Advances in Atomic Spectroscopy

ADVANCES IN ATOMIC SPECTROSCOPY

Volume 7

9 2002

Volumes 1-5"

Published by JAI PRESSINC.

Volume 6:

Special Issue of MicrochemicalJournal Published by Elsevier Science B.V.

Volume 7:

Published by Elsevier Science B.V.

ADVANCES IN ATOMIC SPECTROSCOPY

Editor: JOSEPH SNEDDON Department of Chemistry McNeese State University Lake Charles, Louisiana VOLUME 7

9 2002

2002 Elsevier A m s t e r d a m - B o s t o n - L o n d o n - N e w Y o r k - O x f o r d - Paris San D i e g o -

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Table of Contents Preface ................................................................................................ xiii Contents a n d Contributors to Volumes 1-6 in the series ............................... xv Short B i o g r a p h y o f Contributors to Volume 7 .............................................. xix A b s t r a c t o f Chapters in Volume 7 .............................................................. xxvii

Chapter 1: ~

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Use of atomic spectrometry (ICP-MS) in the clinical laboratory .................................................................................... 1 Introduction ................................................................................ 1 Atomic spectrometry techniques in the clinical laboratory ................................................................................... 3 2.1 Requirements for trace element analysis in the clinical laboratory ........................................................... 3 2.2 Flame atomic spectrometry ............................................. 6 2.3 Electrothermal atomization atomic absorption Spectrometry ................................................................... 7 2.4 Inductively coupled plasma optical emission Spectrometry ................................................................... 8 2.5 Inductively coupled plasma mass spectrometry ........... 11 Inductively coupled plasma mass spectrometry ...................... 11 3.1 Fundamentals and recent development of the technique 11 3.2 Techniques for sample introduction .............................. 13 Determination of trace element concentrations in body fluids and tissues ................................................................................ 15 4.1 Background ................................................................... 15 4.2 Sample preparation ....................................................... 15 4.3 Interferences and their control ...................................... 16 4.3.1 Spectral interferences .......................................... 16 4.3.2 Non-spectral interferences .................................. 18 4.4 Applications .................................................................. 18 Stable isotopes tracers: a tool for research and diagnosis ....... 20 5.1 Background ................................................................... 20 5.2 Biological and analytical constraints for human studies using stable isotopes as tracers ..................................... 22 5.3 Determination of stable isotopes ratios fro tracer studies in humans by ICP-MS ................................................... 23 5.3.1 Copper and nickel ............................................... 24 5.3.2 Calcium ............................................................... 25 5.3.3 Iron ...................................................................... 26

5.3.4 Selenium ............................................................. 27 5.4 Other applications of isotope measurements ................ 28 6. Speciation ................................................................................ 29 6.1 Background ................................................................... 29 7. Reference methods and reference materials for trace element analysis .................................................................................... 30 References ............................................................................... 30 C h a p t e r 2: New developments in hydride generation-atomic spectrometry ............................................................................. 53 1. Introduction ............................................................................. 53 2. Novel hydride generation ........................................................ 54 2.1 Electrochemical hydride generation ............................. 54 2.2 HG utilizing fast gas-liquid separation ......................... 60 2.3 HG with immobilized borohydride on ion-exchange column and moveable reduction bed ............................ 62 2.4 Vesicle-assisted hydride generation .............................. 64 3. Advances of methods of atomization ...................................... 68 3.1 Atomization interferences in the gas phase .................. 68 3.2 In-situ trapping HG/electrothermal atomic absorption spectrometry .................................................................. 71 4. Chemical interferences in liquid phase and pre-reduction ...... 74 5. Hyphenated techniques ............................................................ 80 5.1 HPLC/on-line treatment/HG/atomic spectrometry ....... 80 5.2 CE/HG/ICP-AES (or ICP-MS) ..................................... 84 6. Applications ............................................................................. 87 6.1 Arsenic .......................................................................... 87 6.2 Selenium ........................................................................ 92 6.3 Antimony and bismuth .................................................. 99 6.4 Germanium, tin and lead ............................................. 102 6.5 Miscellaneous .............................................................. 103 7. Conclusion ............................................................................. 103 References ............................................................................. 104 C h a p t e r 3: Analysis of biological materials by double focusing-inductively coupled plasma-mass spectrometry (DF-ICP-MS) ............... 117 1. Introduction ........................................................................... 117 2. Instrumentation ...................................................................... 121 2.1 Magnetic and electrostatic mass analysers ................. 121 2.1.1. Magnetic mass analysers .................................. 121 2.1.2. Electrostatic analysers ...................................... 123

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Double focusing: forward and reverse Nier-Johnson geometries ................................................................... 124 2.3 Performance of commercial D F - I C P - M S instruments 125 2.4 Peak shapes and sensitivity ......................................... 127 2.5 Data collection ............................................................ 127 Elemental analysis of biological samples ............................. 129 3.1 Spectral interferences .................................................. 129 3.1.1 Blood, plasma and serum samples ................... 129 3.1.2 Urine samples ................................................... 133 3.1.3 Tissue samples .................................................. 134 3.1.4 Arsenic and selenium ........................................ 135 3.1.5 Noble metals ..................................................... 135 3.1.6 Rare earth elements, scandium and yttrium ..... 136 3.2 Matrix interferences .................................................... 137 3.2.1 Serum and urine samples .................................. 137 3.2.2 The case of selenium ......................................... 138 3.3 Sensitivity and limits of detection .... 139 3.4 Biomedical applications .............................................. 141 3.5 Applications of food samples ...................................... 143 3.6 Application to environmental biological samples ...... 148 3.7 Determination of radionuclides in biological s a m p l e s . 149 Isotope ratio measurements ................................................... 150 4.1 Accuracy of isotope ratios by D F - I C P - M S ................. 150 4.1.1 Mass bias ........................................................... 150 4.1.2 Detector dead time ............................................ 151 4.1.3 Blanks ............................................................... 152 4.1.4 Isobaric interferences ........................................ 153 4.2 Precision of isotope ratio m e a s u r e m e n t s .................... 153 4.3 Resolution of spectral interferences ............................ 155 4.4 Tracer studies ............................................................... 156 4.5 Paleoanthropological applications .............................. 157 4.6 Isotope dilution analysis ............................................. 157 Trace metal speciation ........................................................... 158 5.1 High performance liquid chromatography ( H P L C ) .... 159 5.1.1 Size exclusion ................................................... 159 5.1.2 Ion exchange ..................................................... 161 5.1.3 Selenium speciation .......................................... 161 5.1.4 D N A adducts quantification ............................. 165 5.1.5 Organic solvents-induced interferences ........... 167 5.2 Gas chromatography ................................................... 167

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5.3 Capillary electrophoresis (CE) .................................... 169 5.4 Off-line strategies ........................................................ 170 5.5 Future of D F - I C P - M D S for speciation ....................... 171 References ............................................................................. 172 C h a p t e r 4: Field-flow fractionation-inductively coupled plasma-mass spectrometry .......................................................................... 179 1. Introduction ........................................................................... 179 2. General overview .................................................................. 182 2.1 F F F modes ................................................................... 186 2.2 F F F sub-techniques ..................................................... 187 2.2.1 Sedimentation F F F (SdFFF) ............................. 188 2.2.2 Thermal F F F (ThFFF) ...................................... 190 2.2.3 Electrical F F F (E1FFF) ..................................... 192 2.2.4 Flow F F F (FIFFF) ............................................. 194 2.3 Instrumentation and optimization ............................... 198 2.3.1 Instrumentation ................................................. 198 2.3.2 Optimization ..................................................... 199 2.4 Quantitative analysis by F F F ...................................... 200 3. Selected applications ............................................................. 201 3.1 Sedimentation F F F (SdFFF) ....................................... 201 3.2 Thermal F F F (ThFFF) ................................................. 202 3.3 Electrical F F F (E1FFF) ................................................ 203 3.4 Flow F F F (FIFFF) ....................................................... 203 4. Comparison with SEC .......................................................... 204 5. Atomic spectrometry as element specific detection .............. 205 5.1 Literature ..................................................................... 205 5.2 F F F - I C P - M S for biological and environmental analysis ........................................................................ 210 5.2.1 Metal binding proteins ...................................... 210 5.2.2 Humic substances . ......... 211 5.2.3 Tissue and foodstuffs ........................................ 212 5.3 Quantitative analysis by F F F - I C P - M S ....................... 214 6. On-channel flow-fff preconcentration with atomic spectrometric detection ................................................................................ 215 6.1 Frit outlet ..................................................................... 216 6.2 Opposed-flow sample concentration ........................... 217 6.2.1 General overview and application .................... 217 6.2.2 On-channel matrix removal and pre-concentration ............................................................................ 221 7. Conclusion and future trends ................................................. 223

Acknowledgements ............................................................... 225 References ............................................................................. 226 C h a p t e r 5: Slurry sample introduction in atomic spectrometry : application in clinical and biological analysis ......................................... 237 1. Introduction ........................................................................... 237 2. Overview and nomenclature .................................................. 238 2.1 Slurry preparation ....................................................... 239 2.2 Particle size ................................................................. 240 2.3 Slurry concentration .................................................... 241 2.4 Chemical (matrix) modification .................................. 241 2.5 Calibration techniques ................................................. 241 2.6 Precision and accuracy ................................................ 242 2.7 Nomenclature .............................................................. 242 3. Slurry sample introduction .................................................... 242 3.1 Atomic absorption spectrometry (AAS) ..................... 243 3.1.1 Flame atomic absorption spectrometry(FAAS) .247 3.1.2 Electrothermal atomic absorption spectrometry (ET-AAS) .......................................................... 244 3.1.3 Flow injection techniques ................................ 246 3.2 Flame atomic emission spectrometry (FAES) ............ 247 3.3 Direct current plasma .................................................. 247 3.4 Inductively coupled plasma-atomic emission spectrometry (ICP-AES) ............................................. 247 3.5 Inductively coupled plasma-mass spectrometry (ICP-MS) ..................................................................... 248 3.6 Microwave-induced plasma-atomic emission spectrometry (MIP-AES) ............................................ 248 3.7 Atomic fluorescence spectrometry (AFS) .................... 249 3.8 Thermal vaporization (TV) techniques ........................ 249 4. Analytical figures of merit .................................................... 251 5. Practical applications of slurry sample introduction ............. 255 6. Conclusions ............................................ :.............................. 256 7. Suggestions for future studies ............................................... 257 Acknowledgements ............................................................... 258 References ............................................................................. 258 C h a p t e r 6: Application of laser-induced breakdown spectrometry in biological and clinical samples ............................................. 287 1. Introduction ........................................................................... 287 2. Fundamental studies .............................................................. 290 2.1 The interaction of a laser beam with target materials.. 290

2.2 2.3

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Laser-induced plasma production ............................... 293 Factors influencing plasma formation ........................ 295 2.3.1 Laser parameters ............................................... 295 2.3.1 A Influence of the irradiation wavelength.295 2.3.1 B Influence of irradiation energy ............. 297 2.3.2 Physical properties of the target material .......... 298 2.3.3 Ambient conditions ........................................... 300 2 . 3 . 4 Influence of electric and magnetic fields .......... 302 2.3.5 Plasma shielding ............................................... 303 2.3.6 Effect of sampling geometry ............................ 304 Excitation temperatures and electron densities ..................... 304 3.1 Excitation temperature calculations 304 3.2 Electron density calculation ......................................... 306 3.2.1 Electron number densities from stark broadening calculation .......................................................... 306 3.2.2 Electron number densities from Saha-Eggert ionization calculations ...................................... 307 3.3 Experimental results .................................................... 308 Spectral and analytical characteristics of LIBS .................... 310 4.1 Basic principles of LIBS ............................................. 310 4.2 Analytical characteristics ............................................ 311 Instrumentation ...................................................................... 314 5.1 Excimer laser and CO~ laser based LIBS .................... 316 5.2 Nd: YAG laser based LIBS instruments ..................... 316 5.3 Fiber-optic based LIBS instruments ........................... 316 5.4 Field instrumentation .................................................. 319 5.5 New approaches to LIBS ............................................ 322 5.6 Echelle spectrometer ................................................... 325 Applications ........................................................................... 326 6.1 Environmental applications ........................................ 327 6.2 Metallurgical samples ................................................. 333 6.3 Applications to liquids and solutions .......................... 339 6.4 Applications to aerosols and gases ............................. 342 6.5 Applications to non-metallic solids ............................ 343 6.6 Applications for advanced materials ............................. 345 6.7 Miscellaneous applications ......................................... 347 Conclusion ............................................................................. 348 References ............................................................................. 348 7: Application of graphite furnace atomic absorption spectrometry in biological and clinical samples ......................................... 361

~

Introduction ...................................................................................... 361 1.2 Spectroscopy ............................................................... 362 1.2.1. Introduction to atomic spectroscopy ................. 362 1.3 G F A A S analytical signal: absorbance ........................ 363 1.4 The nature of the transient G F A A S signal: m e c h a n i s m of atom formation in a graphite furnace ..................... 365 1.5 Instrumentation ........................................................... 366 1.5.1 Graphite furnace ............................................... 367 1.5.2 Graphite tube material and design .................... 368 1.5.3 Furnace heating cycle ....................................... 370 1.5.4 Methods of atomization .................................... 373 1.6 Sample preparation and sample introduction .............. 374 1.6.1 Liquids ........................................................... 375 1.6.2 Solids ................................................................ 376 1.6.3 Wet decomposition ........................................... 376 1.6.4 Combustion ....................................................... 378 1.6.5 Fusion ................................................................ 379 1.6.6 Solids analysis with slurry sampling (see Chapter 5 ) ................................................. 379 1.6.7 Direct solid sampling ....................................... 381 1.6.8 Laser ablation .................................................... 381 1.6.9 Preconcentration/separation methods ............... 381 1.6.9.1 Extraction ........................................ 382 1.6.9.2 C h r o m a t o g r a p h y ............................. 383 Flow injection analysis ................... 384 1.6.9.3 1.6.9.4 Other preconcentration/separation methods ........................................... 387 Metal speciation .............................. 387 1.6.9.5 1.7 Determination of elements by G F A A S ....................... 388 1.7.1 Applicability ..................................................... 388 1.7.2 Sampling, sample storage, and sample preparation ........................................................ 390 1.7.3 Quality control procedures ............................... 392 1.7.4 D e v e l o p m e n t of G F A A S methods .................... 393 1.8 Applications ................................................................. 396 1.8.1 Multielement continuum source G F A A S ......... 396 1.8.2 Determination of lead in blood by tungsten-coil AAS ................................................................... 397 1.8.3 Determination of arsenic and tin ...................... 398 1.8.4 Determination of c a d m i u m and zinc by double

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resonance laser-excited atomic fluorescence in an electrothermal atomizer ..................................... 400 1.8.5 Copper determination in biological materials by ETAAS using W-Rh permanent modifier .... 400 1.8.6 Determination of urinary lead, cadmium and nickel in steel production workers by GFAAS 401 1.8.7 Determination of platinum in clinical samples. 401 Conclusion .................................................................. 402 References ................................................................... 403 INDEX ....................................................................... 405

PREFACE

As of Volume 6, Elsevier Science has taken over the publication of this book series, previously published by JAI Press, Inc., CT, USA. Volume 6 was published as a special issue of Microchemical Journal, 2000, vol. 66, nos. 1-3, pages 1-172. The contents of the previous six volumes follow this Preface. This volume continues the tradition of the previous volumes with cutting-edge and current advances in atomic spectroscopy. A new development in the book series is that this volume and subsequent planned volumes have a focus in the area of atomic spectroscopy. This volume focuses on the application of atomic spectroscopy in biological and clinical samples. Where appropriate, the inclusion of other samples is provided to ensure complete coverage of a particular topic. Certain topics, e.g., LIBS in Chapter 6 are just beginning to find an application in this area and so its potential is discussed. Graphite furnace atomic absorption spectrometry (GFAAS) is well established and has a long use in this area. Chapter 7 discusses the technique and focuses on more recent applications A brief biography of all the contributors to this volume is given and a short abstract of each chapter of this volume is provided at the beginning of each contributed chapter. The editor of the book series (Joseph Sneddon) would like to thank the patience of all contributors and the reviewers for their excellent comments which have greatly enhanced this volume.

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Contents and Contributors to Volumes 1-6 in the Series

Volume 1 (1992) Chapter 1: Analyte Excitation Mechanisms in the Inductively Coupled Plasma Kuang-Pang Li, University of Massachusetts-Lowell, Lowell, Massachusetts, USA, and James D. Winefordner, University of Florida, Gainesville, Florida, USA. Chapter 2: Laser-Induced Ionization Spectrometry Robert B. Green and Michael D. Seltzer, Instrumental Chemical Analysis Branch, China Lake, California, USA Chapter 3: Sample Introduction in Atomic Spectroscopy Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Chapter 4: Background Correction Techniques in Atomic Absorption Spectrometry Gerald R. Dulude, Thermo Jarrell Ash Corporation, Franklin, Massachusetts, USA Chapter 5: Flow-Injection Techniques for Atomic Spectrometry Julian F. Tyson, Department of Chemistry, University of Massachusetts, Amherst, USA Volume 2 (1995) Chapter 1: Laser-Excited Atomic and Molecular Fluorescence in a Graphite Furnace David J. Butcher, Western Carolina University, Cullowhee, North Carolina, USA Chapter 2: Electrothermal Vaporization Sample Introduction into Plasma Sources for Analytical Emission Spectrometry Henryk Matusiewicz, Politechnika Poznanska, Poznan, POLAND Chapter 3" Hydride Generation Techniques in Atomic Spectroscopy Takahara Nakahara, University of Osaka, Sakai, Osaka, JAPAN Chapter 4" The Excimer Laser in Atomic Spectrometry Terry L. Thiem, United States Air Force Academy, Colorado Springs, Colorado, USA, Yong-Ill Lee,

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Konyang University, Nonsan, Chungnam, KOREA, and Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Chapter 5" Recent Developments in Analytical Microwave-Induced Plasma Robert C. Culp and Kin C. Ng, California State University, Fresno, California, USA Volume 3 (1997) Chapter 1: Plasma Source Mass Spectroscopy Andrew S. Fisher and Les C. Ebdon, University of Plymouth, Plymouth, Devon, England, UNITED KINGDOM Chapter2: Multielement Graphite Furnace and Flame Atomic Absorption Spectrometry Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA and Kimberly S. Farah, Lasell College, Newton, Massachusetts, USA Chapter 3: Direct Current Arcs and Plasma Jets Rudi Avni, Nuclear Research Center-Negev, Beer-Sheva, ISRAEL, and Isaac B. Brenner, Geological Survey of Israel, Jerusalem, ISRAEL Chapter 4: Direct and Near Real-Time Determination of Metals in Air by Impaction-Graphite Furnace Atomic Absorption Spectrometry Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Volume 4 (1998) Chapter 1: Electrostatic Precipitation and Electrothermal Absorption Spectroscopy: A Perfect Combination for the Determination of Metals Associated with Particulate Spectroscopy Giancarlo Torsi, Clinio Locatelli, Pierluigi Reschiglian, Dora Melucci, and Felice N. Rossi, University of Bologna, Bologna, ITALY Chapter 2: Chemical Modification in Electrothermal Atomic Absorption Spectrometry Dimiter L. Tsalev and Vera I. Slavekova, University of Sofia, Sofia, BULGARIA

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Chapter 3" Recem Developments in Flow-Injection Atomic Spectroscopy Maria Delores Luque de Castro and L. Gameiz-Garcia, University of Cordoba, Cordoba, SPAIN Chapter 4: Determination of Mercury by Atomic Spectroscopy Joseph Sneddon and Mary Gay Heagler, McNeese State, Lake Charles, Louisiana, USA Volume 5 (1999) Chapter 1 9Speciation Studies by Atomic Spectroscopy Miguel de la Guardia, M.L. Cervera and A. MoralesRubio, University of Valencia, Valencia, SPAIN Chapter 2: New Types of Tunable Lasers Xiadeng Hou, Jack X. Zhou, Karl X. Yang, Peter Stchur, and Robert G. Michel, University of Connecticut, Storrs, Connecticut, USA Chapter 3: Developments in Detectors in Atomic Spectroscopy Frank M. Pennebaker, Robert H. Williams, John A. Norris and M. Bonner Denton, University of Arizona, Tucson, Arizona, USA Chapter 4: Glow Discharge Atomic Spectrometry Sergio Caroli, Oreste Senofonte and Gialuca Modesti, Instituto Superiore di Sanita, Rome, ITALY Chapter 5: Laser Induced Breakdown Spectrometry Yong-Ill L e e , Changwon National University, Changwon, KOREA, and Joseph Sneddon, McNeese State University, Lake Charles, USA Volume 6 (2000) Chapter 1: Capillary Electrophoresis Inductively Coupled Plasma Mass Spectrometry Vahid Majidi, Los Alamos National Laboratory, Los Alamos, New Mexico, USA Chapter 2: Thermospray S a m p l e Introduction to Atomic Spectrometry Xiaohua Zhang, Ding Chen, Rob Marquardt and John A. Korpchak, Southern Illinois University, Carbondale, Illinois, USA

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Chapter 3: The Real-Time Analysis of Gases by Direct SamplingMass Spectrometry: Elemental and Molecular Applications David J. Butcher, Western Carolina University, Cullowhee, North Carolina, USA Chapter4: Use of Atomic Absorption Spectrometry for the Determination of Metals in Sediments in South-West Louisiana James N. Beck, Nicholls State University, Thibodeaux, Louisiana, USA, and Joseph Sneddon, McNeese State University, Lake Charles, Louisiana, USA Chapter 5" Field Instrumentation in Atomic Spectroscopy Xiandeng Hou and Bradley T. Jones, Wake Forest University, Winston-Salem, North Carolina, USA Chapter 6: Microwave Plasma Torch Analytical Atomic Spectrometry Wenjun Yang, Hanqui Zhang, Aimin Yu, and Qinhan Jin, Jilin University, Changchun, PR CHINA

Short Biography of Contributors to Volume 7

Joseph Sneddon is Professor in the Department of Chemistry at McNeese State University, Lake Charles, Louisiana. He attended the University of Strathclyde in Glasgow, Scotland obtaining a B.Sc. (honors) in Chemistry in 1976, M. Sc. in Instrumental Methods of Analysis in 1978 and Ph. D in Chemistry in 1981. He was a postdoctoral research fellow at the University of Strathclyde in 1980-81 and has served on the chemistry faculty at New Mexico State University, Las Cruces, New Mexico, California State University, Pomona, California, and University of Massachusetts, Lowell. He was Department Head at McNeese State University from 1992-1995. His research interests are in the general area of atomic spectroscopy, more recently in its application to environmental and biological samples. He has authored or co-authored over 140 papers and original articles in this area. He has edited several books, most recently Lasers in Atomic Spectroscopy (1997), Practical Guide to Graphite Furnace Atomic Absorption Spectrometry (1998), and Laser-Induced Breakdown Spectrometry (2000). He has been the editor of Microchemical Journal since 1990. Chapter 1 Marina Patriarca holds a Degree in Chemistry from the University of Rome, Italy and a M.Sc. in Medical Sciences from the University of Glasgow (UK). She currently holds the post of Senior Research Scientist at the Department of Clinical Biochemistry, Istituto Superiore di Sanit~ (ISS, Italian Institute of Health), in Rome (Italy), where she has been working as a Research Scientist since 1988. A large part of her research activity in the field of clinical biochemistry has been devoted to the development of methods for trace element analysis in human body fluids and tissues and investigations of their biological role in population studies. Further insight into human metabolism of trace elements has been obtained using stable isotopes as tracers in clinical studies, as part of the projects carried out during Dr. Patriarca's long-term collaboration with the Department of Pathological Biochemistry of the University of Glasgow (UK). Dr. Patriarca

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has authored more than 70 publications and has lectured in several international and national conferences and training courses on analytical and quality issues related to laboratory medicine. Antonio Menditto is a senior research scientist and Chief of the Section of Clinical Chemistry at the Department of Clinical Biochemistry of the ISS. Dr Menditto received his Degree in Medicine and Surgery from the University of Rome in 1984. He has presented over 80 lectures and seminars, and has published over 70 papers on various topics of biomedical research, human health, environmental toxicology, and laboratory medicine issues. He has served on the organizing and scientific committees of various national and international conferences. He has served on national and international environmental and human health committees including OECD and UNEP. Dr. Menditto and Dr. Patriarca have undertaken several activities for the promotion of metrology and quality assurance in the field of preventive, environmental and occupational laboratory medicine, among which the organization of the Italian national external quality assessment schemes (EQAS) for trace elements and metabolites of organic substances in body fluids, participation in the activities of the Thematic Network of European EQAS organizers in occupational and environmental laboratory medicine and collaboration to European Union projects for the certification of reference materials. Barbara Rossi received her Degree in Biology from the University of Rome I (Italy) in 1998. Since 1999 she collaborates with the Section of Clinical Chemistry, Department of Clinical Biochemistry at the ISS in the field of trace element analysis by atomic spectrometry and the promotion of quality assurance.

Chapter 2 Hiroaki Tao graduated from the Department of Chemistry of the University of Tokyo, Toyko, Japan in 1980. He received his Ph. D from the same university in 1986. He joined the National Institute of Advanced Industrial Science and Technology (AIST) in 1982. From 1993 to 1993 he was a visiting research fellow with the Institute for Environmental Chemistry, National research Council of Canada, where he worked with Dr. J.W. McLaren. H e has been the Group Leader of the Measurement Technology Group, Institute for Environmental Technology, AIST since 1999. His current research interests include elemental speciation using hyphenated methods such as gas chromatography-inductively coupled plasma-mass spectroscopy (GC-ICP-MS) and liquid chromatography-

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inductively coupled plasma-mass spectroscopy (LC-ICP-MS), sample preparation for atomic spectrochemical analysis, chemical sensors, and ETAAS. He is the author of about fifty publications including book chapters and research papers. He is a member of the editorial board of Bunseki Kagaku (Journal of the Japan Society for Analytical Chemistry) since 1998. Taketoshi Nakahara graduated from the Department of Applied Chemistry of Osaka Prefecture University, Osaka, Japan in 1965. He completed his Ph. D thesis at the same University in 1972. From 1976 to 1977 he was a visiting research fellow with the Department of Chemistry, Carlton University, Ottawa, Canada, where he worked with Professor of C.L. Chakrabarti. He was promoted to Associate Professor in 1985 and Professor in 1993 at the Osaka Prefecture University. His research interests include atomic absorption spectrometry, atomic fluorescence spectrometry with low temperature flames, atomic emission spectrometry with inductively coupled plasma and microwave induced plasma, inductively coupled plasma-mass spectrometry, and gas phase sample introduction techniques with vapor generation (e.g., hydride generation methods) for all kinds of analytical atomic spectrometry. He is the author of some one hundred and eighty publications including book chapters and research papers. Dr. Nakahara was the editor of Spectochimica Acta Reviews, Associate Editor of Applied Spectroscopy and a member of the editorial board of Spectrochimica Acta, Part B, and is currently a member of the editorial boards of Journal of Analytical Atomic Spectrometry, Atomic Spectrometry Updates, Canadian Journal of Analytical Sciences, Spectroscopy, and Microchemical Journal.

Chapter 3 Juan Manuel Marchante-Gay6n obtained a B.Sc. in Chemistry in 1990, and Ph. D in Analytical Chemistry in 1995 from University of Oviedo, Oviedo, Spain. He became an Assistant Professor at the University of Oviedo in 1995. His research interests and experience are centered mainly in the field of atomic spectrometry, with special emphasis in the areas of trace metal analysis and speciation in biological samples. He has published twenty papers. He is a member of the Spanish Society for Analytical Chemistry and Grupo Espectroquimica Espanol. Christina Sariego-Mufiiz obtained a B.Sc. in chemistry in 1995 from the University of Oviedo, Oviedo, Spain. She studied as an Eramus student at the University of Plymouth, Plymouth, United Kingdom in 1996. She started her Ph. D in 1997 at the University of Oviedo in the field of trace

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metal analysis and speciation in biological samples using inductively coupled plasma-mass spectrometry. Jose Ignacio-Alonso obtained a B.Sc. in Chemistry in 1980 and Ph. D in Analytical Chemistry in 1985 from the University of Oviedo, Oviedo, Spain. He was a postdoctoral research fellow at the University of Plymouth, Plymouth, United Kingdom between 1986 and 1987. He returned to University of Oviedo in 1987 as a postdoctoral fellow. In 1990 he became a scientific officer of the European Commission and was appointed to the Transuranium Elements, Joint Research Center, in Karlsruhe, Germany. After five years (in 1995) became a senior lecturer at the University of Oviedo, and in 1996 became head of Mass spectrometry Analytical Services at the University of Oviedo. His research interests and experience are centered mainly on the field of inductively coupled plasma-mass spectrometry, with special emphasis in the areas of trace metal speciation and Isotope Dilution Analysis, both for environmental and biological samples. In recent years, his research has concentrated on the development of semi-quantitative methods for trace metal analysis in environmental samples, the application of isotope dilution analysis for the analysis of biological materials and development and applications of interfaces for coupling gas chromatography to inductively coupled plasma-mass spectrometry for trace metal speciation. He has published around sixty papers. He is a member of the Spanish Society for Analytical Chemistry and fellow of the Royal Society of Chemistry and serves on the Editorial Board of Journal of Analytical Atomic Spectrometry Alfredo Sanz-Medel has been a Professor of Analytical Chemistry since 1982 at the University of Oviedo, Oviedo, Spain. After completing his Ph. D in 1973 at University of Zaragoza, Zaragoza, Spain in 1973, he was a postdoctoral research fellow in 1974 at Imperial College of Science and Technology, University of London, London, United Kingdom with Professor Tom S. West. He was an Assistant Professor of Analytical Chemistry at Complutense University, Madrid, Spain for four years and in 1978 joined the chemistry faculty at the University of Oviedo. He is the author of two hundred and forty publications, and several patents and books. He is a well-known speaker in his country and abroad about his group's research at the University of Oviedo. His research interests include three lines of analytical technologies; (a) new atomic detectors and methodologies for ultra-trace metals elemental analysis, particularly the use of plasmas (microwave induced plasma, glow discharge, inductively coupled plasmaatomic emission spectrometry and inductively coupled plasma mass spectrometry), (b) new molecular sensors, usually based on luminescence

xxiii

and fiber optic techniques for biological and medical applications, and (c) hybrid techniques for toxic metal analysis and speciation in biological and environmental samples, particularly the use of high performance liquid chromatography and capillary electrophoresis coupled with plasma detection. He was President from 1989 to 1999 of the Grupo Espectroquimico espanol and serves on the Editorial Board of Journal of Analytical Atomic Spectrometry, Microchimica Acta, and the Royal Society of Chemistry (United Kingdom) "Book Section" of monographs in Analytical Spectroscopy. Recently he has been appointed Associate Member of the Commission V of IUPAC. He also serves on the Editorial Board if ICP Information newsletter, Atomic Spectrometry Updates (RSC), Talanta, Anales de Quimica International and was a past member of the advisory board of Analytica Chimica Acta.

Chapter 4 Atitaya Siripinyanond is an analytical chemistry Ph. D student at the University of Massachusetts, Amherst since 1997. Her research (under the supervision of Professor Ramon M. Barnes) is focused on elemental speciation in biological and environmental samples using field-flow fractionation coupled to inductively coupled plasma mass spectrometry. She graduated with a B.S. in chemistry in 1994 and M.S in Chemistry in 1996 from Mahidol University, Bangkok, Thailand. She is supported by a fellowship from the Thai government funded through the Ministry of University Affairs. Ramon M. Barnes is Professor Emeritus of Chemistry at the University of Massachusetts, Amherst, where he served on the faculty since 1969. He has been conducting research on ICP and other discharges since 1968 when he spent one-year (1968-69) at Iowa State University, Ames, Iowa at a Postdoctoral Associate after receiving his Ph. D from University of Illinois, Urbana, Illinois in 1966. He serves as the chairman of the Winter Conference on Plasma Spectrochemistry, produced the monthly ICP Information Newsletter, and is director of the University Research Institute for Analytical Chemistry in Amherst, Massachusetts.

Chapter 5 Henryk Matusiewicz is Professor of Chemistry in the Department of Analytical Chemistry at Poznan University of Technology, Poznan, Poland. He received his Ph. D in 1973 and Dr. Sc (habilation) in 1987 in analytical chemistry from Poznan University of technology and University of Warsaw, Warsaw, Poland, respectively. In 1996 he was promoted to Professor of

xxiv

Chemistry. Since 1994 he has been the Head of the Analytical Chemistry Department at Poznan University of Technology. He was a Postdoctoral Research Associate at Colorado State University in 1975-1977 and the University of Massachusetts, Amherst in 1982-1984, visiting scientist at US Food and Drug Administration, Maryland, USA, Elemental Analysis Research Center in 1984-1985 and at NRCC, Institute for Environmental Chemistry, Canada in 1988-1996, and visiting professor at the University of Hanover, Germany, University of Dortmund, Germany, Max-Planck Institut fur Metallforschung, Germany, (1992), and University of Oviedo, Spain (1997).

Chapter 6 Yong-Ill Lee is an associate professor in the Department of Chemistry at Changwon National University, Changwon, Korea. He received a M.Sc. in Polymer Science in 1991 and Ph. D in Analytical Chemistry in 1992 from the University of Massachusetts, Lowell. He was a visiting research professor in the Department of Chemistry at Purdue University in West Lafayette, Indiana for 2000/2001. His main research interests are in analytical spectroscopy in general, laser and molecular spectroscopy and more specifically the development and application of new analytical techniques for atomic spectroscopy of advanced materials such as metals and ceramics. Recently he has started work on mass spectrometry in biological applications. He is a member of the editorial board of Microchemical Journal, Spectroscopy Letters and Applied Spectroscopy Reviews. Kyuseok Song is principal researcher of the Laboratory of Quantum Optics at Korea Atomic Energy research Institute (KAERI), Taejon, Korea. He received a M.Sc. in Physical Chemistry in 1982 from Korea University in Seoul, Korea and Ph. D in Physical Chemistry from Iowa State University in Ames, Iowa, USA. His major research interest has been in laser spectroscopy in general, analytical applications of atomic and molecular spectroscopy, and the development of new optical as well as mass spectroscopic techniques in the analysis of environmental samples. He has a strong interest in developing ultra-sensitive detection techniques for rare isotopes. He has authored over sixty scientific papers, six book chapters and a co-author of Laser-Induced Breakdown Spectrometry (2000). Joseph Sneddon see earlier

XXV

Chapter 7 David J. Butcher is professor in the Department of Chemistry and Physics at Western Carolina University, Cullowhee, North Carolina. He obtained a B.Sc. in Chemistry at University of Vermont at Burlington in 1984 and Ph. D in Analytical Chemistry from the University of Connecticut at Storrs in 1990 with Dr. Robert G. Michel. He is a member of the editorial board of Spectroscopy Letters, Microchemical Journal, and Applied Spectroscopy Reviews. He was the program chair of FACSS 2001. His research interests are in laser spectroscopy for chemical analysis, graphite furnace atomic absorption spectrometry, mass spectrometry, molecular spectroscopy and the application of these techniques to conifer forests and the environment in the western part North Carolina, Eastern part of Tennessee and southwestern part of Virginia. He is the co-author with Joseph Sneddon on the recent book, Practical Guide to Graphite Furnace Atomic Absorption Spectrometry (1998). Joseph Sneddon-see earlier

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Abstract of Chapters in Volume 7

CHAPTER 1 Use of Atomic Spectrometry (ICP-MS) in the Clinical Laboratory Since its introduction as an analytical technique, atomic spectrometry has found wide application in the clinical laboratory. More than 25 elements are important to human life, most of which present at trace or ultratrace levels. Several trace elements are routinely determined in body fluids and tissues for the diagnosis and monitoring of genetic diseases, nutritional deficiencies and occupational or environmental exposure. The choice of the method to apply for the determination of a specific trace element in a human sample requires a clear understanding of the clinical question and the relative performances and limitations of the available techniques. Inductively coupled plasma mass spectrometry (ICP-MS), the latest development of atomic spectrometry, has the capabilities for the fast and simultaneous determination of trace and ultratrace elements, with detection limits in most cases superior to graphite furnace atomic absorption spectrometry. High resolution ICP-MS can be used for the determination of most elements in body fluids and tissues, requiting only minimal sample pretreatment. Some of the interferences limiting the application of quadrupole ICP-MS to biologically important elements have been overcome using alternative methods of sample introduction, such as electrothermal vaporization and hydride generation, on-line chromatographic separation of interfering species and modified plasma conditions (cool plasma). New instrumental developments (collision/reaction cell technology) have been shown to reduce substantially the extent of major argide based interferences. Beside the determination of the total content of trace and ultratrace elements in clinical samples, the identification of their chemical species is necessary

xxvii

xxviii

in order to evaluate their bioavailability and relative toxicity. The on-line coupling of ICP-MS with separation techniques (HPLC, capillary electrophoresis) has been applied to the speciation of essential and toxic elements, such as As, Se and I, and to pharmacokinetic studies of metallodrugs. Stable isotopes are used as tracers in human studies to provide a direct assessment of the absorption, distribution and elimination of labelled compounds. In comparison with other techniques for the identification of isotope composition, ICP-MS allows faster sample throughput with. minimal sample preparation and it is therefore more suitable for studies of mineral metabolism. In addition, the development of ICP-MS reference methods based on isotope dilution can give an important contribution to the improvement of the quality and traceability of analytical data for trace elements in laboratory medicine.

CHAPTER 2 New Developments in Hydride Generation-Atomic Spectrometry Recent advances in hydride generation techniques in atomic spectrometry are overviewed. Fundamental research on novel hydride generation and chemical interferences and their elimination are described. Instrumental developments for speciation of the hydride forming elements, based on chromatographic or electrophoretic separation, post-column on-line sample pre-treatment and hydride generation followed by atomic absorption spectrometric or inductively coupled plasma-mass spectrometric detection are presented. Applications of these techniques in biological and clinical materials are also reviewed. Emphasis is placed on speciation.

CHAPTER 3 Analysis of Biological Materials by Double Focusing Inductively Coupled Plasma-Mass Spectrometry Inductively coupled plasma-mass spectrometry (ICP-MS) is (arguably) the most powerful detector in atomic spectrometry being the quadrupole mass filter the most popular analyzer in ICP-MS due to its relatively low cost and easy handling. However, the full potential of ICPMS cannot be exploited by conventional quadrupole-instrumentation because of spectral interferences. There are a variety of approaches by which such interferences may be compensated for in a practical analysis. However, the only general method to overcome limitations from spectral interferences is high mass resolution. Such high mass resolution can be obtained by Double Focusing-ICP-MS (DF-ICP-MS) instrumentation which combines a magnetic and an electric sector field analyzer. Although

xxix

available since 1988, DF-ICP-MS has not found widespread acceptance until recently, when the high cost of initial generation on instrumentation was considerably reduced with the introduction of a second generation DF-ICPMS instrumentation. This gave a strong impetus to the development of DFICP-MS applications in the analytical community. This is reflected in the increasing number of publications and an international conference devoted exclusively to high resolution sector field ICP-MS, and in general, a growing interest in the analytical performance of this technique. The aim of this chapter is to highlight the major areas of biological research where DF-ICPMS can provide an important contribution by reviewing both basic concepts of DF-ICP-MS and also recent developments in elemental analysis, isotope measurements and speciation of trace and ultratrace elements in biological and clinical samples. CHAPTER 4

Field-Flow Fractionation-Inductively Spectrometry

Coupled

Plasma-Mass

This chapter provides a current view of field-flow fractionationinductively coupled plasma-mass spectrometry (FFF-ICP-MS) applied to the biomedical, environmental, nutritional, and polymeric materials. Primarily the chapter is written to introduce practical information about FFF to the spectroanalytical chemist. The chapter begins with a section describing elemental speciation using chromatographic and non-chromatographic separations coupled with element specific detection techniques. A brief history of FFF and a general overview of different techniques in the FFF family follow. Four fields (i.e., sedimentation, thermal, electrical, and crossflow) that can be used for FFF are discussed. Selected applications of each FFF technique to biomedical and environmental samples are reviewed. After describing essential FFF features, (e.g., how it works, basic principles and physicochemical measurements, applications and application ranges, instrumentation and optimization), FFF is briefly compared with sizeexclusion chromatography especially for macromolecular characterization. The application of atomic/mass spectrometry as elemental detection for FFF is treated next with an emphasis on speciation in ICP-MS. A novel feature of flow-FFF (flFFF) for on-channel pre-concentration with either a frit outlet or opposed-flow sample concentration also is described. In the final section, FFF-ICP-MS is identified as an important growth area both for practical applications and research. Selected presentations made at the international conferences are presented.

XXX

CHAPTER 5 Slurry Sample Introduction in Atomic Spectrometry: Clinical and Biological Analysis

Application in

A short overview of slurry sample introduction in atomic spectrometry is presented, including both fundamental and physical considerations of slurry sample introduction. Methods for slurry sample introduction into atomic absorption spectrometry (AAS), inductively coupled plasmas (for atomic emission and mass spectrometry-AES, and MS, respectively), microwave induced plasmas (MIP-AES), direct current plasma (DCP), atomic fluorescence spectrometry (AFS) are reviewed and critically evaluated and the performance of these atomic sources for real sample determination is evaluated. Brief comparisons of detection limits for analytical atomic spectrometric methods that utilize slurry sampling as presented in most published reports are discussed. Finally, the literature on the application of the selected results from an updated application of slurry sampling techniques to clinical and biological materials are discussed and presented. CHAPTER 6

Laser-Induced Breakdown Spectrometry : Potential in Biological and Clinical Samples When the output from a pulsed laser is focused on to a small spot of a sample, an optically induced plasma, called a laser induced or laser ablated plasma is formed at this sample surface. This will occur when the laser power density exceeds the breakdown threshold value of the surface. When the laser created plasma is used as a source for atomic emission spectrometry, it is frequently called laser induced breakdown spectrometry (LIBS). In recent years this technique has attracted a great deal of interest from the analytical community, particularly in its application to situations where it clearly has advantages over conventional analytical atomic spectroscopic techniques. This chapter will give a brief overview of the basic principles and instrumentation for LIBS and will focus on the application to clinical and biological samples.

xxxi

CHAPTER 7

Application of Graphite Furnace Atomic Absorption Spectrometry in Biological and Clinical Samples Graphite furnace atomic absorption spectrometry (GFAAS) is an established, and reliable analytical technique for trace and ultra-trace metal determination in may samples. Despite its wide acceptance and maturity, it continues to find new applications. This chapter will primarily focus on these new applications as it applies to clinical and biological samples. Following a brief overview of the technique and instrumentation, the results and recent applications will be presented.

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Chapter 1

Use of atomic spectrometry (ICP-MS) in the clinical laboratory Marina Patriarca, Barbara Rossi, and Antonio Menditto Laboratorio di Biochimica Clinica, Istituto Superiore di Sanit/l, viale Regina Elena 299, 00161 Rome, Italy I. INTRODUCTION Of the elements in the periodic table more than 25 are important to human life [1-2]. Along with the constituents of organic matter, electrolytes (Na +, K +, Ca 2+, Mg 2+ and Cl) and trace elements (Co, Cr, Cu, Fe, I, Mn, Mo, Se and Zn) participate in biochemical processes necessary to maintain life and perform essential functions. Some of the non-essential elements pose threats to human health, when exposure occurs at the workplace or from the general environment. Other potentially toxic elements are deliberately administered as a therapy in severe illnesses (e.g., Li+ in manic depression, Pt complexes in cancer, Au in rheumatoid arthritis, Bi in gastric ulcer) and other metallodrugs are under development [3]. Alterations of the concentrations of trace elements in body fluids and tissues occur in pathological conditions, nutritional deficiency, following drug administration and as a result of occupational or environmental exposure. Measurements provide essential information for the prevention, diagnosis and monitoring of diseases and therapy [4-6]. Besides the determination of the total content of trace and ultratrace elements, increasing interest is paid to speciation, as the occurrence of an element in separate identifiable forms affects its bioavailability, metabolism and/or toxicity [7]. Atomic spectrometry has been applied in laboratory medicine since its introduction as an analytical technique. Electrolytes and some trace elements can be detected in biological fluids and tissues by simple and rapid flame atomic spectrometry methods. The development of electrothermal atomic absorption spectrometry (ETAAS) in the late '70s improved the detection limits by 10 to 100-fold and allowed the investigation of the biological role of trace and ultratrace elements [8]. The limitations of ETAAS" single-element analysis, time consuming

ADVANCES IN ATOMIC SPECTROSCOPY Volume 7, ISSN 1068-5561

1

Copyright 9 2002 Elsevier Science B.V. All rights reserved

M. PATRIARCA, B. ROSSI, and A. MENDITTO

procedures, prone to severe matrix interferences, stimulated research for alternative sources of sample atomization, which led to the development of plasma source atomic spectrometry [9]. Most elements are efficiently ionised in an argon plasma and may be detected on the basis of their optical emission or mass spectra. Both inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) can perform fast, multielement determinations and can be coupled on-line with separation techniques [9-10]. However, the concentrations of most trace elements (or their species) of interest in laboratory medicine are beyond the detection limits of ICPOES. In addition to its superior detection power, ICP-MS also has the ability to determine the isotopic composition of a sample, requiring much less sample pretreatment than other mass spectrometric techniques. Over the last 15 years, several innovative developments have occurred in ICP-MS and led to improved performances, especially for elements below mass 80, determination of which is affected by severe interferences. Although the cost of purchasing and rtmning an ICP-MS, including special laboratory requirements and trained personnel, is still high, the number of applications of ICP-MS in laboratory medicine is continually and rapidly increasing. According to manufacturers [11], the number of ICP-MS insmnnents sold each year is increasing, owing to the improvement in instrument performance, the development of simplified operating procedures and userfriendly software and the ever-increasing demand for the measurement of a greater number of elements and their chemical species at lower concentration in several fields of human activities. These considerations suggest that the use of ICP-MS in the clinical laboratory will continue to grow. Several papers and books have reviewed technical aspects of atomic spectrometry [9-10, 12] and its biomedical applications [8, 13-16]. Updates of new applications to clinical and biological samples are published regularly[ 17-20]. This contribution aims to give an overview of the use of atomic spectrometry in the clinical laboratory, with special reference to applications of ICP-MS. The suitability of different analytical atomic spectrometric techniques for their application to specific problems in the clinical laboratory is discussed. The technical features of ICP-MS and its latest development are reported in more detail. Particular attention is paid to the application of atomic mass spectrometry in laboratory medicine for the determination of trace element species for clinical and biological monitoring purposes and for the study of the metabolism of elements in humans using stable isotope tracers. Other important developing areas are elemental speciation and the use of isotope dilution ICP-MS (ID-ICP-MS) for the establishment of the traceability chain for the results of measurements of elements in biological materials.

Atomic Spectrometry in the Clinical Laboratory

2. ATOMIC SPECTROMETRY LABORATORY

TECHNIQUES

IN THE

CLINICAL

2.1 Requirements for trace element analysis in the clinical laboratory The specimens to be analysed are typically blood, serum or plasma and urine. The analysis of hair, nails and tissues (generally liver and bone biopsies) may also be required. Other less common specimens include cerebrospinal fluid, saliva and seminal fluid. Typical reference ranges for the electrolytes and trace elements most commonly measured in the clinical laboratory are reported in Table 1 [6, 8, 14-15, 21-26]. The determination of one or more elements may be necessary in the same sample and the level of accuracy required may vary from simple screening procedures to confirmatory tests. The choice of the method to apply to the determination of a specific trace element in a human sample requires a clear understanding of the clinical problem and the performances and limitations of the available techniques. Beside appropriate detection limits and reliable analytical performances, the size of sample needed, the throughput time, the ability for multielemental analysis, the level of operator skill needed and capital and running costs are all important variables to be taken into account. The fimess for purpose of analytical methods applied in laboratory medicine should be evaluated according to clinical needs [27-29]. It is generally agreed that criteria based on clinical efficacy or biological variation should be used whenever possible to set standards of analytical performance [30-31]. Recommendations of experts (individuals or groups), standards set by laws, regulatory bodies or organisers of external quality assessment schemes (EQAS) and the state of the art, judged from the results of interlaboratory comparisons or the literature, can be used when other data are not available [30-31]. In Table 2, standards of desirable performance for imprecision, bias and total allowable error, estimated from available data on intra- and interindividual biological variation, are reported for some electrolytes and essential trace elements [32-33]. Table 3 shows the maximum allowed errors, in terms of deviation from the target values, set by the organisers of the Italian EQAS for trace elements in biological fluids [34].

M. PATRIARCA, B ROSSI, and A. MENDITTO

Table 1 Reference ranges for electrolytes and trace elements in biological fluids [6,8,14-15]

Element

Specimen, unit

i?

General population

A1

Sa'",

As

U b, ~tg L l U, lag L1

1-30 1-60

Au

S/P, lag L "l

<0.1

Ba

U, jag L~

Be Bi

U,/ag L "l S/P, lag L l U, ~tg L "l S/P, mg L ~ U, mg L ~ WW, btg L l U, ~tg L "l WB, lag L "1 S/P, ~tg L 1 U, ~tg L "l WB, ~tg L ~ S/P, lag L "l U, lag L "1 S/P, mg L -~ U, lag L "l Liver, ~tg g-i d w f S/P, mg L l U, btg L "l Liver, ~tg g-1 dw WB, lag L- 1 U, lag L "1 U, lag L l S/P, mmol L l U, mmol L "1 S/P, ~tg L l S/P, mmol L "l U, mmol L "l

Ca Cd Co

Cr

Cu

Fe

Hg I K Li Mg

0.5-10

Reference ranges Subjects Occupational undergoing medicine therapy

30

1o-i 50 10-200 15-300

0.4-5 mg L l

<1-10 0.03-2 <0.1-3.5 <0.3-5 80-110 70-200 0.3-1.2 d , 0.6-3.9 e <0.1-2.5 <0.1-1 0.1-0.4 4-10 <0.2-3.0 <0.1-0.5 <0.05-0.5 <0.1-3.0 0.7-1.6 4-70 10-35 0.4-1.7 <40 500-900 <0.5-8 <0.2-20 100-200 3.5-5.1 20-80 <1 4-10 mg L ~ 0.65-1.05 2-3.5

10-100 1-10

1-50 1-100

2-15 10-600

1-25 1-300 1-35 1.5-50

5-.100 5-200

>5

Atomic Spectrometry in the Clinical Laboratory

Table 1 (cont. 'd) Reference ranges for electrolytes and trace elements in biological fluids [6,8,14-15]

Element Specimen, unit Mn

Mo Na Ni Pb Pt Sb Se Si T1 V Zn

WB, lag L "1 S/P, lag L "1 U, lag L -1 S/P, lag L "1 U, ~tg L -1 S/P, mmol L 1 U, mmol L -1 WB, ~tg L "l U, ~tg L l WB, ~tg L "1 U ~tg L "1 U lag L "1 WB, ~tg L l U, ~tg L l S/P, lag L l U, ~tg L "1 S/P, mg L l U, mg L "1 WB, ~tg L "1 U, lag L "1 S/P, lag L l U, ~tg L "~ S/P, mg/1 U, mg L "l

General population

Reference ranges S u b j e c t s Occupational undergoing medicine therapy

4-12 0.1-3 0.1-3 0.2-1.2 6-60 135-145 40-220 <0. l- 1 0.1-10.0 86.0 (m), 53.5 (0 g 4-40 <0.001-0.020 0.2-3 0.1-3 40-150 3-70 <0.2-4 <0.3-32 <0.1 <1.0 <0.2-1.0 <0.1-2.0 0.7-1.5 0.3-0.9

10-100 2-200

1.5-30 1.5-50 200-800 30-150 1-50 1-200

1-100 0.5-100

as/P=serum/plasma; bU--ufine; eWB----~hole blood; d non-smokers; * (smokers); f dw=dry weight; g observed in male (m) and female (f) adults, respectively, in a general survey of the Italian populatioL carried out between 1994 and 1996 [35]. Current values may be lower following the phase-out of leaded petrol.

M PATRIARCA, B. ROSSI, and A MENDITTO

Table 2 Analytical goals based on intra- (CVintra) and inter- (CVinter) individual biological variability for precision (CVa), bias and total allowable error (TAE%) of determinations of electrolytes and trace elements in serum [33]

Element

Specimen

Na Ca Cu Cu Fe K Mg Se Zn Zn

Serum Serum Plasma Serum Serum Serum Serum Plasma Serum Plasma

CVintr a

CVinte r

CV a

Bias

0.7 1.9 8.0 4.9 26.5 4.8 3.6 12.0 9.3 11.0

1.0 2.8 19.0 13.6 23.2 5.6 6.4 14.0 9.4 14.0

0.4 1.0 4.0 2.5 13.3 2.4 1.8 6 4.7 5.5

0.3 0.8 5.2 3.6 8.8 1.8 1.8 4.6 3.3 4.5

TAE% p=0.05 0.9 2.4 11.8 7.7 30.7 5.8 4.8 14.5 11.0 13.5

TAt~% p=0.01 1.1 3.1 14.5 9.3 39.7 7.4 6.0 18.6 14.1 17.3

2.2 Flame atomic spectrometry Flame atomic spectrometry can be used for the determination of electrolytes (Na +, K +, Ca 2+, Li+ and Mg 2+) and major trace elements (Cu, Zn, Fe) in biological fluids and tissues. For these elements, detection limits range from 1 to 100 ~tg L~, i.e. well below the concentrations to be measured. As a single element technique, it is best used when the determination of only one or few elements is required per sample. Instnanentation is relatively inexpensive and easy to operate. The technique is well established in the clinical laboratory and requires little or no method development. Typical interferences in clinical samples have been identified and can easily be kept under control, allowing the use of simple aqueous standards for calibration. Samples of biological fluids are generally diluted at least 1+4 prior to analysis, to avoid matrix effect generated from differences in nebuliser aspiration rates of samples compared to aqueous standards. In addition, dilution overcomes the effects of high salt and organic content which cause clogging of the sample introduction system and deteriorate the stability and repeatability of the signal. Determinations are fast (typically a few seconds) but use rather large sample volumes (typically 1-2 ml). Examples of the analytical performance for the determination of Ca, Li, Mg and Zn in serum are given in Table 4 using data obtained in our laboratory on reference materials. Vapour generation techniques are applied for the determination of Hg (cold vapour, CV) and As (hydride generation, HG).

Atomic Spectrometry in the Clinical Laboratory

Table 3 Analytical goals (TAE%) set in the Italian External Quality Assessment Scheme for the determination of trace elements in biological fluids [34]

Element A1

Specimen Serum

Cu

Serum

Se

Serum

Zn

Sertun

Cd

Whole blood

Pb

Whole blood

Cr

Urine

Ni

Urine

Concentration, lag L "l 10 120 500 1500 30 120 500 1500 1 15 100 800 1 10 1 10

TAE% 30 10 15 10 20 10 15 10 60 10 20 10 60 15 60 15

2.3 Electrothermal atomization atomic absorption spectrometry The detection limits for ETAAS range between 0.01 and 5 lag L ~ and are from 10 to 100-fold better than those achievable in flame atomic absorption spectrometry. This technique is suitable for the determination of minor essential elements and for the biological monitoring of environmental and occupational exposure to several trace elements, which concentrations in biological fluids typically range from less than 1 to several hundreds pg L l, according to the degree of exposure. Determinations require only 5 to 50 lal of sample for each analysis: therefore ETAAS is the technique of choice when the sample size is limited as in paediatric specimens. Matrix interferences affect ETAAS to a much greater extent than in flame atomic spectrometry. For the analysis of biological samples, the use of one or more of the now well established methods for interference control, such as Zeeman background correction, platform atomisation, matrix modifiers and matrix matched standards, is mandatory to achieve accurate and for reliable results.

M. PATRIARCA, B. ROSSI, and A. MENDITTO

Table 4 Performance of FAAS for the determination of some electrolytes and trace elements in serum

Element

Specimen

Calcium

Serum

Lithium

Serum

Magnesium

Serum

Zinc

Serum a

estimated

Concentration Repeatability mmolL l (%) 2.20 0.34 2.49 0.30 0.52 0.32 0.98 0.30 1.15 0.63 1.85 0.30 0.014 1.53 0.021 0.65 as percent difference from target value

Bias ~ (%) 0.2 0.8 -1.0 -1.0 1.3 0.1 1.1 -0.6

These precautions however, may not prevent the deterioration of the detection limits actually achievable for an element/matrix pair. The operation of ETAAS is more complex than flame atomic spectrometry and involves a certain amount of method development and validation. Personnel should have a higher level of training and should clearly understand both the technique and the need for strict contamination control associated with higher detection power [36]. Both the purchase and the nmning costs of ETAAS are more expensive than those for flame atomic spectrometry. A major drawback of ETAAS is that the sample throughput is very slow: generally, only one element at a time can be determined in each sample and each determination requires between 2 and 5 minutes. Some insmunentation is available for the simultaneous determination of up to 4 elements, but in the case of complex matrices, the quality of results is strongly dependent on the optimisation of the temperature programme, which must be tailored for the matrix/element pair. Typical analytical performance is illustrated in Table 5 for the determinations of various elements in whole blood, serum and urine using data obtained in our laboratory as examples.

2.4 Inductively coupled plasma optical emission spectrometry Argon plasma sources provide high temperatures (4000-10000~ in a chemically inert environment, where the processes of molecular dissociation, excitation and ionization of all species present in the sample occur with a much

Atomic Spectrometry in the Clinical Laboratory

higher efficiency than in a flame. Emission spectra are generated simultaneously for all elements, which can be rapidly determined in the same sample over a wide range of concentrations, owing to a linear dynamic range expanding from three to six orders of magnitude. Although chemical interferences are strongly reduced, spectral interferences are common and may be difficult to overcome, because of the Table 5 Performance of ETAAS for the determination of some trace elements in biological fluids

Element A1 Cu Se Cd Pb Cd Cr Ni a

Specimen

Concentration Repeatability pgL "~ (%) Serum 39 3.2 122 1.8 Serum 962 1.0 1236 0.9 Serum 96 1.6 116 4.0 Whole blood 1.4 15.3 6.6 3.0 Whole blood 212 1.0 501 1.2 Urine 0.52 12.3 6.6 1.19 Urine 1.2 2.5 6.7 2.7 Urine 2.5 3.7 11.0 3.0 estimated as percent difference fi'om target value

Bias a (%) -1.4 1.9 0.8 0.0 -0.5 -0.7 -3.7 1.7 5.7 3.0 -3.8 -3.3 -0.9 -4.2 -10.6 4.8

complexity of optical spectra. At low concentrations, achieving accurate results depends on the choice of appropriate methods for the correction of the continuum background emission. Instrumentation is moderately expensive and relatively easy to use, but training and experience of the operators is mandatory for optimal results [37]. The detection limits of vertical ICP-OES range from 0.5 to 50 lag L l for most elements of clinical and toxicological interest, but axial view instruments can achieve detection limits up to 10-fold lower. In the clinical laboratory, ICP-OES is applied to the simultaneous determination of electrolytes (Na +, K § Ca 2§ Li§ and

M. PATRIARCA, B. ROSSI, and A. MENDITTO

10

Mg 2.) and major trace elements (Fe, Cu, Mn, Zn,) in biological fluids and tissues [38-43]. In most cases, biological fluids can be analysed without sample digestion or pretreatment other than dilution. Introduction of digested samples by HG allows the determination of As [44], Se [45] and other hydride forming elements. Examples of analytical performance are given in Table 6. Table 6 Performance of lCP-OES for the determination of electrolytes and trace elements in biological specimens Element

Specimen

Concentration

Repeatabilit y(%) A1 S 8-54 ~tg L ~ 9-2.7 S 27-160 ~tgLl 10-3 B S 2.3 ~tmolL 1 2 Ba S 0.67 ~tmol L 1 0.6 Ca S 2.45 mmol L l 1.1 U 3.3 mmol L l 1.7 Cu S 1300 lag L -~ 0.6 S 630-1600 lag L ~ 0.9-1 U 200 ~tg L "1 5 Fe S 18 ~tmol L -1 0.5 S 11 ~tmolL -l 7 K U 53 mmol L l 1.2 Li S 1.54 lamol L ~ 9 Na U 160 mmol L l 1.3 Mg S 1.05 mmol L l 4.4 U 5 mmol L l 1.2 P U 5 mmol L l 0.6 Se S 130 lag L -~ 9 Si U 8-39 mg L ~ 0.8-1.7 Zn S 600-1600 ~tg L ~ 2.1-0.8 a expressed as percent recovery of spiked concentrations

Bias a Ref. (%) 98-108 [46] 72-113 [47] 91-99 [42] 99-116 [42] 99.4 [38] 103 [40] 88-106 [42] 98-115 [46] 105 [38] 73-116 [42] 102 [38] 100 [40] 106-113 [42] 104 [40] 101 [38] 102 [40] 91-103 [39] 78-104 [42] 100-110 [50] 96-102 [46] or target values

Because of its speed, ICP-OES can be preferred to ETAAS when only the identification of samples exceeding action limits is required, such as in programmes for the monitoring of A1 levels in dialysis fluids and serum from patients undergoing haemodialysis or total parental nutrition [46-47]. Finally, ICPOES plays an important role for the determination of P and S, which are difficult to

Atomic Spectrometry in the Clinical Laboratory

determine by other techniques, and for the investigation of the fate of several elements used in therapy and implants. For example, considerable interest has been paid in the last few years to the measurement of the levels of Si in blood, serum, breast milk, and body tissues [48-50], as suitable biomarkers of the leakage of silicone gel breast implants, although, up to now, none of them has been shown to have a significant predictive value [51]. Increased Si levels have also been observed in serum of tmdialysed patients with chronic renal failure [48]. Boron and metal-based compounds are used in medicine as drugs (e.g. the anticancer drugs sodium borocaptate, cisplatin and carboplatin) and contrast media in magnetic resonance imaging (e.g. Gd as Gd-DTPA and gadobenate dimeglumine; Mn as mangafodipir). Their concentrations in biological fluids and tissues are measured by ICP-OES to determine the absorption, distribution, and elimination profiles in experimental, preclinical and clinical studies [52-61 ]. Investigations of the distribution of major essential trace elements [62-63] and metallodrugs [64-65] among different species in biological fluids can be performed by on-line coupling to separation techniques.

2.5 Inductively coupled plasma mass spectrometry Because of the importance of ICP-MS and its rapid development over the last 15 years, details of the technique and of its applications are dealt with in more detail in the following Sections. The most important features of the technique are superior detection power and the ability to determine the isotope composition of a sample, in addition to the capabilities shared with other plasma source techniques, such as fast, multielement determinations, a wide linear dynamic range and easy on-line coupling to separation techniques. Although the imtrtmaentation is expensive and requires special laboratory facilities and skilled operators, ICP-MS has already found numerous and unique applications in the clinical field.

3. INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY

3.1 Fundamentals and recent development of the technique Inductively coupled plasma mass spectrometry was developed as an analytical technique in the early 1980s [66-67]. Over the last 15 years, several instrumental and methodological developments have expanded fuaher its versatility and power of detection [68-74]. Instrumentation consists of an inductively coupled plasma torch, placed horizontally and interfaced to a mass analyser via two especially designed metallic cones (skimmer and sampler). This design allows the extraction into the mass analyser of the central zone of the ion beam, where the concentration of ions from the sample is higher. In the ICP, the

12

M. PATRIARCA, B. ROSSI, and A. MENDITTO

species present in the sample solution are dissociated into their atoms and up to 90% of most elemems are ionised, with a small fraction of multiple charged ions (ca. 1%). A system of ion lenses, placed behind the cones, direct ions to the mass analyser, whereas uncharged atoms and molecules are expelled. The ions entering the mass analyser are separated and detected according to their mass/charge (m/e) ratio. The region between the two cones, the ion lenses system and the mass spectrometer are evacuated to a low pressure by cryogenic and mechanical pumps. Samples are generally introduced into the ICP torch as liquids by pneumatic nebulisation. A computerised system provides automatic control of the various functions of the instrumem, data acquisition and handling. Several instrumental configurations are commercially available. Instnunems equipped with quadrupole mass analysers (Q-ICP-MS) are less expensive and easier to use, but have a limited resolution power (Am/m ~ 300), only permitting the separation of species on a m/e unit basis. This is often insufficient for adequate resolution of the analyte signal from those of isotopes of neighbouring elemems, doubly charged ions and molecular species. For the exploitation of the full potential of the technique, the ICP source has to be coupled to a high resolution sector field mass analyser (HR-ICP-MS) [75-76]. These instrumems, although considerably more expensive and complicated to operate, can achieve resolution (Am/m) between 7500 and 12000 and have lower background (<0.1 ions/s) and higher sensitivity than Q-ICP-MS. Sector field instnmaems equipped with multicollector devices (MC-ICP-MS) provide highly accurate determinations of isotope ratios with a precision at least a factor of 3 better than that of the sequentially operating ICP-MS, and by faster and simpler methods than are used for thermal ionisation mass spectrometry (TIMS) [77]. Another technique of mass spectrometry is based on the measurement of the time of flight (TOF) of ions of known energy over a known distance [78]. Plasma source TOF mass spectrometers (TOF-ICP-MS) have been developed during the past few years, with both orthogonal [79-81 ] and axial off-axis detection [82-83]. Since the TOFs of all ions presem in the source at the starting point are measured almost simultaneously, within a few ~ts, the method is especially attractive for the analysis of rapid transient signals, such as those generated by electrothermal vaporisation (ETV), and for work with hyphenated techniques. The latest developmem in Q-ICP-MS are insmunems that include collision (CC) [84-86] or dynamic reaction (DRC) [87-89] cells, i.e. ion-guiding quadrupoles or hexapoles enclosed in a gas-filled cell. Collision cells are already used in organic mass spectrometry, to study controlled processes of fragmentation of organic molecules. Their application in inorganic mass spectrometry has been shown to reduce greatly most spectral interferences affecting the determination of elemems in Q-ICP-MS, by inducing the dissociation of major molecular ions to

Atomic Spectrometry in the Clinical Laboratory

13

their atomic species. In addition, the reduction in ion energy spread resulting from the collisions improves ion transmission efficiency, sensitivity of elements and precision of isotope ratios [90-92]. In the first insmmaent of this kind (Platform ICP-MS), introduced by Micromass Ltd., a hexapole collision cell, which works as an ion optical lens system, is inserted between the interface and the quadrupole mass analyser. The hexapole is mounted off-axis in the collision cell chamber to reduce the background in mass spectra by photons from the ICP. Collision induced reactions, such as charge transfer and proton transfer reactions, take place between the ions from the plasma and the gas molecules (HE or He), causing the dissociation of major Ar-based molecular ions, such as ArO § ArC +, ArN +, ArNH +, ArIT, Ar2+, and the neutralization of Ar+ [93]. A new concept was developed by Perkin-Elmer with the Dynamic Reaction Cell (DRC). In this case, a quadrupole based reaction cell is inserted between the ion optic lens system and the quadrupole mass analyser. The DRC operates under lower energy (thermalized) conditions, so that reactive gases (NH3, CI-h) can be introduced in the cell. Both collision or chemical reactions can be used to destroy or create new ions to meet the analytical needs and enhance the analytical performance. NHa, CH4, HE and He have been successfully used as the reaction/collision gas for different applications. To eliminate secondary reaction products, the instrtmaem exploits the quadrupole bandpass filtering ability, operating the DCR quadrupole bandpass in concert with that of the quadrupole mass analyser. Applications of these newly designed instruments have already been reported, some of which on biological specimens [94-97].

3.2 Techniques for sample introduction Samples are generally presented to ICP-mass spectrometers as solutions. Solid sampling can be performed by means of laser ablation, [98] but has been rarely used for the analysis of clinical specimens. The choice of the best method for sample introduction depends on the analytical problem at hand. In some cases, an alternative method of sample introduction permits significant improvements in the performance of the method. In general, ICP-MS shows poor tolerance towards high salt and organic content of the solution, because the introduction system can easily become clogged and deposits accumulating on cones lead to greater imprecision and signal instability. Pneumatic nebulisation is the conventional method of sample introduction into a plasma. The sample solution is pumped into the ICP via a peristaltic pump, to maintain a constant flow rate, despite between sample differences of viscosity and density. A fine aerosol is obtained by means of devices of various design

14

M. PATRIARCA, B. ROSSI, and A. MENDITTO

(Meinhard, Cross-flow, Babington, V-groove). An argon flow carries the finer droplets into the plasma, whereas the larger ones condense over the walls of the spray chamber. The sample uptake rate is generally 0.8-1.0 ml min but only 1 to 4% of the sample solution finally reaches the plasma. Several new nebulisers have been designed (microconcentric nebuliser, MCN [99]; direct injection nebuliser, DIN [100-101 ]; hydraulic high pressure nebuliser, HHPN [102]; high-efficiency cross-flow micronebuliser, HECFMN [103]) which use lower sample volumes (<0.5 ml) and generate smaller and more uniform droplets, thus achieving a much higher efficiency of analyte transport to the plasma. The MCN typically uses sample volumes between 30 and 100 laL min~, with an efficiency of about 90% [104]. The HECFMN can operate with sample uptake rate between 5 and 120 laL minl, achieving a transport efficiency between 24 and 95% without loss of performance when compared with conventional cross-flow nebulisers consuming 1 mL min-1 sample. A finer aerosol is obtained with ultrasonic nebulisers (USN). The sample transport into the plasma is from 3 to 10 times more efficient than with conventional pneumatic systems, resulting in 10 to 50 fold improvement of detection limits. The USN requires aerosol desolvation, to avoid cooling of the plasma and increased oxide formation. Its application to biological samples is hampered by memory effects and poor tolerance of high salt content [ 105]. For sample introduction by means of electrothermal vaporisation (ETV), a graphite fin'nace, similar to those used for ETAAS measurements, is connected to the plasma torch via an argon stream. The sample, usually 5 to 50 lal, injected into the fiamace, undergoes a multistep temperature programme. Water and other matrix components are sequentially evaporated and the resulting dry vapour is transferred to the plasma. This method of sample introduction allows the elimination or considerable reduction of most oxide and chloride based interferences (e.g. ArO on Fe), and improves absolute detection limits to fg [106]. Although ETV is prone to similar problems as in ETAAS, with effects on signal reproducibility, the accuracy of ETV-ICP-MS methods can be greatly improved by using isotope dilution [107108]. The principle of hydride generation is used also in ICP-MS for elements such as As and Se, to eliminate matrix interferences and achieve better limits of detection. After complete mineralisation of the sample, the elements are reduced by NaBH4 to their volatile hydrides and transported into the plasma [109-112].

