distribution, i.e. the value about which most of the data arc grouped. The chief measures of loe
Mean The ITK~an (denoted Y and also referred to as the arithmetic mean) is the avemge value of the data. It is oblaincd from the sum of all lhe indi\~dual data values divided by the number of data values (in symbolic terms, E YIII). The menn is a good measure of the centre of symmetrical frequency distributions of qualitative variables. It uses all of the numerical values of the Analysis and presentatiol1
of data
265
Descriptive statistics
Definitions An Ollttlet - any datum whk:h has '8 value mllCh smaller or bigger than ITI06t of the
data. Rank - the position of a dlltl'l value when all the data are placed in order of ascending magnitude. If ties occur. an average rank of the tied variates is used. Thus. the rank of the d8tum 6 in the sequence 1.3.5,6.8.8.10 is 4; the rank of each datum with value 8 Is 5.5.
Box 40.2
sample and thert.-fore incorporalCS all of the inlormation content of the
Met/ian "nle median is the mid-point of the observations when ranked ill im.Teasing order. For odd-sized samples. the mcdilln is the middle observlltion; for (:vcnsized samples it is the mean of the middle pair of obscrYo:l.tions. For 11 qUllntitative variable, the mL'dian may represent thc 100000tioIJ of the main body of data bellcr than the mean when the distribution is asymmetric or when thL'l"C arc outliers.
Three examples where simple arithmetic means are inappropriate
.....n
n
6 7
7
•
1. tf means of samples arl'! themeolves mellned. an eR"Ot can arise if the samples are of different stze. For example. th~ arithmetic mean of the means in the table shown left is 7, but this does not lake account of the different 'retiabiliUes' of each mean due to th8ir sample sizes. The correct weighted mean" is obteiT'lEld by multiplying Ilach mean by its sampl. size tn) (a "Weight') and dividing the sum of these values by the total number of observations. i.e. In the case shcMm. (24+ 49 + 81/12 = 6.75.
4
,
2. When making a mean of ratios (e.g. percenlagesl for sever.1 groups of different sizes, the latio for the combined total of 1J11 the groups is not the mean of the proportions for the individual groups. For exampkl, if 20 students from a batch of 60 ere male. this implies 40% are male. If eo students from a ba.tch of 120 are male,. this jmpl\es 50% are male. The mean percentsge,ot rnele.s
(50+4O)/2=46'Y(I is not the percentage of males in the two groups combined, because there are 20 -+' 60 = 80 males- in a total of 170 students = 47.1% approx.
pH v"lue 6 7
1 )< Ute' 1 )< 10- 1 1 )( 10-'
•mean
3.7
-lo{ho mean
R43
)(.10~1
3. If the measurement scale is not linear, arithmetic means may gfve a false value. For example. if three media had pH values 06, 7 and 8, the appropriate mean pH is not 7· becaulO8 the pH scale is logadthmK:. The definition of pH is -logWlfHI. where '(HI is expressed in moll- l l'molar'); therelore. to obtain the lrue mean, convert data into LHI values (i.e. put them on a linear scale) by calcu6ating 1()l-pH.......) as shown. Now calculate the mean of these values and convert the answer back into pH units. Thus. the appropriate answer is pH 6.43 rather than 7. Note that a similar procedure is necessary when calculating statistK:S of dispersion In such cases. so you will find these almost: certalnly asymmetric
about .the mean. Mean values of Iog--rransfonned data ere- encountered in analyticat chemistry. e.g. the particle size of the dropfets formed 'by a nebul'lzer used f9r flame or induetiv8"( coupled plasrM UCPI spectroscopy, whe.re. log-transformed velues are plotted 1F,ig:; 40.4a -lind b). The use of geomelrk: means in 9IJCh circumstantces s:erves to reducE! the effects of outliers on·the mean.
266
Analysis and presentation of data
Descri ptive statistics
'40
Mode
120
11te mode is the most common value in the sample. The mode is C'clSily found
., j"
frOlTl a tabulated frequency distribution as the most frequent value. The mode provides a rn have the same or similar values for a given frequen(.")' distribution depends Oil lhe symmctry alKI shape of the distribution. If it is llC
'00
40
2lJ
,.,
2
3 jMide
we
•
5
•
'00
Measuring dispersion Here. tlte objective is 10 qualltify the spread of the data about the centre of lhe distribution. The principal measures of dispersion arc lhe range, variance, standard deviation and coefficient of variation.
o
'<>5
''I Fig 40.4 {a} All approximnlelV Iog.transformed distribution: pnrticle size of droplets in flame
spectroscopy. (b) The results in tal plotted against the logarithm of particle size.
Rallge -nle rdllb'C is the difference bch\CCn the largest and smallest data values in the sample (Ihe extremes) and has the same unit.s as the measured variable. The range is e"&1' to detenninc, bUI is greatly affected by outliers. Its value may also depend on sample size: in gencrd1. the larger lhis is. thc greater will be the range. These features make Ihe mnge a poor measure of dispersion for many practical purposes.
Variance alld standard del'iatioll For symmetrical frequency dislributions of quantitative data, all ideal measure of disper.;ion would take into aocount each value·s deviation from the mean and provide a measure of the a\ICrage deviation from the mean. Two such statistics arc the sample variance, which is the sum of squared deviations from the mean (E(Y- fl) divided by n- I (where n is the number of data values). and the sample standard deviatioll, which is the positive square rOOI of the sample variance. The sample variance (~) has units which are the square of Ihe original units. while the sample standard dcv1ation (.f) is expressed in the original units, one reason s is often preferred as a measure of dispersion. Calculating s or ~ longhand is a tedious job and is best done with the help of a calculator or computer. If ),ou don't have a calculator that cakulat.es s for you, an alternative fonnula that simplilies cak:ulations is: Using a calculator for statistics - make sure you understand how to enter individual data values and which buttons wilt give the sample mean (usuallv shown as X or xl and sample standard deviation (often shown as ""n-1). In general. you should not use the population standard deviation (usuallV shown a~
s~+ VIL;Y'-(L;Y)'/" n I
[40.1)
To calculale s using a calculator:
L: Y, square it, divide by n and store in memory.
I.
Obtain
2.
Squ~ Yvalues, obtaill
3. 4.
E
y2, subtr
267
Descriptive statistics
Take atre to retain signifICant figures, or errors in the final valuc of .\' will result. If continuous data have been grouped into c1usscs. the dass mid·valu~ or their squares mllst be multiplied by the appropriutc frequencies before summation. When datu values are large, longhand atlcul:uiolls can be simplified by coding the data, e.g. by subtracting a constant from each datum, and decoding when the simplified cak.:ulations arc completc.
Coefficie"t of I'an'aljo" The coefficient of variation (CoY) is a dimcnsionless measurc of variability relative to location which eXptCSSL'S the sample standard deviation as a percentage of the sample mean, i.e: CoY = 100s1 J'(%)
Relative stend.rd deviation IRSDI is sometimes used inslead of coefficient of variation.
[40.2J
111is statistic is useful when l'Omparing the relative dispersion of data sets with widely dilTer;ng mealls or where different uniLS have been used for the same or similar quantities. A useful appliattioll of the CoY is to compare dilTerent analytiealmethods or procedures, so that you can decide which involves the lcast proportional error - create a standard stoek solution, then compare the results from several su~samplcs analysed by e"deh method. You may find it uscfullO usc the CoV to compare the precision of your own results with those of a manufacturer. e.g. for an autopipcuor (p. II). The smaller the CoV, the more precise (repe-dlable) is the apparatus or technique (note: this doc'S not mean that it is necessarily more am/rale. see p. 65).
Measuring the precision of the sample mean as an estimate of the true value Understanding statistical symbois - note tt1at sample 8tat1lltics fire given latin character symbols while poputerioo statistics are given Greek syrnbo+s.
Most practical exercises are based on a limited number of individual data valu<:s (a sample) which are used to make inferences about the population from which they wcr~ dmwn. For elrnmplc. the lead !.-'Ontent might be mcasurt'd in blood samples from 100 adult females and used as an estimate of the adult fL,nale lead !.-'Olltent, with the sample mean (Y) and sample standard deviation (s) providing estimates of the true values of the un(k-rlying population mean (P) and the population standard devilltion to} "Ibe reliability of the sample ITIC"dll as an estimate of the true (population) mean can be assessed by calculating the standard error of the sample mean (often abbreviated to standard error or SE), from: SE =
ExarripIe Summary statistics for the sample,mean and standard error for the data showrl in Box 40.1 W'Ould be quoted as 4.95 ± O.280ln = 21j.
268
Analysis and presentation of dilta
slVii
[40.3)
Strictly, the standard error is an estimate of the standard deviation of the means of II-sized samples from the population. At a practical level, it is clear from eqn 140.3] that the SE is directly afTectL'd by sample dispersion and inversely related to sample size. This means that the SE will decrease as the number of data values in the sample increases, giving incrcast'd precISion. Summary dcscriptiv~ statistics for the sample mean are often quoted as Y ± SE (11), with the SE being given to one significant figure more than tile mean. You clln use such infonnation to call)' out a I-test between two samples (Box 41.1): the SE is also uscful because it allows calculation of confidence limits for the sample meaD (p. 278).
Descriptive statistics
Describing the 'shape' of frequency distributions Frequency distributions may differ ill the following ehardcterislics: • • •
number of peaks: skewness or asymmetry: kurtosis or pointedness.
The shape of a frequency distribution of a s.mall sample is i\ITected by chance V'driation and mlly not be a fair rcncction of the underlying population frequency distribution: check this by compuring repcatt'd sampk'S from the same population or by increasing the sample size. If the original shape were due to random evcllLS. it should not appear consistently in repeated samples and should become k'SS obvious as s.ample size illcteaSl:S. Genuinely bimodal or polymodal distributions may result from the l:ombinllttoll of two or more unlimited distributions, indit:ating that more than one underlying population is being sampled (Fig. 40.5). An example of a bimodal distribution is the hcight of adult humans (fcmales and malcs L'Ombincd).
Fig. 40.5 FreQuency distributions with different numbers of peaks. A unimodal distribution la) mav be symmetrical Of asymmetrical. The dashed lines in (bl indicate how a bimodal distribution could arise from a combination of two underlying unimodal distributions. NOle here how the term 'bimodal' is applied to any distribution with two major peaks - their frequencies do not have to be exactly Ihe same.
llepende"lmeDsufl!dl VllriDbl,
Fig 40.6 Examples of the two types of kurtosis.
A distribution is skcv.'cd if it is not symmetrical. a symptom being that the mean, ml:dian and mode are not equal (Fig. 40.3). Positive skewness is where the longer 'lail' of the distribution occurs for higher valucs of the measured variable; negative skewness where the longer tail occurs for lower valucs. Skewed distributions frequently occur in the study of chemical measurements, owing to the presence of occasional gross errors. Kurtosis is the name given to the 'pointedness' of a frlXjuency distribution. A platykurtic frequency distribution is one with a flattcned peak, whilc a Analysis and presentation of data
269
Descriptive statistics
lcptokurtic frequency distribution is one with a pointed peak (Fig. 40.6). While descriptive terms can be used. based on visual observation of the shape and direction of SkL'W. the degree of skewness and kurtosis can be <.juantified and statistical tests exist to lest the 'significdnce' of observed values. but the calculations required are complex and best done with the aid of a computer.
270
Anatysis and presentation of data
-------8
Choosing and using statistical tests This chapter outlines the philosophy of hypothesis-testing statistics, indicates the steps to be taken when choosing a lest, and discusses features and assumptiolls of some important tests. For details of the mechanics of tests, consult appropriate tcxlS (e.g. Miller and Miller. 20(0). Most tcsts arc now available in statistical packages for computers (sec p. 315). To carry Ollt a statistical tcst L 2. 3.
Decade what it is you wish to test (crealI..' a llull hypothesis and its allenlative). Dctcnnine whether your data fit a standard distribution pattem. Select a test and apply it to your data.
Setting up a null hypothesis
.,
mlIlIlU 0
I
Hypothesis,testing statistics are used to compare the properties of samples L;ther with other samples or wilh some theory about them. For instance, you may be intercstctl in whether two samplcs can be rcgardctl as having dinl::rent means, whether the concentralion of a peslil..;de in a soil sample can be regarded as randomly distributed, or whether soil organic matter is linC'
~
You can't use statistics to prove any hypothesis. but they can be used to assess how likely it is to be wrong.
Statistical testing operatcs in what at first S(.'ems a rather perverse manller. Suppose you think a 1fC'.Hment has all effocL The theory you actually test is that it has no effoct; the test tells you how improbable your data would be if this theory were true. 'nlis 'no effect' theory is the null hypothesis (NH). If yom data are very improbable undLT the NH, then yOIl may suppose it to be wrong, and trus would support your original idea (the 'alternative hypothesis'). The concept can be illustrated by an example. Suppose IWO groups of subjects were treated in differctlt ways, and you observed a difference in the mean value of the measured variable for the two groups. Can this be regarded as a 'tnle' difference? As Fig. 41.1 shows. it could have arisen in two ways:
.,
I.
2.
depend&nt (meaSUfeen vanable
Fig. 41.1 Two explanations for the difference between two means. In case (a) the two samples happen by chance 10 have come from opposite ends of the same frequency distribution, Le. there is no true difference between the samples. In case fbI the two samples come from different frequency distributions, I.e. there is a true difference between the samples. In both cases, the means
of the two samples are the same.
Because of the way the subjects were allocated to treatments, i.e. all the subjects liable to have high values might, by chance, have been assigned 10 one group and those with low values to the other (Fig. 41.la). Because of a genuine effect of the treatments, i.c. each group came from a distinct frequency distribution (Fig. 41.1 b).
A statistical test will indicatc the probabilities of these options. The NH states that the two groups come from the same population (i.e. the treatment effects arc negligiblc in tllC context of random variation). To test this, you cakulate a tcst statistic from the data, and compare it with tabulated critical values giving the probability of obtaining the observed or il more extreme result by chance (sec Boxes 41.1 and 41.2 below). This probability is sometimes called the significance of the test. Note that you must take into account the degrees of freedom (d.£.) when looking up critical values of most test statistics. The d.C is related to the sizc(s) of the samples studied; formulae f~ calculating il depend on the test being used. Chcrnists normally usc two-tailed tests, i.e. we have no certainty Analysis and presentation of data
271
Choosing and using statistical tests
Definition ModuIus- the absolute value of a number, e.g. modulus -3.385 = 3.385.
Choosing between parametric and noo-
parametric tests - always plot your data graphically when determining whether they are suitable for parametric tests as this may savo a lot of unnece6sary effort
tater.
beforehand thai the trealment will have a poSitIve or negative effect compared with the control (in a one-tailed lest we expect one particular trealment 10 be bigger than tbe other). Be sure to use critical values for the correct type of test. By convention, the critical probability for rejecting the NH is 5% (i.e. P = 0.05). This means we rejt.'C1 the NH if the observed result would have come up less than I time in 20 by chance. If the modulus of the lest statistic is less than the tabulated critical value for P = 0.05. then we accept the NH and the result is said to be 'not significant' (NS for short). If the modulus of the test statistic is greater than the tabulated value for P = 0.05, then we n..j eet the NH in favour of the alternative hypothesis that the treatments had different effects and the result is 'statistically significant'. Two types of error are possible when making a conclusion on the basis of a statistical test. '111e first occurs if you reject the NH when it is true and the second if you accept the NH when it is false. To limit the chance of the first type of error, choose a lower probability, e.g. P = 0.01, but note that the critical value of the test statistic increaSl-'S when you do this and results in the probability of the second lv'TrOr increasing. The conventional significance levels giv<:n in statistical tables (usually 0.05, 0.01, 0.001) are arbitrary. Increasing use of statistical computer programs is likely to lead 10 the actual probability of obtaining the calculated value of the test statistic being quoted (e.g. P = 0.037). Note that if the NH is rejected, this docs not tell you which of many alternative hypotheses is true. Also, it is important to distinguish between statistical and practical significance: identifying a statistically significant difference betwccn two samples docs not mean that this will carry any chemical importance.
Comparing data with parametric distributions A pammetric test is one which makes particular assumptions about the mathematical nature of the population distribution from which the samples were taken. If these assumptions are not true, then the test is obviously invalid, even though it might give the answer we expect! A non-parametric test docs not assume that the data fit a particular pattern, but it may assume some things about the distributions. Used in appropriate circumstances, parametric tests are better able to distinguish between true but marginal differences between samples than their lion-parametric equivalents (i.e. they have greater 'power'). The distribution paltern of a set of data values may be chemically relevant, but it is aL~o of practical importance because it defines the type of statistical tests that can be used. The properties of the main distribution types found in chemistry are given below with both rules of thumb and more rigorous tests for deciding whether data fit these distributions. Billomiallli~'trihutions
These apply to samples of any sizc from populations when data values occur independently in only two mutually exclusive classes (e.g. type A or type B). They describe the probability of finding the different possible combinations of the alllibute for a specified sample size k (e.g. oUl of 10 samples.. what is the chance of 8 being type A'f). If P is the probability of the attribute being of type A and fJ the probability of it being type B, then the expected mean sample IllUl1bcr of type A is kp and the standard deviation is .j(kpq). Expected frequencies can be calculated using mathematical expressions (see Z12
AnalysIs and presentalJon of data
Choosing and using statistical tests
"
lclP-01
•
. . 0.4
•
Z
0.3
t ., 02
o o
, , •
5
0
,
2
3
no. of in6voduals oIlype
4
A"
5 sample
0
, , •,
Fig.41.2 Examples of binomial frequency distributions with different probabilities. The distributions show the expected freqoerlCy of obtaining n individu81s of type A in 8 sample of 5. Here P is the probability of DO individual being type A rather thon type B.
Miller and Miller, 2000). Examples of the shapes of some binomial distributions arc shown in Fig. 41.2. Note thai they are symmctrical in shape for the special car.e p = q = 0.5 and the greater the disparity between p and q, the morc skewed the dil;tribution. O'lC'lniC"
Poissoll distrihutiolls Tendency towards the normal
distribution - under certain conditions, binomial and Poisson distributions can be treated as normally distributed: •
where samples from a binomial distribution are large li.e. > 151800 P
and
Q
are dose to 0.5:
• for Poisson distributions, if the number of counts recorded in eactl outcome is greater than about 15.
~ 5 7 /Y. This is an alternative measure of dispersion to the coefficient of variation (p. 2681
Coefficient of dispersion =
These apply to discrete chara<.1eristics which can assume low whole-number values. such as counts of events occurring in area, volume or lime. TI1C events should be 'mre' in that the mean number observed should be a small proporlion of the total that could possibly be found. Al'\(), finding one count should not influcnce the probability of finding another. l1Je shape of Poisson distributions is described by only olle paramcter, the mean nwnber of events observed. and has the special chanlcterislic that the variance is equal to Ihe mean. The shape has a pronounced positive skewness al low mean connts, bUI becomes more and more symmetric
• •
Use the mle of thwnb that if the coefficicnt of dispersion :::::: I, the distribution is likely to be Poisson. Calculate 'cxpected' frequ<.."Jlcics from the equation for the Poisson distribulioll and compare with actual values using a Y:-tCSI or .a G-tesl.
It is sometimes of inlerest to show that data are not distributed in .a Poisson fashion, If Sl/y > I, the data are 'clumped' and occur together more than would be expected by chance; if s2/y < I, the data arc 'repulsed' and occur together less frequently than would be expected by chance, Analysis and presentation of data
273
Choosing and using statistical tests
leI
r.. 12
0.' o o
I
I
I
I
I
2.
3 4
5
I
o
I!
I , I
I
,
1345678
1111, ..
I I I j I I J I I I l l 1 2 3 4 5 6 1 8 9 10 11 12 13 14 15 16 17 18 19 2D 21 no. of iodMdllll. of lyPll A in UfIlfIle I!!
,
•
I
I
I
!
!
I
n
I
i
ZJ 24
Fig.41.3 Exomples of Poisson frequency distributions differing in mean. The distributions ore shown 8S line charts because the independent variable levents per sample) is discrete.
Normal distributiOlls
(Gaus.~;oll (!i3'/J"ihutioIlS)
These occur when rdndom events act to produce variability in a continuous charactcnstic (quan,itativc variable). This situation occurs frequently in chemistry. so normal distribulions are very useful and much llsed. The belllike shape of nonnal distributions is specified by the population mean and standard deviation (".g. 41.4): it is symmetriC"dl and configured such that 68.27% of the data will lie within ±l !>tandard deviation of the mean. 95.45% within ±2 standard deviations of the mt:"dn. and 99.73% within ±3 !>t.andard deviations of the me-an. Some chLmical examples of dala likely to be distributed in a normal fashion arc pH of natural walers; melting point of a solid compound. To check whether data come from a normal distribution. you can:
•
•
•
c
Use the rule of thumb thaI the distribution should be symmetrical and that nearly all the dala should fall within ±3s of the mean and about t\\·o-thirds within ±Is of the mean. Plot the distribution on norm:!1 probability graph paper. If the distribution is normal. the dala will lend 10 follow a straight line (sec Fig. 41.5).
.,
.,
:; ~
deflllOOI!fW variobls
Fig. 41.4 Examples of normal frequency distributions differing in mean and standard deviation. The horizontal bars represent population standard deviations for Ihe curves, increasing from fal to lel. Vertical dashed lines are population means, wtlile vertical solid lines show positions of values ±1, 2 and 3 standard devintions from the means.
274
Analysis and presentation of data
Choosing and using statistical tests
..
Deviations from linearity rf.'Vcal skewness and/or kurtosis (Sl.'C p. 269). the signiricance of which can be tcsted ~;tatistiC'dlly (see Miller and Miller,
o
20(0).
•
" " rtf
1
r
'"
1 r_.
5
2
o I
I
I
2
3
•
, • , I
5 UPpilf
5
I
I
I !O
Use a suitable statistical computer program to gl.'tlerate prcdielt'd normal curvcs from the rand.f valucs of your samplc(s). Thl.'SC C'dll be comp
TI1C wide availability of tests based 011 the nomlal distribution and their relativc simplicity means you may wish to transform your data to makc them more likc a normal distribution. Tablc 41.1 providl.'S lnlOsformations that can be applied. TIle lnlllsfomlcd data should be tl.'Stcd for normality as described above before proceeding - don't forb'Ct thllt you may lIctxl to check that transfonncd variances are homogeneous for certain tests (sec below). A very important theorem ill statistics. the ccntral limit theorem, states that as samplc size increases. the distribution of a series of means from any frequency distlibulion will become nonoally distributed. This fact can be used to devise an expc:..Timl.'tlllll or sampling stnltegy that cnsures that data are normally distributed, Lc. using mC-dllS of samples as if t1}(:y wcrc primary data.
clan ~miI
Fig. 4J.5 Example of a normal probllbility plot. The plotted points are from a small data sel where the mean Y ... 6.93 and the standard deviation s "" 1.895. Note that values corresponding to 0% and 1ClO% cumulative frequency cannot be used. The straight line is that predicted for a normal distribution with Y = 6.93 and s = 1.895. This is plotted by calculating the expected positions of points for Y ± s. Sinco 68.3% of the distribution falls wLthin these bounds, the relevant points on the cumulative frequency scale are 50 ± 34.15%; thus this line was drawn using the points (4.495, 1S.8Sland (8.285, 84.15) as indicated on the plot.
Choosing a suitable statistical test Compal'ing location (e.g. meall!J') If you can assume that your data are normally distributed, the main test for comparing two means from independent samples is Student's I-test (see Boxes 41.1 and 41.2, and Table 41.2). This assumes that the variances of the dala sets arc homogcneous. Tcsts based Oil the I-distribution are also a.....lilable for comparing paired data or for comparing a samplc mean with a ch()S(.'tl valuc. When comparing means of two or more samples, analysis of variance (ANOVA) is a very useful technique. TIlls method also assumes data arc Ilonnally distributed and that lhe variances of the samples arc homogcncous. The samples must also be independent (e.g. not sub-samples). The ncsK-J types of ANOVA are useful for letting you know the relative importancc of different sources of variability in your data. Two-way and multi-way ANOVAs are useful for studying intCf'dctions bet....-ecn treatmcnts. Table 47.' Suggested transformations aM:ering different types of freQuency distribution to the norm&l type. To use, modify data by the formula shown: then examine effects v.;th the tests described on p. 272-275.
Proportions (including percentages); binomial
lIrcsine .jx (also called the angular transformation)
Scores; Poisson
.jx or .jex + 1/21 if zero values present
Measurements; n6glltively skewed
Xl,
Measurements; positively skewed
x 3 , x·, etc. lin order of increasing strength)
l/.jx, .jx, In x, 1/ x (in order of increasing strength)
Analysis and presentation of data
275
Choosing and using statistical tests
Box 41 1 How to carry out a Hest The t-test was devised by a statistician who used lhe penname 'Student', so you may see it referred to as Studenrs t-test. It is used when you wish to decide whether two samples come from the same population or from different ones (Fig. 41.1). The samples might have been obtained rrom two different sources or by applying two different treatments to an originally homogeneous population. The nutl hypothesis INHl is that the two groups can be represented as samples from the same overlying population (Fig. 41.1al. If, as a result of the test, you accept this hypothesis, you can say that there is no significant difference between the groups. The altemative hypothesis is thai the two groups come from different populations (Fig. 41.1 bl. By rejecting the NH as a result of the test, you can accept the alternative hypothesis and say that there is a significant difference between the samples, or, where an experiment has been carried out. that the two treatments affected the samples differently. How can you decide between these two hypotheses? On the basis of certein assumptions (see belowl. and some relatively simple calculations, you can work out the probabilitY that the samples came from the same population. If this probability is very low. then you can reasonably reject the NH in favour of the altemative hypothesis, and if it is high, you will accept the NH. To find out the probabilitY that the observed difference betv.oeen sample means arose by chance, you must first calculate a 'r value' for the two samples in question. Some computer programs (e.g. Minitab") provide this probabilitY as part of the output, otherwise you can look up statistical tables (e.g. Table 41.2). These tables show 'critical values' the borders between probabilitY levels. If your value of r exceeds the critical value for probabilitY P, you can reject the NH at this probability ('level of significance'l. Note that: •
•
tor a given difference in the means of the two samples, the value of r will get larger the smaller the scatter within each data set; and for a given scatter of the data, the value of r will get larger. the greater the difference between the means.
So, at what probabilitY should you reject the NH? Normally, the threshold is arbitrarily sct at 5% - in scientific literature you often see descriptions like 'the sample means were significantly different (p < 0.051'. At this 'significance lever there is still up to a 5% chance of the t value arising by chance, so about 1 in 20 times, on average, the conclusion will be wrong. If P tums out to be lower, then this kind of error is much less likely. Tabulated probability levels are generally given for 5%, 1% and 0.1% significance levels (see Table 41.2). Note that this table is designed for two-tailed' tests, i.e. where the treatment or sampling strategy could have resulted in either an
276
Analysis and presentation of data
increase or a decrease in the measured values. These are the most likely situations you will deal with in science. Examine Table 41.2 and note the following: • The larger the size of the samples It.e. the greater the 'degrees of freedom'). the smaller t needs to be to exceed the critical value at a given signiflC80Ce level. • The lower the probability, the greater t needs to be to exceed the critical value. The mechanics of the test A calculator that can work out means and standard deviations is helpful.
Y, and Y;r (Vt - Y:tl.
1.
Work out the sample means the difference between them
2.
Work out the sampkl standard deviations Sol and ~. (Note: if your calculator offers a choice, chose the 'n -1' option for calculating s- see p.267.1
3.
Work out the sample standard errors SEt - 51/ v'n1 and SE2 = 5.2/,/iii; now square each, add the squares together, then take tl\8 square root of this (n, and 02 are the respective sam Ie sizes, which may, or may not. be eQual) (SE 1 ) + (SE l ) I.
4.
Calculate t from the formula:
and calculate
)', - Y2
t
~ -J"F(I~SE~'~I')~+~(~<S:::E'~"~)
[41.1]
The value of t can be negative or positive, depending on the values of the means; this does not matter and you should compare the modulus (absolute value) of r with the values in tables.
5.
Work outthe degrees of freedom - (n, -1) + (112 - 1).
6.
Compare the t value with the appropriate critical value in a tabte. e.g. Table 41.2.
Box 41.2 provides a worked example - use this to check that you understand the above procedures. Assumptions that must be met before using the test The most important assumptions are: •
The two samples are independent and randomly drawn (or if not. drawn in a way that does not create bias). The test assumes that the samples are quite large. • The underlying distribution of each sample is normal. This can be tested with a special statistical test, but a rule of thumb is that a frequency distribution of the data should be (a) symmetrical about the mean and (b) nearly all of the data should be within 3 standard deviations of the mean and about two-thirds within 1 standard deviation of the mean (see p. 2741. • The two samples should have eQual variances. This again can be tested lby an F·testl. but may be gauged from inspection of the two standard deviations.
Choosing and using statistical tests
Box 41.2
Worked example of at-test
Suppose the following data were obtained in an experiment (the units are not relevant); Control: 6.6, 5.5, 6.8, 5.8, 6.1, 5.9 Treatment: 6.3, 7.2, 6.5, 7.1, 7.5, 7.3
V2 = 6.9833: means = V, - Y2 = -0.8666
-0.8666 0.280277 = -3.09
5. dJ.=(5+5)=10
Using the steps outlined in Box 41.1, the following values are obtained (denoting control with subscript 1, treatment with subscript 2):
1. V, =6.1167;
-0.8666 4. t= V(0.202348 2 + 0.193934 2)
difference
between
2. 51 = 0.49565; S2 = 0.47504 3. SE, = 0.49565/2.44949 = 0.202348 SE 2 = 0.47504/2.44949 = 0.193934
6.
Looking at Table 41.2, we see that the modulus of this 1 value exceeds the tabulated value for P = 0.05 at 10 degrees of freedom (= 2.23). We therefore reject the NH, and conclude that the means are different at the 5% level of significance. If the modulus of t had been < 2.23, we would have accepted the NH. If the modulus of t had been> 3.17, we could have concluded that the means afe drtferent at the 1% level of significance.
Table 41.2 Critical values of Student's r statistic (for two-tailed tests). Reject the null hypothesis at probability P if your calcutalBd t value axceeds the value shown for the appropriate degrees of freedom = lnl - 1) + (r1
Degrees of freedom 1 2 3 4 5 6 7 8 9
10 12 14 16 20 25
30 40 60 120 00
Checking the assumptions of a testalways acquaint vourself with the assumptions of a test. If necessary, test them before using me test.