Atomic Spectrometry in the Clinical Laboratory

15

4. DETERMINATION OF TRACE ELEMENT CONCENTRATIONS IN BODY FLUIDS AND TISSUES

4.1 Background Soon after the introduction of ICP-MS as an analytical technique, initial work was carried out on clinical samples, such as human serum, urine, plasma protein solutions and tissues [113-117], and confirmed its potential as a versatile and powerful tool for applications in the clinical laboratory, although limited to some extent by the occurrence of polyatomic interferences. Following developments in insmnnentation some of these difficulties have been overcome and the applications of ICP-MS to elemental analysis in body fluids and tissues have continuously grown. To date, most elements of interest in medicine can be determined by Q-ICP-MS or SF-ICP-MS. The ability of ICP-MS to determine in principle any element and its improved limits of detection have been exploited to investigate reference values and biological role of elements, such as Mo, V and the rare earth elements with concentrations in biological fluids and tissues that are beyond the power of other atomic spectroscopy techniques. The capability of plasma sources to determine more than one element in the same sample allows for fast screening procedures, with an accuracy of +30% [118], and reduces the time and cost per analysis, exploiting the amount of information available from a single specimen. This feature can be particularly valuable for the analysis of clinical specimens available in small quantities, such as biopsies and paediatric samples, provided that sufficient sensitivity can be obtained.

4.2 Sample preparation Biological fluids cannot be introduced directly into the plasma, as their high salt and organic content causes signal suppression, deterioration of signal stability and clogging of nebulisers. Sample preparation is therefore necessary, but may be limited to a simple 1+4 or 1+9 dilution for most elements of clinical interest in blood, serum, urine and other biological fluids. Sample digestion is mandatory for tissues and is recommended even for biological fluids, on the grounds that both spectral and non spectral interferences are reduced and long-term stability is improved. Microwave aided sample mineralisation, in the presence of minimal amounts of oxidising agents (HNO3, H202, etc.) has proven successful in most cases. For simpler matrices, such as urine, serum or blood, UV digestion has been reported [119-120]. With Q-ICP-MS, further specific sample pretreatmem may be required to eliminate matrix interferences and achieve reliable results and lower limits of detection (see 4.3).

16

M PATRIARCA, B. ROSSI, and A. MENDITrO

4.3 Interferences and their control

4.3.1 Spectral interferences Spectral interferences in ICP-MS arise from the overlapping between isotopes of different elements (isobaric interferences), doubly charged ions or molecular species formed at high temperature from the reactions between matrix components, including water, and the carrier gas (polyatomic interferences) [121-122]. The occurrence of spectral interferences drastically increases the limits of detection for several elements, especially in complex matrices such as biological fluids and digests, and reduces the number of isotopes available for measurement. Common spectral interferences affecting the determination of elements in clinical samples are reported in Table 7. Among the methods used to overcome these problems, SF-ICP-MS provides the greatest advantages. Most interferences hampering the determination o f trace elements in clinical samples are separated using a resolution factor of about 4000, although a loss in sensitivity is associated with resolution factors > 3000. This may be a problem, if the size of the sample available is small or the element concentration low [ 16, 76]. In Q-ICP-MS, collision or dynamic reaction cells provide a new means to overcome polyatomic interferences and are expected to find wide application in the future. With current Q-ICP-MS insmmaents, some interferences can be eliminated by choosing methods of sample introduction, which allow the chemical or physical separation of the analyte from the matrix. Interferences from ArC1+ on the determination of As and Se in biological matrices is generally avoided by generating their volatile hydrides, which can be transported directly into the plasma. Sample introduction via ETV is effective in reducing most oxide-based interferences, such as those affecting clinically important elements (e.g. Fe, Se). The choice of the fimaace temperature programmes must be tailored for the specific analyte/matrix at hand, thus posing limitations to multielemental analysis in biological matrices and to internal standardisation other than by isotope dilution. Alternatively, the analyte can be separated from interfering species prior to analysis by off- and on-line chromatographic methods, provided that the dilution factor does not prove prohibitive, or by simple chemical procedures. Most transition elements form stable neutral chelates, which can be extracted in organic solvents. This approach removes interferences from major components of biological matrices, such as Na, K and C1, and increases sensitivity by concentrating the analyte, but it is rather time-consuming and prone to contamination. As most transition elements are likely to be co-extracted, isobaric interferences with other overlapping isotopes are not excluded.

AtomicSpectrometryin the ClinicalLaboratory

17

Table 7 Typical isobaric and polyatomic interferences affecting the determination of elements of clinical or toxicological importance in biological matrices

Element

24Mg 27A1

Interferences

12C2+

285i

14N2+spread 14N2+, 12C160+

31p 32S 39K 4~ 51V 52Cr 55Mn 56Fe 58Ni 58Ni 59C0 63Cu 64Zn 67Zn 7~ 75As 77Se 79Br 8~ 95Mo

14N16OH+ 1602+ 38ArH+ 4~ + 35Cl160+ 4~ 36Ar160+, 36S160+, 35Cl16OH+ 4~ + 4~ 58Fe+ 4~ 42Ca160+, 41Cal6OH+, 4~ 43Ca160+ 4~ +, 31p1602+ 32S1602+ 35Cl1602 + 35C12+ 4~ +, 38Ar37C1+ 4~ + 4~ 40AF2+ 79Br160+

97Mo

81Brl60+

Required resolution ~,1600 ~ 1600 ~1600 ~970 ~ 1800 ~5700 ~193000 ~2600 ~3000 ~3000 ~2500 ~28000 ~3000 ~3000 ~3000 ~--~000 ~,~4000 ~6000 ~7800 ~9000 ~ 10000 ,~10000 ~12000 ~18000

Operating the plasma under 'cold' conditions, i.e. at low power with a high nebuliser flow rate, reduces the occurrence of Ar-based interferences and elements with low ionization energy, such as K, Ca and Fe can be determined with lower detection limits [123]. However, there is little effect on non Ar-based interferences and additional polyatomic species may be generated from complex matrices when operating under these conditions [ 124].

18

M. PATRIARCA, B. ROSSI, and A. MENDITTO

The addition of other gasses (e.g. N2, He or CH4) to the carder gas [ 125-126] or organic solvents, such as methanol or butanol, to the diluent [127-128] have also been shown to reduce some of the Ar-based interferences and enhance the response for elements with ionisation energy in the 9-11 eV range, such as As, Se and Hg.

4. 3.2 Non spectral interferences Other, non spectral, interferences occur in ICP-MS which are due to physical influences on the sample introduction system, fluctuation of the plasma stability affecting the intensity of the signal, differences between samples and standards affecting sample introduction, sample transport and the properties of the plasma. These can be generally controlled using an internal standard. Ideally the element chosen as intemal standard should be monoisotopic, unlikely to be found at concentrations above the limit of detection in the sample itself, unaffected by spectral interferences and of mass and ionisation energy close to those of the elements of interest. In practice, compromises are inevitable. In multielemental analysis more than one internal standard is often necessary, spread across the mass range scanned: Sc, for masses up to 80 amu, Rh or In, for the range between 80 and 150 amu and Ir, for masses above 150 amu are frequently used.

4.4 Applications For several elements, ICP-MS provides lower detection limits than other techniques and for some, it is the only technique capable of achieving reliable results at the concentrations found in biological fluids and tissues. Even when these concentrations are much lower than those associated with known clinical effects, establishing their ranges for unexposed subjects is essential to serve as references in the assessment of exposure to environmental pollutants. It should be borne in mind that became of this higher power of detection, control of contamination is of the utmost importance, requiring in some cases, specialised handling facilities, such as Class 100 laboratories. Reagents should be of the highest purity available, as the actual limits of detection depend on the blank values. Calibration is generally performed using aqueous standards, however, matrix matched standards should be used if matrix effects are not negligible. All analytical procedures should be thoroughly validated, whenever possible by analysing matrix-matched certified reference materials. Quadrupole ICP-MS instnunents are by far the most popular, due to their lower cost and easier handling. Several elements of interest in environmental and occupational medicine can be measured by this technique in blood (Be, Cd, Hg, Pb, Sb) sertun (Ba, Bi, Cd, Cs, Hg, Pb, Mo, Sb, Sn) or urine (Ba, Be, Bi, Cd, Hg, Pb, Sb, Sn, Te, Th, T1, U, W) with limits of detection between 1 and 500 ng L "~ [14, 113-115]. At least fourteen elements of clinical interest (B, Br, Ca, Cu, 54Fe, I, K,

Atomic Spectrometry in the Clinical Laboratory

19

Li, Mg, 98Mo, Pt, Rb, Sr, Zn) can be reliably determined in serum samples, in some cases with an appropriate choice of isotopes and mathematical corrections of interferences [113-115, 129]. Generally, procedures for the simultaneous determination of elements in blood, serum or urine have been implemented, which significantly increase the efficiency of routine laboratories and exploit the amount of information obtained from a single sample. The interferences affecting the determination of As in urine and Se in serum and urine can be overcome using HG-Q-ICP-MS, after complete digestion of the sample, with limits of detection of about 0.1 lag L 1 for As and between 0.6 and 1.8 ~tg L "1 for Se in serum. Alternatively, accurate determinations of As in urine can be performed with mixed plasmas (N2) or diluting the samples with 1% ethanol [ 130]. A similar direct procedure for the measurement of Se in serum, whole blood and erythrocytes by ICP-MS aiier dilution with a solution containing 1.0% v/v butan- 1-ol has been described [ 128, 131 ]. With careful selection of analytical masses, chemical modifiers and temperature programmes, elements overlapped by polyatomic interferences can be measured reliably by ETV-Q-ICP-MS and improved absolute limits of detection can be obtained for other elements. S e l e n i u m (77Se and 82Se) was determined in 10 ~tl of 20-fold diluted blood serum, with a limit of detection of approximately 0.1 lag L 1 and a long-term precision of 3.8% [132]. Aluminium, Ti and V were measured in serum with limits of detection of 0.7, 0.4 and 0.1 ~tg L "1 [133]. Improved limits of detection have been reported for the determination of Mo in serum (10 ng L "l) [134], Pt in urine (1 ng L "l) [135] and rare earth elements in urine (from 1 to 10 ng L -l) [136]. Became of the variability of transient signals, the best results are achieved using isotope dilution for standardisation. The determination of Cd and Pb in twine by ETV-ID-ICP-MS yielded limits of detection of 20 and 5 ng L l, respectively, and precision < 11% [ 137]. Several papers have reported the simultaneous determination of trace elements in autopsy tissues by Q-ICP-MS. However, the level of accuracy varies from element to element, depending on concentrations and the analytical conditions adopted. According to an investigation carrried out on certified reference materials (CRMs), including Bovine Liver NIST SRM 1577b and Human Hair NCS DC 73347, Ba, Cd, Cr, Cu, Li, Mn, Ni, Na, Pb, Sr, and Zn can be reliably determined in sample aliquots ranging from 1 to 50 mg, with limits of detection ranging from 0.02 to 0.38 ~tg g-i [ 138]. The analysis of foetal and paediatric tissues is one of the more demanding as the concentrations of most elements are low. In a study of 157 paediatric livers, the concentrations of Ag, Cd, Co, Pb and Sb were measured by QICP-MS aiter pressurised digestion with HNO3, with limits of detection ranging from 0.14 to 3.8 ng g-1 wet mass [ 139].

20

M. PATRIARCA, B. ROSSI, and A. MENDITTO

Since several elemems of clinical and toxicological interest suffer from more or less severe interferences in Q-ICP-MS, the applications of SF-ICP-MS to biological and clinical samples are growing, especially when lower limits of detection are required [15, 140]. A recent report described the simultaneous determination of 57 elements in 10-fold diluted digested whole blood. The limits of detection were less than 1 ng L "1 for Cs, Dy, Er, Eu, Gd, Ho, Ir, Lu, Pr, Pt, Re, Sm, Ta, Tb, Th, Tm, U and Yb; between 1 and 10 ng Ll for Ag, Au, Ce, Cd, Ga, Hf, La, Nb, Nd, Rb, Sc, Sb, T1, Y and W; and between 10 and 100 ng L1 for Be, Ba, Bi, Co, Ge, Hg, Li, Mn, Mo, Pb, Sn, Sr, Te, V and Zr. Higher limits of detection were observed for As, Cu, Cr, Ni and Ti (between 0.1 and 0.5 lag L l) and A1, B, and Se (between 1 and 2 ~tg L "l) [141]. Similar results were obtained for Co, Cr, Mo and Ni in whole blood simply diluted with (1+9) with a solution containing Triton X- 100, EDTA and NH3 [ 142]. Other papers [ 143-145] have reported the determination of several elements of clinical interest in diluted (1+4, 1+7) serum, including Ag, A1, Ca, Cd, Co, Cr, Cu, Fe, Mn, Mo, P, Pb, Rb, S, Si, Sn, Sr, Ti, U and Zn. Paediatric reference ranges (~tg g~ creatinine) for the concentrations of Cr (0.07-0.76), Ni (0.20-1.23) and V (0.02-0.22) in urine were estimated from 131 subjects by means of SF-ICP-MS. Urine was diluted 1+19 and spectral interferences were resolved using a resolution factor of 3000 [ 146]. Cadmimn, Cu, Pb, and Zn were measured simultaneously in 10-fold diluted urine, using a higher resolution factor (3000) for the determination of Cu and Zn, whereas Cd and Pb were measured at lower resolution (300) for better sensitivity. The trace elements most frequently determined for clinical and toxicological purposes (AI, As, Cd, Cr, Co, Cu, Hg, Mn, Ni, Pb, Sb, Se, T1 and Zn) could be measured simultaneously in 10-fold diluted urine, as well as in digested blood and 100-fold diluted serum [147]. In addition, the determination of several essential (Co, Cr, Cu, Fe, Mn, Ni, Se, and V) and non essential (Ag, AI, As, Au, Pt, Sc and Ti) elements in human milk by SF-ICP-MS has been reported [148]. 5. STABLE ISOTOPE DIAGNOSIS

TRACERS:

A TOOL FOR RESEARCH AND

5.1 Background Both radioactive and stable isotopes are used in biochemistry and medicine as tracers of mineral metabolism in animal models and humans. Tracer studies provide a direct assessment of the absorption, distribution and elimination of electrolytes or trace elements, which is necessary in several branches of medicine, such as nutrition, toxicology, pharmacology and clinical biochemistry. Stable isotopes are a safer alternative to radioactive isotopes as tracers for studies in humans. The safety of their use allows the extension of investigations to all

Atomic Spectrometry in the Clinical Laboratory

21

population groups, including children and pregnant women, for whom critical information on mineral and trace element metabolism is often lacking. The availability and sensitivity of analytical techniques for their determination is however a key issue for the exploitation of the potential of stable tracers for human studies. Inductively coupled plasma mass spectrometry has several advantages for such studies because of the minimal sample preparation required and the high sample throughput. Precision of the measured isotope ratios is acceptable for most biological tracer studies (typically <1% for Q-ICP-MS), but well-known interferences hamper the determination of isotope ratios of biologically important elements, such as Ca, Fe and Se. Some of these problems were overcome using separation techniques, alternative methods of sample introduction or higher resolution instruments. The latest developments in ICP-MS are expected to expand further its application to tracer studies in humans. Methods to study the absorption, distribution and elimination routes of electrolytes and trace elements in humans and animal models are needed in nutrition, toxicology, phaxmacology and clinical biochemistry. The exploitation of isotopes started soon after their discovery, with deuterium being used as an in vivo tracer of intermediary metabolites.[149] Use of isotope tracers was later extended to the investigation of electrolyte and trace element metabolism. These experiments, performed in controlled conditions, answer questions regarding, for example, optimal intakes of minerals, [150-152] factors affecting mineral absorption and bioavailability, [153-155] and routes and kinetics of elimination of toxic metals. [156, 157] Clinical tests based on tracer studies have also been developed. Stable isotopes were the first to be used in studies of human metabolism but later radioisotopes were preferred since they are less expensive and much easier to measure. Concern over safety has since limited the use of radioisotopes in human studies to short-lived nuclides. Suitable radionuclides are not always available for the elements of interest and can provide information only over a short period of time, generally on one element at time. Critically important population groups, such as children and pregnant women, for whom optimal nutrition is most important, are excluded for obvious reasons fi'om studies with radionuclides. Stable isotopes are safe and easier to handle, since they have no damaging radioactive decay. Over the last 20 years, their use as tracers in human studies has been growing, in part as the result of a larger availability of stable isotope on the market and wider access to techniques (Q-TIMS and ICP-MS) for their determination [158-160]. A search of the literature, including electronic databases and other sources, suggest at least 25 papers/year were published on this subject during the last 5 years. A comprehensive review of this growing field is outside the scope of

22

M. PATRIARCA, B. ROSSI, and A. MENDITTO

this paper and only some examples will be given of the contribution of ICP-MS to the study of mineral metabolism in humans.

5.2 Biological and analytical constraints for human studies using stable isotopes as tracers The use of stable isotopes instead of radioactive atoms poses some limitations upon what can be achieved by tracer studies in humans [161-162]. Monoisotopic elements, including biologically important metals, such as Co and Mn, are not amenable for investigation. Information on the distribution of the tracer within the body can only be obtained by means of the analysis of accessible body fluids and excreta. The feasibility of the study depends on several factors, such as isotope availability, their relative abundance, their cost and the size of the dose to be administered to achieve a measurable enrichment in blood or urine. Large doses are likely to perturb the system under study and the safety of exposure to a single large dose of a mineral may be questionable. In addition, the cost of the isotopes may be prohibitive. All stable isotopes of an element are present contemporarily and are uniformly distributed according to their natural abundance. Therefore, the tracer must be measured against a 'background level' in accessible fluids or compartments. The percentage of enrichment in any body compartment depends on the dose administered and/or absorbed, the body content of the mineral under study (limited in most cases to the readily exchangeable pool) and the natural abundance of the isotope chosen. If the percent enrichment of isotope A measured against reference isotope B (EA,B~ is expressed as: EA,B% = (RA,Bm- RA,Bn)/RA,Bn

(1)

where RA,Bn is the natural ratio between the isotopes A and B and RA,Bm is the measured ratio, and, if the uncertainty of the determination of the theoretical value of the RA,Bn is negligible, then the uncertainty on EA,B% will depend only on the uncertainty of the measured ratio RA,Bm. The percentage of enrichment that can be measured with a stated level of uncertainty is determined by the performance of the analytical technique. Therefore, an improvement of the precision of the measured ratio determines a reduction of the dose of the tracer needed to achieve a measurable enrichment at a stated time. The techniques available for the determination of stable isotopes, neutron activation analysis (NAA) or mass spectrometry (MS), provide variable degrees of precision in biological media, ranging fi'om 0.1 to 10% [158-160, 163-164]. Lowman and Krivit [165] were the first to apply stable isotopes to studies of

Atomic Spectrometry in the Clinical Laboratory

23

mineral metabolism, using NAA to determine 5SFe in biological fluids and excreta. The first application of MS in this field was the study of the metabolism of Pb in normal humans [156]. The comparison of the performance of TIMS and NAA on the determination of stable isotopes of Fe, Zn and Cu in human body fluids demonstrated superior precision of TIMS over NAA [166-168]. Alternative methods, requiring the synthesis of stable volatile chelates of the elements of interest, were developed to take advantage of insmanemation currently available in many clinical laboratories, such as electrospray ionisation (EI) MS and GC-MS [159]. The determination of the stable isotopes of Se [169-173] and Cr [174-175] by these methods found application in human studies of Se bioavailability [176177], Cr metabolism [ 178-179] and as a method to determine blood volume and red cell survival [ 180]. Precision of measurement of isotope ratios has been reported as less than 2% for both elements, but sample preparation requires skill and is timeconstuning. Soon aiter the appearance of Q-ICP-MS, the performance of the new technique for the analysis of stable isotopes in biological fluids was tested and found satisfactory (precision <2%) for the requirements of human tracer studies [181-183]. ICP-MS requires considerably less sample preparation and allows a higher sample throughput (up to 100 samples/day) than other techniques. In addition, the capability for simultaneous multielemental determination allows investigation of competition between elements and use of the double isotope method. Over the last 16 years several insmunental and analytical methods have addressed the problem of polyatomic interferences affecting or preventing the determination of isotope ratios for biologically important elements at mass <80 a.m.u.. With the latest insmunental developments, the problems hindering applications of ICP-MS to tracer studies in humans should be reduced ft~her. 5.3 Determination of stable isotope ratios for tracer studies in humans by ICPMS Initial applications of ICP-MS, reported as early as 1986, described the determination of Zn isotope ratios in blood and faeces to determine Zn bioavailability [181] and the incorporation of ingested 58Fe into erythrocytes [184]. ICP-MS methods for the determination of isotope ratios in clinical samples for several other elements of interest in medicine have since been applied to tracerstudies in humans 157, 159-160, 162, 185-189]. As for measuring the total content of an element, the determination of stable isotope ratios by Q-ICP-MS is affected by several interferences, sometimes hampered by the low abundance of the isotope to be determined. Among the methods used to overcome the interferences (see 3.1), SF-ICP-MS provides the greatest advantages but it is scarcely used because of the loss in sensitivity associated with resolution factors > 3000, the limited availability of instrumentation and high capital and nmning costs [76].

24

M. PATRIARCA,B. ROSSI, and A. MENDITTO

However the dilution factor associated with chromatographic methods may be prohibitive for stable isotope analysis and techniques based on chelation and extraction, are tedious and time-consuming. The newest instruments including collision (CC) [190-192] and dynamic reaction (DRC) [193, 194] cells have been reported to greatly reduce most interferences affecting the determination of stable isotope ratios in biological matrices. Some examples will be discussed of how these approaches were applied to the determination of stable isotopes for tracer studies in humans.

5.3.1 Copper and nickel Copper plays an essential role in medicine and biology. In humans, Cu participates in more than 25 enzymes performing essential functions such as scavenging of free radicals and incorporation of Fe in haemoglobin. Inherited defects of Cu metabolism cause the fatal Menke's disease and Wilson's disease (WD), for which severe toxic effects of excessive Cu accumulation can only be prevented by early diagnosis and life-long therapy. In 1966, following observations of impaired Cu incorporation in the major serum Cu protein (caeruloplasmin) in WD patients, the measurement of the incorporation of radioactive 64Cu in serUlTI caeruloplasmin was proposed as a laboratory test for the identification of WD cases [195]. Both the radiation risk and the short half-life of this isotope limited its application in the most critical subjects: children. Although the less abundant stable Cu isotope (65Cu) was used as a tracer in human experimental studies since 1988, the determination of Cu isotope ratio by TIMS is too complex to find application as a clinical test. The determination of Cu isotope ratio in blood serum by ICP-MS suffers from a serious interference caused by ArNa + overlapping with 63Cu. Since more than 95% of Cu in serum is bound to the protein caeruloplasmin, Lyon and Fell [186] used size exclusion chromatography performed on disposable Sephadex 25 columns to separate protein-bound Cu from interfering Na. Residual interferences from 32S1602I-1"+ on 65Cu were eliminated by acid precipitation of the sulfur-containing proteins from the eluate. The 65Cu:63Cu isotope ratio was measured with an internal precision of 0.2%, as compared with typical TIMS precision of 0.05-0.1%. Incorporation of orally administered 65Cu, 3 mg, into caeruloplasmin, could be detected by comparing the 65Cu:63Cu ratio in a series of timed serum samples obtained over of the next week [196]. The analysis of the whole series of samples could be completed within 3 h, including the preparation step. The method is currently applied as a laboratory test for the screening of WD cases [196-197]. Under working conditions, an average precision of 0.3%, with isotope ratios varying from 0.447 to 0.953, was obtained when 214 serum samples fi,om patients undergoing the test, were reanalysed in two different days over a six month period [198].

Atomic Spectrometry in the Clinical Laboratory

25

In other biological samples, not suitable for such an approach, Cu can be separated from the matrix with selective chelating agents and solvent extraction. Using this method the precision of Cu ratios determined in faecal material collected over 4 days in a tracer study carried out at the Glasgow Royal Infirmary was, on average, 0.3% (Table 8) [199-200]. The faecal samples were freeze-dried and acid digested. Neutral chelates of Cu with ammonium pyrrolidine dithiocarbammate (APDC) were formed at pH=7 and extracted in 4-methyl-2-pentanone, evaporated to dryness and dissolved in HNO3 1% prior to ICP-MS analysis. The procedure suits several elements, which can be determined simultaneously. The same approach was used to remove the Na20 + interference on 62Ni in the first tracer study of Ni metabolism [201]. The method recovery, assessed by the analysis of blood, serum, faeces and urine samples spiked with 62Ni, was 99.8 + 3.2% [187]. Nickel metabolism was studied in 4 subjects, each ingesting a dose of 10 ~tg 62Ni~g body weight. Blood, urine and faecal samples were collected for 5 days after ingestion of the 62Ni dose. The direct assessment of Ni elimination by a tracer method indicated 11.0 • 3.0% of the ingested dose was retained within the body 5 days atter dosage. A three compartment mathematical model generated from. these data suggested complete elimination of ingested Ni could take up to one month [202].

Table 8. Repeatability o f 65Cu:63Cu isotope ratios (IR) in faeces by Q-ICP-MS. Sample

pretreatment: acid digestion and solvent extraction of Ni-APDC chelates.

Sample code

1st replicate IR

BF-0 BF-1 BF-2 BF-3 BF-4

0.446 1.596 1.260 0.730 0.541

RSD % 0.3 0.8 0.3 0.2 0.4

2 nd replicate IR 0.447 1.597 1.252 0.733 0.540

RSD % 0.5 0.3 0.2 0.2 0.3

5. 3.2 Calcium

One of the major fields of research involving investigations of Ca metabolism is the identification of risk factors for osteoporosis. Both genetic and environmental factors are reckoned to affect adequate Ca absorption and bone ttn'laover in children [203]. Assessment of the efficiency of the intestinal absorption

26

M. PATRIARCA, B. ROSSI, and A. MENDITTO

of Ca is an important approach to research in this field and can be performed using stable Ca isotopes [204-205]. Currently, in most experimental studies Ca isotope ratios are determined by thermal ionization (TI) or fast atom bombardment (FAB) MS. Measurement of Ca isotopes by ICP-MS is a challenge, because of the overlap with argon ions and argon-based molecules. The determination of Ca isotopes (42Ca, 43Caand 44Ca) in urine by SF-ICP-MS requires a resolution factor of 4000, to allow the separation from some of the interfering peaks (Ar2IT, ArH2§ SiO§ CO2+), and mathematical correction for Sr2§ [206]. Under these .conditions, the isotope ratios 42ca:a3ca and 44Ca:43Ca could be measured with a relative standard deviation of 0.33% and 0.41%, respectively. Patterson et al. [ 188] recently reported the performances of Q-ICP-MS under 'cool plasma' conditions for the determination of Ca isotopes in human biological fluids (urine, blood serum and milk) and faeces. Sample preparation involved ashing of serum, milk and faeces, followed by selective precipitation of Ca as Ca oxalate. The same separation step was used for urine. Precision was about 0.25% for both the 42Ca:43Caand the 44Ca:43Ca ratios, i.e. about 1.5-fold the estimated precision attributed to counting statistics alone. Data on the performance of either CC- or DCR- ICP-MS for the determination of Ca stable isotopes ratios are not yet available, but the measurement of4~ can be performed at concentration levels as low as 5 ~tg L "1 by CC-ICP-MS [ 191 ], while a limit of detection as low as 0.4 ng L ~ has been reported for DCR-ICP-MS [ 193]. 5.3.3 Iron

Evaluation of incorporation of SaFe into erythrocytes was one of the first applications of ICP-MS to tracer studies in humans. The ratio 5SFe:57Fe was measured by ICP-MS with a precision of + 1%. The method required 1 ml of blood and sample preparation was demanding. Before analysis, the sample was ashed, Fe was precipitated with APDC, the precipitate dissolved in concentrated HNO3 and diluted to Fe concentrations of approximately 10 mg L l [ 184]. The contribution of polyatomics to the signal was minimised by maintaining a high concentration of Fe in the analysed samples. Notwithstanding the complexity of the sample preparation, the method was readily applied to the assessment of the absorption of haem and non-haem iron fi'om meals in children [207-208] and infants (<1 year) [209-210]. These studies have shown children less than 1 year old are much less effective than adults in utilising newly absorbed Fe. Erythrocyte incorporation of 5SFe measured 14 days after dosage was less than 40% compared with 80-100% in adults [211 ]. Reduced or slower Fe incorporation into erythrocytes occurs in other physiological and pathological conditions and may influence the results of studies of Fe absorption based on timed determination of the ingested tracer in erythrocytes. For

Atomic Spectrometry in the Clinical Laboratory

27

a more accurate estimate of Fe absorption, a double isotope method has been proposed, based on the simultaneous administration of two tracers by the oral and the intravenous route and the determination of both isotope ratios to a third 'reference' isotope. Barrett and co-workers studied Fe absorption in pregnancy by this technique [212], alter devising an ICP-MS method to determine both the 5SFe:56Fe and 57Fe:56Fe isotope ratios I107]. Samples of whole blood were diluted to Fe concentrations between 10 and 20 mg L ~ with a solution containing ammonia, Na2EDTA and NH4HEPO4 prior to analysis. The precision of the 5SFe:56Fe and 57Fe:56Fe isotope ratios in diluted blood samples at Fe concentrations of 9 and 18 mg L "~ was <0.6% and <0.32%, respectively. In pregnant women, absorption of orally administered Fe increased on average from 7% to 66% between 12 and 36 weeks of gestation and reamaed to 11% at 16-24 weeks aiter delivery. The need to reduce sample size prompted Whittaker and co-workers to explore sample introduction by ETV [213]. They were able to determine Fe stable isotopes in 5 pl of blood serum, without previous preparation, within 20 min. Polyatomic interferences from ArN +, ArO + and ArOH + on 54Fe, 56Fe and 57Fe were drastically reduced and both isotope ratios 54Fe:56Fe and 57Fe:56Fe have been determined simultaneously with a CV% of 2.9 and 3.8%, respectively. Further improvements and a wider application of ICP-MS to studies of Fe metabolism can be expected in the near future. Instruments equipped with CC can provide a reduction up to three order of magnitude of the argon based polyatomics interferences on Fe isotopes [191 ] and a detection limit as low as 0.15 ng L q has been reported for 56Fe in DIW by DCR-ICP-MS in class 100 conditions [193]. 5.3. 4 Selenium

Since its recognition as an essential element, there has been an enormous interest in Se, became of reports supporting a role in the prevemion of cancer and cardiovascular diseases, based on its participation in the antioxidant system [214]. Several aspects of Se metabolism are still unclear. Both organic and inorganic Se species occur in food and are absorbed at different rates in the gastrointestinal tract. In blood plasma Se is bound to three major protein fractions, albumin, selenoprotein-P and glutathione peroxidase, of which a different form is found in erythrocytes. In the liver, Se compounds undergo other biochemical transformations, partly depending on the chemical species, leading to incorporation into selenoproteins (as Se-Cys and Se-Met), and elimination of excess Se in breath and urine mainly as trimethylselenonium, although more than 20 Se species appear in twine and their identification is still underway.[215] Tracer studies of Se metabolism are aimed to investigate Se bioavailability from food and supplements, the biochemical transformations of Se-species and potential biomarkers of Se status [216-224].

28

M. PATRIARCA,B. ROSSI,and A. MENDITTO

Since the measurement of elements with high ionization potential by TIMS is difficult, the first studies on Se metabolism by stable isotopes were carded out using RNAA. Determination of 74Se, 76S0, 8~ in faeces, plasma, red blood cells and urine by RNAA was reported with a precision < 10% [225]. Volatile chelates of Se and their isotope ratios were later measured by GC-MS with a precision between 1 and 7% I169-173]. Sample introduction by HG allows the determination of the 74So:77Se and 825e:77Se isotope ratios in biological matrices by Q-ICP-MS with a precision of 1% [185]. Both GC-MS and HG-ICP-MS are currently used in tracer studies of Se metabolism. Although considerably simpler than other methods, HG-ICP-MS still requires complete mineralisation of samples. Faster and simpler methods are necessary, requiring smaller samples and less preparation. Turner et al. recently described the determination of two Se isotopes (77Se and 82So) in l0 pl of 20-fold diluted blood serum, using ETV-ICP-MS, but did not report on the precision of the isotope ratio [132]. Both CC- and DCR-ICP-MS allow substantial reduction of the interferences of the argon dimers on the main Se isotopes (78Se and 8~ Using CH4 as the reaction gas, the ratios between 82Se, 78Se, 77Se and 76So to 8~ could be measured with a precision < 0.44% at a Se concentration of 10 pg L ~ and <1.21% at Se concentration of 1 pg L "~ in aqueous samples [226]. These findings are most promising for the developments of improved methods to determine of Se isotope ratios in biological matrices.

5.4 Other applications of isotope measurements For elements, such as Pb and Sr, whose isotopes were partly generated by the radioactive decay of other elements, the ratios between isotopes are characteristic for different geographic locations. Therefore, measurement of isotope ratios in biological materials may provide useful information to identify sources of exposure, even in the past [227]. Measurements of Pb isotopes ratios have been used to demonstrate the impact of leaded petrol in various countries. In Japan, comparison of Pb isotope ratios in biological remains (bones, teeth) of prehistoric, historic and contemporary Japanese indicated that petrol was the major source of Pb exposure in modem days before leaded petrol was phased out [228]. In the teeth of children bom between 1985 and 1988, the Pb isotope ratio was closer to that in airborne paniculate matter and waste incineration ash. Whereas, in Mexico City, comparison of Pb isotope ratios, measured in ceramic cookware, leaded petrol and whole blood samples by ETV-ICP-MS, showed that leakage from the ceramic ware used in cookery was the predominant source of Pb exposure [229]. Similar methodologies can be applied to individual cases, when exposure to Pb is suspected but the source is unknown [230]. This is otten the situation in domestic settings where there may be several possible sources. In one such case, measurement of Pb isotope ratios by SF-ICP-MS identified ingestion of dust as the source of Pb

Atomic Spectrometry in the Clinical Laboratory

29

exposure [231]. Similarly, traditional remedies, rather than leaded petrol, were shown to be responsible for toxicity in children in Saudi Arabia [232]. 6. SPECIATION

6.1 Background It is a well known concept that the chemical activity of an element is modified by its surrounding and therefore, the essentiality or toxicity of an element critically depends on its chemical form. The understanding of the bioavailability, metabolic pathways and detoxification mechanisms for an element in living organisms is improved by the identification and quantification of the relevant elemental species in appropriate biological fluids or tissues. The challenge for the analyst is to identify and quantify ever smaller amounts of substance, without disturbing the existing balance in a sample, but interpreting such information may prove an even bigger challenge [233-234]. Speciation analysis in biological systems, i.e. the "analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a [biological] sample" [235], is a fast developing area [236], in which both the lower limits of detection of ICP-MS and its ability for a relatively straight-forward on-line coupling with separation techniques, such as chromatography [237-238] and capillary electrophoresis [239240], are being exploited. Coupling ICP-MS with HPLC is a relatively easy task as flow rates commonly used in both instrtmaents are compatible. However, high concentrations of salts in the mobile phase may clog nebulisers and cones, whereas the organics content of the mobile phase may cause plasma instability. Separation can be achieved according to different principles, such as reversed-phase liquid chromatography, ion-pairing, micellar chromatography, ion-exchange and size exclusion chromatography. To achieve reliable results, the species present in the sample should have sufficient thermodynamic stability and kinetic inertness to remain unchanged when injected onto the column. The coupling of capillary electrophoresis to ICP-MS can be achieved by direct insertion of the separation capillary into the nebuliser, followed by an additional sheath flow to compensate for the back pressure produced in the nebuliser capillary. Sensitivity and resolution are influenced by the capillary characteristics as well as the nebuliser type and the gas flow rate. Specific areas of interest for speciation in biology and medicine are oxidation states of an element, biological and man-made organometallic compounds, metallodrugs, transport proteins, metalloenzymes and nucleic acids. The state-ofthe art in bio-inorganic speciation has been recently reviewed by Szpunar and Lobinski in a series of papers [237, 241-243]. Currently most work deals with the

30

M. PATRIARCA, B. ROSSI, and A. MENDITTO

analytical aspects of bio-inorganic speciation, but work in this field has demonstrated the feasibility for the speciation of several elemental species in biological materials. The validation of the analytical procedures, however, remains a problem, due to the lack of representative reference materials certified for the relevant species. Only a limited number of CRMs for trace element species exists, mainly for environmental (trialkyltin compounds, methylmercury and crm/Cr vI) and food analyses (As species and methylmercury). Further developments are needed, especially in the fields of biomonitoring of exposure and phannacokinetics, but may require formidable efforts because of the challenges of production of species specific CRMs [244]. 7. REFERENCE METHODS AND TRACE ELEMENT ANALYSIS

REFERENCE

MATERIALS

FOR

The availability of a relatively simple and rapid technique to obtain isotopic information provides the opporttmity to extend the use of stable isotopes for accurate analytical methods based on isotopic dilution (ID) [245]. Isotope dilution analysis involves the measurement of isotopic ratios in two aliquots of a sample, one of which has been spiked with a known amount of an isotope of the element to be determined. The concentration of the element in the sample can be calculated from the measured isotopic ratios, the amount of the spike and the weight or volume of the sample. The uncertainties of these quantities, generally small, are the main sources of error affecting the results. Extensive sample pretreatment is acceptable as losses of analyte after the sample has been spiked do not modify the isotopic ratios. The combination of ID with ICP-MS improves the accuracy of analytical procedures for inorganic analysis [246-247] and, under appropriate conditions, can be used to assign traceable values to reference materials [248]. This work is especially relevant for analyses in the field of environmental and occupational medicine, where reference materials are few and traceability of the measurement results to SI units is harder to achieve.

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171. V. Ducros and A. Favier, Gas chromatography-mass spectrometric methods for the determination of selenium in biological samples. J Chromatogr., 583 (1992) 35-44. 172. S.K. Aggarwal, M. Kinter and D.A. Herold, Determination of selenium in urine by isotope dilution gas chromatography-mass spectrometry using 4nitro-o-phenylenediamine, 3,5-dibromo-o-phenylenediamine, and 4trifluoromethyl-o- phenylenediamine as derivatizing reagents. Anal. Biochem., 202 (1992) 367-374. 173. P. Van Dael, D. Barclay, K. Longet, S. Metairon, and L.B. Fay, Determination of selenium stable isotopes by gas chromatography-mass spectrometry with negative chemical ionisation. J Chromatogr. B Biomed. Sci. Appl., 715 (1998) 341-347. 174. C. Veillon, K.Y. Patterson, M.R Rubin and P.B. Moser-Veillon, Determination of natural and isotopically encriched chromium in urine by isotope dilution gas chromatography-mass spectrometry. Anal. Chem., 66 (1994) 856-860. 175. C. Veillon, W.R. Wolf and B.E. Guthrie, Determination of chromium in biological materials by stable isotope dilution. Anal. Chem., 51 (1979) 10221024. 176. C.A. Swanson, D.C. Reamer, C. Veillon, J.C. King, and O.A. Levander, Quantitative and qualitative aspects of selenium utilization in pregnant and nonpregnant women: an application of stable isotope methodology. Am. J. Clin. Nutr., 38 (1983) 169-180. 177. V. Ducros, A. Favier and M. Guigues, Selenium bioavailability as selenite (74Se) and as a selenium drug (76Se) by stable isotope methodology. J. Trace Elements & Electrolytes Health Dis., 5 (1991) 145-154. 178. F.Y. Mohamedshah, P.B. Moser-Veillon, S. Yamini, L.W. Douglas, R.A. Anderson and C. Veillon, Distribution of a stable isotope of chromium (53Cr) in serum, urine and breast milk in lactating women. Am. J. Clin. Nutr., 67 (1998) 1250-1255. 179. M.A. Rubin, J.P. Miller, A.S. Ryan, M.S. Treuth, K.Y. Patterson, R.E. Pratley, B.F. Hurley, C. Veillon, P.B. Moser-Veillon and R.A. Anderson, Acute and chronic resistive exercise increase urinary chromium excretion in men as measured with an enriched chromium stable isotope. J. Nutr., 128 (1998) 73-78. 180. C. Veillon, K.Y. Patterson, D.A. Nagey and A.M. Tehan, Measurement of blood volume with an enriched stable isotope of chromium (53Cr) and isotope dilution by combined gas chromatography-mass spectrometry. Clin Chem., 40 (1994) 71-73.

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181. R.E. Serfass, J.J. Thompson and R.S, Houk, Isotope ratio determinations by Inductively coupled/plasma mass spectrometry for zinc bioavailability studies. Anal. Chim. Acta, 188 (1986) 73-84. 182. B.T. Ting and M. Janghorbani, Inductively coupled plasma mass spectrometry applied to isotopic analysis of iron in human fecal matter. Anal. Chem., 58 (1986) 1334-1340. 183. BTG. Ting and M. Janghorbani, Application of ICP-MS to accurate isotopic analysis for human metabolic studies. Spectrochim. Acta, 42B (.1987) 21-27. 184. M. Janghorbani, B.T. Ting and S.J. Fomon, Erythrocyte incorporation of ingested stable isotope of iron. Am. J. Hematol., 21 (1986) 277-288. 185. B.T. Ting, C.S. Mooers and M. Janghorbani, Isotopic determination of selenium in biological materials with inductively coupled mass spectrometry. Analyst, 114 (1989) 667-674. 186. T.D.B. Lyon and G.S. Fell, Isotopic composition of copper in serum by inductively coupled plasma mass spectrometry. J. Anal. At. Spectrom., 5 (1990) 135-137. 187. M. Patriarca, T.D.B. Lyon, B. McGaw and G.S. Fell, Determination of selected Nickel Isotopes in Biological Samples by Inductively Coupled Plasma Mass Spectrometry With Isotope Dilution. J. Anal. At. Spectrom., 11 (1996) 297-302. 188. K.Y. Patterson, C. Veillon, A.D. Hill, P.B. Moser-Veillon and T.C. O'Haver, Measurement of calcium stable isotope tracers using cool plasma ICPMS. J. Anal. At. Spectrom., 14 (1999) 1673-1677. 189. S. Stfimp, Application of HR-ICP-MS for the simultaneous measurement of zinc isotope ratios and total zinc content in human samples. J. Anal. At. Spectrom., 15 (2000) 315-321. 190. I. Feldmann, N. Jakubowski and D. Stuewer, Application of hexapole collision and reaction cell in ICP-MS Part I: Instnnnental aspects and operational optimization. Fresenius J. Anal. Chem., 365 (1999) 415-421. 191. I. Feldmann, N. Jakubowski, C. Thomas and D. Stuewer, Application of hexapole collision and reaction cell in ICP-MS Part II: Analytical figures of merit and first applications. Fresenius J. Anal. Chem., 365 (1999) 422-428. 192. Q. Xie and R. Kerrich, Isotope ratio measurement by hexapole ICP-MS: mass bias effect, precision and accuracy. J. Anal. At. Spectrom., 17 (2002) 69-74. 193. D.R. Bandura, V.I. Baranov and S.D. Tanner, Effect of collisional damping and reactions in a dynamic cell on the precision of isotope ratio measurements. J. Anal. At. Spectrom., 15 (2000) 921-928.

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194. S.D. Tanner, V.I. Baranov and U. Vollkopf, A dynamic cell for inductively coupled plasma mass spectrometry (ICP-DRC-MS). J. Anal. At. Spectrom., 15 (2000) 1261-1269. 195. W.N. Tauxe, N.P. Goldstein. R.V. Randall and J.B. Gross, Radiocopper studies in patients with Wilson's disease and their relatives. Am. J. Med., 41 (1966) 375-380. 196. T.D.B Lyon, G.S Fell., D. Gaffney, B.A. MGaw, R.I. Russell, R.H.R. Park, A.D. Beattie, G. Curry, R.J. Crofion, I. Gunn, G.C. Sturniolo, R. D'Inca' and M. Patriarca, Use of a stable copper isotope (65Cu) in the differential diagnosis ofWilson's disease. Clin. Sci., 88 (1995) 727-732. 197. M. Merli, M. Patriarca, G. Loudianos, C. Valente, O. Riggio, G. De Felice, F. Petrucci, S. Caroli and A.F. Attili, Use of the stable isotope 65Cu test for the screening of Wilson's disease in a family with two affected members. Ital. J. Gastroenterol. Hepatol., 30 (1998) 270-275. 198. Di Felice G. PhD Thesis. University of Rome I, Rome (Italy), 1998. 199. T.D.B. Lyon, S. Fletcher, G.S. Fell and M. Patriarca, Measurement and application of stable copper isotopes to investigations of human metabolism. Microchem. J., 54 (1996) 236-245. 200. W.S. Watson, T.D.B. Lyon and G.S. Fell, A metabolic model for copper in humans. Vth COMTOX Symposium on Toxicology and Clinical Chemistry of Metals, Vancouver, British Columbia, (Canada), 10-13 July 1995. 201. M. Patriarca, T.D. Lyon and G.S. Fell, Nickel metabolism in humans investigated with an oral stable isotope. Am. J. Clin. Nutr., 66 (1997) 616621. 202. M. Patriarca, W.S. Watson, T.D.B. Lyon and G.S. Fell, A metabolic model for nickel in humans. Vth COMTOX Symposium on Toxicology and Clinical Chemistry of Metals, Vancouver, British Columbia, (Canada), 10-13 July 1995. 203. S.K. Ames, K.J. Ellis, S.K. Gunn, K.C. Copeland and S.A. Abrams, Vitamin D receptor gene Fokl polymorphism predicts calcium absorption and bone mineral density in children. J. Bone Miner. Res., 14 (1999) 740-746. 204. S.A. Abrams, Using stable isotopes to assess mineral absorption and utilization by children. Am. J. Clin. Nutr., 70 (1999) 955-964. 205. F. Bronner and S.A. Abrams, Development and regulation of calcium metabolism in healthy girls. J. Nutr., 128 (1998) 1474-1480. 206. S. Stiimp, M. Hansen and C. Molgaard, Measurements of 44Ca: 43Ca and 4ZCa: 43Ca isotope ratios in urine using high resolution indctively coupled plasma mass spectrometry. J. Anal. At. Spectrom., 12 (1997) 919-923.

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207. J.C. Woodhead, J.M. Drulis, R.R. Rogers, E.E. Ziegler, P.J. Stumbo, M. Janghorbani, B.T. Ting and S.J. Fomon, Use of the stable isotope, 58Fe, for determining availability of nonheme iron in meals. Pediatr. Res., 23(1988) 495-499. 208. J.C. Woodhead, J.M. Drulis, S.E Nelson, M. Janghorbani and S.J. Fomon, Gender-related differences in iron absorption by preadolescent children. Pediatr. Res., 29 (1991) 435-439. 209. S.J. Fomon, M. Janghorbani., B.T. Ting, E.E. Ziegler, R.R. Rogers, S.E. Nelson, L.S. Ostegaard and B.E. Edwards, Erythrocyte incorporation of ingested 58-iron by infants. Pediatr. Res., 24 (1988) 20-24. 210. S.J. Fomon, E.E. Ziegler, R.R. Rogers, S.E.. Nelson, B.E. Edwards, D.G. Guy, J.C. Erve and M. Janghorbani, Iron absorption from infant food. Pediatric Res., 26 (1989) 250-255. 211. S.J. Fomon, E.E. Ziegler, R.E. Serfass, S.E. Nelson, R.R. Rogers and J.A. Frantz, Less than 80% of absorbed iron is promptly incorporated into erythrocytes of infants. J. Nutr., 130 (2000) 45-52. 212. J.F Barrett, P.G. Whittaker, J.G. Williams and T. Lind, Absorption of nonhaem iron from food during normal pregnancy. Br. Med. J., 309 (1994) 7982. 213. P.G. Whittaker, T. Lind, J.G. Williams and A.L. Gray, Inductively coupled plasma mass spectrometric determination of the absorption in normal women. Analyst, 114 (1989) 675-678. 214. M.P. Rayman, The importance of selenium to human health. Lancet, 356 (2000) 233-241. 215. R. Lobinski, J.S. Edmonds, K.T. Suzuki and P.C. Uden, Species-selective determination of selenium compounds in biological materials (Technical Report). Pt~e Appl. Chem., 72 (2000) 447-461. 216. C.A. Swanson, D.C. Reamer, C. Veillon, J.C. King and O.A. Levander, Quantitative and qualitative aspects of selenium utilization in pregnant and nonpregnant women: an application of stable isotope methodology. Am. J. Clin. Nutr., 38.(1983) 169-180. 217. N.W. Solomons, B. Torun, M. Janghorbani, M.J. Christensen, V.R. Young and F.H. Steinke. Absorption of selenium from milk protein and isolated soy protein formulas in preschool children: studies using stable isotope tracer 74Se. J. Pediatr. Gastroenterol. Nutr., 5 (1986) 122-126. 218. A.R. Mangels, P.B. Moser-Veillon, K.Y. Patterson and C. Veillon, Selenium utilization during human lactation by use of stable-isotope tracers. Am. J. Clin. Nutr., 52 (1990) 621-627.

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219. R.A. Ehrenkranz, P.A. Gettner, C.M. Nelli, E.A. Sherwonit, J.E. Williams, B.T. Ting and M. Janghorbani, Selenium absorption and retention by verylow-birth-weight infants: studies with the extrinsic stable isotope tag 74Se. J. Pediatr. Gastroenterol. Nutr., 13 (199 l) 125-133. 220. C. Veillon, K.Y. Patterson, L.N. Button and A.J. Sytkowski, Selenium utilization in humans - A long-term, self-labeling experiment with stable isotopes. Am. J. Clin. Nutr., 52 (1990) 155-158. 221. J.W. Finley, A. Duffield, P. Ha, R.A. Vanderpool and C.D. Thomson, Selenium supplementation affects the retention of stable isotopes of selenium in human subjects consuming diets low in selenium. Br. J. Nutr., 82 (1999) 357-360. 222. J.W. Finley, R.A. Vanderpool and E. Korynta, Use of stable isotopic selenium as a tracer to follow incorporation of selenium into selenoproteins. Proc. Soc. Exp. Biol. Med., 210 (1995) 270-277. 223. K.T. Stmt~ and M. Itoh, Metabolism of selenite labelled with enriched stable isotope in the bloodstream. J. Chromatogr. B Biomed. Sci. Appl., 692 (1997) 15-22. 224. J.W. Finley, The retention and distribution by healthy young men of stable isotopes of selenium consumed as selenite, selenate or hydroponically-grown broccoli are dependent on the isotopic form. J. Nutr., 129 (1999) 865-871. 225. M. Janghorbani, B.T. Ting and V.R. Young, Use of stable isotopes of selenium in human metabolic studies: development of analytical methodology. Am. J. Clin. Nutr., 34 (1981) 2816-2830. 226. J.J. Sloth and E.H. Larsen, The application of inductively coupled plasma dynamic reaction cell mass spectrometry for measurement of selenium isotopes, isotopes ratios and chromatographic detection of selenoamino acid. J. Anal. At. Spectrom., 15 (2000) 669-672. 227. C. Latkoczy, T. Prohaska, M. Watkins, M. Teschler-Nicola and G. Stingeder, Strontium isotope ratio determination in soil and bone samples after on-line matrix separation by coupling ion chromatography (HPIC) to an inductively coupled plasma sector field mass spectrometer (ICP-SFMS). J. Anal. At. Spectrom., 16 (2001) 806-811. 228. J. Yoshinaga, M. Yoneda, M Morita and. T. Suzuki, Lead in prehistoric, historic and contemporary japanese - stable isotopic study by icp mass spectrometry. Appl. Geochem., 13 (1998) 403-413. 229. M. Chaudharywebb, D.C. Paschal, W.C. Elliott, H.P. Hopkins, A.M. Ghazi, BC. Ting and I. Romieu, ICP-MS determination of lead isotope ratios in whole blood, pottery, and leaded gasoline - lead sources in Mexico City. At. Spectrosc., 19 (1998) 156-163.

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230. M. Viczian, A. Lasztity and R. M. Barnes, Idemification of potential environmental sources of childhood lead poisoning by inductively coupled plasma mass spectrometry. Verification and case studies. J. Anal. Atom. Spectrom., 5 (1990) 293-300. 231. R.H. Gwiazda and D.R. Smith, Lead isotopes as a supplementary tool in the routine evaluation of household lead hazards. Environ. Health Perspect., 108 (2000) 1091-1097. 232. I. A1-Saleh, C. Fellowa, T. Delves and A. Taylor, Identification of sources of lead exposure among children in Ara, Saudi Arabia. Ann. Clin. Biochem., 30 (1993) 142-145. 233. T. M. Florence, Trace element speciation in biological systems, in "Trace element speciation: analytical methods and problems", G.E. Batley (ed.), CRC Press Inc., Boca Raton, FL, USA, 1989, pp.319-341. 234. P.H.E. Gardiner, Species identification for trace inorganic elements in biological materials, Topics in current chemistry, 141 (1987) 147-173. 235. D. Templeton, F. Ariese, R. Comelis, L.G. Danielsson, H. Muntau, H.P. van Leeuwen and R. Lobinski, Guidelines for terms related to chemical speciation and fractionation of elements. Definitions, structural aspects, and methodological approaches (IUPAC Recommendations 2000). Pure Appl. Chem., 72, (2000) 1453-1470. 236. R. Comelis, J. De Kimpe, X. Zhang, Trace elements in clinical samples revisited--speciation is knocking at the door. Sample preparation, separation of the species and measurements methods, Spectrochim. Acta, 5313 (1998) 187-196. 237. J. Szpunar, Trace element speciation analysis of biomaterials by highperformance liquid chromatography with inductively coupled plasma mass spectrometric detection. Trends Anal. Chem., 19 (2000) 127-137. 238. E. H. Larsen, Method optimization and quality assurance in speciation analysis using high performance liquid chromatography with detection by inductively coupled plasma mass spectrometry, Spectrochim. Acta, 53B (1998) 253-265. 239. J.W. Olesik, J.A. Kinzer, E. J. Grtmwald, K. K. Thaxton, S. V. Olesik, The potential and challenges of elemental speciation by capillary electrophoresisinductively coupled plasma mass spectrometry and electrospray or ion spray mass spectrometry, Spectrochim. Acta, 53B (1998) 239-251. 240. C. Vogt and Klunder G. L., Separation of metal ions by capillary electrophoresis- diversity, advantages and drawbacks of detection methods. Fresenius J. Anal. Chem., 370 (2001) 316-331.

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241. R. Lobinski and J. Spuznar, Biochemical speciation analysis by hyphenated techniques. Anal. Chim. Acta, 400 (1999) 321-332. 242. Szpunar J., Bio-inorganic speciation analysis by hyphenated techniques. Analyst, 125 (2000), 963-988. 243. J. Szpunar and R. Lobinski, Species-selective analysis for metal biomacromolecular complexes using hyphenated techniques (Technical Report). Pure Appl. Chem., 71, (1999) 899-918. 244. R. Comelis, H. Crews, O.F. Donard, L. Ebdon and P. Quevauviller, Trends in certified reference materials for the speciation of trace elements. Fresenius J. Anal. Chem., 370 (2001) 120-125. 245. P. De Bi~vre, Stable isotope dilution: an essential tool in metrology. Fresenius J. Anal. Chem., 350 (1994) 277-283. 246. J.R. Encinar; J.I.G. Alonso, A. Sanz-Medel; S. Main and P.J. Turner, A comparison between quadrupole, double focusing and multicollector ICP-MS instrtmaents. Part I. Evaluation of total combined uncertainty for lead isotope ratio measurements. J. Anal. Atom. Spectrom., 16 (2001) 315-321. 247. J.R. Encinar, J.I.G. Alonso, A. Sanz-Medel, S. Main and P.J. Turner, A comparison between quadrupole, double focusing and multicollector ICPMS. Part II. Evaluation of total combined uncertainty in the determination of lead in biological matrices by isotope dilution. J. Anal. Atom. Spectrom., 16 (2001) 322-326. 248. J. Diemer, J. Vogl, C.R. Qudtel, T. Lisinger, P.D.P. Taylor, A. Lamberty and J. Pauwels, SI-traceable certification of the amount content of cadmium below the ng g-1 level in blood samples by isotope dilution ICP-MS applied as a primary method of measurement. Fresenius J. Anal. Chem., 370 (2001) 492-498.

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Chapter 2 New developments in hydride generation - atomic spectrometry Hiroaki T a o 1 and Taketoshi Nakahara 2

1- National Institute of Advanced Industrial Science and Technology, 16-1, Onogawa, Tsukuba, Ibaraki, 305-8569, JAPAN,2- Deparanent of Applied Chemistry, Crmduate School of Engineering, Osaka Prefecture University, Sakai, Osaka 599-8531, JAPAN. 1. INTRODUCTION For some time, the generation of volatile hydrides of As, Sb, Bi, Se, Te, Ge, Sn and Pb has been used for sample introduction in atomic spectrometry. Since the work by Holak [1], numerous investigations have been carried out, and the hydride generation techniques may be considered to be mature. However, in recent years, there has been more fimdamental research on new hydride generation methods, such as eleclmchemical hydride generation, vesicular hydride generation, and the production of trustable hydrides such as those of cadmium and copper. Mechanisms of chemical interferences in solution, arfl atomization interferences in flames, have been studied in depth, and methods to overcome these interferences have been proposed. For application to biological and clinical samples, elemental speciation has become a steady trend since it is essential to identify and quantify individual chemical species, in order to know the biological and toxicological effects of the elements. Hydride generation has been used for improving the detection limits of hyphenated methods, which combine selxuation methods, such as high-performance liquid chromatography (HPI~) and capillary electrophoresis (CE), with element-specific detection methods such as atomic absorption spectrometry (AAS) and inductively coupled plasma mass

ADVANCES IN ATOMIC SPECTROSCOPY Volume 7, ISSN 1068-5561

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Copyright 9 2002 Elsevier Science B.V. All rights reserved

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H. TAO and T. NAKAHARA

spectrometry (ICP-MS). Many investigations conceming the on-line photooxidation or microwave-assisted digestion of organoarsenic or organoselenium compounds prior to hydride generation have expanded the applicability of hydride generation to a variety of compounds. Since 1993 the number of papers on hydride generation in atomic spectrometry has exceeded 600. Hence, it seems almost impossible to cover all aspects of hydride generation. For more comprehensive infonmtion, the following recent reviews and books are available: DMina and Tsalev [2], Tsalev [3], Yan and Ni [4], Nakahara [5] and Matusiewicz and Sturgeon [6] for hydride generation, Camli [7], Welz [8], Burguem and Burguera [9], Szpunar4~bif~ka et al. [10], Mufioz et al. [11 ] and Dauchy et al. [12] for speciation. In this chapter, a presentation is made of novel developments in hydride generation, chemical intereferences and their elimination, insmnnental developments for elemental speciation, and practical applications to biological and clinical samples, all of which have occtmxxt since a previous review in this series [13]. 2. NOVEL HYDRIDE GENERATION 2.1. Electrochemical hydride generation One major shortcoming associated with use of the sodium borohydride (NaBHa)--acid redt~on technique for hydride generation (HG) is its susceptibility to interferences from transition metals. 1his system is dependent on the oxidation state of the analyte, and NaBH4 is also a potential source of contamination, which may limit detection power. It is expensive and is unstable in solution fom~ which generally necxr,sitates daily preparation of fresh reagent. Electrochemical HG (EcHG) is a possible altemafive to the NaBH4 reaction. It offers the potential advantage of requiting fewer reagents, therefore less chance exists for sample contamination by traces of analyte in the reagents. Tne generation of ~ c hydride by electrochemical reduction was first reported by Sand and Hackford in 1904 [14]. Rigin and coworkers demonstrated the use of a batch type EcHG for the detemaination of As and Sn, and found this method

New Developments in Hydride Generation- Atomic Spectrometry

55

to be remarkably free from chemical interferences [15, 16]. In recent years, a number of investigations on a new flow type EcHG have been done to realize the above-mentioned potential advantage of EcHG. Selected examples ofrec~nt research on EcHG are given in Table 1. Lin et al. [17] and Brockmann et al. [18] reported on the interference from nickel on the determination of arsenic using a platinum cathode, and concluded that there was no effect in the presence of 100 and 10000 lag ml"~, respectively. However, Hueber and Wlnefordner demonstrated that many of the tramition metals that interfere with lhe NaBHn-acid HG methods also affect the EcHG method [19]. Brockmann et al. have shown that no signal can be obtained from As(V) and Se(V0 using a Pt cathode [18]. In conWastto this, Htmng reported responses from these oxidation states (30% and 76% relative to those of As(HI) and Se(IV), respectively), but it is unclear which cathode material was ttsed in their experiments [20]. Denkhaus et al. demonstrated that interferences could be reduced in comparison to wet chemical HG by use of NaBH4, and the sensitivity for As or Sb was independent of the oxidation state ofthe analyte, if cathode materials with a high hydrogen overpotential (Pb, Cd, Hg/Ag) were used [21]. They did the determination of total As and Sb without a pre-reduction step. A HG efficiency of 86-100% was obtained for As and Sb. 1he detection limit was limited by the relatively high blank value induced by EcHG. This relatively high blank value is caused by the formation of electrochemical alloys (PbAsx-phase), which are desorbed by an equilibrium reaction. Schamnl6ffel and Neidhart constructed a flow injection (FI) / EcHG / AAS system for the determination of As(m) and total inorganic As [22]. They used an on-line pre-reduction of As(V) using KI or L-cysteine as the reducing agent, qhe efficiency of reduction of As(V) to As010 by KI was p H - d ~ d e n t and the teAuction was complete only at high acid concentrations, whereas the efficiency of reduction by Lcysteine was independent of acid concentration of the carrier stream. A 10% solution of L~ysteine achieved a total pre-reduction at 95~ in 40 s even in the on-line mode. Experimental results concerning the interference and the dependence

u l

m

Table 1 Electrochemical Hydride Generation Species

Dctcction Dctcction Method Limit

Catliodc / Catholyte / Application Diaphragm

Comments Vitreous carbon, Pt and Ag-Hg were tested as cathode material and H2S04, HC104, HNOl and HCI were tested as electrolyte. Experiments with Pt (hydrogen overpotential: 0.10 V), vitreous carbon (0.82 V) and Ag-Hg (1.42 V) cathodes showed that a cathode with a higher overpotential gave a higher signal. Interference from transition metals varied with the cathode material.

I7

3 I ng n11-l Pt / diluted H2S04 or NlST SRhl Anolyte: 2 M H2S04.As(V) and Se(V1) gave no signal response. No 363,365 ICP-AES for As, 4 0 ng HCI / ion exchange depression of the analyte signal is observed even at concentrations of membrane (Nafion) alloy steel the interfering ions (Ni and Co) of up to 10000 pg rnl?

18

AAS

3 1 11g m1-I vitreous carbon / 1 M for AS, 4 0 ng H2S04 / ion exchange nll-l for se membrane for 100 111 sample

polyester film, mangrove leaves, medicine

volume AAS,

Pt, Ag, Pd, Pb / 1 M H2S04, NaOH, H,P04 / porous glass frit plienylarsi ne oxide

Ref.

NlST S R M 1648 urban Particulate matter, 1646 estuarine sediment

fibrious carbon / I M water, H2SO4 / fritcs madc of mcdicine glass-wool or silicagel and potassium silicate

Cell was operated in the voltage regulated mode. 22 V for As and Se; 18 V for Sb. No significant difference between the effectiveness of Pt, Ag and Pd cathodes for AsHl generation. Pd gave the best results for H2Se. The Pb cathode gave the largest signal for SbH3, but corroded quickly in 1M H2S04 above 18 V. Efficiencies of HG were as follows: As(III), 50-98% As(V), 10% of that of As(Il1); phenylarsine oxide, 25% of that of As(II1); Se(IV), 65-98%; Se(VI), 0%; Sb(V), 0%. Se(V1) and Sb(V) gave no signal under any conditions.

19

Several cathode materials were tested and efficiency of ASH3 generation increases in the following order: Pt
22

,z

I % 0

.d

iT

Sb(lll), Sb(V)

in situ 20 pg ml ~ for Pb / 0.3 - 2.4 M HCI trapping/E 2 ml sample or 0.35 -0.55 M TAAS volume H2SO4 / ion exchange membrane (Raipore 1010 or Neosepta CM- 1)

NRCC SLRS-2 and 3 river water, NASS-4 seawater

Cell was operated in the constant-current mode (150 mA cm'2). Efficiencies of SbH3 production from Sb(lll) with Pb, pyrolytic graphite and Pt cathgode were 90%, <45% and 0%, respectively. When C12 could be visibly detected in anolyte solution, siganal would drop dramatically. To reduce the C!2 and to recover the signal, hydroxylamine hydrochloride was added to the anolyte. Efficiency of SbH3 generation from Sb(lll) or Sb(V) was same.

23

Mechanism of direct interference or memory interference by Cu 2.

24

Z7 O

,.<

As(Ill), Sb(lll), Se(IV)

in situ trapping/E TAAS

Pb, pyrolytic graphite, vitreous carbon, Pt / 1.2 - 7 M HCI or 0.54 M H2SO4 / ion exchange membrane (Raipore 100 or Neosepta CM- 1)

As(ill), As(V), So(IV), Se(VI), MMAA, DMAA, AB, AC

in situ As: 84 pg ml Pb / 0.3 - 2.4 M HCI NASS-4, trapping/E I for 1 ml or 0.18- 1.44 M CASS-3 TAAS sample H2SO4 / ion exchange seawater

As(lll)

in situ 15pg ml l for Pb (10 x 100 mm, trapping/E 200 ~tl thickness 1 mm) / 0.1 TAAS sample M H2SO4 / ion volume exchange membrane (Nation type NAF117, Dupont)

vohlme. Se: 7.5 pg ml j

membrane (Raipore 101() or Ncoscpta

for 10 ml

CM-I )

sample volume.

and Ni 2+ was described.

C~

O I

> O

Pb, Zn and Pt were evaluated as cathode materials for the production of H2Se. The efficiencies with Pb and Zn were about 60% and that with Pt was 0%. The reproducibility of H2Se production with Zn cathode was very poor. With Pb cathode, the efficiencies of HG from various species were as follows: As(Ill), 86%; As(V), 73-86%; MMAA, 86%; DMAA, 56%; AB and AC, 0%; Se(IV), 60%; Se(VI), 30%. Pre-reduction of As(V) and Se(VI) was performed with the addition of 1% cysteine and boiling with HCI, respectively.

25

Current density: 40 mA/cm 2

21

,.... t~

O

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58

H. TAO and T. NAKAHARA

of oxidation state in EcHG ~ to be inconsistent and sometimes contradictory. Probably, this is because the efficiency of EcHG is susceptible to the operating conditions, such as the nature of the cathode materials and electrolyte. Ding and Sturgeon performed an in-depth investigation of these problems [23-25]. Their EcHG system is illusWated schematically in Figure 1. The plexiglas| body of the electrolytic cell consists of cathode and anode blocks, each of which contains tubular inlets and outlets for solutions. A platinum anode and a cathode electrode are fixed in channels of the anode and cathode cells, respectively. A cation-exchange membrane is used to physically isolate the cells. An auxiliary Ar purge gas is introduced to the cathode comparanent to facilitate mass transfer throughout the system. This system provides a large ratio of cathode surface area to cathode cell volume. They reported that the direct interference or memory interference by Cu and Ni was severe when using Pb, pyrolytic graphite or vitreous carbon as the cathode. For example, the presence of as little as 7.5 lag mll Cu severely interfered with stibine generation, resulting in a 50~ suppression of the signal compared with that fi'om a Cu-free solution. Nickel, at a concenWation of 10 lag ml 1, interfered even more severely than Cu. Fm~ermore, severe memory interference occtu'ted when a subsequent solution which contained only Sb0ID was rtm through the EcHG cell. This continued to result in a suppressed response relative to that obtained in a cell which was never exposed to the presence of a solution of Cu or Ni. In conWast, 600 lag ml~ of Ni exhibited no effect when a Pt caflaode was used. However, the production efficiency for arsine itself was too low to be analytically useful with a Pt cathode (<8%). EcHG consists of at least three sequential events: reduction and deposition of the analyte onto the stkrface of the cathode, the stepwise reaction of the deposited metal with nascent hydrogen (H.) generated on the surface of the cathode, and the subsequent desorption of the analyte hydride. The half-cell reduction potentials for the Ni2+-Ni, Cu2+-Cu, As-AsH3 and Sb-SbH3 systems are -0.47, 0.10, -0.78 and -0.75 V, respectively, relative to a satt~ed calomel elecaxxte (SCE). As long as the hydrides are electrochemically formed, the interfering metal ions would be reduced to the

New Developments in Hydride Generation - Atomic Spectrometry

59

elemental state on the surface of the cathode, with the possibility that they might also be subsequently dispersed as fine metal particles within the cathode comparanent. Such a deposit of metal would change the physico~hemical sta-face characteristics of the Pb cathode and, consequently, destroy the favorable nature of Pb for EcHG and lower the efficiency of riG. Additionally, any dispersed metal which is present in the solution phase may also scavenge the generated hydrides, thus reducing the response. They suggested that the major interference occurs, not in the sohrdon phase, but, rather, on the sta'fa~ of the cathode. An examination of the cathode stu'face using dynamic secondary ion mass spectrometry (SIMS) confirmed that the Cu was not delx~ited, as a uniform layer, but as subanicron sized islands, dispersed over the sm'face. It is possible that these islands are formed at active s ~ sites on the cathode, and are probably the same sites which the major portion of the hydrides are formed. Although an increase in the concentration of HC1 is effective in deoeasing the interference caused by transition metals in the NaBH4-acid reduction system, an increase tiom 1.2 to 7 M HCI had no effect in alleviating interference with EcHG. The addition of I~ysteine was also ineffective. The electrodeposition of Pb onto the calhode surface to regenerate the active s ~ continuously was useful for reducing the interference, but caused a very high methodological blank, due to contamination. Also, an unsucy.essful attempt was made to generate Pb2+, inten~ttently and electrochemically, directly from the surfacx of the Pb cathode. They suggested to take advantage of the differences between half-ceU reduction potentials of the metals to effect a preliminary separation of the analyte fi'om the interferent, lhrough control of the cathode potential by use of a tandem cell arrangement. For EcHG to be applicable to real world samples, alleviation of the interference will be indispensable.

H. TAO and T. NAKAHARA

60

Graphite Furnace

t

H2 + Hydride

I

[

Valve# 1

Cation Ion-Exchange Membrane

~

_ 1 ....

/

Hydride +

I

Waste

"~'~Porous Membrane GasLiquid Separator

Electrochemical Cell IAnolyte] !

_3

Cathode Cell

...(~Valve # 2

(~Valve # 3 Liquid Waste

Pump mL loop

Cath

~

.

_

.5

Figure 1 Sdcmaie diagram of e ~ hydrideg ~ . ~uced the Elsevier Science B. V. with acknowledgementto Ding and Sturgeon [25].

by permissionot

2.2. HG utilizing fast gas-liquid separation A major shortcoming of HG is that the presetr~ of high concentrations of transition metals, such as Ni, Co and Cu, severely suppress the formation and release of the analyte hydride. 1his suppression effect is thought to be mainly the result of the reaction between hydrides and reduced forms of interfering metals or their borides. Minimization of the contact time

New Developments in Hydride Generation - Atomic Spectrometry

61

between the analyte hydrides and the interfering species would be e ~ to result in a redt~on in tile severity of the transition metal interference. Sturgeon and co-workers developed a novel HG method which involves very short ~ r times (below 100 ms) and a rapid separation of the gaseous p r o d t ~ [26-28]. Rapid separation is achieved by processing the reaction solutions as a spray during or alter HG. A schematic diagram of the hydride generator is shown in Figure 2. Ding and Sturgeon modified a Meinhard nebulizer by inserting a capillary tube into the sample introduction channel [26]. The acidic sample, introduced through the capillary, and the NaBH4 solution introduced through the normal sample introduction channel of the nebulizer were mixed just prior to nebulization. A membrane gas-liquid selxuator was originally used, but later this was changed to a conventional Scott-type spray chamber due to the long-term instability of the performance [27]. This HG system was interfaced to an ICP, quartz tube atomizer or graphite tin.ace. Under these conditions, the suppression effeOs on the generation of H2Se, arising from high concentmlions of transition metals, can be virt~_~allybe eliminated, i.e., no redt~on of the Se signal is observed in the presence of 50 g 11Ni, 25 g 1l Co or 20 mg 1-1 Cu. qhey also reported on a system which utilized a cross-flow nebulizer and a modified Scott-type spray chamber for HG/ICP-MS. The volume of the reaction zone at~r mixing the acidified sample and NaBH4 solution at the tip of the nebulizer was only 1.6 lal and the reaction time was about 60 ms. An important modification in the spray chamber involved the addition of a second gas inlet to permit i n d ~ d e n t optimization of both the gas flow rate for the nebulizer and that for the transport of the reaction products to the plasma. Analytical results generated by external calibration and isotope dilution methodologies are in good agreement with the certified values for Se in biological reference materials. D e ~ o n limits for Se and other hydride forming elements, i.e., As, Sn and Sb, are below 10 pg m1-1.Volatile species ofCu, Rh, Pd, Ag, In, Au, Hg, T1 and Pb are also produced, and the estimated detection limits for these elements are presented.

62

H. TAO and T NAKAHARA

ICP

"Spray"Chamber . 1.5 "-~ cm

;as Phase

Sample~

i

t AuxiliaryInlet--*NaBH4 CarderGas

. ~ Pump

PorousMembrane

LiquidPhase

-1 ! Pump

=_Waste Figure2 Schematicdiagramof hydrktegeneratorutilizingfastgas-liquid~ ~ced bypermissionoftheAmericanChemicalSocietywithacknowledgementtoDingandSturgeon[26]. 2.3. HG with immobilized borohydride on ion-exchange column and movable reduction bed Although an aqueous solution of NaBH4 is typically used in a conventional HG, other forms of bomhydride have been employed. Tesfalidet and Irgum [29] and Namsaki et al. [30] showed that arsine could be get,rated by passage of an acidified sample through an anion-exchange column on which BH4 had been previously immobilized. Cao and Namsaki extended this technique to the HG of Sb and Se [31]. Can~ro and Tyson reported on a method for the cx)-immobilization of Se(IV) and BH4- on an anion-exchange resin followed by the passage of acid to generate H2Se [32]. Since the co-immobilization method suffered interferences, if the sample solution contained metals which were precipitated from an alkaline solution, they fiarther developed a method for s u ~ i v e retention of Se(IV) an BH4 [33]. The successive retention method not only circumvented the problem of the precipitation of transition metals, but also decreased the amount of BHnused, therefore, the blank contribution fi'om this reagent could be decreased with a consequent improvement in the detection limit.

New Developments in Hydride Generation - Atomic Spectrometry

63

A movable reduction bed hydride generation (MRBHG) system has also been developed by Tian et al. [34, 35]. The schematic diagram of the

Figure 3 Schematicdiagram of movable reduction bed hydride genev~r. Repratuced by izennission of the Royal Society of Chemistry with acknowledgement to Tian et al. [34].

MRBHG system is shown in Figure 3. The physical set-up of the device resembles a VCR cassette. The re,action bed is circulated by two rollers that are placed in a sealed organic glass cassette and moves around by ttm~g a tape miler. The bed is constructed with a mixture of KBH4 and solid tartaric acid powder at a certain ratio onto the surface of a long, naxrow, adhesive tape. The tape crosses the two holes on the stopper into and out of a reaction chamber. The most important advantages of the technique over conventional methods is the elimination of the use of a complex gas-liquid separator. A similar hydride generator has been reported by Inui et al. [36, 37] and Bamett et al. [38, 39]. The principal difference between the MRBHG, and the device described by Inui et al., is that KBH4 and solid organic acid are pre-mixed as the reducing reagent in the MRBHG. This means that the sample solution does not need to be acidified prior to analysis. In addition,

64

H. TAO and T. NAKAHARA

the MRBHG has the advantage that the reducing reagents can be renewed continuously, which facilitates continuous on-line operation. They reported that m ~ interferences from hydride-forming elements are greatly eliminated or s ~ t i a l l y redtr.M with the MRBHG, because of the excess amounts of reducing reagents. In addition, the kinetics of the reaction may play a role in minimizing suppression effects, since hydride-forming elements are reduced faster than the interfering metals, and the volatile hydrides are immediately removed from the reaction stwface by a flow of gas. "lhey also reported that tl~ MRBHG gave the almost same signal intensity for all the As species tested, such as As(I[0, As(V), MMAA and DMAA, and Se species, such as Se(IV) and Se(VI), although the reason for this was not clearly stated.

2.4. Vesicle-assisted hydride generation Traditionally, the elements that can be determined by HG are limited to eight elements, namely As, Sb, Bi, Se, Te, Ge, Sn and Pb. Therefore, the development of a new HG system for the determination of additional elements is one of the important research areas in atomic spectrometry. A list of recent investigations ofthe HG of cadmium is provided in Table 2. Cacho et al. reported the generation of a volatile Cd species (presumed to be a hydride) in N,N'-dimethylformamide in the presence of diethyldithi~amate using NaBH4 [40]. Sanz-Medel and co-workers proposed the ttse of organized media, particularly didodecyldimethylammonium bromide (DDAB) vesicles, which enhance the efficiency of riG in the presence of NaBH4 [41-46]. They concluded that the analytical sensitivity and selectivity achieved with HG could be improved by the use ofsurfactants for the following reasons. (a) Sttrfactants may concentrate reactants at tt~ molecular level and, thus, change the thermodynamic and kinetic reaction constants; thtts, the analytical sensitivity can be substantially changed, and perhaps improved, in an appropriate medium. (b) Surfactants may solubilize, in a selective manner, analytes and reactants (e.g., NaBH4) in organized "aggregates"; thus, the special m i ~ v i m n m e n t which exists in or on these aggregates may change the

Table Media /

2

Hydride Generation of Cadmium Analytical Detection Limit Application

Catalyst Method DMF")/DDTc b), Quartz Tube 9.1 ng 55oc AAS tea infusion

Z Sample Preparation and Brief Comments

..... R~'f.

The speed of HG increases with increasing iemperature. Batch generation ,'node.

40

Tea leaves: extracted with hot water for 30 min. The efficiency of CdH2 generation and transport increases with decreasing temp. Continuous flow generation mode. Organized media improved the kinetics of CdH2 generation. Among various organized media, DDAB vesicles gave tile best results. Tile only interferences were those of Zn and Ni and no interference from Cu and Pb was observed.

41

0.()1 M DDAB ~) ICP-AES vesicles, room temp. (0 - 60~

1 ng ml ~

ibid.

1 ng ml 1

An overview of surfactant-based organized media, such as micelles and vesicles, on the generation of volatile species for atomic spectrometry is presented.

42

ICP-AES

1 ng mi l

Cold vapor generation of monoatomic Cd ~ was accomplished by reduction of Cd 2§ with

43

C VJ)/AAS

80 pg ml ~

NaBH4 using vesicles. This cold vapor were not thermodynanfically stable, bul the Cd ~ lifetime was long enough to be measured. This provides the basis for AAS measurement of Cd at room temp.

ICP-AES CV~)/AAS

ibid

O

)..

t~

O

ibid.

in situ 60 pg ml ~ for trapping/ET 1.4 ml sample AAS volume

0.01 MDDAB ~ vesicles, room temp. (spray chamber, 2~

insitu trapping/ET AAS ICP-MS

ibid.

15 - 28 pg for anion50 Ill sample exchange HPLC/ICP- volume MS

NIST SRM2670, 14 pg for 1.4 ml sample human urine volume 3.5 pg for 50 ~tl sample volume

O

Volatile Cd species were trapped in a graphite platform coated with Pd, pre-heated at 150~

44

Urine: filtered through a 0.45 pm membrane; diluted 1+1 with H20. Use of silicone as

45

an antifoam agent for vesicular HG allowed a throughput of about 20 samples h ~. Concentration of Cd in urine was evaluated directly by aqueous standard calibration as no matrix interferences were observed.

standards of Urine: filtered through a 0145 lam membrane. Separation was carried out using a rabbit liver MTe) concentration gradient (2 - 200 mM) of Tris-HCl buffer at pH 7.4 and a polymeric and human urine anion-exchange column. The efficiency of CdH2 generation for Cd bound to the main isoforms of rabbit liver was the same as for inorganic Cd 2+ from a vesicular medium.

)

48

O

ibid.

vesicular8 - 17 pg for 50 standards of mediated Id sample rabbit liver MTe) HPLC/ICP- volume and cylosol MS samples of eel liver and kidney

!% Ihiourea and AFS

8 pg m l-I

1 jig ml l Co,

room letup.

ibid.

CVa)/AAS

20 pg ml 1

NIST SRM 1577a bovine livcr, SRM 1645 reiver sediment, tap water ibid.

Eel livers and kidneys: homogenized in 3 volumes of 10 mM Tris-HCl containing 250 mM sucrose (pH 7.4); centrifuged at 100000g (60 min, 4~ supernatant fraction (cylosol) was filtered through a 0.45 lam membrane. Separation was carried out using a concentration gradient (2 - 200 mM) of Tris-HCl buffer (pH 7.4) containing DDAB

49

vesicles and C~8 reverse-phase column. Chromatographic behavior of Cd MT ':) was similar to that observed by anion-exchange I-[PLC. Bovine liver: digested with HNO3-HCIO4-HCi. Effects of 28 diverse ions on the

50

generation of Cd volatile species were investigated. Serious signal depressions were observed from Cu and Pb but these were decreased by using KCN as masking agcnl for Cu and by coprecipitation with BaSO4 for Pb.

Vald6s-Hevia y Temprano el al. [41] reported that 50 lag ml l of Cu and Pb caused no

51

interference in the determination of 100 ng ml ~ of Cd. The different results obtained in this paper may be caused by different conditions uscd for the generation. 0.5 [tg ml -~ Ga, room temp.

CVa)/in situ 4 pg ml ~ for trapping/ET 500 lal sample AAS volume

2% thiourea and isotope 26 pg ml ~ 2 jig ml 1 Co, dilution/ICP room 91 -MS

none, 5~ (2 10~

CVa)/AAS

50 pg ml ~

53

NIST SRM2704, NRCC BCCS-!, PACS-1 sediment, NIST SRM2670 urine

52

Freeze-dried urine: reconstituted and spiked with enriched isotope; digested wilh HNO.~ in a closed PFA vessel by MW digester. Spectral interferences from MoO and ZrO on Cd were separated by vapor generation technique and the intereference from transition metals on the efficiency of vapor generation was alleviated by isolope dilution method.

NIST SRM1643, A gas-liquid separator with a cooling system eliminated water vapor by condensation seawater, tap and prevented CdH2 from decomposing owing to its thermal instability. Since a matrix water effect was observed, standard addition method was used. Identical deleclion limit and precision were obtained without surfactants or metallic species as catalysts.

waste water, Interferences from transition metals (Cu, Ni, Zn and Pb) were ~ompletely eliminated by sewage sludge addition of KCN to reaction medium, removing the need for slandard addition method. a) N,N-dinlethylformamide. b) diethylditlliocarbamate, c) didodecyldimethylammonium bromide, d) cold vapor, e) metallothionein. none, 3 - 5~

CVd)/AAS

54 pg ml ~

NRCC NASS-4 Volatile Cd species were trapped in a graphite tube coated with lr. Co(+40%), Si(+25%) seawater, SLRS- and Ga(+50%) exhibited a significant catalytic effect on the Cd cold vapor generation. 2 riverine water

Z: ,--] >, 9 54 -.] 2~ >, 55

ZZ >.

New Developments in Hydride Generation - Atomic Spectrometry

67

reactions (interferences) observed in the bulk aqueous phase. This vesicle-assisted Cd hydride generation has been utilized to flaker increase the AAS sensitivity for Cd by in situ trapping in a graphite fumace [44, 45, 47]. This highly positive effect of vesicles for HG could be also combined with their potential in vesicular HPLC separations for CA speciation in human urine and fish cytosols [49]. Sanz-Medel et al. also reported on the cold vapor generation ofmonoatomic Cd~by reduction of Cd2+solution with NaBH4 using vesicles [43]. They proposed the mechanism shown below for the cold vapor generation of Cd: BH4"+

3H20 + H + --+ H3BO3 + 8H"

(1)

8H- + Cd 2+ ~ CdH2 + 2H2 +2H +

(2)

CdH2

(3)

-->

Cd 0 + H 2

where H. is "nascent" hydrogen. Since CAH2 is volatile, this should be the transportation mechanism for cadmium. However, at room tempeman~, during transport, the volatile hydride which is formed would decompose according to reaction 3, and the resulting Cd ~ which is not volatile, would detx~it all along the connecting tubing. 1his decomposition is more efficient at higher temperatures. In any case, the great excess of H2 formed by reaction 2 would prevent the complete decomposition of CdH2. Thus, at least a ceaain amount of CdH2 would ultimately reach the absorption cell. Once there, according to reaction 3, a substantial proportion of CdH2 seems to be able to form monoatomic Cd~ Although this cold valor would not be thermodynamically stable, the lifetime of monoatomic Cd~ appears to be sufficiently long to be measured. 1his provides the basis for the AAS measurement of Cd at room tempemau~. Guo and Guo also developed HG (cold vapor generation) using thiourea 'and Co as calalysts for the reaction, resulting in an increase in sensitivity [50, 51]. The detection limits obtained with this method were 20 pg m1-1 by AAS and 8 pg m1-1 by AFS. Hwang and Jiang used the same media for HG and determined CA in urine by

68

H. TAO and T. NAKAHARA

ICP-MS [52]. An isotope dilution (ID) technique was used to alleviate the depressing interference from the concomitant elements, qhe detection limit was 26 pg m1-1.Bennejo-Barrem et al. generated the hydride in the absence of organic w.zction media by adding Ga as a catalyst, resulting in a detection limit of 4 pg ml~ with the in situ trapping / ETAAS [53]. Cmnara and cxr-workers showed that it was not ner.essary to add surfactants and metallic species as catalysts in order to generate the volatile Cd species [54, 55]. They achieved a detection limit of 50 pg ml~ with cold vapor AAS and applied the p ~ u r e to file analysis of a number of standard reference water samples; but, the method of standard additions was needed to alleviate interference from the concomitant elements, qhey later extended the applications to the analysis of waste water and sewage sludge, and interference from co-existing metals such as Cu, Pb, Ni ar~ Zn was overcome via the addition of potassium cyanide. (Caution: This must have produced considerable amounts of hazardous HCN on merging with the acid carrier stream.) 3. ADVANCES OF METHODS OF ATOMIZATION 3.1. Atomization interferences in the gas phase In addition to graphite fumaees, diffusion flames and quartz tubes are also employed to atomize hydrides for AAS. In quartz tubes, there are two types: 'flameless' extemally heated quartz tubes and flame-in-tube a t o ~ . Atomization interferences are intimately coupled to the mechanism of hydride atomization and to the fate of free atoms in the observation volume of the atomizer. Two types of atomization interferences are currently proposed: 1) A radical population interference due to the depletion of hydrogen radical population in the atomizer. 2) An analyte decay interference due to acceleration of the decay of free analyte atoms in the atomizer. Welz and Stauss investigated the mechanism of atomization interferences in the 'flameless' externally heated quarlz tube atomizer [56]. They reported that in the batch HG system, the radical deficiency constittaed the main reason for the low tolerance of other hydride-forming elements and that in the flow injection HG system, the prevailing mechanism was analyte

New Developments in Hydride Generation - Atomic Spectrometry

69

decay interference due to sm'hc~ alterations catts~ by the deposition of the interferent in the quartz tube atomizer. D'Ulivo and ~ dina investigated the mechanism of atomization interefere~ in flame-in-mt~ and in diffusion flame atomizers [57]. For this purpose, they developed a hydride atomizza" which was able to operate in lhese two modes. Liquid phase interferences were eliminated by using a twin-channel continuous-flow hydride generator. They repotted that only the analyte decay interference was significant in unheated flame-in-tube atomizers and ditSasion flames. 1hese atomizem, which were characterized by a relatively large supply of oxygen, were inherently resistant to the radic~ population interference since hydrogen radic~ production was proportional to the oxygen supply. They also repotted that the removal of free analyte atoms at high analyte concentrations or in the presence of the interfetent was dominated by the reaction with polyatomic species and/or particles formed at high c o ~ t m t i o n s of analyte and interferent atoms. 13~dina and D'Ulivo developed an argon-shielded highly fuel-rich hydrogen-oxygen diffusion microflame, refened to as a flame-in-gas-shield atomizer, for AFS [58]. The sensitivity was at least 2 times higher and the noise was lower lhan the miniature diffusion flame. Sensitivity .was controlled by interaction of the analyte (Se) with atmospheric gases. Analyte free atoms were removed from the observation volume by chemical re,actions with oxygen penetrating fi'om the ambient atmosphere. No significant quenching effect due to the intem~on of excited Se atomic levels with nitrogen or hydrogen was found. I~ dina et al. investigated the mechanism of hydride atomi~tion and the fate of free atoms in the miniattae diffusion flame [59]. Selenium hydride was used as a model for other hydrides. The spatial temtmature distribution was highly inhomogeneous ranging from 150~ to 1300~ The entire flame volume was a c a ~ y a cloud of hydrogen radicals, which maintained the analyte in the flee atom slate, sirm~ hydrogen radicals which were formed in outer zone of the flame ditfused to its cooler inner regions. Tesfalidet et al. determined hydrogen radicals in a miniaturized oxygen/hydrogen flame, which is similar to a flame-in-tube atomizer, by means of electron spin resonance (ESR) spectt~copy to investigate the

70

H TAO and T. NAKAHARA

atomization mechanism [60]. By using this technique, they were able to monitor the production of hydrogen radicals in the hydride atomization step and the decline and consumption of these radicals when AsH3 was intaxtuced into the flame as a model compound. These results provided the direct experimental evidence needed to support the proposed mechanism for the atomization of hydrides, i.e., that atomization is brought alx~ by hydrogen radicals. Matott~ek et al. measured the cross-sectional distribution of free Sb atoms in quartz tube atomizers by AAS using a CCD camera [61]. They confirmed that the highest fi~ atom concenWations were found near the tube axis, decreasing towards the walls, in the ~ e a t e d flame-in-tube atomizer. In the extemally h~aed atomizer, the most widely used in routine analysis, the free atom d'~tribution was much more homogeneous compaw.d to the unheated atomizer under analytical conditions, although pronounced inhomogeneity was obtained at high Sb concentrations in a roll-over part of the calibration curve, qhis was explained on the basis of free atom decay on the surface ofpolyatomic particles formed at high analyte concentrations. Grinberg et al. developed an externally heated quartz tube atomizer which diffened from the usual T-tube design only in having 5 holes drilled along the length of the optical tube, which was heated by the acetylene-ak flame [62]. q'hey investigated mtmml hydride-forming element interference and found that the holed quartz tube a t o ~ was able to induce larger tolerance limits for the interference in 9 of the 16 mtmml interference possibilities studied. D&lina and Matou~ek developed a multiple microflame quartz tube atomizer which consisted of two concentric tubes [63]. The inner tube had multiple tiny orifices over its length in the wall and the otaer tube, which was devoid of orifices, was externally h~/ed as the conventional quartz tube atomizer. They demonstrated that lifts set-up reduced the poor resistance to atomization interferences and the unsatisfactory linearity of calibration graphs. Numemm fundamental studies have been ~orted on atomization mechanism and many improvements ofqtmrtz tube atomizers and diffusion flames have been made in the past few years. By using newly developed techniques such as ESR spectroscopy, fia~er progress on the atomization

New Developments in Hydride Generation - Atomic Spectrometry

71

mechanism and elimination of atomization interferences is expected.

3.2. In situ Trapping HG / electrothermal atomic absorption spectrometry The most atWactivc advantages of the in situ trapping HG / ETAAS technique are the very low detection limits and the decrease of the kinetics interferences in HG and interferences in the atomizer. Matusiewicz and Sturgeon presented a review of this methodology [6]. Palladium is both a well-recognized chemical modifier in ETAAS and an efficient collector for the trapping of volatile hydrides. One of the main problems of a Pd-treated atomizer is that a Pd modifier solution must be applied before every hydride trapping nm because of its thermal instability during the atomization step. Ni et al. used a graphite atomizer coated with Ag to trap the hydrides of Se and Te [64]. The advantage in this approach is that atomization occurs at 1800~ lower than that for a Pd-coated surface (2000~ thereby increasing the lifetime of the tube. Shuttler et al. proposed the in situ trapping of hydrides of As, Se and Bi in a graphite tube coated with a Pd-Ir modifier as a "permanent modifier", which allowed up to 300 complete trapping and atomization cycles [65]. Other "permanent modifiers" tested included Ir sptater-c~ated tubes [66], carbide coatings onto which Ir had been deposited [67-69] and other carbide-forming elements [70, 71]. Haug and Yiping investigated two groups of trapping reagents, i.e., carbide-forming elements (Zr, Nb, Ta or W) and noble metals Or, Pd-Ir) [70]. The effective trapping of germane was possible on Zr-coated tubes and more than 400 complete trapping and atomiz~ion cycles were possible, k-coated graphite tubes allowed trapping at lower temt~'ature but the signals were small and the stability was low, compared with those for the Zr coating. C r a t ~ et al. also investigated a graphite tube crated with Zr [72]. The Zr coating employed was relatively stable; and, once formed it withstood about 80 firings without any significant change in the efficiency of hydride collection. Tsalev et al. evaluate~ an Ir-Zr-treated and k-W-treated platform as a permanent modifier and found that Ir was much more promising than Pd, being an efficient thermal stabilizer for numerous volatile elements

72

H. TAO and T. NAKAHARA

during more than 800 firings [67]. Although Ir was better stabilized on a W-treated platform than on a Zr-treated one, the vaporization and atomization temperature for volatile analytes were also higher and double peaks were observed for B i and Te, when an k-W-treated platform was ttsed, and therefore, an Ir-Zr-treated platform was the most promising. Tsalev et al. also performed an optimization study for HG and collection and investigated the behavior of some organometallic species of As, Sn and Se [68, 69]. Optimum HG conditions differed substantially for As(m), As(V), MMAA and DMAA, unless L-cysteine was added. Organoelement species of As, Sn and Se were thennMly stabilized in a similar manner on both Ir-Zr- and k-W-treated platforms, the least stable species being selenomelhionine and trimethylselenonium. Do~ekal et al. presented a table with available publications conceming the p r e ~ e n t of the graphite surface (Pd and/or Ir modifier) and the resulting efficiencies of riG and trapping till 1995 [73]. Efficiencies of riG and in situ trapping in the transversely heated graphite fumace with a L'vov platform by using Pd modifier were studied for selenium hydride, arsine and stibine by means of the radioWac~rs of 75Se, 76Asand lz2Sb. This ra~otmcer study proved that optimum conditions for H2Se were achieved with a broad range of Pd modifier mass, trapping t e x t u r e , carder gas flow rote and capillary distance. This is very important for routine applications of in situ trapping of hydrides to the determination of hyddde-fomaing elements by AAS, as variations in these parameters cannot negatively influence accuracy. Murphy et al. reported a comparison of the applicability oflr- and Zr-coated graphite tubes to simultaneous multi-element (As, Sb, B i, Se and Te) hydride trapping and found that Ir was well suited to this application [74]. They also developed a purification method for the NaBH4 solution using an on-line and off-line active alumina column. The purification is very important because the ~ detection limit is not limited by the instrumental sensitivity but by impurities in the NaBH4 reagent. HG and in situ trapping on the inner surface of the graphite tube, treated with Pd or Ir, was coupled with the determination by furnace atomic nonthennal excitation spectrometry (FANES) [75] or ICP-MS [76, 77], as

New Developments in Hydride Generation - Atomic Spectrometry

73

well as by ETAAS. 1hese methods were validated using standard reference materials of biological tissues and seawaters. Marawi et al. attempted to me strips of W, Ta, Mo and Re coated (electroplated/sputtered) with Pd as platforms inside the graphite ftmaace to trap the hydrides [78]. Scanning electron microscopy (SEM) showed a smoother Pd layer coveting the metallic substances when compared with the graphite. The Pd-sputteted W platform showed a superior performance over tt~ other platforms. In situ trapping with an untreated graphite furnace was coupled wilia the determination by Ar and He MIP-AES [79, 80]. A number ofpal~rs on flow injection (FI) / HG coupled to graphite fumace AAS with the in situ trapping of hydrides has been reported [72, 81-86]. Burguera and Burguera [81] and Burguera et al. [87] ttsed the Fleitmann reaction, in which arsine was generated by the reduction of arsenic with metallic aluminum in a basic medimn, instead of using NaBH4 in HC1. Walcerz et al., using a Pd-coated graphite furnace, reported that TritonX-100, added together with the Pd solution, improved sensitivity and reproducibility, likely by promoting a more even distribution of Pd over the s~ of the graphite tube [85]. Willie developed FI / on-line photo-oxidation / HG / in situ trapping / ETAAS system for the "fl~ order" speciation of As in biological tissue and urine samples [86]. The in situ trapping method was also applied to the detenuimtion of Ge in garlic [82], Sn in hair and serum [83], Pb in biological samples and ionic organolead compounds [84, 88], and Se and As in highly minemliz~ water [89]. Liao and Li trapped indium hydride into a pre-heated graphite ~ a c e coated with Pd and obtained greatly improved sensitivity for the determination of In by HG/AAS [90]. Tyson et al. investigated the effect of smile volume on the limit of detection in FI / HG / in situ trapping / ETAAS to evaluate the claim that a decrease in the limit of detection might be achieved by increasing the sample volume [91]. They observed that as the sample volume was ino'eased, the detection limit improved significantly up to a volume of 500 pl, but for volumes larger than 1000 lal no ~ r significant improvement was obtained.

74

H. TAO and T. NAKAHARA

4. CHEMICAL INTERFERENCES IN LIQUID PHASE AND PRE-REDUCTION Interference effects, especially by wansition metals, are widely realized to be very severe. A number of papers have attributed interferences to the capture and decomposition of hydride by finely dispersed concomitant metal precipitates [92-94] or metal borides [95-97]. Measures taken to minimize such interferences include use of masking agents, matrix isolation, and the introduction of flow-injection and membrane-based gas liquid separator systems. Wickstrom et al. reported on an approach to minimization of transition metal interferences for H2Se generation [98]. They ttsed complexing agents, such as EDTA and diethylenetriamine~taac~ic acid (DTAP), and reduced selenite to selenide with NaBH4 in an alkaline medium, followed by acidification of the solution to produce H2Se. The reduction of transition metal ions could be avoided before the hydride was released from the liquid phase, and high concentrations of interfering Wansition metal ions which were present in the solution were acceptable without causing interferences, 8000 mg 1-1for Ni2§ and Co2+, 5000 mg 1-1for Cr3§ 500 mg 1-1for Fe3+, and 20 mg 1-1 for Cu2§ Uggerud and Ltmd [99] and Bowman et al. [100] investigated the use of thiourea as an agent for the pre-reduction and masking of interferences in the multi-element detemfination of As, Sb, Bi, Se and Te by HG/ICP-AES and of As, Sb and Se by HG/ICP-MS, respectively. Martinez~ria et al. made a comparative study of the magnitude of the interference in different inorganic acid media during the determination of Sb by HG/AAS [101]. They proposed a scheme, which accounts for all the different interference mechanisms. In particular, the me of conditional reduction potentials, and of their variation with pH, provided a basis for deducing, in a general way, whether or not a particular chemical species was capable of producing interference. L~ysteine has proved to be very effective reagent in both reduction of interferences and enhancement of signals. In addition, L-cysteine is fairly

New Developments in Hydride Generation - Atomic Spectrometry

75

soluble in water, has low toxicity and has little smell compared with most other thiol compounds. The optimum concentration of acid required for the procedure can be reduced, resulting in a reduction in the acid-derived blank contribt~ons to the analyte response. Brindle and coworkers previously reported that Lcysteine s ~ s f u l l y reduces matrix interferences in the determination of As, Sb, Ge and Sn when HG techniques are used [102-105]. The pentavalent forms of Sb and As are ttst~y ~duced to their trivalent oxidation state before their determination by HG, and KI alone or mixed with ascorbic acid is most frequently ttsed for that purpose. However, KI can only be ttsed in strong acid media, typically 5-6 M HC1, and KI solutions are unstable and must be prepared fresh daily. For these reasons it would be desirable to find an alternative reagent for the pre-reduction of As(V) and Sb(V) to their lrivalent states and as an interference releasing agent. Chen et al. demonstrated the utility of L~ysteine over KI as an efficient pteteducing agent for As(V) and Sb(V) [106, 107] and Brindle et al. showed that L-cysteine could be ttsed for on-line reduction in a continuous-flow system [108]. Welz and Sucmanov~i optimized file analytical parameters for using L~ysteine as a preteducing and releasing agent in FI / HG / AAS [109, 110]. They showed that L-cysteine provided greater freedom from interference and much better stability of solution with low concentrations ofanalyte and the reagent consumption and particularly the acid concentration were significantly lower than KI, which pemaitted a change in the me of highly corrosive solutions to solutions of low acidity and low toxicity. Tolerance limits (less than 10% interference) of at least 250 and 500 mg 1-~ were found for Ni and Cu, respectively, in the presence of 1% L-cysteine, in the determination of Sb. For the determination of As, the corresponding tolerance limits were 200 mg 1-1 for Ni and more than 1000 mg 1-1 for Cu. Only 100 mg 1~ of Cu could be tolerated when KI was used. Lugowska and Brindle investigated the nature of precipitates formed by the reaction ofFe01), Ni(/I) and Co01) with NaBH4, which interfere with the determination of Se by HG, by potentiomelric titration, direct current plasma atomic emission spectrometry and positive ion FAB mass spectrometry [111]. The precipitates obtained by the reaction of acidic

76

H. TAO and T. NAKAHARA

solutions of Fe0I) with NaBH4 did not contain boron, whereas precipitates obtained fi'om Ni0I) and Co01) contained metal (55-68%) and boron (2.6-4.3%) in a ratio of 3-5 91.1he two hypotheses presented in the litemane [97, 112] can both be supported by the results of this work. (i) The hypothesis that the hydride is not formed explains the mechanism of the interferences in terms of the prevalerr.e of the reaction of NaBH4 with M(I1) (the higher rote and NaBH4 consumption, where MOO is the transition.metal ion) over the reaction of NaBH4 with IT and in terms of changing the mechanism of the HG under these conditions. (ii) The hypothesis conceming the decomposition of the previously formed hydride by the precipitates is based on the thermodynamic instability of H2Se (the potential of Se(0)/H2Se couple for 10-6 M H2Seis alx)ut -220 mV) in the presence of the boride-like species (steady redox potentials between 0 and -250 mV). The oxidation of HzSe during the formation of the boride-like species is exemplified by equation (4) for the reaction of the precipitate of the boride: 4Ni2++ 2BH4 + 3H2Se+ IT --~ 2Ni2B+ 3Se + 7.5H2

(4)

Interference in HG have mostly been studied with regard to total metal determination, but some have been investigated for speciation using HG / cryogenic trapping / C~ / AAS. Sulfur compounds and pigments have been sttspected of having depressive effects on HG [ll3]. Martin et al. investigated the effect of different organic comlxxmds (organic pollutants, organic solvents, complexing agent and humics) and a mixture of 14 inorganic compounds on organotin speciation (MMT, MBT, DBT and TBT) by HG / cryogenic trapping / C~ / AAS [114, 115]. Organic compounds had little effect on signal suppression for any of the organotin species. Interference effects by inorganic elements were found to be significant, but they could be successfully controlled by the use of masking agents such as EDTA and L-cysteine. Le et al. [116] and Guo et al. [ 117] reported that pre-reduction of the As species by marion with Imysteine equalized the sensitivities of the technique to As which was present as A s ( ~ , As(V), MMAA and DMAA.

New Developments in Hydride Generation - Atomic Spectrometry

77

Brindle and Le proposed that enhanced performance in the presence of Imysteine results from the formation of an H3B-SR intermediate from the reaction between Lmysteine and NaBH4 which is a more efficient reductant than NaBH4 alone [104]. ~B NMR spectra showed evidence of formation of an intermediate slxxfies, BH3SR-. On the other hand, Howard proposed that the enhancement has more to do with file formation of the reduced arsenic-cysteine complexes than to the formation of a super-reductant such as H3B-SR- [ 118]. In the conventional NaBH4 trMuction of As species those arsenic oxy-aniom which contain arsenic in the pentavalent state must fLrst be reduced to the tfivalent state. R,As(O)(OH)3~ + I-I+ +BH4" --~ Rd~OH)3-n + H20 + BH3

(5)

(in most cases, R is a methyl group and n ranges li'om 0 to 3.) A subsequent reaction with the NaBH4 converts the As compot~ to the corresponding arsine: Rdks(OH)3.. + (3-n)BH4 +(3-n)H + ~ ~ 3 - .

+ (3-n)BH3 + (3-n)H20

(6)

The pH characteristics of the NaBI-h reaction lead to the conclusion that for the re~.cfion to p ~ rapidly, the target species must not be present in solution in the form of a negatively charged species. This indicates that the arsenic oxy-anions must be fully protonated if they are to be conveaeA to arsine. Acid dissociation constants of several As and Se species are given in Table 3 and the pH dependency of arsine generation yield from various arsenic oxy-anions is shown in Figure 4. Since the pK1 for arsenic acid is 2.3 the reaction must therefore be carried out at very low pH and 1-2 M HC1 is typically used. However, in the case ofthiol compounds such as L-cysteine and thioglycollic acid (denoted as T-SH), it is hypothesized that strong acid conditions are tmnecessary, as the As species are pre-teduced from the pentavalent to the trivalent state prior to the NaBH4 reduction step. The standard oxidation potential of the cysfine-cysteine system is 0.074 V [69].

78

H. TAO and T. NAKAHARA

Thus, it is possible to reduce As(V) and Sb(V) to their trivalent states, and MMAA and DMAA to their trivalent thiolates" 2T-SH + Rd~O)(OH)3-n~ T-S-S-T+ Rnms(OI--I)3.n+ H20

(7)

The trivalent species can then react to give As0ID-thiol complexes: RnAs(OH)3.n + (3-n)T-SH~ Rdks(-ST)3-n+ (3-n)H20

(8)

The three co-ordinate As(m)-thiol complexes are both uncharged and less sterically hindered to attack by NaBH4. 1his would lead to the potentially faster and more uniform production of mines. On the other hand, L~ysteine also reduces Sn(IV) to Sn(ff), Se(IV) to Se(0) and Te(IV) to Te(0), and therefore, Se and Te cannot be detemfined. Moreover, interference by Se on Ge and Sb may ~ m e stronger presumably due to coprecipitation of these analytes with the elemental form of the Se. Howard and Salou investigated the pre-reduction of As(V), MMAA and DMAA with a range of sulfur compounds and found that pre-reduction with thioglycollic acid was more rapid than with L-cysteine and was better suited to continuous flow pre-reduction procedures [ 119]. The pre-reduction of Se(VD to Se(IV) is generally accomplished by heating the analyte in the presence of HC1, usually at an acid strength of 6 M. Hill et al. investigated the kinetics of Se(VI) reduction and reported that the activation energy for the reduction of Se(VI) to Se(IV) was calculated to be 90.4 kJ moll using 6 M HC1 [120]. This result indicates that the full reduction of Se(Vi) to Se(IV) may be achieved in as short a time as 6 min at 70~ Brindle and Lugowska explored mild conditions for reduction of Se(VI) to Se0V) and repotted that the most effective pre-reduc~t for Se(VD was bromide ion in acidic solutions [121]. They concluded that bromide ion was about 19 times more effective than chloride ion under the same conditions and that the reduction of Se(VI) was complete in 2 M HBr solution atter 2 min of boiling.

New Developments in Hydride Generation - Atomic Spectrometry

79

A~ENATE + ARSENIT-~ @.SEN,T 1.2-

amenite[ 0.8

-

.,

',,,

(D

:=0.4 m

\,

,....,.

MeAsO(OH ":, ", - .

arsenal 0.0

-I

-2.0

_.,

0.0

\ \

..p=Kp. / " .pK ~ ._- "IPK' "~_K~_ . ,~-.--~ ~--~- , 2 4 6 8

pH Figure 4 pH dependency of the amine yield obtained from the reaction of a number of arsenic oxy-anions wilh NaBH4. Small vertical arrows indicate the pH values at which pH=pK, for each acid. Reproduced by permission of the Royal ~ of Chemisa3' with acknowledgement to

Table 3 Acid dissociation constants of some acids ,,

,,|

_

,

,

,,,

,

Abbreviation

Acid ,|,

,

pK

,,

As(O)(O~

.......

AsiI As(V)

(CHs)ns(OXOH~

MMA

(CH3)As(O)OH Se(O)(OH)2

DMAA

Se(O)~(OH~

Se(VI)

Se(W)

....

P K 1 = 9.2" pK1 =2.3 pK 2=6.8 pK3 = 11.6 pK 1 = 4.0 pK 2 = 8.6 pK = 6.3 pK 1 =2.5 pK2 = 7.3 pKl=-3(estimate) pK2 = 2.0

80

H. TAO and T. N A K A H A R A

5. HYPHENATED TECHNIQUES

The biological and toxicological effects of an element often d ~ d on its chemical form or oxidation state. The determination of total element concentration does not provide this vital information. Coupling chromatography to an element-specific detection method such as AAS, ICP-AES and ICP-MS can pen~t the discrimination of various species. HPLC is usually the method of choice as a separation technique rather than GC because the latter requires a previous derivatization step to produce volatile substances, which is not always feasible. Recently, capillary electrophoresis (CE) has attracted extensive attention, ~ e of its high separation capability. Unfortunately, the detection limits obtained by simply coupling HPI~ or CE to AAS and ICP-AES have been less than adequate for speciation at trace level in te~ samples. The hydride generation technique has been used as the most successful melhod for improving the sensitivity of these hyphenated methods. Since the oxidation state and chemical forms which can form volatile hydrides are limited, it is also necessary to incorporate the on-line conversion system from non-hydride forming ~ i e s [e.g., AB and Se(VI)] to hydride forming species [e.g., As(ill) and Se(1V)] into the HPI~ / HG / AAS or CE / HG / ICP-MS systems in order to expand the applicability of these mefl~ods to a variety of species. 5.1. HPLC / on-line treatment / HG / atomic spectrometry A number of investigations c o n ~ g on-line oxidation systems, assisted by thermal [122, 123] or microwave (MW) heating [86, 124-129] or by UV in~ation [130-138], have been carried out for the determination of non-hydride forming organoarsenie species. Rieei et al. had previously reported on the on-line K2S2Os-HC1 oxidation step of p-aminophenyl arsonate incorporated in an ion chromatography / HG / AAS system [139]. L6pez et al. employed thermooxidation to b~lk down or~'x)arsenie eompotmds [122]. The t h e r m o - ~ o r consisted of a loop of PTFE tubing dipped in a

New Developments in Hydride Generation - Atomic Spectrometry

81

powdered-graphite oven heated to 140~ The thenno-conversion etticiencies were above 96%. On-line photooxidation combined with HG/AAS for the determination of organoarsenic eon~unds originated from the work by Atallah and Kalman [140], although on-line photoo~'dation itself had been used previously in the field of voltammetry for heavy metals [141] and in colofimetry for total organic carbon (TOC) and total nitrogen (TN) [142]. This photooxidation was also applied to organotins, since they give rise to hydrides of restricted volatility. These organotins could be converted to inorganic Sn(IV) by UV inadiation, which then gave stannane when reacted with NaBH4 [143]. The UV degradation of the As species in the presence of persulphate pemaits the tmasformation of all As sixties into As(V) to be cznied out in a short time. Rubio et al. reported on the detennimtion of the six arsenic species [As0ID, As(V), MMAA, DMAA, AB and AC] by HG/ICP-AES after HPLC separation and on-line photooxidation and reported that the detection limits (2~) were 7.9 ng As ml~ for AB and 6.1 ng As mll for AC [130]. The same technique has been ~klgorted for As speciation in marine biological materials [132, 135, 144]. Zhang et al. developed the technique of argon segmented flow in the post-column eluent, and obtained a substantial improvement in chromatographic resolution [135]. Guo and Baasner employed 'knotted' VIFE tubing reactor to reduce peak broadening [145]. Slejkovec et al. reported a dtml As speciation system, which combined HPLC and purge and trap (PT) / C~ separation with AFS detection [134]. Using HPI~ / UV photooxidation / HG / AFS, it was possible to sepm~ up to six As species with detection limits of ca. 0.5 ng ml-~As (100 lal injected). Using selective HG / PT / C~ / AFS, up to four As species with detection limits of ca. 2.5 pg ml-l (for 100 ml-sample size) could be determined. A limitation in the UV inadiation method would be the use of a mobile phase of organic character in HPLC separation, which could be decomposed during the photooxidation procx~ and, thus, the yield of analytes oxidation would be decreased significantly. Rubio et al. also reported on a HPLC / photo~duction / HG system using ICP-AES or QCAAS (Quartz Cuvette Atomic Absorption v

82

H TAO and T NAKAHARA

Spectrometry) as detection methods for Se(IV) and Se(VO determination [146]. The detection limits (2o) were 6.8 ng ml ~ for Se(IV) and 16 ng ml~ for Se(V1), when using ICP-AES, and 15 ng mll for Se(IV) and 33 ng ml"l for Se(VI) when using QCAAS. Separation was performed using an anion-exchange column and phosphate buffer as the mobile phase. The eluate from the column entered the photore,actor, where it underwent UV inadiation for 120 s by 15 W low-pressure Hg lamp. Effects ofthe pH ofthe medium and the presence of various reducing agents on the teAuction efficiency fi'om Se(VI) to Se(IV) were investigated. Reduction efficiency was highest in water and in 1% NaOH without the addition of any reductant, showing that neutral or alkaline media are the most suitable. A reduction efficiency in excess of 50% was obtained. Vilan6 and Rubio extended this approach to the determination of Se(IV), Se(VI), selenocystine (SeCys) and selenomethionine (SeMet) [147]. 1he detection limits (3o) for Se(1V), Se(VI), SeCys, and SeMet were 0.6, 14.5, 0.9, and 5.9 ng ml1, respectively. The method was validated by analyzing two water certified reference materials, in which only Se(1V) was detected. Although they concluded that, after photoreaction, all the Se species were converted to Se(IV), the conversion efficiencies from Se(VI), SeCys and SeMet to Se(IV) were not given in this paper. On-line MW oxidative digestion instead of photooxidation has been used for the conversion of organoarsenic compounds to As(V) by the reaction with KES2Os-NaOH. Compared with photooxidation, MW digestion appears to be subject to less interference from organic substances, which penrfits the use of a wider variety of mobile phase in HPLC. However, bubble formation sometimes leads to irregular flow rates and increased baseline noise with the MW digester. The cooling loop is n ~ a r y to condense gas bubbles and to prevent water vapor from entering the AAS detection unit [137]. On-line microwave aeamaent has also been used for pre-reduction fi'om Se(VI) to Se0V) by the reaction with HCI. Pitts et al. reported that the detection limits (3o) were 0.2 ng ml1 for Se(1V) and 0.3 ng mll for Se(VI), respectively, by using HPI~ / MW reduction / HG / AFS system [148].

New Developments in Hydride Generation - Atomic Spectrometry

83

Cobo-Femfi.ndez et al. reported on an HPLC / MW oxidation / MW reduction / HG / AAS system for the determination of Se(1V), Se(VI) and tfimethylselenonium ion (TMSe+) [149]. In this case, TMSe+ was first oxidized to Se(V1) with KzS2Os-NaOH and then reduced to Se(IV) by HC1 in a microwave oven. G6mez et al. extended this technique to the detem~ation of Se(IV), Se(VI), TMSe +, SeMet and SeCys [150]. M a r c h a n ~ y 6 n et al. ttsed a reverse-phase column modified with a vesicular mobile phase ofdodecylammonium bromide (DDAB) to separate Se(IV), Se(V1), SeMet, SeCys and selenoethionine (SeEt) [151]. Since only Se(IV) forms H2Se, the prior transformation of other Se compounds into Se(IV) is required. For this purpose, they and Gon~ez-laFuente et al. [152] used a MW-assisted one-step p r e ~ e n t by KBrO3-HBr, and Ellend et al. [153] used a MW-assisted one-step pretreatment by KBr-HC1. Since the use of acetonitrile, methanol or a vesicular fonning reagent, which are typically used in reverse-phase HPI~, sometimes presents problems owing to their organic nature, the mobile phase must be an appropriate medium for MW-assisted pretrealment and HG. Gon~ez-laFuente et al. repoaexl on the detemalna"tion ofSe(IV), Se(VI), SeMet, SeEt and SeCys in urine by HPI~ / MW prear.aanent (KBrO3-HBr) / HG / ICP-MS (double-focusing) [154]. A double focusing ICP-MS was operated in the 'low resolution' mode, to increase the sensitivity. They obtained a sensitivity 23-59 times higher than that obtained with a quadmlx~le ICP-MS. However, the limits of detection were only 1-8.7 times ~ than those found with the quadmt~le ICP-MS due to a serious increase in background noise from polyatomic ions of Ar and from Se contamination of the reagents. Tsalev attempted to evaluate three techniques, i.e., a thermostated bath (TB), a microwave-assisted digestion (MWD) and UV irradiation (UV) [3]. The efficiency in digesting organoelement compounds was ranked as follows: MWD .~UV + heating .~UV >> TB. For routine analysis HPI~ / on-line pretreatrnent / HG / AAS appears to be the more affordable instrumental approach over HPLC coupled with more expensive detectors such as ICP-MS or electrospray ionization MS.

84

H. TAO and T. NAKAHARA

5.2. CE / HG / ICP-AES (or ICP-MS) The key point to s u ~ f u l on-line coupling of CE with ICP-AES or ICP-MS is the design of the interface. The i n t e ~ should be able to provide the minimum band broadening of CE peaks. Since CE may only provide
New Developments in Hydride Generation - Atomic Spectrometry

85

Other designs of HG systems for coupling CE and ICP-MS for the speciation of As [155] and Se [156] have been reported. The compatibility of HG with CE requires rapid on-line conversion of Se(VI) into Se(1V) to reduce diffusion broadening. A schematic diagram of CE / on-line reduction

I

CE INTERFACE ! I i ! ! !

NaBH4

Concentrated HCI I I 3 0mm

Capillary ~I~~Jbe I

Umt .

,

/

PTFE manifold

.

PTFEcross

pump I aJbe J9l t .

out to mixing ...~manifold above

M=Make_up flow

Platinum ground wire

Figure 5 S d a m ~ c diagram ofCE/On-line ~ C P - M S . Reproduced by penmssion ot the Royal Society of Chemistry with acknowledgement to Magnuson et al. [156].

/ HG / ICP-MS for Se speciation is shown in Figure 5. The Pt ground wire for the CE circuit and the HCI for on-line reduction of Se(VI) enter through opposite ports of a FIFE cross, qhe isolating peristaltic pump serves to isolate the CE interface from the HG reaction, which produces large volumes of HE gas. The volume of gas induces a back-pressure which tends to came a large, undesirable flow in the capillary. It also serves to draw the HC1 fi,om a ventilated reservoir. The HC1 mixed with capillary effluent is then diluted in a three-way VITE manifold by make-up flow (0.81 ml min-~ of distilled water) supplied by mother peristaltic pump. The isolating peristaltic pump delivers this solution into anotl~r PTFE manifold where it is mixed with NaBH4. The evolved hydrides are separated by passing them through the membrane gas-liquid separator and they are then inaxxiuced into the ICP by Ar camer gas. Conversion efficiency from Se(V1) to Se(IV)

86

H. TAO and T NAKAHARA

increased with increasing reaction time, but the CE peak shape was likely to be distorted. This limited the conversion of Se(VI) to Se(1V) to alx~ut 30% even when concentrated HCI was ttsed. In this system, a high negative voltage (-22 kV) induc~ an EOF away from the detector, but the application of the hydrodynamically modified electroosmotic flow (HMEOF) at a low pressure (<3 psi) made the bulk flow to move towards the detector. The retention time of the analytes was then d ~ d e n t on the HMEOF rote and the electrophoretic mobilities of the analytes. For low HMEOF separation pressure, the peak shape was not excessively broadened by laminar flow induced by hydrodynamic pressure. The use of HMEOF allows flexibility in the choice of buffer, thereby potentially decreasing the analysis time. For an injection volume of 250 nl, the detection limits (3.14~) for Se(VI) and Se(IV) were 10 and 24 pg, respectively. This HMEOF can also be utilized for sample introduction into the capillary [155]. To increase the amounts of analyte injected into the capillary, electrokinetic injection is often utilized. However, this technique is limited because the high voltages of injection produce an EOF within the capillary, which causes broadening of the analyte zone with increasing injection time. This limitation can be eliminated by using a small hydrodynamic pressure applied optx~ite to the dhe~on of the EOF. The use ofHMEOF o~ets the EOF, thereby allowing an arbitrary choice of electrokinetic injection times which, in ttun, lowers the detection limits. The detection limits (3.14~) for As(m), As(V), MMAA, and DMAA were 25, 6, 9, and 58 pg m1-1, respectively, based on a 2.0 min electrokinetic injection with HMEOF. For extemal calibration, it was necessary to con'ect for changes in the efficiency of the electrokinetic injection caused by the ionic strength of the malrix. Germanium(IV) was used as a sun,gate to correct for malrix effects because it forms a hydride and is present in low natural abundance. The use of Ge(IV) allowed the direct detemaination of As(m), but Ge(IV) was not appropriate for As(V), possibly becattse of electrophoretic mobility differences.

New Developments in Hydride Generation - Atomic Spectrometry

87

6. APPLICATIONS 6.1. Arsenic

Arsenic species differ greatly in toxicity, which mainly depends on their ability to bind to thiol ftmctions of proteins [137]. 1he median lethal dose (LDs0) values in rats for some As compounds are (in mg kg-1): potassium arsenite 14, calcium arsenate 20, MMAA 700-1800, DMAA 700-2600, and AB>10(~ [157]. Methylation of inorganic As compounds is known as a detoxifying mechar~m of the human organism after ingestion of inorganic As compounds. Most seafoods contain lag g~ e o n ~ t ~ o n s of As, with AB being the major As species in erustaeeam and arsenosugars in seaweeds [158]. Both AB and arsenosugars have been found in bivalves [159-161]. Other organoarsenieals, such as AC, TeMA+, TMAO, and trimethylarsine (TMA), have also been reported to be present in some seafotxts at much lower concentrations than AB and arsenosugats [162]. AB is very stable and is rapidly excreted unchanged into the u~e. The metatx)lism of arsenosugars is not well understood and the effect of arsenosugar ingestion on urinary As excretion is not widely known. Arsenic concentrations in u ~ e have a short half-life ar~ have been commonly used as a biomarker o f ~ t exposure to As. Arsenic in hair is a useful indicator for longer-term exposure ~ e As is believed to accumulate in hair and fingernails because of the high content of keratin. For these reasons, it is essential to identify and quantify individual chemical forms of As to assess health risks associated with its exposure. Speciation of As has been discussed in depth in several recent reviews [9, 163] and books [2, 7]. A list of recent applications of HG to the determination of As in biological and clinical samples is given in Table 4. A non-chromatographic procedure has been proposed to determine 'toxic arsenic' as a summation parameter. Since fish-derived organoarsenic species (AB, AC, etc.) do not form volatile hydrides, the sum of inorganic As [As(III)+As(V)] and its organic metalx)lites (MMAA and DMAA) may be detemained directly by HG/AAS. Le et al. [124] and Guo et al. [117] determined the toxicologically relevant As species in urine [the sum of

. . . . S e c c t e d Applications to As Determination in Biological and Clinical S a m p l e s . Sample Preparation and Brief Comments Rei'. Analylical Detection Matrix Method l.imit Urine: stored at 4';C a'nd analyzed within 48h. The a'ddition of I'-cysteine enabled 124' sum of[As(Ill), FI/MW/IIG/AA urine As(Ill), As(V), MMAA and DMAA to give the same sensitivity. Analysis of urine after As(V), MMAA, S pre-reduction with L-cysteine by HG/AAS gave the sum of As(Ill), As(V), M AA and DMAA], total As DMAA. After MW digestion (KzSzOa-NaOH), the analysis gave total concentration of all As compounds.

Table 4 /Mlalyte

As(Ill), As(V), llPl.C(ion-pair) 0.4 - 0.8 ng urine, NIST Column, Phenomenex ODS-3 (30 x 4.6 mm x 5 lxm); Mobile phase, 10 mM letrabutylammonium hydroxide + 1 mM malouic acid + 5% C1t3Ott (pH 6.0). ml 1 for 20 l.tl SRM2670, MMAA, DMAA, /IIG/AFS arseuosugars sample natural water, volume mt,ssel

175

As(III), As(V), 1) FI/I IG/AAS MMAA, DMAA, 2) tlG/cryogenic total As trap/AAS

llrille

116 Urine: MW digested. Pre-reduction of As species with L-cysteine prior to their determination by a cryogenic trap/HG/AAS procedure led to more uniform sensitivities to As(lll), As(V), MMAA and DMAA.

As(Ill), As(V), I tPLC (ion-pair, MMAA, I)MAA, ion-exchange) AB /MW/! lG/AAS (or ICP-MS)

urine 10 - 20 ng mi l for 20 !~1 sample volume

125 Urine: filtered through 0.45 om nylon filter. Ion-pair HPLC: Column, Phenomenex C~8 (300 x 3.9 mm); Mobile phase, 10 mM heptanesulfimate + 0.1% C! 13011 (pll 3.5). Anion-exchange HPLC: Column, BDH PolySphere SAW (120 x 4.6 mm); Mobile phase, 50 mM phosphate or carbonate buffer (pH 7.5, 9.0, 10.3). As(Ill) and AB were partially separated by increasing the mobile phase pll from 7.5 to 10.3. ('omplete separation o1"the 5 As species was achieved on a reversed-phase Cls column by using sodium heptanesulfonate as ion-pair reagent.

As(Ill), As(V), HPLC (ion-pair) 10 ng m1-1 for seaweed, MMAA, DMAA, /MW/ItG/AFS 20 ~tl sample urine AB, AC, TeMA +, wflume arseuosugars

Sample preparation; same as Ma and Le [157]. Column 1, Phenomenex ODS-3 (250 x 4.6 mm x 5 ~m); Mobile phase, 10 mM tetraethylammonium hydroxide + 4 mM malonic acid + 0.1% CH3OH (pH 6.8) (for arsenosugars and their metabolites). Column 2, Phenomenex Cls (300 x 3.9 mm x 10 I.tm); Mobile phase, 1 mM tetraethylammonium hydroxide + 10 mM sodium hexanesulfonate + 0.5% CIt3OH (pH 4.0). A nearly baseline resolution of the 7 As species except for arsenosugars was achieved by using elevated column temperature and mixed ion-pair reagents with column 2.

126

As(Ill), As(V), HPLC (ion-pair)MMAA, DMAA, /MW/tlG/AAS

urine

AB, AC, TeMA +, arsenosugars As(Ill), As(V), HPLC(ion-pair) 0.7 - 1.2 ng urine, NIST MMAA, DMAA, /MW/IIG/AFS ml -~ for 20 ~tl SRM2670, arseuosugars sample seaweed volume

Column, Phenomenex Cls (300 x 3.9 mm x 10 ~m); Mobile phase, 1 mM tetraethylammonium hydroxide + 10 mM sodium hexanesulfonate + 0.5% CH3OH (pH 4.0).

176

Urine: filtered through 0.45 ~tm nylon filter; Seaweed: extracted with CH3OH:H20 (1:1) by sonication for 20 min (5 times); evaporated to dryness; redissolved in II20. Column, Phenomenex ODS-3 (250 x 4.6 mm x 5 ~tm); Mobile phase, 5 mM tetrabutylammonium chloride + 4 mM malonic acid + 5% CH3OH (pH 6.0). Direct analysis of urine samples enabled to maintain a good DI~. Ion-pair I1PI.C enabled to separale no! only anionic species but also neutral and cationic species.

157

O t~

C~ .=1 o

I

As(Ill), As(V), I1PLC (iou0.45 - 1.6 ng urine MMAA, DMAA, exchange)/UV, m1-1for 100 AB, AC MW/ttG/AAS ~tl sample wflume

total As

FI/HG/AAS

137 Urine: filtered through 0.22 l.tm membrane; high-molecular weight matrix components were removed by solid-phase extraction using a Cls-syringe filter. Column, Bio-Rad UNO S-1 cation-exchange; Mobile phase, 3 mM NaI-IzPO4 (pit 2.1); HPLC using a continuous bed chromatography (CBC) technology, which permitted 4 times higher flow rates than conventional HPLC. Mobile phases with organic constituents could not be used for online UV digestion. Carbonate buffers which release CO2 by acidification could not be used. Although the oxidation to As(V) was not complete (besides As(V), MMAA and DMAA are Ibrmed), the resulting species patterns were reproducible and led to linear calibration function. The S/N ratio was 2 times better for UV photolysis than MW digestion.

31 pg mf 1 for serum, packed Serum (1 ml) or packed cells (lg): digested with HNO3-HCIO4-HzSO 4 mixture at about 500 ~1 cell 200~ till dryness (only H2SO4 left); pre-reduced to As(Ill) with HCI-KI-ascorbic acid" sample diluted to 10 ml. MW-assisted digestion with HNO3 in a closed vessel was difficult to volume obtain meaningful recoveries because NOx fumes absorbed in the digest caused erroneously high results for HG/AAS determinations.

177

o o

,-t o ,=.l

90

H TAO and T NAKAHARA

As0IO, As(V), MMAA and DMAA] by an off-line pre-reduction with L~ysteine combined with FI / HG / AAS. In addition, Le et al. also determined total As concentration in urine by FI / MW digestion (K2S2Os-NaOH) / HG / AAS [124]. Welz et al. determined inorganic As [As0ID + As(V)] in seaun by FI / MW oxidation (diltaed HNO3-HC104) / MW reduction (L-cysteine) / HG / AAS [164]. However, the purpose of this MW oxidation with diltrted HNO3-HCIO4 was not to break down the organoarsenic compounds, such as DMAA and AB, but, rateher, to break down the organic matrix of seama to the extent that it was no longer able to interfere with HG. Therefore, file degree of which the organoarsenic compounds are attacked by this p ~ u v e was not investigated. Le et al. [116] and Howard and Salou [165] concluded that the off-line pre-reduction of the As species with L-cysteine prior to their detemaination by the HG / cryogenic trap / C~ / AAS prcr~dure led to uniform sensitivities to As0~, As(V), MMAA and DMAA. Thtts, a single As species could be used for calibration. Ritsema et al. reported on the off-line photooxidation followed by FI / HG / AAS for total As concentration in urine samples [166]. Vuchkova and Arpadjan reported on the HG of As, Sb, Bi, Se and Sn when present as d i t h i ~ a t e complexes in methanol solution [167]. This may pennit the development of an effective solid phase extraction preconcentration procedure combined with direct HG from the methanol eluate for the simultaneous determination of these elements. Le et al. determined As0ID, As(V), MMAA, DMAA and AB in urine by ion-exchange or ion-pair HPLC / MW digestion (K2S2Oa-NaOH) / HG / AAS (or ICP-MS) [125]. The complete separation of the five As compounds was achieved on a reverse-phase C~s column by using sodium heptanesulfonate as an ion-pair reagent. Zhang et al. applied this method, substituting the MW digestion by UV photolysis, but obtained a very poor sensitivity for the AB [ 135]. The reason for this may be that ion-pair reagent ofheptanesulfonate was also decomposed during the on-line der~omposifion of AB, consuming the free radical oxidants produced from the persulfate and causing a decreased efficiency of AB digestion. 1his phenomenon also occun~ when using tetmbutylammonium as the ion-pair reagent or

New Developments in Hydride Generation - Atomic Spectrometry

91

methanol as the solvent for the mobile phase. They selected cation-exchange HPLC and determined As species in htmaan serum. Only AB and DMAA were significantly detected. Ma and Le determined As(HI), As(V), MMAA and DMAA by ion-pair HPI~ / HG / AFS [157]. They demonstrated that arsenosugars were metabolized in the human body and that the commonly used biomarkers of exposure to inorganic As, based on the measurement of As(m), As(V), MMAA, and DMAA, were not reliable when arsenosugars were ingested from the diet. Zhang et al. determined the low molecular weight As species by ion-exchange HPLC / HG / AAS [136]. The high molecular weight As species were separated by fast protein liquid chromatography, either size-exclttsion, ion-exchange or ~ t y chromatography, and the fractions were digested and m e a s ~ off-line with HG/AAS. They reported that the major As species in the senma of tl~ uraemic patients were DMAA and AB and that the inorganic As species were bound to proteins, mainly tmnsfenSn (about 5-6% of total As in serum). This binding may play an important role in the detoxification of arsenic. Zhang et al. also op"tlmized deproteinization of the low molecular weight fraction before injecting the sample onto the HPLC column [168]. Protein precipitation wilh an orgmaic solvent cattsed a serious decrease in the AAS signal and precipitation with an acid, such as HCIO4 and CC13COOH, greatly changed the pH of smile matrices, and shifted the retention time. Moreover, As0II) may also be trustable in this strongly acidic medium. A disposable Cls-type Sep-Pak cartridge for clean-up was also not applicable because of limited sample volume. They chose membrane ultmfil~on with a 10 kDa molecular weight cut-offfor the purpose. Mufioz et al. developed a method for the selective and quantitative determination of inorganic As [As010 + As(V)] in seafood [169]. They tested various proc~ures for solubiliTation and extraction with methanol [170], methanol-chloroform-water [ 171] and HC1Oa-HCl-chloroform [ 172], quoted in the literature and found that none provided satisfactory recoveries for As(m) and As(V) in ~ samples. A newly developed method included solubilization with HC1 and subsequent extraction with chloroform. The As

92

H. TAO and T. NAKAHARA

was solubilized in 9 M HC1. Alter reduction by HBr and hydrazine sulfate, the inorganic As was extracted into the chloroform phase, back-extracted into 1 M HCI, dry-ashed, and quantified by HG/AAS. The recovery was 99% for As(m) and 96% for As(V). In optimized conditions, other As species (DMAA, AB, AC and TeMA+) were not co-extracted. Although MMAA was quantitatively extracted, MMAA concenWations in seafood products are generally low, therefore, quantification of MMAA would not lead to a significant overestimate for inorganic As levels. It should be noted that, compared with ~ samples, As(ill) in some certified reference materials (CRMs) such as I~RM-1 is thought to be more weakly bound to the proteins because I~RM-1 is treated with acetone during its preparation as a CRM, producing total or partial degradation of its constituent proteins. As a result, it is easier to extract As0IO in CRMs compared to ~ samples. L6pez et al. [173] and Mufioz et al. [174] proposed a rapid method for the determination of inorganic As [As(III)+As(V)] in seafood products. The inorganic species were distilled as AsCI3 fi'om the matrix by micmwave-assist~ distillation and determined by HG/AAS. Although minor organoarsenic compounds, such as MMAA (109%), DMAA (11%) and TMAO (0.2%), were distilled, AB, AC and TeMA+ were not distilled. Palacios et al. evaluated the stability of As(V), MMAA, DMAA, AB and AC in deionized water, urine and dry clean-up residue of urine, stored in dark at -20~ 4~ and ambient temperature [129]. At -20~ all species were stable in water and untreated urine. At 4~ and ambient temperature, they were stable during the 67 days of testing in the urine dry residue alter the clean-up procedure. The dry urine residue seems to be a good matrix for use as reference materials for As species. 6.2. Selenium

In recent years, the chemistry and biology of Se and its various species have been the subject of increasing attention, due to the importance of Se both as an essential and a toxic element. The inorganic stx~cies,selenite [Se(IV)] and selenate [Se(VI)], are very important in the biological cycle of Se. Because of their different chemical and biological properties, these two

New Developments in Hydride Generation- Atomic Spectrometry

93

species exhibit quite different chemical and biological properties. Organic species of Se, as selenoaminoacids, take part in the biological Se cycle and are incorporated into proteins. Several selenoaminoacids have been identified in tissues and body fluids. Selenocystine (SeCys) is part of the active site of the enzyme glutathione peroxidase. In addition, Se seems to prevent several types of cancer in ~mals, as well as toxic effects cattsed by As and Hg. In humans, the assumed normal values for total Se are 80-120 ng ml"l in blood and around 50 ng mll in urine [11]. Chemical forms of various Se species encountered in biological and environmental samples are shown in Figure 6. From a nutritional point of view, Se is found mainly as selenoproteins, selenosugars, Se-lipids, Se-peptides and Se-aminoacids, and through these species, the organism takes up the necessary amount of this element, removing the excess through the usual pathways of excretion, urine being the most representative. Selenoaminoacids [e.g., selenomethionine (SeMet)] are ttsed as Se supplements in the diet of man and animals. The Se concentration in urine is used as an indicator of Se stattts and a strong correlation apwaazs to exist between dietary Se intake and daily Se excretion. Tfimethylselenonium ion (TMSe+) is a minor metabolite of Se which is useful in predicting excess Se intake and the detoxification mechanism in Selenite (Selenious acid), [Se(IV)] Selenate (Selenic acid), [Se(VI)] Selenocystine, [SeCys] Selenocysteine Selenomethionine, [SeMet] Selenoethionine, [SeEt] Trirnethylselenoniumion, [TMSe~J Selenoniobetaine Selenoniocholine Selenourea Sekmob Dimethyl selenide, [DMSe] Dimethyl diselenide, [DMDSe] Figure 6

SeO~SeOZHOOC-CH(NH2)-CH2-Se-Se-CH2-CH(NI-~)-COOH H-Se-CHz-CH(NH2~3OOH CH3-Se-CHzCH2-CH(NH2~3OOH C2Hs-Se-C~H2-CH(NH2)-COOH (CH3)~O)e§ (CH3)2Se+-CH2-COOH (CH3)~Se+-CH2-CI-12OH Se--C(NH2)2 R-Se-H CI-13-Se-CH3 CI-13-Se-Se-CH3

Chemicalforms of selenium species encountered in biological and environmental samples.