Critical values for
Critical values for
P=O.05
P= 0.01
12.71
63.66 9.92
4.30
3.18 2.78 2.57 2.45
5.84
2.36
3.50 3.38
2.31 2.26 2.23
2.18 2.14 2.12 2.09 2.06 2.04
2.02 2.00 1.98 1.96
4.60 4.03
3.71 3.25 3.17 3.08
2.98 2.92
Critical values for P '='" 0.001 636.62
31.60 12.94 8.61 6.86 5.96 5.40
5.04 4.18 4.59 4.32
4.14 4.02
2.86
3.86
2.79 2.75 2.70 2.66 2.62
3.72
2.58
3.86 3.55 3.46
3.37 3.29
For dala satisfying the ANOVA requirements, the least significant difference (LSD) is useful for making planned comparisons among several mean... Any two means that differ by more than the LSD will be significantly different. The LSD is useful for showing on graphs. The chief non-parametric tests for comparing locations are the MannWhitney V-test and the Kolmogorov-Smimov test. The fanner assumes that the frequency distributions of the data selS are similar, whereas the latter makes no such assumption. 10 lhe Kolmogorov-Smirnov lest, significant differences found with the test may be due to differences in 1000.Hion or shape of the distribution, or both. Suitable non-parametric comparisons of location for paired quantitative data (sample size ~ 6) include Wilcoxon's signed rank test, which assumes that the distributions have similar shape. Analvsis and presentation of data
277
Choosing and using statistical tests
Non·pantmetric comparisons of location for three or more samples include the Kruskal-Wallis II-test. Here. the two data selS can be unequal in size. but again the underlying distributions are assumed to be similar,
Comparing dispersiom; (e.g. I'ariances) If you wish to compare the variances of two sels of data that arc normally distributed. use the F·lcst, For comparing marc than two samples, it may be suffICient to use the F......·test. on the highest and lowest vanan<.'eS. The SchelT'e-Box (Iog-ANOVA) test is recommended for tcsting the significance of differences bet\\.oeen severdl variances. Non.paramelrie tesls exisl but are nOI widely available: you may need to transform the data and usc a test based on the normal distribution.
Detet-minillK wlletller ft'eqllellcy ohse"I'atiolujit theoretical expectatioll The i-test is useful for tests of 'goodness of f"it', e.g. comparing expe<;ted and observed progeny frequencies in genetical experiments or comparing observed frequency distributions with some theoretical function. One limitation is that simple fommlae for calculating i assume that no expected number is less than five. The C·test (21 test) is used in similar cireumstaoc'eS.
CompUl'inK p"oportion data When comparing proportions bet\veen two small ~roups (e.g. whether 3/10 is significantJy different from 5/10), you can use probability tables such as those of Finney el (d. (1963) or calculate probabilities from fomlUlae; however, this can be tedious for large sample sizes. Certain proportions can be tmnsformed so that their distribution becomes normal.
Placing confidence
Confidence limits for statistics other than the mean - consult an advanced statistical text (e.g. Miller arK! Miller, 2000) if you wish to indicate the reliability of estimates of, for example, population variances.
/imit~
on all estimate of a popil/atioll parameter
On many occasions, sample statistics are used to provide an estimate of the population pammeters. It is extremely useful to indicate the reliability of such estimates. This can be done by putting a confidence limit on the sample statistic. The most common application is to place confidence limits on thc mean of a sample from a nonnally distributed population. This is done by working out the limits as Y - (/1'\,,-11 x SE) and Y + ('11" I) x SE) where ''''''-11 is the tabulated critical value of Student's t statistic for a two-tailed test with 11 - I degra:s of freedom and SE is the standard error of the mean (p. 268). A 95% confidence limit (i.e. P = 0.05) tells you that on average. 95 times out of 100. this limit will contain the population mean.
Regression Ulld con'e/atiOIl These methods are used when testing relationShips between samples of two variables. If olle variable is assumed to be dependent on the other then regression techniques are used to find the line of best fit for your data. This does not teU you how well the data fit the line: for this. a correlation coefficient must be calculated. If there is no a priori reason to assume dependency between variables, correlation methods alone are appropriate, If grnphs or theory indicate a line'"d( relationship between a dependeD! and an independent variable, linear regression can be used to estimate the 218
Analysis and presentation of data
Choosing and using statistical tests
,., • ••
,
• •
•
'"•
, • •
•• • • •
• •
,,' ,
•
• • • • • • • •• • •
• • •
•
'"
, • •• • •
• • • ••
..•
Fig.41.0 Examples of correlation. TIle linear
regression line is shown. In (aland (bl, the correlation between x and y is good: for l81 there is a positive correlation and the correlation coefficient would be close to 1: for lbl thare is II nogative correlation 1Ind the correlation coefficient would be close to -1. In (c) there is a weak positive correlation and r would be close to O. In (dl the correlation coefficiont may be quite large, but the choice of linear regression is clearly ir'lappropriate.
equation that links them. If the relationship is not linear, a tmnsformation may give a linear relationship. For example, this is sometimes used in analysis of chemical kinetK:s. However, 'Iincariutions' can lead to errors when carrying out regression analysis: take care to ensure (i) that lhe data arc evenly distributed throughoul the rdnge of the independellt variable and (ii) lhat the vlirianccs of the dependCllt variable are homogeneous. If thcse criteria cannot be met. weighting methods may reducc errors. In this situation, it may be better to usc non-linear regression using a suitable computer program. Model I linear regression is suitable for experiments where a dependent variable Y varies with an error-fi'ee independent variable X and the mean (expected) value of Y is given by (J + bX. This might ()(.:cur whcre you have carefully controlled the independent variable and it (;an therefore be assumed to have zero error (e.g. a calibration curve). Errors can be calculated for estimates of a and b and predicted wlues of Y. The Y values should be nonnally di~1ributed and thc variance of Y constant at all values of X. Model II linear regression is suitable for experiments where a dependent "ariablc Y "aries with an independent variable X which has an error associated wilh it and. the mean (expected) value of Y is given by a + bX. This might occur where the experimenter is measuring t.....o variables :md believes there to be a causal relationship betwccn them; both variables will be subject to errors in this Qlse. The exact method to usc depends on whether your aim is to estimate the functional relationship or to estimate one variable from the other. A correlation coefficient measures the strength of relationships but does not dCSLTibc the relationship. "Ibesc coefficients are expressed as a number between -I and I. A positive coefficient indicates a positive relationship while a negative (;oefficient indicates a negative relationship (Fig. 41.6). The nearer the cocflieient is to -lor I. the stronger the relationship between the variables.. i.e. the less scalier there would be about a tine of best tit (note that this does not imply that one variable is dependent on the other!)_ A coefficient of 0 implies that t~re is no relationship between the variables. The importancc of graphing data is shown by the C'"dSC illustrated in Fig.41.6d. Pearson's product-moment t"Orrelation coefficient (r) is the most commonly used correlation coefficient. If both variables are normally distributed. then r can be used in statistical tests to test whether the degree of correlation is significant. If one or both variables are not normally distributed you call usc Kendall's coefficient of rank correlation (t) or Speannan's coefficient of rank correlation (rs). They require thal data are ranked separately and calculation can be complex if there are tied mnks. Spearman's coefficient is said to be bcth.;r if ttx:rc is ulla;rtainty about the n:liability of dosely ranked data values.
Analvsis and presentatioo of dsta
279
-0
Drawing chemical structures Omwing the structure of a chemical compound is probably one of the first
Fig. 42.7
"I c /1' " "" Methane.
",N_-'' '__'''' '"
H~"""I"""NH,
""
Fig. 42.2 Structure of hexaamminecobaltate.
--
~ --,
,
,
, , "
"'" "
"
'r~
, "," J
• •
•
"", ' \. "
~,-,-- -~...,
Co
a
'~
•
I
'--l-~I% ,,, -'
H,N ,-" , ',
basic requirements of any chemisl. It requires knowledge of the chcmi/':al composition of the structure 10 be drawn, an understanding of the type of bonding. and frequently a menial visualization of the arrangement of atoms (or iOllS). Once this has been assimilated il is not uncommon to dmw a representation of the structure on paper. What is often lacking is the reali7.alion that lhe molecule should be rcpres<.'llcd in three clinlcnsions. To some extent it is po:;siblc 10 represent a thrre-dimension
~'
Fig. 42.3 An octahedron.
• •
• • •
•
Always dmw chemical structures in ink (pencil fades with timc). Always ensure that the chemical structure is dmwn large enough, so that no ambiguity is possible. If drawing by hand. ensure that each atom is clearly identified. ntis may require the usc of coloured pens. Try and keep the structure as simple as possible, highlighting only the key features. Any tcxt should appear in a clear script (by hand or word processed). When possible. it is advisable to use computer software packages to gencmte chemical structures. For example. Figs 42.1-42.4 and 42.6 were genemted using ChemWindow·..·. AI....-ays indicate the number of chemical bonds. Make sure no confusion is possible between different leiters of the alphabet representing elements in the Periodic Table. If necessary, show the number of electrons (pairs or individual electrons) dearly. Remember, a . (full SlOp) may be mistaken ror a mark in the paper. It may be necessary to indieate lhe structural fonnula of a molecule, e.g. in isomerism.
Selected examples of drawing chemical structures Lewis st"IIct"res When dmwing LeWls structures it is important to show the position and number of c1eclTOns. For example. the Lewis structure for Coj- is shown in Fig. 42.4a. Note the posilion of the double oond. Also, both carbon and 280
AnalysIs and presentation of data
Drawing chemical structures
oxygen obey the octet rule, i.e. the number of electrons around each alom adds up to eight (a single bond is composed of two electrons. a double bond of four electrons). Lewis structures sometimes give rise to canonical fonns. The three possible canonic-al [anTIs for C~- are shown in Fig. 42.4b. It is noted that as the position of the double bond moves the number of lone pairs of electrons changes. In reality, experimental evidence indicates thai the C-O bond in carbonate is composed of neither single nor double bonds but is intenncdiate in bond length and strength A more appropriate method of representing this structure is by delocalization of bonding ek'Ctrons (Fig. 42.4c).
J' C
Ionic st"uctllres In solid-slate chemistl)' it is often necessary to draw a 'unit celr. 1ne most commonly found arc the hexagonal close-packed (HCr) crystal structure and cubic close-packed (CCP) crystal structure. In the HCr structure atoms are arranged in an ABABA repeating pattern. while in the CCI) structure the arrangemenl is an ABCABCA pattern. In both (:
I'
O
0,)'0 I"
Fig. 42.4
Lewis diagrams for
C~-.
Isomerism Common chemical drawing packages: ChemDraw furl: http://WWW.camsoh.coml ChemWindow R (URL: hnp:/twww.sohshell. coml IsislDraw (UAL: http://WWW.mdli.coml
The importance of drawing ehemiC
~
It is often important when drawing c:hemic:al structures to impart some structural identity.
c
A
B
B
A
A
I.'
Ibi
Fig. 42.5 Close-packing arrangement for (al
hexagonal close packing (ABABA etc.) and (bl
cubic close packing (ABCABCA etcJ.
Atomic OI'hita/!)' YOli may need to draw a visualization of atomic orbitals, usually the s, p and d orbitals. This (:
E/C£'tronic configuration It is often difficult to fC(.-ollect the order of filling of the electronic struclUres
of atoms of different elemeDts. Fig. 42.8 shows the usual order of filling of Analvsis and presentation of data
281
Drawing chemical structures
,
•
:::=::g,. ----a.. ~
f c.....!!a
" So
.
f -!!..o
• "
"
"
" " f-2'-o
o
o
'" "
•
I---!'a Fig. 42.8 Elactronic configuration.
o ""'-
e-
O-
Fig. 42.6 Geometric isomers of dichlorocyclopropane. Fig. 42.9 Mnemonic for electronic configuration determination based on the Aufbau principle.
s
,
,
+. +. +. ,
•
•
,
,
-¥. L
-~,
l
the orbiUtls of ftn atom according to the Aufbau principle (lowest energy first). However, a simple mnemonic exists to facilitate the correct order for each element in the Periodic Table (Fig. 42.9). In order to usc the mnemonic all you need 10 remember is that: • • • •
s orbitals can have up to 2ep orbitals am have up to 6 ed orbitals can have up 10 IOef orbitals can have up to 14 e~ .
Then, il is simply a case of addition. In general terms, the order of electron filling is as follows (simply tmnslating Ihe mnemonic into an order): Is~2p3s3p4sM4p5s~5p~4f~6p7s
...
For example. tbe atomic number of calcium u; 20 (corresponding to 20 e1cctrons). Therefore the electronic configuration is: Is 2 2i 2 p6 3s2 J p6 4~
Valence shell e/ect"oll pair "eplilsion (VSEPRj theory Fig.42.7 Atomic orbitels.
282
Analysis and presentation of data
1lUs is used 10 predict Ihe shape of molecules. In order 10 be able to predict the geometry (or shape) of a molecule several simple steps arc required. For example, consider the case of BrFJ • In this case \\>"(: have indicated by
Drawing chemical structures
_ 'V ~
'V
""
""
underlining that the central atom is bromine. The first step is to detennine the number of valence electrons for bromine; this is done by establishing the electron configuration for bromine, i.e. Is 2 2s 2 2p6 3~ 3p6 4s2 3d lO 4p5. We can determine that the number of outer elcctrons is seven (from 4s2 and 4p5). We then determine how many atoms are attached to the central bromine atom - the answer is three. By simple addition we have 7 + 3 = 10 electrons. We know that two electrons are required per bond; therefore we have enough electrons for five bonds. Then, it is simply the C'ase to determine a geometry that allows for five bonds to be at the maximum distance from each other. Commonly. five different arrangements of atoms are found: linear, trigonal planar. tetrahedral. trigonal bipyramidal and octahedral. These arrangements are shown in Fig. 42.10. We can therefore see that the gwmetry of I3rF) is trigonal bipyramidal. VSEPR also works for anions and cations using the same procedure. for a cation, a positively charged species, simply deduct one electron from the total; similarly for an anion, a negatively charged species, add one electron to the total. In all l-'aSCS the total number of electrons obtained will be an even number.
octahedral
-jj
Fig. 42.10 Geometries of molecules.
-I-
AO
MlJ
M)
~
o
~::
0i
It7+-
H o~
,
t'
0
-I-
0" -.....: II
II
<:,
II
< :: "/C>-tt-
II
~
*'" "
0"
h'
0,
0- elec\rOllic (::(Il1iglullilln: Is2 2s'l 2p4
Fig. 42.11
Molecular orbital diagram for oxygen.
Motc('utar Of'hital diagrams Molecular orbital diagrams (see Fig. 42.11) are required in the study of chemical bonding. For a diatomic molecule these consist of two atomic orbitals (AOs) and two molecular orbitals (MOs). In the case of the two MOs, one is the bonding MO and the other the anti-bonding MO. An asterisk (..) is used to represent an anti-bonding MO. Bonding MOs (of lower energy) are always oc"Cupied first. Two types of MO are shown, (J and n, using oxygen, O 2 • as an example. The first task is to determine the e1cctronic configuration for each oxygen atom, i.e. ls 2 2s 2 2p4. An outline of the MO diagram is constructed such that one AO is located on the lefthand side of the page and the other on the right-hand side (the use of ' indicales that a different AO is represented) with the MOs positioned in the centre (bonding MOs in the lowest position). You then have an MO diagram composed of three different energy levels corresponding to the Is, 2s and 2p orbitals. Then. by simply adding the correct number of electrons into the two AOs first total up the number of electrons. Electrons are then placed into the MO in the following sequence; lowest available position first; and then, individual electrons prior to pairing of electrons. The completed diagram hi shown in Fig. 42.11 where all solid lines (in both the Analysis and presentation of data
283
Drawing chemical structures
AD and MD positions) represent places where up to two electrons C'dn he paired up (dashed lines simply indicale association wilh a particular AD). Arrows (TO indicate that the eleclrons are spinning (paired electrons have opposite spins). The numbering of bonding and anli·bonding MOs is merely for numerical sequencing and has no olher significance. It is also worth noting that O 2 has unpaired electrons in the
264
Analysis and presentation of data
-8 Table 43.1
Melting points for naphthalene
m.pt.
rei
7'81
A
B
C
80 T7 80 80
o E F G H
7'76
8'
I
J
80
7'78
K
l M
82 81
N
Mean melting point of naphthalene is 79.5 C.
':LiJjJ
i
2
-
1
--
' -
-
o
16
77
18
19
SO
81
fTIIlltirJ,J p(liIll loel
Fig. 43.1 Histogram: melting point of naphthalene.
82
Chemometrics Chemometrics has been dcfim...d as the chemical discipline that uses mathcmatiatl and statistical methods 10 design or select optimal measurement procedures and expcrimcnlS and to provide maximum chemical infonnation by analysing chemical data (Kowalski. 1978). It is a relatively new discipline thai assists with (i) the planning of experiments, aDd (ii) the manipulation and interpretation of large data sets. Some aspeclS of chcmomClrics can be done using an appropriate spcadshcci but the majority of applications require the usc of dedicated software. The fundamcmal prin<;iples of most of the processes involved in chemometrics are those of slatistics. You are therefore advised to become familiar with the material in Chapters 40 and 41 before proceeding. When carrying out any experimental work, e.g. an undergraduate practical, you should always read the entire practi~1l1 script before starting the experimentation. This is important as it allows you to plan each step of the pr(x:ess and to organize space and time to perform the experiment. This initial planning is further complicated in project work and research projects when, often. there is no labomtory script to follow. In these situations. you linally come down to planning the initial experiments after background research (e.g. reading' the appropriate scientific literature on the subject area to be investigated), purchasing/obtaining the appropriilte chemicals/reagents, etc. (see also Box 10.1). It is at this sta~ that chcmometrics can be of some assistance. Assuming that you are able to identify the dependeot variables in the experiment, then you can apply 'experimenlal design' which allows you to gain the maximum amount of knowledge about the system you are investigating from a limited number of experiments. Once the experimental work has been completed you then need to consider how to interpret the results., i.e. how to maximize the chemical information inherent io the dala. Initial attempts are onen centred around plotting the data, to visualize trends and to allow conclusions to be drawn. The simplest form of data visualization is simply to tabulate the results (Chapter 38). As an example, if a class of students has determined the melting point of naphthalene, it is a relatively simple matter to tabulate the data (sec Table 43.1). One possibility for the dala is then to calculate the me-dn and standard deviation. Another approach would be to plot the data as a histogram. as in Fig. 43.1, so we are then able to make a visual interpretation of the quality of this univariate (one-variable) data. However, what if we had more than one variable to consider? In other words., we have multivariate data. For example, what if we want to identify trends in the propcnies of a range of organic molecules? The variables we might want to consider could be: melting point, boiling point, M" solubility in a solvent and vapour pressuTC. We can, of course, tabulate the dala, as before, but this does not allow us to consider any trends in the dala. To do this we need to be able to plot the data. However, once we exceed three variables (which we need to be able to plot in three dimensions) it becomes impossible to produce a straightforward pial. II is in th.is COnlcxt that chemometrics offers a solution, Tt.'ducing the dimensionality to a smaller number of dimensions and hence the ability to display multivariate data. The most impOltant technique in this context is called principal component analysis (PeA). Analvsis and presentation of data
165
Chemometries
Useful sources of information
Joumats: Annual Rel/iew of Physical Chemistry Chemical Physics Chemical Physics Letters International Joumal of Quantum Chemistry Journal of Chemical Physics Journal of Computational Chemistry Joumal of Moleculer StructurelTheochom Journal of Physical Chemistry Rel/iews in Computational Chemistry Theoretica ChimiCB Acta
The following discussion highlights only the basic principlcs. For more detailed information you are advised to (:onsult the literature and dedicated chemometric sortw:uc p:lckal;es. It should always be bornc in mind. however. that tile choice of which variables to optimize should be selected (i) by someone with prior knowledb>e or the system/instrument under im'estigation. or (ii) arter performing preliminary experiments to determine which are tile most imponant ,ariables.
Experimental design There arc (wo main multi,'arialC optimization strategies; those ba.. .e d on sequential designs and those based on simultaneous designs.
Useful web sit..:
Sequential desipn
Brookhal/en databank (protein structures) http:\\www.pdb.bnl.gov
SeqUt.'1ltial optimization is based on the onc-at-a-time approach. The major limitation of this approach is that it assumes that no interaction effects occur between the variables. Unfortunately this is not always the case. A. StXJ~ntial design stnuegy invol,'eS carrying out a f~w experiments at a lime and u~ng the re.<>ults of those experiments to determine tile next experiment to be done. The best known of tile sequentiSite direction. By reflecting point 2, we can obtain point 4. By perfonning tile experiment described by point 4 we obtain its response, thereby perpetuating tile simplex. Four rules can be described for a simplex design:
Cambridge Crystallographic Data Centre http:\\csdvx2.ccdc.cam.ac.uk\
,
I.
" Fig. 43.2 Simplex optimization.
,
,
,
2.
•
Fig. 43.3 Step-size simplex.
,
3.
4.
A new simplex is fanned by rejeding the point with the lowest response and replacing it with its mirror image acro~ thc line defined by the two remaining points. If the new point in the simplex has tile lowest response, return to th~ preceding simplex and create the new simplex by using instead the point with the second-lowest response. If a point is retained in three consa'"Uti,-e simplexes, then it can be assumed that an optimum has hem reached. (Notc: it may be that this optimum is not the true optimum. but that the simplex has been trapped al a false optimum. In this situalion, it is necessary to start thc simplcx again, or use a modified simplex in which the slepsize is not fIXed but ,ariable. see J-Ig. 43.3.) If a point is suggested by tile simplex algorithm thai is beyond the limit of the variables, i.e. it is beyond tile safc working limits of an instrument, then the point is rejected and an artiftcially low response is assigned to ;1. and tile simplex is continued wilh rules 1-3.
Simultaneous desig" In a simultancaus approach the relationship between variables and results is studied as follO\ys: carry out an appropriatc design, apply a mathematical model to tile design. and then apply a response surface mcthod to the data. 286
Analysis and presentation of data
Chemometries
Appropriate designs might be baS(.'d on factorial designs (full or fractional) or a central composite design. Response surface melhod'! frequently rely on visualizalion of the data for interpretation.
Factorial design In b'CllCral terms, consider the case of two variables at two levels, e.g. a high value find a low value. This is teoned a two-Ievd desig.n or a (full) 2l ' f
--
Table 43.2 Two-level factorial design
1
2' 3
.'
5' 6 7 6'
Volurneof~
-1 +1 +1 1 1 +1 +1 -1
... ... -1 -1
"
+1 -1 -1 +1 +1
-1 -1 -1 -1 +1 +1 +1 +1
_h
Y, Y, Y, Y, Y, Y, Y, Y,
The limitation of the Iwo-Ievel factorial design approach is that no estimation of curvature aln be delennined. In order to lake Ihis into account the use of dl'Signs with at least three levels is required. Three-level designs arc therefore often known as response surface: designs. Probably the most important design in this context is the central composite design (CCD). Central composite designs consist of a full (or fraliional) factorial design onto which is superimpoSl.'d a star design. The number of experiments 10 be done (R) can be worked out as follows:
[43.1J Box 43.1
Example of a two-level factOrial design
The recovery of phenols by liquid-liquid extrac1iol1, from 1 litre of river water, and subsequent anatysis by high-perfon-nance liquid chromatography is to be optimized. It has been determined thet the following are critical to achieving an optimum extraction: the volume of extraction solvent,. the mass of salt added and the pt:l of the water sample. Therefore.·& z3 design is required. The experimental'leveis are: volume of extraction solvent (5 and 50 mW; mass of salt added 10.0 and 1.0g); and pH (4 and 7). The coded values for the experiment
•
are shoYo'n in Tatile 43'.2. The +1 values represent the higher value, e,g. pH7, whDa -1 represents the 1000000r value, e.g. pH 4. The numberof experiments can be reduced if a fractional factorial design is u99d. For example, in thts situation the fractional factorial design would become 2]....1, 4 experiments. In this situation the experiments labelled with an asterisk in Table 43.2 woold be done.
Analysis and presentation of data
287
Chemometrics
. ,, . ,
,, ~~/~
p-
, -+---
j Slll"design
where k is the number of vmiables. and no is the number of experiments in the centre of the design. For a design with three variables we would require [21 +(2 x 3) + I) = 15 experiments. In order 10 obtain repeatability information it is necessary to run an experiment several times. This is done by perfonning the centre point experiment twice. The total number of experiments would therefore be 16. The list of experiments is shown in Table 43.3 while Fig. 43.4 shows a diagrammatic representation of the CCD. The CeD is composed of a 3k factorial design superimposed with a star design (+ 0:, -0:). In order to minimize systematic eror (bias) it is necessary randomize the experimental run order. This is shown in Table 43.4.
Response surface methodology Fig. 43.4 Central composite design.
Response surface methodology allows the relationship between the responses and variables to be quantified, using a mathematical model, and to be visualized. Thus the equation for a straight-line graph can be written as:
y=mx+c
[43.21
Table 43.3 Central composite dosign for three variables
........... ,
Factt:lfilM design,
v_, +'
4
5
• 7
•
I.
Star design, 2k 9
+1 -1 -1 +1 +1 -1
-.
.-0
11
Variable 3
Reo.h
-,-1
-,-,-1
Y, Y, Y, Y, Y,
z3 -1
2 3
Variable 2
•• •
12 13 14 Centre points
15
0
•
I.
+1 +' -1 -1 +1 +1
• -.•
+.
•• ••
-, +' +' +1 +1
V,
Y, V,
•• ••
-, .-
••
Y, Y" V" V"
Y" Y" Y" Y"
where TIl is a constant and {' is the intercept. Thi<; describes the relationship between a single variable (x) and its response (.1'). Using the previous example. with three variables (Xl. X2 and Xl) it i<; pos.<;ible to extend this mathematical model. First of all we can consider how each of the variables influences the response (y) in a linear manner. However, the relationship between.v and Xl. x2 and x] may not be linear. so it is necessary to consider the possibility of curvature. This is done in tenns of a quadratic variable. i.e. a squared dependence (xf. x3 and ~). Finally. it is also important to consider the effects of possible interactions between the variables. Xl -+ X2 -+ Xl. i.e. XIX2, XIX] and X2XJ. The overdIJ general equation can therefore be written as:
y= bo +b1xI + b}X2 +bJX] + b 4 .q +bsx~+ bsXIX]
288
Analysis and presentation of data
+
1x} x 2X ]
b 6 xj +b7XIX2+
[43.3]
Chemometries
-- "_ Ori_
Table 43.4
(from Table
<3.3)
8 3
" 6
12 13 4 I
7
I. "
'New' 1 2 3 4
• 6 7
8 9 I.
"
2 5
"
"
Varillble 1
Variable 2
V. . . . . 3
-1 +1
+1 +1
+1 -1 +. +1
.......
NO """"
9
16
Fig. 43.5 Example of a response surlace.
A typical randomized ceo
12 13
15 16
• ••
+1
-1 -1 1
..
• •
~
+1 -1
•
• • -, •• •• -1 +. +1
1
-I -I
-,
-.•
-1 -1 1
••• • •
-1
+1
Y, Y, Y, Y. Y, Y, Y,
Y, Y, Y" Y" Y" Y" Y" Y" Y"
where bo is the intercept 1X1rdmeter and b l - b<} are the regression coefficients for linear, quadratic and intcmction effects. This equation can be analysed using multiple linear regression and tcsted for statistical significance at, for example. the 95% confidence interval (see p. 278). In addition. the response can be explored by plotting a threedimensional graph. Unfonunately, in the above example, three variables are present. lbis immeditltcly constrains what it is possible to plot On the graph (one of the axes must be the response). One way 10 select the two variables to plot is by considering Iheir slali<;lical significance and then selecting two variables which are significant at the 95% confidence interval. An alternative approach might be simply to plOl the two variables yoU might wish to discuss in your experimental report. A typical response surface is shown in Fig. 43.5. It can be seen that thc 'timc' variablc has a maximum at 8-12min while the 'temperature' variable has a maximum at 160-180°C. Further experiments might be carried out at these two maxima to determine the repeatability of the approach. However, it is neressary to plot all variables consecutively to identify all maxima. In general, it is important to consider the following issues when carrying out an experimental design:
• • •
•
Carry out repeat measurements for a particular combination of variables, to determine the repeatability of the approach. To remove systematic error (bias). you should randomi7.e the order in which experiments are done (p. 66). It is important to eliminate intervariable effects (confounding), i.e. the situation where one variable i" inter-related to another. Often, the large number of experiments to be carried out makes it impossible to run all of them on the same day. If this happens run your experiments in discrete groups or 'blocks'.
Principal component analysis The use of modern automated instrumentation allows the acquisition of large amounts of chemical data. As well as simply tabulating the data, other forms Analvsis and presentation
of data
289
Chemometrics
of 'analysis' are required to interrogate the chemiC<11 inforrrmtioll I;ontaincd within the data. One such approach, enabling the simplification of large data sets by reducing the number of independent variables, is principal componenl analysis (PCA). The basis of this approach is: •
• •
To reduce the number of original independent variables into new axes, so-calk'CI 'principal components', PC", each of which can be c.:stimllled unambiguously. The dut;:l contained in lhese new PCs, lind which lire expressed as 'scores', are uncorrellitoo with each other. To express. in a few PCs, the amount ofvliriation in Ihe dlitli. To Illl\'e eadl new PC express a decreasing amount ofvuriation.
An example of lhe application of PeA is shown in Box 43.2. Box 43.2
Example of principal component analysis
Consider a chemical reaction where reactants, X and Y, produce product Z. The yield of Z is dependent upon the temperature of the reaction and its pH. And suppose that lhe reaction has been carried out by a class of students, providing a large amount of data. By ~otting temperature against pH (Fig. 43.6). we can identify a single new variable, PC" which obviously contains aspects of pH and temperature, i.e. PC I = a(temperature) + b(pHI
Fig. 43.6 Ootcrminalfon of principal component 1.
'" '" Fig. 43.7
290
'.'.
"
'"
Principal component analysis.
Analysis and presentation of data
143.4]
PC, can then be used to replace the original two variables. In addition, the direction of PC I indicates where the greatest variation in the data lies. The other information that can be obtained Is the scatter of the data on either side of the regression line. This is due to random varletion rather than 8 trend. In this example therefore it is not possible to extract any further PCs. In eqn 143.4], the coefficients a and b indicate the relative importance of the two variables to PC" and are called factor loadings. In general, therefore, the plotting of factor loadings betvlleen any two Pes can provide useful information as to the relationship of variables to the Pes. Fig. 43.7 shows such a plot. It is seen that variables 1-3 contribute strongly (positively) and variable 5 (negatively) to PC,. Variable 6 contributes strongly to PC2 • whereas variable 4 contributes significantly to both PCs.