94

H. TAO and T. NAKAHARA

living organisms. A list of recent applications of HG to the determination of Se in biological and clinical smnples is given in Table 5. One of the main problems in the determination of tmee amounts of Se in biological samples is represented by the possibility of systematic errors due to the difficulties associated with the complete mineralization oforganoselenium compounds. Strong oxidizing acid mixtures such as H2SO4-HCIO4-NHO3 have proved to be the most effective digestion media in achieving both complete mineralization of organoselenium compounds and avoiding Se losses by volatilization. The use of such acid mix'tta~ requires final digestion temperatures of up to 310~ and may create some risk to the operator due to the presence of HC104. Furthermore, because of the high oxidation potential of the mixture, part or most of the Se present is conveaed into Se(VI) which is inactive to HG. Recknagel et al. developed a method of on-line ultrasonic-assisted wet digestion (H2SO4-HC104-HNO3)/ HG / ICP-AES for the determination of total Se in blood seama [183]. The cartier solution and injected sample (300 lal) were merged with an acid stream (80% H2SO4, 12% HCIO4, 8% HNO3) and passed through a reaction coil which was heated to 240~ in an ultrasonic bath. Li et al. reported on the optimization of batch-type closed Teflon vessel MW digestion for the determination of total Se in urine [184]. Urine was digested wilh HNO3 and H202 and then reduced with HC1 in a closed Teflon vessel with the aid of MW energy. Urea was useful to e"hminate NOx fumes, which might be absort~ in the digest and interfere in the determination of Se by FI~G/AAS. The recoveries for TMSe+, SeMet and SeEt added to urine were 96.5-105%. SeMet and SeEt were unstable during the MW heating ttsed to reduce Se(VI) to Se(IV). Such a MW reduction procedure should be used with caution to distinguish Se(VI) from Se(IV) in malrices which might contain organoselenium compounds. In order to set up a simplified digestion procedure for total Se determination in biological or clinical samples, D'Ulivo et al. investigated an off-line HBr-Br2 wet digestion system and showed that quantitative recoveries were obtained for TMSe+, SeMet, SeCys, 6-selenopufine (SePu),

Table 5 Analyle

Selected Applications to Se D e t e r m i n a t i o n in Biological and Clinical Samples Analylical Method Detection Limit Matrix Sample Preparation and Brief Comments

St'(IV), Se(VI), l lPLC(ion3 - 8 ng ml q for 100 TMSe § SeMel, exchange) lal sample volume ScCys /MW/pre-reductiou /HG/AAS

urine

Se(IV), Se(VI), I II~LC (vesicle1 ng ml ~ lor 500 tal Se(.'ys, ScMct, mediated)/MW/llG/ sample volume Selit AAS

urine

Urine: clean-up by passing through Cls cartridge. Colunm, Hamilton PRPX100 anion-exchange column (250 x 4.1 mm x 10 ~tm); Mobile Phase, 100 mM phosphate buffer (pH 6.8).

Z Rt~f.

190 O t~

Urine: filtered through 0.45 btm membrane. Column, Spherisorb Cla bonded silica, modified with 1 mM DDAB (250 x 4.6 nun x 10 lain); Mobile Phase, 10 mM sodium acetate buffer (pll 5), 0.5% CII3OII, 0.01 mM DDAB. Online MW digestion of selenocompounds with K.13rO3-11Br. The high ionic strength of urine produced a great change in retention times of Se(IV) as compared with aqueous solution.

151

Se(IV), Se(VI), IIPLC (vesicleFor Se(IV) 6.8 ng ml "1 urine SeMet, ScCys, mcdiated)/MW/! i(;/ by AAS, 30 ng ml l by Sci~l AAS, ICI~-AES or ICP-AF.S and 0.16 ng ICP-MS mi ~ by ICP-MS lot 100 lal sample volume

Urine: filtered through 0.45 ~tna nlenlbrane. Column, Sphcrisorl~ (.'~ I• silica (250 x 4.6 mm x 5 Ixm); Mobile Phase, 100 mM sodium acetate buffer (pit 4.5). As CIt3OH produced high carbonaceous deposits in the ICP-MS sampler, its use was abandoned. KBrO 3 was needed to achieve complete conversion of organocompounds into Se(IV).

152

So(IV), Se(VI), IIPI.C (reversedca. 1 ng mi "l for 50 lal urine SeMeI, SeEr, phase, vesiclesample volume SeCys medialed)/MW/l IG/ ICP-MS(quadrupole or double-tbcusing)

Urine: diluted 1:1 with H20; filtered through 20 ~tm membrane. Reversedphase column, Spherisorb C1s bonded silica (250 x 4.6 mm x 5 p.m); Mobile Phase, 100 mM CH3COONH4 buffer (pH 4). Vesicle-mediated column, Spherisorb Cls bonded silica, modified with 0.001 mM DDAB (250 x 4.6 mm x 5 I,tm); Mobile Phase, 10 - 200 mM CIt3COONH4 buffer (pH 4 - 6.5), 0.5% CH3OH, 0.01 mM DDAB. MW treatment with KBrO3-HBr.

154

Se(IV), Se(VI), Batch closed vessel 3 ng ml 1 for 500 p.l TMSe § SeMet, MW digestion sample volume ScCys, total Se /reduction/Fl/llG/A AS

m ,'=t

t~ ,'=t O

i

> O ,..,.

NIST SRM2670,

Urine: digested with HNO 3 + H202; reduced with IICI in closed Teflon vessel 184 by MW digester. Urea was useful to eliminate NOx fumes, which was

urine

absorbed in the digest and interfere with IIG/AAS. Recovery ofTMSc § SeMet and SeEt added to urine was 96.5-105%. ScMet and SeEt were unstable during MW heating used to reduce Se(VI) to Se(IV). Such a MW reduction procedure should be cautiously used to distinguish Sc(VI) from Se(IV) in the matrices which might contain organosclenium conq~ounds.

t~ ,'=t O

,.7

total Se

isotope dilution /I IG/N 2 MIP-MS

10 pg lnl l

NIES SRM

Serum: added with Se spike; digested with IINO 3 and 11202. Interferences of

No.4 freezedried human blood serum

Ar-associated polyalomic ions on 78Se and 8~ MS.

191

Se(IV), total Se llG/in still Irapping 18 pg lnl 1 for 2 ml /ETAAS sample volume

orgauoselenium For total Se determination, selenosugar was completely decomposed to (selenosugar) inorganic Se by treatment with K2S2Os in a water bath at 85~ for 15 min oral nutrition without UV irradiation.

Se(IV), Se(VI), HG/in situ trapping 60 pg ml 1 for 1 ml Salllllh' Vllilllile TMSc +, ScMcl, /I~TAAS SeCys, ScPur, (corrcspoding to 3 ug total Se ml l for urine)

NIST SRM2670, urine

Urine (1 ml): digested with 10 ml of HBr + 0.5 ml of 0.35 M KBrO3 at 150 ~ C filr 2 h; destroy excess bromine by adding hydroxylamine hydrochloridc; diluted to 50 ml with 10% HCI.

189

total Se

blood serum, On-line ultrasonic-assisted wet digestion with H2SO4-HCIO4-|INO 3. The SRM Seronorm oxidation state of Se from the digested SeMet was +4. A cross flow nebulizer 116 worked as gas/liquid separator and allowed the simultaneous determination of other elements of clinical interest.

183

on-line ultrasonicassisted digestion /HG/ICP-AES

Se(IV), Se(VI) MW pre-reduction /HG/AAS

So(IV), Se(Vl), I I(;/AI:S TMSe +, SeMet, SeCys, SePur, total Se

5 ng mi 1 tor 300 ~tl sample volume

1.0 ng ml ~ for Se(IV) citric fruit an,l 1.5 ng ml 1 for Sc(VI) lor 300 lal sample volu me

{I.5 and l.{I ng ml l lor blood serum, 100 lal original urine urine, NIST and serum sample SRM 2670 wfl u me, respect ivcl y

Se(IV), SeMet HPLC(ion-exchane) 0.73 ng ml "l for /IlG/N2 MIi'-MS

juices, geothermal waters

Se(IV) and 8.7 ng ml "1 tor SeMet for 100 ~tl sample volume

urine

o~

were eliminated in N2 MIP-

195

MW-assisted thermoreduction of Se(VI) to Se(IV) with 12 M HCI. A knotted 196 reaction coil was used.

Urine (1 ml): digested with 10 mi of llBr + 0.5 ml of 0.35 M KBrO 3 ;it 122 ~ C for 90 min; destroy excess bromine by adding hydroxylamine hydrochloride; diluted to.50 ml with 10% HCI. For the analysis of samples like serum with a protein and fatty material content higher than urine, preliminary dissolution with HNO3 was necessary lbr quantitative recoveries.

185

192 Column, Hamilton PRP-X100 anion-exchange column (250 x 4.1 m m x 10 ~tm); Mobile Phase, 15 mM phosphate buffer (pll 7.0). With N2 MIP-MS, the polyatomic interference related to Ar was eliminated, and the major isotopes of 7SSe and S~ were used for the analysis. SeMet directly generated wdalile DMSe and DMDSe when reacted with NaBII4 and I ICI. No pretreaiment of SeMet prior to HG was necessary.

,.q 9 ,..q Z >

New Developments in Hydride Generation - Atomic Spectrometry

97

Se(IV) and Se(VI) in less than 90 min using 0.01 M Br2 in 48% HBr at a 122~ solution tempema~ [185]. The paxedure was s u ~ f u l l y adapted to the digestion of urine and blood serum samples, although a preliminary dissolution step with HNO3 was necessary to obtain quantitative recoveries for serum. The redox-buffer properties of the HBr-Br2 solution were originally employed to convert all the inorganic Se species into Se(1V) with the aim of determining the total inorganic Se in environmental samples [186, 187]. D'Ulivo et al. also investigated the mechanism of breakdown of organoselenium compounds in a HBr-Br2 digestion system by using HG and C~, both coupled with atomic f l u o ~ c e detection, polarography, and 1H and 77SeNMR spectrometry [188]. The roles played by Br2 and HBr in the conversion of organoselenium into inorganic Se(IV) were identified as (i) the oxidative addition of Br2 to divalent selenium to form bromoselenonium intermediates and (ii) the dealkylation of selenonium compounds by the bromide ion. The fact that, at the end of the HBr-Br2 digestion, the selenium is converted into Se(1V), and the abserr.e of potentially interfering acids such as HNO3, HC104 or H2SO4, make this digestion method attractive for the determination of total Se not only by HG/AFS but also by other methods such as C~ and polarography. Tyson et al. retorted on an off-line sample preparation method using HBr-KBrO3 for total Se determination in urine and found that for TMSe§ it was necessary to increase the digestion temperature to 150~ for 2 h to increase its percentage recovery to above 90% [189]. Marchante-Gay6n et al. [151] and Gonz~ez-laFuente et al. [152] detem~ed Se(IV), Se(VI), SeMet, SeCys and SeEt in urine by HPLC / MW prelw..alment (KBrO3-HBr) / HG / AAS (or ICP-AES, ICP-MS). Gon~ez-laFuente et al. reported on the determination of Se0V), Se(V1), SeMet, SeEt and SeCys in urine by the same HPLC / MW pretreatment / HG system using double-focusing ICP-MS [154]. G6mez et al. detemfined Se(IV), Se(V1), TMSe +, SeMet and SeCys in urine by HPLC / MW oxidation (K2S2Os-NaOH) / MW reduction (HC1) / HG / AAS [190]. They reported that Se(VI), SeMet and TMSe + were stable in the cleaned-up urine for at least two days. However, significant losses of Se(IV) were observed,

98

H. TAO and T NAKAHARA

probably due to its co-adsorption into the colloids that were formed with time and 60% of SeCys was transformed in 5 h to an unknown Se organic compound. Therefore, to avoid losses of the Se species studied, the fresh urine had to be cleaned-up and analyzed on the day of collection, especially if the s m i l e potentially contained Se0V) or SeCys. They concluded that most Se excretion occtmvd in the first 12 h alter ingestion of the sample. Ingested Se(VI) was partially excreted and partially transformed and excreted as a species that behave as SeCys. SeMet was completely transformed and excreted to species that behaved as Se(VI) and SeCys. Ohata et al. developed a method for the accurate detefinination of total Se in blood sennn by HG coupled with isotope dilution analysis using high-power nitrogen microwave-induc~ plasma mass spectrometry (N2 MIP-MS) [191]. Interferences of Ar-associated polyatomic ions on 78Se and 8~ were eliminated in N2 MIP-MS. Chatterjee et al. detennined Se(IV) and SeMet in urine by HPLC / HG / N2 MIP-MS [ 192]. Flow injection (FI) employing on-line microwave (MW) reduction followed by HG / AAS [193] or AFS [194] has been developed for the differential deten'nination of Se(IV)and Se(VI). "I'he Se(V1)concentration was given as the difference between total Se concentration and Se(IV). A mini-column was used in FI / MW reduction / HG / AFS for retention and specific elution of Se(IV) and Se(V1) with fomaic and hydrochloric acids, respectively [194]. A detection limit (30) of 0.04 ng mll and reproducibility of <5% as RSD were obtained. The method was applied to tap water and haemodialysis samples. De-qiang et al. detemained trace levels of inorganic Se in organoselenium (selenosugar) oral nutrition liquids using HG/ETAAS, taking advantage of the fact that this selenosugar did not generate a volatile hydride upon reduction [195]. B u ~ et al. developed an on-line FI system for the detennination of Se(1V) and Se(VI) in citric fruit juices by HG/AAS with MW reduction of Se(VI) to Se(IV) with 12 M HCI [196]. Gonz~ez-laFuente et al. developed FI / MW digestion (HBr-KBrO3) / HG / AAS for total Se detemaination in tap water [197]. Chatterjee and Irgolic showed that TMSe +, selenocholine (SeCh) and other selenoamino acids had the potential to form bomhydride active volatile Se compounds directly,

New Developments in Hydride Generation - Atomic Spectrometry

99

without any p ~ t m e n t when reacted with NaBH4 and HC1 [198]. Cabon and Erler proposed an experimental p m ~ l to differentiate Se species in seawater by considering the difference responses of Se(IV), Se(VI) and SeMet to a thermal treatment in 5 M HC1 or UV irradiation ttr.atments in acidic or basic medium [199]. Certified reference materials (CRMs) available for the analysis of biological and environmental samples are listed in a review by Mufioz Olivas et al. [11]. However, no CRMs certified for the content ot~ e.g., Se(IV), Se(V1) and methylated and organoselenium species are available. Mufioz Olivas et al. [11] and Dauchy et al. [12] presented reviews on analytical methodology for the speciation of Se in biological and environmental samples.

6.3. Antimony and Bismuth The determination of total Sb in plant materials [200] and in lipid-rich animal tissues [201] by FI / HG / AAS was reported. The plant materials and animal tissues were digested with HNO3-H2SO4-HF-HC104 and HNO3-H2SO4-HC104, respectively, in open digestion vessels made of glassy carbon in a heating block. The accuracy and precision was evaluated by analysis of reference materials. For a better risk assessment, total concentrations of Sb alone are not sufficient. Generally, trivalent Sb compounds have greater toxicity than pentavalent compounds. In addition to the two inorganic Sb species, methylated Sb compounds have also been found in environmental samples. Men6ndez Cmrcia et al. developed a non-chlvmatographic method for Sb(m) and Sb(V) speciation in water based on a continuous tandem on-line separation device in connection with ICP-AES [202]. Only Sb(IIl) was extracted into MIBK with APDC and on-line, SbH3 was directly formed from the organic phase and continuously introduced into the ICP. Sb(V) was then determined in the remaining aqueous phase, via a continuous mode, atk~r pre-reduction with KI and stibine generation/ICP-AES. Rond6n et al. reported on the selective determination of Sb0Ii) and Sb(V) in liver tissue by optimizing the acid conditions for stibine generation with NaBH4 [203].

100

H. TAO and T. NAKAHARA

Sb(m) was selectively obtained from an acetic acid medium with good recovery with the aid of microwave energy without any change in the oxidation state of the analyte. Total Sb was obtained atk~r MW-assisted reduction of Sb(V) to Sb(III) with 0.5 M H2SO4 and 10% KI. Microwave energy was necessary to quantitatively release Sb species from sample slurries. Smichowski et al. proposed a method based on anion-exchange HPLC / HG / AAS with detection limits of 5 and 0.6 ng per 100 lal sample for S b ( ~ and Sb(V), respectively, or HPLC / HG / ICP-MS with detection limits of 0.04 ng for Sb(lII) and 0.008 ng for Sb(V) [204]. Some problems associated with the column length (long retention time and chromatographic peak broadening) can be overcome by using a miniaturized column coupled to a HG/AAS detector [205] or a HG/AFS detector [206]. Sayago et al. ttsed a miniaturized anion-exchange colmam, 12 mm in length, for the separation of Sb(Ili) and Sb(V) [206]. qhe retention times were 0.45 and 3.5 min for Sb(V) and Sb(/I1), respectively, and the detection limits were 0.8 and 1.9 ng mll, for Sb(V) and Sb(lll), respectively. The efficiency of riG with testx~t to inorganic Sb is well known to be dependent on oxidation state, a fact that is ttsed for the off-line speciafion of Sb(llI) and Sb(V). However, in these papers, lower detection limits for Sb(V) were obtained despite the fact that the detection limit for Sb(l[l) should be superior due to the kinetics of the HG prtx:ess. The chromatographic peak broadening for the Sb(l]/) signal leads to a higher limit of detection. Krachler and Emos reported on the speciafion of Sb(lll), Sb(V) and trirnethylantimony dichloride (TMSbCI2) by HPLC / HG / AAS [207]. They compared the efficiency of separation of five anion exchange coltunns. The detection limits of 0.4, 0.7, and 1.0 ng ml-l for TMSbCI2, Sb(lll), and Sb(V) were obtained. Dodd et al. retx)rted on Sb speciation in freshwater plant e x a a ~ using HG/C~-MS and reported that molecular reanangements occun~ during HG of TMSbCI2causing demethylation of the generated trimethylstibine [208, 209]. This demethylation leads to the formation of (CHa)~SbH, (CH3)SbH2, HaSb and (CHa)aSb when a standard of TMSbCI2 or trimethylantimony oxide (TMSbO) is introdtr.ed into a HG system. As a

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101

consequence, earlier saflies, which tetx)rted the existence of organoanfimony species in the aquatic environment [210, 211 ] must now be reconsidered. Koch et al. investigated the issue of molecular rearrangements and found that demethylation was enhanced with deo'easing pH when using two different analytical methods: semi-continuous flow HG / C~ / AAS, and batch-type HG / C~ / ICP-MS [212]. They also reported that an increased amount of teanangement took place when HCI was ttsed instead of acetic acid, but a citrate buffer was b e ~ than either, and the malrix in a fungal extract sample enhanced demethylation. Craig et al. demonsa~at~ that the rigorous exclusion of oxygen combined with rapid purging of riG products into a cold trap led to a reduction in this molecular rearrangement to undetectable levels, and reported that levels of dirr~thylantimony found in some UK plant samples were in the 100-200 ng g-l range [213]. In a recent review, Smichowski et al. repotted on analytical methods for Sb speciation in water samples, in which a number o f ~ including HG were cited [214]. qhey pointed out that most of the p ~ u r e s developed had been applied to spiked samples, since the detection limits were not sufficiently low to determine Sb species or even total Sb in real samples. They also concluded that ft~re work must be performed on the production of certified reference materials for specific chemical forms to validate the developed methods and on the preparation of standards of organoantimony compounds to evaluate the different forms of antimony in natural matrices. Matusiewicz et al. determined Bi in clinical samples by HG / in situ trapping / ETAAS [215]. A detection limit of 20 pg m1-1was obtained with 5-ml sample volumes. Microwave-assisted Teflon bumb digestion procedures using HNO3-H202 were used to decompose clinical samples. The method was used to determine Bi in human blood, sertma, urine and tissue before and att~ an intake of therapeutic doses of colloidal bismuth citrate. Cadore et al. reported on the determination of Bi in wet-oxidized urine samples and prescription medicines by FI / HG / AAS [216]. The detection limit was 0.32 ng ml1 Bi with an analytical frequency of 150 hl. Interferences c a t t ~ by Ni0I), Co0I~, Cu0I), Ag(l), Se(1V), Sb(IID and

102

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Hg(ii) could be controlled by using a masking solution of thiourea (0.2%)-KI (10%). Marrem et al. investigated the effect of hydrochloric, tartaric, citric, oxalic, acetic and sulfosalicylic acids on the HG of Bi in a continuous flow system in conjunction with ICP-AES [217]. The interfering effect of transition metals, other hydfide-fomfing elements and Hg on the Bi signal using different acids was evaluated. Tarlaric acid was the most suitable reaction medimn, in temas ofetticiency in the HG p ~ and in the control of interferences. Smichowski and Marrero also investigated opfimizeA conditions for Ge and obtained similar results [218]. 6.4. Germanium, Tin and Lead Tao and Fang determined Ge in garlic and Sn in hair and serum by HG / in situ trapping / ETAAS [82, 83]. Erber et al. determined Pb in biological samples and ionic organolead compounds [84, 88]. Ni and He developed a method for tt~ determination of Ge by HG / in situ trapping / ETAAS [219]. qhey reported that, among tl~ acids investigated (HCIO4, HCI, HNO3 and H2SO4), only HCIO4 provided a wider range of acidity (0.15-0.6 M) giving maximum efficiency for the production of GeH4. Interferences fi'om Ni, Cu, Fe, Mn and Sn on the generation of the hydride fi'om 0.5 M HCIO4 were minimized by the addition of0.1% EDTA. Quevauviller et al. reported on the detemaination of mono-, di- and tri-butyltin (MBT, DBT and TBT) by HG / C~ / AAS in various matrices such as mussel and algae [113]. Riepe et al. reported on the selective detection method for organotin compotu~ by elimination of inorg~c Sn using HG / in situ trapping / ETAAS [220]. An acid concentration of 7.6 M HCI permitted, not only the complete suppression of stannane generation but also a minimum influence on organotin hydride generation. They also reported that severe contamination was caused from some tube materials which contained diorganofin compounds for stabilizing the tube materials, to achieve better resistance towards aggressive reagents, such as acids. Bettmer and Cammann reported the selective determination of organolead in inorganic Pb malrix [221]. Inorganic Pb was complexed with EDTA and only the hydride of organolead was generated.

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6.5. Miscellaneous

Although the number of elements which form volatile hydrides is limited (e.g., As, Sb, Bi, Ge, Sn, Pb, Se and Te), the application of riG has been extended to In [222] and TI [4], and the technique has recently been extended to Cu [223], Cd and various types of metals [28]. The mechanisms of riG of these elements are not very clear, and their generation conditions are very difficult to control. Ebdon et al. reported that a 1500-fold improvement over the previous sensitivity for batch HG was obtained by the use of continuous flow HG methodologies for TI [224]. Liao et al. reported on the in situ tr-~ping of volatile TI hydride in a preheated graphite furnace coated with Pd and showed that the presence of Te led to an increase in the generation efficiency of T1 hydride [225]. Wei and Jiang reported on the determination of Tl in seawater by FI / HG / isotope dilution / ICP-MS [226]. The detection limit was 0.01 ng ml-~ for TI in seawater. Although the detection limit is not as low as is currently possible using direct solution nebulization, this vat~r generation method alleviated non-spectroscopic interferences which were err~ountered when the pneta~atic nebulizer was used for sample introduction. 7. CONCLUSION The use of hydride generation in atomic spectrometry is steadily increasing due to the increasing needs for the determination of the hydride-forming elements at tm~ levels. In particular, the incorporation of on-line sample aeamaent and hydride generation in hyphenated methods, which are based on chromatographic or electophoretic separation and element-specific detection is growing rapidly. As an element-specific detection atomic absorption spectrometry has been the method of choice to date, but other methods such as, atomic fluorescence spectrometry and ICP mass spectrometry, may well be ttsed more frequently in the fiatae. The high detection capability oflCP-MS is expected to make the detection limit of the hyphenated method sufficiently low to determine analytes in biological and clinical samples. However, as shown in this review the detection limit has

104

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not been improved as much as expected from the point of view of improving sensitivity due to impurities in the NaBH4 reagent. To take full advantage of ICP-MS, the development of a purification method for NaBH4 is indispensable. It is also encouraging to find fundamental progress in hydride generation, for example, eleclax:hemical hydride generation, vesicular hydride generation, and hydride generation utilizing fast gas-liquid separation, are still on-going. These new methods will open broad fields of research and applications in a near future. The chemistry and biology of hydride-forming elements, such as arsenic and selenium, have been the object of increasing attention, due to their importance as both essential and toxic substances. However, the accurate s~iation of these elements remains as a major challenge for analytical chemists, and a knowledge of toxicity, bioavailabilty and metabolism in living organisms is still limited. Further improvements are necessary in the area of analytical chemistry if more information conc.eming these problems is to be accumulated. REFERENCES o

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Acta, 360 (1998) 209. 226. M. -T. Wei and S. -J. Jiang, J. Anal. At. Spectrom., 14 (1999) 1177.

Chapter 3

Analysis of biological materials by double focusing-inductively coupled plasma-mass spectrometry (DF-ICP-MS) Juan Manuel Marchante-Gay6n, Christina Sariego Mufiiz, Jose Ignacio Garcia Alonso, and Alfredo Sanz-Medel. Department of Physical and Analytical Chemistry, University of Oviedo, Juli~in Claveria 8, 33006 Oviedo, Spain 1. INTRODUCTION Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is probably the most powerful detector today in atomic spectrometry. This is due to: (1) its extremely high sensitivity, (2) its ability to make multielemental measurements, (3) the capabilities to measure isotope ratios, (4) its comparatively simple-tointerpret spectra, (5) the wide linear dynamic range obtainable, (6) the high sample throughput and (7) its easy coupling as specific detector with different chromatographic separation techniques [1 ]. The quadrupole mass filter is clearly the most popular mass analyser in ICP-MS due to its relatively low cost and easy handling. However, the full potential of ICP-MS for the analysis of biological materials cannot be exploited by conventional quadrupole-based instrumentation because of severe spectral overlap for many low mass isotopes. The main problem of a quadrupole-based mass spectrometer for the analysis of biological materials is its limited resolution in the mass spectrum (resolving powers of ca. m/Am = 300) and so, the presence of spectral interferences, caused by atomic or molecular ions having the same nominal mass as the analyte of interest [2] can be troublesome. Species generated in the ICP ion source can produce isobaric (two isotopes of different elements with the same nominal mass/charge ratio) or polyatomic spectral overlaps, especially below m/z 80. Of special interest are those polyatomics arising from molecular species formed by a combination of sample, plasma gas and matrix or solvent constituents. For example, it is difficult to determine trace levels of As in a chloride matrix by conventional quadrupole-based ICP-MS instrtmaents because chlorine combines with argon ions to produce a polyatomic ion (4~ +) which has the same nominal mass as the only isotope of arsenic (75As). Thus, 4~ + contribute to the analyte signal unless those masses are resolved

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Copyright 9 2002 Elsevier Science B.V. All rights reserved

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adequately. The most important spectral interferences, nominal masses, and the instrumental resolution required for the separation of the analyte signal from the spectral interferent in an analysis of biological materials are shown in Table 1. Table 1 Resolving power to separate analyte ions from interfering ions [3] in biological materials Isot. lnterf.

24Mg 12C2 27A1- 12C15N 13C14N 12C14NIH 2SSi 14N2

12C160 3lp 14N1601H 15N160 325 1602 39K 3SAtin 4~

4~ 4~ 42Ca 4~ 44Ca 12C1602

455c 13C1602 12C16021H

4STi 32S160 51V

34S14N 35C1160

37C114N 52Cr 4~ 36Ar 160 3SAr14N

35C1170 35C116OIH

53Cr 37C1160 54Cr 4~ 54Fe 4~ SSMn 4~ 37C1180 4~

39K160 56Fe 4~ 4~ 57Fe 4~ SSFe SSNi

Res. 1600 1500 1500 920 960 1600 970

Isot. Interf. ~SNi a~

42Ca160

59Co 4~

43Ca160 36Ar23Na

44Ca160 23Na37C1 1500 62Ni 23Na2160 1800 38,Ar-24.. Mg 5700 63Cu 4~ 6~

Res. 2250 2900 1650 2900 2400 3100 2400

Isot. 74Ge

74Se

lnterf. 4~ 36Ar38Ar 37C12

4~ 75As

76Se

1300

3200 2800 77Se 193000 31p1602 1900 29000 23Na216OIH 1200 78Se 2400 15N1603 1100 1300 64Zn 32S1602 2000 S~ 1200 32S2 4300 1100 48Ca160 3500 2600 64Ni 54000 2100 4~ 3300 S2Se 2600 36Ar12C160 1900 2100 65Cu 31p180160 1600 2400 32S170!60 1700 24 00 31p1702 1600 2100 33si602 1900 96M0 1900 33S32S 1900 l~ 1700 66Zn 4~ 3500 2700 49TiI601H 2700 2100 132Ba2+ 2500 l~ 2100 34SI602 1600 2300 67Zn 134Ba2+ 2700 2100 68Zn 4~ 4700 l~ 1600 136Ba2+ 2500 l~ 2700 36si602 2100 l~ 2500 4~ 1600 l~ 2500 69Ga 37C11602 2300 1900 7~ 35C12 5600 28000 14~ 2600 4~ 2000

74Ge 4~

4~ 4~ 76Ge 4~ 4~ 78Kr 4~ 4~ S~ 4~176

32S1603 1HSlBr S2Kr 4~

3451603 66Zn160 4~

87Sr160 2~

63Cu4~ 2~ 8SSr160 64Zn4~

65Cu4~ 66Zn4~

67Zn4~ 68Zn4~

Res. 8200 9500 7900 9500 57000 7800 7100

7400 35000 9200 5700 25000 10000 9700 550000 9500 2000 11000 25000 3500 2300 19000 13000 61000 1300 7600 1200 29000 2000 7100 7100 6900 6500

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For elemental analysis of biological samples these spectral interferences may constitute one of the most important limitations of Q-ICP-MS systems. There are several approaches to overcome such interferences in practical analysis. Those approaches can be physical in nature i.e. optimisation of plasma parameters, addition of supplementary plasma gases, the use of alternative plasma gases or, more recently, collision gases [1 ]. Chemical means may also be used, such as separation of the analyte from the matrix either during sample preparation or by using an appropriate sample pre-treatment accessory (e.g. an electrothermal vaporiser) or, altematively, using corrections involving the use of mathematical algorithms implemented within the insmanent software [1]. In each case the compensations rely to a certain extent on a rule-of-thumb approach, since the exact extension of the interferences is often not fully understood or even quantified. The use of mathematical corrections can be simple to implement in mass spectrometry, relying both on the availability of an uninterfered isotope and on the natural abundances of the elements, around which simultaneous equations can be derived in order to minimise the effect of an interference. Alternatively, corrections can be applied based on experimental information. In each case, assumptions are made which can propagate further errors. In complex samples the correction procedures are limited by the ambiguous assignment of the interferent and it was mainly this fact which prompted the development of an instrument capable of spectrally separating several species at very closed nominal masses. Thus, it seems that the only general method to overcome limitations from spectral interferences from polyatomic ions is high mass resolutions (resolving powers up to 10000 have been described) [4-8]. Such high mass resolutions can be obtained with Double Focusing-ICP-MS (DF-ICP-MS) insmmaents that combine a magnetic and an electric sector field analyser. Many molecular interferences in ICP-MS could be eliminated by using such instruments, since a resolution of only 2000-5000 is required to overcome most molecular ion interferences (Table 1) in biological samples. However, resolution of some isobaric overlaps has generally been considered to be difficult, since the resolving power required, (e.g. 11000-1000000) is much higher than the resolution required for molecular interferences [9]. Furthermore, as resolution increases the sensitivity decreases. Thus for the analysis of ultratrace elements in biological samples the sensitivity required could not be always achieved. Reviews have appeared in the most recent literature covering various aspects of the applications of ICP-MS in the analysis of biological materials [1013] but the literature on the applications of DF-ICP-MS in this field is so far rather scarce, even if its analytical potential for biological research is extremely

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high. Although available since 1988, DF-ICP-MS has not found widespread acceptance until recently, when the high cost of the initial generation of instruments were considerably reduced with the introduction of a second generation of DF-ICP-MS instruments. This gave strong impetus to the development of DF-ICP-MS applications in the analytical community, which is reflected in an increasing number of publications, the celebration of an international conference devoted exclusively to high resolution sector field ICPMS [14] and, in general, a growing interest in the analytical performance of this technique. Figure 1 shows that the number of publications on DF-ICP-MS for biological materials has increased drastically since its introduction in 1989 [15] with the first publication appearing in 1994. The aim of this chapter is to highlight the major areas of present biological research with DF-ICP-MS. Both, basic concepts of DF-ICP-MS and recent developments in elemental analysis, isotope ratio measurements and speciation of trace and ultratrace elements in biological material by DF-ICP-MS will be revised.

Figure 1. Number of publications of DF-ICP-MS in biological analysis

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2. INSTRUMENTATION A mass resolving power of up to 10000 can be achieved with double focusing ICP-MS instruments on the basis of the combination of a magnetic and an electrostatic sector field. These instruments have a longer tradition in MS than quadrupoles, although they are technically more sophisticated. They are based on the simultaneous use of a magnetic sector (mass or momentum focusing) and an electrostatic sector (energy focusing). There are a number of designs which will provide double focusing at all masses. Most double focusing ICP-MS instruments use the Nier-Johnson geometry [16]. We will discuss briefly this arrangement. 2.1.

Magnetic and electrostatic mass analysers

2.1.1. M a g n e t i c m a s s analysers

A magnetic mass analyser is formed when a flat flight tube at high vacuum is subjected to a strong, homogeneous magnetic field perpendicular to the flight path of the ions. The magnetic field is usually provided by an electromagnet. There are different designs of magnetic analysers being the 60 ~ sector developed by Nier [17] the most popular for many years. This analyser is shown schematically in Figure 2. The ions produced in the ion source are accelerated into the magnet through the entrance slit which, in this configuration, is equidistant to the exit slit. The magnetic field is applied perpendicular to the direction of the ions and has a mass dispersing and direction-focusing effect on ions. It can be shown that when an ion is accelerated through a voltage U acquires a kinetic energy Ue provided that the initial kinetic energy can be neglected. This can be expressed as 1

- m v 2 = Ue 2

(1)

where v is the velocity of the ion after acceleration and m its mass. When this ion enters the magnetic sector, with field B, experiences a force evB perpendicular to the magnetic field and to the motion of the ion. This results in a circular orbit of radius r where the centrifugal force equals the deflection force. This can be expressed as equation (2): mv 2 r

-evB

(2)

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J.M. MARCHANTE-GAYONet al. Magnetic Sector

....... N

~ ~

Ion Source

Entrance

Silt' .......

"''''. . . . . "'\

//;

"..

Exit

Siii"

Momentum rsion Plane

........~ ?

Detector

\/: .....

Figure 2. Magneticsector analyser

Substituting equation (1) into equation (2) we obtain: m

r2B 2

e

2U

(3)

Ions with different mass to charge ratio (m/e) will describe orbits with different radii (r). For a particular magnetic field (B) and acceleration voltage (U), only monocharged ions (as mainly provided by an ICP) with a given mass (m) can pass through the exit slit, and by varying the magnetic field or the acceleration voltage, it is possible to focus ions with different masses through this slit. Considering equation (2) in the following form: m_vv= B r

(4)

e

it can be seen that the magnetic analyser separates charged particles according to their momentum to charge ratio. In equation (1) we have assumed that the initial kinetic energy of the ions can be neglected. However, ions of identical mass and charge do not necessarily have the same energy and this is particularly true for the ICP as an ion source. According to equation (4), ions with different masses but having the same momentum, will be focusing together. Because the ICP ion

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123

source produces ions of constant medium velocities with a wide distribution, a wide energy spread is observed for the ions (2-10 ev). There are several ways in which such energy spread of ions can be eliminated, for an ICP ion source for instance, Turner et al [18] have shown that using an hexapole collision cell between the source and the magnet most of the initial energy of the ions can be suppressed. However, the most general form of energy focusing is the use of an electrostatic analyser. 2.1.2. Electrostatic analysers

An electrostatic analyser (ESA) consists of two curved plates (a cylindrical condenser) with a voltage E of the order of 0.5 to 1 kV applied between them and its design is given in Figure 3. Generally, the outer plate is positive, the inner plate is negative, and the centreline is at ground. For a given ESA configuration with mean radius r and gap d operated with voltage E, a singly charged ion of mass m and velocity v will be deflected and focused through the exit slit only if: mv 2 .

.

e

.

Er .

(5)

d

This means that the radius r of the orbit of the ion is determined by its kinetic energy. If the ESA voltage (E) or the ion velocities (v) are varied, ions with different energy will be swept across the exit slit of the energy analyser.

+E/2 ~./.~ ....................~ . ~ ............ Energy / J ~ J ~ ~ ~ I D ! s P e ~ rsiOn Plane

Entrance Slit

\,~.//////

~,

Figure 3. Electrostatic energy analyser

Exit slit

Detector

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J.M. MARCHANTE-GAYON et al.

2.2.

Double focusing: forward and reverse Nier-Johnson geometries A double focusing instrument provides both energy and momentum separation. In that sense the combination of a magnetic sector with an electrostatic sector will provide separation only by mass to charge ratio. Additionally both sectors provide angular focusing so the instrument is considered to be double-focusing. Mass spectrometers using the double-focusing principle were first constructed in the 1930s for atomic mass determinations. The Mattauch and Herzog design was the fast to provide double-focusing simultaneously for all m / e ratios [19]. Later, Nier and Roberts [20] constructed a mass spectrometer which provided not only first-order energy-focusing but both first- and secondorder directional focusing at all masses using a 90 ~ electrostatic sector and a 60 ~ magnetic sector arranged in series. This design is now known as the NierJohnson geometry and is used in multicollector instrtunents. For single collector-double focusing ICP-MS instruments the preferred geometry is the "reverse" Nier-Johnson in which the magnetic sector is located fast, followed by the electrostatic sector, is illustrated in Figure 4. Two schematic trajectories are shown in Figure 4 for ions of the same mass and charge but having two slightly different energies. Ions having higher energy will be focused at the exit of the magnetic sector at a point which is slightly offset from the focusing point of the lower energy ions. At this intermediate slit the distance of the focusing point from the axis will be proportional to the difference in energy from those ions to the mean energy ions. Electrostatic sector Magnetic sector .................................

. .......................... "--.......

Entrance slit

Figure 4. Reverse Nier-Johnson double-focusing mass spectrometer

--

- - E x i t slit

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

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As the ions pass through the electrostatic sector, both high and low energy ions are forced back into the axis by the focusing action of this sector and both trajectories come together at the position of the collector slit. The reverse NierJohnson geometry is used in most DF-ICP-MS instruments. This is because this geometry minimised collision effects which could occur in the mass spectrometer causing high noise levels in the detector system and a degradation of the abundance sensitivity (tailing on the low or high mass side). The main advantage of double focusing instruments against single focusing or quadrupole based instruments is their ability to provide higher mass resolution and, hence, the separation of the analyte peaks from interfering peaks. Mass resolution, Am, is defined as the mass difference necessary to achieve a valley of 10% of the maximum intensity between two neighbouring respectively. An alternative peaks of identical intensity at mass m and m• def'mition for Am can be used when the intensities of both peaks are very different. In these cases, Am is computed from the peak width for a single interference-free peak measured at 5% of its peak height. Mass resolving power will be then: R= ~m Am

(6)

The resolving power required to separate the interfering components for a number of well-known difficult elements for ICP-MS range from 1500 to 10000 (see Table 1) and such R values are within the capability range of modem DFICP-MS insmnnents. Resolution can be modified according to the needs of the analyst by changing the widths of the entrance and exit slits. When the slits are wide open R approaches that obtained by quadrupole instruments (ca. 300-500) while by narrowing the slits R can be as high as 10 to 12 thousand. Usually a resolving power of 2000-4000 is required to separate most polyatomic interferences in ICP-MS (see Table 1). It is clear, however, that while resolution increases transmission decreases proportionally. For example, when increasing the resolution from 300 to 3000 this will result approximately in a 10-fold drop in ion transmission. 2.3. Performance of commercial DF-ICP-MS instruments The first commercial DF-ICP-MS instrument was the "Plasmatrace I", introduced in 1988 by VG (now Thermo Elemental) [15]. This instnunent was a modified version of a previous instrument designed for organic MS. In this design the accelerating voltage was applied to the extraction interface while

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keeping the rest of the MS at ground potential. This instrument was the only instrument commercialised worldwide for many years. In 1991 Jeol introduced the JMS PLASMAX 1 but this instrument was not commercialised outside of Japan (now they have introduced the JMS PLASMAX 2). The big breakthrough in DF-ICP-MS instrumentation came with the introduction of the "Element" from Finnigan MAT in 1993. This instrument was based on a reverse NierJohnson geometry, in a similar way to the other two instruments, but the acceleration potential was applied further down the mass spectrometer keeping the extraction interface grounded. A compact design, an attractive price and good performance were the reasons for the high success of this instrument. Most of the applications of DF-ICP-MS to the analysis of biological materials have been published using this instrument. In the last 6 years improved versions of those and new insmanents were released. Their main specifications, as indicated by the manufacturers, are summarised in Table 2. The performances of commercial DF-ICP-MS systems are similar in terms of high sensitivity, low noise and low detection limits. However, the instruments have some differences in the slit systems and the MS configuration. Table 2 Comparison of commercial single collector DF-ICP-MS instruments

MS Configuration

Element2 PlasmaTrace 2 VG Axiom (Finnigan M A T ) (Micromass) (Thermo Elemental) Reverse Nier-Johnson Reverse Nier-Johnson Forward Nier-Johnson

Mass range

2-260 Da

2-300 Da

2-260 Da

Mass resolution settings

300; 4000; 10000

400-10000

300-20000 (continuous)

Ion detection

Analog/Ion counting Analog/Ioncounting Analog/Ioncounting

Noise

<0.2 counts.s-~

<0.2 counts.s-~

<0.2 c o u n t s . s

Linear dynamic range 10 (orders of magnitude)

>9

>9

Multiple collector model of the same manufacturer

IsoProbe

Optional on the same instrument

Neptune

-I

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127

For example, the Element has three fixed resolution settings, the PlasmaTrace 2 five and for the VG Axiom the resolution can be changed continuously. It is worth noting that the manufacturers offer multiple collector versions of their instruments which can be operated in high resolution. However, only the VG Axiom offers the multicollector as an option in the basic model instrument.

2. 3.1. Peak shapes and sensitivity One of the advantages of the double focusing instruments is the possibility of changing the widths of both the source and collector slit to control the resolution of the instrument. This will also modify the shape of the peaks. Figure 5 shows the peak shapes which can be obtained with one of those instruments (Element) for indium at mass 115 at three different nominal resolution settings: 300, 3000 and 7500. As can be observed, at low resolution (R=300), it is possible to obtain peaks with trapezoidal shapes (flat-topped peaks). This is due to the fact that the width of the entrance slit is adjusted so the beam width is smaller than the collector slit width. That means that over a small range of masses the intensity of the ion beam measured at the collector is independent of the position of the beam at the collector. These settings have shown to be useful for isotope ratio and high sensitivity measurements. By decreasing the slit widths, the resolution will increase at the cost of lower sensitivity. As can be observed in Figure 5 a loss in sensitivity of 16 times is observed by changing from 300 to 3000 nominal resolution. A further loss of 10 times appears when changing to resolution 7500. For most of the modem double-focusing single-collector ICP-MS instruments a sensitivity around 109 counts per second per ppm of indium can be obtained at low resolution. This means that for non-interfered isotopes of medium to high mass instrumental detection limits in the ppq range can be obtained. Of course, in most cases detection limits will be blank limited and not instrument limited any more. 2.3.2 Data collection There are two general modes of data acquisition in single collector DFICP-MS instruments. According to equation (3) ions of different mass to charge ratio will follow different trajectories (different r) in a magnetic analyser depending on the magnetic field (B) and the acceleration voltage (U). By changing the magnetic field strength, in the so-called B-scan, a whole mass spectrum can be acquired in less than 300 ms. Alternatively, an electric scanning

128

J.M. MARCHANTE-GAYON et al.

(E-scan) can be realised by variation of the accelerating voltage, with B and r at fixed values. R=300

~

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,

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~ 3000.

0 114.87

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Figure 5. Effect of slit widths on peak shapes and sensitivity for 115In(10 ppb) Electric scanning is faster than magnetic scanning but, for practical purposes, it can be performed only for a limited mass range (ca. 30% of the magnetic set mass). When a large number of isotopes have to be measured coveting a large portion of the mass spectrum a combination of magnetic jumping and electric scanning is usually performed.

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

129

3. ELEMENTAL ANALYSIS OF BIOLOGICAL SAMPLES The accurate measurement of trace metals in biological samples, such as blood, serum or plasma, is vitally important to nutritional and toxicological studies. Metal ions play a key role in the function of many biomolecules, as well as adversely affecting a number of important biological/biochemical processes. Thus, is important to have sensitive, precise and accurate analytical methods at our disposal, so that the narrow division between the concentration at which the metal is considered deficient, optimal or toxic, can be measured with confidence. There are numerous analytical procedures available for the analysis of trace elements in biological samples. Nowadays, if available, DF-ICP-MS is probably the method of choice for trace multi-element analysis. However, there are still numerous limitations of which the analyst should be aware for a successful application of DF-ICP-MS for the analysis of such samples. Therefore, problems of spectral and matrix interferences will be discussed first, before individual literature reported applications are eventually be reviewed.

3.1. Spectral interferences Biological materials are particularly difficult matrices for ICP-MS. They contain large amounts of proteins and inorganic salts plus small organic molecules. For ICP-MS, the high concentration of dissolved matter causes serious matrix effects that make dilution or even digestion prior to the analysis desirable. In addition, polyatomic species as oxide and argide ions are always present, and they add to isobaric and polyatomic interferences from the major elements in the samples (C, Na, P, S, CI, K, Ca). Figure 6 shows the resolving power, R, required to resolve all possible polyatomic interferences which can be observed in the analysis of biological materials [12]. As can be observed, such interferences are so common and difficult to avoid that the determination of some bioelements in biological samples by ICP-MS becomes difficult [21 ]. As it can be also observed in Figure 6, for many biologically important elements R values of 3000 to 4000 are largely sufficient. At that resolution level the sensitivity of DF-ICP-MS instruments is similar to modem Q-ICP-MS systems. 3.1.1. Blood, plasma and serum samples The fhst analysis of a biological sample by DF-ICP-MS was published by L. Moens et al [22] in 1994. In this work, Fe, Cu and Zn were accurately and precisely determined in a certified human serum sample after a 8-fold dilution of

130

J.M. MARCHANTE-GAYON et al.

A) 20000

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+Ca

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& I

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Figure 6. Resolving power required to eliminate possible polyatomic interferences arising from combination of plasma gases (Ar) and major matrix components (O, H, C, N, P, S, Si, CI, Na, K, Ca and Mg) in biological fluids: A) 24-53 amu and B) 54-88 amu [From ref. 12, with permission]

Double Focusing-InductivelyCoupled Plasma-Mass Spectrometry

131

the sample and monitoring of 56Fe+, 63Cu + and 66Zn +-~sotopes. The interferences from 4~ and 4~ § on 56Fe+ and 4~ + on 63Cu+ were eliminated by using a resolving power setting of 3000. In addition, 5~V+ was also measured at such resolution which allowed the resolution of the 1000 times higher peaks of 35C1160+ in a 4-fold diluted serum. Later work has demonstrated the usefulness of DF-ICP-MS for direct multielement analysis of blood and serum samples [23-25]. Using external calibration, with internal standards in order to overcome matrix interferences, both the non-spectrally interfered elements (Cd, Sn, Ag, U, Sr, Rb, Mo and Pb) at R=300 and the spectrally interfered (AI, Si, P, S, Ti, Cr, Mn, Fe, Cu, Zn, Ca and Co) elements at R=3000 were determined in certified five-fold diluted sertun samples [23,24]. The required resolution and the internal standards used in each case are summarised in Table 3. Table 3 Internal standards and resolution settings used for multielement analysis in serum samples Monitored Isotope 27AI 28 Si

InternalStandard*

Internal Standard**

59C0

45Sc

59Co

.....

31p 32S 44Ca 47Ti 49Ti

59Co 59Co

..... .....

Resolution 3000 3000 3000 3000

.....

69Ga

3000

59Co 59C0 59C0

..... ..... 69Ga

59C0 59C0 ..... 59C0 59Co 59C0 ..... .....

69Ga 69Ga 69Ga 69Ga ..... 69Ga 89y 89y 89y

3000 3000 3000 3000

l~

~75~

.....

11lCd 12~

l iSin

89y

52Cr 55Mn 56Fe 59C0 63Cu

64Zn 66Zn

85Rb 88Sr 98Mo

ll5In ..... 2~ ..... 2~ 23SU 2~ 2~ *Data obtained from reference 23. ** Data obtained from reference 24.

3000 3000 3000

3000 3000 300 300 300 300 300 300 300 300

132

J.M. MARCHANTE-GAYON et al.

As can be observed in the table both publications use the same resolution settings and masses for the same elements but the choice of internal standards differ. However, internal standards are selected in both cases with masses similar to those of the corresponding analytes. The selection of the right resolution mode is important for the elimination of isobaric interferences. For example, Figure 7 shows the typical spectrum of a 5-fold diluted human serum measured at R-3000 for masses 54 to 58 showing that all Fe isotopes (54, 56, 57 and 58) are interfered by polyatomic ions. Fortunately, at the resolution set used all isotopes could be measured free from spectral overlap. This fact is of particular interest for isotope ratio measurements as more than one isotope of the same element has to be measured. DF-ICP-MS has been used by Dunemann et al [26] for the quasisimultaneous determination of AI, Co, Cr, Cu, Fe, Mn, Ni, Pt, V and Zn in human serum and urine. In the case of urine, Pb, Cd and Tl were additionally determined. Sample pre-treatment was restricted to UV photolysis and to a subsequent dilution with 0.5% HNO3 solution without using any chemical separation or enrichment. Calibration was carried out by the standard addition procedure. Owing to spectral interferences, the determination of AI, Co, Cr, Cu, Fe, Ni, V and Zn was performed at R-3000 while the determination of noninterfered elemems was carried out at a 300 resolving power in order to preserve maximum sensitivity. More recently, the rapid simultaneous determination of more than 50 elements in microwave digested human blood samples using Sc, In and Lu as internal standards has been reported [25,27]. Three different approaches have been used to overcome spectral interferences. The tailing of the 1602+interfered on 9Be but it was nearly resolved even in low resolution mode. It was shown that by the simple use of a narrow acquisition window this interference could be completely eliminated. In the mass region from 27 to 81 amu (A1, Si, P, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga and Br) analyte peaks could be completely resolved from interfering molecules at medium resolution settings (R=3000). In the higher mass region mathematical correction, based on measured rates of formation (ROF) for interfering species, was applied for analytes suffering from interferences (Se, Ru, Rh, Pd, Cd, Ir, Pt, Au, Hg and Bi).

133

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

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Figure 7. Mass spectra of a five-fold diluted human serum measured at R=3000 for masses 54, 56, 57 and 58 [From ref. 12, with permission]

3.1.2. Urine samples For the determination of Cr, Ni and V in 20-fold diluted certified urine [28] a DF-ICP-MS instrument was used at 3000 to separate the signal of the analyte from severe polyatomic interferences caused by high concentrations of bulk elements (C, Ca and CI). DF-ICP-MS was also used to determine Cu, Zn, Cd and Pb in urine [29]. In this latter case a sample dilution of 1:10 and external calibration (with In as internal standard) were found to offer an acceptable compromise between analytical accuracy and sample throughput. A 3000 resolving power was used to separate Cu and Zn isotopes from interferences commonly found in this kind of samples (see Figure 6), while a resolution of 300 was used for Cd and Pb. With the exception of Zn, all measured concentrations for the fLrst row transition metal (Sc, V, Cr, Mn, Fe, Co, Ni and Cu) in a certified urine sample were found to lie within the certified range at R=3000 [30]. According to the author, Zn may suffer from ionisation suppression in urine samples as a result of its relatively high ionisation potential (9.39 eV). Elevated concentrations wel~ found for Sc, V, Cr, Ni and Cu using low resolution setting (R=300) in comparison with those obtained using R=3000 [30]. Elevated Sc values may arise frorr~ 29Si160 and 13C1602 based polyatomics, with the latter being more likely for this type of sample. The major interference

134

J.M MARCHANTE-GAYt3N et al.

of 35Cl160 would account for the highly inaccurate V concentration measured in low resolution, while 4~ and 37~C1160polyatomics may explain the elevated Cr values at masses 52 and 53 respectively. High Ni and Cu values were also found using resolution 300, with Ca and Mg polyatomics possibly accounting for the elevated Ni values, and the well known 4~ and 31p1602 interferences contributing to 63Cu [30]. Better agreement between both resolutions was observed for 6SCu and for most Zn isotopes, indicating that interferences on these isotopes were small [30].

3.1.3. Tissue samples A crucial poim in the analysis of solid biological samples as tissues by ICP-MS is to find the appropriate digestion method in order to avoid incomplete digestion. On the other hand, a too complex digestion mixture may cause severe spectral interference problems. However, most of these problems can be overcome by using DF-ICP-MS. Thus, Ni and Co were reliably determined by Hinners et al [31] in a digested certified bone ash sample, without matrix separation. The use of R=3000 allowed to resolve all interferences arising from different CaO polyatomic ions. First row transitions metals (Sc, V, Cr, Mn, Fe, Co, Ni, Cu and Zn) have also been determined in certified oyster tissue and tomato leaves samples [30]. Accurate concentrations were measured for almost all transition metal isotopes considered using R=3000. An exception was Cr in the oyster tissue sample, with both isotopes analysed providing consistently lower values than those certified by the supplier. Elevated Sc and V concentrations found using low resolution mode (R=300) can be explained by interferences noted also in urine samples [30]. However, the lower CI concentrations found in tomato leaves allowed the accurate quantification of S~V at R=300. Elevated 45Sc and 6~ concentrations were also found in this sample using such low resolution, most likely resulting from Ca or Si polyatomics [30]. The capabilities of DF-ICP-MS for the determination of 71 elements in hair and nails have been evaluated by Rodushkin and Axelsson [32]. In this work, a microwave-assisted digestion procedure with nitric acid and hydrogen peroxide was compared with the direct sampling by laser ablation (LA). Method detection limits below ng/g were obtained for 39 elements investigated by using high-purity reagents and by taking special care to prevent contamination during sample preparation. However, these detection limits were insufficient for detection of some platinum group elements in the majority of the samples. The average reproducibility (assessed from replicate analysis and including sample preparation) was found to be, as average values for all elements, 9-10% and 1819% RSD for hair and nails, respectively. Contribution from exogenous

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

135

deposition was also evaluated by analysing samples before and after washing, as well as by studying spatial element distribution along hair and nails. It was found that even after sample washing, many elements are enriched in the surface of the nail [32]. 3.1.4. Arsenic and selenium

The interference-free determination of As and Se in biological samples requires resolution settings above 7500 (see Figure 6). It should be mentioned that when using R=3000 the signals drop to about 10% of that measured using resolution 300, while the signal measured using resolution 7500 is only about 1% of that recorded at the low resolution setting. In fact, at such high resolution the signals for low concentrations of As and Se could not be measured [33] and so previous matrix separation and/or preconcentration was mandatory. Thus, As and Se determinations in biological SRMs after acid digestion of samples and matrix removal via cation exchange (using a Chelex-100 column) was described by Narasaki et al [33]. The determination of arsenic and selenium in some microwave-digested biological reference materials such as tomato leaves or urine using DF-ICP-MS in high resolution mode (R=7500) has been recently reported [34]. Despite near baseline spectral separation, 75As and 778e were still found to be influenced by ArCI at high CI concentration, the effect being most pronounced for 778e. 3.1.5. Noble metals

The determination of Rh and Pd in microwave digested blood samples [35] and in ultraviolet photolysis digested urine samples [36,37] at R=300 is interfered by the formation of doubly charged and polyatomic ions originating from Pb, Sr, Cu and Zn (Table 1) occurring in blood and urine at comparatively high concentrations (lag.Ll to mg.L l levels). These interferences cannot be eliminated by using a DF-ICP-MS instrument in the high resolution mode for two reasons: first, the high resolution capability of the DF-ICP-MS is not sufficient to separate the Pd, Rh and Ag signals from all spectral interferences, and, secondly, the sensitivity at R=7500 mode is only about 1% of that at R = 300. Thus, ICP-MS measurements are generally performed in the low resolution mode and the potentially interfering elements are additionally determined in each standard and sample, so that the relevant interferences are dealt with by mathematical corrections. In contrast, the determination of Au and Pt in microwave digested blood samples [35], Ir, Pt and Au in ultraviolet photolysis digested blood samples [38] and Pt and Au in ultraviolet photolysis digested urine samples [36,37,39,40] is unaffected by spectral interferences.

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3.1.6. Rare earth elements, scandium and ytrium Nowadays, there is an increasing demand for rare earth elements (REE) determinations in new technology applications. The bioavailability and toxic properties of these elements are under investigation. As a consequence, in the last years attention has been drawn to the development and steady introduction of analytical methods suitable for the quantification of REE in biological samples at very low concentration levels. The analysis of REE in biological samples by ICP-MS is limited due to the interferences by oxide, hydroxide, chloride molecular ions and multiple charge ions [41,42]. Of course, the general approach to deal with the problem of several spectral interferences in ICP-MS is to separate the analyte peak and interference peak in the mass spectrum by using high mass resolution. However, as the REE levels in biological samples are extremely low, the sensitivity required could not be achieved. The combination of DF-ICP-MS working at low resolution after separation and preconcentration of the REE is one of the most promising alternatives [42]. All these elements have at least one isotope free from isobaric interferences which is sufficiently abundant and hence suitable for quantitative measurements. Some isotope-pairs with isobaric overlap require resolving powers much higher than obtainable with DF-ICP-MS instrtunents (from 75000 for ~44Nd/144Sm to 5900000 for 164Dy/64Er). Therefore, those isobaric overlaps cannot be resolved. However, they can be corrected for by measuring the intensity of an isotope of the interfering element, which is itself free from interferences. In the case of unknown matrices, the isotopic pattern obtained allows additionally the estimation of other possible spectral interferences. It is important to point out that even correct isotopic patterns for at least two different isotopes do not guarantee the absence of spectral interferences. The elements causing spectral interferences at half-mass (doubly charged ions) were not found usually to be present at high levels in biological samples. 45Sc and S9y can be interfered by doubly charged Zr and Hf ions requiring R values of 13000 and 1300 respectively. However, in some plant materials high levels of Ba have been observed resulting in a 135Ba3+interference on 45Sc [41 ]. REE determination in plant materials is known to pose an analytical challenge since problems arise due to high levels of Ba and extremely low levels of REE. Thus, 135Ba160 interference on 151Eu have to be corrected for mathematically, although the separation of the 151Eupeak and the 135Ba160peak in the mass spectnan is possible with a resolving power of 7800 [41 ]. However, the low concentration of Eu in plant materials makes the quantification in the high mass resolution mode problematic due to the low sensitivity. As oxide

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

137

formation strongly depends on the plasma and operation conditions, these must be optimised in order to obtain low oxide formation rates. On the other hand, the use of a microconcentric nebuliser in combination with a membrane desolvation unit has been proved to reduce oxide formation rates [41 ]. Nevertheless, the high salt transfer to the nebuliser leads to a blockage of the membrane after some consecutive measurements and subsequently to an increase of the oxide ratio. The signal for 45Sc is influenced by a significant 2Ssil6oIH and 29Si160 interference due to high levels of Si in plant materials [41 ]. Furthermore, the use of HF as digestion agent leads to an increased background of Si released from the quartz torch by some remaining free HF (even if H3BO3 was used as complexing agent). Since the required resolving power is 1900, it is evident that higher mass resolution is a key prerequisite for separation of the Si spectral interferences from the 45Sc peak. 3.2. Matrix interferences Besides spectral interferences, systematic errors occur due to non-spectral interferences which are often referred to as '~natrix-induced signal variations" (suppression or enhancement) or "matrix effects". These matrix interferences can be caused by irreversible processes (e.g., clogging the nebuliser, cones, torch) or reversible processes occurring only during the measurement of the sample (e.g. change of nebulisation efficiency and transport, ionisation in the plasma, ion extraction). These matrix interferences have been usually corrected by internal standardisation, standard additions or isotope dilution analysis. The use of one or several internal standards is the most attractive method for the correction of matrix interferences in routine analysis (see Table 3) because instrumental fluctuations are also corrected at the same time. The method involves the addition of a specified amount of one or several elements, which (i) are not present in the sample, (ii) have no obvious spectral interferences and (iii) show the same effect of signal variation as the analyte of interest. Typically, monoisotopic rare elements covering the mass range of interest are used (for example Be, Sc, Co, Y, Rh and Tb).

3.2.1. Serum and urine samples Important matrix interferences have been reported in the analysis of biological materials by DF-ICP-MS. The high salt content and the viscosity of sermn samples makes it a difficult sample for virtually all analytical techniques. In a previous work [24] we observed that the suppression of the ICP-MS signal by the presence of the serum matrix was independent of the considered element mass but it did depend on the dilution factor used for serum analysis. To evaluate this effect the slope of an aqueous calibration graph was compared with

138

J.M. MARCHANTE-GAYON et al.

the slopes obtained for standard additions calibrations at different dilution factors and with or without the use of internal standards. The results obtained are presented in Figure 8. As can be observed, severe suppression effects were present both at 1:5 and 1"10 dilution factors and, with the exception of selenium, the suppression was independent of the mass of the analyte. However, the use of internal standards compensated for most of the matrix interferences observed in this biological fluid even at the 1:5 dilution factor. Severe problems of sensitivity changes of DF-ICP-MS applied to the direct analysis of human body fluids (especially for diluted blood-serum and urine samples without any other pre-treatment) have also been reported recently by Schramel et al [43]. Very fast clogging (atter a few minutes) of the torch, followed by a dramatic loss of sensitivity was observed analysing diluted urine and serum samples. This effect was ascribed to the obstruction of the injector channel of the torch. The torch injector capillary had a diameter of 4-5 mm throughout the whole torch length except for the last 5 cm where it was reduced to 1 mm diameter. These authors [43] explain this behaviour because the last part of the torch capillary is located fully in the UV-radiation area of the plasma. Turbulence and the UV-radiation destroys the organic matter (specially proteins) in these samples and the products are deposited at the beginning of the thin part of the capillary. This effect can also be observed to a lesser extent for digested samples, because this reduction may also act as a zone of crystallisation and therefore the deposition of growing residues in the capillary in case of a relatively high salt content in the solutions (_>1%). This means that urine and serum should be diluted 1:5 or even better 1:10 to prevent this effect. Higher salt concentrations may cause difficulties also by clogging of the cones. Torch constructions where the diameter reduction of the capillary is nearly at the beginning of the torch (and therefore far away from the influence of the UV-radiation) improved these effects dramatically [43]. Also flow injection or microwave digestion have been proposed to eliminate this matrix problem and concomitant memory effects [23]. 3.2.2. The case o f selenium Many authors have described the peculiar behaviour of selenium in ICPMS in the presence of carbon containing solvents or compounds. Enhancement of the selenium signal has been observed in the presence of organic solvents in the liquid sample by different authors [44,45]. This sensitivity changes, well known in the Q-ICP-MS determination of Se in carbon containing matrices, have also been noticed [24] by DF-ICP-MS (see Figure 8 at mass 82). The enhancement effect of carbon can not be corrected by the use of internal

139

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

standards. It seems that the ionisation efficiency of selenium increases drastically in the presence of carbon due to charge transfer between C § and Se ~ [24]. Such matrix interferences could be compensated by using constant carbon levels in the plasma. Following this philosophy a method for the determination of Se by Q-ICP-MS in human serum has been recently published [46]. However this methodology has not been transferred to the analysis of Se in biological materials by DF-ICP-MS yet.

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140

J.M. MARCHANTE-GAYON et al.

areas, has been clearly demonstrated. Adsorptive voltammetry offers comparable limits of detection, but DF-ICP-MS is faster, less sensitive to interferences, does not require total mineralisation of the sample and is capable of multi-elemental analysis [35,36,39]. Reference "values or concentrations" for environmental contaminants serve an important role in environmental health investigations and studies. They provide information about the prevalence and magnitude of exposure, which can be used as a basis for comparing concentrations in subjects who have suspected or known exposure to a given point source. To illustrate this, let us suppose that a community is located near a hazardous waste site or a plant where metals are processed. By comparing the urinary metal concentrations in people of that community with the reference values for normal people it is possible to determine if that community has had an elevated or unusual exposure. In this context, limits of detection of 1.0 ng/L and 0.85 ng/L for uranium and thorium respectively in human urine have been reported recently [48] after a simple 1:10 dilution of the samples followed by analysis using DF-ICP-MS. In this study, uranium was detectable in 96.6% of the 500 specimens (at a mean level of 11.0 ng/L) while thorium was detectable in only 39.6% (at a mean level of 1.01

ng/L). However, it should be stressed here that the limits of detection attainable by DF-ICP-MS at low resolution are so low that reagent blanks and memory effects must be drastically reduced to make full use of the possibilities of these powerful instruments. Thus, L. Moens et al [22] found that the limits of detection for Ag in serum were 10-100 times higher than the instrumental limits of detection due to the high blank values caused by memory effects. In order to minimise blank signals it is crucial to ensure minimal exogenous contamination by resorting to clean room facilities and thorough purification of all the acids by sub-boiling distillation [23,28,29,36-42]. Moreover, continuous checking for the sought element(s) in any reagent used for final analysis is mandatory. Limits of detection obtained in our laboratory [49] in five-fold diluted human serum for 14 elements at both 300 and 4000 resolving power are given in Table 4. As can be observed, most detection limits are near or below 0.1 ng/g with the exception of 27A1 and 64Zn which are limited by blanks and not by instrumental detection limits. For heavy elements, such as uranium, detection limits below pg/g levels can be obtained.

141

Double Focusing-InductivelyCoupled Plasma-Mass Spectrometry

Table 4 Detection limits obtained in the direct analysis of five-fold diluted serum samples [49] for 14 trace elements (either at R= 300 or 4000, as required) Monitored isotope 27A1 43Ca 52Cr 5SMn SaFe

59Co 63Cu 64Zn 8SRb 8SSr 98Mo

ll4Cd 2~

238U

R used 4000 4000 4000 4000 4000 4000 4000 4000 300 300 300 300 300 300

Detection limit (ng/g) 0.35 0.01 0.01 0.02 O.12 0.02 0.09 0.34 0.009 0.006 0.02 0.003 0.02 0.0005

3.4. Biomedical applications. One of the important applications of the extreme sensitivity afforded by the DF-ICP-MS instruments could be the confirmation/establishment of "reference values" for trace element content in organs and tissues of "normal" populations and using such data to be able to diagnose health and disease status, related with anomalous trace element total contents [24,25,50]. For example, DF-ICP-MS has been applied to measure serum AI basal levels in healthy people [51 ]. In order to avoid spectral interferences of the CN § ions not resolved from the A1 peak at low resolution, it is necessary to work at R=3000 (see Figure 9). There, the limits of detection for AI determination in human serum by DF-ICP-MS are one order of magnitude better than those obtained by Electrothermal Atomic Absorption Spectrometry (ETAAS) which has been for many years the preferred technique for these determinations. It appears that by minimising exogenous contamination and lowering instrumental detection limits by resorting to these of DF-ICP-MS a fin'ther lowering of "normal" or basal levels of AI in human serum from the present accepted 2 ~tg.L value to values below 0.35 ~tg.L"l is possible [51 ]. The amazing capacity of healthy kidneys to clear up aluminium from the body is even more astonishing in the light of these fmdings [51 ].

J M. MARCHANTE-GAYON et al.

142

1400

12ClSN+ 13C14N+

1200

1000

~, 8oo v

" 600

27A1+

_c 4O0

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0 26.96

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Figure 9. Mass spectrum of basal AI in a five-fold diluted human serum from a healthy individual showing the resolution of CN+ type spectral interferences at R=3000. [From reference 51, with permission] Single and multielemental analysis of body fluids (blood, serum, urine) using DF-ICP-MS has been undertaken by several authors [23-25,50,52] but routine measurements of large numbers of clinical samples have not been published so far in relation to "normal" or "abnormal" clinical situations, e.g. for uraemic patients [53,54]. In this vein, in our laboratory we have recently completed a study of the levels of 14 trace elements in 59 healthy subjects (41 male and 18 female, blood donors) by DF-ICP-MS and compared such levels with those found in 14 renal failure patients undergoing haemodialysis [49]. The results obtained are summarised in Table 5. As can be observed, lower values for the essential elements Fe and Zn and also for Rb and higher values for Cu, Sr, Mo, Cr and AI were detected in the haemodialysis patients. To give an insight of the ranges found, Figure 10 shows the plot of the concentrations measured for Mo and Zn for both types of sera. As can be observed, there is a clear distinction between healthy individuals and uraemic patients in their Fe and Zn content.

143

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

12.0

10.0 Uraemic patients

9

8.0 O

~-

6.0

#

g~ 0

o

4.0

Blood d o n o r s

Q~

2.0

o

o 00

.000

i

i

1

.200

.400

.600

9800

1.000

1.200

Zn concentration (pg/g)

Figure 10. Concentrations found for Mo and Zn in serum from blood donors (0) and uraemic patients (*).

3.5. Applications to food samples The analysis of trace elements in the diet is very important in order to better protect the health of the consumers. In this context, a study was undertaken by Caroli et al [55] to investigate the average levels of a number of key elements in several types of honey, with special regard to the influence of the various honey processing steps. Thus, As, Cd, Pb, Pt, Sn and V were determined by a DF-ICP-MS instrument working at R=300 while Cr, Cu, Fe and Ni were determined working at R=3000. Multielemental analysis of a wide range of elements in milk whey, human milk and infant formulae using DF-ICP-MS has been recently published by two independent groups [56,57]. In the First work, milk samples were diluted 1+4 for minor and trace elements and 1+1999 for major elements, with ultrapure water and the addition of Ga, Y, Rh, In and TI as internal standards (Table 6). In the second paper [57], milk samples were microwave digested with nitric acid and hydrogen peroxide and addition of Rh, In and Re as internal standards was used (Table 6). In both eases, DF-ICP-MS at R=3000 demonstrated that polyatomic interferences in milk whey samples were well separated, with the exception of

144

J M. MARCHANTE-GAYON et al.

Table 5 Trace and ultratrace concentrations found in blood donors and haemodialysis patients. Values are given as mean and standard deviation or concentration range when the lowest value is below the detection limit

Element

Concentration units

Ca Fe Cu Zn Sr Rb Mo Cr Mn Cd Pb U Co AI aLOD = Limit of detection

~tg/g ~tg/g l-tg/g ~g/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g ng/g

Healthy people (n=59) 92.3 + 7.3 1.47 + 0.38 0.90 + 0.15 0.83 + 0.11 16.3 + 6.0 141 + 34 0.81 + 0.35
Haemodialysis patients (n=l 4) 88.4 + 12.0 0.97 + 0.63 1.01 + 0.15 0.61 + 0.10 57.2 + 30.7 106 + 32 5.8 + 2.3 0.49 - 3.00 0.12 - 0.55 0.012 - 0.090 O.17 - 1.60 0.006 - 0.043 0.15 - 1.30
1HSlBr and 4~ interferences on S2Se and 75As, respectively (they would require higher mass resolution). As an example, Figure 11 shows the mass spectra o f 6~ and 62Ni recorded from a milk whey sample diluted 1+4 in the DF-ICP-MS at resolution 3000 [56]. As can be seen, nickel isotopes on such sample matrices would be interfered by Ca, Mg and Na polyatomics for 6~ and 38Ar24Mg and 23Na2160 for 62Ni) unless higher mass resolution is employed. As it was mentioned previously, matrix effects in blood serum were minimised by internal standardisation. For the analysis o f milk whey signal enhancement was observed both at 1+4 and 1+1999 dilution factors. However, those matrix interferences could be also corrected by the use of internal standards as shown in Figures 12 and 13 [56]. A microconcentric nebuliser in combination with a membrane desolvation unit was also tested with respect to signal enhancement and reduction o f interferences [57]. In general, a factor o f 5 increase of the signal intensities was observed, whereas not all spectral

(44Ca160

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

145

Table 6 Internal standards and resolution settings used for multielement analysis in milk samples

Monitored Isotope , 23Na 24Mg 26Mg 27 AI 42Ca 44Ca

Internal Standard* 71Ga 71Ga 71Ga 71Ga 71Ga 71Ga

45Sc

.....

47Ti

.....

51V

52Cr 53Cr 55Mn 56Fe 57Fe 59Co 6~ 62Ni 63Cu 65Cu 66Zn 68Zn 75As S2Se 86Sr 88Sr l~ ll~ lllCd l l4cd

71Ga 71Ga 71Ga l~ l~ ..... 71Ga 71Ga l~

Internal Standard** ..... ..... ..... l15In ..... ..... lS7Re ilSin l~ l~

l~

..... 115In 11Sin ..... l~ l~ ..... ll5In .....

l~ l~

..... .....

..... 71Ga 89y 89y ..... ~lSln 115in .....

llSIn ..... .....

187Re ..... ..... 11Sin 195pt ..... 187Re 197Au ..... 187Re 2~176 2~ ..... 2~ 2~ ..... 2~ 2~ ..... 2~ 2~ lS7Re *Data obtained from reference 56. ** Data obtained from reference 57.

R 3000 3000 3000 3000 3000 3000 4000 4000 4000 3000 3000 3000 3000 3000 4000 3000 3000 3000 3000 3000 3000

8000 3000 300 300 400 300 300 400 400

400 300 300 300 300

146

J.M. MARCHANTE-GAYONet al.

r.~

~., (.)

16000 14000 12000 10000 8000

-I -~ -

6~

44Ca16O

6000 4000 2000

L

~

-

-

I

-

59.92

t

i

59.94

59.96

i

59.98

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-

62Ni

|

38Ar24Mg

23Na216 0

2000 -

&

1500 -

L) 1000 5 0 0

0

-

-

-

I

61.92

I

t

I

61.94

61.96

61.98

Mass(amu)

Figure 11. Resolution of polyatomic interferences on nickel isotopes in milk whey samples using R=3000. [From ref. 56, with permission]

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

147

_

:$

o o"

<

O

fith IS.

l k whe y dilut( d l + 4

1.6-

|e-~ ~o

1.4-

=in c0

1.2-

o

l k w h e y dilutt d l + 4 .

1.8-

(

--

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1

-o -~

0.8

Er

0.6

ct~ O9

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a,

...........

I

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0.2

I

0

50

1 O0

150

200

250

Mass (amu) Figure ]2. Observed effect of internal standard correction on the analysis of minor 66 68 and trace elements in milkwhey: 27AI, 52,53 Cr, 55Mn, 56 57 9 Fe, 60'62Ni, 63.65 ' Cu, ' Z n , 82 86 88 110 111 200 202 206 208 Se, ' Sr, ' Cd, ' Hgand ' Pb. Dilution factor l+4. [Fromref. 56, with oermissionl

o=

1.61

=

1.4

= =

1.2

<

C).. t-~

oo

r er 0 0:~ "0 r ~ =

0 0

t

|

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m

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0 "r0

9

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0 Milk whey dilutr d l +

1999.

9 Milk whey dilut~ d l +

1999 ~ith IS.

0.4

20

30

40

50

Mass (amu) Figure 13. Observed effect of internal standard correction on the analysis of major elements in milk whey: 23Na, 24'26Mg and 42'44Ca. Dilution factor l +1999. [From ref. 56, with permission]

148

J.M MARCHANTE-GAYON et al.

interferences could be reduced to a negligible amount (only metal oxides to some extent). Also, the use of nitrogen as the make-up gas did not improve the sensitivity but led to additional N-containing polyatomic interferences (e.g., for V, Cr and Mn) [57]. The results reported show considerable differences in trace element content in human, cow and formula milk whey [56] while trace element levels in instant milk formulae are significantly influenced by the quality of tap water used for preparation [57]. The determination of rare earth elements (REE) in wines is another interesting application of DF-ICP-MS for studying wine origin [58]. It is well known that REE patterns are widely used in geochemistry as a useful soil characteristic. For instance, the REE pattern of vineyard soil should be reflected in the corresponding wine composition; thus, determination of the REE in wines could permit identification and geographical location of the wines origin. Different ICP-MS instruments, including DF-ICP-MS, were compared by Augagneur et al [58] for the determination of REE in undiluted wine samples. At maximum resolution setting of the DF-ICP-MS instrument used (R=7500) they still found serious spectral interferences by oxide ions. In any case at R=300 much lower detection limits (up to two orders of magnitude) than those obtained by Q-ICP-MS were reported [58].

3.6. Applications to environmental biological samples Ultra low ambient blood lead concentrations (0.13+0.06 ~tg/dL) in northern elephant seals have been measured by Owen et al [59] using DF-ICPMS. These results support previous estimates of ultra low blood lead levels in preindustrial humans. Such estimates remained unsubstantiated until two years ago, since blood lead levels in this range had never been measured in any organism. The marginally higher blood lead levels and rates of lead exposure in contemporary marine mammals are consistent with lead isotopic composition analyses that indicate how their blood lead levels have been elevated after exposure to industrial lead. A hydride generation system combined with DF-ICP-MS was used by Feng et al [60] to determine tin in biological marine materials. In this method, a strongly basic anion exchanger was used in order to remove the interferences from transition element ions. DF-ICP-MS has been also used in the simultaneous determination of V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Cd, Sn and Pb in microwave digested Antarctic krill samples [61 ]. The Antarctic krill is a small shrimp-like crustacean, which is the staple food of many fish, birds and mammals in the Southern Ocean. Most of the elements (V, Cr, Mn, Fe, Ni, Co, Cu, Zn, Se and Sn) were measured at R=3200 in order to avoid spectral interferences, while Cd and Pb were determined at a resolving power of 300.

Double Focusing-InductivelyCoupled Plasma-MassSpectrometry

149

3.7. Determination of radionuclides in biological samples The common techniques to analyse radionuclides in biological samples are of course radioanalytical techniques. However, the determination of longlived radionuclides are not very sensitive and very long counting times are required. Also, the separation of the monitored radionuclides from other elements is usually required before the f'mal measurement of the activity. The low limits of detection of DF-ICP-MS, working at low resolution, turned out to be very useful in order to measure long-lived radionuclides in environmental and biological samples directly without chemical separation [62]. An interesting application is the determination of the long lived 13-emitting 798e nuclide with a half-life of 6.5x104 years. According to the legislation in many countries, extremely low concentrations of this radionuclide have to be determined in radioactive waste samples prior to their permanent disposal. The determination of 798e by ICP-MS is interfered by polyatomic ions (4~ 4~ 63Cu160+) +, and by direct overlap from 79Br+. Even the maximum resolution setting of current DF-ICP-MS instruments was insufficient to avoid all spectral interferences. In order to reduce the level of 79Br+, Hoppstock et al [63] have developed a hydride generation method for sample introduction permitting separation of the selenium from the majority of matrix components. The presence of 4~ + could not be eliminated so the limit of detection was only 0.1 ng.mL "l with the instrument operated at the low resolution setting (R=300). Digested certified biological reference materials (mussel tissue and apple leaves) were used to verify the method with respect to the determination of natural selenium [63]. Kerl et al [64] have designed an equipment for the introduction of gaseous iodine into a DF-ICP-MS working at R=300, using flow injection, on-line oxidation with concentrated perchloric acid of the iodide present in microwave digested tissue sample solutions and f'mal iodine gas/liquid separation. For 129I determination an on-line standard addition was performed reporting a limit of detection of 50 pg.mL 1. This limit of detection is inadequate for analysis of radioactive ~29I in biological samples and its mainly limited by the l:Z9Xe impurity in the argon used. On the other hand, accurate determination of natural 1271 in the same materials was performed by on-line isotope dilution reporting a limit of detection of 100 pg.mL. In this latter case the microwave digested certified tissue sample solution was injected in a continuous flow of a 129I containing solution. The obtained limit of detection was influenced by contamination in the oxidation reagent, but it seems to be sufficiently low for the determination of iodine in most biological materials.

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J.M. MARCHANTE-GAYON et al.

4. ISOTOPE RATIO MEASUREMENTS The measurement of isotope ratios using single collector DF-ICP-MS has been applied to a number of problems including Pb and Sr source characterisation, Ca metabolic studies, tracer experiments and isotope dilution analysis of different trace elements. In all cases, studies had to be carded out on the performance of DF-ICP-MS for isotope ratio measurements in terms of precision and accuracy. We will summarise briefly those parameters which affect the attainable precision and accuracy before commenting on the applications.

4.1. Accuracy of isotope ratios by DF-ICP-MS It is generally agreed that ICP-MS can provide isotope ratios of high accuracy when four factors, namely mass bias, detector dead time, reagent blanks and isobaric interferences, are under control. We will discuss those factors separately. 4.1.1. M a s s bias-

It has been observed that in most ICP-MS instruments the efficiency of transport of ions through the mass spectrometer is not constant but increases as a function of the mass of the considered ion. This has been also observed for DFICP-MS instruments [65]. The effect of this increased transmission with mass on the measurement of isotope ratios is the so-called mass bias. Isotope ratios measured by ICP-MS are usually biases with respect to the heavier isotope because of its increased transmission. This effect can be easily observed by the measurement of isotope ratios on a certified isotopic standard or on a natural element with well characterised and constant isotopic composition. For example, Table 7 shows the measured and natural isotope ratios obtained for a Mo standard on a DF-ICP-MS instrument working at low resolution. As can be observed, the relative errors on the measured isotope ratio increased with the mass difference between the measured isotopes. It can be also demonstrated that there is a linear correlation between the relative error, ( R m ~ - R~t)/Rn,t, of the measured isotope ratio and the mass difference, AM, between the isotopes used for the calculation of the ratio. For the data shown in Table 7 the equation is Relative error = -0.0185 x M a s s difference. This means that an error of-1.85% is made per unit of mass difference between the measured isotopes.

151

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

Table 7 Mass bias effect observed for molybdenum in a DF-ICP-MS instrument Ratio 92/98 94/98 95/98 96/98 97/98 100/98

Measured 0.5442 0.3553 0.6222 0.6667 0.3896 0.4122

Natural 0.6150 0.3833 0.6598 0.6913 0.3958 0.3991

Relative error -0.1151 -0.0731 -0.05 71 -0.0356 -0.0157 0.0328

Massdifference 6 4 3 2 1 -2

Once this correction factor (the '~nass bias factor", K) is computed, the measured isotope ratios can be corrected using the expression:

Rc~

Rmeas = 1+ KAM

(7)

4.1.2 Detector dead time Pulse counting detectors, such as the electron multipliers used in most ICP-MS instruments, may suffer from lack of linearity at high counting rates due to the dead time of the detector and associated electronics. When an ion arrives at the detector and generates a pulse there is a finite time, of the order of nanoseconds, in which the detector is incapable of recording another event. For high counting rates pulse pile-up may be considerable and count losses occur in the detector. As the arrival of ions to the detector can be modelled by Poisson statistics, the losses due to detector dead time can be corrected using the expression:

Icorr =

Imeas 1- rlmeas

(8)

where r is the "dead time" of the detection in seconds, lmeas is the experimental count rate and Lo,, the true count rate. For isotope ratio measurements the losses occurred for one isotope will not be the same as for the other isotope unless the isotope ratio is 1. Hence, the effect of the dead time has to be corrected for accurate isotope ratios to be obtained. The determination of the detector dead time can be carried out by measuring the isotope ratios of a given isotope pair at different concentration levels. When dead time losses occur, the isotope ratios measured at high

152

J M. MARCHANTE-GAYON et al.

concentrations will deviate from those measured at low concentrations. This can be illustrated for the measurement of the dead time of the "Element" at 3000 resolution for the 65Cu/63Curatio. The results obtained are presented in Figure 14 both, before and after dead time correction. As can be observed, the isotope ratios measured for 65Cu~3Cuincreased with the concentration of copper. By testing different values of dead time in equation (8) the best dead time estimate will be that giving the lowest standard deviation between the different isotope ratios measured at different concentration levels. In our case, a dead time of 49 ns provided the best agreement between the measured isotope ratios. It is important to clarify that detector dead time correction has to be applied before mass bias correction for accurate ratios to be obtained. 0.478

0.476

e / ~

0.474 en 0.472 ~ ~ 0.47 .o~

9

9

0.468 O,, O

~O 0.466 ~

0.464 0.462

9 0.46 [ 0.458

- ~ .

0

0

0

200

. 0

.

. 400

. 600

.

. 800

1000

1200

1400

Cu concentration (rig/g)

Figure 14. Determination of dead time for DF-ICP-MS using Cu isotope ratios at different concentration levels. (o) Experimental data. (O) Data atter correction for 49 ns dead time.

4.1.3. Blanks Contamination of the instrument itself or during the chemical pretreatment used can give rise to signals in the ICP-MS instrument coming from the same element to be measured. Generally, however, different isotopic composition of blanks and samples are observed. Instrument contamination can be due to memory effects in the sample introduction system (nebuliser, spray

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

153

chamber) or in the interface. External contamination can also occur during the sample preparation step due to impurities in the reagents used or leaching from the containers. At any rate blanks have to be minimised in order to obtain accurate isotope ratio measurements paying special attention to memory effects, purity of the reagents and pre-cleaned containers. It is important to obtain a blank value which is stable and representative of the chemical procedure used. The correction for blanks is normally performed simply by subtracting the intensities measured at both masses used to calculate the ratio between sample and blank. However, this correction will increase the uncertainty of the isotope ratio measurement. It is very important to keep the blank values as low as possible. 4.1.4 Isobaric interferences Spectral interferences due to polyatomic ions cannot be easily corrected matemathically. In those cases it may be necessary to resolve the interferences by working at high resolution settings. For example, for the measurement of Fe isotope ratios in human serum all Fe isotopes are interfered by polyatomic ions (see figure 7). However, those interferences can be eliminated working at R=3000. There are cases that even high resolution cannot resolve isobaric interferences. In general, those interferences arise from other metals with isotopes of the same nominal mass as the analyte (e.g. 5SNi and 5SFe). In those cases mathematical corrections can be applied by measuring the intensity of a non-interfered isotope of the isobaric element (for the example 6~ and calculating its contribution at the interfered mass by using the known natural isotopic composition of the elements. 4.2. Precision of isotope ratio measurements Thermal Ionisation Mass Spectrometry (TIMS) is the technique that offers a priori the best precisions for isotope ratio measurements. However, work on TIMS is lengthy and cumbersome because the analyte has to be separated from the sample matrix. That is, TIMS sample preparation is tedious and timeconsuming. ICP-MS offers a much faster approach [66], although the Q-ICP-MS does not provide TIMS precisions. One of the major advantages of the DF-ICP-MS compared to Q-ICP-MS is the flat top shaped peaks obtained in the low-resolution mode (Figure 5). Those flat peaks are more tolerant to instrumental fluctuations (so common with all mass spectrometer types) and could grant a better overall stability of the measurements. This feature would provide enhanced isotope ratio precisions, as shown in Table 8 for lead isotope ratio in bones [31 ]. Its is worth noting here that modem multicollector ICP-MS insmunents can provide isotope ratio

154

J.M. MARCHANTE-GAYON et al.

precisions similar or even better than those obtained by TIMS. However, we will focus the discussion here only on single collector DF-ICP-MS instruments. DF-ICP-MS, working at the low resolution mode has been recently applied to obtain precise isotope ratio measurements for Pb, Cu and Zn in biological samples [67-69]. Lead isotope ratios in biological samples, e.g. human body fluids, could reflect the primary source of Pb exposure and uptake. Q-ICP-MS measurements at low Pb concentration levels did not provide sufficient precision to adequately distinguish environmental Pb sources based upon their isotopic composition, since the isotope ratios of contaminant environmental Pb vary typically in a very narrow range. Also, most published Q-ICP-MS measurements of Pb isotopic compositions in environmental and biological matrices have not included ratios for 2~ (1.4% natural relative abundance) which is required for complete assessments of Pb sources using isotopic composition analysis. However, after careful optimisation of the DF-ICP-MS operating parameters, insmnnental bias factors including mass bias and dead time, internal standardisation with 2~ (in order to obtain information about the Pb concentration) and mathematical correction of the ~~ interferences, lead isotope ratio precisions lower than 0.1% in real biological samples (blood, bone ash or bovine liver) can be achieved [67,68]. Table 8 Preeisions obtained by TIMS, Q-ICP-MS and DF-ICP-MS in the determination of Pb isotopic ratios in a NIST SRM 1400 certified bone ash sample (data obtained from reference 31) isotoPe Ratios 2~176176 2~176 2~176

ZIl~S a

Zi]~S b

0.0087% 0.016% 0.0064%

0.014% 0.012% 0.012%

Q-ICP-MS a 0.15% 0.10% 0.091%

Q-ICP-MS b 1.3% 1.2% 1.6%

DF-ICP-MS c 0.066% 0.12% 0.057%

Geological Survey of Canada, Ottawa, Ontario (Canada). b Geochemisches Institut, Universit~t GSttingen, Grttingen, Germany. c Institute of Marine Sciences/Environmental Toxicology, University of California, Santa Cruz, USA.

a

In a recent publication, some preliminary results show the existence of Cu and Zn variations of up to several per mil amongst natural samples of silicates, ores, sediments, and biological materials, which paves the way for the use of Cu and Zn isotopes as geochemical and biochemical tracers [69]. In this work, a multi-collector DF-ICP-MS was used in order to obtain precise Cu and Zn isotope compositions. As this instrument can only be operated at low resolution

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

155

mode, spectral interferences were compensated by separating the elements from the matrix and using mathematical corrections.

4.3 Resolution of spectral interferences. One of the main limitations encountered in the isotope ratio measurements by Q-ICP-MS is the interference of polyatomic species which determine the need of chemical separations. However, these time-consuming separations could become redundant if DF-ICP-MS at medium or high resolution settings could resolve the peaks. On the other hand, as the resolution increases the precision in the isotope ratio measurements decreases (typical precision ranges are: lower R <0.1%, medium resolution <0.5% and higher resolution <1%). Careful selection of the measurement conditions [70] allowed a 6 3 C u / 6 5 C u isotope ratio precision close to 0.1% for n=10 in a 10-fold diluted human serum reference material using R=3000 in order to avoid interfering signals from 4~ +, 31pl602+and 2.3~ N a 16 2 0 1H + for 63 Cu+ and 32S 1602 1H + for 65Cu.+ Isotope ratio measurements of Mg, K and Ca have been used to investigate the transport phenomena of nutrient solutions in ~lants by tracer experiments using highly enriched 5Mg, 26Mg, 41K, 42Ca and Ca isotopes. In order to resolve the ArH + ions from the 39K+and 41K+analyte ions for potassium isotope ratio measurements [71], a DF-ICP-MS insmunent was used at a resolving power R=9000. The precision of such potassium isotope ratio measurements in biological samples was 0.7%. Isotope ratios for Mg and Ca at R=3000 were also reported and precisions of 0.4% and 0.5%, respectively, were obtained [71]. A new method has been reported by Sttirup [72] for the simultaneous measurement of zinc isotope ratios (64Zn/66Zn, 67Zn/66Zn, 68Zn/66Zn and 7~ and total zinc content in microwave digested human faeces and diluted urine and serum samples using DF-ICP-MS. In Table 9 all spectral interferences found in microwave digested faeces and diluted urine and serum samples are listed [72]. All 15 spectral interferences were to some extent found in the faecal solution, while silicon and chlorine based interferences were absent in sertma. Barium, cerium and chlorine based interferences were absent in the urine solution. The only completely interference-free zinc signal was the 67Zn signal in urine. Nevertheless, using a mass resolving power of 6000 all zinc isotopes could be measured free from interferences, except for 64Zn where the overlap from 64Ni had to be corrected for mathematically. At such resolution, RSDs of 0.7% were found for 67Zn/66Zn,68Zn/66Znand 7~ while a RSD of 1.2% was found for 64Zn/66Zn.

J.M. MARCHANTE-GAYON et al.

156

4.4 Tracer studies. The application of ICP-MS to measure stable isotopes, used as tracers to study the absorption and metabolism of essential or toxic elements in living organisms is of great interest in Biology and Medicine. In these experiments the diet is labelled with one or two enriched stable isotopes. The resulting change in isotope ratio with time is followed by measurement of metal isotope ratios in, for example, faeces and urine. In this way, the biological detrimental effects of the radioisotopes traditionally used for this purpose can be avoided. Measurements of 44Ca/4aca and 42Caflaca isotopic ratios in 50-fold diluted urine using DF-ICP-MS were used to study the uptake and excretion of Ca by the human body [73]. Relative standard deviations of 0.33 and 0.41% were found for 44Caf13Ca and 42Ca/43Ca respectively. Using a resolution setting of 4000, the calcium peaks were resolved from interfering polyatomic ions of diluted urine. However, interferences from Sr 2§ were not resolved even at this resolution. Thus, mathematical corrections were carried out for this element. Table 9 Spectral interferences on zinc in faecal, urine and serum samples [72] Isotope ~Zn

Interference ~li 4~

66Zn

4~

36Ar12C160 49Ti16OIH 132Ba2+

3451602 67Zn 68Zn

134Ba2+ 4~ 136Ba2+

3651602 7~

4~ 35C12

14~ 4~

Faeces Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Serum Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes No Yes Yes

Urine Yes Yes Yes Yes Yes No Yes No Yes No Yes Yes No No Yes

In a recent study [74], an interesting adult primate model of human Pb exposure was developed. This model uses a sensitive stable Pb isotope (2~ tracer methodology to determine the efficacy of succimer for reducing Pb in specific brain regions resulting from chronic (5 weeks) and short term (3-4 days) Pb exposures. The extent to which blood Pb can serve as a surrogate of brain Pb,

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry.

157

over the Pb exposure period and following succimer chelation treatment is discussed.

4.5 Paleoanthropological applications. Using a DF-ICP-MS insmunent operated at the low resolution setting, Latkoczy et al [75,76] determined 86S1"/87Srisotope ratios in prehistoric human bone samples. The isotopic composition of Sr varies in nature as a result of natural radioactive decay of 87Rb to 87Sr. Since the Sr isotopic composition of vegetation is in equilibrium with that of the local geology, the Sr isotope analysis of prehistoric human bone samples permits to obtain information about the provenance of single individuals. Any paleoanthropological interpretation of such analysis require a highly accurate isotopic ratio determination with a precision of at least 0.1%. Thus, by careful optimisation of the instrument operating parameters such as sampling time, scan duration and instrumental bias factors including mass bias and dead time, precisions of about 0.05% were achieved allowing the distinction of humans individuals of different origin.

4.6 Isotope dilution analysis. Another field where precise and accurate isotope ratios are needed is the determination of trace metals in biological materials by isotope dilution analysis. For that purpose an enriched isotope of the element to be determined is spiked to the sample altering the natural isotopic composition of the element to be analysed. By measuring the altered isotope ratios the concentration of the element in the sample can be measured with high precision and accuracy. In other words, since another isotope of the same element represents the ideal intemal standard for that element, isotope dilution results are expected to be highly accurate and precise even when the sample contains high concentrations of concomitant elements and/or there is sample loss during the preparation or pre-treatment processes. Therefore, isotope dilution mass spectrometry (IDMS) can play an important role in accurate routine trace analysis. Of course, validation of the results for trace elements is more and more demanded and IDICP-MS has been proposed as a convenient approach to the establishment of traceability of analytical measurements [77]. For the analysis of Fe, Cu and Zn in human serum applying isotope dilution analysis the use of a DF-ICP-MS is necessary to avoid spectral interferences and obtain accurate results as recently reported by our group [78]. As it can be seen in Figure 15, at R=3000 allowed the complete resolution of polyatomic ions overlapping the main isotopes of these elements The RSD values obtained for the isotope ratios of Fe (57/56), Cu (65/63) and Zn (67/64) were 0.7, 0.2 and 0.8%, respectively, for a natural standard aqueous solution

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containing 50 ~tg.L"~ for each element. The measured isotope ratios were corrected for dead time of the detector and mass discrimination errors [79]. The sample to spike ratio was also optimised to minimise the relative error on the isotope ratio measurement [79]. Serum samples were diluted 1+19 with ultrapure water after the addition of the 57Fe, 65Cu and 67Zn spikes. The method was validated by the analysis of NIST SRM 1598 Inorganic Constituents in Bovine Serum. The levels found were in agreement with the certified values. The method was applied to the analysis of sertma samples from 59 healthy blood donors and 14 uraemic patients showing clear differences in their Cu, Fe and Zn content (data given in Table 5 for Fe, Cu and Zn were obtained by isotope dilution analysis). The work carried out in Gent by Moens" group [80] seems to go in the same direction. An increase of applications of Isotope Dilution DFICP-MS for more accurate multielemental analysis of biological materials is to be expected in the near future. A detailed examination of the performance of a DF-ICP-MS instrument working at R=300 for the ID-ICP-MS determination of cadmium in biological samples has been recently developed in our laboratory [81], comparing the results with those obtained by using a quadrupole based instrument. Systematic errors, including dead time, mass bias effects and spectral interferences could be easily corrected in the two instruments. Typical isotope ratio precisions of 0.20.3% could be obtained routinely with both instruments but the precision of the measurements in the DF-ICP-MS insmunent could be improved (<0.1%) by increasing the number of scans. Moreover, for very low cadmium concentrations, the DF-ICP-MS instrument at low resolution setting (R=300) could provide better isotope ratio precision than the quadrupole-based ICP-MS insmnnent because of its higher sensitivity. Nevertheless, both ICP-MS instruments were successfully applied to the determination of very low levels of cadmium by ID-ICP-MS in certified reference biological materials. 5. TRACE METAL SPECIATION Speciation of toxic and essential trace elements in biological systems is of increasing interest [82]. DF-ICP-MS has been shown to be a most powerful detector for trace element speciation using High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and, more recently, Capillary Electrophoresis (CE) separations because of its selectivity (elimination of spectral interferences working at high resolution settings) and extremely high sensitivity (working at low resolution). In contrast to other popular atomic techniques used for biological materials (e.g. ETAAS), DF-ICP-MS offers the advantage of on-line coupling ability with the chromatographic system, more

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159

sensitivity, its multielement ability and the isotopic measurements capabilities. All those features are very important to tackle the modem problem of identification and determination of metal-biomolecules in living organisms.

5.1. High Performance Liquid Chromatography (HPLC) As the effluent flow rate of an HPLC column matches the typical liquid flow rate of the most commonly used nebulisers, the coupling of both techniques is quite straightforward. Also, as a result of the possibility to use reversed phase, reversed phase ion pairing, ion exchange or size exclusion chromatography for separations, the potential of the coupling HPLC-ICP-MS is enormous [82] for the speciation of trace elements in biological materials. 5.1.1. Size exclusion

Speciation and metabolism of platinum in biological systems was investigated by Jakubowski et al [83]. Using size exclusion chromatography (SEC). They concluded that more than 90% of the Pt found in a grass sample was present in an inorganic fraction while the remaining 10% occurred in 4 different organic fractions. ~3C, 34S were also monitored at R=3000 in order to obtain some molecular information. A DF-ICP-MS instrument coupled to SEC has been applied by Wang et al [84] to investigate the identification of inorganic elements in proteins of human serum and DNA fragments. Metals able to bind biomolecules (Zn, Cu, Se, Cd, Pb, Th and U) were studied, using R=300 in the analysis. Cr, Mn and Fe required a R=3000 setting. In both cases, good limits of detection were achieved. For example, Th and U were determined in human serum reference material, at levels in the low pg.mL "~ in several fractions of molecular masses ranging from 10 to 630 kDa (Table 10). On the other hand, the interaction of Cr(VI) added to DNA fragments at ppb levels was also investigated. It could be shown that Cr binds as a cation in a specific DNA fragment during or after oxidation of the DNA, and just this oxidation process could possibly explain its carcinogenic behaviour. In a more recent paper, the distribution of various elements (V, Mo, Fe, Co, Mn and lanthanides) in human and bovine serum samples using SEC-DFICP-MS has been also studied by the same group [85]. Resolving power of 4000 was used to resolve spectral overlap interferences for 5IV (35C1160), 52Cr (4~ 55Mn (39K160 and 37C1180) and 56Fe (4~ Vanadium was found in two protein fractions with average molecular weights of 70 and 30 kDa. Like V, Mo was also found in two distinct protein fractions (100 and 30 kDa).

J.M. MARCHANTE-GAYON et al

160

a) lOooO

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Figure 15. Mass spectra of (a) 56Fe, 57Fe, (b) 63Cu,65Cuand (c) 64Zn, 67Zn for a human serum sample diluted 1+l 9 at nominal resolution settings of R-3000. [From ref. 78, with permission]

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161

Lanthanides are primarily bound to proteins in human serum in the mass range 70-90 kDa (probably albumin), and a fraction bound to small molecules. Alkali metals and TI are present primarily as free metal ions and are not bound to proteins. Two protein fractions containing Co and Cd was observed while Mn was observed in three protein fractions (440, 80 and 25 kDa) and also in the fraction containing small molecules. EDTA did not remove Fe, Pb, Sn or Th from the proteins but did extract Mn from some proteins studied.

5.1.2. Ion exchange Comparative chromatograms obtained by coupling a Fast Protein Liquid Chromatography (FPLC) column, using ammonium acetate buffer as the mobile phase, with Q-ICP-MS and DF-ICP-MS for AI speciation in an unspiked human serum [86] (below 5 ~tg.L"l) are shown in Figure 16. As Figure 16.a shows, the use of the quadrupole instrument proved to be inadequate in this particular application due to the AI spectral interference from 13C14N§ ions. Conversely, the coupling of the FPLC separation with the DF-ICP-MS detector solved the problem because that spectral interference is well separated at R=3000 (see Figure 9 and 16.b). In this way, only the coupling FPLC with DF-ICP-MS offered the possibility of AI speciation in basal human serum samples (less than 5 ~tg.L"1 total AI) for the first time [87]. Using the same approach, the speciation of Ca, Fe, Cu, Se, Sr, Sn, Zn, Cr, and Mn in uraemic and control human serum was undertaken in our laboratory [88]. Investigations in that line have been reported in a Conference by Riondato et al [89]. A mixture of 49 elements was given during 3 weeks to rabbits in order to establish, by DF-ICP-MS multielement analysis at R=300 and 3000, the bioavailability of the studied elements at a concentration far below their toxic levels. Speciation techniques as ultrafiltration and size exclusion, cation and anion exchange and inmunological affinity chromatographies were evaluated for multi-element protein binding speciation. 5.1.3. Selenium speciation The latest discoveries in selenium biochemistry [90] have increased the interest for the speciation of this element in biological materials. In this case, however, the performance of ICP-MS is restricted: the sensitivity for Se is comparatively low due to its high first ionisation potemial, and for all isotopes the determination suffers from interferences, isobaric as well as polyatomic [91 ]. Polyatomic interferences in general require high resolution (see Table 1). The isobaric interferences from Ge and Kr require extremely high resolution. However, Ge is usually not present at elevated concentrations in biological matrices and Kr as an impurity in the gas supply is present at only low levels.

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Table 10 Metal ions in proteins from human serum obtained by coupling SEC with DF-ICP-MS. Cu, Zn, Pb and Cd measured simultaneously. Also U and Th measured simultaneously [84] Element

Main peaks Minorpeaks Resolution Observations (kDa) (kDa) 200 to 20 600 300 Mainly bound to albumin (maximum at 75) (75 kDa) 200 to 20 600 300 Mainly bound to albumin (maximum at 75) (75 kDa) 200 to 20 <10 300 Mainlybound to albumin (75 kDa) (maximum at 75) and a little to erythrocytes 600 and 80 300 Not bound to metallothioneins (10 kDa) or metallothioneins bound to large proteins 80 10 300 80 630 and 20 300 10 140 3000 Possible bound to gluthatione 140 and 80 760 and 20 300 peroxidase (80 kDa) and selenoprotein P (75 kDa)

.

Cu Zn Pb Cd U Th Cr Se

.

.

.

.

.

.

Only the dimers of argon may be a real limitation, because the necessary resolving power of up to about 10000 is at the upper end of the accessible range of commercial DF-ICP-MS instruments. Operation of a DF-ICP-MS instrument at high mass resolution may be useful for reduction of the spectral background. (One should be aware, however, that this is always accompanied by an important loss of sensitivity). The sensitivity problem may be overcome to a certain extent by modem high efficiency sample introduction techniques, such as hydraulic high pressure nebulization (HHPN) [91,92] or alternatively by hydride generation [93]. These sample introduction techniques can be additionally used to reduce spectral interferences. Thus, the speciation of selenocystine, selenocystamine, selenomethionine and selenoethionine has been performed by Jakubowski et al. [92] by on-line coupling of reversed phase HPLC with DF-ICP-MS using HHPN for sample introduction. With this coupling, absolute detection limits below 5 pg were achieved and an improvement of 25-50 in the sensitivity can be achieved comparing with pneumatic (Meinhard) nebulisation.

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry 5000

163

'

(a)

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r~ "~

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0

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1000 0 0

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20

T i m e (m i n ) Figure 16. Comparative chromatograms obtained for AI speciation in an unspiked human serum by coupling FPLC column, using ammonium acetate buffer as the mobile phase, with: (a) quadrupole instrument (R=300) and (b) double focusing instrument, R-3000. [From ref. 86, with permission]

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The separation of selenium species (selenite, selenate, commercially available selenoaminoacids and trimethylselenonium) in human urine has been carried out in our laboratory by both reversed phase and vesicle mediated HPLC coupled on-line with DF-ICP-MS via conventional nebulisation and via an online microwave digestion-hydride generation interface (see Figure 17) [93]. This latter interface has important advantages over conventional nebulisation: (a) sensitivities 30-100 times better depending on the chromatographic system and (b) elimination of interference problems from the urine matrix or from the components of the mobile phase. Unfortunately, the hydride generation system increases substantially the background noise, observed using conventional nebulisation, so the limits of detection were only 2-10 times better depending again on the chromatographic system. Nevertheless, the use of a DF-ICP-MS instrument (Element) for selenium detection working at low resolution (R=300) enhanced the sensitivity 20-60 times, as compared with a quadrupole-based instrument (HP-4500) [93]. However, the limits of detection were only 1-9 times better than those with the quadrupole-based instrument due to serious background noise from polyatomic ions of Ar. This problem can be overcome by the use of hexapole collision and reaction cells instruments. With the use of hydrogen as reaction gas and helium as collision gas the 4~176 and 38Ar4~ polyatomic interferences with 8~ (the most abundance selenium isotope) and 78Se, respectively, could be eliminated. The relative limits of detection for some selenocompounds (selenite, selenate, selenocystine, selenomethionine and selenoethionine), previously separated by reversed phase HPLC and monitoring isotope 8~ were in the range 35-90 pg.mL 1 [94,95]. This means 20-35 times better detection limits (DL) than the best DL obtained by double focusing ICP-MS at R=300 and coupling microwave digestion-hydride generation at the exit of the chromatographic system [93]. Another application of the same microwave digestion-hydride generation interface was the separation of the D,L-Selenomethione enantiomers by HPLC on a ]3-Ciclodextrin column [96]. The DF-ICP-MS instnament (working at R=300) provided a sensitivity one order of magnitude higher than that of a quadrupole ICP-MS instrument but the limits of detection obtained with fluorimetric detection was at least one order of magnitude lower than those using the DF-ICP-MS instrument. This drawback is compensated for by the much higher selectivity of the atomic detector. The method was applied to a commercial "pure" L-selenomethionine and a natural selenium-enriched ("selenized") yeast used as a supplement in human nutrition [96]. Very recently, a most successful "chiral" speciation and determination of selenomethionine enantiomers in selenized yeast by HPLC-ICP-MS using a

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165

teicoplanin based chiral stationary phase has been reported [97] and would be useful with DF-ICP-MS detection.

Quadrupole

CONVENTIONALNEBULISATION HPLCpump " !

i""l i l C > c ~ ~ '

.,1

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~,

:,~:==3~~

phases

Double Focusing

~ HBr[~~ KBrOj

aste

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N,B~ "~,T,,I

Figure 17. Schematic diagram of the coupling MW-HG-DF-ICP-MS

5.1.4. DNA adducts quantification The main obstacle for the quantitative determination of adducts of DNA components is the impossibility of combining quantitative determinations and the elucidation of unknown structures. Known components can be determined precisely (using accelerator mass spectrometry) or newly formed unknown adducts detected (with post-labelling), but to detect and quantify unknown adducts without synthetic standards is not possible. With electrospray ionisation mass spectrometry (ESI-MS) only, the structures of unknown adducts could be elucidated, but again without pure synthetic standards quantitative results are difficult to obtain [98]. Only by combining both HPLC-ESI-MS and HPLC-ICPMS it is possible to elucidate the structure and determine the concentration of unknowns [99] and so of DNA adduct in the same sample [98]. The natm,ally common feature of all nucleotides, the phosphate group, can be employed for the purpose. The detection of phosphorus at mass 31 can be used in combination with HPLC separation of the DNA adducts using simply inorganic phosphate for quantification as the retention time for phosphate is different to that of the DNA adducts. In this context, the use of DF-ICP-MS is mandatory in order to avoid interferences from 15N160 and 14N16OIH by resorting to the use of 1500 as resolving power [98].

J.M. MARCHANTE-GAYONet al.

166

Table 11 Identification of organic solvents-induced interferences in ICP-MS.

Isotope

24Mg 25Mg 26Mg 27A1 3~p 39K 42Ca 44Ca 45Sc 5~ ' 5~ 51V 52Cr 53Cr 54Fe' 54Cr SSMn 56Fe 57Fe

A~COz

CxH~

CxHyNOz

CN CN, CHN

CH30 C3H3 C2H20

CO2 38Ar12C 4OAr12C 4~

5SNi' 5SFr

C4H2 C4H3 C4H5 C4H6 C4H7 C4H8 C4H9

59Co

6ONi 61Ni 62Ni 63Cu 64Zn 65Cu 66Zn 67Zn 68Zn 69Ga 7OZn 71Ga 72Ge 73Ge 74Se 75As 76Se' 76Ge 77Se 78Se 79Br 8OSe

CxnyOz

C2 12CI3C, C2H C2H2 C2H3

36Ar!2C160

4~

C5 CsH C5H2 C5H3 C5H4 C5H5 C5H6 C5H7 C5H8 C5H9

C6H C6H2 C6H3 C6H4 C6H5 C6H6 C6H7 C6H8

CHO2

C3HO C3H20 C3H30 C202, C3H40 C3H50 C3H60, C2H202 C3H70, C2H302 C3H402

C4HO C4H20 C4H30 C4840, C302 C4H50, C3HO2 C4H60, C3H202 C4H70, C3H302 C4H80, C3I"I402 C3H502

CsHO C5H20 C5H30 C5H40

C2NO C2H2NO

Double Focusing-InductivelyCoupled Plasma-Mass Spectrometry

S~Br 82Se 85Rb 86Sr

C6H9 C6Hlo

167

C5H50 C5H60, C4H202 C4H502 C4H602,C3H203

5.1.5. Organic solvents-induced interferences DF-ICP-MS is an effective means to elucidate the influence of organic solvents, used as mobile phases in HPLC, in ICP-MS analysis Using higher resolutions the analytical determinations derived from low resolution measurements can be checked for accuracy. In Table 11 most of the polyatomic interferences observed and identified by precise mass measurement are compiled [91]. All of them may impede application of reversed phase chromatography or introduce problems for the direct investigation of organic matrices with quadrupole-based instrumentation. However, most of these interferences could be overcome by using higher mass resolution settings in a DF-ICP-MS instrument. In the mass interval 24-86 amu the possible appearing polyatomic ions in the presence of organic solvents can be sub-divided into four groups: (i) ArCOz with z=0 or 1, (ii) CxHy with x = 1-6 and y--0-10, (iii) CxHyOz with x = 1 or 2, y=08 and z=l-3 and (iv) CxNHyOz with x =1 or 2, y=0-2 and z-0 or 1. As it can be seen, these interferences continuously cover the whole interval 24-86 amu, and some of them may appear even above 100 amu. Their distribution depends strongly on the operation conditions chosen, such as generator power, nebuliser flow rate, sample uptake rate and organic solvent concentration, and also on the instrument itself, the sample introduction system used and its operational conditions [91 ].

5.2. Gas chromatography (GC) In comparison with HPLC-ICP-MS, GC-ICP-MS offers a higher chromatographic resolving power and 100% sample introduction efficiency (higher sensitivities); it allows a more stable plasma and gives origin to fewer spectral interferences as a result of the plasma being dry; moreover, this coupling leads to less sampling cone and skimmer wear. Of course, GC-ICP-MS can only be used for the separation and detection of volatile and thermally stable compounds or compounds that can be derivatised into a volatile form. Unfortunately, no applications of its use with DF-ICP-MS have been reported so far. Also the coupling GC-ICP-MS is somewhat more complicated as a heated transfer line is usually required, such that condensation of the species and, hence, peak broadening can be avoided. In the last years a new interface for the coupling of GC to the ICP-MS has been described [ 100] which does not require

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the heating of the transfer tube between the gas chromatograph and the ICP-MS simplifying the coupling and decoupling of both instruments [100]. The coupling of GC to a DF-ICP-MS instnnnent is under way in our laboratory for the speciation of sulphur compounds in saliva and its relation to bad breath. For example, Figure 18 shows the separation of 8 volatile sulphur compounds by GC as detected by DF-ICP-MS at mass 32 at R=3000 to eliminate the interference from 1602+. In this case the injection was 1 ml of air containing about 200 ng of each compound. In the same figure the chromatogram obtained for a sample of saliva fermented anaerobically for 24 hours is presented. As can be observed, many sulphur compounds can be SH 2

MeSH EtSH

MeS-SMe I MeSMe MeSEt

EtSE t

l

.1 Standard

Sample

Figure 18. Separation by GC and detection of volatile sulphur compounds at mass 32 using DF-ICP-MS at resolution 3000.

Double Focusing-Inductively Coupled Plasma-Mass Spectrometry

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detected in the real sample and some of them can be readily identified by their retention time. Unfortunately, many other compounds remain unidentified so far. This is an unexploited area worth of future research.

5.3. Capillary Electrophoresis (CE) In principle, the field of application of CE is similar to that of HPLC, but CE has several advantages such us higher efficiency, small sample volume requirement, shorter analysis time, minimal buffer consumption and higher sample throughput. In addition, CE is especially suitable for the separation of biological macromolecules, e.g. proteins. The major drawback in the application of CE to the speciation of real samples is the small sample volume (up to 50 nL) which requires a very sensitive detector to match the (low) naturally occurring analyte concentration levels. The critical need for sensitivity of the specific detector coupled for speciation points to DF-ICP-MS, working at low resolution, as the preferred choice [101 ]. The key to the successful realisation of a coupled system for trace metal speciation is the design of the interface. This is especially true for CE-ICP-MS [102]. Whereas HPCL-ICP-MS coupling is relatively easy to achieve (flow rates in HPLC fit well with the nebulisation flow rate in ICP-MS) for a CE-ICP-MS interface we have to provide (a) an electrical connection; (b) to adapt the flow rate in the capillary with the flow rate of the nebuliser, to prevent suction effects in the nebuliser ruining the separation and (c) preserve a good sample transport (for sensitivity). The analytical characterisation of a CE-DF-ICP-MS system for the separation and determination of three chemical species of Hg (CH3CH2Hg +, CH3Hg+ and Hg 2+) has been recently applied in our laboratory [103]. The three Hg compounds were separated as mercury-cysteine complexes by CE at 20 kV using a 20 mM sodium tetrabomte buffer (pH--9.3). In this interface, the capillary was inserted through a T-piece into the Meinhard nebuliser and held in place, after optimisation of its position, with a ferrule fitting. Grounding of the capillary was achieved by the use of a coaxial '~nake-up liquid" flow which was mixed with the CE effluent prior to nebulisation. This solution was pumped through the vertical arm of the T-piece via a teflon tube using a peristaltic pump. To complete the electrical circuit, a cathodic connection was made through the same ann of the T-piece by placing a platinum wire between the teflon tubing and the ferrule[ 104]. The self-aspirating nature of the commercial concentric nebulisers induce a laminar flow (suction) in the capillary which can ruin the performance of the CE separation. Thus, the aspiration rate in the nebuliser, strongly influenced by the nebuliser gas flow rate, is a critical parameter. The optimum nebuliser gas

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flow rate will depend upon a combination of its effect on the electrophoretic migration time and resolution and on the ICP-MS signal intensity. If the nebuliser gas flow is too low, the generation and transport efficiency of a f'me aerosol into the ICP will be compromised. The concentration detection limits observed are compiled in Table 12 [103]. From this Table, it is apparent that the detection limits obtained using conventional UV absorption are significantly higher than using ICP-MS detection. Moreover, as expected, the use of a DF-ICP-MS detector (working at resolution 300) gave improved detection limits in all cases as compared with quadrupole-based ICP-MS intruments. Table 12 also shows that the "sample stacking injection" [103] can be used to improve the detectability in the CE separation and f'mal determination of mercury compounds. Much more work is needed to render CE-ICP-MS techniques a real competitor in trace element speciation of HPLC-ICP-MS. Table 12 Detection limits (~tg/L) for CE-UV, CE-Q-ICP-MS and CE-DF-ICP-MS for mercury species[ 103] Method CE-U:v (injection volume = 0.163 ~tL) CE-Q-ICP-MS (injection volume = 0.350 ~tL) CE-DF-ICP-MS (injection volume = 0.450 laL) SS-CE-DF-ICP-MS (injection volume of 3 ~tL with sample stacking)

Hg2§ 500 81 25 4

CH3Hg+ CH3CH2Hg+ 680 750 128 275 54 84 7

5.4. Off-line strategies The capability of flatbed electrophoresis combined with DF-ICP-MS has been reported by Comelis et al [105]. Rabbit serum samples were separated by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Rabbits received intraperitoneal injections of Ga (III) and In (III) in physiological buffer. Each rabbit serum sample was rtm twofold and, aider separation, one gel was silver stained (in order to detect and identify serum proteins) and the other replicate was cut into segments, which were digested with aqua regia and measured for Ga and In at R=7500 (in order to avoid spectral interferences). Using such strategy, it was observed that both elements are bound exclusively to transferrin. Similar strategy has been used by the same group [106,107] in order to develop native two-dimensional electrophoresis methods for the separation and detection of platinum carrying serum proteins. In the first dimension IEF was

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performed using immobilised pH gradients (IPGs), while PAGE was done in the second dimension. Detection of proteins was achieved again via silverstaining. For the DF-ICP-MS determination of platinum, one gel was cut into small pieces and the element extracted with aqua regia. 5.5. Future of DF-ICP-MS for speciation It should be pointed out that the main intrinsic disadvantage of DF-ICPMS for speciation of biological materials is, as for other elemental detectors, its atomic character which prevents any molecular information to be obtained. Its coupling with a powerful separation technique (chromatography or capillary electrophoresis) alleviates this limitation via hybrid techniques. However, identification of the metal binding biomolecules using such techniques, in real samples relies only in the observed retention time. Such parameter is not enough because it depends on the chromatographic conditions and, what is more, an adequate standard (appearing at the same retention time in the same conditions) should be available for final identification of a given biometallic compound in the samples. While this is not usually a difficult problem in trace element speciation of organometallic compounds in the environment (because we know virtually all metal species concerned) it can be a terrible headache in biological systems where the possible metal-biomolecules forms are unknown [93]. In the organisms, the sought metal or semimetal has been usually integrated in the biological material by the living organism, transformed into often tmknown compounds and so buried into a very complex matrix. Awareness of the need for complementary techniques providing matching results [82] for such problems is important at this stage. We are needing, more and more, reliable tools for identification and confirmation of metal-biocompounds before going into the mandatory last step of quantification of the sought species in a given biological sample. For identification purposes, the use of retention times provided by a single chromatography-detector system is not enough; different-principle-based separations with different detectors for the same problem should give matching results [87] and, therefore, could constitute a helpful approach to solve the speciation problem. Confirmation techniques and methods are eventually required to be sure that the separated species are the compounds we expected (and just a pure compound). Isolation, purification, preconcentration, etc. of the unknown "pure" species is advisable before final characterisation and confirmation of its nature/purity by molecular techniques (preferably common organic mass spectrometry tools, NMR etc.).

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It is opportune in this paper to point out that "high resolution" achieved in the DF-ICP-MS instrument refers only to isotopic masses considered (it is atomic). Of course, in speciation different molecular species of the same element not separated adequately in the chromatographic column will coelute and our "high resolution" setting in the atomic detector will be useless. All the above considerations matter in the search for quantification strategies and final validation of new speciation methods developed. Adequate quantification of the species requires first a "neat" compound separation, then a sensitive and element-specific detection and finally a validation of the determination method developed. In the search for validating strategies we can resort to the use of Certified Reference Material (CRMs) for a given species. These "speciated" CRMs are unfortunately difficult to produce [108] and scarce today. An alternative strategy for validation is the use of alternative well established ("reference") methods producing statistically identical results. It seems that this second approach is more flexible and can be advantageously used in many laboratories lacking the CRMs for validation. In this vein, we believe that such highly qualified "reference" methods for speciation results validation could be provided by Isotope Dilution ICP-MS, considered as definitive or "primary" measurement methods [109]. The application of postcolumn isotope enriched spikes or of synthesised isotope enriched species, as described by Heumann [110], are approaches of a great potential for accurate quantification of metalbiomolecules in biological systems [82]. In this vein, the ability for high precision isotope ratio measurements afforded by DF-ICP-MS along with its extreme sensitivity at R=300 warrant a brilliant future for this detector, particularly in trace element speciation of biological materials. REFERENCES ~

.

.

,

.

0

.

A. Montaser (ed.), Inductively Coupled Plasma Mass Spectrometry, Wiley-VCH, New York, USA, 1998. N. Jakubowski, L. Moens and F. Vanhaecke, Spectrochim. Acta Part B, 53 (1998) 1739. N.M. Reed, R.O. Cairns, R.C. HuRon and Y. Takaku, J. Anal. At. Spectrom., 9 (1994) 881. L. Moens and N. Jakubowski, Analytical Chemistry News & Features, 1 (1998)251. A. Sanz-Medel, J.I. Garcia Alonso and J.M. Marchante-Gay6n, Anales de Quimica, 94 (1998) 149. J. Sabine Becker and H.J. Dietze, J. Anal. Atom. Spectrom., 12 (1997) 881. J. S. Becker and H.J. Dietze, Spectrochim. Acta Part B, 53 (1998) 1475.

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9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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Chapter4

Field-flow fractionation-inductively spectrometry

coupled

plasma-mass

Ramon M. Barnes, and Atitaya Siripinyanond Department of Chemistry, Lederle Graduate Research Towers, University of Massachusetts, 710 N. Pleasant Street, Amherst, Massachusetts 01003-9336, USA, and University Research Institute for Analytical Chemistry, 85 N. Whitney Street, Amherst, Massachuetts 01002-1869 USA I. INTRODUCTION In the elemental analysis of biological, biomedical, environmental, geological and other natural materials, three key objectives are often sought: (1) identification and quantification of elemental concentrations, (2) identification and quantification of compounds containing metals, and (3) evaluation of bioavailability and health effects, mobility, transport, fate assessment, and toxicity [1,2]. Nowadays, information on elemental concentration is insufficient for these objectives owing to the increasing knowledge of metabolism and biological effects of elements. The determination of chemical species is essential for characterizing the biogeochemical cycle and contaminant transport in terrestrial and aquatic ecosystems, and assessing their risk to biota and humans [3]. The primary goals in many elemental analysis of biological and environmental substances are identification and quantification of their chemical forms. "Elemental speciation" underpins elemental bioavailability, in vivo absorption and distribution, mobility and toxicity studies. Over the past 2 decades, various definitions of speciation have been proposed. This ambiguity was officially validated by the International Union for Pure and Applied Chemistry (IUPAC) definition which denotes speciation as "the process yielding evidence of the atomic and molecular form of an analyte" [4]. The term "speciation" has been used in different ways, however. Only recently, three IUPAC Divisions (represented by the Commission on Microchemical Techniques and Trace Analysis, the Commission on Fundamental Environmental Chemistry, and the Commission on Toxicology) attempted to provide clear definitions of concepts related to speciation of elements. Speciation of an element has been defined as "distribution of an element

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amongst defined chemical species in a system", whereas ffactionation has been defined as "process of classification of an analyte or a group of analytes from a certain sample according to physical (e.g., size, solubility) or chemical (e.g., bonding, reactivity) properties [5]. Challenges and trends of analytical speciation in biological, environmental, and other natural systems have been surveyed [4,6-12]. Elemental speciation is otten achieved by combining two complementary techniques since a single one is often insufficiently selective or sensitive. One provides an efficient and reliable separation procedure, whereas the other provides adequate detection and quantification. Ideally, technique coupling requires sample introduction compatibility. Minor instrumental modifications with maximum interface efficiency and response of each technique is desirable. In addition, real-time data acquisition for the separation process is expected. Separations include both chromatographic and nonchromatographic techniques. In elemental analysis, inductively coupled plasma (ICP), microwave induced plasma (MIP), and glow discharge (GD) sourceatomic emission and mass spectrometry (ICP-AES, ICP-MS, MIP-AES, MIPMS, GD-AES, GD-MS) are of primary importance, owing to their multielement detection capabilities [13,14]. On-line coupling between separation procedures and ICPs have been assessed and occasionally reviewed [15-18]. Commonly, the analytical technique selection criteria used for elemental speciation depend primarily on the study objective. For most biomedical and environmemal samples, reversed-phase (RP), ion-exchange (IE), size exclusion chromatography (SEC), and electrophoresis are preferred liquid separation methods. The three former are chromatographic techniques that typically provide moderate resolution. Became their mobile phase flow rates are wellsuited to ICP-MS sample introduction flow rate (e.g., ~0.1-2 ml min-~), coupling between these chromatographic techniques with ICP-MS is physically straightforward. The methods are subject to artifacts, however [2]. In contrast, capillary electrophoresis (CE) is a popular high-resolution, nonchromatographic technique for analytical separation [19]. Despite its high resolution, CE operates at low flow rates (e.g., nl min'~), which sometimes leads to unstable ICP-MS operation. An appropriate interface design is required to provide an electrical connection, to adapt the electro-osmotic flow rate in CE to the sample introduction flow rate of ICP-MS by using a make-up solvent and to prevent a suction effect between the nebulizer and the CE capillary [20]. The combined CE-ICP-MS technique has been spearheaded by Tomlinson et al. [21], Olesik et al. [22], and Lu et al. [23]. Following these three pioneered articles, CE-ICP-MS applications have developed. Other interface configurations have been evaluated [24] and commercial interfaces and nebulizers are now available [25] (Meinhard | SB-30-A3 and DIHEN nebulizer

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for CE (J.E. Meinhard Associates, Inc., Santa Ana, CA, USA),Microneb 2000 and MCN 1000 microconcentric nebulizers (CETAC Technologies, Omaha, NE, USA), and a MicroMist AR30-1-F02 nebulizer (Glass Expansion Pty Ltd., Australia) ). A modified microconcentric nebulizer MCN 100 developed by Prange and SchaumlOffel was reported and commercialized recently [20,26]. Majidi et al. demonstrated an approach to improve analytical sensitivity and detection limits using multicapillary parallel separation interfacing to ICP-MS [27]. The multicapillary comprises several individual capillaries with identical internal diameters and lengths placed in a loose bundle. An increase in signal intensities proportional to the number of capillaries was observed. Instnarnentation development and applications have been reported continuously [28-34]. For interested readers a series of contemporary papers by Michalke et al. is recommended [35-37]. In their on-line CE-ICP-MS, a laboratory modified commercial concentric glass nebulizer initially employed with a cyclonic spray chamber was fabricated [35-37]. However, as with SEC, artifacts from instrumental, column or packing materials, in CE might limit trace and ultra-trace metal quantitative analysis for complex polymer systems, colloids or biomolecules. Therefore, as predicted by Barnes [1,2] an alternative separation approach, like field-flow l~actionation (FFF), for elemental speciation in biomedical, biogeochemical, and natural samples might provide less artifactdependent separations. Field-flow fractionation is a non-chromatographic elution-based separation technique that is capable of separating and characterizing materials in the macromolecular and colloidal size range and larger. Since ICP-MS is a rapid and sensitive elemental detection technique, the combination of FFF with ICP-MS should provide detailed elemental characterization of macromolecules and particles. Field-flow fractionation-ICP-MS was first reported in 1991 [38], and a number of publications has appeared since [39-42]. In these papers natural suspended particulate matter, clay minerals, and soil were analyzed. In contrast, many FFF applications with conventional detectors (i.e., spectrophotometry, light scattering, etc.) have examined biological macromolecules [43-49]. Now FFF is a demonstrated practical on-line separation tool for ICP-MS elemental detection. However, FFF-ICP-MS for biomedical research and clinical analyses is still in its infancy and unexplored. Only a single report demonstrating the feasibility of linking F1FFF with ICPMS for analysis of several protein standards has appeared [50]. As FFF provides gentle separation with delicate and shear-labile species with minimal loss of biological activity, FFF-ICP-MS should give unique separations and characterization of natural biopolymers (e.g., humic acid, hyaluronic acid). Therefore, the FFF-ICP-MS technique is worth examining as a complementary

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tool for elemental speciation. Considering the increasing number of publications and presentations made at several international conferences, the exciting FFF-ICP-MS journey has begun. In this chapter, the basic FFF principles and a general overview of FFF techniques are summarized. Different FFF modes are described briefly. Selected applications to environmental and biological samples are reviewed, and specific FFF-ICP-MS applications are highlighted. Recent experimental results obtained from our research laboratory are presented as possible applications of the technique for biological and environmental analysis. A forecast of the future direction of FFF-ICP-MS is also presented. 2. GENERAL OVERVIEW

The FFF concept was first envisioned and documented in 1966 as a threepage short communication by the late Professor J. Calvin Giddings[51]. The FFF idea emerged late in 1965 during his vacation in Wyoming, where he spent a night in a motel in Evanston, a cowboy town [52]. A banging radiator kept him awake, so that he started thinking about the diffusion problem [53]. He imagined using some kind of force field to restrain a mixture within a narrow layer while differential diffusion acted to allow some species to escape further from the layer than others [52]. Thermal diffusion was the first appropriate field that came to mind. Then, the idea was first implemented experimentally at the University of Utah, Chemistry Department. For the interested readers brief accounts of the early FFF history are given by Giddings et al. in a worthwhile paper, published in 1981 [52]. Field-flow fractionation is a versatile technique capable of separating and characterizing materials with an application range from less than 103 daltons (Da or g mol l) to particle diameters of 100 pan [53]. Similar to chromatography, FFF is an elution-based technique. The fractionated components can be detected on-line and/or collected off-line. Unlike chromatography, however, FFF is achieved in a thin ribbon-like, open channel with no stationary or liquid phase. By avoiding a stationary phase, retention is not induced by a distribution between two or more phases, and FFF is not a chromatographic method [54]. The essence of FFF is to apply a force perpendicular to the channel flow stream (Figure 1). This force drives molecules toward an accumulation wall where they encounter slow-moving laminar fluid streamlines. Different size sample components are driven different average positions in the channel flow, causing them to migrate at a characteristic rate. Thus each component leaves the channel at a different time related to its molecular weight. Fundamentally, these primary driving forces

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can be generated by a number of fields or gradients. The field is applied to compress sample components against an accumulation wall while the channel flow is stopped temporarily, immediately after sample is introduced. This results in numerous FFF sub-techniques that are generally applicable to different materials (Table 1). Table 1 Applicable range of particle diameters by FFF techniques FFFsubtechnique Sedimentation Thermal Flow Electrical

Applicable particle size range (nm) 100-100,000 2-100 1-100,000 40-1,000

Applicable MW range ..(g mo1-1, Da). 106-10 ~5 104-10s 103-1015 102-106

Four retarding fields have been widely studied. The most common is sedimentation [55], usually generated in a centrifugal force but sometimes by gravity [56]. The centrifugal or gravity force acts perpendicularly to the flowseparation axis. This sub-technique is known as "sedimentation FFF (SdFFF)". Other important fields include a temperature gradient (thermal FFF, ThFFF), in which thermal diffusion is the separation driving force [57]; electrical field (electrical FFF, EIFFF), in which force depends on particle charge [58]; and a cross-flow stream of carder liquid (flow FFF, F1FFF) [59], in which the force originates in the friction of the cross-flow stream moving across the components. Among them, F1FFF is most universal, because of its wide applicable range, as illustrated in Table 1. Broad range of FFF applications in the Giddings' research group are listed in Table 2 to illustrate the scope. Since the first paper appeared in 1966, published results have continually grown (Figure 2). Thus far, eight international FFF symposia have been held. The most recent one was held in 1999 in Paris, France. The next will be held in Golden, Colorado, June 2001. The general principle of FFF techniques is given in many reviews [43,61-69] and textbooks [70-72]. Several useful FFF related web sites are also available. Among them, the FFF webspace "http://dns.unife.it/~rskf' maintained by Pierluigi Reschiglian is very web page "http://www.rohmhaas. com/fl~' by Mark R. Schure at Rohm & Haas, where all the publications of FFF and related techniques are compiled and listed for

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Channel inflow

Outflow ,

AppliedS

side view

o"

... \

Parabolic flow profile Flow r----

.s

.%

/

Inflow and sample injection

Outflow (to detector)

_.

top view Z

Y

X

Figure 1. Principle o f FFF separation X are the smallest sample particles, having highest diffusion coefficient Y are the moderate size sample particles, having moderate diffusion coefficient Z are the largest sample particles, having lowest diffusion coefficient ly is a mean sample layer thickness of distance of a cloud of particles Y Briefly, separations are performed within a flat open channel with a rectangular cross-section and triangular end pieces where the sample and cartier liquid enters and leaves. Fractionation is achieved by the balance between the field force applied on sample particles toward the accumulation wall and the diffusivity of sample particles acting against the field force. The field force pushes X, Y and Z into thin clouds or layers of different mean thickness. Owing to the highest diffusivity of sample X, a mean sample layer of particles X is thicker than that of Y and Z. Therefore, a cloud of particles X meets the higher speed region of parabolic profile, thus particles X elute earlier than particles Y and Z.

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185

each year, is convenient. A discussion forum "the FFF mailing list" maintained by Frank von der Kammer, is where questions and announcements related to FFF are posted. To register, a requested message can be submitted to "[email protected]. de". Table 2 Some particles characterized by FFF methods in the FFF Research Center (FFFRC, University of Utah)and FFFractionation Laboratories (listed in 1990,

[60]) Category Samples Samples Polystyrene Inorganic Selenium Polyvinylchloride Nickel Polybutadiene Glass Polyurethane Silica Hematite Polymethylmethacrylate Styrene-butadiene CLay Grafted polybutadiene-PMMA Limestone Vinyltoluene t-butadiene Biological Red blood cells White blood cells Epoxy-acrylic resin Alkyl resin Human lens particles Teflon Albumin particles Emulsions Soybeanoil Casein particles Safflower oil Viruses Perfluorocarbon Pollen grains Liquid crystal Mitochondria Liposomes Lysozomes Environmental Fly ash Coal liquefaction residue Ground coal Waterborne colloids Reprinted from [60] with kind permission of the American Chemical Society.

Category Polymer

Presentations made in 1996 to 1999 from the University of Ferrara (Italy) research group are available to download on line. The web site "http://www.wsc.monash.edu.au/t~' of Ronald Beckett at the Water Studies Center, University of Monash in Australia also is instructive. Only a few FFF manufacturers exist with many distributors worldwide. These manufacturers include (1) FFFractionation, Salt Lake City, Utah [http ://www. fffract, com] (2) Consenxus, Ober-Hilbersheim, Germany

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BARNES and SIRIPINYANOND

[http://www.consenxus.de], and (3) Postnova Analytics, Munich, Germany [http://www.postnova.com/ueberuns/index.htm].

.~ ..~ =u = t. o -=

110 100 90 80 70 60 50 40 30

f ra U T A H

[] not U T A H I

20 =

10 0 ~

t -~

t ~-

11 .o00=0=H0 tHII

~--

t ~-

t~

O0

O0

O0

O0

O0

~

~

~

~

~

Year

Figure 2. Number of FFF papers. Source: Field-Flow Fractionation web site updated on August 22, 2000 and Web of Science updated on September 12, 2000. Note: Number of FFF publications are striking in 1997. This is probably due to many special issues in scientific journals dedicated to the death of Professor d. Calvin Giddings in 1996. 2.1 F F F M o d e s

For a given field type three basic operation forms arising from different kinds of opposing forces extend versatility and FFF variety. These are normal, steric [73], and liit-hypedayer FFF [74,75] (Figure 3). In normal mode FFF, the center of gravity of the solute zone lies very near the wall, usually extending only a few micrometers from the wall. As the 2-dimensional flow profile is parabolic, flow velocities vary fi'om zero at the walls to a maximum in the channel center. For parabolic flow the velocity at the channel center is twice the average flow velocity. The smaller particle zones extend a greater distance, compared to the bigger particles, into the channel owing to their weaker field interaction and higher diffusivity. Consequently, the smaller particles elute earlier than the bigger ones. However, as particle size increases to a critical value, where the particle cannot intercept flow lines closer to the wall than its radius, diffusion from the wall is strongly suppressed, and it no longer plays a major role in retention. This operation mode is called "steric FFF" [73]. Larger particles occupy an elevated position from the accumulation wall than the smaller counterparts. Therefore, the large particles are swept downstream and leave the channel more rapidly, resulting in elution order trend opposite to that observed for the normal FFF. As the average velocity of the carder in the

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channel increases, particles that were in the steric region begin to experience hydrodynamic lift forces that drive them away from the position at or near the wall. This operation mode is called "hyperlayer FFF", in which the particles gain a significant elevation above the wall where they form hyperlayers [72]. The word "hyperlayer" describes particles forming thin layers above the accumulation wall, where "hyper" signifies over or above [72]. The hyperlayer mode elution trend is similar to the steric mode. A. Normal FFF |

Ill

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Figure 3. Schematic representation of the three major modes (or mechanisms) for FFF separations: (a) normal, (b) steric, and (c) hyperlayer. R is retention ratio, I is mean cloud thickness, Wis channel thickness y is an empirical correction factor which takes into account several non-ideal phenomena a is the particle radius (assumingthat a >>/) Xeq is a distance of a cloud of particles. It depends on the balance of the force generated by the external field and the imperfectly characterized hydrodynamic lift force.