----------18
Computational chemistry A chemist is normally visualized as someone in a white laboratory coat petfonuing an experiment. However, some chemists never actually go ioto a 'v"ct chemictll' laboratory but utilize computers to perform experiments. Why do chemistry on computt.'rs? •
•
•
Computational chemistry uses commercially available software 10
enhance chemical knowledge.
This branch of chemistry, sometimes referred to as theoretical chemistry, molecular modellinJ:; or computational chemistry, used to be restrir..1ed to a few researchers with access to expensive computers. This has chanboOO in recenl years with the aWlilability of low-cost, high-powcr computcrs, coupled with the availability of software packages that require no knowledge of computer programming, making computational chemistry accessible to undcrgmduate students. The new user of computational chemislry will quickly discover Ihc ease wilh which it is pos.<;ible to observe complex molecules on the screen usinJ:; the software's computer gr'tlphics (Fig. 44.1). However, computational chemistry is much more than pretty pictures. II aUows the chemist to perform theoretical experimcnts in three distinct areas: I.
2. 3. Definitions An ab initio method is a quantummechanical approach which attempts to
calculate. from first principles, solutions to the Schrtklinger wave equation.
Motecular mechanics describes a system in which the energy depends only on the nuclei present.
Quantum mechMic:s describes a molecular system in which both electrons and nuclei are involved. A semi-emplric&l mtrthod is a type of quantum-mechanical theory that incorporates information derived from
experimental data.
Safely: invariably all labo....dtory cxperiments carry some risk, associated with the chemicals or apparatus 10 be used. It is usual to perform a COSHH ii~ment (p. 7) prior to experimental work. Computational chemistry allows the user to carry out work on 'dan£!.crous' chemicals with no risk! Cost: apart from the financial outlay on a computer and associated peripherals together with the appropriate software, no further costs are involved, unlike the experimental laboratory where most chemicals require disposal lIftcr use. Understanding: computational chemistry has the ability to provide a basis for understanding chemical principles.
single molecule calculations molecular interactions reactions of molecules.
In all cases, the basis of the calculation is the determination of the energy of the system. Two approaches are used: quantum mechanics and molecular mechanics. Molttular mcdmnics is best suited to large molecules, c.l;. protcins, whereas quanlum mechanics, while offering a more fundamental approach, is restricted to smallcr molecules.
~ Just as in spectroscopy or chromatography, where
not every spectrum or peak is resolved, so in computational chemistry do not assume every computed number is exact. However, computational chemistry can allow a qualitative or approximate insight into chemical processes provided the user understands the basis behind each approach and can Interpret the resuhs.
Quantum mechanics A quantum-mechanical calculation commences with the SchrOdinger wave equation: }}lJ!
= tl!'
(44.11 Analysis and presentation of data
291
Computational chemistry
,.,
,.,
where H is called the Hamiltonilln operator (it describes the kinetic energies of the nuclei and electrons and the electrostatic interactions fell between individual particles). t is the energy of the system, and \11 is Called a wuvefunction ~~ de$(.nbes the probability of finding an electron at a p
Molecular lIIl'chauics
,,'
Most moleeular modelling packages allow the use of empirical methods which only consider the nuclei. These arc called molecular mechanic-dl methods and are faster than the quantum-mechanical methods. They are based on c1assic
The cOlllputat;onallaborato,'J' Like all laboratory classes it is important to go prepllred so thllt you will get the most out of your time in the laboratory. This might includc background reading, making an outline of the experimental procedure, a sketch (or photocopy) of the chemical structure of all molecules to be worked on, and a plan of how you will draw each molecule - obviously the morc complex molecules may require more thought than a simple molecule. Onre there, it is imporlant to:
,., Figure 44.1
Computer generated images of sb< amino acids using dffferent representations Ii'll stick, (bl balls, leI balls and cylinders and ldl sticks and dots.
• •
• •
292
Analysis and presentation of data
Get a comfortable chair ~ you may be sitting in it for quite a while. Make yourself feel at ease and relax. Pilln your work - a considerable amount of lime in the laboratory will be spent constructing models, selling up calculations and evaluating resuJts. II is therefore important to maximize your time on the computer by pl
Computational chemistry
•
Save all your results for rechecking by yourself at assessment by }'our tutOf.
It
later date or for
Computer softwore A typical software package used 10 pcrfonn computational chemistry should be able to:
• • • • • •
Build and display molecules. Optimize the Slructure of molecules. Im>estig
II is inappropriate to describe any particular sortwarc system. Nevertheless, all software is usually accompanied by a user manual or a "help' lile to make the use of commercial sofiware packages user-friendly.
Applications Computational chemi"try is being appHed to a wkle variety of areas, in part)cu!ar ils ability to predict chemical properties. such as: • • • • • • •
molecular structure reaction enlhalpics dipole moments and infrared intensities vibrational frequencies reaction free energies relative acid constants reaction rates.
In addition, computational chemistry is \videly applied in drug design in the phannaceuticaJ indw.1l)', where it can allow the modeller to invesLigate whether a molecule \vill bind tightly to an enzyme or receptor. In addition. it is possible Lo assess whether a molecule will fit into an active site of an enzyme, how well the molecule will bind. and. ultimately, to assess whether it is possible to design the 'best' molecule.
Analysis and presentatiOfl of data
293
Resources
Resources for analysis and presentation of data /J()ok~' on
.ftat;st;es and ehemometries
Main .wpplementmy text: Miller, J.N. and Miller, J.e. (2000) Sra/6-/ics ar/(/ ChemomR.tricsfor Analytical Chell/istl}',4th Edn, Prentice Hall, Harlow, &,-o;ex. Other U5eful :wurce.~ (clwOlwlogicul oriler): Adams, MJ. (1995) Chemometr;('s in Arut!yt;t:al Chemiwry, Royal Society of Chemistry, Cambridge.
Dc u.'Vie, R. (2001) !JOII' to V.te excel in AnalyriiXIl Chert/i.ttr)' and in Geneml Sciemific Data Ana/ysi.t, Cambl'idge Uni\'ersity Press.
Doggett, G. and Sutcliffe. BT. (1995) MOlhemnticsfor Clwm;.)·rry. Longman, Harlow, Essex. Fammt, T.J. (1997) Practical Statit/ic.t for the Analytical CIJemi.tl, Royal Society of Chemistry, Cambridb>e.
Gardiner, W.P. (1997) Statistical Andy.tis Metluxlr for Cllemist.\'. A sof1ll'arebased approach, Royal Society of Chemistry, Cambridge. Gormatly. J. (2000) £tselltial Marhemat;c.t for Chemist.f. Prentice Hall. Harlow, Essex.
Massart, D.L, Vandeginste. B.G.M .. Buydens. L.M.C.. de Jong, S.. Lcwi, PJ. and SmeyL'Ts>Verbeke. J. (1997) !Jandbook of Cllemometrics (md Qualinretrics: Part A. Elsevier Science B. V .• Amsterdam. Meier, P.e. (2000) Statistical Meth{)(1s in Analytical ClU?mistry, 2nd Edn, John Wiley and Sons Ltd. Chichester.
Books on compmat;onal c:hem;.~try Main supplementQry text: Goodman, J. (1999) C1wmical Applications of MoleClilar Moclelling, Royal Society of Chemistry, Cambridge. OtiJer useful soarces (chrono/ogicDl order): Allen, M.P. and TLldeslcy, DJ. (1991) Computer Sinlll101iotl of LiquitJs. Clarendon. Oxford. Brooks, c.L.. Karplus, M. and Pettitt. 8.M. (1991) Proteins: A theore/ical Perspeclire of Oynamics. StrlICtlll"e anil Thenllodyrulntics. Wilcy-Interscit:nct:, New York. llirk, T. (1985) A Handbook afComfJIltotional Chemistry, Wilcy-Interscicncc. New York. Comba, P. and Hamblt:y. T.W. (2000) Molemlar MOiJelling of Inorganic Compounds, John Wiley and Sons Ltd, Chichester.
Foresman, J.B. and Frisch, A.E. (1993) Exploring Chemisfry witll Electronic Structllre Methoils, Gaussian Inc.. Pittsburgh. Grant, G.H. and Richards, W.G. (1995) Computational ClU?mistry, Oxford Univer.;ity Press. Oxford.
Haile, J.M. (1992) Molecular Dynamics Si/fwlation: Elementary Metllmls. John Wiley and Sons, New York. Hehre, WJ., Radom, L., Schleyer, P.V.R. and Pople. J.A. Ab Initio Molecl/lar Orbital 111l!0rr, Wiley-intersciell<:e, New York.
294
Analysis and presentation of data
Resources
Hehre, W.J., Shustennan. AJ. and Huanl:!, W.W. (1996) A U/OO"l/foq Book of COlllputfl/iO",11 Org,,,/ic Chemistry, Wa\'efunction Inc., Irvine. Hehre, WJ., Shustl'fman, AJ. and Nelson, J.E. (1998) Molecular /l!lJi/elli"g. WOIkbookfor Org,mic Chemistry, Wiley, New York. Jensen, F. (1999) In/rotll/clion to Complltational Chemistry. Wiley, london. Jensen. P. and Bunker. P. (2OOCl) Cmll/mill/;Ollal Molecular Spectroscopy, John Wiley and Sons ltd, Chichester. Leach, A.R. (1996) Molecillar Afotlelfing. Principle.f antl Applicillions, Longman, Harlow.
McCammon. J.A. and Harvey. S.c. (1987) Dynamic.f of PrOiei/ls anti Nut:leic Acids, Cambridb'e University Press, New York. Schlecht, M.F. (1998) A'JokCllwr Modelling on the PC, Wiley.VCH, New YOrk. Szabo. A. and Ostlund.
.S. (1989) MOlli!m Quallt/un Chemistry. Introduction to A(lI'allC;efl Electronic Strl/{;ture 11/{!ory, MeGmw·Hill Publishing Co.. New York.
Software/or dJ'aH';ng chemical slrlf(:t"re.~ ChemDnlw. Adept Scientillc pk:, letchworth, Hert... UK. ChemSketch. AdvdI'lttd Chemical De...elopment. Inc.• Toronto. Canada (www.aedlabs.com).
Software for' SlQf;SI;cJj' Q/ld cllemomelr;CJj' MultiSimplex. KarlskroIlli., Sweden (htlp:l/www.multisimplcx.com). Statisticd, 6.0. Statsofi (http://www.statson.com). StatiSlics for the Analytical Chemist Softbook. Royal Society of Chemistry. Cambridge.
Analysis and presentation of data
295
The Internet and World Wide Web DefInitions
BrOWMf' - a program to display ..... b pagEl$ and other internet resources.
FAa - Frequently Asked Question;
sometimes used as a file extension (.faq) for downloedable files. FTP-FileTransferProtocol; means of dowr'\k)ading files. URL.,. Uniform Resource lOClltOr: the 'address' for \NWW resources.
Making the most of information and communic-
se'drching databases and Internet resources, e.g. llsing Web 'browsers' such as Netsc:apelf and Interr~t Explorer,a; retrieving network resources, e.g. applying FTP to obtain copies of files; storing, modifying and iUUllysing infomlatlon, e.g. using databases. sprC'"
The Internet as a global resource
g desktop PC
I modem'
I~I
rotllef
The key to the rnpid development or the Internet was the evolution and cxpansion of networks - coUcclions of computers which can communicate with each other. They operate at various scales such as local arCl:t networks (LANs) and wide area networks (WANs) and these can be connected 10 the Internet, which is a complex network ofnelworks (Fig. 45.1). The lnternt:l is loosely organiZl.-'d; no one group runs it or owns it. Instead, many private org<'lIlizations, universities and government departments pay ror and run discrele paris or it. Private organizations include commercial online service providers such as America Online\!: and CompuServe't!. You can gain access to the Internel either through a LAN at your place of work or rrom home via a modem connceLL-'d Lo a dial-in service provider over the telephone line. You do nOI need to understand the technology of the network to use it - most of it is invisible LO the user. However, if you do wish to understand more, sources sn::h as Gralla (1996) and Winship and McNab (2000) are re<:onunended. What }OU do need to know are the options available to you in temlS of using its facilities and their relative merits or disadvantages. You also need LO understand a lillie about the nature of Internet addresses.
~ Most material on the Internet has not been subject to
peer review, vetting Of" edrting. Information obtained from the WWW Of" posted on newsgroups may be inaccurate, biased Of" spoof; do not assume that everything you read is true or even legal.
Fig. 45,1
Diagram of B network system.
Communicating on the Internet: e-mail and newsgroups Using e-mail. you are able to send messages 1,0 anyone who is connecll'd to the Internet, directly or indireclly. You can attach lext files. data files, pictures., video clips. sounds and cxccutable files to your messages. The messages themselves are usually only very simple in format but sufficient for most purpa;es: fonnalled material can be attached as a file if necessary. The uses that can be made of the system vary rrom personal and business related LO the submission of work to a tulor in an educational Systl"lll. Note, however, that the S)'Stem is not secure (confillcnlial) and the transmission of Information technology lind library resources
299
The Internet and World Wide Web
Junk and chain mail on the Internet - do not get involved in distributing junk or
chain mail (the electronic equivalent of chain letters). This activity is likely to be against your institute's rules for the use of its computer facilities because it can foul up the operation of networks.
Examples Common domains and subdomains include: .com .edu .gov .mil .net
commercial (USA mainly) education (USA mainly) government tUSA only)
.org
organization
.uk
United Kingdom
military (USA only) network companies
sensitive information should be done with caution. Another downside of email is the junk mail Ihat you may receive once your e-mail address is distributed, e.g. in newsletters. Specific address information is required to exchange information and mail between computers. Allhough the computer actually uses a complex series of numbers for this purpose (the 'I.P.' address), Ihe Domain Name System/Service (DNS) was developed to make this easier for users. Each computer on the Internet is given a domain name (= Internet address) which is a hierarchy of lists and addresses. Thus, 'unn,ac,uk' is the DNSregistered name of the University of Nonhumbria at Newcastle: the top (root) level domain is 'uk', identifying its country as the United Kingdom, and the next is 'ac', identifying the academic communit), sub-domain. The final sub-domain is 'unn' identifying the sJX'Cific academic institute, For the purposes of c-mail, the names of individuals at that site rna)' be added before the domain name and Ihe @ sign is used 10 separate them. Thus, Ihe e-mail address of the first author of this book is 'John.Dean@ unn.ac.uk'.
Internet tools There are various facilities available for use on the Internet depending on your own system Bnd method of accessing the system, The best way to learn how to use them is simply 10 try them out.
rhe WOI'ld Wide Web (WWW) locating information on the WVI/W useful searching systems are located at the following UALs (some may be directly accessible from your browser);
http;J/www.altavista.com http://w.vw.goto.com} http://www.lycos.comJ http://INww.webcrawler.comf hnp://www.yahoo.com http://WNW.google.com
http://vvww.askjeeves.com
The 'web' is the most popular Internet applicalion. II allows easy links to information and files which may be located on computers anywhere in the world. The WWW allows access to millions of'home pages' or 'websites', the initial point of reference with companies, institutes and individuals, Besides their own teXI and images, these comain 'hypertext links', highlighted words or phrases that you click on to take you to another page on the same website or to a completely different site with related subjcct material. Certain sites specialize in sudl links, acting like indexes to other websites: these are p<1l'ticularly useful. When using a web browser program to get to a particular page of information on the web all you require is the name and location of thaI page. The page location is common I)' referred to as a URL (Uniform Resource Locator). The URL always takes the same basic format. beginning with 'http:// and followed by the various terms which direcl the system to the resource pages. If you don't have a specific URL in mind but wish to lind appropriate siles, use a 'search engine' within the browser. enter appropriate and limited key words on which 10 search and note the site(s) which may be of interest. There is so mllCh available on the web, from company products through library resources 10 detailed infonnatioll on specialist chemical and environmenIal topics, thaI much time can be spent searching and reading information: try to stay focused!
FTP(File TI'amfer Pl'Otocolj alldfile
tralls",is.~ioll
FrP is a method of transferring liIes across the Internet. In may cases the files are made available for 'anonymous' FrP access, i.e. you do not need previously arranged passwords. log in as 'anonymous' and give your e-mail address as Ihe password. Use your web browser to locale Ihe file you wanl and then use its FTPsoftware to transfer it to your computer. 300
Information technology and library resources
The Internet and World Wide Web ~ The transfer of files can resuh in the transfer of associated viruses. Always check your files for viruses before running them.
When using Icr. including the Internet, always remember the basic rules of using compulers aod networks (Box 45.1).
Box 45 1
Important guidelines for using computers and networks
Hardwar.
F'" management
• •
Don't drink or smoke around the computer. Try not 10 turn the computer off more than is neces·
•
sa",.
•
• • •
Never tum off the electricity supply to the machine while in use. Rest your eyes 8t frequent intervals if working for extended periods at a computer monitor. Never try to reformat the hard disk except in special circumstances.
•
Always use virus-checking programs on imported files before running them. Make backups of all important files and at frequent intervals (say every half hour) during the production of your own work. e.g. when using a word processor or spreadsheet Periodically clear out and reorganize your file storage areas.
Netwo
•
Protect floppy disks when not in use by keeping them in holders or boxes. Never touch a floppy disk's recording surface. Keep disks away from dampness, excess heat or cold. Keep disks well away from magnets; remember that these arE.! present in loudspeakers, TVs, etc. Don't use disks from others unless first checked for viruses. Don't insert or remove a disk from the drive when n is operating (drive light on). Try not to leave a disk in the drive when you switch the computer off.
• • •
Be polite when sending messages. Never anempt to 'hack' into other people's files. Don't play games without approval- they can hinder the operation of the system. • Periodically reorganize your e-mail folder!s). These rapidly become filled with ecknowledgements and redundant messages which reduce server efficiency. • Remember to log out of the network when finished: others can access your files if you forget to log out.
The golden rule: atways make b8ekup copies of important diMs/fifes and stora wei away from your WOfking copies. Be sure that the same accident cannot happen to both copies.
t""
Information technology and library resources
301
~--------jG
Internet resources for chemistry A common way to find jofonmllion on the Internet is by browsing {'surfing') the World Wide web (WWW). However, as Ihis can be time consuming and wasteful. browsing should be focused on relevant sites. Many of the most useful wehsites are those providing detailed lisls and hypertext links 10 ol.her localions. A useful place to find out what the Internet orrers the chemist can be found at the URL http://www.chemdex.org.
~
Remember that the lntemet should not be viewed as a substitute for your univer-sity libt'ary and other Jocal resources. but should complement. rather than replace. more traditional printed texts and CD-ROM material.
General information Revisiting websites - 'bookmark" sites of
interest, so that you can return to them at a later date. Make a copy of your bookmark file occasionally, to avoid loss of relevant information.
Some of the principal resources you can utilize via the WWW are: •
Using Internet addresses - note that the locations given in the chapter may change as sites are updated: you can make a key-word search using a
searching system or 'search engine' to find a panicular website if necessary.
•
302
Information technology and library resources
Libraries, publishers and companies. These organi7..ations recognize the significance of the Internet as a means of communication; for example. the Addison Wesley Longman higher etIucation website at hUp:// www.awl-he.com/allows information on specific catalogues and books [0 be requested online. A large number of scientific journals are also available in electronic format "ia iogema.com (hup://www.ingenta.com). Ingenta journals offer a single point of access to over 2500 academic and professional journals from over 30 publishers. Access to browse and search the database of articles is free, as is the ability to display tables of contents. bibliographic information and flbstraets. Full-text articles require a fee for access - check whether your institution subscribes. A broad range of scientific journals published by Academic Press is available I';a the site http://www.apnet.com/. and students at UK colleges and universities should have full access without additional subscription (via hup://www.janetidealibrary.comf). You can keep up to date using Nell' Sdell1ist pages (al hup://www.newscientis(.comf).Scientific American (at http://sciam.com/) or Nalllre (at hllp:/www.nature.comf). A few of the newer titks are available only in electronic format. e.g. lhe ejournal CryslEngComm can be found al hnp:/iwww.rsc.org/is/journals/ currcnt/crystengcomm,'cecpub.hlm. Electronic publishing is likely to develop further in future y~rs. In<;litutlons. Many research organizations. societies and educational institutions around Ihe world are now online. with their own web pages. There is a detailed list of scholarly chemical societies at http://www. chemdex.orgfchemdexfleamed-society.htmL You can use the websites of these organizations to oblain specific infonnation. They rrequenl1y provide hypertext links 10 other relevant sites for particular groups or topics: for example. the Royal Society of Chemistry web page (hltp://www.rsc.org) has links to various sites of interest to chemists. Use the WWW to obtain details of the activities of research organi7..ations (e.g. GlaxoSmithKline, at hnp://www.glaxosmithkline.co.ukl or individual laboratories and researchers at specific universities. Some other relevant websites are given in Table 46. L
Internet resources for chemistry
Table 46.1 websltes
Selected eKampies of usefUl
•
American ChemiQI Society http:tlwww.acs.org The Royal Society of Chemistry
•
hnp:Jlwww.rsc.orQ
Society of Chemical Industry hnp:t/sci.mond.org International Union of Pure and ~ied
Chemistry UUPACI hnp:tliupac.chemsoc.org laboratory of the Government Qlemist hnpitwww.lgc.co.uk Oaresbury labot'atory http:,li'Nww.dl.ac.uk
Brookhaven National Laboratory
Data and pictures. Archives of leXI m:lterial. pholographs arxl video clips c.an be a<x:cssed and easily downloaded. However. downloading graphical images m:lY (:Ike quite a while. especially for remote links or at busy times, leading to high costs. Newsgroups (Usenet). News :lrticl~ 011 a wide runge of topics are 'posted' al appropriate siles, where Ihey are pLaced into subject groups (newsgroups). Any user can contribute to the discussion by posting his/hcr own message. to be re'.Id by other users via appropriate news soflw:lre. While lhe number of rJe\\Sgroups is very large. some of them m:ly include relevant infonnmioll. Further informalion on topiQi of c1u:mistry interest is :lwilable at hup://www.chemdex.org/index.html. Student access to newsgroups may be limiled at some universities and colleges.
~ Remember that the information from Internet news·
http-J/Vvww.chemi&try.bnl.gov
groups and similar websites may be unedited and may represent the personal opinion of the author of the article.
United States Environmental Protection A9ftocy
•
http://WINw.epe.gov
Nationsllnstitute of Standards and
Tec::hnotogy IMsn laboratory hnpJ/WINw.cstl.niSlgov
Nationallrn:tltute of Standards and Technology IMSn WebBook hnp:Jlwcbbook.nist.gov Univer'5ity of Sheffield
hnp:,l/Www.chemdeK.Ofg Specialized Information Services - Chemical
•
Information page 00 drugs. pesticides, environmental poIhrtants and other potential tOlCins hnp:Jlchem.sis.nlm.nih.gov
Reaetm Reports - A chemistry web magazine hnpJJw,t.m.acdlabs.com/'olYebzine WebElements Periodic Tabte http:/twv.w.webelementIi.com
Table 46.2 EKamples of chemical databases
Bei'stein http://wvvw.beilstein.com Sdentlflc and TeeMkallnfOt'mltion Netwolil: http://www.fiz-kartsruhe.de/onlin_db.htmt et.emEllper Chemical Directory hnp:,lJw,t.m.chemexper.belmain.shtml
Cambridge Crystallographic Data Centre
http.J/Iwww.ccdc.cam.ac.uk The Chemical Database Service httpJlcds3.dl.8C.uklcdsA:ds.htmt National Institute of Standards and
Technology (NtsTI WebBook http:,l/webbook.nislgov The Wored Chemist hnp:J/wulfenite.fBndm.edu
Mailing lists. Messages sent to the lisl address arc distributed aUlomatically to all membt-rs of the mailing list, I'ill their personal e· mailbox, keeping them up to date on the particular topic of the mailing list. To receive such messages. you will m:ed to join lhe mailing list. Relevant mailing lists for chemistry can be found at hllp:/lwww. chemdex.org/chemdelt/listserv.html. Take care not to join 100 m:my lists. as you will receive a large number of mess.,1ges, and many are likely 10 be of only marginal interest. A number of mailing lists also have archived liles, offering a more selective means of locating relevant material. Dalabases. Sites such as Bath Information and Data Services (BIDS) Hnd web of Science (WOS) provide access to abstmcts of recent publications: use lhese to find relevant literature for specifK: topics. Access is via the ....-ebsitcs at hllp:{!www.bids.ac.uk/orhup:f!wwv...webofscience.com; you will neW a username and pass....'Ord - check wilh your department or IibrdJ)'. In the case of BiDS. it provides access to dalabases covering subjects from science, engineering and medicine to economics. politics, education and the arts. Specific databases offered include: lSI Litalion indexes; EMBASE (internalional biomedical infomlation); TNSPEC (physics. electronic engineering and computing); international bibliography of the social sciences (IBSS); The Royal Society of Chemistry databases: and edtlC:\lion databases. See also Table 46.2.
The BIDS Royal Society of Chemistry service The BIDS RSC service provides access to four bibliogmphic dalabllses and a further two databases (Chemical Safety Data Sheets and the UK Nutrient Databank via l.he WWW al hup:ffwww.bids.ac.uk/websearch.html) are supported by The Royal Society of Cht:misiry (URL http://v.ww.bids.ac.ukf Tfcd0C8/rsc.html). The dalabases are: • • • • • •
Analytical Abslracts OJemical Business NewsBase Chemical Safety NewsBase Mass Spectmmeuy Bulletin Chemical Safety Data Sheels UK Nutrient Databank. InformaliOfl technology and library resources
303
Internet resources for chemistry
TIle BIDS RSC bibliogmphic databases can be searched by u!>;ng: • • • • • •
Words or phrases in :tnides, key v.'Ords or ab'>lfacis AUlhor names and/or addresses Journal names CAS registry numbers ChemiC'"cl.ljproduct names Specilliist index terms.
Infonnation (e.g. abslrclds) can be displayed. downloaded or e-mailed 10 your personal address. In addilional. electronic full-Ie'lt articles may al~o be a,~dilable via ingetlta jourmlls (hltp:/Iwww.ingenta.com).This is normally offered in PDF Vill a viewer, e.g. Adobe Acrobat Reader. In contrast, the Chemiwl Sllfety Dala Sheet database is searchtxl using chemical name or synonyms, while Ihe UK Nutriem databank is searched by food name. A nalytirol Abs/mclS (illtp:/Iwww.rsc.org/isj
Muss Spec/rametr.1' Blllle/;n (hnp:/jwww.rsc.orgJis/dalabase/msbhome.hlm) This is a current awareness bullelin providing information on mass spectrometry and related ion processes. The bulletin is sub-divided as follows: instrument design and techniques; isotopic analysis., precision mass measurement, isotope sepamtion, age determination, etc.; chemical analysis; organic chemistry; atomic and molecular pr~; surface phenomena and solid-state studies; and thermoclynamics and reaction kinetics. The database 304
Information teehoology and library resources
Internet resources for chemistry
Table 46.3 EXllmple recOl'"d from Analy1ical Abstr&Cls
AA Accession No.: 60-07-H-00251 DOC. TYPE: Journal
Kinetic determination of ultrauace amoonts of ascorbic acid with spectrofluorimetric detection.
AUTHOR; Feng. S. L; Chen, X. L; Fan. J.; Zhang, G.; Jiang, J. H.; Wei, X. J. CORPORATE SOURCE: Dept. CHern., Henan Normal Univ.. Xinxiang, Henan, 453002, China JOURNAL: Anal. lett.,IAnaJytical lettersl, Volume: 31, Issue: 3, Pagels): <63-474
CODEN: ANALBP ISSN: 0003·2719 PUBUCATlON DATE: Feb 1998198(200) lANGUAGE: English ABSTRACT: To the test solUlion. l.2mL of phosphate buffer of pH 2.6, O.3mL of 0.1 mM·modamlne 6G, 1.2 mLof 10mu.glmL vanadium(V) solution and 1.5mL of o.lM·potassium bromate were added. The mixture was made up to 25 mL with H2O end alklwed to stand for 4 min. The reaclion WllS Quenched by addition of 1 mL of 1m-sodium acetate and the fluorescence intensity was measured at 548.. nm (excitcltion at 348.4 nml. calibration graphs were linear for the renge 1.6 to 28nglmL Detection limit was O.62nglmL None of the common ions and organic compounds investigaled interfered. The method was used in the analvsis of phermaceutical preparations, fruit juJce5 and vegetables. Results were in good agreement with those obtained by the iodimetIic method. ANALYTf: ascorbic acid (50-81-71 -detmn. of, in fruit juices. pharmaceuticals and vegetables. by fluorimetry MATRlX; pharmaceutical preparations -detmn. of ascorbic acid in, by fluorimetry fruit juices -detmn. of ascorbic acid in, by fluorimetry vegetables -detmn. of ascorbic acid in. by fluorimetry SECT1ON: H-B7000 (Environmental, Agricutlure and Food) SECT10N CROSS REFERENCE: G001 (Pharmaceutical Anatyslsl
is updaled monthly. More than 900 sources are accessed 10 provide Up-IOdale information.
Cllemical So/ely Dow Sheets (hnp:llwww.bids.ac.ukifedocs/TSC_safetydata. html) TIleSe provide safety information for over 550 common chemicals. The dalabase is ammged alphabetically to enable easy access. UK Nutrition Datalxmk (hltp:ffwww.rsc.orgjisfdatabasefnutshome.htm) This provides nutrient composition dllta on foods consumed in the UK. The database provides data for 1188 foods covering all food groups, providing values per IOOg (or IOOmL) for over 40 nutrienls. In addition. a food index, lisling ahemative and taxonomic names and ra.ipe infonnalion, is also provided. The particular food groups covered include: cereals and cereal products; milk prodUCIS and eggs; vegelables, herbs and spices; fruit and nuts; ,'egelable dishes; fish and fish products; miscellaneous foods; meat, poultry and game; meat products an
The Royal Society of Chemistry: Bibliographic Databases and Reference Databanks In addilion to the BIDS RSC service, The Royal Society of ChemistI)' (http:// www.rsc.org/isfdatabase/sec_serv.hlm) also offers a range of bibliographic dawbases and reference databanks. These are (excluding the ones available via the BIDS RSC service) described below. Information technology and library resources
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Internet resources for chemistry
Anal.I·lical ."emislry
• • • •
Analytical Abstracts Chromatography Abstracts Mass Spectrometry Bulletin Window on Chemometrics.
Chemical business
• • • • • •
Chemicals, Fonnulatcd Products and their Company Sources Focus on Catalysts Focus on Pigments Focus on Polyvinyl Chloride Focus on Powder Coatings Focus on Surfactants.