2.2 F F F Sub-techniques

Any field or gradiem capable of providing differential displacement can be used in FFF. Each field impresses its unique selectivity dependence on particle

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BARNES and SIRIPINYANOND

properties. Relationships between force and physicochemical properties, as summarized by Giddings [76], are indicated in Table 3. Instnunental setup and principles o f each sub-technique are described in this section. Table 3 Force equations and physicochemical properties

Force equations J Sedimentafio nFFF (SdFFF) Force = m'G = m(Ap/pp)G = VpApG d~ /6)ApG Thermal FFF (ThFFF) force = DTf(dT/dx) = a k(dT/dx) = kT(Dr/D)(dT/dx)

Electrical FFF (EIFFF) force = qE

=0rE

Properties measurable from FFF retention ,

m' = m = Vp = d =

effective mass (g), G = acceleration mass, pp density, Ap = density difference (gcm -3) volume (cm3) equivalent volume spherical diameter (cm) =

DT = thermal diffusion coefficient, f = friction coefficient ~t = thermal diffusion factor, dT/dx = temperature gradient D = ordinary diffusion coefficient, T = temperature (K) k = Boltzmann constant (1.38 x 1016 gcm2 s'2Kq)

q = effective charge, E = electrical field intensity la = electrophoretic mobility, f = friction coefficient

Flow FFF (FIFFF) force = fU f = friction coefficient, U = cross-flow velocity (cm sq) = 3~ rldhU dh = hydrodynamic diameter,rI = cartier viscosity (g crn"lsq) = (kT/D)U D = ordinary diffusion coefficient (cm2 s-1) Reprinted from [76] with kind permission of the American Chemical Society

2,2,1 Sedimentation FFF (SdFFF) Sedimentation FFF is the most common FFF technique. In principle separation is caused by using either gravitational or centrifugal field forces on the particles suspended in a carder liquid (Figure 4). The gravitational or centrifugal force causes sedimentation o f the separated colloidal sample components according to the product o f their effective volume and density difference between the suspended particles and carder liquid. Like other FFF techniques, separations are performed within a flat open channel with a rectangular cross-section and triangular end pieces where the sample and carder liquid enters and leaves (Figure 1). Sedimentation FFF was first envisioned by Giddings [51] and was fn~t theoretically proposed [77] and experimentally verified by Berg et al. in 1967 [78]. A centrifugal device that bypassed the need

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Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry

for complicated rotor seals was employed. The channel for SdFFF is usually wraped around the inside circumference of a centrifuge rotor basket. Special seals are used to close the inlet from the outlet streams and thus to prevent leakage. The channel assembly can therefore be spun at different rotation rates to control retention in the fractionation system [79]. The separation time sequence of SdFFF is illustrated in Figure 4. The retention parameter depends on the effective mass of the fractionated species and operating parameters. Despite its high resolution, the centrifugal force is too weak to induce retention of small particles (less than 10 to 30 nm). At the highest spin rates available (2500 rpm) some retention begins to occur at a molecular weight of about 106 g mol 1. Beyond this transition value, SdFFF becomes a highly selective technique. Determination of size and density of the separated particles is possible by performing the fractionations in carrier liquids of various densities.

A)

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Figure 4. Schematics of SdFFF A) principle of SdFFF Three particle sizes are shown: smaller particles (X), bigger particles (Y), and floating particle (Z) with a density smaller than that of the solute. (Reproduced from [43] with kind permission of the American Association for the Advancement of Science) B) time sequence of separation using SdFFF (or SFFF): a) sample injection and relaxation before flow; b) sample separation by flow; c) elution of smaller particles; d) elution of larger particles. (Reproduced from [80] with kind permission of the American Association for the Advancement of Science)

2.2. 2 Thermal FFF (ThFFF) In the FFF family ThFFF was the first techniqueimplemented [81,82]. A temperature gradient is the separation driving force in ThFFF (Figure 5). A thin ribbon-like channel is slotted in between two metallic blocks with high heat conductivity such as highly polished copper bars with coated nickel or chromium surfaces (Figure 5). Generally, the copper bars act as the heat transfer source to maintain the temperature gradient between the top (hot) and the bottom *(cold) walls. Several holes are drilled in both copper block sides,

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where thermistors or thermocouples are located to control and regulate the block temperatures and the temperature gradient between the two main channel walls. Electrical cartridge heaters and carder stream inlet and outlet tubes are positioned in the top bar. The lower or cold wall is cooled by flowing a coolant through a heat exchanger unit. A channel is properly cut from a spacer comprising a low thermal conductive material (Mylar| or Teflon| and is placed between the metallic blocks. The tThFFF channel is represemed in Figure 5. Because of thermal diffusion, the separated sample componems migrate toward the cold wall. This well-known phenomenon is called the Ludwig-Soret effect. In IhFFF, unlike other FFF methods, the flow profile is perturbed by the change in solvent viscosity with position in the channel owing to the temperature variation across the channel. Theoretically, ThFFF is the most complicated FFF technique, because of the numerous assumptions and approximations. In practice, retention is linearly related to the temperature difference between the cold and hot walls. This temperature gradiem pushes particles or macromolecules toward the accumulation (cold) wall. Generally, larger particles are driven closer to the accumulation wall than the smaller species. Experimemally, the retention yields the Soret coefficiem, which is the ratio between thermal diffusion and diffusion coefficients. Therefore, thermal diffusion coetticiem can be calculated when the normal diffusion coefficient is determine experimentally one parameter when the other is known independently.

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Figure 5. ThFFF A) channel arrangement (Reproduced from [83] with kind permission of the Wiley) B) basic principle (Reproduced from [84] with kind permission of the Dekker) 11is a mean layerthicknessof low molecularweightfraction 12is a mean layerthicknessof high molecularweightfraction 2,2. 3 Electrical FFF (EIFFF) Although the system setup is seemingly simple (Figure 6), E1FFF is very difficult to implement in practice. Experimental difficulties are encountered, leading to less development of this than the other FFF techniques. Electrical FFF uses an electric field to establish a potential drop across the channel to generate a lateral flux of charged macromolecules or particles [85]. Semipermeable membranes are usually placed inside the channel to allow small ions to flow from the channel. According to Caldwell, one important consideration with early EIFFF channel construction was a gradual droop at the membrane walls [51]. Additionally, small fluctuations in the channel flow cause a wall deformation, which affected both retention and zone spreading [86]. A system was redesigned with membranes cast directly onto a porous rigid polymeric flit support [87]. This design provided a stable channel geometry, yet the electrical resistance was too high causing undesirable heat effects. Later an experimental setup was constructed (Figure 6) in which main channel parts comprise two Plexiglass blocks with chambers that enable flow through buffer solution. Two semipermeable flexible membranes were placed inside the channel to allow small ions passage and separate the channel volume from electrode compartments in the blocks. The two membranes are separated by the channel spacer material. The entire system is clamped and bolted together.

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In the previous design a spacer that determines the channel thickness was 0.356 mm thick [88]. Platinum wire electrodes were placed above and below the spacer and were positioned 51 mm apart. Owing to the thinness of the channel compared to the spacing between the two electrodes, only about 0.356/51 or 0.7% of the applied potential was used for the separation. Later a design was constructed by Caldwell and Gao [89] in which two graphite plates served the dual role of electrical field source and channel wall. A Teflon | spacer is inserted between these plates. Since the channel walls are made of graphite, an upper limit to the practical separation fields exists owing to the electrolytic breakdown of water. Despite this limitation, the voltage generated from a very narrow gap is very high. Typically, the EIFFF channel dimensions resemble those of F1FFF. In E1FFF an external electric field is applied between the two channel walls to force charged analytes to migrate toward the wall of opposite charge. Electrical FFF exploits the differences in the electrophoretic mobility of particles to separate them. This electrophoretic mobility fundamentally depends on particle size, shape, surface charge density, and solution ionic strength. Usually, the apparent retention time is used to determine experimentally one parameter when the other is known independently

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Polarity

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Figure 6. EIFFF A) instrumental setup (Reproduced from [83] with kind permission of the American Association for the Advancement of Science) B) separation principle (Reproduced from [90] with kind permission of the Elsevier) l is a mean layer thickness of particle clouds A is big particles with high charge density B is small particles with low charge density E is field strength

2.2. 4 Flow FFF (FIFFF) Similar to other FFF techniques, F1FFF separation is induced by an external flow field perpendicular to the separation axis (Figure 7). The field subsequently causes components to migrate to the accumulation wall. The physical fluid cross-flow drives all entrained particles and molecules toward the accumulation wall. This makes F1FFF an almost universal FFF technique [91]. Owing to its universality, F1FFF has been exploited as the separation method combined with ICP-MS for elemental speciation. Therefore, mathematical equations related to fi'actionation using F1FFF are given below. Flow FFF theory has been extensively described [59,60,64]. Fractionation in the FIFFF channel is achieved according to the fractionated components diffusion coefficients and hence their molecular weights. In a F1FFF apparatus, a ribbonlike channel is generally cut from a thin plastic spacer (Figure 7). A membrane having a specific molecular weight cutoff is inserted inside the FFF channel. Generally, requirements for the membrane include the following: fiat surface, rigid support, uniform porosity, suitable pore size, inertness to the carder liquids and samples, and suitable back pressure to maintain a uniform crossflow. A channel flow is introduced at one end of the channel where a smallvolume sample is injected. Practically, the channel flow is stopped momentarily alter the sample has entered the channel to allow the sample components to accumulate on the membrane, relax, and reach equilibrium

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distributions. A second stream of liquid, applied perpendicular to the channel, is a cross-flow and serves as the driving force to displace particles across the thin channel toward the membrane. The cross-flow enters the channel by passing uniformly through a porous ceramic flit and then the membrane. This secondary flow is introduced to retard the movement of sample particles in the parabolic channel flow stream. This retardation provides the fractionation between particle sizes, primarily based on their diffusion coefficients from the accumulation membrane (D, cm 2 sl). The F1FFF principle is illustrated in Figure 7. Theoretically, the diffusion coefficient is related to the Stokes (hydrodynamic) diameter (d~, cm) of the component by the Stokes-Einstein relationship [47] D = kT/3xrld~

(1)

where k is Boltzmann's constant (1.38 x 1016 gcm 2 s2Kl), T is the temperature (K), and r/is the viscosity of the cartier liquid (g em'ls-l). For random coil maeromoleeules, d~ is related to molecular weight M (Da) [47] by a, = a 7v/'

(2)

where the constant A" depends upon the macromolecule-solvent system. The constant b depends on the molecular conformation in the solution. In normal-mode FIFFF, the retention time (tr, min) for well-retained components is approximated by [63] tr = W2 Vo/6D V

(3)

where w is the channel thickness (cm), Vc is the cross-flow rate (em 3 min-1), and V is the channel flow rate (cm 3 min'l). When D from equation (1) is substituted into equation (3), t~ becomes [47] t~ =

~

V~J2 k TV

(4)

From equations (3) and (4), tr can be directly controlled by adjusting the flow rates V, and V. Therefore, the system can be readily adjusted to suit the sample as well as to meet analysis speed and resolution goals.

BARNES and SIRIPINYANOND

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A)

To detector flow in Crom lioN in

Porous [fit

,~loar

Mm~ane O r m flow otd

\

Porous fr#

Figure 7. FIFFF A) FIFFF schematic diagram arrangement (Reproduced from [47] with kind permission of the Dekker) B) FIFFF separation principle X represents smaller particles with higher diffusion coefficient Y is bigger particles with lower diffusion coefficient

Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry

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Difficulties in FIFFF arise from the uneven surfaces and compressible membranes used as the FFF channel accumulation wall. This frit surfaces unevenness and membrane non-rigidity leads to some measurement uncertainty [92]. Accuracy in F1FFF, however, can be improved by using a set of calibration standards with known diffusion coefficients, hydrodynamic diameters, and by coupling the F1FFF system to an on-line detector such as a multi-angle laser light-scattering (MALLS) instrument [93-96]. The MALLS provides the absolute determination of size or molecular weight of each fraction. This FFF arrangement is referred to as symmetrical F1FFF, where the cross-flow enters the channel through the upper frit wall and leaves through the lower flit wall. Another F1FFF modification is asymmetric FIFFF, first introduced by Wahlund and Giddings [92]. In this configuration the top wall is impermeable to the liquid flow. Only one permeable wall at the bottom of the channel allows the carrier liquid to leave the channel and thus generate a crossflow. The channel and the cross flows are introduced from the inlet flow to channel. Aider its introduction in 1987, several publications using asymmetric FIFFF have since appeared [97-100]. Early channels were confined with the same rectangular geometry as the symmetrical FIFFF. The channel breadth remains constant along the entire length. With this geometry, however, a gradual fall in volumetric flow rate occurs between the inlet and outlet, became of the continuous loss through the membrane of carrier fluid as it moves downstream to the channel outlet. This leads to a gradient in the mean channel flow velocity. As a result, in the interpretation of sample component properties from the observed retention times requires correction. To overcome or compensate for this situation, a tapered channel is preferably used to maintain constant channel flow velocity. With a trapezoidal geometry where the breadth decreases toward the outlet, the velocity gradient is altered by the ratio between the breadth of the inlet and the outlet. Moreover, this breadth ratio also affects sample zone dilution [ 101 ]. Generally, the asymmetrical F1FFF experiment consists of three different steps: (1) relaxation/focusing; (2) elution; and (3) channel back-flushing. In the first step, the counteracting flows are introduced to the channel from both the inlet and outlet. The flow leaves the channel only through the membrane. By using the counteracting flows samples ar,~ focused at a certain point, called the focusing point, where the axial velocity becomes zero. The focusing point is determined by the relative rates of the forward and backward flows. The forward flow refers to the flow that is directed from the channel inlet to the channel outlet. Likewise, the backward flow refers to the flow that is introduced from the channel outlet moving upstream to the channel inlet. A

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BARNES and SIRIPINYANOND

sample is concentrated at the focusing point, where an exponential concentration profile is established during the relaxation step. Next is the elution step, when the flow enters the channel from the inlet and leaves the channel through both the membrane and the channel outlet. The former is regarded as a cross-flow and the latter is termed an outlet flow. The balance between the cross-flow and outlet flow rates is regulated by a control valve attached to the detector outlet. After the sample components have been eluted from the channel, back-flushing is required to clean the channel thoroughly. In this step, the flow enters the channel from the outlet and flushes the retained materials out of the channel [101]. Asymmetrical F1FFF provides improved separation efficiency compared to symmetrical design. Yet some limitations in versatility occur, since the cross-flow and the outlet flow rates need to be controlled precisely. Despite the complexity in mathematical and theoretical derivation, several advantages result from asymmetrical F1FFF. These include the possibility of focusing the sample into a very sharp band before separation and the simplicity of channel construction. The first feature results in higher separation resolution and improved accuracy of particle size measurements. The latter results fi'om the technical simplicity of channel construction. Only one frit (at the bottom wall) is required. Therefore, the heterogeneity resulting from the uneven permeability of the upper flit, as well as the surface irregularity, is avoided. A further advantage, pointed out by Litzen and Wahlund [97], results from decreasing the breadth along the trapezoidal channel, whereby the sample zone dilution is reduced.

2.3 Instrumentation and optimization 2.3.1 Instrumentation The experimental setup of an FFF insmanent is similar to a conventional liquid chromatograph. Instead of employing a chromatographic column as separation cell, an FFF employs a thin ribbon channel and its support. Generally, the FFF arrangement is composed of a liquid carrier reservoir, a liquid delivery system, an injection system, an FFF channel, and a detector. Typically, sample volumes are in the range of 5-200 lxL and channel volume is 1.2 mL. In this chapter only the detector is described, and complete description of other ancillary parts including the channel is found elsewhere [70,72]. Since FFF is an elution-based method, many on-line and off-line detection techniques can be used to acquire information. Most liquid chromatograph detectors can also be applied in FFF systems. Molecular absorption spectrophotometers operating in the ultraviolet or visible range are

Field-Flow Fractionation-InductivelyCoupled Plasma-Mass Spectrometry

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widely employed [69]. Fluorescence detectors also have been used, with limited numbers of applications, because most polymers and colloids do not fluoresce [69]. Therefore, samples need to be dyed or fluorophores added before the analysis. The universal refractive index detector has been applied for several macromolecules. Several light scattering detector types also have been used. These include low-angle laser light scattering (LALLS) [102], multiangle laser light scattering (MALLS) [93-96], and evaporative light scattering [103]. Direct data on molar masses of the eluted sample was obtained using LALLS in combination with a concentration detector. Nowadays, MALLS detectors have become fashionable, since they enable absolute measurement of molar masses and molecular dimensions determination. In addition, viscometric detectors can be used [ 104-106]. Recently, electrospray mass spectrometry (ESI-MS) was employed as a F1FFF detection system [107]. Fractionation of poly(styrene sulfonates) using different carrier solutions were tested. Molecular weight distribution of individual polymers was obtained by their mass fractograms. Nevertheless, salt clusters forming at high ionic strength, that leads to complicated spectra, can be troublesome. To avoid this situation, amounts of salt introduced to the electrospray should be kept below 1 mmol 11. On-line ICP-MS, pioneered by Beckett [38], has also been used. Details are given in Section 5. Many possibilities for the combination of FFF with off-line detectors also were described [108,109]. Off-line scanning electron microscopy (SEM) detection of fractionated acrylate latex from FFF channel was reported [108]. Electrothermal AAS (ETAAS) was also used for off-line detection of elemental concentrations in geological samples [ 109].

2.3.2 Optimization An FFF instrument can be operated with a constant or programmed applied field. A constant field is comparable to the isocratic elution, whereas a programmed field is analogous to the gradient elution in chromatography. For polydispersed samples, field programming during elution is necessary [110]. Generally, a high external field strength must be introduced to fractionate the least retained macromolecules or particles. Normally, well-retained species leave the channel atter an excessively long retention time. To overcome this problem, the force intensity should be decreased gradually. Typically, operation at a constant field provides the maximum resolution between the sample components at this field. Field programming can shorten the analysis time and increase the detection limit without loss of resolution, however. Once an FFF measurement has been completed, investigating the same sample separation under different experimental conditions (such as field strength) is

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BARNES and SIRIPINYANOND

worthwhile. If the same distribution is obtained at reasonably differem retemion times, the absence of artifacts is guaranteed. Lastly, to avoid samplesample interactions or overloading effects, introducing the sample with the lowest detectable concentration is desirable. Considering the F1FFF experiment, the carder liquid, membrane type, and channel and cross flowrates need to be optimized. Clearly, the membrane should have desirable molecular weight cut-off (MWCO) value. Membrane type should be carefully selected to minimize interaction between membrane surface and samples. Consequemly, the carder liquid should be well-suited for both the sample and the membrane to minimize the effect of electrostatic forces. Generally, adding salt to the carrier liquid is required to reduce the retention perturbations caused by repulsive electrostatic interactions. Since the channel and cross flow rates affect retention and fractionation power, these parameters should be optimized to obtain desirable fi'actionation resolution without excessively long retention time (or analysis time). Usually at fixed channel flow rate, increased cross-flow rate leads to longer retention time and hence improved resolution. Sample adsorption to the membrane surface may become problematic, however. Lastly, the effect of channel dimension and geometry should also be considered. This effect is beyond the scope of this chapter and described in references [97,101 ].

2.4 Quantitative Analysis by FFF Most FFF experiments exploit UV spectrophotometry as detection means, owing to its simplicity, availability, and price. For large particles (supermicron-size particles), however, dependence of the analytical response upon carder liquid composition, and the relationship between sample size and optical properties, is quite complex. For the supermicron-size range light attenuation is caused mainly by scattering (Mie scattering) rather than absorption [72]. For submicron-size particles, however, the degree of Mie scattering is negligible. Regardless ef these complications, the UV detector signal is typically used as a direct measure of the mass concentration of sample in the eluent. Nonetheless, extraction of quantitative information from an FFF experiment requires care. As a result, absolute quantification of the fractionated sample has not yet been fully demonslmted. In addition to the problems from detector calibration, complete sample recovery must be guaranteed. Practically, sample losses occur because of irreversible adsorption in different parts of the apparatus. The degree of irreversible adsorption can be controlled by changing the carder liquid composition. Reschiglian et al. have reviewed these difficulties and proposed a standardless method of analysis to determine the extinction efficiency of the particulate samples fractionated by FFF [111,112].

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The standardless analysis was defined as "a method through which a signal can be related to the concentration or quantity of the analyte by an exact equation that is reliable to allow for direct calculation of the desired quantity from a single measurement" [111]. The authors demonstrated that the lack of information on particles' optical properties were overcome [ 112]. 3. SELECTED APPLICATIONS Since a FFF separation takes place in a single phase without the participation of second phase, alteration of biological materials or other macromolecules is minimized. Owing to its open channel characteristic, shear degradation of fragile high molecular weight species becomes less significant than chromatographic techniques [47]. Molecular integrity and global structure of macromolecules are maintained. Although FFF is not well known in most fields related to biology, geology, or environmental science, sufficient applications have appeared in a few key areas to suggest its broader potential [ 113]. These applications span a billion-fold mass range from small proteins to cells and starch granules. Between these extremes, FFF has been applied to protein aggregates and conjugates, protein particles, lipoproteins, viruses, DNA, subcellular particles, milk colloids, cell lysates, polysaccharides, liposomes, bacterial cells, and pollen grains, among other materials [53]. Although quantitative analysis by FFF is possible, the majority of FFF applications have been done qualitatively. General overview of FFF in biomedical analysis was also reported [113]. In this chapter, selected applications of FFF to biological and environmental samples are reviewed. 3.1 Sedimentation FFF (SdFFF) Since its first introduction, sedimentation has been successfully applied to fractionation and characterization of particles and macromolecules in environmental and biological samples. Those applications include serum albumin microspheres [114], liposomes [115], cartilage proteoglycans [116], viruses [117,118], starch granules [119,120], DNA [121,122], bloodstream trypanosomes [123], cellular species [124], red blood cells [ 125], totoplasma gondii [126], bacteria [127-130], bacterial cell wall [131,132], and corn root membranes [133,134]. Specifically, SdFFF was exploited to analyze Creutzfeldt-Jakob disease infectious fractions [133]. In addition, protein inclusion bodies from Escherichia coli lysates were investigated [134]. Applications of SdFFF in food science and technology have also been reported. These include fat emulsion [137], pharmaceutical fat emulsion [138,139], and yeast cultivations [140]. Aggregation of nonfat dry milk proteins was also

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BARNES and SIRIPINYANOND

examined [141]. Validation procedures of SdFFF techniques for biological applications were reported [142]. For environmental samples, colloidal particles in fiver water [143,144], and aggregations of colloidal materials [145] also were investigated. Several cellular species were separated with the SdFFF with gravitational field (gravitational FFF, GFFF). These include cells [146], red blood cells [ 147-153], Pneumocytis carinii cysts [ 154], living Trichomonas vaginalis [ 155], and hemopoietic stemcell from the mouse bone marrow suspension [156]. Fractionation of starch materials from barley also was reported [ 157]. Sedimentation FFF can handle samples in both aqueous and organic solvents with satisfactory separation resolution. Polymers of larger than 106 g mol ~, and colloids or particles of larger than 30 nm can be characterized. Fractionation in the gravitational field can be applied to particles of larger than 1 ktrn. This technique has a wide range of applications including particulate materials and biological molecules. 3.2 Thermal FFF (ThFFF) Thermal FFF is applicable to samples in both aqueous and organic carder liquids. However, thermal diffusivity is strongest in organic solvents without hydrogen bonding. In other words, the separation driving force (thermal diffusion) is very weak in aqueous fluids. Therefore, ThFFF is the preferred technique for characterizing organic soluble, synthetic polymers and copolymers, as well as determination of the average molecular weight and molecular weight distributions. In addition, information about polydispersity, and the polymer Brownian diffusion coefficient also can be obtained. Since the use of organic solvents cause extensive conformational changes and even sample denaturation, the sample application range is very limited. Nonetheless, applications of ThFFF for biomacromolecules have been reported. Myers et al. first investigated fi'actionation of a water-soluble blue dextran using ThFFF in water and mixed water-DMSO carder liquids [ 158]. No retention in water was observed, owing to the weak thermal diffusion effect in water, as confirmed by Kirkland and Yau [159]. Separations of dextrans, ficools, pullulans, cellulose, and the starch polymers amylose and amylopectin using DMSO as carrier liquid were investigated [84]. These polysaccharide samples have a wide application ranges in industries and are difficult to separate by SEC. These SEC difficulties arise from sample adsorption, shear degradation and clogging of the column. Lastly, application of ThFFF to the characterization of natural rubber also was reported I160]. In stmunary, size and chemical composition can be characterized using ThFFF. The technique is favorable for lipophilic samples, but not hydrophilic

Field-Flow Fractionation-InductivelyCoupled Plasma-Mass Spectrometry

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samples. Thermal FFF is suitable for very high molecular weight macromolecules, macromolecular assemblies subject to shear degradation, and copolymers prone to surface interaction. Since the thermal diffusivity in aqueous carrier liquid is weak, applications to water soluble macromolecules are limited. 3.3 Electrical FFF (EIFFF) The E1FFF was first investigated by Caldwell et al. for its capability to separate proteins [88]. In this early investigation, albumin, lysozyme, hemoglobin, and gamma-globulin were separated. The fractionation between albumin, hemoglobin and gamma-globulin was achieved within 240 minutes. Baseline separation was not obtained with the flow rate of 60 ml min1, however. Slower carder flow rate was suggested to improve resolution. Later, human and bovine serum albumin, gamma-globulin (bovine), cytochrome C (horse heart), lysozyme (egg white), and ribonucleic acid, as well as denatta,ed proteins, were fractionated [87,161 ]. Both flexible membrane [ 161 ] and rigid membrane [87] channels were tested. Furthermore, sugars [162], colloids and particles [163] using E1FFF were separated. So far, E1FFF has been in limited use. Since it is particularly sensitive toward differences in surface charge, the technique can be applied to study the adsorption of materials to colloidal substrates [72,164]. EIFFF should be an informative tool for biological and environmental research. 3.4 Flow FFF (FIFFF) In 1977 Giddings et al. first proposed the F1FFF as a method for protein separation and characterization [91]. Proteins, plasmids, plasmid fragments, polysaccharides, unicellular algae [165], nucleic acids, viruses [166], and monoclonal antibody aggregates [167] were fractionated. Flow FFF also was applied for water soluble synthetic and biological macromolecules separation [47,168]. Linear and circular DNAs [169], and red blood cells of diverse size, shape and origin [170] were separated. Wijnhoven et al. studied the retention behavior of proteins as a function of injected mass and ionic strength using hollow-fiber FIFFF [171]. Li and Giddings evaluated a modified F1FFF technique called "membrane selective F1FFF" used for the isolation and size distribution measurement of colloids in human plasma [172]. Fractionation of lipoproteins from human serum was examined [173]. Moreover, effect of ionic strength and pH on size characterization of liposomes was investigated [174]. In addition, F1FFF was used to characterize humic acids in solution [175]. Applications of FIFFF in food and dairy technology were reported. These include characterizing high molecular weight proteins present in glutenin [176],

204

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wheat proteins [177,178], whey proteins, casein micelles, and fat globules in dairy products [179], and colloidal components in reconstituted skim milk [180]. Carbohydrates (like dextran in seawater [181] and pullulan [182]) also were characterized using F1FFF. Conformational change and aggregation of kcarrageenan were studied using F1FFF and MALLS [183]. Flow FFF was applied to process monitoring in biotechnology. Ribosomes and their subunits in Escherichia coli during production of glucose isomerase were monitored [ 184]. Polymeric wheat proteins were also characterized [ 185]. Asymmetrical F1FFF also has been used for proteins characterization. Wahlund and Litzen reported a rapid high performance fractionation of five proteins coveting molecular weight ranging from 12,000 to 669,000 Da with baseline separation [165]. The technique also was used for molar mass characterization of modified celluloses using on-line MALLS detection [186]. Furthermore, drug-plasma protein interaction was studied [ 187]. Like other FFF techniques, FIFFF exhibits both advantages and disadvantages. Flow FFF is universally applicable to both macromolecules and particles of biological and environmental origins. The disadvantage of the technique is due to its low resolution, which can be improved using asymmetric FIFFF configuration. 4. COMPARISON WITH SEC Size exclusion chromatography (SEC) was first established three decades ago as the standard method for polymer separation and molecular weight distribution (MWD) determination [84]. Even so, SEC is not suitable for separating ultrahigh molecular weight polymers (>1,000 kDa), owing partly to the difficulties in preparing sufficiently large porous packing to allow the permeation of large macromolecules. Shortcomings of SEC include the possibility of shear degradation of large, fragile macromolecules in porous media and column clogging by large particles. Because of the column clogging, filtration before SEC is generally required. In terms of shear degradation, random-coil macromolecules at least as large as 2 x 106 MW might be successfully separated with columns of 0.5-1am particles without serious difficulty [186]. However, the shear imposed by flow in packed columns ultimately limits the upper molecular weight separation range of fragile macromolecules. Because of these persisting problems, alternative approaches like non-chromatographic size separation techniques should be explored for certain samples (e.g., hyaluronic acid, pullulan). As mentioned earlier, FFF is a "gentle" separation technique, owing to its open-channel characteristic. Shear degradation and adsorption are minimized,

Field-Flow Fractionation-InductivelyCoupled Plasma-Mass Spectrometry

205

since no stationary phase or packing material exists inside FFF channel. Global architectures of macromolecules are preserved as their native forms. Therefore, MWD information obtained from FFF is expected to be more accurate than that obtained from SEC. In addition in comparison to SEC, no MW calibration is required as long as channel dimensions are known. This is beneficial since difficulty arising from finding a good match between sample and standard macromolecules is substantial. Considering F1FFF, the cross-flow can be finely tuned permitting a single channel applicable to a very broad size range. This cannot be achieved with SEC, in which one column usually can work within only a certain size range. In other words, F1FFF has a higher upper MW limit than SEC [187]. Size exclusion chromatography gives higher resolution for low MW (< 50 kDa) materials, however. Another disadvantage of FIFFF is the band broadening. This is more serious in F1FFF than in SEC, thus the peaks resulting from an FFF separation tend to be broader than those from SEC separation. Some literature compares FFF, SEC, and electrophoresis [ 186,188]. 5. ATOMIC DETECTION

SPECTROMETRY

AS

ELEMENTAL

SPECIFIC

5.1 Literature

Field-flow l~actionation-ICP-MS (FFF-ICP-MS) is a relatively new technique for size separation with elemental analysis. Promising preliminary results have been reported for FFF coupled to ICP-MS. In 1991 Beckett first introduced the concepts and described initial experience in linking FFF separation techniques with ICP-MS [38]. According to Beckett, the initial idea of direct coupling between FFF and ICP-MS was arose during discussions with Howard Taylor of the U.S. Geological Survey in Denver, Colorado (Figure 8). Few publications applying FFF-ICP-MS have appeared since then [39-42]. In these papers natural suspended particulate matter, soil, and clay minerals were analyzed by SdFFF-ICP-MS [39-42,189,190] and by FIFFF-ICP-MS [191]. The applications of FFF-ICP-MS are summarized in Table 4.

206

BARNESand SIRIPINYANOND

tN IlmtlL"ll~t

t___._ --

)k

t 9

lil m

i

Figure 8. Instrumental setup of SdFFF-ICP-MS. [Reproduced from [40] with kind permission of the American Chemical Society] A modified Sciex Elan Model 250 ICP-MS (quadrupole), Perkin Elmer was used. Table 4 Applications of FFF-ICP-MS

,,Technique Sedimentation

Samples Colloids

Sedimentation

Soil colloids

Sedimentation

Complex colloid

Sedimentation Sedimentation

Kaolin Natural colloids

Sedimentation Sedimentation

River Po particles Colloids

Sedimentation

Natural co lloMs

Flow

Colloids

Flow

Proteins

Comments Minerals and river-bornes colloids characterized AI, Fe, and Mg determined in several soil colloid samples (K monitored by ICP-AES) Kaolinite, illite and particulates characterized Off-line ETAAS tested Effect of hydrous iron oxide comings on adsorption of orthophosphate studied A1, Cd, Cr, Cu, Fe, and Pb analyzed A1, Ba, Ce, Fe, Mg, Nd, Rb, Sr, and Ti determined Adsorption studies of Cs, La, and Pb conducted A 50-mL sample introduced into the channel using opposed-flow sample concentration. Feasibility studies for biological samples

Year [Ref] 1992139] 1992 [192]

1993 [40] 1995 [ 109] 1996 [193]

1997 [41 ] 1999 [42] 1999 [190] 1999 [191]

1999 [50]

Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry

207

Generally, less than 1 mg of sample is required for fractionatiorL High sample concentration may cause overloading effects in the FFF channel. With low sample concentration (e.g., < 0.5 mg mll), however, very sensitive analytical methods must be used in the subsequent chemical characterization step. Since ICP-MS provides excellent sensitivity, low detection limits, extended linear dynamic range, and also it can be applied to many elements with limited interferences, ICP-MS is an ideal elemental specific detector for FFF. In addition, ICP is a source capable of producing atoms from solid-phase particulate matter and ionizing all elements including those with high ionization potentials. The first evidence of FFF-ICP-MS application to size fractionation with elemental specific detection was published by Taylor et al. in 1992 [39]. A schematic diagram of SdFFF-ICP-MS is illustrated in Figure 8. In their work, a Babington-type pneumatic nebulizer was used to introduce and nebulize suspended particulates without the risk of clogging. In their experiment, major, minor, and trace element composition of the size-separated colloidal (
208

BARNES and SIRIPINYANOND

carded out in our laboratory in collaboration with other research groups at the University of Massachusetts [ 194]. To reveal how elemental composition varies with particle size, mathematical treatment of data is necessary. If the concentration of element E in the eluent (din "F/dV) is monitored by the ion currem of the mass spectrometer (IE), then the element based size distribution is obtained by plotting dm'F/dd versus d, as shown in equation (5) [195]. (dm "e/dd)

= (dm "r_,/dV)*(d V/dd) oc IE *b'V/&I

(5)

The concentration of E in the particles (dm "Fj'dm") for a given particle diameter can be plotted from (dm "F_/dm)

= (dm "e/dV) *(d V/ dm ") oc lr]UV detector response

(6)

With this deciphering process, a plot between the elemental composition of the particles as a function of particle size is obtained. Recently, Ranville et al. examined the applicability of SdFFF-ICP-MS to the analysis of horizon soils collected from Mountain Bold in South Australia [42]. They used 1 mmol ll sodium pyrophosphate with 0.02% sodium azide as carder liquid for SdFFF with 100 ng ml l In as internal reference for ICP-MS to correct for noise and drift. A simple Teflon | T connector was used to split the flow to the ICP-MS. Attempts to determine Si (m/z = 28), another major element present in the sample, were unsuccessful owing to spectral interferences from the presence of diatomie species (N2+ and NO+). They compared the results of direct slurry with digested sample nebulization. Disagreement between the two approaches occurred when larger than 1 jam particles were introduced into the ICP-MS, indicating that either the transfer of particles through the spray chamber or the ionization of the particles in the plasma was incomplete. Yet the results of <1 ~rn particles by the two sample introduction techniques agreed, confirming the quantitative nature of direct particle analysis by ICP-MS for colloidal samples. They also demonstrated changes in clay mineralogy. Hassell6v et al. studied the application of SdFFF-ICP-MS to trace metal adsorption processes [190]. Experiments at different pH distinguished between surface complexation (pH dependent) and ion exchange adsorption (pH independent). They expected that the ability to relate metal uptake to the size and composition of colloids would lead to new insights into uptake processes and into the transport and fate of trace metals in aquatic systems.

Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry

209

In 1995 a group of researchers from Italy and Spain first introduced ETAAS as an off-line detection for SdFFF of clay analysis [109]. Aluminum and Si were analyzed quantitatively, and the limits of detection were 63.6 and 212.4 ng for A1 and Si, respectively. Two years later, the same research group together with Beckett extended their work to the elemental characterization of water-borne fiver particles (Po fiver) [41]. Recently, ETAAS also was used as an off-line element detector atter separation using a split-flow thin (SPLITT) channel [196], another mode of FFF not described in this chapter. (Details of SPLITT can be found elsewhere [196-198]). The advantages and drawbacks of FFF-ETAAS coupling compared with ICP-MS are as follows:

1) FFF-ICP-MS

gives higher resolution and faster analytical information. Nonetheless, the limited availability of ICP-MS and its high cost reduced its broad applicability. 2) Despite the disadvantage from its off-line feature, ETAAS is a very widely used technique and available in most laboratories. As indicated in Table 4, most FFF-ICP-MS has been reported using SdFFF. In 1999 the first application of F1FFF-ICP-MS was described by Hassell6v et al. for elemental size characterization in natural water [191]. Twenty-eight elements in freshwater from a small creek in Sweden were determined, and results of six selected elements were shown. In their experimem, an on-channel preconcentration procedure allowing large volume sample introduction was used to provide preconcentration factor of about 1000 times. The on-channel preconcentration procedure will be discussed separately in another section (Section 6). Although FFF has been used successfully for separation of several biological materials, only one application of FFF-ICP-MS to biological samples has been reported [50]. In 1993 Barnes briefly predicted that FFF-ICP-MS should be an alternative technique for elemental speciation in biomedical samples [1]. The idea was also suggested in the review on analytical plasma source mass spectrometry in biomedical research in 1996 [2]. Only recently, however, a feasibility study of FFF-ICP-MS was reported for several protein standards (e.g., metallothionein, ceruloplasmin, carbonic anhydrase, ferritin and thyroglobulin) [50]. In this preliminary work, the effect of cross flow rate on fractionation between carbonic anhydmse and 13-amylase was studied. Became of the straightforward linkage between FFF and ICP-MS and the protein fractionation capability of FFF, FFF-ICP-MS is expected to be a valuable tool for speciation of metal binding proteins.

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BARNES and SIRIPINYANOND

5.2 FFF-ICP-MS for biological and environmental analysis

5.2.1 Metal binding proteins According to Comelis [199] trace element species in biology can be classified into five groups as follows: (1) small organometallic molecules; (2) exposure biomarkers; (3) elements with different valence states; (4) essential building block of biomolecule; and (5) elements forming a metal ligand complex. Clearly, FIFFF or SdFFF-ICP-MS cannot differentiate elements with different valence states. Nevertheless, information on elemental speciation of the last two groups, which are essential building block and metal forming ligand complex elements, can be acquired using FFF-ICP-MS. Complexation studies of elements with metal transport or metal storage proteins are vital to understand the behavior of those elements in biological tissues. Recently, binding of uranium and other elements to serum albumin, transferrin and ferritin was investigated in our laboratory [200]. Experiments were planned and conducted to study the preferential binding of the aforementioned proteins and change of the binding patterns with the contact time. Ion fractograms of Co, Fe, La, T1, and U in ferritin are shown in Figure 9. All elements studied are proportionally distributed to the ferritin-monomer (400 kDa),-dimer (800 kDa), and -trimer (1200 kDa). Limit of detections are calculated to be 68 ng 59Co, 3400 ng 57Fe, 21 ng 139La, 11 ng 2~ and 7 ng 23SU in 1 milligram of ferritin. Experimental results for other proteins will be reported elsewhere. In brief, study of the biodistribution measurement of essential and toxic elements is now possible. New and/or confirmed information can be obtained using FFF-ICPMS. Since information about metal distributions in different proteins in biological fluids is important to pathological implications, FFF-ICP-MS should be ft~her explored for its potential capability of providing such data. Investigation of the interaction between metallodrugs with serum proteins should be conducted, for example. Nonetheless, when practical samples are to be analyzed, preseparation technique should be used to isolate the interested protein from complex matrices.

211

Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry

70000 400 kDa

"t~La 2381I-

60000 w 50000

Q.

40000 .=,,,,

r

30000

57Fe

"= 20000

890 kDa

59Co

mE

1200 kDa

10000

0

-.

0

1.

10

.

.,

20

.

,

30

40

.

~

50

'

I

60

Time (min)

Figure 9. Ion fractograms o f Co, Fe, La, T1, and U in ferritin. [Experimental conditions: A 2000 p,g mllof ferritin (from horse spleen, Sigma) was equilibrated in vitro with 10 ~g ml"l of elements studied for 110 minutes. A 30 mmol 11 of TRIS*/nitric, pH 7.5, was used as carrier liquid and solvent. A sample volume of 20-p.l was introduced into the FFF channel by a channel flow rate of 1 ml min"l. A cross-flow rate of 2 ml minl was employed. A regenerated cellulose membrane, 10 kDa MWCO, was used. Three distinct peaks were obtained corresponding to ferritin monomer (400 kDa), dimer (800 kDa) and trimer (1200 kDa), respectively.] * Tris(hydroxymethyl)aminomethane

5.2.2 Humic substances

Humic substances are well known for their ability to bind and form complexes with metal ions. In addition, they regulate metal ions in the terrestrial and aquatic environments. Owing to their binding capacity, humic acids are considered as important contaminants carrier in the environment. Data on elemental distribution in humic substances is still limited and further investigation is desirable. Development of analytical procedures to provide this information is undoubtedly worthwhile. Although SEC is the most known technique using for humic acid characterization, precautions must be taken to minimize adsorption effect and ionic interaction between humic substances and packing materials in SEC column [201]. Furthermore, only non-humic model compounds are available for SEC calibration. Accurate detemaination of humic acid molecular weight is difficult to achieve. In contrast, Beckett and Hart illustrated that FFF channel MW calibration using poly(styrene sulphonate) standards was suitable for humic acid characterization [195]. Reasonable correlation between diffusion coefficient and molecular mass of some humic acid reference materials (International Humic Substances Society) was obtained.

212

BARNES and SIRIPINYANOND

Therefore, molecular weight characterization of unknown humic substances using F1FFF shows great promise for reliable humic material MW determinations. Flow FFF coupled with ICP-MS enables the simultaneous characterization of molecular weight distribution of humic acid macromolecules and investigation of heavy metals bound to them [202]. Elemental size characterization of several soil-derived humic acids was reported recently by Amarasiriwardena et al. [202]. Ion fractograms of leonardite and Michigan soil-derived humic acids are shown in Figure 10. Hydrodynamic diameter, molecular mass, and diffusion coefficient of two humic acids studied were calculated from these data. Reproducible, monomodal ffactograms with different polydispersities were obtained. Ion fractograms demonstrate good conformity with fractograms detected by UV spectrometry. Moreover, possibilities of using FFF-ICP-MS to study the aggregation of humic acids in the presence of divalent metal ions also were suggested. Clearly, FFF-ICP-MS is an additional tool for studying the role of metal ions in the formation or transportation of biogeochemical and environmental substances.

5. 2. 3 Tissue and foodstuffs The presence of essential and toxic elements in food has long been a health concern issue. Field-flow fractionation-ICP-MS in food science and nutrition has not yet been documented. Presently, element speciation of fruit, food, tissue and plant extracts are being explored in our laboratory. Several fruit juices, including fresh orange, fresh strawberry (Figure 12), and tomato, were characterized. Spinach, garlic, seaweed, grass and mung bean, extracted with hot water and TRIS buffer, also were examined. Bovine and turtle liver, as well as shrimp and yeast extracts, were investigated. In addition, elemental distribution profiles in milk, infant formula, and whole egg were observed. Fractions of _< 5 kDa was found in garlic extract, flesh orange and tomato juices. Fractions of 10-200 kDa was detected in tomato juice, grass, mung bean, mushroom, spinach and seaweed extracts, as well as in whole egg, yeast, bovine liver, and seafood extracts. In grass a fraction of _> 400 kDa was also found. Several fractions were detected in a milk sample, (i.e., 3, 40, and 330 kDa). Ion fractograms of fresh strawberry juice are shown in Figure 11 as an example. For all samples studied, only monomodal or bimodal peaks were obtained. By converting the signal obtained from the ICP-MS to concentration, the concentration versus retention time curve is obtained. This curve is then integrated to give the total peak area. Subsequently, the area is translated to the amount of metal by multiplying by the flow rate and the dilution factor in the FFF-ICP-MS interface. Finally, the amount of metal elements found in the

213

Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry

HAMI

HALN

a)

0.06 l

0.12 0.1 o 0.08 m 0.06

0.05

o

o

0.04

<~9

0.02 0 -0.02

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,.. 0.03 0

.n 0.02 1

< 0.01 0

0

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10

15

5

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t,.-

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0.08

eo ,a

o

,, 0.02 '< 0.01 0 0

10

5

i

0

15

5

Hydrodynamic diameter (nm)

Hydrodynamic diameter (nm) 0.12 0.1 o o 0.08 0.06 .m Q <=9

15

,- 0.03

0.02 0 -0.02

o

10

0.05 ,- 0.04

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L-

15

0.06

0.12 0.1 0.08

L

= <

i

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Time (min)

o

. . . . .

i

0

0.06

0.05 c 0.04 0.03

0.04

0

.~ 0.02 < 0.01

0.02 0 -0.02 0

10000

20000

30000

40000

50000

|

|

i

J

|

10000

20000

30000

40000

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40000

50000

MW (Oa)

MW (Da) IOO0

1000 A

800

r

800

O

600

w

6130

'~ r

4OO

"~

40O

_=

200

..E

O m

t-

0 0

|

|

i

i

i

10000

20000

30000

40000

50000

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200 0 0

10000

200(X)

30000

MW (Da)

Figure 10. Fractograms of leonardite (HALN) and Michigan soil-derived (HAMI) humic acids with (a) UV detection (b) calculated hydrodynamic diameter distribution (c) calculated molecular weight distribution (d) ion fractograms with ICP-MS detection. (Reprinted from [202] with kind permission of the Royal Society of Chemistry) Experimental conditions: Fractionation was carried out using a 30 mmol 1-1 of TRIS/nitric, pH 7.5, as carrier liquid. A 20-1al of 200 lag ml-~was introduced into the FFF channel by a channel flow rate of 1 ml mm-]. A cross-flow rate of 2 ml mm-~ was used. A regenerated cellulose membrane, 3 kDa MWCO, was used.

BARNES and SIRIPINYANOND

214

5000 w

-I

I I

Q. 4000 -1

< 5 kDa

~/I

64Zn

~ 5 kDa

o~ 3000 .~_ "~ 2000 t'Q

,-'9 1000

0

I

I

I

I

I

2

4

6

8

10

Time (min) Figure 11. Ion fractograms of fresh strawberry juice. Experimental conditions: A 30 mmol 1-~of TRIS/nitric, pH 7.5, was used as carrier liquid. A 20-~1 sample was introduced into the FFF channel by a channel flow rate of 1 ml mm-~. A cross-flow rate of 2 ml rain-~ was employed. A regenerated cellulose membrane, 1 kDa MWCO, was used. Remarkably, Cu is bound to both small (< 5 kDa) and large (-~5 kDa) molecules, whereas other elements are bound to only small molecular fraction. This investigation suggests that Cu might act as a center metal ion betweentwo or more units of macromoleculein strawberryjuice.

fractionated peak can be then computed. To differentiate the elemental concentration in the fractionated species from total elemental concentration, quantitative elemental analysis of samples without fractionation is necessary. A s s t m ~ g that sample recovery during fractionation is 100 percent, a difference between elemental concentration in the fractionated species and total elemental concentration is an elemental concentration of unbound species. Therefore, the FFF-ICP-MS can be used to quantify the elemental bound and the unbound fractions.

5.3 Quantitative Analysis by FFF-ICP-MS Generally, element quantification by ICP-MS of the separated species from chromatographic techniques is performed by using external calibration. In column chromatography the experimental setup is designed to allow a column by-pass injection of elemental standards for ICP-MS calibration purpose. Similarly, a channel by-pass injection of standard solution can be used in FFFICP-MS to permit an on-line calibration, as reported by Hassell6v et al. [ 191 ]. Most of the published FFF-ICP-MS works have reported using an off-line external calibration and internal standardization, however. According to

Field-Flow Fractionation-InductivelyCoupled Plasma-Mass Spectrometry

215

Hassellfv et al., elemental quantification was done by translating the ICP-MS signal (y-axis) to concentration and retention time (x-axis) to elution volume. Consequently, integrated peak area represented the amount of metal elements found in the fractionated peaks. Yet, method detection limits (MDLs) were not given. Assuming that the sample flow rate in FFF is the same as sample introduction flow rate in the ICP-MS, and the limit of detection (LOD) of element analyzed by ICP-MS is known, the MDLs in FFF-ICP-MS analysis can be predicted theoretically by Equation 7, as MDL(ng ltg~9 = LODlcp(ng mf t) *Atw(min) * V(ml min~)/injectedsampleOzg)

(7)

where dtw is the peak width at base of the fractogram between signal response and elution time. Nonetheless, quantification in elemental speciation is not straightforward. The difficulty arises from non-spectroscopic interference effects, which may differ among different fractions of the sample during fractionation or separation. This leads to non-reliable quantitative elemental distributions. To overcome this problem, Heumann et al. successfully demonstrated a method of on-line isotope dilution with a species-unspecific spike for determination of heavy metal complexes macromolecules by HPLCICP-MS [203-206]. To obtain exact calibration and accurate quantification by FFF-ICP-MS, the on-line isotope dilution method should be verified. 6. ON-CHANNEL FLOW-FFF PRECONCENTRATION WITH ATOMIC SPECTROMETRIC DETECTION Generally, FFF detectors require sufficiem sample quantity and reasonable sample concentration to provide adequate detector response and linear range. In contrast, minimum sample concentration is favorable for separation techniques, since risk of channel overloading is minimized. In reality, sample preconcentration is usually required for adequate detection. Samples can be preconcentrated either before or after separation. In chromatography the former is referred to as "pre-column preconcentration" and the latter is regarded as "post-column preconcentration". Like liquid chromatography, FFF preconcentration can be achieved both ways. Before FlFFF separation preconcentration can be achieved "on-channel" by exploiting a feature of FlFFF similar to cross-flow ultrafiltration [207]. A large sample volume can be introduced into the FFF channel by two counteracting flows. This technique is called "opposed-flow sample concentration" [207-209]. Preconcentration after F1FFF separation is also possible with a flit outlet mechanism [210,211]. This is desirable when small amount of sample is

216

BARNES and SIRIPINYANOND

available and sample overloading needs to be avoided. Details of ffit outlet and opposed-flow sample concentration are described in the following sections. 6.1 Frit outlet Generally in FFF the sample zones are localized in the bottom portion of the channel approximately 10% next to the accumulation wall (Figure 13). Owing to this unique feature, the excess uncontaminated fluid at the top part of the channel can be skimmed off, and the sample can be thus concentrated in the remaining stream for improved detection. The flit outlet utilizes these features. Splitting the flow stream into two parts, one on the channel top and one on the bottom, allows removing excess carder fluid through one outlet and retrieving all sample through another. The sample-free fluid on the top part can be collected through the flit outlet. The sample concentration in the remaining flow stream at the bottom part is enhanced [ 187,210]. This allows for increased detection of the distribution tails or for species detection present in low concentration such as higher order protein aggregates. From the mechanical viewpoint, a small section of flit (flit outlet in Figure 13) is isolated from the main frit. The outlet frit element is located at the end of the channel to split off the excess, sample-free carder fluid. Using pressure restrictors the amount of flow can be balanced and controlled. The flow from the flit outlet goes to waste, whereas the flow from channel outlet continues to the detector. The frit outlet arrangement is illustrated in Figure 12.

Figure 12. Frit outlet arrangement with flow [After Hansen et al. [187] with kind permission of the FFFractionationCompany, Utah]

Field-Flow Fractionation-InductivelyCoupled Plasma-Mass Spectrometry

217

Theoretically, the detection signal is enhanced according to the ratio of the two flow streams, from the frit outlet and the detector stream flows rates. However, with excessive use of the flit outlet, the detector stream flow rate decreases to ~tl min~. This slows the sample flow rates and hence increases the sample transfer time to the detector. Therefore, the signal enhancement obtained experimentally does not correspond exactly to that calculated theoretically. At some ratio of the two flow streams where the channel outlet flow becomes too slow, the dead time between channel and detector increases. As a result, the flit outlet loses its utility as the dead volumes become significant. Extra-channel band broadening and excessive analysis time overwhelm the advantages of enhanced signal detection. Nevertheless, many detection systems benefit from the slow flow rate. The interface to a conventional mass spectrometer, and the interface to ICP-MS using direct injection nebulizer (DIN), high efficiency nebulizer (HEN), microconcentric nebulizer (MCN) and oscillaing capillary nebulizer (OCN) are well-matched with the microliter flow rates, for example. As suggested by Hansen et al., the FFF with flit outlet operation should be useful for interfacing with ICP-MS [187]. Preliminary tests in our laboratory showed promising results with humic acid and human serum albumin samples. Further investigation should be completed to improve detection limit by ICP-MS for several samples.

6.2 Opposed-flow sample concentration 6. 2.1 General overview and application Ultrafiltration technology can be used as sample preconcentration technique for large macromolecular species [212]. As a membrane filtration technique, ultrafiltration is capable of concentrating species that have molecular weight of greater than 10,000 Da. Owing to the similar feature of FFF and ultrafiltration, on-channel preconcentration with FFF unit is possible. The idea was proposed by Lee and presented at the 5th International Symposium on FFF in 1995 [208]. The analysis procedure consists of three steps" sample loading and focusing; sample relaxation; and fractionation, as illustrated in Figure 13. Sample can be loaded through either the ~ont or back ends of the FFF channel while the carrier liquid enters the channel through both front and back ends with constant ratio between the forward and backward flow rates. During sample loading and focusing, the carder solution leaves the channel only through the cross flow outlet. Similar to asymmetrical F1FFF a cross flow is not introduced externally during sample loading and focusing. The sample is focused in a small region where the two flow streams balance. The ratio between the forward and backward flow rates determines the focusing point. Ideally, the

218

BARNES and SIRIPINYANOND

accumulation or focusing point is located as close to the beginning of the channel as possible to exploit the full channel length for an optimum separation without introducing channel end effects. Typically, a flow ratio of 1"12 is selected, giving a focusing point about 2 cm from the channel front end (when the channel length is about 27 cm). Once the entire sample is introduced into the channel and migrates close to the focusing point, the sample zone created must be sharpened by allowing carder solution to traverse the channel for an appropriate length of time (the focus time) to minimize zone broadening. During relaxation, only cross-flow is introduced through the channel and flows to the detector and waste. Practically, the relaxation step is long enough to allow approximately two void volumes to pass through the FFF channel [207]. In the final elution step, the channel and cross flows revert to the standard F1FFF configuration, the sample is fractionated and data acquisition is started. In the opposed-flow sample concentration technique, the focus time and flows should be considered. Usually, the total focus time consists of the time for the sample to travel from the sample loop to the channel plus the time to accumulate and focus the sample in the focus zone. Generally, the sample travel time depends on the flow through the sample loop and the sample loop volume whereas, the zone focus time depends not only on the focus flows, but also direction of sample loading and the molecular weight of the sample. Therefore, the total focus time should be optimized either theoretically or experimentally. The optimum focus time is defined by attaining a constant retention time or theoretical plate height. Once the constant theoretical plate height is established, to proceeding further with focusing is not advisable. With an unnecessarily excessive focus time, sample loss becomes significant and degrades the advantageous sample preconcentration. At fixed focus flows, sample loss as a function of focus time is illustrated in Figure 14. To optimize the focus flows, loss rates should be minimized (indicated by absorption peak area). Lyv6n et al. showed that loss rates increase significantly with increasing flow rates [207]. This results because colloids are less easily forced through the membrane under low flow conditions. We also measured loss rates for human serum albumin (HSA) as a function of focus flows (Table 5). The degree of loss rates of HSA are in good agreemem with those reported for PSS in Lyv6n's experiment [207].

219

Field-Flow Fractionation-InductivelyCoupled Plasma-MassSpectrometry i Channel

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-Jl ]

end

,

-

:>

Back end

~1 Waste '.1 ~1

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t l oete~orI

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Crossflow Pump

~

! Detector i

t

U

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I

~ Waste I . ....

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,

>

Back end

~] Waste"i

7

I

Figure 13. Schematic of opposed-flow sample concentration (A. sample introduction and focusing, B. sample relaxation, C. sample elution)

220

BARNES and SIRIPINYANOND

Focus time (min) 0

2

4

6

8

10

12

0.08

0.06 A

2:)

0.04

6:2) < v

rc .r

tll

5 2

0.02

O

.8

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o.oo -0.02 0

100

200

300

2 400

Retention time (s) Figure 14. Sample loss as a function of focus time. The filled squares and regression line shows the peak areas for 10 mg 1l 1430 MW PSS standard, injected backwards, with different focus times. The solid lines show the corresponding UV detector response. A 1000 MWCO Millipore membrane with 25 mn~l 1l TRIS as carrier liquid was used with 1.74 ml sample, 0.5/6.0 ml min q as focusing flows, 0.5 ml min l channel flow and 3.8 ml minl crossflow. [Reproduced from [207] with kind permission of the Elsevier]

Table 5 Losses for different backward and forward focusing flows at forwarding loading

Forward:backward flow (ml min1) 0.1"1.2 0.2:2.4 0.5:6

Loss rate (% min'l) a 0.4 1.3 5.6

Forward:backward flow (ml min "1) Loss rate (% min'l) b 0.08:1 0.1 0.16:2 0 0.24:3 0.01 0.32:4 2.5 0.40:5 7.3 a) Conditions: 1000 MWCO membrane; 25 mmol r I TRIS as carrier liquid; 1430 MW PSS standard (10 ~tg ml"1) 1.74 ml as sample (Results: reprinted from [207] with kind permission of Elsevier) b) Conditions: 10000 MWCO membrane; 30 mmol 1"~ TRIS as carrier liquid; 67000 MW human serum albumin (70 ~lg ml"l) 1 ml sample (Results: obtained from our laboratory)

Field-Flow Fractionation-Inductively Coupled Plasma-Mass Spectrometry

221

An on-channel preconcentration of natural colloids before ICP-MS detection was investigated by Hassell/Sv et al. [191]. In their experiment 28 elements were examined. Results of six elements are shown in Figure 15, where hydrodynamic diameter and molecular weight are plotted. Carbon signals conform closely to the UV signal. Markedly different distribution patterns are shown for each element, indicating their dissimilar behaviors in aquatic environment. According to HassellCiv et al., colloidal nickel was mainly associated with the carbon, and possibly with the smaller iron fraction [191]. Lead was associated with the larger iron-based colloids, while lanthanum was bound to both the organic and inorganic colloids. Considering the molybdenum elution time, however, Mo co-emerged with the void peak. This suggests that Mo occurs as dissolved molybdate ions, which are partially retained in the channel during the preconcentration step by charge repulsion from the membrane surface. Similar investigation using an on-channel preconcentration FFF before ICP-MS detection were conducted in our laboratory for sewage water from a local wastewater treatment plant. Three types of water, including influent sewage water; sewage atter primary treatment, and effluent water, were tested for 10 different elements [213]. As expected, reasonable trends of decreasing elemental and colloidal concentrations as the water treatment process proceeded, were achieved. In addition, elemental distribution patterns also changed along the course of the treatment. Clearly, FFF-ICP-MS should be regarded as an indispensable tool for elemental speciation in natural waters and other aquatic media. Potential applications of FFF-ICP-MS to industrial process and its wastewater treatment monitoring are suggested. 6.2.2 On-channel matrix removal and preconcentration The similarity of FIFFF to ultrafiltration suggested use of FIFFF as an onchannel sample preconcentration-matrix removal arrangement. Conceptually, the sample is introduced to FFF channel, focused and concentrated by using an opposed-flow sample concentration. Large volume of samples can be introduced into the FFF channel and focused at a certain point. Thus sought analyte elements can be retained in the channel, if their molecular sizes are larger than the effective membrane pore size. To meet this requirement, chelating the analytes with high molecular weight complexing agents is suggested. Consequently, the complexed analytes are retained in the channel during sample introduction and focusing. The uncomplexed matrix elements, however, permeate through the membrane and leave the channel. As a result, an on-line preseparation of analyte and matrix elements is achieved before ICPMS analysis. This enables elimination of matrix effect during the analyte

222

BARNES and SIRIPINYANOND

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Field-Flow Fractionation-lnductively Coupled Plasma-Mass Spectrometry

223

determination. The idea has been tested experimentally in our laboratory [214]. In the study, preconcentration and matrix elimination in the presence of sodium and calcium matrix elements were examined, since these elements occur often in environmental and biological samples. A commercial FFF system was modified to accommodate an on-channel preconcentration procedure allowing 50-ml sample injection. The matrix-flee, preconcentrated sample was then introduced directly to the ICP-MS. The method was tested using 10-ml sample aliquots that contained 5 pg 1~ As, Cd, Cu, Mo, Pb, Re, Sn, Te, T1, Y, Zn, and Zr analytes and 5000 pg ml-I Ca or Na matrices and ethylene imine polymer complexing agent. Copper and Re isotopic ratio determinations in reference standards were also tested after preconcentration and matrix element removal. Details of experiments and results are described elsewhere [214]. Briefly, the proposed procedure is useful for the analysis of ultratrace analytes in samples containing high matrix concentrations. For the elements studied, recovery range of 60-100% was obtained depending on the strength of the complex formed. Preconcentration factor of at least 50 fold was achieved. Furthermore, isotopic ratio measurement in practical samples were improved. Application of this proposed idea to practical clinical, biological and environmental samples is worth exploring. 7. CONCLUSIONS AND FUTURE TRENDS The marriage between FFF and ICP-MS is practical. Field-flow fractionation-ICP-MS enables continuous elemental determination as function of molecular size. Some differences between FFF and chromatography are based on different type of forces used to induce retention. In chromatography forces are highly selective but at the same time are so powerful that they cause irreversible adsorption and structural disruption, including denaturation. In contrast, the FFF and electrophoresis field-based driving forces are much more diffuse in nature. For that reason, molecular integrity of macromolecules is maintained during separation. In addition owing to the absence of solid support in FFF and electrophoresis, biological activity loss becomes less significant as compared to chromatography where interaction between analyte molecules and stationary phase is likely to occur. Therefore, FFF and electrophoresis are considered gentle separation techniques. Since FFF-ICP-MS provides information only on elemental size distribution but not the functionality of the analytes, the method is found to be less popular compared to CE-ICP-MS. Nonetheless, for researchers interested in studying the relative binding of specific metals to macromolecules, FFF-ICP-MS is considered as an alternative approach. In term of technical simplicity, less effort is required for FFF-ICP-

224

BARNES and SIRIPINYANOND

MS than for CE-ICP-MS, where major modifications is necessary. This is due to the sample introduction incompatibility between CE and ICP-MS flow rates. Several keynote lectures and presentations describing FFF-ICP spectrometry have been made at the international conferences. Some of the recent presentations, contributed from three or four research groups, are listed in Table 6. Considering the number of publications devoted to FFF-ICP-MS each year, this technique has attracted the increased interest only recently. Therefore, FFF-ICP-MS can be regarded as a growth research area at beginning of this millennium. Essentially, cross-checking between SEC-ICP-MS and FFF-ICP-MS should be considered essential to guarantee the correctness of the information obtained, since the artifacts may present in one technique but not the other. The technique should therefore be regarded as a for elemental speciation. Similar to other hyphenated techniques with ICP-MS, enriched isotopes can be used to study the fate of elements in biological and environmental samples. Studies of nutrition and drug metabolism in animals and human, as well as metal adsorption processes in both terrestrial and aquatic environments are suggested. Considering the growing need to acquire new scientific information nowadays, one analytical technique alone is not sufficient to serve the purpose. Offemimes, many analytical methodologies should be used to answer one question, or analogously to put many pieces of information together to fill in a jigsaw-ptmzle. Several analytical techniques like flow injection analysis (FIA), CE, and HPLC can be used and symbiotically combined together. Flowinjection is employed to facilitate sample pretreatmem before CE or HPLC separation, and it can also be used to deliver sample efficiemly in a flowing stream to CE or HPLC, for example. Additionally, FIA is used as an interface between HPLC and CE. In a similar fashion way, sample delivery to FFF by FIA or coupling between FFF-FIA-CE also should be possible. Orthogonal combination between FFF-ICP-MS and FFF-CE or FFF-HPLC could also be applied for added information, where elememal size distribution and its functionality information could be obtained. Currently, analytical instrumentation development has been progressing toward minituafization. Looking to the future, downsizing of FFF or FFF-ICP-MS (with a microflow nebulizer) should also be considered.

Field-Flow Fractionation-InductivelyCoupledPlasma-MassSpectrometry

225

Table 6 FFF-ICP-MS and FFF-ICP-AES presentations made at conferences

Conference (year) Title A (1999) FFF-ICP: an efficient and effective means for the analysis of environmental colloids A (1999) Coupling of FIFFF with on-channel preconcentration to ICPMS for size distribution determinations of trace elements associated with natural aquatic colloids A (1999) Characterization of groundwater colloids using PCS and difference FFF methods A (1999) Determination of phosphorous distributions in environmental colloids using sedimentation field-flow fractionation-HR-ICPMS A (1999) Application of F1FFF-ICP-AES for characterization of fine clay particles A (1999) Colloidal metal size distributions and concentrations in 10 Swedish rivers by FIFFF with on-channel preconcentration coupled to ICP-MS B (1999) Mobilization of lead by extracting agents in highly weathered natural porous media C (2000) Application of FIFFF-ICP-MS for elemental speciation of biomolecular complexes C (2000) Application of FIFFF-ICP-MS for the investigation of trace metal bound to humic acids C (2000) Flow FFF with on-channel preconcentration coupled to ICP-MS for comparing size distribution determination of trace metals associated with natural aquatic colloids in different fresh waters C (2000) On-channel simultaneous sample preconcentration-matrix elements removal using FFF coupled on-line with ICP-MS D (2000) Field-flow fractionation-ICP-MS for the characterization of environmental colloids

Ref 215 216

217 218

219 220

221 222 223 224

225 226

A- International Symposiumon FFF, Paris, France B - American Geophysical Union Fall Meeting, San Francisco, California C - 2000 Winter Conference on Plasma Spectroch~nistry, Ft. Lauderdale, Florida D - Whistler 2000 Speciation Symposium on Speciation of Elements in Biological, Environmental and Toxicological Science, Whistler, British Columbia, Canada

ACKNOWLEDGMENTS Preparation of manuscript is supported in part by ICP Information Newsletter, Inc., Hadley, M A and University R e s e a r c h Institute for Analytical Chemistry (URIAC), Amherst, MA. A.S. gratefully thanks the government of Thailand for the student fellowship funded through the Ministry o f University

226

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Affairs, Bangkok. Comments from Dr. Dula Amarasiriwardena on humic substances (section 5.2.2) and Dr. Assad A1-Ammar on on-channel matrix removal and preconcentration (section 6.2.2) are sincerely appreciated. REFERENCES o

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152. P.J.P. Cardot, J.-M. Launay and M. Martin, J. Liq. Chromatogr. Relat. Technol., 20 (1997) 2543. 153. E. Assidjo and P.J.P. Cardot, J. Liq. Chromatogr. Relat. Technol., 20 (1997) 2579. 154. C.Bories, P.J.P. Cardot, V. Abramowski, C. Pos, A. Merino-Dugay and B. Baron, J. Chromatogr., 579 (1992), 143. 155. A. Bernard, C. Bodes, P.M. Loiseau and P.J.P. Cardot, J. Chromatogr. B, 664 (1995) 444. 156. E. Urbankova, A. Vacek and J. Chmelik, J. Chromatogr. B, 687 (1996) 449. 157. J. Chmelik, A. Knamlova and J. Caslavsky, Chem. Pap., 52 (1998) 360. 158. M.N. Myers, K.D. Caldwell and J.C. Giddings, Sep. Sci., 9 (1974) 47. 159. J.J. Kirkland and W.W. Yau, Macromolecules, 18 (1985) 2305. 160. S. Lee and A. Molnar, Macromolecules, 28 (1995) 6354. 161. L.F. Kesner, K.D. Caldwell, M.N. Myers and J.C. Giddings, Anal. Chem., 48 (1976) 1834. 162. R. Mori, T. Nakagama and T. Hobo, Kuromatogurafi, 15 (1994) 270. 163. M.E. Schimpf and K.D. Caldwell, Am. Lab. 27 (1995) 64. 164. M. Dunkel, N. Tri, R. Beckett and K.D. Caldwell, J. Microcolumn Sep., 9 (1997) 177. 165. K.G. Wahlund and A. Litzen, J. Chromatogr., 461 (1989) 73. 166. A. Litzen and K.-G. Wahlund, J. Chromatogr., 476 (1989) 413. 167. A. Litzen, J.K. Walter, H. Krischollek and K.-G. Wahlund, Anal. Biochem., 212 (1993) 469. 168. J.C. Giddings, M.A. Benincasa, M.K. Liu and P. Li, Polym. Mater. Sci. Eng., 65 (1991)21. 169. M.K. Liu and J.C. Giddings, Macromolecules, 26 (1993) 3576. 170. B.N. Barman, E.R. Ashwood and J.C. Giddings, Anal. Biochem., 212 (1993)35. 171. J.E.G.J. Wijnhoven, J.P. Koom, H. Poppe and W.T. Hok, J. Chromatogr., A, 732 (1996) 307. 172. P. Li and J.C. Giddings, Phar. Sci., 85 (1996) 895. 173. P. Li, M. Hansen and J.C. Giddings, J. Liq. Chromatogr. Relat. Technol., 20 (1997) 2777. 174. M.H. Moon, I. Park and K. Yonghee, J. Chromatogr. A, 813 (1998) 813. 175. M.E. Schimpfand K.-G. Wahlund, J. Microcolumn Sep., 9 (1997) 535. 176. K.-G. Wahlund, M. Gustavsson, F. MacRitchie, T. Nylander and L. Wannerberger, J. Cereal Sci., 23 (1996) 113. 177. S.G. Stevenson and K.R. Preston, J. Liq. Chromatogr. Relat. Technol., 20 (1997) 2835.

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220. K. Andersson, M. Hassell6v, B. Lyv6n, C. Haraldsson and D.R. Turner, P20, Eighth Intemational Symposium on Field-Flow Fractionation, Paris, France (1999). 221. V.M. Vulava, J.C. Seaman and B.P. Jackson, American Geophysical Union Fall meeting, San Francisco, California (1999). 222. A. Siripinyanond, R.K. Gupta, D. Amarasiriwardena and R.M. Barnes, TP76, Winter Conference on Plasma Spectrochemistry, Fort Lauderdale, Florida (2000). 223. D. Amarasiriwardena, A. Siripinyanond, R.K. Gupta and R.M. Bames, TP58, Winter Conference on Plasma Spectrochemistry, Fort Lauderdale, Florida (2000). 224. K. Andersson, M. Hassell6v, B. Lyv6n, C. Haraldsson, and D. Turner, TP59, Winter Conference on Plasma Spectrochemistry, Fort Lauderdale, Florida (2000). 225. A. A1-Ammar, A. Siripinyanond, D. Gumerov and R.M. Barnes, TP11, Winter Conference on Plasma Spectrochemistry, Fort Lauderdale, Florida (2000). 226. R. Beckett, Whistler speciation symposium: Fourth International Symposium on Speciation of Elements in Biological, Environmental and Toxicological Sciences, Whistler, British Columbia, Canada (2000).

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Chapter 5

Slurry sample introduction in atomic spectrometry: application in clinical and biological analysis Henryk Matusiewicz Politechnika Poznafiska, Department of Analytical Chemistry, 60-965 Poznafi, Poland I. INTRODUCTION Conventional sample preparation of clinical and biological materials prior to atomic spectrometry involves complete solubilization of the analyte and matrix, which is achieved typically by oxidative (destruction, decomposition) of the organic matter. These aspects of sample treatment have been dealt with in several books [1-5] and review papers [6-8]. Mineral acid decompositions typically use nitric acid as the main oxidizing acid, often in combination with H202 and HC104 and non-oxidizing acids such as HF and HCI. However, these digestion procedures can be labor intensive and prone to contamination errors. Frequently, the time required for sample preparation can exceed the actual instrument time by two or more orders of magnitude. Moreover, the more labor intensive the sample pretreatment, the more prone to errors the analysis becomes. In consequence, there is a continuing interest in the development of simplified sample preparation techniques. In recent years, the slurry sampling technique has been extensively employed for the analysis of biological tissues and clinical samples by atomic spectrometry, in order to simplify sample preparation procedures and to avoid some inconveniences related to wet decomposition and dry ashing procedures. As early as the sixties, an interesting new method of solid sampling appeared: Gilbert [9] obtained flame emission spectra from a soil suspension, and Mason [10], determined alkali metals in plant materials by flame emission spectrometry. Subsequently, Brady et al. [11,12] proposed an interesting method of solid sampling, the dispensing of water suspended powdered sample into the atomizer, using a micropipet. These authors obtained satisfactory results for Pb and Zn determinations in plant samples (leaves) and marine sediments.

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Introduction of slurry samples combines the advantages of direct solid sampling (shortening of sample preparation time; in sample contamination; decrease in analyte losses through volatilization prior to analysis and/or associated with retention by insoluble residues) and liquid sampling (sampling dispensing by using a conventional liquid sample handling apparatus; straightforward automation; flexibility in slurry preparation and the advantage that slurries may be prepared in advance). Several reviews on important aspects and techniques of slurry sample introduction in atomic spectrometry in general have been published [13-24]. However, no review or book has dealt with the slun'y sample introduction methods unique to clinical and biological analysis to a given atomic spectrometric techniques. Although it is very difficult to refer to every paper published in this area, the enlisted bibliography of this chapter could give a comprehensive coverage of advances of the topic made to date. This chapter is an attempt to fill this void and will focus on slurl'y sample preparation methods of clinical and biological samples unique to atomic spectrometric techniques. The techniques covered are: atomic absorption and emission spectrometry, direct current plasma, inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry, microwave induced plasma atomic emission spectrometry and atomic fluorescence spectrometry since these are most commonly used in trace element analysis of clinical and biological materials. The intent is not to present the procedural details for the various tissues or elements, but rather to highlight the methods which are unique to each insmmaent and sample. This chapter gives an overview of slurry sample introduction, its combination with AAS, AES, AFS, DCP, ICP and MIP and recent developments and applications of the direct analysis of clinical and biological materials. Other sample introduction system solid sampling in atomic spectrometry is beyond the scope of this review and will not be discussed here. 2. O V E R V I E W AND N O M E N C L A T U R E

The primary objectives of slurry analysis is to reduce the time and effort involved in solid sample preparation. Therefore, successful slurry analysis requires the characterization of: (i) the preparation of the slurry, (ii) the concentration range of the slurry required to obtain reproducible and representative sampling, (iii) the liquid medium by the addition of chemical modifiers, (iv) the possibility of using aqueous standards for calibration and (v) the determination of the precision and accuracy.

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2.1 Slurry preparation Preparation of the slurry consists of adding a liquid diluent to the solid material that has been previously ground and sieved (when necessary), weighed and placed into a container, in which the slurry is stable during the time required for sampling. The amount of solid material that is weighed depends on the concentration of the analyte and the dilution in the final volume of the slun~. The powdered samples are generally suspended directly in autosampler microvials in water or in a diluted acid solution (which ensures a partial solubilization of the sample, thereby improving its homogeneity). It has been shown that only very fine granulometry of the slurry may ensure correct results: concerning the representativeness of the sampling, it is evident that the presence of large particles in the sample was found to be the most critical factor in the analysis. The importance of an intensive grinding of the sample prior to the analysis is thus obvious. To achieve a particle size distribution that would yield results similar to those of an equivalent, equimolar aqueous solution, a wide range of manually or automatically operated grinding techniques have been employed. Biological materials may be ground by using PTFE beads in polyethylene bottles (the "bottle and bead" method). As alternatives, mortar grinding, grinding in porcelain, agate ball mills, a "mixing mill" (impact grinder and blender) and vibration millls and dental mills might be used. For most elements of interest, cryogenic grinding at the temperature of liquid nitrogen (-196 ~ is a straightforward and almost contamination free grinding technique if devices made of PTFE and metal (Ti, Zr) are used. These techniques have been developed during the last decade and successfully used for grinding and homogenizing of various fresh and dry biological materials and for the preparation of heat sensitive biological reference materials. Some of the sott tissues of human and animal origin can be disintegrated and homogenized in water at maximum speed. Preparing slurries in aqueous solution alone is unsuitable for the majority of biological samples owing to flocculation effects which result in rapid sedimentation of the finely powdered material. It is therefore essential to prepare a stable and homogeneous slurry, this is achieved by employing stabilizing agents, commonly termed "dispersants" or "surfactants". The slurry can be stabilized using a highly viscous liquid medium. So far, Viscalex, glycerol, non-ionic surfactants and organic solvents of high viscosity have been used as slurry stabilizing agents. Apart from stabilizing agents, the addition of wetting and antifoaming compounds can improve the dispersion of the slurry. In slurry analysis Triton X-100 is useful to disperse solid particles that might otherwise tend to float on the top of the liquid. Important for final homogeneity of the slurry and reliable results is effective agitation (stirring) and mixing after addition of the various reagents mentioned above to the material so that sedimentation, at least during the time of sampling,

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can be avoided or as far as possible minimized. Magnetic agitation and vortex mixing have been widely used, mainly during the early work carried out using the slun'y technique. After magnetic agitation or vortex mixing, slurries are usually introduced with a micropipet became of the difficulty of incorporating these systems in an autosampler. The recently most frequently and (with manually and automated slurry introduction) almost exclusively applied agitation technique is ultrasonification with appropriate power settings applied for a relatively short time of, typically 15 - 30 s. An advantage of this elegant and versatile approach in comparison with magnetic agitation and vortex mixing is that the analyte o f interest is partly extracted into the liquid phase, owing to the ultrasonic action, when the slurries are prepared in an acidic medium. Ultrasonic agitation is more effective than other agitation systems. Other technique in which effective homogenization of the slurry can be achieved is gas mixing (passing an Ar stream through a narrow capillary tube introduced into the slurry medium). This system is easy to handle and it does not require the use of stabilizing agents or special devices for agitation. Pre-digestion of the slurry can be helpful to extract the analyte of interest into the liquid phase. In contrast to the extensive sample pre-treatment inherent in digestion procedures that are conventionally used, pre-digestion of a slurry only requires a partial decomposition of clinical and biological samples, hence it is not time-consuming. The pre-digestion step significantly improve both the precision and accuracy. 2.2 Particle size

The mean particle size of a slurry is of particular importance for the representativity of the analytical results as well as for the uninterrupted passage of the slurry through tubing and pipet orifices. It is obvious that smaller particles facilitate sample preparation and improve recovery. Errors associated with large particles (diameter >100 jam) arise from the difficulty of maintaining a homogeneous distribution of the large particles in slurry and the lower pipetting efficiency for large particles. To ensure a good repeatability of the measurements a representative number of particles must be analyzed. Slurry nebulization into atomic sources requires that both the analyte transport efficiency of the slurry particle through the sample introduction system and the atomization/excitation efficiency of that particle in the source must be identical with those of a solution. If these criteria are fulfilled then simple aqueous calibration may be used and precision of analytical results may be attained.

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2.3 Slurry concentration Another important factor in the slurry technique is the slurry concentration. Slurries can be diluted, but only within a limited range as the precision may be degraded with slurries that are highly diluted. This is because of the smaller number of particles in the total volume which remain after dilution has been performed. Another factor to be taken into account is the increase in the matrix effects that can arise when the slurry concentration is increased. Conventionally, acid dissolution procedures produce solutions with a 1% sample content. Clearly, the ability to use slurries with a sample content of 20% or more yields major advantages in trace analysis. 2.4 Chemical (matrix) modification Sltm'y sampling in atomic spectrometric techniques allows the use of chemical modifiers. However, the matrix inferences are problematic owing to the different forms of the aqueous standards and biological sample suspensions. The interaction between the chemical modifier and the particles of the solid sample is closer than for direct solid sampling. Most of the work on chemical modification for the slurry technique has been carried out in order to stabilize highly volatile elements such as Cd and Pb.

2.5 Calibration techniques Different calibration methods can be employed for the direct analysis of slurries. Simple aqueous calibration may be used successfully. This technique has been used by most analysts who have achieved the desired mean particle size (<2 ~tm) and stability of the slurry. It is possible with the slurry technique to calibrate by standard additions with aqueous solutions. However, this technique assumes identical transport characteristics between solutions and slurries. When reliable solid standards are available, with certified values for the elements to be determined, and with matrices corresponding to those of the clinical and biological samples, measurements against such materials are to be preferred. Unfortunately, the selection and the availability of such standard reference materials, in particular for the analysis of trace components, are limited with respect to both materials and elements. Alternative calibration methods in emission spectrometry can be used when analyzing slurries to improve analytical accuracy and precision. This can be done by employing an internal standard, use of empirical correction factors and use of intrinsic internal standarization.

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2.6 Precision and accuracy

Several factors may affect the measuremem precision when analyzing slurry sample preparations. One of them is the small amount of the solid sample used to make the slurry, which in fact, must be homogeneous and representative of the whole material. Others are an even distribution of particle sizes which is necessary in the mixed slmay, inherent uncertainties in the measurement technique or varying degrees of the dissolution of the analyte into the solvent. The last may depend on various parameters such as particle size and time between sample preparation and analysis. In general, the errors can be minimized by working with smaller particle sizes, larger total masses of material and narrower particle size distribution. The accuracy of the present method for the analysis of clinical and biological samples can be checked by different approaches. These include: recovery test and standard addition, use of independent analytical methods of proved validity and verification of the method by means of standards or certified reference materials, the latter two methods are mostly applied. In the specific case of clinical and biological samples, a variety of CRMs such as those issued by the NIST (USA), IAEA (Austria) or NIES (Japan) are available. In consequence, the accuracy of the present technique can be mainly checked against these standards. 2.7 Nomenclature

The term "slurry sample introduction" is widely utilized in the literature on this subject; hence "slurry sampling technique/method" will be used throughout this chapter. It should be noted that descriptors such as slun3, nebulization, slurry atomization, slurry formation, slurry injection, slutTy analysis and direct dispersion used be numerous authors occur frequently in the literature and are synonymous with the term slurry sample introduction, and are occasionally used here to avoid confusion. 3. SLURRY SAMPLE INTRODUCTION As mentioned earlier, a convenient way to introduce solid material into the measurement cell of atomic spectrometry is to prepare a slurry, suspension or an emulsion. The sample is not digested as in conventional sampling but is finely ground and suspended in a solvent and is thus introduced as an aerosol of fine, hydrated, solid particles. Conventional liquid sample handling devices such as autosamplers and pipets may then be used to inject material into the atomic sources for analysis. Therefore, slurry sample introduction has been implemented using a variety of atomic spectrometric techniques.

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In the literature survey presented here, the main characteristics of the slurry sample introduction method will be emphasized. A brief discussion of subgroups of these methods, identified by the atomic spectrometric unit, will be presented.

3.1 Atomic Absorption Spectrometry (AAS) Most of the work done on the slurry sampling method applied to clinical and biological samples is based on atomic absorption spectrometry (AAS) using either flame (FAAS) or electrothermal atomization (ET-AAS), the latter is better adapted to ET-AAS and more and more applications of this technique appear in the literature.

3.1.1 Flame atomic absorption spectrometry (FAAS) Conventional flame nebulization systems are prone to blocking when slurries of large particle size are continuously nebulized. The effect is partially attributed to sample transport effects. Solid particles and agglomerates generated from slurries with large particle sizes will be rejected as they pass through the spray chamber. The poor analytical response with slurries is also explained, in part, by incomplete atomization of the particulates when they reach the flame. Flame composition and flame temperature have also a significant effect on the atomization temperature. However, some authors [25-29] have used methods of atomic absorption spectrometry based on atomization flame. It appears from the literature that few determinations have been made on biological materials with this technique. Fry et al. [25,26] described a direct, clog-free production of high density finely dispersed aerosols from tissue samples through use of Babington nebulizer. A conventional air-acetylene slot burner and the method of transient nebulizer sampling were used to avoid burner slot clogging. Excellent agreement was found between the results of the overall 5 min slurry atomization procedure and those of lengthy conventional dry/wet ashing methods for the determination of trace elements in diluted (13% solids) homogenate. A particle-size independent method for the determination of calcium in powdered oyster tissue was proposed [27]. The procedure was based on treatment of the powder with acids to solubilize the elements to be determined and dilution with distilled water to obtain a solid-in-water suspension, which was directly aspirated into the flame using aqueous solutions as standards. Results were similar to those obtained by FAAS aiter dry ashing the samples. Methods for the determination of major metals (Ca, Cu, K, Mg, Na, Zn) in human scalp hair have been developed by FAAS, using high performance nebulizer [28]. The use of wetting agents was only found adequate for dilute slurries, and thus it use cannot be considered a good approach for general purposes. A total mobilization of the analytes to the liquid media of the slurry can be reached

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with the use of nitric acid. The developed methods can therefore be considered as alternative procedures for the determination of major elements in human hair based on wet acid or alkali digestions. A method, based on slurrying of solids, is described for the direct, routine flame AA spectrometric trace determination of Cd, Cu and Zn in solid biological samples [29]. The sample powder (40 - 100 mg) is firstly wetted with a 0.5% sodium hexametaphosphate solution and is then dispersed in a 5.6% v/v nitric acid solution. Both magnetic and ultrasonic stirring were evaluated. The latter proved to be the more effective especially when dealing with the higher concentration suspensions.

3.1.2 Electrothermal atomic absorption spectrometry (ET- AAS) Electrothermal atomization atomic absorption spectrometry (ET-AAS) is a highly sensitive technique for element determination, typically 1 0 - 100x lower detection limits than conventional flame AA. Atomization efficiency obtained from a slurry with ET-AAS seems not to vary with particle size as that observed with FAAS atomization. Greater tolerance to particle size of electrothermal atomization may be one of the reasons of wider applications. Hoenig and Van Hoeyweghen [30] suspended powdered lyophilized animal tissues in a mixture of glycerol, MeOH and HNO3 together with matrix modifier for the determination by platform ET-AAS. The suspension was stable for about l h. The procedure was shown to give reliable results for the determination of Cd and Pb in eight NBS and IAEA SRMs. According to Olayinka et al. [31] solid samples can be reduced to a median diameter size < 44 ~tm with a pestle and mortar to prepare samples for slun'y atomization by ET-AAS. Results within experimental error were the same whether determined by wet- and dry-ashing procedures or slurry sampling. In situ oxygen ashing in ET-AAS was considered to be of critical importance for success of the determination and reduction of the spectral background and avoiding the build up of carbonaceous residue in the tube. The procedure was applied to the determination of Cd in animal tissues. In contrast, the method consisting of partial wet oxidation of small samples with conc. H2SO4 and subsequent direct analysis of the carbonaceous slurry thus formed, was employed for the determination of Cd and Pb in animal tissues [32]. Suspensions of SRMs in 0.01% v/v Triton X-100 were analyzed for Cu by Ebdon and Evans [33] using ET-AAS. Powdered solids prepared as slurries were analyzed by Miller-Ihli [34] using continuum source multi-element AAS (SI-MAAC) with electrothermal atomization. The slurries were prepared from 5 - 10 mg of sample in 5 ml of 5% v/v HNO3 containing 0.04% m/v Triton X-100 as a wetting agent. Slurries were homogenized with an ultrasonic probe just prior to sampling. With platform

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atomization and measurement of integrated absorbance, matrix effects were minimal for the determination of trace elements allowing calibration with standards in 5% HNO3 solution. The technique was tested with a range ofNBS SRMs. A conventional autosampler was modified to permit magnetic stirring of the slma3, samples in the autosampler cups [35]. Stirred slurries can be accurately and precisely pipetted into a graphite furnace up to concentrations of 5% m/v. Above this, systematic errors occur. The procedure involves calibration with aqueous standards, palladium matrix modification and platform atomization. The Perkin-Elmer ultrasonic agitation system for slurry sampling has been described by Jordan and co-workers [36] with examples of its application to biological samples. Ebdon et al. [37] explored the advantages of using an air-ashing stage in the analysis of slurries by ET-AAS. Higher ashing temperatures were possible (e.g., for Pb) allowing removal of organic material and hence reducing background absorbance from smoke. It also reduced chemical interferences. Slurry sampling was used by Robles et al. [38-42] in a preconcentration procedure using bacteria, which retained toxic elements in the pH range 4 - 10. The bacteria were dispersed in dilute HNO3 and the slurry were introduced directly into the graphite fiamace. It would appear that the slurry sampling and preparation procedure was identical in nearly all cases. A method for the selective determination of Sb m and Sb v in liver tissue using ET-AAS was described by Rond6n et al. [43]. Lyophilized liver tissue was slurried with 0.5 mol 1l H2SO4- 10% K I - 0.1% ascorbic acid for quantification of total Sb, or with 1 M CH3COOH for quantification of Sb m. The slurry samples were microwave digested and reacted with the aforementioned acids and 0.1% NaBH4. The stibine generated was carded in an N2 stream to a 10 cm quartz tube heated in an air-CEn2 flame. Antimony (V) concentrations were calculated from the difference between total Sb and Sb m concentrations. The use of microwave energy proved to be beneficial for the release of both antimony species from the matrix. High-pressure homogenization was evaluated for the preparation of slurries suitable for the determination of trace elements by ET-AAS in soit organ tissues and CRMs of biological origin [44-47]. The results indicate that high-pressure homogenization is capable of generating emulsions/dispersions of samples. Attractive features of this approach include the speed (60 s blending followed by four passes through the homogenizer, total less than 3 min) and simplicity of sample processing and the apparent stability of the preparation. Another instntmental modification for the analysis of slurries, was described by L6pezGarcia et al. [48]. This modification to an autosampler allows argon gas to bubble directly into the sample cup to homogenize the sample as aliquots are removed to the atomizer for analysis.

246

H. MATUSIEWICZ

A major series of studies have been carried out by Bermejo-Barrera et al. [49-55]. Slurry sampling was successfully applied to aluminum, Cd, Cr, Hg, Mn, Ni and Pb hair determination as alternative methods to introduction of solutions in ET-AAS. As the slurry preparation is rapid, only 20 min are necessary to pulverize the hair samples and to reduce the particle size, glycerol was added as a stabilizing agent, the proposed method is attractive for routine analysis in clinical determinations. The effect of different inorganic constituents was studied and no significant interferences were found. However, Meeravali and Kumar [56] found that slurries of biological CRMs were not stable for most elements in glycerol but were stable in 5% H N O 3 , which extracted a high percentage of the elements Cd, Cu, Mn and Pb that they studied. Chen et al. [57] claimed that the homogeneity and stability of the results obtained for the determination of Se in serum is improved when ultrasonic mixing of the samples takes place prior to injection. However, in this author's opinion, ultrasonic mixing of serum samples for the determination of Se is not necessary, but the results found by these workers are interesting. Cadmium in solid biological CRMs was determined by Fuyi and Zucheng [58] using ET-AAS aiter slurrying the sample with PTFE, H N O 3 and plant glue solution using ultrasound to help mix and extract the cadmium. The PTFE acted as a chemical modifier. Amoedo et al. [59] considered the extraction of Pb from solid samples using ultrasound with subsequent analysis of the slurries. With this sampling technique, sedimentation and volumetric errors inherent in the slurry technique are avoided. For samples in which non-quantitative analyte extraction occurs, the ultrasonic slurry sampling technique must be used in order to achieve accurate results.

3.1.3 Flow injection techniques Flow injection (FI) techniques complement atomic spectrometric approaches to trace analysis. Economy of sample, increased throughput and enhanced detection power coupled with the ease of replicating precise chemical manipulations are attractive features which have been utilized in conjunction with AAS techniques. In recent work [60], the slurry formation by sonication and FI cadmium vapor generation and AA detection has been proposed as a rapid and effective technique to determine Cd in different solid samples. It is worth to mention here as a main achievement the fact that Cd is readily leached out from almost all solid samples studied even with diluted acids. This method was applied to the determination of Cd in krill and human hair.

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3.2 Flame Atomic Emission Spectrometry (FAES) A method for the determination of potassium in powdered milk is proposed [61 ]. The procedure consists in weighing 0.1 g of milk, adding 10 ml of a solution of La (4% m/v) and dispersing the mixture in water to give a volume of 100 ml. The dispersed samples are then directly introduced into an air-C2H2 flame and K is determined by FAES. Methods for the determination of major metals in hair have been developed by FAES, using a high performance nebulizer [28]. 3.3 Direct Current Plasma (DCP) There was very little work in this area. The only publication of any great novelty described the adaptation of Babington nebulization and the more general method of slurry atomization to DCP spectrometry [62]. The final system reported herein has vimmlly no remaining points of restriction through which suspensions of homogenates must pass. If sufficient sample volume is available, continuous nebulization of homogenates may be used without clogging, which will allow longterm electronic integration for the purpose of detection limit improvement. The system was applied to the direct nebulization and aerosol transport of homogenized solid samples such as animal tissue. Other useful published applications include the slurry atomization to DCP spectrometry (Beckman Spectrospan dc plasma spectrometer) and determination of elements in milk [63]. 3.4 Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) Introduction of solid samples as slurries into ICPs received attention [64-67]. Fagioli et al. [64] used carbonaceous slurries which are prepared chemically rather than by grinding, avoiding the contaminations which may be introduced during the grinding process. Particle size below 5 Jam was obtained for six different SRMs. The method required 2h, so it does not yet rival with microwave digestion in terms of speed; however, it may be a valuable alternative if the investment for a microwave digestion system cannot be made. A simple and rapid method was developed for the determination of elements in maternal milk samples [65]. The emulsified samples were diluted 10-fold with HNO3 and to 1% v/v. Aqueous solutions with the same amount of emulsifier and acid were used as calibration standards. The accuracy of the proposed method was assessed using the NIST SRM 1549 Non-Fat Milk Powder. In other work [66], samples of milk and infant formula were prepared as slurries and analyzed using aqueous standards. The instrument incorporated a charge transfer device detector, which allowed simultaneous acquisition of multi-element and multi-line measurements.

248

H. MATUSIEWICZ

Matrix removal and preconcentration of metals from liquid samples were obtained with a solid-phase extraction/slurry sampling procedure [67]. A polymeric, adsorbing resin, dispersed in the sample, was used for total sorption of analytes previously complexed with a dithiocarbamate ligand. After filtration, the resin was recovered and re-dispersed by means of a non-ionic surfactant; metals retained were determined with the slurry sampling technique. All parameters were optimized to yield a solution-like signal fi'om the final dispersion. 3.5 Inductively Coupled Plasma- Mass Spectrometry (ICP-MS) Inductively coupled p l a s m a - mass spectrometry (ICP-MS) has become accepted as a powerful tool for elemental and isotopic analysis. The technique is characterized by a linear dynamic range of 5 - 6 orders of magnitude, rapid multielement analysis, comparative freedom from spectral interferences and detection limits in the range of 10 - 100 pg ml~ for most elements. This makes it ideal for the multi-element analysis of milk on a routine basis. Dried milk powder is naturally suited to slurry analysis, as the product is spray-dried during production and has a particle size of <5 lam. Dean et al. [68] used ICP-MS to determine Pb isotope ratios in samples of Australian and European dried milk powders (0.25 - 1 g)) by direct slurry nebulization of the powders suspended in a dilute solution of Triton X-100 to which Mg was added as an internal standard and using one or two drops of ammonia solution to prevent the precipitation of protein from the milk. The slurried suspension was continuously stirred and introduced via a peristaltic pump and a high-solids nebulizer. Excellent results were obtained for A1 and Pb. In the case of Pb, the stable isotope ratio was also measured in the slurry using isotope dilution analysis ICP-MS. Sample contamination and sample heterogeneity represent two major disadvantages with slurry atomization. The application of slurry nebulization/iCP-MS to trace elemental analysis of biological samples has been investigated [69]. Three standard samples of the NIST were dispersed in 1% aqueous Triton X-100 solution by grinding with a planetary micronizing mill. The resulting slurries were nebulized into an ICP without any additional treatments. The 1% (m/v) slurry of the NIST Bovine Liver showed no significant influence on cone blockage and signal suppression/enhancement. However, one of the fundamental difficulties in the application to biological samples is their fibrous nature and/or fat content.

3.6 Microwave Induced Plasma-Atomic Emission Spectrometry (MIP-AES) Given the generally poor tolerance of M1Ps to high total sample loadings and their poor performance as an atomizer, it is intriguing to note that one paper has even reported the introduction of slurries into an MIP [70], despite the significantly lower kinetic temperature of this plasma compared with most ICPs. The use of

Slurry Sample Introduction in Atomic Spectrometry

249

slurry sample introduction has been reported by Matusiewicz and Sturgeon [70], where a V-groove nebulizer was used to introduce suspensions of tissue reference material to an MIP. Direct calibration with aqueous standards gave recoveries of 40 - 70 %, the use of correction by standard additions gave acceptable recoveries in agreement with certified values for Cd, Cu, Fe and Zn. 3.7Atomic Fluorescence Spectrometry (AFS) The analytical attraction of AFS to analysts lies in its potential, in favorable circumstances, for very great sensitivity and a long linear range. The contribution of AFS to the determination of elements by slurry sampling atomic spectrometry is, however, very modest, primarily because it is very vulnerable to matrix interferences. The difference between slurry sampling and slurry digestion seem to be becoming less clear. In recent work [71], an on-line microwave digestion system for the determination of total mercury in solid biological samples was developed. The slurried samples were injected into a carder stream of HC1 and mixed with a solution of potassium bromide-bromate before passing through a PTFE coil situated in the microwave cavity of the microwave digester. Aiter mixing with a solution of hydroxylammonium chloride, the determination of mercury was undertaken by on-line cold vapor atomic fluorescence spectrometry (CV-AFS). Method was validated using the NRCC CRM DORM-2. An unmodified graphite fiamace utilizing from-surface illumination has been investigated as an atomizer for AFS using laser for excitation [72]. Biological solid NIST standard reference samples (bovine liver, non-fat milk powder) were dried and vortexed or ultrasonically mixed with 5% HNO3 containing 0.04% of Triton X100 to form a slurry. In general, results (determination of Mn, Pb, T1) agreed with certified values. 3.8 Thermal Vaporization (TV) Techniques TV (electrothermal and in-torch vaporization) has developed into a popular tool for the determination of trace elements in recent years. This technique allows the use of slurries and permits the possible separation of matrix constituents using particular temperature and time components before transportation of the analyte into the plasma excitation sources via a stream of gas. Several papers on slurry sampling TV coupled with ICP-AES have been published, in addition to the use of ETV'ICP-MS, which offers superior limits of detection. Long and Snook [73] were the first to utilize this technique in the form as practiced today. The major element composition of vitamin pills formulated as multimineral capsules were determined by vaporization of a 10 ~tl slurry into an ICP via a polyethylene connecting tube (0.5m) from a resistively heated graphite

250

H. MATUSIEWICZ

rod ETV device, contained in a 1 1 cylindrical glass manifold. They examined the following parameters" sample transport, effect of viewing height and plasma operating power, and detection limits and precision. In spite of the dilution of picogram amounts of vaporized sample in the large-volume manifold, adequate signals were produced in the plasma. A further extension of their work, volatilization of refractory elements, and compounds via their more volatile metal halides, has recently been reported. Bin et al. [74] and Chen et al. [75] have used PTFE as a slurry fluorinating reagent in order to avoid the formation of refractory carbides by converting the analytes into their corresponding fluorides and to facilitate a direct analysis (slurry direct injection into the graphite furnace) of refractory elements in different biological matrices by ETV-ICP-AES. Karanassios et al. [76] evaluated several vaporization chambers for the introduction of micro-samples to an ICP. The sample was deposited onto a Re filament forming a three-coil loop and inserted into the vaporization chamber attached to the ICP torch. The feasibility of direct elemental analysis of powders as slurries has been demonstrated. Example include sample of biological origin (bovine liver). A fttaher extension of Karanassios work [76] has been reported [77]. A new direct sample insertion (DSI) device that can be used vertically with ICP-AES has been designed, and an automated ICP-AES system has been developed around this DSI device. Sample handling, weighing, and homogeneity concerns were addressed by using slurries. Slurry DSI-ICP-AES for biological powdered reference materials was described. Carbide formation is a key chemical limitation of graphite-cup DSIs, and it was addressed by using in situ chemical modification by mixing SF6 with the plasma gases. Ultrasonic slurry sampling (USS)-ETV-ICP-MS has been applied to the determination of Cd, Cu, Hg, Pb and Zn in CRMs of biological origin [78,79]. Palladium was used as modifier to delay the vaporization of mercury in this study. Results of analyses by use of the standard addition method agreed satisfactorily with the certified values. The method should be useful for the direct analysis of other volatile elements in various solid samples. Lanthanides are not an obvious candidate for ETV as they are refractory but Buseth et al. [80] showed that the addition of Freon-23 (CHF3) to the Ar purge gas lowered the vaporization temperature and reduced memory effects. A method was developed for the determination of endogenous concentrations of the lanthanides in tissues with ETV-ICP-MS. Analyses of undigested biological tissues (oyster tissue, bovine liver, human hair) by slurry sampling by direct injection were performed. For undigested samples, hydrogen peroxide injected directly onto the sample inside the graphite furnace was found to be effective in preventing the build-up of ash in

Slurry Sample Introduction in Atomic Spectrometry

251

the furnace. The analytical results for slurry and digested samples of the same material were similar with respect to accuracy and precision. Application of the magnetron rotating DCP with graphite furnace sample introduction to the analysis of suspension samples was described [81]. Analytical data were presented for a number of NIST biological reference materials. 4. ANALYTICAL FIGURES OF MERIT The analytical performance of slurry sample introduction- atomic spectrometry is characterized by figures of merit, such as detection limit, linear dynamic range, and precision and accuracy of measurements. It is usual practice to quote the detection limit pertaining to a particular technique or method, and to draw comparisons between the detection limits obtained using similar techniques. The detection limits for slurry sample introduction- analytical spectrometric techniques are summarized in Tables 1-3. This approach was adopted became the range of reported values is a reflection of differences in instrumental applications and a variability in the slurry samplingspectrometric technique capability. Detection limits are presented in terms of both mass or concentration to simplify comparison. The compiled data refer mainly to the determination of the elements in pure aqueous solutions containing only the analyte in question. Became of this simplification, any application of the data to practical trace analysis must be subject to some restrictions. The limit of detection is only one of several figures of merit characterizing a technique and should not be used alone as a criterion of choice. Nevertheless, the data compiled here may be useful as an initial survey of the effectiveness of slurry sampling atomic spectrometric techniques with respect to the determination of trace levels of these analytes. In addition, became of the wide range of slurry sample introduction techniques and atomizers/excitations and the resulting differences in optimized experimental conditions, it is very difficult to accurately compare published data with regard to analytical performance of the slurry s a m p l i n g - atomic spectrometry. Moreover, there is much confi~ion over the definition of the term "detection limit", so users of such data as shown in Tables 1-3 and in the literature should always check the definition applied in the original papers. Acceptable precisions in most instances, reported as percent relative standard deviations (RSDs) usually range from 1% to slightly higher than 10%. Thus, in general, slurry sampling elements can be detected at concentrations below 1 ng ml" 1 and concentrations than are 10 or more times the detection limit can be measured with precisions less than 5% RSD. The accuracy of the present method for the analysis of clinical and biological samples has been checked by different approaches. These include: recovery test

252

H. MATUSIEWICZ

Table 1 Summary of detection limits for slurry sampling with AAS and EAS

Elem. A1 As Au Be Ca Cd Cr Cr Cs Cu Fe Hg K Mg Mn Mn Na Ni Pb Sb Se Si Zn

FAAS ng/ml Ref.

5000

ng/ml 900 22 0.004 0.05

ET-AAS pg 23

Ref. 53 86 38 39

15 3

87 82 83 82 82

100

55

1

1.7

3500

FAES ng/ml Ref.

28 0.017 0.75

3500 3100

FI-AAS ng/ml Ref.

28 28

0.6

60

20000

28

0.6

28

28

28

20

56 82

273.3 0.097 0.1 0.1 30 500

52 85 43 41 84 82

Slurry Sample Introduction in Atomic Spectrometry

253

Table 2 Summary of detection limits for slurry sampling with plasma sources

Elem. .

ICP-AES ng/ml Ref. ,

Ag A1 As Br Ca Cd Ce Co Cr Cu Eu Fe

23

ICP-MS ng/ml 0.002 0.16 0.01 0.37

Ref. 69 69 69 69

AFS

0.006 0.003 0.007 0.24 0.01 0.001 0.34

69 69 69 69 69 69 69

8.9

65

7.9

65

12000

64

23 300

65 64

9000

64

55

65

50

Ref.

0.013

71

65

Hg K La Mg Mn Mo Na Ni P Pb Rb Sb Se Sr Th U Zn

ng/ml

65

0.004 0.25 0.03 0.01

69 69 69 69

0.20

69

0.01 0.01 0.002 0.02 0.001 0.0006 0.0001 0.52

69 69 69 69 69 69 69 69

H. MATUSIEWICZ

254

Table 3 Summary of detection limits for slurry sampling with TV

Elem.

n

m

Be

Cd Cd Ce

278

Cr CU

13 4.7

ETV-ICP-AES Pg

ns/ml

0.1

75 76

3

89

10

0.0 1 0.07 3.2 0.21 0.005 0.003 0.008 0.04 2 0.0 1 0.002

80 89 89 89 80 80 80 80 78 80 80

0.06

89

0.01 0.24 6 0.006 0.0 1

80 94 93 80 80

0.002

80

0.0009

80

0.006 200

80 79

co 75 75

DY Er Eu Gd Hg Ho Lu Mg

Mn Mn Mo Nd Ni Pb

3 10

0.3 0.7

76 75 76 74

15

76

0.08

76

Pr Sm Sr

Tb Ti

6.3

75

Tm V Yb Zn

ETV-ICP-MS Ref

Ref, 76

10 10

76 76

Slurry Sample Introduction in Atomic Spectrometry

255

and standard addition, use of independent analytical methods of proved validity and verification of the method by means of standards or certified reference materials (CRMs), the latter two methods being mostly applied. In the specific case of clinical and biological samples, a great variety of CRMs such as those issued by the NIST (formerly NBS) and IAEA are available. In consequence, the accuracy of the present technique has been mainly checked against these standards. From the survey of the literature it is evident that the accuracy of the slurry sample introduction technique compares favorably with the accuracy of other techniques for these kind of materials. Linear dynamic ranges for the slurry sampling- atomic spectrometry vary from two to five orders of magnitude, depending on the particular method used. Since the figures of merit for solution nebulization are comparable to those for slurry nebulization, and the operating procedure is simpler, slurry sample introduction is the present "method of choice" for sample introduction of the elements. 5. PRACTICAL APPLICATIONS OF SLURRY SAMPLE INTRODUCTION Illustrative applications of slurry sample introduction in atomic spectrometry have been summarized in Tables 4 - 6 at the end of this chapter. These applications are listed for sample type, and elements determined, the analytical technique used and dispersant employed to stabilize the slurries, homogenization technique and particle size, the methodological approach are given. In addition, the tables includes the reported standard deviation. The RSD is only an informative value and does not differentiate between in-slmTy and between-slurry precision, because this is often not stated in the literature an because of very variable numbers of individual measurements. The aim of this section is to examine representative publications, not merely to present potential users with established methods, but rather to point to the reasons why slma3, sampling/atomic spectrometry has been used to solve particular problems and to stimulate further interest in its application. The references cited may contain additional determinations, or trials, for a particular sample type. A wide range of applications is clearly evident, showing that slurry sampling is applicable widely throughout clinical and biological analysis. The variety and number of samples indicate that future studies involving slurry sample introduction would be readily applied to the analysis of more complex samples.

256

H MATUSIEWICZ

6. CONCLUSION Several sample preparation methods unique to each instrumental technique utilizing both AAS (flame, furnace) and AES (plasma atomization using either optical or mass spectrometric detection) exist for the elemental analysis of clinical and biological specimens. In the last decade increasing attention has been placed on the development of slurry sample introduction techniques into flames, furnaces and plasmas. Thus, this sample introduction method should complement conventional pneumatic nebulization for solutions and solid amounts of materials. The technique, though being inexpensive and holds considerable promise; is in the author's opinion yet to be firmly established. It is evident from this review that there is considerable research being done on the problem of introducing different clinical and biological sample types into various sources. It would be fair to say that most of the fundamental parameters and requirements of this sampling technique have been established and virtually all of the work examined in this chapter mainly concerns applications (reports are summarized in Tables 4-6). There have been many different procedures described to generate a slurry for sample introduction into an absorption or emission sources; however, based on this review, three procedures are the most popular. These are: suspension in a suitable solvent; mixing using either a magnetic stirrer or an ultrasonic bath/probe (in the absence of stirring, the settling of the solid in the water-suspended samples may be also overcome by the preparation of a more stable slurry by using a viscous medium or thickening agents); or automatic generation of the slurry with an ultrasonic probe attached to the autosampler. Chemical modification to minimize the effects of the matrix components is easy to perform in the slurry technique, because of more effective contact between the sample particles and the modifier. The increasing tendency of using direct calibration with aqueous standards and rapid procedures (slurry analysis is as fast as analysis of solutions) with a little sample preparation as possible would be useful in hospital and clinical laboratories (application of clinical diagnosis or biological monitoring) and makes the slurry method very promising, mainly when certified reference materials are not available and also the time-consuming standard addition method should be avoided. The advantage of the method is its freedom from time-consuming steps and potential contamination associated with sample preparation (i.e., decomposition) and the freedom from the loss of volatile elements in the sample preparation process (no volatilization). It has repeatedly been shown that only very fine granulometry of the slurry may ensure correct results" concerning the representativeness of the sampling, it is evident that the presence of large particles in the sample was found to be the most critical factor in the analysis. The importance of an intensive grinding of the sample prior to the analysis is thus obvious.

Slurry Sample Introduction in Atomic Spectrometry

257

In general, detection limits are better than those obtained with conventional pneumatic nebulization. The reported overall precision is satisfactory, 1 - 30%, but in most instances is worse than that of solution nebulization with absorption and emission source analysis. Some of the authors reporting highly poor precision have found their error sources and most of them have suggested ways to overcome them. Because slurry sampling-DCP/ICP/MIP is used in an emission mode, simultaneous multielement detection is possible. Very few observations have been reported concerning the speciation of solid samples, thus making it difficult to draw any conclusion on this matter, although encouraging preliminary results were obtained for the speciation of solid samples. However, there is little widespread use of slurry sampling-absorption/emission source spectrometry with application to practical samples for speciation. The present approach allows direct sample analysis, that is, no sample preparation is needed. Furnace vaporization techniques has shown that ETV-ICP-AES/MS can be used successfully for the direct analysis of slurries and that the technology developed for the analysis of slurries by ET-AAS is directly transferrable to ETVICP-AES/MS. Clearly, the future of ETV-ICP/MIP applied to the analysis of slurries is promising. The present technique may be subject to a number of positive and/or negative systematic errors, which depend on the element to be determined, the analytical instrumental technique, the matrix composition and other factors. However, it appears from the survey of the literature that the slurry sample introduction technique compares favorably with the accuracy of other methods for the determination of trace (and major) elements in clinical and biological materials. 7. SUGGESTIONS FOR FUTURE STUDIES The slurry analysis capabilities should encourage their adoption and be consistently useful in analytical atomic spectrometry. It seems probable that most future developments will arise in trying to minimize blank levels for samples which must be ground. The challenge for slutTy sampling remains in the area of rapid particle size reduction without "wear-metal" contamination from homogenizer, mill, or bead methods of slurry preparation. The cryogenic preparation procedures as an alternative to wet grinding should be considered in some situations. The ultimate goal is development of a fast, accurate procedure which can handle large numbers of heterogeneous solid clinical and biological samples efficiently, which is free of contamination, and which can be calibrated with nothing more than an aqueous standard.

258

H. MATUSIEWICZ

In the case of ICP-AES/MS or MIP-AES, the electrothermal vaporization is gaining momentum and has the potential to become a useful sample introduction system for ICP/MIP, in particular, when only micro-samples are available for analysis. Examples include samples of clinical, biological or forensic origin. Further work is required on the study of chemical modifiers which could either be used to remove matrix components and/or stabilize analytes, such that vaporization of analyte occurs after matrix components have left the graphite tube. A further area of growing interest is speciation of elements to supplement the total element figure, which will help scientists in the biomedical materials field to obtain a better understanding of the role of metals, especially toxic metals, in life processes. In situ hydride generation procedures is especially suitable for speciation work, because this approach allows direct slurry sample analysis without sample preparation. In conclusion, as time passes and the number of investigators pursuing this area increases, the author is confident that slurry sampling methods will evolve into a series of widely practiced standard procedures for clinical, biological, pharmaceutical and forensic sample analysis.

Acknowledgement The author acknowledges, with thanks, the financial support by the State Committee for Scientific Research (KBN), Poland Grant t09A 129 17.

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Slurry Sample Introduction in Atomic Spectrometry

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90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101.

Slurry Sample Introduction in Atomic Spectrometry

The following tables are representative of the applications of slurry sample introduction.

263

Table 4 Applications of slurry sampling atomic spectrometry systems to the analysis of clinical materials

Elements Instrumentation Dispersant Sample type determined used Milk Cu,Mn,Zn FAAS . . . . concentrate

Homogenization -

Particle Size/pm <5 5-9

Whole blood NBS-SRM 1577 Bovine liver

Cu,Mn,Zn

Ca,Cu,K, Human scalp hair, Mg, Na,Zn BCR CRM 397 Human hair, NRCC DORM- 1

FAAS

FAAS/FAES

Viscalex HV30 glycerol

Zirconia beads vibrating ball

0.8

Slurry approach

Ref.

i ml sample is delivered by Babington nebulizer yielding a transient signal 500 gl of sample is adequate for transient sampling, a clog-free Babington nebulizer was employed. RSD=5% Acid (1% HNO3) predigestion used as a pre-treatment stage, slurry concentration 0.20.35% m/v. Wetting agents were only

25

26

28

Dogfish muscle BCR CRM Cd 185 Bovine liver

ET-AAS

0.5% plant glue solution

Ultrasonic

Human Ni scalp hair, BCR CRM 397 Human hair, NRCC DOLT- 1 Dogfish liver NRCC DORM-I Dogfish muscle Serum, Se SRM 1598 Bovine serum Secondgeneration

ET-AAS

Glycerol

Vibrational zirconia mill ball

ET-AAS

0.5% Triton X-100

Ultrasonic probe

beneficial fox"dilute slurries. RSD=8.7-18.6% 10-35 mg sample were mixed with 1 ml 60% PTFE slurry (as modifier) and 0.4 ml HNO3. RSD= 1.9-7.8% 0.1 g hair was suspended in 25 ml H20. Mg(NO3)2 was used as chemical modifier. RSD-9.5%

To the sample 0.2% HNO3 were added. Found that absorbance decreased with time after dilution if

58

52

57

BRM of freezedried human

samples were not mixed.

serum

Human Ag,AI,Mn scalp hair, BCR CRM 397 Human hair, NRCC DOLT-1 Dogfish liver NRCC DORM- 1 Dogfish muscle Liver ET-AAS

ET-AAS

Glycerol

Zirconia vibrational mill ball

Si

0.004% Ultrasonic bath Triton X- 100 and probe

<0.8

Samples, 100 mg, suspended in water and diluted to 25 ml. Pd and Mg nitrates employed as chemical modifiers. RSD-7.9% (Al); 16.0 % (Mn)

53,90

10-800

Comparison of analysis of slurries prepared from original sample and those prepared from ashed specimens. Fewer matrix interferences observed from ashed samples. Best

84

sensitivity obtained when using Pd(NO3)2Mg(NO3)2 as modifier. Human Cd,Mn,Pb scalp hair, BCR CRM 397 Human hair

ET-AAS

Human scalp hair

ET-AAS

Cd

Bacteria" Au,Be,Cd, Escherichia Se

0.4% m/v glycerol

Vibrating zirconia ball mill

Pd-Mg(NO3)2 used as chemical modifier for Pb and Mn and Pd alone used for Cd. Aliquots of slurry were taken for analysis by "fast furnace" ET-AAS program.

0.4%

Vibrating zirconia ball mill

Washed hair was 50 pulverized and suspended (0. I g) in 25 ml H20, (NH4)2PO4 was added. Standard additions calibration was employed. RSD-2.5-6.9% The pH of 38-42 solub!lized samples

glycerol

ET-AAS

Ultrasonic

51

Coli and Pseudomon as Putida

Liver and kidney tissues, NRCC DORM- 1 and DORM-2 Dogfish muscle, NRCC DOLT-1 Dogfish liver

Cd,Cu,Pb

ET-AAS

0.25% TMAH in 10% C2HsOH

High pressure flat valve homogenizer

was adjusted to 410 and slurries of either E. coli or P. putida added. Au, Be,Cd,Se was retained on the bacteria cell walls and a slurry in diluted HNO3 was prepared for transfer into the graphite furnace. RSD=2.6 % Slurries were stable for at least 6 d. NH4H2PO4 and NH4NO3 used as modifiers. Results in good agreement with analysis by ICP-MS after acid digestion. No significant differences observed between aqueous calibration and method of analyte additions.

44

Liver tissue Sb(III), NIST SRM Sb(V) 905 and 906 Whole blood

HG-FAAS

Pb Human hair, BCR CRM 397 Human

ET-AAS

Ultrasonic

0.4% glycerol

Vibrating zirconia mill ball

Contamination from the homogenizer (Cu and Pb) caused high blanks but accurate results were obtained with CRMs. RSD=9.6% Homogenized, lyophilized specimens were suspended in 1 M CH3COOH or in 0.5 M H2SO4-10% KI0.1% ascorbic acid tbr determination of Sb m or total Sb, respectively. The slurries were sonicated and microwave heated before formation of stibine. RSD=3.0% "Fast .furnace" program used with mixed modifier of Pd-Mg(NO3)2.

43

49

hair Liver and kidney, NRCC DORM- 1 and DORM-2 Dogfish muscle

Cr, Cu,Fe, Mn, Ni,Se

ET-AAS

Hair

Cd,Cr, Mn, Pb

ET-AAS

Bovine liver NIST SRM 1577

Cd,Pb

ET-AAS

Bovine liver NBS

Cd

ET-AAS

0.25% TMAH in 10% C2HsOH

High pressure homogenizer

Ultrasonic

Glycerine, methanol

10

Stirrer

Magnetic stirrer

<44

RSD=1.7-5.3% Slurries prepared and determined as described in ref. 44. Capping flat valve head of homogenizer with a ruby disc reduced but did not eliminate contamination. Addition of EDTA to solvent increased mobilization of Fe, but increased contamination from homogenizer. Graphite cup atomizer and oxygen ashing used. RSD=2% Graphite platformtube atomizer and matrix modifier used. RSD=0.45-13% In situ ashing to avoid the build UP

45,47

83

30

31

SRM 1577, Pig kidney Bovine liver NBS SRM 1577a, Oyster tissue NBS SRM 1566 Bovine liver NBS SRM 1577

Cd,Pb

ET-AAS

Cu

ET-AAS

0.01% Triton Ultrasonic X-100

Fe Bovine liver NBS SRM 1577a

ET-AAS

0.04% Triton Ultrasonic X-100

<250

Pb Bovine liver NBS SRM 1577, kale

ET-AAS

5% ammonia Magnetic stirrer solution

<50

ET-AAS

0.04% Triton Ultrasonic

Bovine

Mn

of carbonaceous residue RSD=3.7% Partial wet oxidation with cone. H2SO4, direct analysis of the carbonaceous slurry thus formed RSD--3-6% Direct platform atomization and calibration RSD=4.9% Platform atomization, calibration standards prepared in 5% HNOa RSD=12% Calibration with aqueous standards, Pd matrix modification, platform atomization RSD=4% No matrix modifier

32

33

34

35

36

liver NBS SRM 1577a Human hair CRM 397, Dogfish liver NRCC DOLT-l, Dogfish muscle, NRCC DORM-1, human hair samples Human hair NIES 5 Human hair BCR CRM397, NIES 13, IAEA 085, human hair samples Human hair BCR CRM 397 Haemodial ysis

X-100

Slurry Sampler 0.8

or pyrolysis step was used W or Zr-coated graphite tubes as permanent chemical modifiers were used RSD=-~16%

Cr

ET-AAS

Zr vibrational ball mill

Co,Cr, Mn, Pb Hg

ET-AAS

The use of and airashing stage Pd chemical modifier was used RSD=l.8-8.8%

37

ET-AAS

Mechanical shaker Mechanical shaker

Cd

FI-CV-AAS

Ultrasonic

60

Cd, Co,Cu, Fe,Ni,Pb

ICP-AES

Ultrasonic

Samples were suspended in HCI, direct calibration RSD=6-12% Precomplexation with APDC pH 2 or

30

54

55

67

solution

1% Triton X- Magnetic stirrer 100

Bovine liver NBS 1577a

18 elements

ICP-MS

Bovine liver NBS SRM 1577 Chinese traditional medicine Loulu Bovine liver NIST SRM 1577b, Oyster tissue NIST SRM 1566a, Serum Seronorm, Human hair GBW0760 1, Blood

Mo

ETV-ICP-AES

Cd,Cr, Cu, Mn,Ti

ETV-ICP-AES

60% PTFE emulsion

Lanthanides

ETV-ICP-MS

0.004% Ultrasonic Triton X- 100

Ultrasonic

Ultrasonic

4.5 and adsorption of analytes on Amberlite XAD-2 The PTFE Babington-type nebulizer was used for the introduction of slurries PTFE was used as a fluorinating agent RSD=3.2% PTFE emulsion was used as a fluorinating agent RSD=1.5-4.0% H202 injected directly onto the sample inside the GF to prevent the build-up of ash. Freon-23 was used as an chemical modifier RSD=<25%

69

74

75

80

samples Dogfish liver NRCC DOLT-l, Dogfish muscle NRCC DORM-2, Oyster tissue NIST SRM 1566a Dogfish muscle NRCC DORM-2, Oyster tissue NIST SRM 1566a, Bovine liver NIST SRM 1577b Bovine liver NIST SRM 1577a Dogfish

Cd,Cu,Pb, Zn

ETV-ICP-MS

0.1% Triton X-100

Ultrasonic

Partial decomposition with HNO3

79

Cd,Pb,Se

ET-AAS

0.1% Triton X-100

Ultrasonic

Suspensions prepared in a medium containing 30% H202, 1% HNOa and matrix modifier RSD=--.3%

91

Mn,Zn

ETV-ICP-AES

76

Pb

ET-AAS

In coiled-filament in-torch vaporization sample introduction International

0.005%

.

.

.

.

.

.

.

94

t,,a

liver NRCC DOLT-1 Milk powder SRM A-11, Milk samples Dogfish muscle NRCC DORM-2 Dogfish liver NRCC DOLT-l, Dogfish muscle NRCC DORM- 1 Bovine liver NIST SRM 1577b, Bovine liver BCR SRM 185, Pig kidney BCR CRM 186

collaborative study

Triton X- 100 ET-AAS Cd,Co,Cr, Cu,Fe,Ni,Pb

0.1% Triton X-100

Ultrasonic

A mixture of HNO3-H202 was used

96

Pb

ET-AAS

0.1% Triton X-100

Ultrasonic

97

Cd

ET-AAS

0.2% Triton X- 100

Mechanical stirrer

Pd and ammonium nitrate were used as the modifier RSD=I6% (NH4)2HPO4 was used as the modifier

Cd,Cu,Pb

ET-AAS

0.04% Triton Ultrasonic X-100

10

<30

W-Rh permanent modifier was used

98

99

Dogfish muscle NRCC DORM-2, Lobster tissue NRCC TORT-2

As

ET-AAS

0.01% Triton Ultrasonic X-100

Automated ultrasonic slurry sampling and ultrasound-assisted extraction were developed RSD=l-6%

100

Table 5 Applications of slurry sampling atomic spectrometry systems to the analysis of biological materials .

.

.

.

.

.

,

,

,

Sample type IViilk and infant formula NRCC TORT- 1 Lobster tissue

Elements lnstrumentat. determined Ca, Cu,Fe,K, " ICP-AES Mg,Mn,Na,P, Zn Cd,Cu,Fe,Zn MIP-AES

Milk powder,

Cd,Cr, Cs,Cu, Mo,Pb,Zn

ET-AAS

Ca,Cu,Fe,K, Mg,Mn,Na,P, Zn

DCP-AES

IAEA- 11

Milk Powder Milk

i

.

Dispersant used

10% HNO3

-

.

.

.

.

.

.

.

Homogenization

.

.

.

.

,

Particle Slurry approach size/gin ......... Simultaneous analysis of slurry samples Ultrasonic A 1% slurry of lyophilized sample was fed into an MIP via a V-groove Babington nebulizer. Good agreement with certified results. RSD=4.4% (Cd); 3.5% (Cu); 6.9% (Fe); 4.6% (Zn) Stirring Samples (0.5 g) were mixed with 3 ml H20, then heated to 50 ~ RSD=5-20% <8 '

Ref. 66

70

82

63

Ca,K,Mg,Na Milk powder, IAEA- 11 Milk Powder Maternal Ca,Cu,Fe,K, Mg,Zn milk, NIST SRM 1549 NonFat milk powder

FAAS, FAES

Genapol PF 10 surfactant

ICP-AES

Ethoxy nonylphenol (0.03% m/v)

Dogfish muscle NRCC DORM-2

Hg

CV-AFS

NRCC LUTS-1 Lobster tissue

Co,Cr,Ni,Pb

ETV-ICPMS

Ultrasonic

<50

NRCC

Cu

ET-AAS

Ultrasonic

<125

Sample (0.1 g) was mixed with water and solution of lanthanum (4% ~w'v) The maternal emulsified milk samples were diluted 10-fold with H N O 3 (1% v/v). Good accuracy was obtained. RSD=0.3-2% Batch open focused microwave digestion and an online microwave digestion procedure were developed RSD=l.5% Reference material was provided as 10.3 g o f homogeneous slurry. Method of standard additions was applied. Sonochemical

61

65

71

89

l 01

DORM-2 Dogfish muscle

t,~

'~

NIST-SRM Cd,Cu,Zn 1566 Oyster tissue NRCC TORT-1 Lobster tissue NIST-SRM As 1566a Oyster tissue

FAAS/ICPAES

0.5% m/v (Na3PO4)6

ET-AAS

0.1% m/v Ultrasonic, Triton X- 100 manually stirring

Magnetic, ultrasonic

<20

extraction of the analyte from slurried sample was advantageous over slurry sampling RSD=-..3.4% Suspension in 5.6% v/v HNO3. Good agreement with certified values obtained using aqueous calibration. RSD=7 % Samples, suspended in 5 ml solution containing 1% v/v HNOa, 20% v/v H202, 0.3% v/v nickel nitrate. 20 ktl aliquots taken for analysis during stirring. Fast furnace program used. Method of standard additions used. RSD=5.7%

29

86

NIST-SRM Cd,Cu,Mn, 1566a Pb Oyster tissue

ET-AAS

Glycerol

Magnetic

NIST-SRM Cr,Pb,Se 1566a Oyster tissue

ET-AAS

0.1% m/v silicone antifoam

Argon mixing

NRCC

Se

TRIS buffer

Mechanical

ET-AAS

Dried, powdered specimens were suspended in 5% HNO3. No chemical modifiers or pyrolysis step employed. The slurry was analyzed using very rapid heating programs. Only Cu required the method of analyte addition. Loss of Cd was observed in slurries stabilized with glycerol. RSD=0.2-2% Modification to commercial furnace autosampler provide s a stream of H20 saturated Ar to homogenize slurry in autosampler cup. RSD=4.4%(Cr); 2.0%(Pb); 5.7%(Se) Samples were

56

48

46

homogenizer

DORM- 1 Dogfish muscle, NRCC TORT-1 Lobster tissue

NRCC DORM- 1 and DORM-2 Dogfish muscle, NRCC DOLT- 1 Dogfish liver Mussel tissue BCR CRM 278 Non-Fat milk powder NIST SRM 1549,

Hg

ETV-ICPMS

Pb

ET-AAS

Ca,Cu,Fe,K, Mg, Mn,Na, Zn

ICP-AES

homogenized and partially enzyme digested using crude protease either alone, or in conjunction with lipase or cellulose. Results agreed with certified values. RSD=15% Pd chemical modifier was employed, to delay Hg vaporization. Using standard addition the RSD was 7%

1% v/v Ultrasonic bath Triton X- 100

Ultrasonic

<50

Sedimentation was avoided RSD-~-7% Nebulizing carbonaceous slurries, partial oxidation with conc.

H2SO4

78

59

64

t',9 OO

,,-,

Bovine liver NIST SRM 1577a Dogfish muscle NRCC DORM-2 Oyster tissue NBS SRM 1566, powdered milk Bovine liver BCR CRM 185, Milk powder BCR CRM 150 and 151 Bovine liver NIST SRM 1577a, Non-fat milk powder NIST SRM

RSD=<5%

Cd,Hg, Pb

ETV-ICPMS

Ultrasonic

EDTA was used as the modifier RSD=14%

88

Ca

FAAS

Manual shaking

Aqueous solutions as standards RSD=3%

27

Cd

ET-AAS

Manual shaking

Pd matrix modifier was used. Direct calibration with aqueous standards RSD=I 5%

92

Ag,Cu,Fe,Mn, Pb,Zn

ET-AAS

Direct calibration with aqueous standards RSD=3-13%

93

0.04% Triton Vortex mixer X-100

1549, oyster tissue NIST SRM 1566a Whole milk Zn

FAAS

1% NaDBS

Tube stirrer

Method of standard additions was used RSD=-4%

95

Table 6 Applications of slurry sampling atomic spectrometry systems to the analysis of drugs and pharmaceuticals materials

Sample type Calcium drugs

Elements Instrumentation determined Pb ET-AAS

Calcium drugs

Cd

ET-AAS

Dispersant used -

Homogenization Ultrasonic bath

Particle size/pro 3

-

Ultrasonic bath

3

Slurry approach

Ref.

Suspensions of the powders were atomized from a Mo-tube with thiourea chemical modifier. Marked changes in absorbance were seen with changes in particle size. Results in good agreement with those obtained from acid digested samples. RSD=3.2% A Mo-tube atomizer was employed with thiourea as chemical modifier. Results in good agreement with

85

87

Formulat.

Ca,Cu,Fe, Mg,Zn

ETV-ICP

Whole capsule

Ca, Cu,Fe, Mg, P

ETV-ICP

0.2% Triton X-100, 4 M HNO3 0.2% Triton X-100, 4 M HNO3

those obtained from acid digested samples. RSD=5 - 17% 10 lal pipetted onto the graphite rod. RSD=2-4% 10 pl pipetted onto the graphite rod. RSD=2-4%

7

7

This Page Intentionally Left Blank

Chapter 6 Laser-induced breakdown spectrometry: potential in biological and clinical samples Joseph Sneddon l, Yong-lll Lee 2 and Kyuseok Song 3 1- Department Chemistry, McNeese State University, Lake Charles, Louisiana 70609, USA, 2- Department of Chemistry, Changwon National University, Changwon 641-773, Korea, 3- Laboratory of Quantum Optics, Korea Atomic Energy Research Institute, Taejon, Korea 1. INTRODUCTION When the output from a high-powered laser beam is focused onto a small spot of a (solid) surface in a buffer gas or atmospheric environment, the temperature of the locally heated region rises rapidly to the vaporization temperature of the (solid) material and then an optically induced plasma, frequently called a laser-induced plasma (LIP) or laser-ablated plasma (LAP) or laser spark is formed at the surface. The plasma will be formed when the laser power density exceeds the breakdown threshold value of the solid surface. Although different materials have different breakdown threshold values, an optical plasma is produced when the laser power density exceeds several megawatts per centimeter squared (106 - 109 W/cm2). This plasma has been used for sampling, atomization, excitation, and ionization in analytical atomic spectroscopy. When used as a sampling tool, it has shown great potential in laser ablation-inductively coupled plasma atomic emission spectrometry (LAICP-AES) or laser ablation inductively coupled plasma-mass spectrometry (LAICP-MS). The potential and success of this technique has prompted the development and commercialisation of these techniques. LA-ICP-AES and LAICP-MS will not be discussed in this chapter. It has been used primarily for direct elemental or metal determination and appears to have greatest advantage when the sample is a solid. This can be very useful if the solid is a very hard material such as polymer, ceramic or superconductor. These types of material are extremely difficult to digest/dissolve. However, it has been applied to liquid or solutions as well as aerosols (a solid or liquid particle in a gaseous medium). It has also been frequently used and proposed as a source for atomic emission spectrometry. In

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Copyright 9 2002 Elsevier Science B.V. All rights reserved

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this case the technique has been referred to as laser microprobe optical emission spectrometry (LM-OES) developed by Brech and Cross [ 1] in the early sixties or more recently called laser-induced breakdown (emission) spectrometry (LIBS) by the pioneering work of Cremers, Radziemski and co-workers at Los Alamos National Laboratory (LANL) in Los Alamos, New Mexico, USA. Generally, this analytical technique involves two steps. The first is the pulsed focusing laser beam is directed into a gaseous sample or the surface of a solid or liquid to produce a transient laser-induced plasma (LIP), and the second is the measurement of a characteristic atomic emission signal related to some species present in the plasma. The LIP consists of vaporized atoms, ions, electrons, and molecular fragments, and is formed when it is tightly focused. In recent years, this powerful technique as an analytical tool has been recognized by a number of research groups. This has led to an increase in the number of publications on the applications of LIBS both in the laboratory and in industry. This growing success of LIBS is a result of increased research performed to understand the related plasma physical processes, aided by marked improvements in laser systems and photo-detector technology. The principal advantages of LIBS over conventional analytical techniques, for elemental or metal determination such as inductively coupled plasma-atomic emission spectrometry (ICP-AES), inductively coupled plasmamass spectroscopy (ICP-MS) and graphite furnace atomic absorption spectrometry (GFAAS) are speed and simplicity. In particular, the required sample size (typically ~-0.10 microgram to 1 milligram), the analysis time (a few seconds or less), and sample manipulation and preparation are minimal. LIBS can also be coupled with fiber optics for remote, and in-situ measurements. These advantages are particularly valuable in the analysis of chemically and radiologically hazardous samples. Sample pre-treatment procedures for solid analysis by conventional methods are time-consuming, costly, and generate undesirable amounts of chemical wastes, such as perchloric and hydrofluoric acids. They also may increase the potential for incorrect results via contamination. The specific advantages and disadvantages of LIBS for direct spectrochemical analysis over conventional analytical techniques are summarized in Table 1. At present there are no major manufacturer of commercial systems for LIBS instrumentation. However, there is an increasing number of laboratory constructed systems and it is not beyond expectation that a major manufacturer of a system will be realised the not to distant future. The main objective of this chapter is to provide a brief overview of fundamental perspectives about the laser-induced plasma including the interaction of a laser beam with target materials, laser-induced plasma formation, factors influencing plasma formation and excitation temperatures and

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Laser-Induced BreakdownSpectrometry Table 1 Advantages and disadvantages o f LIBS for direct spectrochemical analysis

Advantages 1. No or little s m i l e preparation is necessary (resulting in increased throughput and reduction of tedious and time-consuming preparation procedures which can lead to contamination) 2. Versatile sampling of all media (analyze nonconducting as well as conducting materials)

Disadvantages 1. Increased cost and complexity of the system 2. Difficulty in obtaining suitable standards, (for this reason the technique must be regarded as semi-quantitative) 3. Large interference effects (including matrix and, in the case of LIBS in aerosols the potential interference of particle size)

3. Very small amount of sample (about O.1 Ixg - O.1 mg) is vaporized. (sometimes called "nondestructive" method)

4. Detection limits are generally not as good as established solution techniques.

4. Ability to analyze extremely hard materials which are difficult to digest or dissolve such as ceramics and superconductors

5. Poor precision, typically 5 to 10 % depending on the sample homogeneity, sample matrix, and excitation properties of the laser

5. Local analysis in microregions offers a spatial resolving power of about 1-100 lam

6. Possibility of ocular damage by the high-energy laser pulses

6. Possibility of simultaneous multielemental analysis 7. Potential for direct detection in aerosols (a solid or liquid particle in a gaseous medium) or ambient air using a transient plasma which is only piece for fraction of. 8. Simple and rapid analysis (ablation and excitation processes are carried out in a single step)

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electron densities of the plasma. A section on more recent developments in LIBS instnmaentation will be presented. Finally, the application of LIBS to the area of elemental or metal determination in a wide variety of samples will be presented. The potential in the biological and clinical area will be discussed. 2. FUNDAMENTAL STUDIES It is beyond the scope of this chapter to describe the fimdamental studies which have been performed on the laser ablated plasma used for LIBS. The reader is referred to an earlier text [2]. This part of the chapter provides some information which will provide a fundamental perspective about the laserinduced plasma including the interaction of a laser beam with target materials, laser-induced plasma formation, factors influencing plasma formation and excitation temperatures and electron densities of the plasma. Furthermore, spectral characteristics of the laser-induced plasma and analytical techniques will be discussed. 2.1 The interaction of a laser beam with target materials

The interaction of high-power laser light with a target or solid sample has been an active topic not only in plasma physics but also in the field of analytical chemistry. From a practical stand point, the use of lasers to vaporize, dissociate, excite, or ionize species on solid surfaces has the potential of becoming a powerful analytical tool. When a high-power laser pulse is focused onto a solid target, the irradiation in the focal spot can lead to rapid local heating, intense evaporation, and degradation of the material. The ablated material compresses the surrounding gas and leads to the formation of a shock wave. The incident laser radiation interacts with the partially ionized material vapor and the condensed material clusters embedded therein, and affects the efficiency and quality of the ablation. The interaction between a laser beam and a solid is a complicated process dependent on many characteristics of both the laser and the solid. Numerous factors affect ablation, including the laser pulse properties, such as pulse width, spatial and temporal fluctuations of the pulse, and power fluctuations. The mechanical, physical and chemical properties of the sample also influence the ablation process. The phenomena of laser-target interactions have been investigated by several authors. In 1971, Ready [3] gave a comprehensive description of melting and evaporation at metal surfaces. Anisimov and co-workers [4, 5] related the thermal conductivity mechanism to the boundary condition of free

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vaporization of the solid into a vacuum. Caruso et al. [6] found three differem regions existing in a metal and the hot plasma formed on the outer surface expanding towards the light source at a supersonic speed. The hot vapor plasma interacts with the surrounding atmosphere in two ways and involves (1) the expansion of the high-pressure vapor drives a shock wave into the atmosphere, and (2) energy is transferred to the atmosphere by a combination of thermal conduction, radiative transfer, and heating by the shock wave. The subsequent plasma evolution depends on irradiance, size of vapor plasma bubbles, target vapor composition, ambient gas composition and pressure, and laser wavelength. The history of important quantities such as radiative transfer, surface pressure, plasma velocity, and plasma temperatta'e are strongly influenced by the nature of the plasma, as is the final steady-state nature of the plasma. The three major types of a laser absorption wave are (1) laser-supported combustion waves (LSC), (2) laser-supported detonation waves (LSD), and (3) laser-supported radiation waves (LSR) [7]. The difference in the laser waves arise fi'om the different mechanisms used to propagate the absorbing front into the cool transparent atmosphere. The characteristics in distinguishing the waves are velocity, pressure, and the effect of radial expansion on the subsequent plasma evolution. At low irradiation, laser-supported combustion waves are produced. Razier [8] examined the long-time propagation of the laser-supported combustion waves at one atmosphere. Thermal conduction was assumed to be the primary propagation mechanism. Subsequent work proposed that radiative transfer could contribute [9, 10]. The major mechanism causing LSC wave propagation is radiative transfer from the hot plasma to the cool high-pressure gas created in the shock wave. The plasma radiation is primary in the extreme ultraviolet and it is generated by photo-recombination of electrons and ions into the ground-state atom. At intermediate irradiance, the precursor shock is sufficiently strong so that the shocked gas is hot enough to begin absorbing the laser radiation without requiring additional heating by energy transport from the plasma. The laser absorption zone follows directly behind the shock wave and moves at the same velocity. This is the analog of the chemical detonation wave and has been modeled by Ramsden and Savic [11] and Razier [12]. The propagation of the laser-supported detonation wave (LSD) is entirely controlled by the absorption of the laser energy. Several workers [13-15] studied theoretically and experimentally the ignition and propagation of the LSD wave off metal surfaces. Plasma energy transfer to metal surface and the breakdown times were calculated and modeled.