Health. safelY and loxh'Ology
• • • • •
Chemical Hazards in Industry The Dictionary of Substances and their Effects Environmental Chemistry. Health and Safety Hazards in the Office Laboratory H37.1l.rds Bulletin.
As an example. Chromatography Abstracts (http://www.rsc.orgjis/dawbasei chroabs.htm) provides an update of developments in separation science. It is arrallgt.>d into the following sections: general and miscellaneous techniques; gas chromatography; liquid chromatography; electrophoresis: and thin-layer chromatography. An example of a typical record is shown in Table 46.4. Table 46.4
Example record from Chromatography Abstracts
1854 Bovine whey fractionation based on cation-ekc;h,onge ctlromatography.
Hahn. R.; Shull, P. M.; Schaupp, C.; Jungbauer. A.J. Chromatogr., A, 6 Feb 1998, 795l2J, 277-2fJ1 Four callon"exchange resi05. namely Ii) S-HyperD-F. (ii) S-Sepharose FF.liii) Fractogel EMD S03-650151 and liv) Macro-Prep High 5 Support. were compared for the separation of proteins such as rgG, lactoferrin and lactoperoxidase from bovine whey. The chromatographic method was based on nushing .alpha.lactalbumin through the column and then eluting the proteins by a sequential step gradient with 0.1, 0.2, 0.3, 0.4 and 1M-NaC!. The collected fractions were analysed by GPC and 50S-polyacrylamide gel electrophoresis. The lactoperoxidase activity was measured by the oxidation of o-phenylenediamine. Resin iii exhibited the highest binding capacity for rgG of 3.7 mglml gel at a frow rate of 100crnfh but the lowest proteIn resolution. Resins i and ii exhibited slightly poorer dynamic capacities for IgG but better protein resolution. For i. ii and iii. the binding capacities were not dependent on flow rate for 1Q0....600cmjh and me binding capacities were not improved by removing low molecular weight compounds from the food solution. Resins i. ii and iii were considered suitable for the large scale purification of bovine whey proteins.
.
306
Information technology and library resources
-------10
~ The spreadsheet is one of the most powerful and
()efIn;t;ons ~
Using spreadsheets
- a displ&y of a grid of (l&/Is
into which numbers, text or formulae can , be typed to form 8 worksheet. Each cell is uniquely ldentiflable'bv its column and row number combination (I.e. tts t\Nodimensional co-ordinate£1 and can contain 8 formula which makes 11
wide ranging of all microcomputer applications. It is the electronic equivalent of B huge sheet of paper with calculating powers and pt'"ovides 8 dynamic method of storing end manipulating data sets.
Statistical c:lkulations and graphical presentations are available in many versions and most have scienlific funclions. Sprcndsheetscan be used 10:
possible for an entry to Doe cell to aher the contents of one or more other cells.
•
Ternplate- 8 pre-designed spreadsheet withCIut data but: including all formul8e
•
necessary for (repeated) date an8lys;i&.
Macro - a sequence of user-defined instructions carried out by the spmadsheot. allowing complex repealed tasks to be 'automllted".
• • •
manipulate raw data by removing the drudgery of repeatL'd c.tlculations. allowing easy tr.lIlsformalion of data and calculation of st:ltistics; graph out your data rapidly to gel an instant evaluation of results printout can be used in practical and project reports; carry Qut limited statistical analysis by buill-in procedures or by al10winB construclion of formulae for specific task~ model 'what if siluations where the consequences of changes in data can be secn and evaluated; store data sets with or withoul statistical and graphical analysis.
The spreadsheet (Fig. 47.1) is divided into rows (identified by numbers) and columns (identified by alphabetic characters). Each individual combination of column and row forms a cell which can contain a data item. a formula, or a piece of lext called a label. Fonnuiae c.m include scientific and/or statistical fWlctions and/or a reference to other ce.lls or groups of cells (often called a range). Complex systems of data input and analysis can be constructed (models). The analysis. in part or complete. can be printed out. New data call be added at any time and the sheet recalculated. You ean constnlet templates. pre-designed spreadsheets containing the fonnulae required for repealed data analyses.
Men~ber
E"'tline
Aclive cen (All
Formetting toolbllr
-:JF5?
Rowoomber
Fig. 47.1 A screen dump of a typical spreadsheet, showing cells, rows and columns; 10010015 etc. Screen shot reprinted by permission from Microsoft Corporation.
Information
technology and library resources
307
Using spreadsheets
Templates - these should contain:
• a data input section, • data transformation and/or calculation sections, • •
a results section, which can include graphics, lext in the form of headings and
annotations, •
The power a spreadsheet offers is directly related to your ability 10 create models (hat are accurate and templates that are easy to use. The sequence of operations required is: I. 2. 3.
a summary section.
4.
Constructing a spreadsheet - start with a
5. 6. 7.
simple model and extend it gradually. checking for correct operation 8S you go.
8.
Determine what information/statistics you want to produce. Identify the variables yOli will need to use, both for original data that will be entered and for any intermediate ealculoltions Iholt might be required. Set up areas of the spreadsheet for data entry. calculation of interme(Jillte values (statistical values such as sums of sqll:\re5 etc.). calculation of final parameters/statistics and. if ncccs!;ary. a summary area. 8.1ablish the format of the numeric data if it is different from the default values. This can be done g1ob:llly (affecting the entire spreadsheet) or locally (affecting only a s~fied part of the spre.adsheet). Establish the column widths required for the various activities. Emer labels: usc extensively for annotation. Enter a test set of values to use during formula entry: use a fully worked example to check that formulae are working correctly. Emer the formulae required to make all the calculations. both intermediate and final. Check that results are correct using the tcst data.
The spreadsheet is then ready for use. Delete all the test data values and you ha\~ created your template. Save the template to a disk and il is then available for repeated operations.
Data entry Spreadsheets have built-in commands which allow you to control the layout of data in the reUs. These include number fonnat. the number of decimal places to be shown (the spreadsheet always calculates using eight or more places). the I,.'CII width and the location of the entry within the rell (left, right or centre). An autOo-entry facility assists greatly in enlering large amounts of data by moving the entry cursor either vertically or horizontally as data are entered. Recalculation default is usually automatic so that when a new data value is entered the entire sheet is recalculated immediately. This Colli dr.tmatic.llly slow dO\\'n data entry so select manual recalculation mode before entering new data sets if the spreadsheet is large with many calculations.
The parts of a spreadsheet
Labels These identify the contents of rows and columns. They arc text characters. and cannot be used in calculations. Separate them from the data cells by drawing lines. if this feature is available. Progmms make assumptions about the nature of the entry being made: most asswne that if the first character is a number. then the entry is a number or formula. If it is a letter. then it will be a label. If you want to start a label with a number, you must override this assumption by typing a designalcd character before the nwnber [0 teU the program that this is a label; check your program manual for details.
Numhe,'s Using hidden {or zero-widthl columnsthese are useful for storing intermediate calculations which you do not wish to be displayed on the screen or printout.
308
You can also enter numbers (values) in cells for use in calculations. Many programs let you enter numbers in more than one way and you must decide which rnethOO you prefer. The way you enter the number does not affect the way it is displayed on the screen as this is controlled by the cell fonnat at the
Information technology and library resourt:eS
Using spreadsheets
point of ently. There are usually special ways to enter data for percentages, currency and scientific notation for vcry large and small numbers.
Formulae These are the 'power tools' of the spreadshret be<::ause they do Ihe cakulations. A cell can be referred to by ils alphanumeric code, e.g. AS (colunm A. row 5) and the value containt.'d in that cell manipulated within a formula. e.g. (AS + 10) or (AS + 822) in another cell. Formulae can include a diverse alTay of pre-programmed functions which can refer to a cell. so that if the value of that cell is changed. so is the result of the formula calculation. They may also include limited branching options through the usc of logical opemtors.
Functions
Definition
Function -
8 pre--progfsmmed code for the transfonnation of values (mathematical or statistical functions) Of selection of text characters (string functions).
Example = sin(A5) is an example of a function in Microsoft ~ Excel-. If you write this in a cell, the spreadsheet will calculate the sine of the number in cell A51assuming it to be an angle in radians)
and write it in the cell. Different programs may use
8
slightly different syntax,
Empty cells - note that these may be given the value 0 by the spreadsheet for certain functions. This may cause errors, e.g. by rendering a minimum value inappropriate. Also, an error return may result for certain functions if the cell
cOntent is lero.
A variety of functions is usually offered, but only mathematical and statistical functions will be considered here.
Mathematical funL1iom' Spreadsheets have program-specific sets of prcdetennined functions but they almost all include trigonometrical functions, angle functions, logarithms (p. 262) and random number functions. Functions arc invaluable for transforming sets of data rapidly and can be used in formulae reqUired for more complex analyses. Spreadsheets work with an order of preference of the operators in much the same way as a standard calculator and this must always be taken into account when operators are used in formulae. They also require a very precise syntax- the program should warn you if you break this!
StatiMical functions Modem spreadsheets incorporate many sophisticated statistical functions, and if these are not appropriate, the spreadsheet can be used to facilitate the calculations required for most of the statistical tests found in textbooks. The descriptive statistics normally available include: • • • • •
Statistical calculations - make sure you understand whether any functions you employ are for populations or samples (see p. 263).
In Microsoftll Excel". copying is normally relative, and if you wish a cell reference to be absolute when copied, this is done by putting a dollar ($) sign
sums of all data present in a column. row or block; minima and maxima of a defined range of cells; counts of cells - a useful operation if you have an unknown or variable number of data values; averages and other statistics describing location; standard deviations and other statistics describing dispersion.
A useful function where you have large numbers of data allows you to create frequency distributions using prc-defined class intervals. The hypothesis-testing statistical fW1ctions may be reasonably powerful (e.g. t-tesl, ANOVA, regressions) and they often return the probabilit,l' P of obL1ining the test statistic (where 0 < P < I), so there may be no need 10 refer to statistical tables. Again. check on the effects of including empty cells.
Copying
Example
before and aftS'" the column reference lener, e.g. $C$56.
All programs provide a means of copying (replicating) fonnulae or cell contents when required and this is a very useful feature. Whcn copying, references to cells may be either relative. changing with Ihe row/column as they are copied. or absolute, remaining a rued cell referemx and not changing as the formulae are copied. This distinction between cell references Information technology and library resources
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Using spreadsheets
is very important and must be understood; it provides one of the most common forms of error when copying formulae. Be sure to understand how your spreadsheet performs these operations.
Naming blocks When a group of cells (a block) is carrying out a particular function. it is often easier to give the block a name which can then be used in all formulae referring to that block. This powerful feature also allows the spreadsheet 10 be more readable.
Graphics display Most spreadsheets now offer a wide range of graphics facilities which are easy to use and this represents an ideal way to examine your dllta sets rapidly and comprehensively. The quality of the final graphics oulpUI (to a printer) is variable but is usually sufficient for initial investigation of your data. Many of the options are business graphics styles but there are usually histogram. bm chart. X-Y plotting, line and area graphics options available. Note that some spreadshttt graphics may not come up to the standards expected for the formal presentation of scientifIC data {po 343).
Printing spreadsheets This is usually a straightforward menu-controlled procedure. made difficult only by the fact that yom spreadsheet may be 100 big to fit on ont piece of paper. Try to develop an area of the sheet which contains only the data that you will be priming. i.e. perhaps a summary area. Remember that columns C'.m usually be hidden for printing purposes and yOll can control whethcr the printout is in portrait or landSC'dpe mexle, and for continuous paper or single sheets (depending on printer capabilities). Use a screen prcview option, if available, to check your layout before printing. Most spreadsheets. are now WYSIWYG (What You See Is What You Get) so that the appeanmcc on the SLTeeIl is a realistic impressioo of the printout. A 'print to fit' option is also available in some programs. making the output fit lhe page dimensions.
Using string functions - these allow you to manipulate text within your spreadsheet and include functions such as ·search and replace· and alphabetical or numerical ·sort·.
310
Use as a database Many spreadsheets can be used as databases. using rows and columns to represent the fields and records (see Chapter 48). For many applications in chemistry. the spreadsheet fonn of database is perfectly adequate and should be seriously considered before using a full-feature database progrdm.
Informatie>rl technology and library resources
-0
Word processors, databases and other packages Word processors The word processor hots f:lcilitmed writing because of the ease of revising texl.
Word processing is a lr.msfemble skill valuable beyond the immediate requirements of your chemistry course. Using a word processor should improve
your writing skills :lOd speed because )'ou can create. check Hnd change your text on the screen before printing it as 'hard copy' on paper. Once entered and saved, multiple uses can be madeofa piece of text with littleeffort. When using a word processor you can: • • • • • •
refine material many times before: submission; insert material easily, allowing writing to take place in any sequence: use a spell
The potential disad.....lnlHges of using a word prOC'essor include: • • • • The computerized office - many word processors are now sold as part of an integrated suite. ag. PerlectOffice" and Microsoft'''' Office, with the advantage IhBt thoy share a common interfoce in the
'different components (word processor,
spreadsheet, database, elc.) and allow ready exchange of information fe.g. text
graphics) between component programs.
Using textbooks, manuals and Morialsthe manuals that come with some
programs may not be very user-fTiendly and it i. often worth investing in one of the textbooks that are 8vaiiabAe for most word processing programs. Altflf08tivetv, use an online 'help' tutorial, evailable with the more sophisticated packages.
lack of ready access to a computer, software and/or a printer; time taken to learn the operational details of the progmm; the temptalion 10 make 'trivial' revisions; loss of files due to computer breakdown or disk loss or failure.
Word processors come as 'packages' comprising the program and a manual, often with a lutorial program. Examples are Microsofr H- Word and WordPerfecrH • Most word processors have similar general fealUres but differ in operational deUlil; it is best to pick one and stick to it as far as possible so that you become familiar with it. learning to lise the package is like learning to drive a car - you need only to know how to drive the computer and its program, not to underst'llld how Ihe engine (program) and transmission (data transfer) work, although a little backb'TOund knowledge is often helpful and will allow you to gel the most from the program. In most word processors, the appearance of the screen realistically represents what the printout on paper will look like (WYSIWYG). Word proces..'iing files actually contain large amounts of code relating to text format etc., but these duller the screen if visible, as in non-WYSr\VYG programs. Some word processors are menu driven, others require keyboard entry of codes: menus are easier 1,0 stan with and the more sophisticated programs allow you to choose between these options. Because of variation in operational details, only general and strategic infonnation is provided in this chapter: you must learn the details of your word processor through use of the appropriate manual and 'help' facilities. Before stnning you \\'111 need: • • •
the program (ideally on a hard disk): a noppy dis.k for storage, retrieval and backup of your own files when created; the appropriate manual or textbook giving opemtional details; Infonnation technology and library resources
311
Word processors, databases and other packages
•
• •
a dmft page layout design: in particular you should have decided on page size. pa!,'C margins. typeface (fom) and size., type of text justification, and fonnat of page numbering; an outline of the text content; access to a suitable printer: this need not be attached to the computer you are using since your file can be taken to an office where such a printer is available. providing that it has the same word processing program.
La)'ing out (formatting) ),our document Using a word processor - take full advantage of the differences between
word processing and 'normal' writing (which necessarily follows a linear sequence and requires more planning):
•
Simply jot down your initial ideas for a plan, preferably to paragraph topic level. The order can be altered easily and if a paragraph grows too much it can easily be split.
Although you can fonnat your text at any time, it is good practice to enter the basic commands at the start of your document: entering them later can lead to considerable problems due to reorganization of the text layout If you use a particular set of layout criteria regularly, e.g. an A4 page with space for a letterhead. make a template containing the appropriate codes that can be called up whenever you start a new document. Note that various printers may respond differently to particular codes. resulting in a different spacing and layout.
• Start writing wherever you wish and •
fill in the rest later.
T)'ping the text
Just put down your ideas as you think, confident in the knowledge that it is the concepts that are important to
Think of the screen as a piece of typing paper. The cursor marks the position where your text/data wiU be entered and can be moved around the screen by use of the cursor-control keys. When you type, don't worry about running out of space on the line because the text will wrap around to the next line automatically. Do not use a carriage return (usually the IENTER I or key) unless you wish to rorce a new line, e.g. when a new paragraph is wanted. if you make a mistake when typing, correction is easy. You can usually delete characters or words or lines and the space is dosed automatically. You can also insert new text in the middle of a line or word. You can insert special codes to carry out a variety or tasks. including changing text appearance, such as underlining. bold and italics. Pumgraph indentations can be automated using ITAB I or [§ as on a typewriter but you can also indenl or bullet whole blocks or text using special menu options" The runction keys are usually pre-programmed to assist in many of these operations.
note; their order and the way you •
express them can be adjustecllater. Don't worry about spelling and use of synonyms - these can (and should) be checked during a special revision fun through your text,. using the spelling
checker first to correct obvious
mistakes, then the thesaurus to change wOfds for style or to find the exact word. • Don't forget that a draft printout may be required to check Ii} for pace and spacing - difficult to correct for on a screen; and (ii) to ensure that words checked for spelling fit the required sense.
B
Editill!: feat"res Deleting and restoring text - because deletion can sometimes be made in error, there is usually an "undelete' or "restore" feature which allows the last deletion to be recovered.
312
Word processors usually have an array of features designed 10 make editing documents easy. In addition to the simple editing procedures described above. the program usually allows blocks of text to be moved ('cut and paste'), copied or deleted. An extremely valuable editing facility is the search procedure: this can rapidly scan through a document looking for a specified word. phrase or puncttlalion. This is particularly valuable when combined with a replace facility so that, ror example, you could replace the word 'test' with 'trial' throughout your document simply and rapidly. Most WYSIWYG word processors have a command (e.g. Show/Hide in Microsoft!! Word) which reveals the non-printing chamcters, including paragraph and space markers. This can be userul when editing text, as spacing may alter in apparently mysterious ways ir the precise position of the cursor ~is-ti-I'js these markers is not taken into account.
Information technology and library resources
Word processors, databases and other packages
FOII/.f alld tine spaci"g Most word processors otTer a variety of fonts depending upon the printer being used. Fonts come in a wide variety of types and si:a:s. but they :Ire defined in p
PnMenting your documents - it • good pntetice not 10 mix typefaces too much in a formal d9Curnent; abto the font size shouk! not differ gr68t1v f~ dlffe«tnt
headings, sub-headings and the lext
•
•
•
Preparing draft documents - use doubts spacing 10 allow room for your editing comments on the printed pege.
•
•
Preparing final documents .. for most work, use a 12 point proportional sef"if typeface with spacing dependont upon the specme.tions for th8 . .W ..O .."'.:.
•
Typeface: the term for a family ofch:lracters of a panicular design. each of which is given a particular name. The most commonly used for normal text is Times Roman, as used here for the main text, but many others are widely available, particularly for the better quality printers. They fall into three broad groups: seriffOl\ts with curves and nourishes at the ends of the characters (e.g. Times Roman); saliS serif fonts without such flourishes, providing a clean, modern appearance (e.g. Helvetica, also known as SwiSS); and derorative fonts used for special purposes only, such as the production of newsletters and notices (e.g. r~F J'~). Size: measured in points. A point is the smallest typographical unit of measurement, there being 72 points 10 the inch (:lbout 28 points per cm). The standard si7.£S for text are 10, II and 12 point. but typefaces are oOell available up to 72 point or more. Appearance: many typefaces are 'I\'ailable in a variety of styles and weights. Many of these arc not designed for usc in scientific literature but for desktop publishin!l. Spacing: em be either fixfld, where every chara(.1er is the same width. or proportional, where the width of f:\·ery character, inclw:ling spaces, is varied. Typewriter fonts such as Elite and I'restib'e use fixed spacing and are useful for filling in fonus or tables, but proportional fonts make the overall appearance of text more pleasing and rcadnble. Pilch; specifIeS the number of characters per horizontal inch of lext. Typewriter fonts are usually 10 or 12 pitch, but proportional font..<; are never given a pitch value since it is inherently variable. Justific:::ltion is the tenn describing the way in which text is aligned \~rtically. Left justification is normal, but for fonnal documents. both left and right justification may be used (as here).
You should also consider the \'ertical spacing of lines in your document. Drafts and manusctipls are frequently double spaced to allow room for editing. If your document has unusual font sizes. this may well affect line spacing, nlthough most word processors will cope with this automatically.
Table construction Tables can be produced by a variety of methods: •
•
•
Using the tab key [§] as on a typewriter: Ihis moves the cursor to predetennined positions on the page, equivalent to the start of each tabular column. You can define the positions of these tabs as required at the sLart of each table. Using special table-constructing procedures. Here the table construction is largely done for you and it is much easier than using tabs. providing you enter the correct information when you set up the table. Using a spreadsheet to construct the table and then copying it to the word processor. This procedure requires considerably more manipulation than using the word processor directly and is best resen.'Cd for special circumstances, such as the presentation of a very large or complex table of data, especially if the data are already stored as a spreadsheet. Inrormalion technology and library resources
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Word processors, databases and other packages
Gtaph;c~'
and ~peda{ chm'acters
Many word processors can incorporate graphics from other programs into the texl of a document. Files must be compatible (see your manual) but if this is so, it is a relatively straightfonvard procedure. For highly professional documents this is a valuable facility, but for most undergraduate work it is probably better to produce and use graphics as a separate operation, e.g. employing a spreadsheet. You can draw lines and other graphical features directly w:ithin most word processors and special characters may be available dependent upon your printer's capabilities. It is a good idea to print out a full set of characters from your printer so that you know what it is capable of. These may include s)111bols and Greek characters, orten useful in chemistry. Too{~'
Many word processors also offer you spc<.ial lools, the most important of which are; •
• •
Using a spell-eheck facility - do not rely on this to spot all errors. •
Macros: special sets of files you can create when you have a frequently repealed set of keystrokes to make. You can record these keystrokes as a 'macro' so that it can provide a shortcllt for repeated operations. Thesaurus: used to look up alternative words of similar or opposite meaning while composing text at the keyboard. Spell-check; a very useful facility which will check your spellings against a dictionary provi.ded by the program. This dictionary is often eXIXmdable to include specialist words which you use in yOllr work. "Tbe danger lies in becoming too dependent upon this facility as they all have limitations; in particular, they will not pick up im;orrcct words which happen to be correct in a different context (i.e. 'was' typetl as 'saw' or 'meter' rather than 'metre'). Beware of American spellings in programs from the USA, e.g. 'color" instead of 'colour'. The rule, therefore, is to use the spell-eheck first and then carefully read the text for errors which have slipped through. Word count: useful when you are writing to a prescribed limit.
PrinrbJg from your program
Using the print preview mode - this can reveal errors of several types, e.g. spacing between pages, that can prevent you wasting paper and printer ink unnecessarily.
Word processors require you to specify precisely the t)'PC of printer and/or other style details you wish to use. Most printers also offer choices as to text and graphics quality, so choose draft (low) quality for all but your final copy since this will save both time and materials. Use a print preview option to show the page layout if it is available. Assuming that you have entered appropriate layout and font commands, printing is a straightforward operation carried out by Ihe word processor al your command. Problems usually arise because of some incompatibility between the criteria you have enlered and the printer's own capabilities. Make sure that you know what your printer offers before starting to type: although parameters are modifiable at any time, changing the p'1£C size, margin si:le, font size, etc., all caw;e your text 10 be rearranged, and this can be frustrating if you have spent hours carefully laying oul the pages!
~
It is vital to save your work frequentlv to a hard or floppy disk lor both). This should be done every 10min or so. "you do not save regularly, you may lose hours or days of work. Manv pt'"ograms can be set to 'autosave' every few minutes.
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Information technology and library resources
Word processors, databases and other packages
Databases II. database is an electronic filing systcm whose structure is similar to a manual record card collection. Its collection of records is termed a file. The individual items of infonnation on each record are tenned fields, Once the database is constructed, search criteria can be lL...oo to view lites through various filters according to your requirements. The computerized catalogues in your library are just such a system; you enter the filter requirements in the form of author or subject keywords. You can use a database to OItalogue, search, sort and relaLC collections of infonnation. The benefits of a computerized database over a manual c:ml-lite system are:
• • •
•
•
Choosing between 8 dafabase and a spreadsheet - use a database on~ after careful consideration. Can the task be done better within 8 spreadsheet? A database program can be complex 10 set up and usually needs to be updated regularly.
Thc information content is easily IImended/updated. Printout of relevant itcmJ; can be obtained. It is quick and easy to organize through sorting lind searching/selection criteria, to produce sub-groups of relevant records. Record displays can easily be redesigned, allowing ncxiblc methods of presenting records according to interest. Relational databases can be combined, giving the whole system immense flex.ibility. The older 'nat-file' databases store infonnation in files which can be searcht(! and soned, but cannot be linked to other databases.
Relatively simple database files can be constructed within the more adv:lI1ced spre;Jdsheets lL';ing the columns and rows as fields and records respectively. These are capablc of limited soning and searching operations and are probably sulTlcient for the types of datab..'lse5 you are likely to require as an undergnlduate. You may also make use of a bibliography database especially constructed for that purpose.
Statistical analysis packages Statistical packages vary from small pro!,'Tams designcd to carry out vcry specific statistical tasks to large sophisticated packages (Statgraphics,lt. Minitab'lf, Statistica'iiJ etc.) intended to provide statistical assistance, from experimental design to the analysis of results. Consider the following features when selecting a package: •
• • •
1be data entry and editing section should be user-friendly, with options
for tr.msforming data. Options should indude descripth-e statistics and exploratory data analysis techniques. Hypothesis testing techniques should include ANOVA, regression analysis, multivariate techniques and panl.lnetric and non-parametric statistics. Output facilities should be suitable for graphical and tabular formats.
Some programs have complcx data entry systems, limiting ease of use, The data entry and storage system should be based upon a spreadsheet system, so that subsequent editing and transfonnation operations are straightforward.
~ Make sure that you understand the statistical basis for your test and the computational techniques involved before using a particular program. Information
technology and library resources
315
Word processors, databases and other packages
Graphics/presentation packages ChemWindow" and ChemOraw" allow you to draw chemical structures easily. They contain, for example, beru:ene rings which can be easily sc)ecte
Many of these packages are specifically designed for business graphics mlher than science. They do, howe\'er, have "."onsideHtble value in the preparation of materials for posters and talks where visual quality is an important factor. There are several packages available for microcomputers such as Freelance Graphics'1f, Harvilrd Graphics
• •
Graphics qualily: the built-in graphic.. are sometimes of only moderate quality. Use of anno13tion facilities can imprO\'C graphics considerably. Do nOI use inappropriate graphics for scientific presentation. The production of colour graphics: this requires a good-quality colour printer/ploner. Importing grnphics flies: grnphs produced by spread<;heets or other statistical program.li can usually be imported into graphics programs - this is useful for adding le~nds, anno13tions, etc., when the facilities offered by the original programs are inadequate. Check that the format of files produced by your statistics/spreadsheet program enn be recognized by your gmphics program. The different types of file are distinguished by the three-charncter filename extension.
~ Computer graphtcs are not always satisfactory lor scientific presentation. While they may be useful for exprorato~ procedures, they may need to be redrawn by hand for your final report. h may be helpfUl to use a computer--generated graph as a template for the final version.
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Information technology and library resources
-----------10
Finding and citing published information The ability to find scientific information is a skill required for many exercises in your degree programme. Vou will need to research facts and published findings as part of writing es.<;ays, literature reviews and project introductions, and when amplifying your lecture notes and revising for exams. You must also learn how to follow scientific convention in citing source material as the authority for each statement you make.
Browsing in a library - this may tum up interesting material, but remember the books on the shelves are those not currently out on klan. Almost by definition, the latter may be more up to date and useful. To find out a library's full holding of books in any subject area, you need to search its catalogue (normallv available as a computerized database).
Example The book Inorganic Chemistry, 3rd edn, by D.F. Shriver and P.W. Atkins 11999; Oxford University Press) is likelv to be classified 8S follows: Dewey decimal system: 546 where 500 refers to natu ral sciencos and mathematics S40 refers to chemistry 546 refers to inorganic chemistrY UbrBfY of Congress system: 00146-191 where Q refers to science
00 00146-197
refers to chemistry refers to inorganic chemistry
Computer databases - several databases are now produced on CD-ADM for open
use in libraries le.g. Mcdline. Applied SCience and Technology Index). Especially useful to chemists are Chemical Abstracts and Analytical Abstracts. Some databases can be accessed via the Internet such as BIOS (Bath Information and Data servicel, a UK service providing access to information from over 7000 periodicals (usemame and password required via your library). Each of these databases usually has its own easy-ta-foIlOW menu instructions. It is worthwhile to consider key words for your search beforehand to focus your search and save time.
Sources of information
Fo,. essays and I'evi!,';on You are unlikely to delve into the primary literature for these purposesbooks and reviews are much more readable! If a lecturer or tutor specifies a particular book, then it should not be difficult to find out where it is shelved in your library, as most libraries now have a computerized index system and their staff will be happy to assist with any queries. If you want to find out which books your library holds on a spa;:ified topic, use the system's subject index. You will also be able to search by author or by key words. There are two main systems used by libraries to classify books: the Dewey Decimal systctn and the Library of Congress system. Libraries differ in the way they cmploy thcse systems, especially by adding further numbers and leiters after the standard classification marks to signify, say, shelving position or edition number. Enquire at your library for a full explanation of local u<;age. Example of boofl classification Ubrary of Congress Chemistry Analytical chemistry Chromatography Crystallography Electrochemistry Inorganic Chemistry Organic chemistry Physical chemistry RadiOChemistry Spectroscopy Surface Chemistry Synthesis lorganic)
540 543 543.544 548 541.13 54<3 547
541.1 541.28 543.42 541.18 547.07
001-999 0071-145 00117 .C5; OO79.C4 00901-999 00551-571; 00261; TP25D-261 00146-197 00241-449 00450-731 00601--655 0095-96; QC45Q-467; 00272.56
00506-608 00262
The World Wide Web is an expanding resource for gathering both general and specifil; information (see Chapter 45). Sites fall into analogous categories to those in the printed literature: there are sites with original infonnation. sites that review information and bibliographic sites. One considerable problem is that websites may be fn-'quently updated. so infonnation present when you first looked may be allen-xl or even absent when the site is next consulted. Further, very lillIe of the information on the WWW has been monitored or refereed. Another disadvantage is that the site information may not statc the origin of the material, who wrote it or when it was wriuen. Information technology and library resources
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Finding and citing published information
FOI'/itel"Uture slIn'eys and project wOI'k You will probably need to consult the primary literature. If you are starting a new research project or writing a report from scratch, you can build up a core of relevant papers by using the following methods:
•
•
•
Definitions
•
Joumal/periodical/serial- any publication issued at regular intervals. In chemistry, usually containing papers describing original research findings and reviews of
literature. The primary literature - this comprises original research papers, published in specialist scientific periodicals. Ccrtain prestigious general journals (e.g. Nature,
Sciencel contain important new advances from a wide subject area. Monograph - a specialized lxlok covering a single topic. Review - an article in which recent advances in a specific area are outlined and discussed. Proceedings - volume compiling written
versions of papers rcad at a scientific meeting on a specific topic. Abstracts - shortened versions of papers. often those read at scientific meetings. These may later appear in the literature as full papers.
•
Bibliography ~ a summary of the
published work in a defined subject area.
Asking around: supervisors or their postgraduate students will almost certainly be able to supply you with a reference or two that will start you off, Searching a computer database: these cover very wide areas and are a convenient way to start a reference collection, although a charge is often made for access and sending out a listing of the papers selected (your library mayor may not pass this on to you). Consulting the bibliography of other papers in your collection - an important way of finding the key papers in your field. I n effect. you are laking advantage of the fact that another researcher has already done all the hard work! Referring to 'current awareness' journals or computer databases: these are useful for keeping you up to datc with current research; they usually provide a monthly listing of article details (title, authors, source. author address) arranged by subject and cross-referenced by subject and author. Current awareness journals covcr a wider range of primary journals than could ever be available in anyone library. Examples include: (a) Currellf COlllents, published by the Institute of Scientific Information, Philadelphia, USA, which reproduces the contents pages of journals of a particular subject area and presents an analysis by author and subject. (b) Currellt Ach'w/as, published by Elsevier Science, Oxford, UK, which sub-divides papers by subjtx:t within research areas and crossreferences by SUbject and author. (c) Chemical Abstracts (llltl Al1l1lytkaJ Abstracts, in which each paper's abstract is also reproduced. Papers may be cross-referenced according to various taxa, which is useful in allowing you to find out what work has been done on a particular organism. Using the Science Citation lIulex (SCI): this is a very valuable source of new references, because it lets you see who has dted a given paper; in effect, SCI aUows yOll to move forwan! through the literature from an existing reference, SCI is published regularly during the year and issues are collated annually. Some libraries have copies on CD-ROM: this allows rapid access and output of selected infolUlation.
For .~pecialized infomlation You may need to consult reference works such as encyclopaedias, maps and books providing specialized information. Much of this is now available on CD-ROM (consult your library's information service). Two books worth noting are: I.
2.
318
Information technology and library resourr:es
The Hmulbook of Chemistry (lIul Physics (Lide, 2000): the Chemical Rubber Company's publication (affectionately known as the 'Rubber Bible') giving all manner of physical constants, radioisotope half-lives. etc. The Merck IIIdex (Budavari, 1999), which gives useful infonnation about organic chemicaL~, e.g. solubility, whether poisonous, etc.
Finding and citing published information
Obtaining and organizing research papers
Copyright law - in Europe, copyright rogulations were harmonized in 1993
(Directive 93/98/EECI to allow literary copyright for 70 years after the death of an author end typographical copyright for
25 yeers after publication. This was implemented in the UK in 1996, where, in
addition, the Copyright. Designs and Patents AI:t (1988) allows the Copyright licensing Agency to license institutions
so that lecturers, students and researchers may take copies for teaching and personal reseerctl purposes - no more than 8 single article per journal issue. one chaptet of a book, or extracts to a totel of 10% of a book.
Storing research papers - these can easily be kept in elphabetkal order within
filing boxes or drawers, but if your collection is likely to grow larg8. it will need to be rcfiled as it outgrows the
storage space. An alternative is to keep an alphabetical card index system luseful when typing oot lists of references) and file the papers by 'acression number' as they accumulate. New filing space is only required at one 'en(f and you can use the accession numbers to form the basis of 8 simple cross-referencing system.
Obtaining a copy 11 is usually more convenicnt to havc personal copies of key research articles for direcl consultation when working in a laboratory or writing. The simplest way of obtaining these is to photocopy the originals, For academic purposes, this is nom13l1y aet:eptable within copyright law. If your library does not take the journal. it may be possible for the library to borrow it from a nearby institute or obtain a copy via a national borrowing centre (an 'inter-library loan'). If the latler, you will have to fill in a foml giving full bibliographic details of the article and where it was cited, as well as signing a copyright clcanl.nce statemenl concerning your USC of the copy. Recent advances in electronic publishing mean that full-text copies of research articles Clin be downloaded either from a publisher or via BI OS. In the case of the former you will need to establish whether your institute hlls lin electronic subscription to the publication of inlerest. Viewing of an electronic research paper is done via a particular software programme, e,g, the reader Adobe Acrobat >\. It is oOen possible to download a free copy of the reader to your computer the first time you try this. Your department might be able to supply 'reprint request' postcards to be sent to the designated author of a paper. This is an unreliable method of obtaining a copy because it may take some time (allow al least 1-3 months!) and some request'i will nOl receive a reply. Taking jnto account the wa'ite involved in postage and printing. it is probably best simply \0 photocopy or send for a copy via inter.library loan.
Organizing
paper~'
Although the numbers of papers you accumulale may be small 10 start with, it is worth putting some thought into their storage and indexing before your collection becomes disorganized and unmanageable. Few things are more frustrating than not being able to lay your hllnds on a vital piece of information, and this can seriously disrupt your now when writing or revising.
Cal'd index systems title
date accession of numller publication
allthors
SMITH, Af18M Ja.JES. C.D.
Anat./leT 5m.111 Step
126
1980
lOw.as Ow- ~ ftizz
JoorntJi of lrnp0t"t8nt ~ 14 345--67
Index cards (Fig. 49.1) are a useful adjunct to any filing systern. Firstly, you may not have a copy of the paper to file yet may still wish the reference inlormation to be recorded somewhere for later use. Secondly, a selected pile of cards can be used when typing out different bibliographies. Thirdly. the cards can help when organizing a review (see p. 339). Fourthly, the card can be used to record key points and comments on the paper. The priority rule for storage in card boxes is again first·author name. subsequent author name(s), date. Computerized card index systems simplify cross-referencing and can provide computer files for direct insertion into word processed documents; however, l.hey are vcry time consuming to set up and maintain. so you should only consider using one if the time invested will prove worthwhile.
Making citations in text journal delaits
space lor abslfact Of ComfJ1eftts
There are two main ways of citing articles and creating a bibliography (also referred to as 'References' or 'Literature Oted'),
Fig. 49.1 A typical reference card. Make sure the
index card carries all the bibliographic information of potential relevance.
The Harrw,d system For each citation. the author name(s) and the date of publication are given at the relevant point in the text. "f1te bibliography is organized alphabetically Information technologV and library resources
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Finding and citing published information
and by date of publicution for papers with the same authors. rormat~ normally adopted arc, for example, 'Smith and Jone.
19890; 1989b).
The tIImrerical or Vancolltlel'system Papers are cited via a superscript or bracketed reference number inserted at the appropriate point. Normal formal would be. for example 'computational chemistry4.S has shown thai .. .' or 'Jones (55,821 has claimed lhat .. .'. Repeated citations use the number from the first citation. In the true numerical method (e.g. as in Nafllre, Scierlce). numbers are allocal.ed by order of citation in the text. This is by far the most common approach in chemistry journals. Note that adding or removing references is tedious. so the numbering should be done only when the lext is finalized.
The 'Kat";tzky· .(j'stelll A third system has been popularized in publications involving Professor Roy Katritzky (University of Florida, USA) and uses the best features of both the Harvard and Vancouver systems. For each reference in the text a code is written comprising the year of publication, the journal and the page of the journal. In the reference section the coded references are cited, together with the full reference - authors. journal. year. volume and page - in year order and a list of journal codes in alphabetical order can be provided. For example: ~.
Paper in journal: Smith, A. 8., Jones. C.O. and Professor, A JournaJ of New Results, 11 (2000) 19. BooIc Smith, A. B. 119981. Summary of My Ufe's Work. Megadosh Publishing Corp., Bigcity. C~
in edited book.
Jones. C. D. and Smith. A. B. 119981. Earth-shaUering research from our
laboratory. In: Research Compendium 1998 led. A. Professor), pp. 123-456. Bigbucks Press, Booktown.
Thesis:. Smith, A. B. (19951. In\/8Sligat~ns on mv fawurite topic. PhD thesis:Uniwrsity of life, Fulchoster. Note that underlining used h~e specifIeS italics in print; use an italic font if working with 8 word processor.
320
in the text ... Robinson and Watt (34JCSI536) found ... in the reference section 341C'S1536 R. Robinson and SJ. Watt. J, Chem. Soc., 1934, 1536. TIle advantage of this system is that it is easy to insert missed references into the text, a great disadvantage of the otherwise neat Vancouver system. For delails consull the reference seclion in Comprehensil'e Heleroc)'c/ic Chemistry, ed. A.R. Katritzky and C.W. Rees, Pergamon. Oxford, 1984.
How to list your citations in a bibliography Whichever citation method is used in the text, comprehensivc details are required for the bibliography so that the reader has enough information to rmd the reference easily. Citntions should be lisled in alphabetical order with the priority: first author. subsequent author(s). date. Unfortunately, in temlS of punctuation and layout, there an: almost as many ways of citing papers as there are journals!
Information technology and fibfary resoul'Ces
Finding and citing published information ~ Your department may specify an exact format for
project work; if not. decide on a style and be consistent - If you do not pay attention to the details of citation you may lose marks. Take special care with the following aspects:
• •
•
• • Citing websites - there is no widely accepted format at present. We suggest providing author namelsl and date in the text if using the Harvard system, while in the bibliography giving the above. plus site title and full URL reference (e.g. Hacker, A. (1998) University of Cybertown homepage on aardvarks. http://www.myserver.ac.uk/ homepage).
•
Authors and editors: give details of wI authors and editors in your bibliography, even if given as e( af. in the text. Abbreviations for journals: while there are standard abbreviations for the titles of journals (consult library Slam, it is a good idea to give the whole title, if possible. Books: the edition should always be specified as contents may change between editions. Add, for example, '(5th edition)' after the title of the book. You may be asked to give the International Standard Book Number (ISBN), a unique reference number for each book published. Unsigned articles, e,g. unatlributed newspaper articles and instruction manuals: refer to the author(s) in text and bibliography as 'Anon.'. Unread articles: you may be forced to refer to a paper via another without having seen it. If possible, refer to another authority who has cited the paper, e,g, '". Jones (1980), cited in Smith (1990), claimed thai ... '. AJternal.ivcly. you could denote such references in the bibliography by an asterisk and add a short note to explain at the start of the reference list. Personal communications: information rt'Ceived in a letter. seminar or conversation can be referred to in the text as, for example, ', .. (Smith, pefS. comm.)'. These citations are not generally listed in the bibliogrdphy of papers, though in a thesis you could give a list of personal communicants and their addresses.
Information technology and library resources
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General aspects of scientific writing Written communication is an e!i.'iCnlial component of all scicncl,.'S. Most courses include wriling cxerciso; in which you will learn to OCSl.:ribc ideas and resull~ accurdlcly, sUf...c inctly and in an appropriate style and formal. The following are features common 10 all (omlS of scientific writing.
Organizing time Making a timetable al the outsct helps ensure thaI you give each stage adequate attention and complete the work on time. To creale and uSC: a timetable: I. 2. 3.
4.
Break down the task into stage<;. Decide on the proponion of the total lime each slage should hIke. Set realistic deadlines for completing each stage, allowing some lime for slipp3b'C' Refer 10 your timetable frequently as you work: if you fail to meet one of your deadlines, makc a serious errort 10 catch up liS soon a~ possible.
~ The appropriate allocation of your time to reading,
planning, writing and revising will differ according to the task in hllnd {see Chapters 51 and 531.
Organizing information and ideas Before you write, you need to gather and/or think about relevant material (Chapter 49). You muSt then decide: • •
what needs to be included and whal doesn't; in Whal order il should appear.
Start by jOlling down headings for cvcl)'thing of potcntilll relevance to the topic (this is sometimes called 'brainstorming'). A spider diagram (Fig. 50.1) will help you organi2e these ideas. The nexi stage is to create an outline of your text (Fig. 50.2). Outlines are valuable because they: Creating an outline - an informal outline can be made simplV by indicating (he order of sections on 8 spKIer diagram (as in Fig. 50.1).
• • • •
force you to think about and plan the structure: provide a checklist so nothing is missed out; ensure the material is balanced in content and length; help you organize figures and tables by showing where they will be used.
In an essay or review-, the structure of yow- writing should help the reader to understand your main points. Sub-divisions of the topic could simply be related to the nature of the subject mailer (e.g. levels of organization of a protein) and should proceed logically (e.g. prim.'1ry structures, then secondary, etc.). A chronological approach is good for evaluation of past work (e.g. the development of the concept of aromaticity), whcreas a step-by-step comparison might be best for certain exam questions (e.g. 'OiS(;:uss the differences between solid phase and Soxhlct extraction in the analysis of pesticidcs'). There is lillie choice about structure for practical and project reports (see Chapter 52). as.~imilatc and
Communicating information
32li
General aspects of scientific writing
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Fig. SO., Spider diagram showing how you might 'brainstorm' an essay with the title ·Detectors for Gas Chromatography'. Write out the essay title in full to fOfm the spiders body, and as you think of possible oontent. place headings around this to form its legs. Decide which headirlgs are relevant and which are nOI and use arrows 10 note connections between subjects, if required. This may Influence your chotee of order and may help to make your writing now because the links between paragraphs wilt be natural. You can make an informal outline directly on a spider diagram by addirlg numbers indicating a sequence of paragraphs las shown). This method is best when you must wol1l: quickly, as with an cssay written under exam conditions.
Fig. SO.2 Formal outlines. These are useful for a long piece of work where you or the reader might otherwise lose track of the structure. The headings fOf sections and paragraphs are simply written in sequence \/Yith the type of leltering and level of indentation illdicating their hierarchy. Two different forms of formal outline are shown. a minimal form lal and a numbered form fbI. Note that the headings used in an outline are often repeated wilhin the essay to emphasize its structure. The conlent of an outline will depend on the time you nave available and the nature of the work:. but the most detailed hierarchy you should reasonably include is the subject of each paragraph.
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Writing Adopting a scientific style Your main aim in developing a scientiffC style should be to get your message across directly and unambiguously. While you can try to achieve this through a set of 'rules' (sec Box 50.1). you may find olher requirements driving your WTiling in a contradictory direction. For instance, the need to be accurate and complete may result in textliuered with technical terms. and the flow may be continually interrupted by references fO the literature. The need to be succinct 326
Communicating information
General aspects of scientific writing
,
Box 50.1
How to achieve a clear, readable style
Words and phrases • Choose short clear words and phrases rather than long ones, e.g. use 'build' rather than 'fabricate' 'now' rather than 'at the present time', At certain times, technical terms must be used for precision, but don't use jargon if you don"t have to. • Don't worry too much about repeating words, especially when to introduce an alternative might subtly alter your meaning. • Where appropriate, usc the first person to describe your actions ('We decided to ...'; '1 conclude that ... 'J, but not if this is specifically discouraged by your supervisor. • Favour active forms of speech ('the solution was placed in a beaker") rather than the passive voice (·the beaker was filted with the solution'). • Use tenses consistently. Past tense is always used for the experimental section ('samples were laken from .. :) and for reviewing past work ('Smith 119901 concluded that ... 'j. The present tense is used when describing data ('Fig, 1 shows .. ,'J, for generalizations ('Most authorities agree that .. :) and condusions l'I conclude that ...'). • Use statements in parentheses sparingly - they disrupt the reader's attention to your central theme (see above section for examples).
• Avoid cliches and colloquialisms - they are usually inappropriate in a scientific context. Sentences • Don't make them overlong or complicated, • Introduce variety in structure and length. • Make sure you understand how and when to use punctuation, • If unhappy with the structure of a sentence, try chopping it into a series of shorter sentences.
Paragraphs • Keep short and restrict them to a distinct theme, • Use repeated key words (same subject or verb) or appropriate linking phrases (e.g. 'On the other hand .. :) to connect sentences and emphasize the flow of text. • The first sentence should introduce the topic of a paragraph and the following sentences explain, illustrate or give examples. Note: If you're not sure what is meant by any of the terms used here, consult a guide on writing (see Box 50.2).
also affects style and readability through the use of, for example, stacked noun-adjectives (e,g. 'ring-opening metathesis polymerization') and acronyms (e.g. 'Romp'). Finally, style is very much a mattcr of taste and each tutor. examiner, supervisor or editor will have pet loves and hates which you may have to accommodate.
DevelopiJlg technique Writing is a skill that can be improved, but not instantly. You should analyse your deficiencies with the help of fct.'
Getting started A common problem is 'writer's blo::k' - inactivity or stalling brought on by a variety of causes.. If blocked, ask yourself these question,,: •
Are you comfortable with your surroundings? Make sure you are scated comfortably at a reasonably clear desk and bave minimized the possibility of interruptions and distractiolls. Communicating information
327
General aspects of scientific writing
Box 50.2
Improve your writing ability by consulting a personal reference library
Using dictionaries We all know that a dictionary helps with Spelling and definitions, but how many of us use one effectively? You should: • Keep a dictionary beside you when writing and always use it if in any doubt about spelling or definitions. • Use it to prepare a list of words which you have difficulty in spelling: apart from speeding up the checking process. the act of writing out the words helps commit them to memory. • Use it to write out a personal glossary of terms. This can help you memorize definitions. From time to time. test yourself. Not all dictionaries are the samel Ask your tutor or supervisor whether hclshe has a preference and why. Try out the OKford Advanced Learner's Dictionary, which is particUlarly useful because it gives examples of use of all words and helps 'with grammar, e.g. by indicating which prepositions to use with verbs. Using a thesaurus A thesaurus contains lists of vvords of similar meaning grouped thematically; words of opposite meaning always appear nearby.
• Use a thesaurus to find a more precise and appro-priate word to fit your meaning, but check definitions of unfamiliar words with a dictionary.
•
•
•
•
•
328
Communicating information
• Use it to find a word or phmse 'on the tip of your tongue' by looking up a word of similar meaning. • Use it to increase your vocabulary. Rogers Thesaurus is the standard. Collins publish a combined dictionary and thesaurus.
Using guides for written English These provide help with the use of words. • Use a guide to SaNe grammatical problems such as when to use 'shall' or 'wiJ1', 'which' or ·that', 'effecr or 'affect', etc. • Use it for help with the paragraph concept and the correct use of punctuation. • Use it to learn how to structure writing for different tasks. Recommended guides include the following: Kane, T.S. (1983) The Oxford Guide to Writing. Oxford University Press, New York. This is excellent for the
basics of English - it covers grammar, usage and the construction of sentences and paragraphs. Partridge, E. (1953) You Have a Point There. Routledge and Kegan Paul, london. This covers punctuation in a very readable manner. TIChy, H.J. (1988) Effective Writing (or Engineers, M1Jnagers and Scientists. John Wiley and Sons, New York. This is strong on scientific style and clarity in writing.
Are you trying to wrile 100 soon'! Have you clarified your thoughts on thc subject? Ha\'C you done cnough preliminary reading? Talking to a friend about your topic might bring out ideas or reveal dcficienci(.'S in your knowledge. Are you happy with the underlying structure of your work? If you haven't made an outline. try this. If you arc unhappy because you can'l think of a particular detail at the planning stage, just start writing - it is more likely to come to you while you are thinking or something elsc. Are you trying to be too clever? Your first scntem."C docsn't have to be earth-shattering in content or particularly smart in style. A short statement of fact or a definition is fine. If lhere will be time ror revision. get your ideas down on paper and revise grammar. content and order later. Do you really need 10 start writing at the beginning? Try writing the opening remarks aftcr a more stmightforward part. With reports of experimental work, the materials and methods section may be the eask'St to start al Arc you too tired to work? Don't try to 'sweat it our by writing for long periods at a stretch: stop frequently for a rest.
General aspects of scientific writing
Revising your text Wholesale revision of your first drafi is strongly advised for all writing apart from in exams. If a word processor is available, this can be a simple process. Where possible, schedule your writing so you can h..'ave the lin;t draft to 'setlle' for at least a couple of dliYS. When you return to it fresh, you will sec more easily where improvcments elin be made. Try Ihe following stru<.1un:d revi...ion p~ss. each stage being covered in a separate scan of your text:
I. 2.
3. Revising your text - to improve clarity and shorten your lext, 'distil' each sentence by taking wwav unnecessary vvords and 'condense' words or phrases by choosing a shorter alternative.
4.
5.
Examinc content. Have you included everything yOll need to? Is 1\11 thc material relevant? Check the grammar and spelling. Can you spot any 'howkrs"! Focu... on clarity. Is thc text clear and unambiguou...? Doc'S each sentcnce really say what you wlmt it to say? Try 10 achicve brevity. What could be missed Ollt without spoiling Ihc essence of your work? It might help to imaginc an editor has sel you Ihe targel of reducing the text by 15%. Improve slyle. Could the lext read beller'! Consider the sentence and parngmph slructure and the way your lexl develops to its conclusion.
Communicating information
329
----------j0
Writing essays 1bc function of lin es.<>ay is 10 show how much you understand about a topic and how well you
3. rNd answe, arod mah cOITIlClI0IIlI!5'll.'
1. read question and pl8nanswe,(lO'1l.!
j
CHn
orgllnize and express your knowlt:dgc.
Organizing your time Most CSSCI)'S have a relatively slraighlforwurd slnlClUre and it is best to divide your lime into th~ lTUIin pans (Fig. 51.J). For exam slr.ttcgics, see Chapler 56.
Making
8
plan for your essay
Dissect tile melllling of tlte
e.~suJ' q"e.~t;on
0" title
RC'dd the title very carefully and think about Ihc topic before slarting to wrile. Consider the definitions of each of the impOrtant nouns (Ihis can help in approaching the introductory scction). Also think about Ihe meaning of the vcrb(s) used and try 10 follow each instruction precisely (sec Table 51.1). Don't get sidc-trllCkcd because you know something aboul one word or phrase in the title: consider the whole title and all its ramifications. Ir there arc two or more parts to the qucstion, make sure you give adequate attention to each part. 2, write _ _
185"',
Fig.51.1 Pie chart showing 8 lypiC.!l1 division of time for an essay.
Table 51.1 Instructions often used in essay questions and their meanings. When more than one instruction is given (e.g. compare and contrast; describe and explain). make sure you carry out both or your may lose a large proportion of the available mar1l:s
Account for. Anillyse;
Assess: Comment: Compare: Contrast: Criticize: Define:
Describe: Discuss: Enumerate: Evaluate: ~3in:
lUustrate: Inteq>ret: Justify:
List: Outline:
Prnve: Relate: Review: State: ~mmarize:
Trace:
330
Communicating inforrnatiOfl
gllle the reasons for examine in depth and describe the main characteristics of weigh up the clements of and arrive at a conclusion about give an opinion on and provide evidence fOl" your views bring out the similarities berween bring out dissimilarities berween judge the worth of 19ive both positive and negalive aspects) explain the exact meaning of use words aod diagrams to illustrate provide evidence or opinions about. arriving at a balanced conclusion list in outline form weigh up or appraise; find a numerical value for make the meaning of something clear use diagrams or examples to make dear express in simple terms. pt"oviding a jUdgement Show that an idea or statement Is c()("rect provide an itemized series of statements about describe the essential parts only, stressing the classification establish the truth of show the connection between eIIamine critically. perhaps concentrating on Ihe stages in the development of an idea or method express clearlv without illustrations. provide a brief account of describe a sequence of events from a defined point of origin
Writing essays
Consider possible colltellt and examples The spider dillgntm technique (p. 326) is li speedy way of doing this. If you have time to read several sources. consider their contcnt in relation to the cssay litle. Can you spot different approa<:;hcs to the same subject'! Which do you prefer as a means of treating the lepic in relation 10 your title'! Which examples are most relevanl 10 your CllSC. and why'!
Cons"."c( all olltlille Every essay should hllve
II
struclure related to it.s titk. Most marks for essays
llre losl because Ihe wrillen material is badly organized or is irrelevant. An
essay plan. by definilion. creates order and. if thought about carefully. can cnsurc rclLvdllCC. Your plan should be written down (but SI,.'Ored through later ifwrilten in an exam book). Think llboul an CSSlly'S content in three parts: I.
Essay content - it is rarely eoougb simply to lay down facts for the reader - voo must lInalyae them and comment on their signifiC8nce.
2.
3.
The introdl.K;lory section. in which you should include definitions lind some background infonnation on lhe (.'Ontcxt of the topic being considered. You should also tell your rCllder how you plan to approoch the subjecl. The middle of the cs.'\
~ Use paragraphs to make the essay's structure obvious. Emphasize them with headings and sub-headings unless the material beneath the headings would be too short or trivial.
Now start writing I • Using diagrams - give a title and legend for each diagram so that it makes sense in isolation and point out in the text when the rOlldCf should consutt it le.g. 'as shown in Fig. 1 •. : Of 'as can be seen in the accompanying diagram•... 'I.
•
•
Nevcr lose tmck of lhe importance of contcnt and its rclcVllllce. Repealedly ask yourself: 'Am I really answering this question?' Never warne just to increase the length of lln esSllY. QUlllity mther thlln quantity is importlln!. Illustrate your llnsw'er llppropriatcly. Use examples to mllke your points clear, bul remember Ihal too many similar examples can stille the flow of an cssay. Use diagmms where a written dcscription would be difficult or take too long. Use tublcs to condense information. Take all~ with your handwriting. You can't gct marks if your writing is illegible! Try to culth'llle lln open form of hllndwriting, making thc individual Iclters large and dislind. If there is time, make out II rough draft from which II lidy version can be copied.
Reviewing your answer learning from lecturers' and tutors' comments - ask for further explanations
if you don't understand a comment or why an essay was Ie5s successful than you thought it should have beerI.
Don't stop yct! • Reread thc question to check that you have answered all points. • Rcread your essay to check for errors in punctuation, spelling and contenl. Make any corrections obvious. Don't panic if you suddenly realize you'vc missed a large chunk oul as the reader can be redirected 10 a supplemcntary paragraph if nea..-ssary. Communicating information
331
--------10
Reporting practical and project work PmcticaJ reports. project reports. theses and scientific papers differ greatly in depth. scope and size. but they all ha\'c the same basic structure (BoX 52.1). Some variation is pcnnitted. however (see Box 52.1). and you should always follow the advice or rules provided by your department. Additional parts may be specified: for theses, a title page is often required and a List of Figures and Tables as part of the Contents. When work is submitted for certain degrees. you may need to include certain declarations and statements made by the student and supervisor. In scientific papers. a list of Key Words is often added following the Abstract: this infomlation may be combined with words in the title for computer cross-referencing systems.
~ Department
Of" faculty regulations may specify an ex· act format for pt"oducing your report or thesis, Obtain a copy of these rules at an early stage and follow them closely.
Options for discussing data - the main optional variants of the general structure include combining Results and Discussion into a single section and adding a separate conclusions section. •
The main advantage of a joint results and discussion section is that yOu can
Pract;cal and pmject reports These are exercises designed to make you Utink more deeply about your experiments and to pmctise and test the skills necessary for writing up research work. Special features arc: •
link together different experiments,
perhaps explaining why it particular result ted to a new hypothesis and the next experiment. • The main advantage of having a separate conclusions section is to draw together and emphasize the chief points arising from your work, when these may have been 'buried' in an extensive discussion section.
•
•
Introductory material is genemlly short and unless otherwise specifit.'
Theses Theses are submilted as part of the examination for a degree following an extended period of research. They act to place on record full details about your expetimcntal work and will normally only be read by those with a direct interest in it - your examiners or colleagues. Note the following: • •
•
332
Communicating information
You are allowed scope to expand on your findings and to inelude detail that might otherwise be omined in a scientific paper. You may have problems with the volume of information that has to be organized. One method of coping with Ihis is to divide your thesis into chapters, each having the standard fonnat (as in Box 52.1). A gellcral introduction can be given at the start and a general discussion at the end. Discuss this option with your supervisor as it is nol universally favoured. Thl'TC may be an oml exam ('viva') assodated with fhe submission of the thesis. The primary aim of the examiners will be to ensure that you understand what you did and why you did it.
Reporting practical and project work
Box ~2 1
The structure of reports of experimental work
Undergraduate practical and project reports are generally modelled on this structure or a close variant of it. because this is the structure used for nearly all research papers and theses. The more common variations include Results and Discussion combined Into a single section for convenience and Conclusions appearing separately as a series of points arising from the work. In scientific papers, a list of Key Words (for computer cross-referencing systems) may be included following the Abstract. Regarding variations in positioning, Acknowledgements may appear after the Contents, rather than near the end. Department or facuhy regulations for producing theses and reports may specify a precise fonnat; they ohen require a title page to be inserted at the start and a List of Figures and Tables as part of the Contents. These regulations may also specify declarations and statements to be made by the student and supervisor. Pert (in order)
Contents/purpose
Tit.. Authors plus theU"
Explains \.-\Itlat the project was about Explains who did the \IlIOn; and ~e; also where they can be oontacled now Synopsis of methods. results end conclusion of woril; described. Allows the reader to grasp qufckly the essence of the wor1l: Shows the organizlllion of the text (not required ror stloft papersl Usts all the abbreviations used (but not those of SI, chemical elements. or standard chemical terms) Orientates the read8l". explains why the work has been done and its oontext in the literature. lMly the mothods used were chosen. Indicates the central hypothesis behind the experiments
institutions Abstr8CtJSumm&ly
List of Content. Abbreviations Introduction
Exp8l"imental
Explains how the wort was done. Shoutd contain sufficient detail to allow another competent VIIOn;cr to repeat the wol1;:
Resuhs
Displays and describes the data obtained. Shouk1 be presented in a form lNhich is easily assimilated {graphs rather than tables. small tables rather than large ones}
DiscussionJ Con<:lusions
Discusses the results: their meaning. their importance; compares the results with those of others; suggests what to do next Gives credit to those who helped carry out the work USIS aU references cited in appropriate format provides enough information to allow the reader to find the reference in a library
Acknowtedgements
Ut.-ature Cited (Bibliography)
Doos it eXl)lain what the text is about succinctly? Ase all the details correct?
Does it explain Why the worlc was done? Docs It outline the whole of your work and your findings?
Are all the sections covered?
Ate the page numbers correct? Have they all been explained? Are they aU in the lICCeJ)ted form 1 Are they in alphabetical order? Does it provide enough background Information and cite all
the reievant references? Is it of the oorrect depth tor the readership? Have all the technical terms been defined? Have you explained why you investigated the problem? Have you explained vour methodologlc81 approach to the problem? Is each experiment covered and have vou avoided unnecessary duptiCatlon? Is there suffICient detail to allow r'epelition of the work? Are the cormet names, sources and grades given for all chemicals? Is tt1e sequence of experiments logical? Are the parts adequate1v linked? Are the dala presented In the clearest possible way? Have 51 units been used property throughout? Has adequate ststjstical lInalysis been carried out? Is all the material relevant? Are the figUfes and tlIbies all numbered in Ihe order of their appearance? Are their tides appropriate? 00 the figure and table legends pn:wide all the Information necessary to i01m-pre! the datu without reference to the text? Have you presented the same data more than onea? Have you explained the significance of the results? Have you compared your data with other published work? Are your conclusions justified by the data presented? Have you listed everyone that helped, including any grantawarding bodies? Do all the references in the text appear on the lisl? Do alt the listed references appear 1n the text? Do the yeBrs of publications and authors match? Are the journal detlJils complete and in the rorrect format? Is.the Hst in alphabetical order, or correct numerical order?
Communicating information
333
Reporting practical and project work
Steps in the production of a practical report or thesis Cltom.e the expel';mellls }'OU wish to descrihe and decide how best 10 presellt them
Repeating your experimentsremember, if you do an experiment
twice. you have repeated it only once!
Try (0 stan this process before your lab work ends." bt:call<;C at tile stage of reviewing your experiments, a gap may become apparent (e,g. a missing control) and you might still have time to rectify the deficiency. Irrelevant material should be ruthlessly eliminated, at the same time bearing in mind that negative results can be extremely important. Use as many different fonns of data presentation as are appropriate, but avoid presenting the same data in more than one fonn. Graphs are generally easier for the reader to assimilate, while tables can be used to condense a lot of data into a small space. Relegate 1arb'C tables of data to an appendix and summarize the important points. Make sure that the experiments you describe are representative: always state the number of times they were repeated and how consistent your findings were.
Make lip plans 01' outlines for tlte component parts The overall plan is well defined (sec Box 52.1), but individual parts will need to be organized as with any other foml of writing (see Chapter 50).
Write! Presenting your results - remember that the order of results presented in a report need not correspond with the order in which yOU carried out the experiments: you are oxpected to rearrange them to I 'fOvide a IOLlical SO'lucnce of findifloJs.
The experimental section is often the easiest to write once you have decided what to report. There may be minor variations in style, but the one shown in Box 52.2 will be suitable in the majority of cases. The results section is the next easiest as it should only involve description. At this stage your should be jolting down ideas for the Discussion - tltis may be the hardest part to compose, as you need an overview of both your own work and the relevant literature, It is also liable to become wordy. so try hard to make it succinct. The Introduction should not be too difficult if you have understood fully the aims of the experiments. Write the Abstract and complete the list of references at the end. To assist with the latter, it is a good idea to use or pull out their cards from your index system. Rel';~'e
the text
Once your first draft is complete. try 10 answer all the questions given in Box 52.1. Show your work to your supervisors and learn from their comments. Let a friend or colleague who is unfamiliar with your subject read your text; hefshe may be able to pinpoint obscure wording and show where information or explanation is missing. If writing a thesis. double-check that you are adhering to your institution's thesis regulations.
Prepllre the finall'ersion Markers appreciate neatly produa.'d work but a well-presented document will not disguise poor science! If using a word processor. print the Iinal version with the best printer available. Make sure figures are clear and in the correct size and format.
Submit YOW' work Remember. there is no excuse for sloppy presentation if you use a word processor.
334
Communicaling information
Your department will specify when to submit a thesis or project report, so plan your work carefully to mf..'tt this deadline or you may lose marks, TeU your supervisor f..vdrly of any circumstances that may cause delay,
Reporting practical and project work
Box 52.2 Writing experimental procedures Th4J main purpose of the methods described in the" experimental section of a laboratory report, project report. thesis or paper is to communicate sufficient
usingnC ISiO l ~tes and CHlCI2 as eluent) and calculate the p9Jcentage yield of the product.'
information to allow an experienced chemist to repeat your experiments. One of the goals of writing laboratory reports is to provide you with practice in writing in
ExperimentBl A mixture of 2,4-dimethylbenzoic acid (3.0g, 0.02 mol).
the generally eccepted style required for more professional pvblications. The following points should
be noted: •
Always write in the third person, past tense.
•
Do not copy word for word the instructions gi....en in the experiment protocol. You ere in a leemlng situation In the laboratory and the protocol may contain infonTIation. which msy be new to you, but is general knowledge to experienced chemlsJs.
• •
Condensing the experimental protocol for yOur report will help you to see the important steps in the experiment. All sentences begin with a cepital letter not a number or bracket.
The following examples illustrate the differences between the instructions of the experiment protocol and the accepted style required for the experimental section. Example 1 A preparative experiment: the synthesis of methyl 2,4-dimethylbenzoate Experiment protocol 'In a round· bottom flask (100 mll place 2,4-dimethylbenzoic acid (3.0 g, 0.02 moll, anhydrous potassium
carbonate (3.3g, 0.024 mol) and a small magnetic flea. In tho fume cupboard aod Vllearing protective gloves, carefully weigh out dimethylsulphate (2.8g. O.022mol) into a glass sample tube (10 mW using a Pasteur pipette to transfer the liquid. Using the Pasteur pipette, transfer the dimethylsulphate to the reaction flask and use anhydrous propanone (10 mll to rinse the samp'e tUbe. Add the rinsings to the reaction flask and add more dry propanone (10mL). Fit the flask with a reflux condenser and boil the mixture under reflux for 3 hours using an oil bath an a stirrer hot plate in the fume cupboard. Allow the reaction mixture to cool to room temperature and pour into water (100ml) washing the reaction flask with a few millilitres of water. Extract the aqueous solution three times with dichloromethane 120 mLl. dry the dichloromethane (MgS04 1, filter off the drying agent. remove the solvent on the rotary evaporotor and record the weight of the crude product. Recrystallize aod charcoal the crude ester from ethanol and dry jn a vacuum desiccator. Record the weight and melting point of the purified ester and obtain and interpret infrared and lH_NMR spectra. Check the purrty of your product
dimethylsulphare (2.8 g, 0.022 mol) and anhydrous potassium carbonate (3.3g, 0.024 mol) in dry propanone (20 mW was boiled under reflux (311), cooled, poured into water (100mW and extracted with dichloromethane (3 x20mW. Removal of the dried (MgS0 4 ) organic satvent gave a white solid (2.3 g), which was recrystallized from ethanol, using ch8rcoal to improve the colour, to yield white needles of methyl 2,4-
la) the molar quantities of the reaetents used; fbi the number of moles of each reactant required to make 1 mole of product; (cl the assumption that reactions go 100%; (dl the 'Iimiting quantity' of one of the reactants. To calculate the theoratical yield of a reaction, It IS essential that you recognize the reactant, which is the limiting quantity. If a reaction requires 1 mole of reactant A 'and 1 mole of reactant B to give 1 mole of product AB. Le.:' A+B-AB
if we use A (1 mol) and B (1.5 mol), then only 1 mol of AB can be formed and there is 0.5 mol of B in excess
Communicating information
335
Reporting practical and project work
Box 52.2
(continued)
and unreacted. Therefore the limiting quantity is the amount of reactant A (1 mol). The excess of reactant B may be necessary to ensure complete conversion of A into AB, i.e. 100% reaction. In the example above, the limiting quantity is the amount of 2,4-dimethylbenzoic acid (0.02 mol) and only 0.02 mol of the methyl ester can be formed (164 x 0.02:c 3.28g). Therefore:
% ield = reaction yield x 100 = 2.28 x 100 = 70% Y
theory yield
3.28
Exampte 2 A quantitative experiment: standardization of sodium thiosulphate solution Experiment protocol 'Prepare a standard solution of potassium iodate by dissolving potassium iodate (about 1.34g, accurately weighed) in di!>1il1ed water (l00ml!, quantitatively trans· ferring the solution to a volumetric flask (250.00 mll making up to the mark using distilled water and mixing well. Pipette an aliquot (25.00 mll of the solution into a conical flask (250 ml) and add SUlphuric acid (50 mL 1 M). Weigh out potassium ioclide (1 gl, add it to the conical flask and swirl until it has all dissolved. Titrate the liberated iodine (brown solution) with the sodium thiosulphate solution until a pale straw colour is reached. Add iocline indicator (two drops) or freshly prepared starch solution (two drops) and continue the titration until the colour changes from blue-black to colourless. If the solution does not tum blue-black when you add the iodine indicator or starch, you have overshot the end-point. Repeat the experiment until consistent results are obtained. Calculate the molarity of the sodium thiosulphate solution: Experimental Potassium iodate (1.3402 g) was dissolved in distilled water (100 ml), transferred to a volumetric flask (250.00 ml) and made up to the mark using distilled water. An aliquot (25.00 mll was transferred to a conical flask (250 mll, SUlphuric acid (50 mL. 1 M) and potassium iodide (1 g) were added and the mixture swirled to effect solution. The iodine liberated was titrated with the sodium thiosulphate solution to a pale straw colour. Iocline indicator (two drops) was then added and the titration continued to a blue-black to colourless endpoint. The experiment was repeated until consistent results were obtained and the concentration of the sodium thiosulphate solution calculated. Note 1. The differences in accuracy, indicating the equipment you must use. are shown by the decimal point quoted in the quantities. Note 2. Titrations should be repeated to give consecutive results to within one or two drops of titrant. Do not average widely differing resuhs.
336
Communicating information
Note 3. Balance readings. burette readings and calculations should be included in the results section. Example 3 Reaction kinetics: determination of rate constant and energy of activation using titrimetry Experiment protocol 'The following solutions are provided: A. Potassium persulphate (0.04 M) B. Sodium thiosulphate (0.01 M) C. Potassium iodide (0.4 M). Place solution A (100 ml) and solution C (100 mL) into separate conical flasks and suspend them in a thermostat bath at 25 C. ii. Mix solutions A and C (50 ml of each) in a stoppered flask and immerse in hot water until the end of the experiment. iii. After about 15 minutes from step i, mix the solutions A and C and start the clock. This mixture must be kept in the thermostat bath at 25 'C, throughout the experiment. iv. Just before 2 minutes have elapsed, lake a sample (10.00 mll from the flask by pipette and add it to distilled water (200 mll in a separate flask. This quenches (stops) the reaction. v. Take further samples at 4, 6. 9, 13, 18, 24, 32, 44 and 60 minutes and quench in the same way. vi. Titrate each quenched solution with sodium thiosulphate, including a sample of the 'hot--water' sample. Use a small amount of freshly prepared starch indicator solution and titrate from blue to colourless. The titres for the samples are the values (7) and that for the 'hot-water" sample is T.,.,. vii. Now repeat the whole experiment using a thermostat bath set at 35-40°C. Make an accurate record of the temperature. You do not need to repeat the 'hot-water" sample since this will selVe for both experiments. Since this higher tempera·ture reaction is faster. there is no need to take samples at 44 and 60 minutes. viii. For each temperature plot a graph of -In{T'lC - T) versus time (s).' L
Experimental Solutions (100 ml) of potassium persulphate (0.04 M) and potassium iodide (0.4 M) were equilibrated (0.25 h) in a constant temperature bath (25 "C). The two solutions were mixed in a conical flask (250 ml) in the constant temperature bath and the clock started. After 2 minutes, an aliquot (10.00ml) of the mixture was quenched with distilled water (200 ml) and the sampling process repeated after 4, 6, 9, 13, 18, 24, 32, 44 and 60 minutes. Each sample was titrated with
Reporting practical and project work
Box 52.2
Icontinued)
sodium thiosulphate solution 10.01 M). using iodine indicator, to 8 colourless end-point and the values recorded (T). The experiment was then repeated at a known temperature between 35 C and 4() C but the samples at 44 and 60 minutes were not taken. Solutions (SO mll of the potassium persulphate and potassium iodide were mixed and kept in a water bath (60-70 Cl for 2 hours. An aliquot (10.COmli of this
Box 52.3
Steps in producing
a
solution was quenched in distilled water (200ml) and titrated (T... ) with the sodium thiosulphate solution. A graph of -In(Lx. - T) versus time (s) was plotted. Note 1. Only experimental detail is given. Calculations, results and theory are in the appropriate sections.
scientific paper
Scientific papers are the life-blood of any science and it is a major landmark in your scientific career to publish your first paper. The major steps in doing this should include the following. Assessing potential contsnt The work must be of an appropriate standard to be pUblished and should be 'new, true and meaningful'. Therefore, before starting, the authors need to review their work critically under these headings. The material included in a scientific paper will generally be a subset of the total work done during a project, so it must be carefUlly selected for relevance to a clear central hypothesis - if the authors won't prune. the referees and editors of the journal certainly willI Choosing a journal There are thousands of journals covering chemistry and each covers a specific area (which may change through timeJ_ The main factors in deciding on an appropriate journal are the range of subjects it covers, the Quality of its content and the number and geographical distribution of its readers. The choice of journal always dictates the format of 8 paper since authors must follow to the lener the journal's 'Instructions to Authors'. Deciding on authorship In multiauthor papers, a contentious issue is often who should appear as an author and in what order they should be cited. Where authors make an equal contribution. an alphabetical order of names may be used. Otherwise, each author should have made a substantial contribution to the paper and should be prepared to defend it in public. Ideally. the order of appearance will reflect the amount of work done rather than seniority. This may not happen in practicel
Writing The paper's format will be slmilarto that shown in Box 52.1 and the process of writing will include outlining, reviewing. etc.• as discussed elsewhere in this chapter. Figures must be finished to an appropriate standard and this may involve preparing photographs of them. Submrtting When completed. copies of the paper are submitted to the editor of the chosen journal with a simple covering letter. A delay of 1 to 2 months usually follows while the manuscript is sent to one or more anonymous referees who will be asked by the editor to check that the paper is novel. scientifically correct and that its length is fully justified. Responding to referees' comments
The editor vvill send on the referees' comments and the authors then have a chance to respond. The editor will decide on the basis of the comments and replies to them whether the paper should be published. Sometimes Quite heated correspondence can result if the authors and referees disagreel Checking proofs and waiting for publication If a paper is accepted, it will be sent off to the typeseners. The next the authors see of it is the proofs (first printed version in style of journal), which have to be corrected carefully for errors and returned, Eventually, the paper will appear in print. but a delay of 6 months following acceptance is not unusual. Most journals offer the authors reprints. which can be sent to other researchers in the field or to those who send in reprint request cards.
Communicating informarion
337
Reporting practical and project work
Producing a scientific paper Scientific papers are the means by which research findings are communicated to others. The)' are published in journals with a wide circulation among academics and are "peer reviewed' by one or more referees before being accepted. Each journal covers a well-defined subject area and publishes details of the format they expect. Jt would be very unusual for an undergrdduate to submit a paper on his/her ov..n - this would normally be done in collaboration with your project supervisor and only then if your research has satisfied appropriate criteria. However, it is important to understand the process whereby a paper comes into being (Box 52.3), as this can help you when interpreting the primary literature.
338
Communicating information
-------j8
Writing literature surveys and reviews The literature surveyor review is a specialized ronn of essay which summarizes and reviews the evidence and concepts concerning a particular area of research.
8(5%1
\
~ A literature review should not be a recitation of facts.
The best reviews are those which analyse information rather than simply describe it.
SIS%I 2(45%1
Making up a timetable 3 (15%1
Fig. 53.1
Pic chan showing how you might
allocate time for a literature survey: 1. select a topic;
Fig, 53.1 illustrales how you mighl divide up your lime for wntrng a literature survey. There arc many sub-divisions in Ihis chart because of the size of the task: in general, for lenglhy tasks, il is best to divide up Ihe work inlo manageable chunks. Notc also lhat proportionately less time is allocated to writing itself than with an essay. in a literdture survey, make sure that you spend adequate time on research and revision.
2. scan the literature; 3. plan the review; 4. write first draft;
5. leave to settle; 6. structure-review the text; 7. write final draft; 8. produce top copy.
Selecting a topic You may have no choice in the lopic 10 be covered. but if you do. carry out your sclection as a thn."'C-stage process: I. 2.
3.
Identify a broad subject area that interests you. Find and read relevant literature in that area. Try 10 gain a broad impression of the field from books and general review articles. Discuss your ideas with your supervisor. Select a relevant and concise title. The wording should be considered very carefully as it will define the content cxpected by the reader. A narrow subject area will cut down on the amount of literature you will be expected to review, but will also restrict the scope of the conclusions you can make (and vice versa for a wide subject area).
Scanning the literature and organizing your references You will need to carry out a thorough investigation of the lilerdture before you start to write. The key problems are as follows: •
Using index cards lsee p. 319) - these can help you organize large numbers of references. Write key points on each card - this helps when considering where the reference fits into the literature. Arrange the cards in subject piles. eliminating irrelevant ones. Order the cards in the sequence you wish to write in.
•
•
Gelling an initial toe-hold in the literature. Seek help from your supervL~or, who may be willing to supply a few key papers to get you started. Hints on expanding your collection of references are given on p.317. Assessing the relevance and vdlue of C'dch article. This is the essence of writing :'I review, but il is difficult unless you alre:'ldy have a good understanding of the field (Catch 22!). Try reading C'drlier reviews in your area. Clarifying your thoughts. Sometimes you can't see the wood for Ihe trees! Sub-dividing the main topic and assigning your references to these smaller subject areas may help you gain a better overview of lhe litemturc. Communicating information
339
Writing literature surveys and reviews
Deciding on structure and content The gcncral structure and content of a litcrature survcy arc described below.
IIltrOl!uctioll Thc introduction should givc the gcllcml background to the research area, concentrating on its development and important"t. You should also make a statement about the scope of your survey; as wcll as defining the subject mattcr to be discussed, you Inay wish to restrict lhe period being considered.
Main holly of text The rcview itself should discuss the published work in thc selected field and may be sub-divided into appropriatc sections. Within each portion of a review. the approach is usually chronolog;cal, with appropriatc linking phrases (e.g. 'Following on from this... .'; 'MC'
•
• •
allow the reader 10 obtain an overall view of thc current slalc of the research area. identifying Ihe key areas where knowledge is advandng; show how techniques arc de\'Cioping and discuss the benefits and disadvantages of using particular chemicals or cxperimental systems; assess the relative wonh of dincrcnt types of eviden~ - this is the most important aspecl: do not be intimidated from taking a critical approach as the conclusions you may n:-
Conclusions 11tc conclusions should draw tOb'Cther the thfC'dds of the preceding parts and
point thc way forward, perhaps listing an.-
Reference!)' etc. Making citations - a review of literature poses stYlistic pmblems because of the need to elte large numbers of papers; in Chemical Society Reviews this is
overcome bV using numbered references (see
The references or litcratun: dted section should pro\~dc full details of all papcrs referred 10 in the lext (see p. 320). The regulations lor your department may also specify a formal and position for the title page, list of contents, acknowledgements. ctc.
p. 320).
Style of literature surveys 'rbc Chemical Socicty Review series (available in most university libraries) provides good examples of appropriatc style for rcviews of the chemical scicnces.
340
Communicating infonnation
--------18
Organizing a poster display A scientific JX>Stcr is a visual display of the results of an investigation. usually maunled on a rectangular board. Posters ,ire used at scientific meetings. to communiC"
In a writlcn report you can include a reasonable amount of specific detail and the reader C"dn go back and reread difficull passages. However. if a poster is long winded or contains too much detail, your reader is likely to lose interest.
~ A poster session is like 8 competition - you are com-
peting for the attention of people in a room. Because you need to attract and hold the attention of your audience, make your poster as interesting as pOS5ib1e. Think of h: as an advertisement for your work and you will not go far wrong.
Preliminaries
•
,., ,
,
•
•
Before considering the content of your poster. you should find out: • •
• •
,
,
•
• ,
''I 2
,.,
the linear dimensions of your poster area. typically up to 1.5 m wide by 1.0m high; the composition of the poster board and the method of atlllchment, whether dmwing pins. Velcro'" tape. or some other form of adhesive; llnd whether these will be provided (in any case, it's safer to bring your own): the timc(s) when the jX)ster should be set up and when you should attend; the room where the poster .session will be held.
Design
.J
::J
Fig.54.1 Poster design. (al An uninspiring design: sUb-uoits of eQual area, reading left to right, are not recommended. (bl This design is more interesting and the text will be easier to
rcad lcolumn format). lei An alternative approach, with a central focus and arrows! tapes to guide the reader.
Plan your poster with your audience in mind, as this will dictate the appropriate level for your presentation. Aim to make your poster as accessible as jX)ssiblc to a broad audience. Since a poster is a visual display, you must pay particular attention to the presentation of information: work that may ha\'C laken hours to prepare can be ruined in a few minutes by the ill-considered arrangement of items (Fig. 54.1). Begin by making a drat) sketch of the major clements of your jX)ster. It is worth discussing your intended design with someone else. as constructive advice at the dmft stage will saw a lot of time and effort when you prepare the final version (or consult Simmonds and Reynolds. 1994).
Layout Usually the best approach is to divide the poster into several smaller areas, perhaps six or eight in all, and prepare each as a separate item on a piece of thick carel. Some people prefer to produce a single large poster on one sheet of paper or card and store it inside a protecti~ cardboard tube. However, a sin~ large poster will bend and CfC".tse. making it diflkult to nallen oul. In addition, photographs and text attached to the backing sheel oftcn work
'0=. Sub-dividing your postcr means that each smaller area can be prepared on
a separate piece of paper or card, of A4 s.ize or slightly larger. lrul.king Communicating information
341
Organizing a poster display
transport and storage easier. It also breaks the reading matter up imo smaller pieces. looking less fonnidablc 10 a potemial reader. By using pieces of card of different colours you C"dn provide emphasis for key aspects, or link text with figures or photographs. You will need to guide your readcr through the poster. It is often appropriate to use either a numbering systcm, with large. c1eltr numbers al the top of each p~ of card, or a systcm of arrows (or thin tajX$), to show the relationship of sections within the poster (sec Fig. 54.1). Make sure that the relationship is clear and that the arrows or tapes do not cros.'>.
Title Your ehosen titk: should be coneise (no more than cight words). specific and interesting, to encourdge people to read thc poster. Makc the title large and bold - it should run across the top of your poster. in letters at least 4em high, so that it can be read from the other side of the room. Coloured spiritbased marker and block capitals dr,nvn with a ruler work well. as long as your writing is readable and neat (the colour can be uS<.'d to add emphasis). Altcrnatively. you C"dn use Lctrasct ". or similar lettering. Details of authors. together with their addresses (if appropriate), should be given, usually in the top right-hand comer in somewhat smaller lettering than the title. At conferences, a passport-sized photogmph of the contributor is sometimes useful for identifiC"dtion.
Text Making up your poster - text and graphics printed on good-quality paper can be glued directtv' onto a contrasting mounting card: use photographic spray mountant or Pritt I< rather than liquid glue. Trim carefully using a guillotine to give equal margins, parallel with the paper. Photographs should be placed in a window mount to avoid the tendency for their corners to curl. Another approach is to trim pageS Of" photographs to their correct size, then encapsulate in plastic film: this gives a highly professional finish and is less weighty to transport.
Keep text to a minimum - aim 10 ha\'t: a maximum of 500 words in your poster. Write in short senl(:nces and avoid verbosity. Keep your poster as visual as possible and make efTcctive usc of the spaces between the blocks of text. Your final text should be double spaced and should have a minimum C"dpitalletler height of 5-10mm, prefembly greater. so that the poster can be read at a distance of I tn. One method of obtaining text ofthc required size is to photo-enlarge standard typescript (using a good-quality photocopier). or use a high-quality (laser) printer. It is best to avoid cominuous use of text in capitals.. since it slows reading and makes the text less interesting to the TC"dder. Also avoid italic.. 'balloon' or d(:comtive stylL'S of lettering. Sllb-title~'
and "eading~'
These should havc a capilal Ieltcr height of 12-20mm. and should be restricted to two or three words. They can be produced by photo-cnltlrgement, by stencilling., Letroset ~ or by hand. using pencilled guidelines (but make sure that no pencil marks are visible on your finished poster).
Colour Consider Ihe ovemll visual effect of your chosen display, including the relationship between text. diagrdms and the backing board. Colour can be used to highlight key aspects of your poster. HO\':ever. it is very C'"dSy lO ruin a postCT by the inappropriate choice and application of colour. Careful use of two, or at most three. complementary colours will be C'dsier on the eye and may aid comprehension. Colour can be used to link the texl with the visual images (e.g. by pickin!; out a colour in a photograph and using the same colour on the mounting board for the aa:ompanying text). Use colourt'd inks or water-based paints to provide colour in diagrams and ligures. as felt pens rarely give satisfactory results. 342
Communicating information
Organizing a poster display
Content 1ltc typical farom! is thai of a scientific report (sec Box 52.1), Le. with lhe same he'd-dings, but with a considerably reduced COntent. Never be tempted to spend the minimum amount of time converting a piece of scientific writing into poster formal. At scientific mcctings, the least interesting posters arc those where the author simply displays pages from a written communication (e.g. a journal article) on the poSler board! Keep references within the text to a minimum - interested patties C'dll always ask you for further infomlation.
I"trot!llt:t;o" This should give the fC"ddcr background information on the broad field of study and the aims of your own work. It is vital that this sectioll is as interesting as possible, to capture the intel'C!it of your audience. It is often worth listing your objectives as a series of numbered points. Deslgnll"!(l the materials and methods .section':' photographs or diagrams of apParatus can help to break up the text of the experimental section and provide visual interest. It is sometimes worth preparing this section In e smaller typeface.
Keeping graphs and diagrams simpleavoid composite graphs with different scales fur the same axis. or with several trend lines (use 8 maximum of three trend tines per graph).
Exper;melltal Kcc.:p this short• .and describe only the principal techniques u~. You might mcntion nny speci.al techniques. or problems of general imeCt."Sl. Reslllt!,; Don't present your raw data: usc data roouClion wherever poAAiblc, i,e. figures and simple statistical comparisons. GrAphs, diagrams. histograms and pic charts give clc-df visual imagt:s of trends and relationships and should be used in place of tabulated data (sec p. 251). Final copies of all figuft,"S should be produced so that the numbers can be read from a distance of I m. Each should have a conci~ title and legend. so that it is self-contained: if appropriate. a series of numbcred points c.tn be used to link a diagram with the accompanying text. Where symbols arc used, provide a key on each gr.tph (symbol size should be at Ic-dSt 5mm). Avoid using gr.tphs straight from a wrillen version. e.g. a project report. textbook or a paper, without considering whether they need modification to meet your requirements.
COllcl"s;OllS Listing your concJustons - a series of
numl:!ered -pOints.is 8 useful appfoach, if your findings fit this pattern.
Ths is where many readers will begin. and they may go no further unless you make this SL'Ction sufficiently interesting. This section needs to be th~ strongest part of your poster. Refer to your figures h(:re to draw the reader into the main part of your poster. A slightly larger or bolder ty!=Cfacc may add emphasis. though too many different typefaces call look m<."Ssy.
The poster session
Consider providing a handout - this is a useful way to summarize the main points of yOUr po6ter. 80 that your readers have a permanent record of the information you have presented.
If you stand at the side of your POSI<-'f throughout the session, you arc likely to discoumgc some readers. who may not wish to bocomc involved ill a detailed conversation about the poster. Stand n<''arby. Find something to dotalk to someone else. or browse among the other posters. but remain aware of people rC'dding your pmtcr and be ready to answer any queries they may raise. Do not be 100 discouraged if you aren't asked lots of questions: remember, the poster is meant [0 be a self-contained, visual story, without need for further explanation. A poster display will never fed like an oral presemation, where the nervousness before.hand is replaced by a combination of satisfaction and relief as you unwind after the event. However, it can be a vcry satisfying means of communication, particularly if you follow these guidelines. Communicating intormation
343
------------jG
Giving an oral presentation Most students feel vcry nervous about giving talks. This is natural, since vcry
few people arc sufficiently confident and outgoing thai lhey look forward to speaking in public. Additionally, the technical nalure of your subject matter may give you cause for concern, especially if you feel that some members of the audience have a ~tcr knowledge Ihan you have. Ho\\,-ever. this is a fundamental method of scientific communic-dlion and it therefore forms an important component of many courses. The comments in this chaptcr apply equally to informal tnlks, e.g. those based on assignments and project work, and to more formal conference presentations. II is hoped that the advice and guidance given below will cncoumge you to make the most of your opportunities for public speaking. but there is no substitute for practice. Do not expert to find all of the ansv.'Crs from this, or any other, book. Rehearse, and leam from your own expcnen~.
~ The three 'Rs' of successful public speaking are:
Reflect - give sufficient thought to all aspects of your presentation, particularly at the planning stage. Rehearse - to improve your delivery. Rewrite - modify the content and style of your material in response to your own ideas and to the comments of others.
Preparation learning from experience - use your own experience of good and bad lecturen;; to shape your performance. Some of the
more common errors include: • •
•
,-
speaking too quickly reading to notes and ignoring the audience
unexpressive. impersonal or indistinct
• distracting mannerisms • poorly stn.IctUred material with little emphasis on key information
•
•
factual information too complex and detailed too few visual aids.
P"elim;nar)' ilifol"nlUtioll Begin by marshalling all of the details you need to plan your presentation, including: • • • •
the dumtion of the talk; whether time for questions is included; the size and location of the room; the projection/lighting facilities providt:d, and whether pointers or similar aids are available.
It is especially important to find out whether the room has the necessary equipment for slide projection (slide projector and screen, black-out curtains or blinds, appropriate lighting) or o\'Crhcad projection before you prepare your audiovisual aids. If you concentf'.l.te only on the spoken part of your presentation at this stage, you are inviting trouble later on. Have a look around the room and try out the equipment at the earliest opportunity, so that you are able to usc the lights, projector, etc.. with confidence.
AlldiO)';SIlUI aids
Find out whether your department has facilities for preparing ovcrhead transparencies and slides, whether these facilities are available for your use and the cost of materials. Adopt the following guidelines: • • • • • 344
Communicating information
Keep lext to a minimum: present only th~ kcy points, with up to 20 words per slide/lransparency. Make sure the text is l'C'.l.dable: try out your material beforehand. Usc scveral simpler figures rathcr than a s.ingle complex graph. Avoid too much colour on overllC'.l.d transparencies: blue and black are easier to l'C".l.d than red or green. Don't mix slides and transparencies as this is often distracting.
Giving an oral presentation
• •
Usc spirit~bascd pens for transparencies; usc alcohol for corrections, Transparencies can be produced from Iypewrinen or printed text using a photocopier, often giving a bencr product than pens. Note that you must usc special heat-resistant acetate sheets for photocopying.
Alldience You should consider your audience at the earliest slage, since they will determinc the appropriate levd for your presentation. If you are talking to fellow studcnts you may be able to assume a cornman level of background knowledge. In contrast. a research lecture given 10 your dt.:partment, or a paper at a meeting of a scientifIC socicty. will be presented to an audience from a broader range of backgrounds. An oml presentation is not the place for a complex discussion of specialized information: build up your talk from a low Icvel. The spct:d al which this am be done will vary according to your audience. As long as you are nOI boring 01' patronizing. you can cover basic infomlation without losing the :H1ention of the marc knowledge-dble members in your audicnce:. Thc gencrdl rule should be: 'do not ovcrestimate the background knowledge of your audience'. This sometimes happcn~ in slUdent presentations. where fears about the presence of 'experts' can encourage thc speaker to include too much detail. overloading the audience with facts.
Content While the specific details in your talk will be for you to decide, most oral presentations share some common features of structure. as described below.
Jlltrot!,lctory remarks It is vital to capture the intcrest of your audience at the outset. Consequently, you must make sure your opening comments are strong. otherwise your audience will lose interest before you reach the main message. Remember it lakes a sentence or two for :m audience to establish a relationship wilh a ncw speaker. Your opening sentence should be some fonn of preamble and should not CQntain any key information. For a fonnal lecture, you might begin with 'Mr Qairman, ladies and gentlemen, my talk today is about ...' then restate the title and acknowledge other CQntributors etc. You might show a tmnsparency or slide with thc title printed on it. or an introductory photograph. if appropriate. Thi.s should provide the necessary settling-in period. After these preliminaries. you should introduce your lOpic. Begin your story on a strong note- this is no plalX for timid or apologetic phmses. You should:
• • •
explain the structure of your talk; set out the aims and objectives of your work; explain your approach to the topic.
Opening remarks are unlikely to occupy more than 10% of the talk. However, because ofthcir significance. you might re'.lsonably sp;:nd up to 25% of your prcpamtion time on them. Make sure you have practised Ihis section, so that you can deliver thc matcrial in a nowing style, with less chance of mistakes.
Tile mtu'" menage 11\is section should include the bulk of your experimental results or litemlure findings, depending on the type of presentation. Keep details of methods to t~ minimum needed to explain your dala. This is nol the place for a detailed description of equipment and experimental protocol (unless it is a talk about methodology!). Results should be presented in an easily digested format. Communicating information
345
Giving an oral presentation
Allowing time for slides - as a rough guide you should allow at least 2 minutes per illustration, although some diagrams may need longer, depending on content.
•
Final remarks - make sure you give the audience sufficient time to assimilate your final slide: some of them may wish to write down the key points. Alternatively, you might provide a handout, with a brief outline of the aims of your study and the major conclusions.
Do not eXJX.'Ct your audience to cope wilh large amounts of data; use a maximum of sill numbers per slide. Prescnt summary statistics rather than individual results. Show the final results of any analyses in terms of the statistics calculated, and their significance (p. 271), rather than dweUing on details of the procedures used. Remember that graphs and diagrams are usually better than tables of raw data, since the audiencc will be able 10 sec the trends and relationships in your data (p. 251). However, figures should not be crowded with unnecessary detail. Every diagram should have a concise title and the symbols and trend lines should be clearly labelled. with an explanatory key where necessary. When presenting graphical data always 'introduce' each graph by stating the units for each axis and describing the relationship for each trend line or data set. Summary slides can be used at regular intervals, to maintain the now of the presentation and to emphasize the key points. Take the audience through your story step by step at a reasonable pace. Try not to rush the delivery of your main message owing to nervousness. Avoid complex. convoluted story-lines - one of the most distracting things you can do is to fumble backwards through slidcs or overhead transparencies. If you need to usc the same diagram or graph more than once then you should make two (or more) copies. In a presentation of experimental results, you should discuss each point as it is raised. in contrast to written text, where the results and discussion may be in scparate sections. The main message typiCilly occupies approximately 80% oftre time allOC
Concluding rema,.ks Having captured the interest of your audience in the introduction and given them the dew.ils of your story in the middle section, you must nOw bring your talk to a conclusion. At all costs.. do not end weakly, e.g. by running out of steam on the last slide. Provide your audience with a clear 'take-home mcssage', by returning to the key points in your presentation. 11 is often appropriate to prepare a slide or overhead transparency listing your main conclusions as a numbered series. Signal lhe end of your talk by saying 'finally .. :, 'in conclusion ...., or 11 similar comment and then fini<;h speaking after that sentence. Your audience will lose interest if you extend your closing remarks beyond this point. You may add a simple end phrase (e.g. 'thank you') as you put your noles into your folder, but do nol say 'that's all folks!', or make any similar ofihand remark. Finish as strongly and as clearly as you started.
Hints on presentation Notes Many a<.'COmplished speakers usc abbreviat<.'(\ notcs for guidance. rather than reading from a prepared script. When writing your talk: •
•
•
34ti
Communicating information
Prepare a first draft as a full script: write in spoken English, keeping the text simple and avoiding an impersonal style. Aim to talk to your audience, not read to them. Usc note cards with key phrases and words: it is best to avoid using a fuU script at the final presentation. As you rehearse and your confidence improves. a set of cards may be a more appropriate format ror your notes. Consider the structure of your talk: keep it as simple as possible and announce each sub-division. so your audience is aWClre of the struclure.
Giving an oral presentation
• • •
• Using slides - check that the lecture theatre has a lectern light. otherwise you may have protHems reading your notes when the lights are dimmed.
•
Mark lhe posItion of slides/kcy points. elc.: each notc card should contain dctails of structure. as wcll as contcnt. Mcmorize your introductory/closing remarks: you may prefer to rely on a full wriuen version for these SI.'Ctions, in case your mcmory fails. Usc notes: writc on only one side of the cardfpaper, in handwriting Inrge cnough to be read easily during the presentation. Each card or sheet must be clearly numbered, so that you do llot lose your placc. Rehearse your prtSCntation: ask a friend 10 listCfl and to comment constructively on pans that werc difficult to follow. Use 'split timcs' to pace yourself: following rchcarsal. note the time at which you should arrive at key points of your lalk. These timing marks will help you keep to timc during the 'real thing'.
Image Ensure that the image you project is appropriate for the occasion: • • • •
•
•
•
•
Consider what to wear: aim to be respectable without 'dressing up', otherwise your message may be diminished. Develop a good posture: il will help your voice projection if you stand upright, rather than slouching or leaning over the k-acrn. Project your voice: speak towards the back of the room. Make eye contact: look al members of the audience in all pariS of Ihe room. Avoid talking to your notes, or 10 only OIlC section of Ihc audience. Dcli\'Cr your material with expression: arm movements and subdued body language will hclp maintain the interest of your audiencc. However, you should avoid extreme gestures (il may work for some TV personalities, but it isn't recommended for the bcginner!). Manage your time: avoid looking at your walch as il gives a negative signal 10 the audience. Use a wall clock, if one is provided. or take 01T your wateh and put it beside your notes, so you can glance at it without distracting your audience. Try to idcntify and control any distracting repetitive mannerisms. e.g. repeated empty phrases. fidgeting with pens, keys, etc., as this will distract your audiencc. Practising in front of a mirror may help. Practise your delivery: use the comments of your friends to improve your perfonnance.
Questiolls Many speakers are worried by the prospect of questions after their oml presentation. Once again, the best approach is to prepare beforehand: • •
•
•
Consider what questions you may be asked: prepare brief answers. Do nOI be afraid to say 'I dOll't know': your audience will appreciate honesty, rather than vacillation, if you don't have an answer for a particular question. Avoid arguing with a questioner: suggest a discussion afterwards rather than becoming involved in a debate about specific details. If no questions are forthcoming you may pose a quc;tion yourself. and then ask for opinions from the audience: if you use this approach you should be prepared 10 comment bricny if your audience has no suggestions. This will prevent the presentation from ending in an em-barrassing silence.
Communicating information
347
---------.,8
Examinations You are unlikely 10 have rc
Information gathering Examples include:
3
Commonly used abbreviations
there are, there exist's)
~ To
do well in an examination, you need to put in effective WOf"k long before you go into the examination hall and even before you start to revise. Vou need to base your revision on accurate, tidy notes with an appropriate amount of subject detail and depth.
therefore
because Is proportional to leads to, into comes from, from involves several processes in a
DC
---..-.
seQuence
1·,2 ~. ~
primary, secondary (etc.) approximately, roughly equal to
=,
'I'
equals, not equal to
~.
'1-
equivalent not equivalent to
<, >
smaller than, bigger than
IX)
much bigger than concentration of X
L:
sum
f #
function
00
infinity. infinite
»
number
You should also make up your own
abbreviations retevant to the context. e.g. if a lecturer is talking about resonancestabilized carbocations, you could write 'RSC~'
instead.
Taking notes/rom lectures Taking good lecture notes is essential if you are to make sense of them later. St~rt by noting the date, course, 10pic ~nd lecturer. Number every page in C
~ - c.k.d>"ooI r'" ~"""""""J'~--4S~<:r~c
-"~tf"'f-~"'"'"I~NP ~------",~&-"*~",,,
/?
/"
':,.,2
\ l-1, 2}1'<1
jJlN"~'~~~~
348
Communicating informalion
"""-J,
I
-"",",," -
of ~
,,~
-~
-~~
An example of 'pattern' notes, an alternative to the more commonly 'Iinear' format.
Fig. 56.1
used
'" swrn s,..~fiool(;
Examinations
'Making up' your notes As soon as possiblc afier the lecture., work through your notes. tidying them up and adding detllil where llecessary. Add emphasis [0 any headings you have made. so that the structure is clearer. Compare your notes with material in a textbook and correct any inconsistencies. Make notes from. or photocopy, any useful material you sec in textbooks. ready for revision.
Skimming texts
Seeing sequences - writers often number their points (firstly. secondly. thirdly, etc.) and lOOking for these words in the text can help you skim it Quickly.
This is a valuable means of gaining the maximum amount of information in the minimum amount oftimc. by reading as linle ofa text as is required. It can be used to decide what parts to rc.'1d in detail and to make notes of, perhaps when you have already read the text ill detail at some point in the past. Essentially, the technique requires you to look at the struCture of the text. rather than the detail. In a sense. you arc trying to sec the writer's origin"l plan and the purpose behind each part of the text. Look through the whole of the piece first. to gain an overview of its scope and structure. HCl.ldings provide all obvious clue to structure, if present. Next, look for the 'topic sentcnce' in each paragraph, which is ollen the first. You might then decide that the paragraph contains a definition that is important to note. or it may contain examplcs. SO rn.'1y not be worth readilll; for your purpose.
Preparing for an exam Begin by finding out as much as you can about the exam, including: • • • • • •
its format and duration; the date and location; the types of question; whether any qucstions/SCL1ions are compulsory; whether the questions are internally or externally sct or assessed: whether calculators are required.
Your course tutor is likely to give you details ofeX3m structure and timing well beforehand. so that you can plan your revision: the eourse handbook and past papers (if available) can provide further useful details. OIcck with your tutor that the nature of the exam has nol changed before you consult past papers.
Organizing and using lecture notes. assignments and practical reports Given their importance as a source of material for revision. you should have sorted out any deficiencies or omissions in lecture notes/pral-1ical reports al an early stage. For example. you may have missed a lecture or practical due to iUness etc., but the exam is likely to assume attendance throughout the year. Make sure you attend classes whenever possible and keep your notes up to date. Your practical reports and any assignment work will oontain specific comments from the teaching stafT, indicating where marks were lost, c.:orrections. mistakes. inadequacies, etc. It is always worth reading these oommcnts as soon as your work is returned, to improve the standard of your subsequcnt reports. If you are unsure about why you lost marks in an assignl1lCnt. or about some particular aspects of a topic. ask the appropriate member of staff for further explanation. Most lecturers are quite happy to discuss such details with students on a one-to-one basis and this information may provkie you with 'c1ucs' to the expectations of individual lecturers that Communicating information
349
Examinations
may be useful in exams set by the same members of starr. However, you should nel'er 'fish' for specific infonnation on possible exam questions, as this is likely to be counter-productive.
Revision Begin your revision early, to avoid last-minute pi.mic. Start in earnest about 6 weeks beforehand: •
• • • •
Prepare a revision timetable - an 'action plan' that gives details of specific topics to be covered. Find out at an early stage when (and where) your exams arc to be held. and plan your revision around this. Try to keep to your timetable. Time management during this period is as important as keeping to time during the exam il<;elf. Remember, your concentration span is limited to 15-20 min: make sure you ha\'e two or three short (5min) breaks during each hour of revision. Make your revision as active and interesting as possible: the least productive approach is simply 10 read and reread your notes. Include recreation within your schedule: there is liUle point in tiring yourself wilh too much revision, as this is unlikely to be profitable. Ease back on the revision nC
Active revision The following techniques may prove useful in devising an active revision strategy: •
• • Preparing for an exam - make a checklist of the items you'll need (e.g. pens. pendls, sharpener and eraser. ruler, calculator, paper tissues, watch).
•
• • • • •
Final preparations - try to get 8 good night's sleep before an exam. last-minute cramming will be counter-productive if you are too tired during the exam.
Prepare revision sheets with details for a particular topic on a single sheet of paper, arranged as a numbered checklist. Wall posters arc another useful revision aid. Memorize definitions and key phrases: definitions can be a useful starting point for many exam answers. Use mnemonics and acronyms 10 (;ommit specific factual information to memory. The dafter they are, the beUer they work! Prepare answers to past or hypothetiC<\1 questions., e.g. write essays or work through C<\lculations and problems, within appropriate time limits. However, you should 110l rely on 'question spotting': this is a risky practice! Use spider diagrams as a means of testing your powers of recall on a particular topic (p. 326). Try recitation as an alternative to written recall. Draw diagrams from memory: make sure you can label them funy. Fonn a revision group to share ideas and discuss topics with other students. Usc a variety of dilTercnt approaches to avoid boredom during revision (c.g. record infonnation on audio tape, ll"e cartoons, or any other method, as long as it's not just reading notes!).
The evening before your exam should be sp:nt in consolidating your material, and checking through summary lists and plans. Avoid introoucing new material at this late stage: your aim should be to boost your confidence. putting yourself in the right frame of mind for the exam itself.
The examination On the day of the exam. give yourself sufficient time to arrive at the correct room, without the risk of being late (e.g. what jfyour bus breaks down?). 350
Communicating informmion
Examinations
Tlte exam paper Begin by reading the instructions at the top of the exalll paper carefully, so that you do not make any errors based on lack of understanding. Make sure that you know: • • • • • •
Using the exam paper - unless this is specifically forbidden. you should write on the question paper to plan your strategy, keep to time and organize your answers.
efficlelll use 01_
... Fig. 56.2 EX;lm m;lrks as a function of time. The marks awardod in a single answer will follow the law of diminishing returns - it will be filr more difficuh to achieve the final 25% of the available marks than the initial 25%. Do oot spend too long on anyone question.
how many questions are set; how many must be answerOO; whether the paper is divided into sections: whether any parts are compulsory; what each question/section is worlh, as a proportion of the total mark; whclher dilTerent questions should be answered ill difTerent books.
If you are unsure 3.oout anything. ask! - the e3.sicsl W3.y to lose marks in 3.n exam is to answer the wrong number of questions. or to answer a dilTercnl question from the one set by the examiner. Underline Ihe key phr.l.~ in Ihe instructions, to reinforce their message. Next, read through the set of questions. If there is a choice. decide on those questions to be answered and decide on the order in which you will taekle them. Prepare a timetable which takes into account Ihe amount of time required 1,0 complete each questioo and which renccts the allocation of marks - there is little point in spending one-quarter of the ex.am period on a question worth only 5% of the lotal marks! Use the exam paper to mark the sequence in which the questions will be answered and write Ihe finishing times alongside: refer to this timetable during the exam 10 keep yourself on course. Do not be tempted to spend too long on anyone question: the return in tenns of marks will not justify the los.o;; of time from olher questions (see Fig. 56.2). Take the first IOmin or so to read the papcr and plan your slralegy, before you begin writing. Do not be pul off by those who begin immediately; it is almost certain they arc producing unplanned work of a poor standard. PrOJ'iding QIlSll'crs Before you tackle a partiaJlar question, you must be sure of what is required in your answer. Ask yourself 'What is the examiner looking for in Ihis particular question?' and then SCt about providinl; a relerallf answer. Consider each individual word in the question and highlight. underline or cirele the key words. Make sure you know the meaning of the ternlS given in Table 51.1 (p. 330) so that you can provide the appropriate information, where necessary. Refer back to the qua;lioo as you write. to confinn that you arc keeping 10 the subit.''Ct matter. Box 56.1 gives advkc on writing essays under exam COIlditions. It is usually a good idea to begin with the question that you arc most confident aboul. Tins will reassure you before tackling more dimcult parts of the paper. If you run out of time, write in note fonn. Examiners arc usually understanding, as long as the main components of the question have been addressed and the intended structure of the answer is clear.
Tlte filial stage At the end of the exam, you should allow at least IOmin to read through your script. to ChlXk for: • •
grammatical and spclling errors; mathematical errors. Communicating infonnalion
351
Examinations
Box 56.1
Writing under exam conditions
Never go into an exam without a strategy for managing the available time.
• Allocate some time (say 5% of the totall to consider which questions to answer and in which order. • Share the rest of the time among the questions. Aim to optimize the marks obtained. A potentially good answer should be allocated slightly more time than one you don't feel so happy about. However, don't concentrate on anyone answer (see Fig. 56.2). • For each question divide the time into planning. writing and revision ph__ (see p. 3081. Employ time-saving techniques as much as possible. • Use spider diagrams (p. 326) to organize and plan your answer. • Use diagrams and tables to save time in making difficult and lengthy explanations.
After the exam - try to avoid becoming involved in prolOl'lQed analyses with other students over the 'idear answers to the questions; after all, it is too late to change anything at this stage. Go for a walk, watch TV for a white, or do something else that helpS you relax, so that you are ready to face the next exam with confidence.
• Use abbt'evia1tons to save time repeating text but always explain them at the first point of use. • Consider speed of writing and neatness especially when selecting the type of pen to use - ball-point pens are fastest but they can smudge. You can only gain marks if the eKaminer can read your script' • Keep your answer simple and to the point, with clear explanations of yOur reasoning. Make sure your answer is relevant. • Don't include irrelevant facts just because you memori:z:ed them during revision as this may do you mOre harm than good. You must answer the specifIC question that has been set. • TIme taken to write irrelevant material is time lost from anoth8f question.
Makc sure your namc is on each exam book and on all orner sheets of paper. including graph paper. even if securely attached to your script. as it is in your interest to ensure that your work does not go astray. Nel'(!r leave any cxam carly. Most exams assess work carrictl out over SC\'cral monrns in a timc period of 2-3h and there is always something constructi,
Practical exams: special considerations Thc prospect of a practical examination may cause you more concern than a theory exam. This may be due to a limited experience of p,.dCtical examinations, or the fact that practical and observational skills arc tested, as ".e11 as recall, description and analysis of factual information. Your first thoughts may be that it is not possible to preparc for a practical exam but. in fact. you can improvc )'our pcnormancc by maste.ring the various practical techniques described in this book. The principal types of question you are likely to encounter include: • • •
•
• •
352
Communicating information
Manipulative exercises. onen based on work carried out as part of your practical course (e.g. recrystallization, p. 92). Illlcrpretation of spectra - either provided b)' the cxaminer or obtained during the practical examination. Numcrical exercises, including the preparation of aqueous solutions at particular conccntrations (p. 15) and statistical exercises (p. 271). General advice is given in Chapter 39. Data analysis. induding the prcparahon and interpretation of graphs (p. 251) and numcric-d1 information, from data either obtained during the exam or provided by the examincr. Preparativc exercises - making a compound which will be a~ for yield and purity. Analytical excrcise:,; where the composition of an unknown chemical must be discovered and quantified.
Examinations
Practical reports You may be allowed to take your laboratory rcJXlrts and other texts into the practical exam. Don't assume that tllis is a soO option, or that revision is unn~s.uy: you will !lot have time to read large sections of your reports or to familiarize yourself with basic principles etc. The main advantah'C of 'opcnbook' exams is that you can check specific details of methodology, reducing your reliancc on memory, provided you know your way around your practical manual. In all other respects, your revision and preparation for such exams should be similar to theory exams. Make sure you arc familiar with all of the practical exercises, including any work carried out in class by your partner (since exams are assessed on individual performancc). Check with the teaching staff to sec whether you can be given access to the laboratory, to complete any exercises that you have missed.
The practical exam At the outset, detennine or decide on the order in which you will tackle the questions. A qucstion in the latter half of the paper may need to be started early on in the exam period (e.g. a prep.1.f3tion requiring a Ih reflux in a 3h exam). Such questions are included to tcst your forward-planning and timemanagement skills, e.g. you may need to take samples for measurement of reaction rates at 5 minute intervals, which can be done while the prepamlive part of the examination is occuring. Make sure you explain your ehoicc of apparatus and experimental design. Calculations should be sct oUi in a stepwise manner. so that credit run be given, even if the final answer is incorn,'ci (see p. 261).
Communicating information
353
Resources
Resources for communicating information Lindsay, D. (1995) A Guide to Scientific Writing. Longman. Harlow, Essex. Northcdge, A., Thomas, 8.. Lane, A. and Pcasgood, D. (1997) The Sciences Good STudy Guide. Open University, Milton Keynes. Porush, D. (1995) A Short Guide 10 Writing about Scie1!a, Longman, Harlow, Essex. Rafferty. D. (1999) Gt!IIing tl.e MeSSdge Across. Key skillsfor .ft;ieru:es. Royal Society of Chemistry, Cambridge. Shortland, M. and Gregory, J. (1991) Commlmiroting science. A handbook. Longman, Harlow, Essex. Silyn-Roberts., H. (1996) Writing for Science. A prOClical handbook for science, engineering and ttt/TJlolDgJ' students, Longman, Harlow, Essex.
354
Communicating information
References Anon. (1963) Tabb of Spectrophotometric Absorption Data for Compoflfld~ useel for the Colorimetric Detection of Elements. International Union of Pure and Applied Chemistry. Buttcnvorth. London. Buckingham, J, and Macdonald, F. (1995) The Dictionary of Organic Compound~', 5th Edn, eRe Press, London. Budavari, S. (1999) The Merck Index: An Encyclopedia ofChemical.~, Drugs and Biologicals. 12th Edn. Merck and Co., Inc., Rahway, NJ. Errington. RJ. (1997) Advanced Practical Illorgallic l111d Metalorgwlic Chemistry. B1ackic Academic and Profcs..,ional, london. Finney, DJ., Latscha, R., Bennett, B.M. and Hsu. P. (1963) Tahles for Testing Significance in a 2 x 2 Table, Cambridge University Press, Cambridge. Furniss, B.A., Hannaford, A.J., Smith, P.W.G. and Tatchell. A.R. (1989) Vogel's Texrbook of Practical Organic Chemistry, 5th &In, Longman, Harlow. Gralla, P. (1996) /low the IlI1erllet Works, Ziff-Davis Press, Emeryville, CA, Harwood, LM., Moody, Cl. and Percy, J.M, (2000) Experimental Olgank' Chemistry, 2nd &In, Rlackwell Science, Oxford. Katritsky, A. R. and Rccs, C. W. (1984) Comprehellsil'e Heterocyclic Chemistry, Pergamon, Oxford, Kowlaski, B.R. (1978) Chemicallllthlslr)', 22, 882. Lide, D.R. (<--d.) (2000) CRe /lamlbook ofCllCl11islry and Physic.s, 81st Edn, eRC llress, Boca Raton, FL Loewenthal, H.J. E. (1990) A Guitle jor Ille Perplexed Organic Experimellwlist, 2nd Edn, Wiley, Chichester. Milazzo, G., Caroli, S. and Sharma, V.K. (1978) Tables oj Standartl Electr()(k Potential, Wiley, London. Miller, IN. and Miller, J.e. (2000) Slali~tic.~ and ClIl?1lometric.~fOl· Analylical Chemistry, 4th &In, Prentice Hall, Harlow. ()errin, D.D. and Dempsey, B. (1974) Bliffersfor p/l and Metal/Oil Control. Chapman and Hall, London. Robinson, R.A. and Stokes. R.H. (1970) Electrolyte Solutiolls, BuUerworth. London. Simmonds, D. and Reynolds, L (1994) Data Presentation and Visual Literacy in Medicine alltl Science. Butterworth-Heinemann, Oxford. Winship. I. and McNab. A, t2(00) The Student's Guide to Ihe Inrernet, 2nd Edn, Libr
355
Index
AAS 1611
7~
anaI)1i<:al2J
AbIorbcloe 164-j /ltJiorpcMMl eodTlnaK 1604 AbIorpIJDn ofbaht 164
AbMlrptJ.... y 164 rl'KlYt 16s-6 AbJt~ lrqxwtl) ])J NcUl"K) 6S 66
"
............... '" _" " ....... Band 'II'Idth 16S
IUJna )48 ,n rqxsu]11 ........ lSI nok
""""
Il
AOd d.~hon 171. 119 Aad-t-l: n Aad-baK mdlClUon \q Acb"Jl;y U. 2Xl
--.
Adobe' Acrobat l 119 Adwlrploon du'omat.,.,.phy ~ Ac...... r;c:n«aIM'lOl 114 AIOS IU Amnn)' chtornatoJrllphy 201
_ _ 216
u,bc:s 117 Ccncnfllllt 116 balMa,. rfl&or III Iow-spccd (badl) Il7 CcrmkO\ rIofhI.uon 238 Ccr11foecl mCfUlClr mlItenall71
Ikc.-Lambnl rdabombp 164. 166 8enc~.k,*.
242
fkliI deay 235 80m pI.ftldeIrMliauon 2J5
......
C"hartmI29,]1. 97
Ana]ytical"mcf1lCb J04 Aniom; IH. 136 ANOVA 275. 2n Arl'icllutz milnOl11C,cr 110. III Answering uam qUl:ititliu lSI Anll·bumping 31. 32, 96. 110. 137 Anl;·IOl\Ilti'hm 263
em" Equipment)
ArgCIl\;melric thrall"" 157 g A.ymmclry f"",Of 2t* Atmosphere 243 Atomic .. bsorptiof,
1ipd1romctcr
17(}-4
sp«\rOKOp)'
16l:l 75
Atomic tmiwon speo:trQInClct 115 Atom., orblt"ls 281 Alomic~roKOOttlll7l-2 flame III Ill1lpiutc rurrlaa 171. 174 hydriock CWl'11I11QI1 171 <:old ....pour In AudiovtIual axis l44
""uft.", principle: 2:&2 Aulllf'ipcucn 10 Autonrdq..phy 239 "'nlpdm'. oonslanl 71. 73 A,'Opdro'. numbrr 262 JUes (p1tpN1251 Bad; tllflIbOO 152
Baeqround CXlfnUIOfI 114 8aJaTa::li 11,22-)
(kmUn... •
handl,,.
IS I\.u:Ird I)'J'Ibok 7 bntllCS219 punty 17 aaf~y IS ~n (NMR) 191 ~nJCIURS 280-"
......."""
e-a..:.23l
iliad:: (~tal) 16, 78 8oih... pomll!:ltiINuon 114
Boilinc 5ld. 32 8arId vitnl.lOn lSI
800* da&1ifocaOOn. )17 BooLmar": lnlftftd ..us lD2
"'~ 299
Bro..'iinc]17 8udInt'I"rtm 140 Bud!nt'I" r........ 23--lO .Buffet" 58-61 ca""",ly.58 prepllf'ation 61 ""/IF 61
odrction 57 1;\to;lIerionio:c 60. 61 Bumpms ]1 Buns;m burner]2 3, Ill! Bureue 10. 144. 147
]16
Chooo<sl
8ln
8om~ aJlPl'01lllTIlIuon
Anal)'lli,of.·.ri.. ~(ANOVAI21S
~dt>uI16
Biblqraphy llO,])] BIDS lD] Bomodrol distribulXll'l 269
Bouks 12 BrUnw>nni1ll: ]26 Broadband det'OupIina 199 Il
Amphol)1C Sf;
cbl&n 281
CcnUlfupl>oll 1)6, Il7 suny 117
AI\&f"OE Ed da:"~ 226 Algdln 2fJO Alkali 56 Alp.... <\eIc.2.y l]S Alpha plInide radlallOO 2JS Alter..... Ye hypothl:sii 7S, 271 2
Ampm:lfT1Ctry 232
Cnura1l:OfT1poi1~
&r chart 2S1. 253
Ap'OR'l11
Appal'll!UII
Cclk plaNlC 16j Ctlls r.... IR 1M Cmtrailullli thalRm 275
................ 21
Abm,uo 292 Abbm....lMXIt
<::hmIQl
292
IMineM ~ J04
Olemical SlIC\y Data ~ 304 Chtm>cal Safety ~ 304 0I.em1al 5hirt 191. 19], 194. 195. 197. 199 ChaniI~2J9
CtanDmecm 28j 90 ChnnW'IJ'Idcl'w- JI6
C'h>-sqlW't'd 101 273. 278 """""" llI2 CIItomaTOIfllm 1lJl. 209. 222 Chmmatosraphy 21»-24 lIdIorpt..... 21)5
.mlllty 207
pa-lIquJd (GC) 211-6
ad filtration 206-7. 219 &d IlItlTl1ll'ation 206-1. 219 IU&!"l"'n<)l'n\8""" liquid (IIPLC) 209
lOfI-elCh'ulgc: 206. 219 Jllt~205
pllrlition 205-6 ~U'Wd
phaM' 206
UC_NMR ~pecI""I97-9 Calculations
lJZI::ud,wfm 219 theory 20S 10
prn<MlIres~
,tun la)'O' 216 ChromIC acid bath 16 CiulIIon Illda 3111 Citauons l19. )21.340
usOfIA spn:adslIttt$ ]01 CalculaTors 4. 260. 261 C.bbra,ioo 90 Catibr'won cun'C 251 CalibraTion soIl1llOOS ror GC 212 eatibnot..... Ilanda.-dl S9 Calornd dtctmde no. 231 OononicLl
ronns 281
Q;pac:ily ra:co.- '11T1. 2O\l Cllpilbu)' cc1umn 211 Cap.~~m-I
Cbm.. "
Clark (Rant) 0Jf)F' deccrodl:: 232-] ~ el«trodc 231
CodTlCiall of dIspasooft 273 of
""""011 26llinjtc:tor 2\1
C~ 0II-a)Iumn
Cold ,.. pour ........t>on 112 C(Illipu~
pmpcrtocs 49
Capillary Iubt$ 11 I Capillary ZQIIt ~ 228 C....-borI dalinl 240 Carlxm dior.ido:, 50Iid ]1,llI Cart! indaiDc ]19 Canesian co-ordin.alcl 111 Calions 135. 1)6, lJ8
Column df..,.,.,.,. 2011 C(IlWlUlIm&,h 214--S CoIUJftns 211 CampIc::a stability lSI 2 C(lI'nflIl:ulion rexhons 51 COIIIPUutitlnal damsuy 291-3
Cdi~ :II Cds- .... I6S
COIllJIUkn 2.SS
Col....... chTttnill
Compu~ soC\wan;
29l
357
Index
Concentration 45-8 acti'lly 48 molar 45 pans per million 46 percenla~ 45.
46
SI unit 45 Conclusion (reports) 333
ConOCnsers 116 COl1doctivity deteaion 22l\
Confidence inlcrvlll 289 Confidence limits 271l ConFounding vanahles 18 Conical flasks 10, 12 Conjugate pair 56 Constant temperature bath 33 Corn;l~nls. physical 72
ContllCt len:ses 7 Cooling 37-9 bath>;
37-8
probes 31\-9 in recrystaUisation 98
Coordination cl1emislry 2110 CopYing 310
Copyright law 319 Correlation 218
coefficient 218 COSHII regulaliOO$ 5. 69 Coupling 195 Coupling constam 196 erash-l;ryslallis:llion 98 eRe ~lal\dhook 15
Crudble31.243 Cubic dosed-packed crystal 281 Cupferron 140 Cune 236
Current aware1leS.' jOllrn~ls 3111 Cur\~ 253 CW-NMR 192. 194 Cydic vollammetry 2J4 Dam 66-7, 257 calculations 259--63 collection 66 dcn,'e4-70 DessicalOf 40 Detectors 209-10. 220-2. 228 [kteclorS in GC 215-6 Detergents 13 Deuterium 192 De''elopingtank 216 Dewar llask }7, 38 Dewey Decimal system 317 DiclloMry, U>;e of328 Differcntial.'i<-'liIming colorimetry 243 Differential them.." analysi~ 243 Dilution faclor 20. 1,0 Dilulions 20. 4(i tinear series 20 DimeU,ylglyoxime 140 Diode array delector 221
358
Direct titration 152
Discussion ""pons 33) talks 341 Disintegration (radioactive) 236 Dispersion (statistical) U,7 Dissolution 96 Dislillallon 107-15 equipment 101
Fra<;lional lOS Kuge/rob. III. 112 simple 106. 109
steam 112. 114
,'llCuum 110. In Distribution binotllial269-10 frequency 262-8 normal (Gaussian) 211-2 Poisson 210-1 Domains 300 Double beam spectrophotometer 165 Double-beam instrurnenl 182 Drawing packages 281 Drugs 244 Dry ashing 119 Drying 39--41. 100 ,,~ts41
glassward9.126 liquids 41. 121 solids 39,121 solvents 121 tubes 111 Easily ionizable elements 117 Editing rearures 312 EDTA 151.152 Eketric healed block 89 EJcctroonalylical methods 229-34 EJeclrochcmical analysis 229 Elcctrochcmkal ceU 229 Ekctrochemi~'lJ1 deleCtor 221 Elcctrode S1 calomel 230 carbon dioxide 231 combination 57 gu-sensing 231 glass 51 glucose 233 4 membrane 231 oxygen 232-4 pH 57. 231 petential229-30 rd'er~nce 57 sil'-er/silver chlofide 230 solid'!lIale 232 st.andard hydrogen 230 Eleetro-cndosmosis 2'£1 Electrolysis 2)2 Elo:ctroryt~ 45 Elo:ctrolytic ccll232 Ek:ctromagnetic radilliion 163 Electromagnelic speclrum 163, 180 Elc:etron 194,235 capture 2J5 capture detector. 21 5 Electron impact Electronic configuration 281-2 Electronic publishing 302 Sectron·impact mas.~ spcclromeler 200 Electro-osrttolK: now (EOF) 227 Electrophoresis 225-8 agarose ~I 226 capilla'1" 227-8 media 226-7 polyacrylamide gel 226
Ekettophoreric mobilily 225, 221 e-mail 299-300 End·point 141. 158 Energy spectrum 236, 240 Equipment ooLances 11. 22 3 cnlculators 4 centrifuges 136 chromalographic 205-24 cooling 31-9 eleclrophoresis ill-g gases 125-6 glassware 10-14 heating 31-2 osmometen 49 50 oxygen eleclrodes/probes 232--4 pH meters 59 pipctles/pipeuors IH I scintillation ~'Oumers 231-9 specrrophotometen 164 Equivaknoe point 149 EGUi"lllcnt miOss/weight 48-9 Error blll'S 254 Errors. randonl 65 Essays (ser aI.
design 15-9. gl. 21\5-9 multifaetorial18-9 randomization 18 f<:plicalion 19 Expenenls 262 EXlernal standard 224 Exl....polalion 254 Eye protection 1
n.
FAAS 170-5, 117 hetorial design 281 Figures and diagr.lms 331 Fajans titralion 158. 159 FAQ 299 File n'anagemenl 301 File transfer protocol (FTl') 300 Fi~ng lileralure references )19 Filtralion 27-31. 140 gravity 27. 29 suction 28. 30 fill~r funnc:l 27 filler paper 27. 28 hoi 98 Flame atomic abl;orplion speclroscopy 170-5, 177 ;onisalion detector 215 pholomclry 168, 169, 175 lests 138 flash chromatography 217, 219 Roppy disks 301, 311 tlasks 10-12 ~olumetric 10 conical 10. 12 Buchner 29 C1~;scn
108
round-bollomed 116
Index
Fluorescein 159 f1uore.eencc 167 Fluor=n~'e
Gre'dSC 21 Grignllrd reaction 127
detrtlor 221
Fluoride electrode 212 Fluorimeter 167
Fonts 30 Formatting 312 Formulae matllcma'icaJ 259-61 spreadsh~1
309
'H-NMR spectra 193, 195. 1%, 191, 198 Half-life (radioiwl0p0) 236 HamIltonian operator 292 H,mdbook ofChemislry lind Physics 318 Hardware 301
Harlree-Fock method 2';12 Harvard system 319
Fourier lran6formation 182. 192 FrngmcnlHlion in MS WI. 203 Frequency 163 Frequency distribuhon 252 Frequc"'-)' polygons 253 F-test 278 FT-IR lIa
Hazardous chemiC
Ff-NMR 192. 194. 197
warning symbols 7 Health and Safety 6-8 chemicals IS
fTP299.300 Functiolllll group absorptions 181
Furn.1ions (spreadsheets) 309 Funnel addition 118. 119 Funnel. dropping 118. 12b Funnel. separatolY 103-4, 118. 119 Funnels 26. 31 F",",on 179
Gah'anic cell 229 Gamma counter 237 raY' 235 Gamma-ray speclrometry 239 Gas 'b!ow down' 123 Gas bubblers 126 Gas chromatography 201 Gas chromatography 211--{, Gas<:ylinden; 125 Gas·liQuid chfomalograph~ (GC) 211-6 Gas-sensing glass electrodc 231 Gaussian dislribulion 264. 214 GC 211-16 GC-MS 204. 215-16 Geiger-Mullet lUbe 237 Go! clectrophoresis 226--7 fJJmuion 206, 219 pet'TTleation chromalography 206. 219 Glass membrane ele<:trode 231 Glassware 10-14 dcaning 13 drying 39 glass versus plastic 12 joillled 43 SlIfet~ asf)CCIS 13 seleclinn 12 spttlral cul-off 13 Glo''CS 3(i Glucose eleclrode 233, 234 Gooch crucible 140 Gooch funnel 140 Good buffers 58, 60 Gradicnt elUlion 219. 244 Gradient formers 206 Graphics 310. 314. 316 Graphics packages 4 Graphile furnace alomi1Jef 171 Graphs 251-255 and word-processing 314-15 dm",;ugjplouing 251-2 mterpreting 255 paper types 252 using computer programs 314, 316 Gravimelric analysis 31. 51. 139-40 Gravity chromalography 217, 219 Gray 231
cemrifu!l"S 136-7 ch.emicals 6-8 glass....are 12 radioactivity 241
regulations 6
databases 304--5, J06 glassware 13, 110 hOi glasswan: 36-1 in anal~sis 131 la",'s 6 mercury .'apour 89 rnd;Ollclivit~ 23ti. 241, 242 Healing 31-1.131 HendeNml-Hassdhalch ~uation 58. 61. 206 HETP (height equiv..Jent to a Iheoretical plate) 2Qll
Hexagonal-dose packed crystal 2111 High-performance liquid chromalograrh~ (HI'LCl209.218-22 Hirsch funnel 28 HilY:h lube 29 Hislogram 254. 285 Hollow calhoc\e lamp 170. 114 Hookc's Law HOI air guns 36 Hoi plalc 33-4 HPLC209.218-22 HI'LC columns 220 HPLC syslem 220 Hydlogen ions (pH) 5(,.61 H}'1.ide genennion 111 Hypolhesis 75 alternative 75. 271-2 null 271-2 leslingsmlistics 271-2
Ice 37 lce-sah mix lure Jll Icr 115-7 Ilkal behaviour 45 Image 341 Independenl variable 251 Indi.:alors 148. 152. 153 Induction coil 175 Inductively coupled plasma AES 175--7 lnen almOSphcn: rnelhodsl25-9 Infra-red (IR) spc.'Clroscopy 18l}-9 Inorganic analysis 135-8. 140 Inlegralion ofpe.ah 194 Inlerfen:rteel> in FAAS 174 Interferences in lCP-AES 176-7 Inter-library loan 319 Inlernal standard 224 Inlemet 80. 299-306 Inlcmel add~ 302 Internet resour<:es 302-6 Inlcrpolallon 254 Inre"-.ll scalc (rnc'.lsurement) 65 lnlroduction (repolls) 333
lodinc vapour 218 lOll product 56. 57 Ion seleeti.'e ek:<:trodes 230 lon~xchange chromatography 206, 219 lon.excl1ange reMn 206 Ionic slructures 281 Ionizing rudiations acl 24t lonophore 232 Ion-selective dcctrodcs 230 IR band intensity 187 IR spectromcler 182 IR spectrum 181, 183. 1M, 186. 189 lsocralic separalion 219 Isomerism 281 Isotope dilution analysis 240-1 effecl239 peak!; 201 safety 242 Isotopes 201, 235. 236 loinl clips 44 Journal. 286 Kalril1.ky system 320 KBrdi5ks 184, ISS, 186, 188 KendaJl's coeflkienl of mnk coefficienl 279 Kolmogorov-Smirnov tesl 277 Krusul-Wallis 'i-lest 278 Kurtosis 269 K. "
Labelling posilion 241 Laboralory book 3, 66--7
=" work,
basis rules 7
LANsm Lalin squan: 78 LC-MS 204 Leasl significanr difference 277 Lecture t\OtC$ 348--9 Lewis diagram 281 Lewis SlIuetures 280 library classifIc'.luon sYSlem> 317 currenl awareness journals 31S Library of COllgress systern 317 Ligand 151. 207 Ughl ab;orplion 164 el«lromagneticsp:<;trum 163 measuremenl 164-7 units 70. 164 Lime walcr I 36 Line spa<.ing 31) Linear dynamic muge 210 Linear ~ression 279 Liquid chromalogr.lphy 216-24 liquids 9-14 measuring 9 dispensing 9-11 holding and storing 11-14 Literalure n:views 339~34O Lile,alun: surve}"" 318-9, 339--40 contcnl 340 infonnalion mrieva1318-19 plan 340 slyle 340 topic selection 340 Litmus paper 136 load coil 115 l...ocal area networks 299 Localion (stati,timl, 264-8 Logarilhms 262
35.
Index
Molecular Ofbjlal dill~ 1lIJ-4 MoIocular sones 41 MonochrnmalOr 174. 175. 176
loucifenIJC 1611 loum~16lI
Ma<m 301 M~oc field 190. 191 Magnet:oc llC'l16, III Mlgnetoc nudct 190 Mlgncuc aliITel' 16 MIIplCtOf,)'Iit ratto 190
MS ..... Mulls 184. IllS Mullifaaonal to:pnvrlCIlls 711 9
Mu.1lt·lt.-.r
~
2119
MIIlllmdCl' 110 II
\".cbullm' 172. 17j, 116 Ntptmol ~ 1)$ ~ ftllialU DO Nctworb (C(ICllplJlCrl299. JOI
Manlr.:• •&1.;111)5 6
NrvtnliAtiat curves 148
Mlllinalttl$)O) MaM-WhilllC)' U-1aI1n
IlnlClUTC )3)
f'aratilm" 12,13 P...~craplil )27, ))1
Paramnn- 6S Pln"..,1.... ddlnbUllOlI 2n~s JUtutoc:al II:aU 17S-9 Part.1IOlI diroonIlOIf'lIrily lOS I'1InlOOa codTtdtnu 103 Pans per miJIion 191
...... '"
.....tnrrppclIC9. 114. 12l. 116
PeA 18$. 2'.lO
M_41.1l
Nt-~JOJ
J'cat. llI)'n'lmdry 108
M_
N'i1....,1l, liquid 11 NMR 190-9 NMR spccu..-. 191 2 NMR spcctrunI 192. 19<*
Pt:al5OII's oondallOll 0DI:ff1Qml179 Pfllod~ tabiL' 2.S6.. 2:1.2. ICC inside bKk CO¥n PtnonaI alI1lmllNClllOll J11
Nan-lI..-. rcp-..n 279
pH 56-61
Ml»lfl«lr_ 100 toll» $Il«Ir
MIA sr«c-ry
8dcl:In)()l
MM5liC*tnam 100, 10 I M-"o
fUnc:tionl )09
........ "',
optrattont 2.5'9 6J MC'M 1M, 266, 174 Maa.umnmt 65 6
.....
-""os errol 66
pH $1
".....os.
'lUaIoIIUIoi: 65 qUlnt"IIi"" 65 nldaetivily 1)1 9 I'lInlct16S
_156$ ~mpholometnc: 16) Meuu.;ng cylil\denl 10 Median 266
MEKC 22ll Meltillj pnlnl 81 91. 2li.S IPJlM'llUllll 9
MiccllB' elec:"okmet ic: ehromllll.>gnlphy 228 Mian-Bunscn burner 1)7 Micmbu.~)2
Mia<)plpetlC 216, 217 Mocrotall" F..ux:I" lO9. )10 l'o,",r~m a S Word )11 Minimum dc1eculblc Itl.-d 110 Jl,I,.ed-iOlvmt llia)'5IaI~SlIlion 99 Mobile ph_ lOS. 211. 216., 218
Mode 261 Mndel. mlIlherTL11lica1 7$ Modan 299
Modulu5m Mobr tilration IS&. 1$9 Moialily 46 Mdar abl;orpti...t) 1M Molu conomlration IS. 4S Molant:y 4$. 46 Mokatlar 0llII :!OO.. 2l11. 102
Md=dar maa 2111 M~
MolcaiLu
Non-parvnc:trE ....Iiau:a! teIU 272. 271
~n.j9.60
r-omIII (Ga..-n)lc6ltributtQn 2to4. 114 Normal phaJc: Hf'lC211 ~ pollaWil:)' plot: 17$ NrJnnaIW:y (~) 4'9 NtlIc tabll& 148 9
butJrrs S8-61 UlldK::r.1OI" d)'t:ll S7
....
,.....
mcdwucs 292 moddIift& 291
~=
..
lltration
~
58
Plto$omuklfllin lube 174. 17S. 176
lcc1un: )48 9 p-o;ccb II NIQar mapltl.,
~1S7.j9.2l1
_'"~ """"""',OS
b.b (practical) 66-7
9.1)1
"')'IC'n 23,2 ) prvlC1plcI of 65 8
P·f~lS1
......... ,6) ~
(NM R) IpO,I"O!iCOpy
Nud!ar spn 190
f'tI)'5ICI1 constants 311
Pic dian W. )JO.
))9
PiptluA,. 10. 14S. 146
rolln A. 11.146
Nun IIypochesii 211
Pipette
Nurntln- of.Vl&bIr:s 188
Pipdlon 10 II
NumbrnU9 Number 5CU aDd operations)5'} N........
Ocudecylsillllt 206
PlaI'll:L COIlSWII 16l--4 l"'-Da lon:It 17S PIaIC nwnber 2011. 109 PLar C1Illumn 211 l'Io4ted CUnei 2j1 PoiMotI dlslribuliotl 21) l'ofyllCf)'lamide ""I eltctrophort!;K 227
Ohm's law 221
l'oIydlrortlllor
Oil balh )4....S, Il9 Open IUbularc:apirtary C1Illumlll 211 Optical Demity 164
Polymer mn-nbr.l.ne dmTodt 231 2 I'olymtn 14$ Polymcdal diitribuloon 269 f'opul.lliion 2M. 2n f'05ilmn cmiMion 2)S P/l6U:r dupla)'l34I~) COIlIenl 343 da.;ign and layoul 341~2 1CSSionI343 rults and
""
1)'SItm
"..., (cilll.tions))1I)
Onl prtscn18lionl1l44-1 condudinJ, l46 comml34$~
P'"q>Ilr.l.hon Ind p1annifti 344 pfCSrmation l46 1 Ordinal ~ (mtalurtlllCtlt) M Ordinale axis 1$ I OImoIalily 49. SO Osmotanly 49 O>mornttn- 49-jI)
"""""" '"
...pour prtUUK SO
.. """""'"
0un0Ms4S Osmotic 49 SO
"""""'"
OullieT 266 Outli-s 32S.t;
o.uhcadl~S
0Ud&J.i0Il S I. 129
""""'"' " """,m ekctrode
~qmlS2.ISS ~140
m~3
..... 211 PIpet drr~tovaPh)olOS !'apa's (rc:sardl) step 10 produtt))7
"'""""
eIWN
176
H2
_~7
3)2, )B. 31~ reoordiJll3 J'n:gpla~ 140 Pntipwiort Sl. 52 P_656.,168 Pn::ocntltllOll par:bjIltS ) 16 Praocntationl; (sec Oral pre8tlltationsJ 1'rcssuR' rccuIalor- III Pnmiuy Itandard 141 n:por1S
IUlJlls
Principal COI:lpDDCDI ~ 285. 290
PnnIirc )1Q, )14
Prot:.biloly (oUtisba) 2n Pl"obltrnt., nwnmcal 2S9-6l
PrcEnm 111 f'n'l,ccts 6., til. 80-3, 332-)
Index
I'ro;x1S (NWtltnwd) litel"alure survey JJg II ",,_10 roooniin& dUll 67-9.11 repofU n. 1)2 1 risk_mml6
yfet)'11 lope dlooce AI PrClIcaive doll".., 1 l'r0l0n 1Ill, 194. 195 fa. 2lS ru~lnf_IioIllJI1
rurily Ir1
Rdlltive .tandllrd
~t>on
26Il
.....,
n
c:onDmll'llJoll 7) mtaOOll~ 1J-4
wnilflA 8l. 1)2 1
1NI.1l phywr:aJ mralIInll12
_.... Mn.dv~
))1
Rqlnnl nquau }Ill Racatdl JlaflCI"1 1I II ResoIuuOfI (duomIIlOpptuc) 206. 2 IS, 206-9,
22J
QuanlJlIIlIVt'
........ "
111,14.124
1Ia&i)'Sd
.......
reoordon& 1. fb-1
IqIOJtIn& )))
R.dcntoII:lP lInw 209
pa- HM.C 206. 211
~
Q\arlIUIll number 190 QuendtuIa (f'IIdioKtlvily) Z11
~ r::hn:Iotu~:lOS. 206
RA200 R.diaoon toUrw for AAS 11'0 R.IIdioKll"t decay 2.lS 1S(lI<)JIeS
US
MlbMa_ _ 241
Ilmpfnl,,", 7]
SitPtulCIIlCt'. Mlhsllcal m
QullnIUfll rncdrllfIIO 163, 2'll1
Quidd'i112
....... " """'" '"
Sirp.J.to IllliM: ralJO 1M
-'0) " . lU:spoosc iV~ ~ 2S&, 219
QuIIIillllM vanabla M 19. 14. 214
SI IIIIlIS 1G-14. 2J6
bIISac f....w. 10
RqJeIltion 19 KqlIoles
Reo.... 1IIdInoques)jO Reo--.. t.- Lounc~ M1~1 R,''''211
........
,lljecIor 220 ...lve219 RISk . - n a i l 6 " IS
stIica Fi 19 Siha nilta.1£ 151 SiMI siluerl:...... drnrode 2JO Simplex dt'sIJlI 2IlfI Sunuhflna>ur; dr:5Ip 2S(i SO« ~ drromIItocrIphy 219 St"'"'ll dosIntlulKlll ~
SIido:s 141
-'SO SIoI 1,. bII'ner
Stdoum p/loIpAaIe 61 Sofno-a.,. dalat.uts 110, liS ...pb:r; 110, 31(; ~)0710
RDcarye...,wn_121-2.
I~
JtaIIMoCll lIS ..
,",m~y 24(l-.1
ROliofiow IIOp 104 Sr:oeIy of C1lanMry dM.IIbutli lOS Rubber )6
lSC)Itlpd 23S ITlI'UUrerrotlll ofU7 9
Safety t- Huardt,)
SMobrJrly pmdlld 50-1
lilIli:ly 142
Sak padM:n1 206
l)'pQd23S
SoIu~
Som"
R.IIdM:-u...cy US
ullig 216 "'Qlkill& pnr:Iiot; 2J6 RadJOodaIIf\A 240 RAdi<Mnmllnoasslly 240 1 RadJOb,belbJ CQrnllOllrdli 240
lUdionuclKle 241 RadiolllCl'llP)' 240
Random numbmi 111 RandoniSllliol1 78 RaI\lC 261 RlllioI26) Rt'>lClivilll· 123 Recordina
RD)':III
rlflCClS
handlinA IV iIIjtclion 220
OOII'idc:IIJ .S Sol.UI!OII d1anIIuy .S-SS
injeaJon In GC 211, 212. 21)
Solu1iol'\!!l IS 21
""sutisticlll 204, 268 sub-sllmples
SvnpIinc 81 Scale(s) 6S, lSI Sealift" diqrams 252 Schdfe·80~ tesl Schmdingl'l W:h" rlQllaliorl291 Scieoa Citation Inda (SCI) ) HI SciIontn..:
m
nOies 66-9
ITlI'lhod 1S-9
notation 162 pa.persl31-l! style 316 wriling 32S-9
RllC~lIlli"'iOll 91 101
coI1rx:\I011 9H ooclill& 98
dryin¥ 100 pmbkms 100 1 prOCUl 9S-1oo II:I1Ildting 911 seedilll 911 soIvelllll 92 (; Redox ISS, 2J4 Redox reactiom SI-4 lilrahons SJ OQLllIIJonJ SS Raluci.. II$N n. ISS
"""><>' "16. III 1I1l111yllQI
n
primbry «:oo
Tef«r:nces 2\lII JOD
Scinu.llahOll Md;lIIil2]1 counlft" 237, 2J&. 2J\l SCOT column 211 S<:mocap IldIIptor 43-4 Sealing (ilin 12 Searcll cnPnel JOO
Sc:IIrcltinl )11 SearclUnalht; Web)OO-2
Sdecti,,, dd«Icw 209
Sdcaiviry (chru:nat""",pry) 207, 2ll Stmi.a:lpirical mediad 292
Red~SI,229
SeminIIn (sw0rai
SenoMivity210
Refermas 319-11
Ser-raIicn flldQl" 20&. 209
...... ""
Sq:vIIliorl
~t.alirlm)
~r.x:Ul:li
dlmmalop
10S
~2.'Is-I
_'207
dcIi&n 2lI6
RdlWl 116-24
Sequemial
~J1t'Mion 218
Sm~ SUUdun
Re:b.tiw:.bur!dIIno;e 200
Seneo. of numb::n 261
RdlIJ,,. fl'1lllllll mobiIiIy 217
ShieIdiIlc CDnSIII.DI
121-8 190
butTen SlI (;1 c:aliblllnon. 111 Dml:t'1lI....I>on 17
Jenmll purpoee 16 bellini 17 OlmOO1l1: properlics SO pH .56-1 .-.nnA 18 prtpIration 1S-19 IIlluralcd SO Blandard 2Q lIock 10, 110 1IOnl~ 21 Sotv<:nt delivery 119
Reference dectrode 2JO, 231 lJibIiotrapIty )2()-1 Clition )1')..11 obuinarc J19
bdIa,""""
ideto.l4S
10
Solvenl CJllnlction 102-(, lIcid·base !iq\lid·liqwd 102 soQd..IiquHi 102, 10S--6 St».hlel 106 SoIvatI IdI:cIJoll 9J-4 SoJ.hkt e~lractlOll 106 SpcIrman'. rodTlcienl of rank rodTlcienl 279 Spear", IctMly 217. 211, 241 Spccrf", de!cl:l0I 2C9 Spx:I.... lllI Speclramdt'T 17(; SpxIlomeuy 180 Spea.op/loIon)eIel'l64 ~0!C0p)" 161-9, 180 .sonuc 1M-1S ' - ' 16).--\1 infra-fcd 180-9 IlWlI $pt'CIomeIIy 2()()-.4,
nucbr mqnetlIr::
21S, 122
J'CSOIIafl(:e
190-9
~orl6J
Ifl«lropholon:dcn 164
361
Index
Speclr05COp)' (cmlinwdj
ultnHiclt'l (UV)/viJible 16$ 6 Sp,:rtrOflhOlometcy (~Spectme;c:opy) Sp;der diagram 326. lS2 SpIilJ~ilbli IIljeCtor 211 Spnly chamoo 176 Spretidslleds)07 10 fllOClions 309 grnphics 310
Test tube holder J6 Tnt tubes 11 Thoeon-lal plale 208 Thermal analysis 24l--5 Thecm.R.l conduct"'ily det.ecklr 21S
~"""
Thennova"imdry 24). 24-t 1llmDometer 31. 90
~,,)7
ThesaUfU5 328 Them (.- Q/JQ WritinaJ B2. Bl
prinlill& llO SlrUCluTe J07 8
Thin-byer Chrortllliopaphy 216-17 Thfft-dimo:nsional cnph 2S2
Ilmplaleli
307
II
Stabilily of oomploer 151-2 Stabilily CCIIaUI\IlI 152
-'"
addmom pph 172 de-ubOO Ul. 277 elecIrode poIential229. 230 ~~'" form 262
h)'1~ electrode no sdUlion 20.141.143. 14-t S1andanlisallOf\ 149 $lafl(blrds 224 $lalionary phas.e 2OS, 211. 213. 216. 218, 224 $laUsIIC 65 Sutislnl analysis 315 funcllon~ J09
""""m 271 9 lr$l$
Sialisllci 25t 257. 26). 264-70 clloollill8 Ic$Is 275-9 oorrctallon 2711 dr$cnpli~ 264-70 di5pm;ion. mc3SlJ~ of 261-8 hYpolh~ IWln8 271-2 locaUOII, meaSUres of 265 7 non.parametrlC 272 nutl hypod~?S. 271-2 pa..... '~Irit: dislribulion, 272-5 "l\ression 2711 soflWar1: ll5 Sicam buh l3 Sleam line 34 Stimn8119 S,ocl;. solulion 170 Sh)lchi"melr)' 147 Stoichiometric lil""liQn 141 SloppeTS 12 SklrulfC 21 Slu
362
T_" Tilrand 145
Titr.ulI 145 Titrallon 14)--60 acid-base 148-50 a
,.,tI"
conplacme&ric: 1S1-4 dil'C'Cl 152 ~1417
pcn:ipimtion 1S7-9 mJoa IS.'i 6 sundardi!l.alion 149 Titrauon cu.-..a 148-9 Titration$. redm: 53 Tilrirnetio:: analysis 141, 148 TLC216-17,2111 TLC plato 216
Transmission 182 TransmillanCe 164 T ca~llUll:ic:5 Olol'rhcad 5 I-WlliSlic m
!-tot 115-7
UK Nutrition o...,abanlr. J()j Uniform Resoora LocaIOl' (URL) 299 Unil all 281 Unies base (SI) 70 oonoentralion 45-9 cOll~rsiOll table 72
deri-.:d 71 in probkms 260-1 radioll~1i~it)' 236-7 SI 70--4 Uni\'C'fSal deteclor 209 URL299 UV lighl 217, 219 UVjVisible delc<:lor 220 UVjVlsible Sp"""OVhOlometcc l65 6 UVfvisible >p<elroscopy 1~7 del«lor 220 measuremenl in 166
dependent 76 independC'nt 76 lnterdcpmdenl 76 nuw~76
quali live 6.S quanl!lati~ 6.'i ranltd 6S UI"ICCllIlroilal 76 Varia!x:e 167 Vcnlllli effect 113 Yertocal electrophottsis 226 Virwes, a>mpUl<:r)l)1
void ,dl,lmC 206 Yolharo titration 158. 1.'19 Yollanunetry 232 Yolume 11 VoIlmd~analysis 14\ 1 ""Iculauom 142. 146. 153. 1.56 end-poinl dettnnil\lliion 158 solutions 14\, 144 lUnda"" 143 Volumetric f1W: 10. IS, 144 VSEPR thlocy 212-] WAN5299 Warrnng symbols 7,11 WM/t,ng 102 Water bath ]] W"'C function 292 WavelenElh 163. 165 Wa'l'bsth detection 174 Wavelength sdernon 174 Wa~nl,lfllber III wan coIU1lU1 211
W,b
""""""
snn 286. ,." )I)] Wcigung 2'0 001111: 20 cOI1lainers 2J..-S procedure 24-.'1 Weigltlcd me-..n 266 Wide an:a nctwocb 299 Wiloo~on'l signed cank test 2TI Word proceuing 4 Wocd P<<J<:CI,$()O 311 16.l12 WocrIPerfflCt" III Wockl Wide Web 80. JOO. ]17 Wriler's block ]27 Wrilillil cititq; referen<:cs ]21)-1
csuys 330.. 1
Vancoo>U S)'!il.aIl no
liler;nun: SUl>'Cysj.....·iews 3l9-40 orgllnizing 12H reference library 328 repol1S 332-4 lcvising tal 329.]]4 scientifIC J2S--9 llCIenl,rOC papen 337-41 s.tructure of reports H3 "Yle l26-7 lheK:s In.]]4 under ehrTl condiOOrl5JSI WWW80. 300. 317 WYSIWYG)II
Variables 65. 16 oonfoundirlf. 76
Z..illerion 60
VllCIW.at ~ 110 Vacuum der.sicaloc 40 dlSUllalion 12 _4l Valence Shell Electron-.....ir Rqx!iJOon 'Theory 281-J