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At sufficiently high irradiance, the plasma radiation is so hot that, prior to the arrival of the shock wave, the ambient gas is heated to temperatures where laser absorption begins. In the idealized configuration, laser absorption is initiated without any density change, and the pressure profile results solely from the strong local heating of the gas rather than a propagating shock wave. This configuration is an example of an overdriven absorption wave [12]. These supersonic waves were numerically modeled by Bergel'son et al. [15]. Their numerical results confirm the basic structure that once the transient plasma initiation and formation process is completed so that the quasi-steady approximation is suitable. The laser-supported radiation (LSR) wave velocity increases much more rapidly with irradiance than that of the LSC and LSD waves.

Russo [16] described laser-material interaction on the basis of irradiance: vaporization and ablation. When the laser pulse duration is microseconds or longer and the irradiance is less than approximately 106 W/cm 2, vaporization is likely a dominant process influencing material removal from a target. Phonon relaxation rates are of the order of 0.1 picosecond (ps), and absorbed optical energy is rapidly converted into heat. At higher irradiance, beyond 109 W/cm 2 with nanosecond (ns) and shorter laser pulses focused onto any material, an explosion occurs. Temperature and pressure of the underlying material are raised beyond their critical values, causing the surface to explode. The pressure over the irradiated surface from the recoil of vaporized material can be as high as 105 MPa (106 atmospheres). During an ablative interaction, a plasma is initiated at the target surface. Plasma temperatures are in excess of 104 K, and radiative heat transport can establish a plasma-material inter-action. Schittenhelm et al. [17, 18] studied the wavelength-dependent transmission phenomenon in excimer laser-induced plasma plumes in the range between 440 nm and 690 nm with a spatial resolution of about 50 ~m. The authors concentrated their research to trying to understand the mechanisms that are responsible for the heating and ionization of the vapor at the start of the exeimer laser pulse applying a simplified stationary model. The modeling showed that extinction of the laser light in the plasma with the assumed thermodynamic parameters was dominated by Mie absorption on condensed material clusters for wavelengths less than 430 nm and is dominated by photoionization absorption and inverse bremsstrahlung above 430 nm. A resonance absorption photograph and interferometric investigation were performed to obtain information about these structures within the shock wave and to obtain information about the characteristic behavior of the species contact front, which was identified by resonance absorption spectroscopy.

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2.2 Laser-induced plasma production By increasing the energy deposited into the sample surface, temperatures reach a point where material transfer across the surface becomes significant. In this type of experiment, target erosion appears in the form of craters. The theoretical consideration of plasma production and heating by means of a laser beam has been proposed by several workers [13, 14]. These models produce essentially similar solutions for the plasma temperature, density and expansion velocity and are broadly in agreement with experimental results. Cottet and Romains [19] studied the formation and decay of lasergenerated shock waves by a hydrodynamic model. Measurements of shock wave velocities were performed on copper foils for incident intensities between 3 x 10 l~ and 3 x 1012 W/cm2, with the use of piezoelectric detectors. Balazs et al. [20] calculated the time development of density, velocity, temperature, and pressure profiles below and above the plasma ignition threshold. Below the plasma ignition threshold, the temperature of the expanding plume never exceeds the surface temperature, and in the vapor, thermal ionization is almost completely absent. The plume expands into the vacuum, and its flow becomes supersonic. In the high-fluence case, the energy delivered to the plume through electron-neutral inverse bremsstrahlung processes was enough to elevate the temperature close to the surface value. This gives rise to high electron density as well as intense light absorption. Between the creation of the localized vapor plasma and the steady-state laser-sustained plasma, the plasma evolves through several transient phases. The initiation of the plasma over a target surface begins in the hot target vapor. Absorption generally commences via electron-neutral inverse bremsstrahlung, but when sufficient electrons are generated, the dominant laser absorption mechanism makes a transition to electron-ion inverse bremsstrahlung. Photoionization of excited states can also contribute for short wavelength interactions. The same absorption processes are also responsible for the absorption by the ambient gas. The basic progression of interaction (from absorption through compression) is, however, preserved. In recent years, research has revealed a strong dependence of absorption and scattering processes on the laser wavelength [20]. The aforementioned trend towards short-wavelength research thus implies investigation of plasma pro,zesses at a much higher density where collisional effects will be emphasized. Recent work dealing with the chalacterization of the laser-ablated plasma formed from various pure metals under a controlled atmosphere by various laser system were performed by Lee et al. [21, 22, 23] and Kumiawan et al. [24, 25, 26, 27]. The high-powered laser-induced plasma consisted of two distinct

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regions when the pressure is reduced below 50 Torr in an air or argon atmosphere. In a helium atmosphere, two distinct plasmas or regions are observed even at 760 Torr. One is near the target surface, called the inner sphere plasma [22], or primary plasma [27] and emits a strong background continuum signal. The other is called the outer sphere plasma [22], or primary plasma [27] surrounding the inner sphere and is shown in Figure 1, which gives the brilliant blue-green emission of copper with a relatively low background continuum. The studies indicate that the emission mechanism of the ablated atoms from the target is not due to a recombination process but, rather, is due to the laser-induced shock wave process, as for the case when the plasma is produced with more high power pulse energy. The temporal history

Figure 1. Features of the interaction between the vapor plasma and the ambient gas of the laser-induced plasma in a gas or on a solid target is illustrated in Figure 2. In liquids the temporal history is compressed. In solids in a vacuum, higher stages of ionization are reached for the same intensity on target. Because of the high initial density of free electrons and ions, the spectra broadening is dominated initially by the Stark effect. As time progresses in the single laserinduced plasma, a recombination occurs, the electron density decreases, and

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pressure broadening is otten the main cause of line-width. The pressure and nature of the cover gas influences the absolute line intensities, the line-widths, and in some cases relative line intensities due to near-resonance collisions [28].

IONS NEUTRALS PLASMA LIGHT SIGNAL

MOLECULES 1.2

! lns

10 ns

1O0 ns

!!

t 1 gs

10 gs

I 100 ItS

TIME AFTER LASER FIRING

Figure 2. Typical temporal history of laser induced plasma generated by 50 mJ of 1064 nm Nd: YAG [28]

2.3 Factors influencing plasma formation. 2.3.1 Laser parameters 2.3.1.A Influence of the irradiation wavelength In practice, different types of lasers mainly Nd: glass resonators (~ = 1064 nm), ruby laser (x = 694 nm), Nd: YAG laser (x = 1064 nm), CO2-TEA (carbon

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dioxide-transversely excited atmospheric pressure laser, .~ = 10.6 lam), N2-1aser (Z = 337 nm), and dye lasers (x = 220-740 nm) are used in the production of laser plasmas. The interaction of near and mid-infra-red pulsed laser with metals has been studied extensively. The influence of the laser wavelength on the material removed were studied by Bingham and Salter [29]. They investigated the ion production in mass spectrometry using three differem lasers, CO 2, ruby and Nd: YAG. They obtained the highest sensitivity for the elements (P, S, Ti, V, Cr, Mn, Ni, Co, Cu, As, Zr, Mo, Nb, S, Ta, and W) with a steel standard using ruby (x = 694 nm) laser ablation. However, the CO 2 laser (x = 10.6 lam) showed poor sensitivity for high boiling point elemems (Ti, V, Zr, Mo, Nb, Ta, and W). Fabbro et al. [30] used a Nd: YAG laser that was frequency doubled and quadrupled to give wavelengths of 1064, 532 and 266 ran, to study the effect of wavelength and postulated the following equation for the mass ablation rate, m (kg/s cm2), its dependence on wavelength, ~., and the absorbed flux, F a (W/cm 2) m = l lO (F a 1/3 )/(lO14 )~,-4/3

(1)

They found that the mass ablation rate would increase strongly at shorter wavelengths. Measuremems of the ablation pressure generated by the ablating plasma have been performed at a number of laboratories [31, 32, 33]. These results confirmed the expected higher ablation pressure with shorter wavelength laser irradiation. Kwok et al. [34] investigated the optical emission produced by laser ablation of YBa2Cu30 7 targets using a wide range of laser wavelengths and showed that 193 nm radiation produced mostly neutral atomic species while 1064 nm and 532 nm radiation produced mostly ionic species. The comparative work on the plasma emission characteristics, specifically, self-absorption, line broadening, emission intensity, and metal ion formation, was carded out by the use of three different laser wavelengths (XeC1 excimer, .~ =308 nm, Nd:YAG, x =1064 and 532 nm) [23]. They found that the degree of self-absorption and line-broadening strongly depends on the surrounding atmosphere and irradiation wavelength. This phenomenon was explained by shock wave excitation of a~.oms in the outer-sphere plasma. The wavelength dependence of laser-induced breakdown in air, CO, and CO2 was studied using the four Nd:YAG harmonics (266nm, 355 nm, 532 nm and 1064 nm) [35]. A significant reduction in the breakdown thresholds for both CO and CO2 is apparent when comparing 193 nm with the four Nd:YAG harmonics which is attributed to the resonance enhanced two-photon ionization of metastable carbon atoms.

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2.3.1.B Influence of irradiation energy

There are two main mechanisms for electron generation and growth. The first mechanism involves absorption of laser radiation by electrons when they collide with neutrals. If the electrons gain sufficient energy, they can impact and ionize solids. The electron concentration will increase exponentially with time due to the cascade breakdown. The second mechanism, called multi-photon ionization (MPI), involves the simultaneous absorption by an atom or molecule of a sufficient number of photons to cause its ionization. Multi-photon ionization is important only at short wavelengths (< 1 ~m ). Both cascade and multi-photon ionization requires high laser irradiances, usually in excess of l08 W/cm 2. For most materials the power density required for evaporation is in the range of 104 to 109 W/cm 2. In the range of 104 to 107 W/cm 2, the resulting vapor consists of polyatomic particles [36]. Selter and Kunze [37] studied the degree of atomization in the laser produced vapor from titanium targets. At power densities below 7 x 107 W/cm 2, no atoms were observed in the vapor whereas the evaporated material became partially ionized above 5 x 108 W/cm 2. It was assumed that power density in the range of 106 to 108 W/cm 2, depending on the solid target, was sufficient for analytical measurements in a laser ablated plume [38, 39]. Carroll and Kennedy [38] found that the threshold power density for the formation of a plasma plume by the laser irradiation was typically in the neighbourhood of 108 W/cm 2. The threshold power density varies with the wavelength of a laser primarily because the absorbance of the target surface depends on the wavelength of the incident light. Dyer [40] also determined the threshold energy for the generation of the plasma on a copper target of l08 W/cm 2 with KrF-excimer laser (~. = 248 nm). In order to describe the fate of the laser energy during laser-solid interaction, several processes should be considered. Due to the character of the target, a fraction of the energy is absorbed from the laser pulse while the rest is reflected by the surface. The deposited part of the laser energy is converted into local heat instantaneously, which can in turn diffuse by heat conduction. An increase in temperature may induce appreciable changes in optical and thermal properties of the target material, thus influencing the rate of energy deposition and heat transfer. If the surface temperature is sufficiently high, phase change (melting) may occur and part of the absorbed laser power is expended into the latent heat of transition. Further heating results in the translation of the solidliquid interface into the bulk, while the surface temperature continues to rise until evaporation commences. Hydrodynamic effects in which droplets and particulates are expelled from the molten surface layer and ejection of the melt

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caused by vapor recoil are among the mechanisms which contribute to the ablation of the target. Vertes et al. [41] established a criterion for the plasma ignition threshold as a relation of the plasma absorption coefficient by the adiabatic absorption model for CO2, ruby, and quadrupled Nd: YAG lasers and for different materials. They found that an increase of the threshold for materials with increasing ionization potential. It was also noticed that under similar circumstances the threshold temperature for the frequency quadrupled Nd: YAG (~ = 266 nm) laser were always largest while for the CO 2 laser they exhibit the lowest value. This is in accordance with the widely known observation that ultra-violet (UV) lasers produce sharply etched craters in the target while increasing the laser wavelength creates a molten crater rim. Laqua [42] distinguished two different cases of vaporization depending on the irradiation, at an irradiation of less than l0 s W/cm 9- and higher than 109 W/cm 2. In the first case, the stream of vapor leaves the surface at a velocity of 10 4 cm/sec. Aiter the initial vaporization, the process changed to a meltingflushing mechanism as a result of the heat conduction of the material. At higher irradiation, the temperature of the vapor leaving the surface is higher than the boiling point of the target material. The gas molecules above the target were ionized with a velocity of l 0 6 cm/sec near the surface. Cabalin and Laserna [43] determined the laser-induced breakdown thresholds for Zn, Al, Ag, Cu, Ni, Fe, Cr, Mo and W, using a Q-switched Nd:YAG laser operating at infrared (~ = 1064 nm), visible (~ = 532 nm) and ultraviolet (~ = 266 nm) wavelengths. Russo et al. [44] employed inductively coupled plasma-atomic emission spectrometry (ICP-AES) for studying the behavior of laser ablation at atmospheric pressure, as a function of power density and gas environment. The amount of ablated mass is highest in He and decreases as the ionization potential of the gas decreases. The mass ablation rate exponentially increases in the low power density regime and drops to near unity at high power densities.

2.3.2 Physical properties of the target material The physical properties of the target have an important influence on the shape and size of craters in target materials. The reflection of part of the laser energy is an important consideration in determining the fraction of laser energy absorbed by the sample material. The change in reflectivity may be due in part to the result of phase changes that occur during intense heating. In any case reflectivity measurements indicates that laser energy can be coupled effectively into a target is initially highly reflective, if the irradiance is high enough [45]. Recognition of the fundamental differences of the interaction of burst (single)

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and Q-switched modes of operation with materials led to the conclusion that craters produced by the latter mode might be less material-dependent that those for the former case [46]. However, Klocke [47] and Baldwin [48] found that the laser beam was strongly target material-dependent, whether the laser is Qswitched or not. Allemand [49] showed that the reflectivity of the sample surface, density, specific heat, and boiling point of the pure metal target have an important influence on the shape and size of the craters and derived the relationship to the physical constants of pure materials. D = A (l-R) / p C T b

(2)

D = diameter of total splash (crater) A = proportionality constant (in energy per unit area) R = reflectivity of the surface at 1 mm p = density C = specific heat T b = boiling temperature Ishizuka [50] studied the size and depth of the crater in samples of rare earth oxides, aluminum oxide, and sodium salts by using a Q-switched ruby laser. The crater produced by a laser shot was about 1 mm in diameter regardless of the composition of the matrix, but the depth of the crater depends on the type of matrix. A comparison of the crater size of the homogeneous material revealed that thermal conductivity is an important parameter. The depth of the crater increased with this value. The volume heated depends on the thermal conductivity of the material for the same laser conditions [51]. On the other hand, heating of material around the crater increases with incident light intensity because evaporation only depends on the boiling point of the material at fixed pressure. Dimitrov and co-workers [52, 53] investigated the substance evaporation processes and the kinetics of plasma plume development depending on target orientation with respect to the laser radiation source direction. When the metal target is irradiated by laser radiation, the erosion products emerge nearly perpendicularly to the target surface. When the target surface is inclined with respect to the direction of laser radiation, the path length of the radiation in the plasma is shortened, which results in decreased absorption of the laser produced plasma.

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Lee and Sneddon [54, 55] investigated the ambient gas breakdown phenomena during and after the laser irradiation and developed a relationship between the degree of ambient gas breakdown and the physical properties of metals. The authors deduced the following relationship between breakdown intensity and the properties of metal species with five differem metal species: IB = K { 1/(Cp x CT X 19)}

(3)

Where, IB = intensity of gas breakdown emission (counts), K = proportional constant, Ca = specific heat (JK ~ moll), CT = thermal conductivity (W m "1 K "l) and p = density (Kg m3). Cabalin and Lasema [43] also investigated the correlation between the plasma threshold of metal targets and the physical properties of metal target materials. The studied elements were chosen according to their different thermal and physical properties, particularly boiling point, melting point and thermal conductivity. A correlation of measured plasma threshold fluence and the melting point and boiling point at different laser wavelength (~ = 266, 532, and 1064 nm) was obtained For a given excitation wavelength, the threshold values correlate reasonably well with melting and boiling points of metals. The ablation thresholds increase with melting and boiling temperature. This suggests that the thermal effects are important during the nanosecond ablation process for laser fluence close to threshold fluences. 2.3.3 A m b i e n t conditions The atmospheric influences on the LIP were concerned with the mass loss, crater formation, and plasma emission characteristics. The investigations were mostly performed in air, argon, helium, and nitrogen. Iida and co-workers [56, 57] investigated the emission of the laser induced plasma, with the use of a Qswitched ruby laser of energy 1.5 J in 20 ns duration in an argon atmosphere at reduced pressure. The emission intensities of atomic lines increased several fold in an argon atmosphere, in comparison with those obtained in air at the same pressure. Moderate confinement of plasma and a resultant increase of emission intensities were achieved at 50 Torr. They also used a Q-switched Nd:YAG laser (150 mJ/pulse, 10 ns pulse) to study the effect of atmosphere and power density on plasma generation [58]. They found that tight focusing of laser radiation did not directly bring about the plasma of high emission intensity, became of the absorption of laser energy by the plasma itself. The importance of prevention of a gas breakdown before sample vaporization was also indicated. Grant and co-workers [59, 60] studied the laser induced plasma by irradiation of a steel target with an XeCl-excimer laser (~ = 308 nm) with an energy of 40

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rrd/pulse in an atmosphere of air, argon, nitrogen, and helium at pressure from 0.5 to 760 Torr. The maximum spectral intensity and line-to-backgrotmd (L/B) ratio occur in an atmosphere of argon at a pressure of 50 Tort. The effect of buffer gas type on LIP formed by a Nd:YAG laser (z = 1064 nm, 100 mJ, 7 ns) on a metal target was investigated by Owens and Majidi [61]. They observed an increase in the ratio of AI(II)/AI(I) intensity in helium gas relative to argon gas and air. This increase was attributed to the ability of excited helium atoms to transfer energy to a similar energy level in the aluminum ion. Lee et al. [22, 23] investigated the effect of pressure over the range of 10 to 760 Torr and of atmosphere (air, argon and helium) on an ArF-excimer laser (~. =193nm, 100 mJ/pulse) induced plasma created above the surface of a copper target. With the use of neutral copper lines, reduced pressure from 760 to 10 Torr resulted in a 7-fold increase in air and 11-fold increase in an argon atmosphere. The use of helium was only a 1.5-fold increase over that obtained at 760 Torr. They also observed nitrogen in air and argon breakdown in the plasma and concluded gas breakdown influences the laser energy coupling to the metal target. However helium gas breakdown was not observed because of higher ionization potential and high thermal conductivity compared to argon and nitrogen. Kagawa et al. [62, 63] studied a XeC1 excimer laser (15-70 mJ/pulse, 20 ns pulse duration) induced shock wave plasma on a Zn plate in a surrounding gas at low pressures (0.75 - 11.3 Torr). They defined the role of surrounding gas as only damping material to prevent the free expansion of the propelled atoms and the total emission intensity of the atom emission lines is determined mainly by the amount of propelling atoms and the entire amount of kinetic energy they produce. Mao et al. [64] also demonstrated the shielding effect on the coupling of laser energy to a target surface during the interaction of picosecond pulsed laser beam and target material using a He and Ar gas atmosphere. They concluded that Ar is easier to ionize than He and plasma shielding is more severe in Ar than in He. Kuzuya et al. [65] studied the effect of laser energy and atmosphere on the emission characteristics of LIP with the use of Q-switched Nd:YAG laser over a laser energy range of 20 to 95 rrd/pulse. The experimental results showed that the maximum spectral intensity was obtained in argon at around 200 Torr at a high laser energy of 95 mJ/pulse, whereas the line-to-background (L/B) ratio was maximized in He gas at around 40 Torr at a low energy of 20 lrd/pulse. Singh et al. [66] investigated the emission characteristics of a metal hydride generated by using a NaBI-h-based hydride generation system, specifically Sn and As, and found that temporal behavior of the LIBS signal was affected by gas composition, gas pressure, and intensity of laser beam. The Sn

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neutral atomic emission (284.0 nm) in a N2 atmosphere decreased exponentially with time. In contrast, with a He atmosphere and identical experimental conditions, the Sn signal increased logarithmically with time over the first 100 s. Then the signal maintained a steady-state value until approximately 400 s, atter which it decreased exponentially. It was found that chemical reactions in the laser plasma that might deplete the metal from the gas volume were responsible for the decrease in the signal with time. 2.3.4 Influence of electric and magnetic fields Over the past decade, there has been an interest on the use of electric or magnetic fields for enhancing the analytical characteristics of plasma sources for spectrochemical analysis. Pulsed magnetic fields have been used to alter the properties of microwave plasmas [67], spark discharges [68, 69] and exploding conductor plasmas [70, 71, 72, 73]. A few recent studies have looked at the effect of a static electric field on ultraviolet emission enhancement and breakdown threshold. Hontzopoulos et al. [74] used a 3-20 mJ KrF excimer laser to study the effect of UV emission lines of gold from a laser induced plasma on the surface of gold target in fields up to 13 KV/cm. They found a significant enhancement, up to a factor of 100, for some lines above 6.6 KV/cm, saturating for some lines at 20 KV/cm. They tentatively interpreted this enhancement on the basis of recombination processes taking place near the surface of the gold electrode. Kumar and Thareja [75] studied the breakdown in Ne, Ar, and Xe gas at different pressures using a XeC1 excimer laser (60 mJ, 8 ns of pulse length) ranged up to the maximum field strength of 1000 V/cm. They concluded that their results were similar to those observed using a high power laser alone. Mason and Goldberg [76] designed and constructed specifically a new capacitive discharge system. The pulsed magnetic field, produced by capacitive electrical discharge through a specially designed solenoid, was oriented normal to the laser axis. Temporally integrated emission enhancements due to the magnetic field were found to be most significant when the plasma was formed about 1 mm below the magnetic field axis. The degree of confinement of the plasma increased with magnetic field strength and also found an increase in linebroadening, neutral atom self-reversal, and minor constituent emission intensities [77]. Subsequently their research was conducted on the dynamic effects of a high-intensity pulsed field having a maximum strength of 85 KG, by using time-resolved emission and absorption measurements [78]. Spatial and temporal discrimination of emission enhancement indicated that radial compression was due to static magnetic field interactions with the laser induced plasma and that mild Joule-heating from the small induced current was most

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likely responsible for emission enhancements later in time. They concluded that more efficient coupling of energy from the magnetic field to the plasma would require low-pressure operation in a controlled atmosphere and/or a pulsed magnetic field having a greater dB/dt. (B is the magnetic field intensity). The influence of a magnetic field on the plasma emission of Mg induced by a KrF excimer laser was reported by Dimberger et al. [79]. They observed a dramatic enhancement of emission signal from neutral and ionized species over the fieldfree case, in certain spatial regimes.

2.3.5 Plasma shielding Plasma shielding can be studied by measuring mass ablation rate behavior as a function of laser pulse duration and wavelength, and ambient gas environment. The laser-induced plasma is initiated and sustained by inverse bremsstrahlung absorption during collisions among sampled atoms and ions, electrons and ambient gas species. Argon is easier to ionize than helium because of its higher ionization cross section and lower ionization potential; as a result, plasma shielding is expected to be more severe in argon than in helium. Mao et al. [64] found that copper emission intensity was 16 times higher with helium than it was with argon in the ablation chamber by the use of Nd:YAG laser irradiation (;~ = 1064 nm). Larger crater volumes were measured in the helium atmosphere. The phenomenon supports a reduced plasma-shielding mechanism and enhanced laser energy coupling to the target. The difference of plasma shielding for picosecond and nanosecond laser ablation was observed and can be explained on the basis of collisions among atoms, ions, and electrons in the atmosphere above the target surface. During the picosecond laser pulse, ejected atoms/ions travel only a few hundred angstroms from the surface, assuming velocities on the order of 106 cm/s [80]. In contrast, high-energy (>100 eV) electrons are generated during picosecond interactions, and these electrons can acquire velocities on the order of 109 cm/s. The fast electrons absorb laser photons during collisions with the support gas atoms. On the other hand, the distance that atoms and ions travel from the sample surface is several hundred micrometers during the nanosecond laser pulse. The sample atoms/ions undergo collisions with each other as they expand into the gas and absorb photons from the laser beam. Plasma shielding with picosecond laser pulse is caused by the collision between fast electrons and gas atoms. While, Plasma shielding with nanosecond laser pulse is caused by the collision between vaporized atoms and ions. Collision of gas species with the ejected atoms and ions are negligible during the nanosecond laser pulse. Therefore, the gas environment influence is not significam [16]. Tokarev et al. [81] proposed a new method for the processing of

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experimemal reflectivity data from a target-plume system. It gives the possibility of finding the order of laser radiation absorption in the laser-induced plume (single- or two-, or three-, and so on, photon absorption) during the lasertarget interaction time. The authors found that three-photon absorption is a main mechanism of plume shielding in the case of crystalline silicon ablation by ArF (x = 193 nm) excimer laser. 2.3.6 Effect of sampling geometry Multari et al. [82] investigated several geometric factors, including the lens-to-sample distance (LTSD), the orientation of the sample with respect to the incident laser pulses, the method of focusing the laser pulse to generate the laserinduced plasma and the method of collecting the plasma emission (lens or fiberoptic bundle). These factors can greatly influence the analytical results. Significant changes in atomic emission intensities, plasma temperature, and mass of ablated material was observed with changes in the LTSD. The use of a cylindrical or spherical lens having a long focal length (150 mm) minimized some effects of changes in sample geometry without degrading analytical performance. Temporally and spectrally resolved images of the plasma showed that the collection of the plasma emission, by forming an image of the plasma on a spectrograph slit (a method used in many types of LIBS measurements), will miss a large fraction of the analyte emission. In addition, this configuration was very sensitive to changes in the angle of incidence of the laser pulses focused onto the sample surface. With the use of fiber-optic bundles to collect light from all parts of the plasma, on the other hand, this sensitivity is reduced significantly.

3 EXCITATION TEMPERATURES AND ELECTRON DENSITIES OF THE PLASMA 3.1 Excitation temperature calculations Temperature, as well as pressure and concentration, is a macroscopic property that is related to the microscopic state of the plasma by various statistical distributions, namely, the Saha-Effect, Maxwell-Boltzmann distributions and Planck's law. These distribution functions are sensitive to temperature, therefore, each one gives rise to a particular temperature which is associated with the various species within the plasma. The gas kinetic energy of a particle (Tkin) and the electron temperature (T~) are related to Maxwell's distribution. The Saha expression relates the ion-to-atom population ratio for a given species as a function of the ionization temperature (Tion). The electronic excitation temperature (T~x~) determines the population of each energy level

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within the species (atom or ion) through the Boltzmann distribution. Temperature is one of the most important properties of any excitation source. Knowledge of the plasma temperature is vital to the understanding of the dissociation, atomization, ionization, and excitation processes occurring in the plasma and thus, is helpful when one is attempting to utilize the plasma to maximize analytical potential. Energy described by temperature is distributed in translational, rotational, vibrational, electronic, and ionization states of atoms. Each state can be defined by its own temperature. When the temperature is the same for all states, the system is said to be in thermal equilibrium, and that temperature truly defines the energy of the system. For a system not in thermal equilibrium, temperature values may differ widely according to the type of excited state sued to define the temperature, and the physical significance of these varying temperatures becomes less clear. The absence of true thermal equilibrium in a physical system is often circumvented by the assumption of local thermal equilibrium (LTE). The excitation temperatures measured are very useful when one is comparing different excitation conditions, as well as helpful in providing insight into the excitation mechanisms of importance in the plasma. The methods most frequently used for determination of excitation temperatures are the two-line method [83, 84, 85], and the Boltzmann plot method [25, 86, 87, 88]. In the Boltzmann plot method, the relative intensity of the thermometric species is measured. Following the Boltzmann distribution and neglecting self-absorption, the line intensity is related to Texc as follows: hc g, -E, ) I~=(~Xno)(--XA~) exp(.-~--~

(4)

where Ik! refers to the line intensity for the transition between the upper level k to the lower level 1 of the emitting species, T~x~ is the excitation temperature, gk and go are the statistical weight factors for the excited and the ground states, respectively, no is the population of atoms in the ground state, Akl stands for the probability of the transition per unit of time (Einstein transition probability), E k is the energy of the excited state, and all other variables are as defined previously. If the relative intensity for a given species (the thermometric species) is measured at various wavelengths, equation (4) can be expressed as equation (5) and (6); I~ - ~--~g ~.exp('KTE k)

(5)

J. SNEDDON, Y.-I. LEE, and K. SONG

306

[ I~'n / E~ g~A~ = KT

(6)

The variables have the same meaning as described in equation (4). It can be seen from these two equations that there is a linear relationship between the ~I~/'ga) and the excitation energy (Ek), and that the slope of this linear profile is equal to k'lT'lexc. Therefore, a plot of the ln(I/~/gA) versus Ek gives a means of measuring the electronic excitation temperature. 3.2 Electron density calculation There are two ways to find the density values, one from direct observation of the emission line shape, the second based on the Saha equation. The first method, based upon line shape analysis, requires knowledge of different broadening parameters, which depend on the plasma characteristics. The main processes are the collisional interactions. There are two kinds of particle interactions: electron-neutral atoms and electron-ion. For electron-neutral atom interactions, we talk about Van der Vaal's broadening, for electron-ion interactions, it is Stark broadening (the more important in our experiments). The second method uses the Saha equation and the intensity of emission lines from two successive ionization stages. 3.2.1 Electron number densities from stark broadening calculations The Stark width for the I-~ line is related to n~ by AkS~/~(R)

(7)

ne(R)= (2a,/,(2.61e) } ~ where A

Stark half-width at radius, R,

~S1/2(R

) Reduced Stark profile half-width parameter,

1/2

E

Electrostatic unit of charge.

Equation (7) cannot be used directly unless experimental line profiles have been de-convoluted to account for Doppler and instrument broadening.

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This correction, on a half-width basis, is not a straightforward procedure. A simpler alternate method is to use pure Stark reduced profiles, S(ot), at various ne values and temperatures as base values and then to apply convolution calculations to account for other broadening contributions. 3.2.2 Electron number densities from Saha-Eggert ionization calculations For the emission lines of the neutral atom and first ionized species the radial ne is given by

ne(R) = 4.83 x 1015 J~

E]~

3/2 x exp { E+-E~176

J~176176 [2+

~

(8)

kT(R)

where

0,+)

The neutral atom and ionized species, respectively,

singly

Emission intensity at radius, R,

(R)

Statistical weight of the emitting level,

(R)

Ei 0

Transition probability for spontaneous emission, Wavelength of the emission transition, Excitation temperature at radius, R, Energy of the emitting level, Ionization energy of the neutral atom species, Lowering of the ionization energy

A A E~~ correction was applied to the ionization energy to account for the interaction of free atom states with the electric micro field, which is produced by the charged plasma particles. A number of methods for calculating A Ei 0 have been reported. When the Uns61d formula was applied, a value of A Ei~ 403 cm -~ (0.05eV) was found to be compatible with the temperatures and densities considered in this study. Equation (8) was used to calculate radial ne from the corresponding radial intensities for the atom/ion line combinations and average

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J. SNEDDON, Y.-I. LEE, and K. SONG

(g~176176 ratios. The ratio of the ion number density to that of the neutral atoms is given by nx+(R)_j+(R) g~176U z+[T(R)]xexr~rE+-~E~) nxo(R) - ~---~" g+A+K~ Z~ "[, kT(R)

(9)

where Z[T (R)] is the partition function for the radial temperature T (R). Partition functions for neutral atom and singly ionized species were calculated from the method suggested by Griem [89] which included a correction for the lowering of the ionization energy. Radial number density rations were calculated from Equation (9) for the atom/ion line combinations.

3.3 Experimental results Grant and Paul [58, 59] determined the electron temperattn'e and density of XeCl-excimer laser induced plasma. The relative atomic contents of the eleven Fe(I) lines used in the Boltzmann plot of electron temperature were used. The temperature ranged from 9,000 to 22,000 K depending on the ambient conditions. Temperatta'e decreases with distance from the surface and with decreasing ambient pressure. Electron densities were calculated according to the Saha equation and the fitted values of temperature with assumption of LTE (local thermodynamic equilibrium). The density profile exhibited feature similar to those for temperature, ranging from 3 x 10~9 to --- 10~6 cm-3. Kagawa et al. [62, 63] calculated the excitation temperature in a high-power nitrogen laser induced plasma with the two line method by the line pair of Zn(I). The temperature ranged from 8,000 to 9,000 K, and the region of maximum was at a point some distance from the center of the plasma rather than at the center. Ursu et al. [90] studied the optical breakdown plasma in a gas in front of various solid samples by a TEA-CO2 laser source. They measured the energy absorbed into the blade calorimeter, placed at various distances from the center of the plasma. An upper limit of the vapor temperature of-~14,000 K was inferred from the characteristic darkening curve of the photographic film. They found that the initial maximum corresponding to the breakdown plasma in gas having a temperature of ~20,000 K is followed by a luminescence tail due to vapors acting with the fireball and having temperature of~l 0,000 K. Radziemski and coworkers [91, 92]) measured the temporal variation of temperatta'e and electron density in the air plasma induced by a CO2 laser operating 0.5 and 0.8 J/pulse. The excitation temperature was determined by Boltzmann plot, and ranged from 19,000 K at 1 ~ts to above 11,000 K at 25 Its. The electron density was measured at 500 mJ/pulse and determined from 3.6 x 1017 cm "3 at 1 ~ts and 4 x 1016 cm -3 at 25 lLts. Lee et al. [22, 25] calculated the

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excitation temperature of ArF-excimer laser (100 mJ/pulse, 11 ns pulse length) induced plasma by the Bolzmann plot method with Cu(I) and Pb(I) lines. The temperattwe of the excimer laser induced plasma were quite high, ranging from 13,200 to 17,200 K in the plasma formed with copper and from 11,700 to 15,300 K for the plasma formed with lead depending on the location in the plasma. They also measured the excitation temperatures of the plasma under different pressure and atmosphere. The temperatures ranged from about 14,000 K at 10 Torr to 18,000 K at 760 Torr for an air atmosphere, from about 13,400 K at 10 Torr to 14,200 K at 760 Torr for an Ar atmosphere, and from 12,600 K at 760 Torr to 14,800 K at 200 Torr for a He atmosphere. Measurement of precision and accuracy in the plasma temperature is highly dependent on sample composition, homogeneity, surface condition, and particle size. Precision was typically 5 to 20% but values of less than 1% have been achieved under certain condition [30]. However, the accuracy of the estimation by the Bolzmann equation is uncertain. It depends on the existence of LTE assumed for this method and the accuracy of spectroscopic constants of neutral atom lines. Recently, the energies and number densities of ions emitted from LIP were investigated by Pakhomov et al. [93]. The plasma were formed on solid elemental targets exposed to focused pulses from a Ti:Sapphire modelocked laser and re-gemerative amplifier. The pulse energy and duration were 1.0 mJ and 100 fs, respectively, with irradiance at the target of 2 x 1013 W / c m 2. Observed ion kinetic energies in most cases were on the order of 30-50. Harilal et al. [94] studied the laser-induced plasma emission from a hightemperature superconducting material, viz., Yba2Cu307 (YBCO) and electron temperature and electron density measurements were made from spectral data. The line intensities of the successive ionization states were used for the determination of the electron temperature, and the Stark-broadened profile of the Ba(I) transition at 553.5 nm was used for the measurement of electron density. An initial electron temperature of 2.35 eV and electron density of 2.5 x 1017 c m "3 was observed. Yalcin et al. [95] evaluated the capability of LIBS for quantitative, in-situ metals measurements in air. They used measurements of light emission to determine temporally- and spatially-resolved values of the plasma temperature and electron density at atmospheric pressure. The Stark broadening of atomic nitrogen lines near 414 nm and the hydrogen Balmer a and b lines at 656 and 486 nm for electron density measurements were used. Hydrogen and hydrogenic ions exhibit a linear Stark effect because of the degeneracy for levels with different orbital angular momentum. Other atoms and ions exhibit a quadratic Stark effect, and so have smaller Stark-broadened line-widths.

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4 SPECTRAL AND ANALYTICAL CHARACTERISTICS OF LIBS 4.1 Basic principles of LIBS Elemental analysis based on the atomic emission fi'om the plasma generated by focusing a powerful laser beam on a sample (solid, liquid or gas) is known as laser induced breakdown spectrometry (LIBS). The principles of LIBS are similar to those of convemional plasma atomic emission spectrometry, such as inductively coupled plasma (ICP)-AES, microwave induced plasma (MIP)-AES, Direct currem plasma (DCP)-AES, arc-AES and spark-AES. In atomic emission spectrometry, the light from an excited sample is spectrally resolved and sometimes temporally resolved as in the case of pulsed light sources, to yield qualitative and quantitative information about the elemental constituents. What distinguishes the LIBS technique from convemional plasmaAES is that the sample need not be transported to the plasma source; rather, the plasma is formed in or on the sample in-situ. It is a simple method because the ablation and excitation processes are both performed by the laser pulse in a single step. However, the mechanism of laser ablation is complex and not clearly defined with research into laser/solid interactions continuing In addition, LIBS technique can be used to analyze gases, liquids, and solids directly without sample preparation, because the plasma is produced by optical radiation. This method also provides, in principle, simultaneous multielement analysis without increased instrumental complexity and cost. Atomic ions as well as neutral atoms will be produced in plasma sources having high temperatures. The energy levels are unique for each elemem so, it is possible to perform simultaneous qualitative analysis by examining wavelength in the emission spectrum of an unknown. A spectrometer is used to disperse the plasma emission, and from the wavelengths of individual elemems in the emission spectrum it is possible to determine the elememal composition of sample material. For the quantitative analysis, a measurement involves integration of the analytical signals from a line or set of lines of the elemems of interest over a certain period of time. The amount of an element present is determined by constructing a calibration curve of signal versus concentration by the use of standard reference materials. Over the 1,000 different standard reference materials (SRMs) are obtained from the National Institutes of Standards & Technology (NIST) (Gaithersberg, Maryland, USA). These materials are certified for their chemical composition, chemical properties, or physical properties by various reliable analytical methods. However, the standard references are often not available with some particular or specific matrix. Other standards are available from various international government agencies such as the National Research Council (NRC) (Ottawa, Ontario,

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Canada) and National Institute for Environmental Studies (NIES), Iibaraki, Japan) 4.2 Analytical characteristics Most fundamental studies on spectral characteristics of LIBS have been dealt with atomic, ionic, and molecular temporal and spatial profiles on LIP. LIBS is a simple, rapid, and in-situ analytical technique based on the laserinduced plasma emission. For analytical purposes, the plasma emission is spectrally resolved and the atomic lines are subsequently analyzed in order to determine elemental concentration in the sample. This technique has, however, several limitations such as high continuum background, line broadening, and self-absorption. This consideration limits the use of this plasma emission for practical spectrochemical analysis. Work in minimizing these limitations has been investigated with the use of time-resolved LIBS (TRELIBS) [96, 97, 98, 99, 100] or time-integrated spatially resolved LIBS (TSRELIBS) [22, 23, 24, 101] in a controlled atmosphere. In most LIBS work, TRELIBS has been generally utilized in order to avoid the intense initial continuum emission and improve the line-to-background (L/B) ratio by to gating off the early stage of the plasma emission. A suitable choice of time delays in detecting the emission spectra allows selective assignment of the resolved line emission to different elements. However, it has been noted that spatially resolved signal along the vertical direction from target surface can provide a similar spectrum to that which is time delayed without using the expensive gated detection system required for TRELIBS. Multari et al. [82] verified the resemble properties of the plasma emission between TRELIBS and spatially resolved spectra with the many images with various different geometric factors. This result shows the potential application in the construction of less expensive LIBS apparatus and portable LIBS instruments. Wu et al. [102] investigated the dynamics of copper plasma generated by laser ablation using optical emission analysis. The plasma emission Cu(I) at 427 and 465.1 nm (designated as group a); 324.8, 327.4, 510.6, 515.3, and 521.8 nm (designated as group b); and copper ion line Cu(II) at 283.7 nm were chosen to study the laser-induced plasma formation and evolution. It was found that, even for the same emitting species, the temporal and spatial behavior of the emissions originating from different transitions can be very different. At the initial stage, the plasma involves a large amount of Cu(II) and high energy-excited Cu(I) species (group a), and then evolves to consist mainly of low energy-excited Cu(I) species (group). The results also show that the plasma maintains higher temperature for a quite a long time, and cool electron-impacting excitations determine the plasma behavior while it expands far away from the target.

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A systematic study of the time evolution of the line shape of radiation emitted by the Au plasma and an exact line intensity calculation was carded out by Nemet and Kozma [103]. Asymmetric Lorentz-type profile equation was tested for two Au lines (406.51 and 389.79 nm) as a function of time. A strong broadening, asymmetry and shill was observable up to 800-1000 ns after the laser pulse. Spectral profiles of the delayed (with 0.8-1.0 ~ts) and timeintegrated (gate time of 2.5 gs) measurements were found to be well represemed by a symmetric Lorentz-type curve. Song et al. [104] investigated the linebroadening mechanism for three Cu lines (510.55, 515.32, and 521.82 nm) in the LIP by TRELIBS. Two emission lines observed at 515.32 and 521.82 nm revealed a dramatic decrease in line-width as the delay of the gate was increased, while the line at 510.55 nm showed a small decrease in line-width. The authors suggested that the larger change in line-width depending on gate delay for 515.32 and 521.82 nm lines were attributed to Stark broadening and characteristics of the Rydberg-like atomic state. Recent work by Lee et al. [22, 27] also showed that the selection of the location in the plasma is a crucial for obtaining the best operating conditions, in particular to achieve the highest signal-to background ratio of analytical atomic lines and to avoid self-absorption and line broadening. They also found that the degree of self-absorption and line broadening strongly depends on the surrounding atmosphere and irradiation wavelength. Recently, Issac et al. [105] reported the observation of a twin peak distribution of the electron pulses occurring during the interaction of infrared radiation from a pulsed Nd:YAG laser with a Ag target. The use of prompt electron pulses as sources for electron impact excitation is demonstrated by taking nitrogen, carbon dioxide, and argon as ambiem gases. Nakai et al. [ 106] studied the transient behavior of emissive atoms and dimers formed with Mo(CO)6, measured by using a time-resolved emission spectroscopy (z =355 or 266 nm). Most atomic emissions were found to consist of two distinct transient components, an "early" component with a similar time profile to the laser pulse, and a "late" componem with a peak at around 300 ns after the laser pulse. The fact that the addition of an electron scavenger decreased the late emission sensibly suggests that the late emission was caused by Mo* produced in a neutralization process of ionic species. A plausible mechanism involving electron-ion recombination was proposed for late emission. St-Onge et al. [ 108] investigated the physical properties and emission yield of plasmas in some detail produced by a double-pulse laser beam (x =1064 nm), in comparison with the single-pulse case. As expected, line emissions were enhanced by the use of the double-pulse approach. Regardless of the absolute intensity of a given line for a

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given element, there is a similar improvement in precision when going from single- to double-pulse operation. The other limit for use of LIBS for quantitative elemental analysis is related to the stability of the laser-induced plasma emission which originated from the laser intensity fluctuation (1-5%) and the scattered light depends on the local matrix effects and on physical and chemical characteristics. The most common method to compensate for signal fluctuations in LIBS is based on calculating the ratio of the spectral peak intensity to that of the reference intensity. However, this currently used internal calibration is that relative, rather than absolute, concentrations are obtained. Recently, Xu et al. [109] developed a new data acquisition approach followed by a suitable data analysis for LIBS. It provides absolute (rather than relative) concentration of elements in particular materials, e.g., industrial dusts and soils, using a sequence of signals from single-pulse breakdown events. This method does not require an internal standard element of known concentration; however, standard samples are still needed to obtain calibration curves. Wachter and Cremers [110] examined the effects of the laser repetition rate, the detector gating parameters, and the number of averaged laser shots on the precision of plasma emission lines. They found an increase in precision (RSD) with increasing laser repetition rate, which was attributed to the increase in and stabilization of the concentration of airborne material above the liquid surface at higher laser repetition rates. For the net signals, the RSD decreased from 13.3% for 50 laser shots to 1.8% for 1600 laser shots. Wisburn et al. [111] also found that the RSD was low up to a laser repetition rate of 8 Hz and then increased dramatically. Investigations of the differences in RSD between fresh and same-site measurements indicated that when more than two shots were collected from the same site, the RSD increased. The lowest RSD was obtained when each shot sampled totally new material. Eppler et al. [112] investigated precision as a function of the laser focusing lens for both spherical and cylindrical lenses for the detection of Pb and Ba in soil samples. They found a reduction of RSD when using the cylindrical lens instead of the spherical lens, when the particle size of the ratioed elements varied significantly. Castle et al. [98] systematically attempted to identify and quantify the controllable factors that affect the precision of LIBS measurements. They investigated the effect of the choice of analytical line, emission signal temporal development, sample translational velocity, number of spectra accumulated, laser pulse stability, detector gate delay, surface roughness, and background correction on LIBS precision. All these measurements were made by using a pure copper sample to eliminate the effect of sample homogeneity on precision. The results were presented in two formats; within measurement/shot-to-shot precision (intra-

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J. SNEDDON, Y.-I. LEE, and K. SONG

measurement) and between measurement precision (inter-measurement). For inter-measurement precision, the following variables should be considered (% RSD ranges indicated inside the parentheses): translational velocity (9.5-0.03), laser pulse energy (8.5-1.2), and number of spectra averaged (6.3-1.0). Gate delay, surface roughness, and background correction has only a minimal effect on the inter-measurement precision. For intra-measurement precision, translational velocity (29-0.29), pulse energy (23-11), and gate delay (21-11) should be considered. Surface roughness and background correction also have only a minimal effect on the intra-measurement precision. LIBS signals are usually very noisy. Except for general instrumental noise, most of the noise comes from the LIP. Since the basic characteristics of a LIP are its high temperature and energy propagation in the form of a shock wave, the corresponding signal seriously fluctuates with some kind of background. Younan and Zhang [113] investigated a noise reduction technique based on an improved adaptive line enhancement (ALE) to effectively increase the S/N ratio level in LIBS spectral data 5. INSTRUMENTATION

A recem review by Song et al. [ 114] and book by Lee et al. [2] describes in detail the more recent developments in instrumentation for LIBS. This section is presented to give a general overview of this very important area. A typical LIBS instrument or set-up is shown in Figure 3. It requires a pulsed laser, a sample chamber with rotating device for mounted samples, a collecting optics for the plasma emission and a monochromator with photon detector. The development of this laser technique promoted the presence of a more compact and more powerful pulsed laser system, while the cost of the laser system is becoming less expensive. The development of a compact laser system is more or less oriented to the Nd:YAG or Nd:YLF series. Recently the diode pumped Nd:YAG laser system is becoming popular, even though the cost of the laser is still higher compared to the lamp-pumped Nd:YAG lasers. The excimer laser and CO2 laser have been also developed with many new laser based analytical techniques, but the use of these lasers is not so frequent in LIBS There has been a trend towards ultra-violet (UV lasers) [115, 116] and lasers with a shorter pulse duration [ 117, 118]. Margetic et al. [ 119] investigated the laser-induced plasma of brass samples under an argon gas by 170 femtosecond (fs) and 6 nanosecond (ns) laser pulses using time-resolved LIBS. They observed differences in the temporal evolution of atomic and ionic line intensities for fs- and ns- pulses caused by the effect of the laser-heated buffer gas plasma above the sample surface. The ablation process and subsequent

Laser-Induced Breakdown Spectrometry

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excitation of atoms was more reproducible for fs- than for ns-pulses because of the absence of the laser-heated buffer gas plasma. Meanwhile time-resolved LIBS has been popular in recent years compared to time-integrated space-resolved LIBS. In time-resolved LIBS applications, the intensified charge-coupled device (ICCD) or other type of gateable photon detectors are fi'equently adopted. Since the time-integrated spatially resolved LIBS has not been used frequently in recent years, not much of the works are included in this paper. The typical references can be found elsewhere [120]. The following section discusses the most widely used laser systems for LIBS.

Nd:YAG laser Vacuum Pump

Mirror .J.. ...... ~1............ :'::'":'"'::: ~ ."....................

l~

.w m.

I

Vaccum ~ . Lens Gauge ::

Sample

Optical Fiber

I Spectrograph

'

~

Motor

Mass Flow Controller

~

~ ~ ~ I ~ ~

Gas

Figure 3. A schematic diagram of a typical experimental set-up for laser-induced breakdown spectroscopy

316

J. SNEDDON, Y.-I. LEE, and K. SONG

5.1 Excimer laser and CO2 laser based LIBS Rinaldi and Ferrero [121] studied the analysis of Ca in BaC12 matrix by using a TEA-CO2 laser with laser fluence of 18 mJ/cm 2. This laser was capable of 200 ns pulse and 0.2 J at 10.6 ~rn operating at 1 Hz. Marpaung et al. [122] used a TEA CO2 laser (400 mJ/pulse, 100ns) for the irradiation on glass samples surrounding by air at a pressure up to 760 torr. Li et al. [123] adopted an excimer laser (~ =308 nm, pulse width : 20 ns) for on-line control of selective removal of cobalt binder from tungsten carbide hardmetal by pulsed UV laser surface ablation. The laser fluence was 2.5 J/cm 2. Li et al. [124] also used a pulsed excimer laser operating at 248 nm to diagnose in situ the surface ablation of cobalt cemented tungsten carbide hardmetal by LIB S. Garcia et al. [125] reported an angle resolved LIBS for depth profiling of coated materials by using the XeCI excimer laser (~ = 308 nm). This laser system is capable of 107 W/cm 2 operating at 5 Hz with the pulse width of 28 ns. The average energy was 100-420 mJ. Ng and Cheung [126] used ArF excimer laser (193 nm, pulse width : 15 ns) for the analysis of sodium and potassium in single human red blood cells by LIB. De Giacomo et al. [127] adopted a KrF excimer laser for the ablation of titanium dioxide and monoxides. The applied laser fluence was 1.7 to 6 J/cm 2. 5.2 Nd:YAG laser based LIBS instruments There are many incidences of LIBS application using a Nd:YAG laser system. Gomba et al. [128] adopted a Q-switched Nd:YAG laser (~7-8 ns of pulse width) for the spectroscopic characterization of laser induced breakdown in aluminum-lithium alloy samples. This laser had pulse energy of 50 mJ/pulse at 1064 nm and was operated at 20 Hz of repetition rate. The laser fluence on the aluminum sample was 6.4 J/cm 2. A steel alloy has been analyzed by Bassiotis et al. [129] by adopting a Nd:YAG laser system at 1064 nm as well as 355 nm. St-Onge and Sabsabi [130] applied LIBS technique to depth-profile analysis of annealed zinc-coated steel samples, with the particular goal of examining how LIBS depth profiles can be fully calibrated. A Nd:YAG laser (1064 nm, 5 mJ/pulse) was used for the experiments. Wallis et al. [131] used LIBS in the analysis of the Lignite by adopting a Nd:YAG laser system operated at 1064 nm. 5.3 Fiber-optic based LIBS instruments There has been various applications of LIBS technique in environmental problems and has the potential in biological and clinical samples. In the environmental situation, some of these applications adopted optical fibers to have a remote sensing capability. A typical schematic diagram of a fiber-optic based LIBS instrument is shown in Figure 4 developed by Marquart et al. [ 132].

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Gruber et al. [133] adopted the LIBS technique for the in-situ analysis of liquid steel. They used a fundamental of Nd:YAG laser, which had a pulse width of 5 ns, and operated at 20 Hz with the pulse energy of 360 mJ/pulse. Buckley et al. [ 134] used a Nd:YAG laser, which had pulse width of 10 ns, and pulse energy of 350 mJ/pulse, to continuously monitor toxic metals. The emission signal was collected by a fiber optic cable, 0.25-m monochromator (2400 grooves/mm) and a time-gated CCD array. The laser ablation and the collection of luminescence were done using the same collimating lens. But the collection optics assembly consisted of two lenses, one for collimating emission from the sample surface, the other one to focus the emission to the tip of the optical fiber, and two mirrors for the reflection of the emission lights. This Sandia LIBS system was deployed at the China Lake Naval Sir Weapons station to measure lead emission during a containing bum of Shillelagh anti-tank missiles, for which the Naval Air Warfare Center and Lockheed Martin are working together to develop a demilitarization procedure. The Navy's contained burn facility (CBF) tests of LIBS monitor illustrate the combination of rapid response, large dynamic range, and hysteresis-free measurements that are possible with the Sandia continuous emission monitor (CEM). Palanco and Lasema [135] developed a fully automated LIBS system based on fiber-optics for quality assessment in the steel industry. Nd:YAG laser (1064 nm) and a crossed Czemy-Tumer (F/3.7, focal length=125 mm) were used. A laser-to-fiber robust interface was developed, which permitted safe and reliable transmission of the laser beam without gradual degradation of the fiber optic. Whitehouse et al. [136] developed a remote sensing LIBS system to remotely analyze a steam generator tubes in nuclear power station. They used a small Nd:YAG laser system (Big sky model CFR-GRM), which is operated at 20 Hz with 1064 nm. A fused silica optical fiber with core diameter of 550 micron was adopted to deliver the laser light to the sample as well as to collect emission signals. The spectrometer was 0.5 m of focal length and F-number of 6.9. In order to optimize the analysis system, they used a reference samples. The target element was copper. The real-time detection of halon derivatives, such as CF4, CF3H, CF2H2, C2FsH, are studied by Williamson et al. [137] by adopting a Q-switched Nd:YAG laser system operated at 1064 nm [128]. A fiber optic cable was used to deliver the laser light as well as to collect emission signal for intense fluorine atom lines. An argon sheath was also used to minimize the mixing of the analyte with room air.

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Figure 4. Schematic diagram of experimental setup adopting optical fibers Martin and Cheung [138] used a TRELIBS (time-resolved LIBS) to detect chromium aerosol. A Nd:YAG laser was operated at 1064 nm, 532 nm, 355 nm, 266 nm and 10 Hz of repetition rate. The aerosols were generated by an aerosol generator. A carbon-core fiber optic cable bundle was used to deliver the laser as well as to collect emission signal. A spectrometer (Acton research model Spectrpro-500) with ICCD detector is used to detect the signal. Neuhauser et al. [139] also demonstrated that a rather simple fiber-optic interface suitable for long-term remote sensing via LIPS can be realized if all relevant parameters- i.e., laser, fiber, and optical setup- are taken into account. A Nd:YAG laser (10 Hz, 1064 nm, 200 mJ/pulse or 532 nm) and an optical fiber of 400~1000 micron diameter was used for the signal detection. The fiber is placed slightly behind the focus in the diverging laser beam, so that about 70-80% of the core diameter is illuminated. The detection of the signal was done in some

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degree of angle. Carranza et al. [140] used Nd:YAG laser for the on-line analysis of ambient air aerosols. The detection of the emission signal was performed by a fiber optic cable and collection optics including a pierced mirror. The pierced mirror was also adopted for the works by Buckley et al. [134] and Sun et al. [141,142]. 5.4 Field instrumentation for LIBS There has been considerable work on the development of field or portable LIBS instrumentation. The first real portable instrument was developed at Los Alamos National Laboratory by the Cremers research group [143]. They used a battery power supply and the system weighed only 14.6 kg with the dimensions of 46 x 33 x 24 cm 3. The whole system was small enough to be fit into a small suitcase and was operated by a 12V battery. A passively Q-switched Nd:YAG laser was adopted and the laser system output was 15-~20 mJ/pulse at 1064 nm with 4-~8 ns of pulse width. The system was operated at 1 Hz. The signal analysis and detection was done by an optical fiber, 1/8 m spectrograph and a compact charge coupled device (CCD) system. Barbini et al. [97] also developed a portable LIBS system for remote detection. Their system had limited length of fiber optic cable but used a conventional laser power supply and laser system. Therefore their system cannot be a real portable LIBS system. They used the 3 rd harmonic of the Nd'YAG laser, which had a pulse width of 8 ns, and operated at 10 Hz with a pulse energy of 5-~30 mJ/pulse. They used a monochromator (F-number: 6.4, 0.55 M, 1200, 2400, 3600 grooves/mm) with an ICCD detector (690 x 256) at the wavelength range of 180~550 nm. The fiber optic coupling was adopted with a 10 micron fiber. A peak finder by using Labview software and standard reference data was used to identify the spectra in this study. A more compact LIBS system was developed by Castle et al. [144] in 1998. They adopted a miniature Nd:YAG laser operated at 1/3 Hz and 1064 nm with a pulse width of 3.6 ns. The spectrometer was equipped with a linear CCD detector (2046 pixels) at the operating wavelength range of 339-462 nm. The average power density was 0.92 GW/cm 2 and the system weighed only 13.8 kg with the dimension of 48.3 x 33 x 17.8 cm 3. A notebook computer was adopted for the analysis of the detected signals. The system is shown in Figure 5. MelDok Inc. of Russia and Technical assistance international of United State of America have jointly developed a portable LIBS system called MD5095 laser microanalyzer. They adopted a Nd:YAP laser operating at 1079 nm and a microscope for viewing sample surface. The amount of the ablated samples are normally 100 ng/pulse and the ablating area was 1-~2 micron in diameter. The laser-induced plasma analyzer was developed by adopting an echelle

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spectrometer in LLA instnmaents GmBH. They use a Q-switched Nd:YAG laser (6 ns of pulse width) operating at 10 Hz. A programmable fast pulse generator with a gated microchannel plate (time resolution 950 ns, 1024 x 1024 pixels) are used for the detection of the emission signal at the wavelength range of 200 nm 780 nm. The model name is LIPAN 3002. Advanced power technologies Inc. has developed a LIBS system called Tracer 2100. This system is a bench-top LIBS instrument with a Nd:YAG laser (10 Hz, 1064 nm, pulse width : < 7 ns, Peak power : 40 MW at 5 ns pulse) and conventional power supply for laser system. The spectrometer was a CzemyTurner type with 1800 or 2400 grooves/mm grating. The spectral range of the system was 190 to 800 nm with the resolution of 2 angstrom. An ICCD camera (128 x 1024 pixels) was installed in the spectrometer and the gate resolution was less than 0.1 microsecond. An automated sampler was also installed by adopting a latitudinal stage 12 V DC motorized folded stage. The observed spectra were analyzed by the Tracer software including detailed spectral line library. This system has been tested and results were recently published by Rosenwasser et al. [145]. In this study the target samples are phosphate ore samples and a reasonable calibration curves are obtained for P205, CaO,A1203, MgO and SiO2. A DIAL (Diagnostic Instrumentation & Analysis Laboratory) of the Mississipi State University also developed a LIBS system by adopting a Nd:YAG laser. The required space for the system is 42 in x 24 in x 30 in, while the laser power supply has dimension of 21 in x 11 in x 20 in. The detection of the emission signal was done by a 13 feet UV optical fiber with IDAD (Intensified diode array detector) or CCD. A portable LIBS system was also developed for in-situ application by the Applied research laboratory at Pennsylvania State University. A handheld YAG laser head is used to directly aim the target surface. A collecting lens coupled with an optical fiber is attached to the laser head so that the emitted photons from the plasma can be collected to the spectrometer via the optical fiber. A laptop computer is used for data acquisition and analysis. Mirov and Pitt [ 146] constructed a laser induced breakdown spectrometer. Their design adopts a Nd:YAG laser with pulse energy of 50 mJ/pulse, pulse width of 5 ns, operating at 1064 nm and 10 Hz of repetition rate, or an Alexadrite laser (pulse energy : 80 mJ/pulse, 740 nm, pulse width : 40 ns, 20 Hz) and a fiber optic cables. A 0.75- m spectrometer with a grating of 1200 or 2400 grooves/mm and a thermoelectric cooled Intensified CCD camera (256 x 1024 pixels) are used for the detection of the emission signals. Especially, an elliptical mirror was adopted for the better collection efficiency of the emission. Thermo ARL also developed a LIBS system based on their spectrometer, a Nd:YAG laser system and a photomultiplier tube (PMT).

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Wainner et al. [147]. analyzed the lead comamination by the comparison of LIBS field and laboratory insmunems. Their portable LIBS instrument (ADA LIBS) is based on a prototype designed by Cremers' group at Los Alamos National Laboratory [144]. The complete LIBS instrument is contained within a 23 x 51 x 38 cm aluminum case. This system can be operated from both a standard 12-V snow mobile battery and 115-V AC current. The fiber optic cable collects and transmits the light to a small spectrograph (0.15-m focal length, 2400 grooves/mm, 250-nm blazed) with a thermoelectrically-cooled 250 x 12 element CCD (24 mm pixel). The laser is passively Q-switched and provides a nominal 15 mJ/pulse at 1064 nm. The laser beam can be focused to the size of 60 mm using a 45 mm focal length lens. The laser head and the fiber optics are installed to the probe head. No focusing lens is used in front of the fiber optic to

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collect and the width of the individual fiber defines the aperture for the light input to the spectrograph. Meanwhile their Lab. System is consisted of a actively Q-switched Nd:YAG laser (12 ns pulse, 3.0 mm diameter, 10-300 mJ/pulse) and a 0.3 m spectrograph (1200 grooves/mm, 500 nm blaze). The detection of the light was done by a thermoelectrically-cooled intensified CCD camera which is capable of gating down to 13 ns. The portable system was tested in the field on soil and paint samples at two military sites with the sensitivity to a 100-ppm. 5.5 New approaches to LIBS Recent developments in LIBS technology provides a more versatile application of the technology. The new approaches include adopting dual pulse operation, multi-fibers, resonant ablation, and combining with laser-induced fluorescence spectroscopy, etc. Lewis et al. [148] detected optical emission produced by a pulsed glow discharge laser ablation system. A Nd:YAG laser system was operated at 10 Hz with the pulse width of 10 ns. The output energy of 1064 nm laser beam was lxl09 W/cm 2, while the output of the 532 nm was ---3 mJ/pulse. The glow discharge system was constructed with using a direct insertion probe and 2 ms GD was operated at 10 Hz and 1 kV. The pulse delay generator (SRS DG 535) was adopted for the time synchronization between the laser pulse and the pulse GD. The emission signal was detected by a photomultiplier tube (PMT) (Hamamamatsu model R955) adapted to the monochromator (F-number: 4.1). The experimental results demonstrate the ability to time ablation appropriately to access specific temporal regions of the pulsed plasma. Nakamura et al. [149] reported a result of the determination of an iron suspension in water by LIBS with two sequential laser pulses. In this study two Q-switched Nd:YAG laser were used. One delivered 8 ns pulse of up to 600 mJ/pulse and the other 6 ns and 160 mJ at 532 nm. They were operated at 20 Hz. The luminescence was collected by a telescope and transferred with an optical fiber to a multichannel photodetector mounted on a 32- cm polychromator with a grating of 1200 grooves/mm. The nominal resolution of the system was 0.24 nm. The synchronization of the laser pulses were controlled by a digital pulse generator. The sequential pulse excitation revealed that the S/N was improved compared to the case of using only one laser pulse. They were able to determine iron concentration in water as low as 16 ppb both in FeO(OH) colloidal suspension and in boiler water sampled from a thermal power plant. Stratis et al. [150] adopted a dual-pulse LIBS using a pre-ablation spark for enhanced ablation and emission. Both Nd:YAG lasers were operated at 1064 nm at 5 Hz of repetition rate. The fiberoptic was coupled to a 0.25-m. f/4 spectrograph. The detection system consisted of a gated intensified CCD with

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model PG-200 pulser and Model ST-138 detector controller. In this study the first laser pulse is brought in parallel to the sample and focused above it to form an air plasma. LIBS emission is observed in the plasma that is created by the second laser pulse. They identified 11-33 fold enhancements in LIBS signal by using this technique. Sun et al. [141, 142] applied LIBS for Zinc determination in human skin. A 60 mJ/pulse Nd:YAG laser operating at 1064 nm was used to form the laser induced plasma. The laser beam was focused on the surface of the sample by a focusing lens through the pierced mirror and the emission signal was collected by a pierced mirror and a telescope. A small monochromator (0.5 m) and an intensified CCD was used with the gating and control electronics for the signal detection. The experimental result revealed that human skin absorbs Zn through the skin and its concentration was decreased exponentially with depth into the skin. The same authors also used the same LIBS system for the determination of Mn and S i in iron ore. Samek et al. [ 151 ] applied the LIBS technique for the analysis of certified tissue samples. By adopting a Nd:YAG laser and a standard spectrograph reciprocal dispersion of 0.7 nm/mm) with a gamble, intensified photodiode array detector (PDA). Their main target elements were Ca, Sr, A1, Pb, and Mg. They could also analyze concentration ratio of Mg content to Ca content for a section of teeth. Maravelaski-Kalaitzaki et al. [152] adopted an excimer lasers (KrF, ~. = 248 nm and XeC1,/~ = 308 nm) for the cleaning of encrustation on Pentelic marble. They showed that both lasers are appropriate for achieving sufficient removal of unwanted selected layers without modifying the surface morphology. Wiggenhauser et al. [153] reported the results for non-destructive testing of element distribution on surface. In this study, a Nd:YAG laser with 10 ns pulse and 300 mJ/pulse of energy was used for the sample ablation. The laser beam was focused to the surface of the sample by a f = 500 mm lens, and the luminescence was collected by a optical fiber. The major elements monitored from the building materials are Ca, Si, A1, Mg, Na, Fe, and C. As a result of the investigation, they could distinguish between cement matrix and aggregates in concrete, and differentiate between different types of aggregates (e.g. quartz and marble). They could also measure salt concentration and profiles with high geometrical resolution. A recently developed passively Q-switched Nd:YAG microchip laser is a very compact in size, while the peak power is big enough to induce photoablate materials [154]. Ptnnped with a 1.2 W diode laser, passively Q-switched microchip lasers can produce pulses as short as 218 ps with peak powers in excess of 25 kW at pulse repetition rates greater than 10 kHz. The passively Q-

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switched microchip laser is consisted of two mirrors and gain medium and saturable absorber. Since these laser systems can be focused to intensities in excess of 1 TW/cm 2, it is sufficient to breakdown metals and many other solids. A new approach to enhance the temporal resolution of the spectra was initiated by Bulatov et al. [ 155] by using a simultaneous multifiber spectroscopy. They used a Nd:YAG laser (1 Hz, 7 ns, 100 mJ/pulse) and the laser beam was focused on the sample by a focusing lens (f = 250 mm) with the fluence of 2.25 x 109 W/cm2. A spectrometer and a fiber-coupled ICCD detector with 1352 pixels are used for the detection of the signal. In this study the focal point of the ablating laser beam was arranged to be ---50 mm beyond the sample surface to induce the soft ablation of the sample. Recently new attempts in applying LIBS to the paper analysis or the analysis of pigments in the painting have been pursued by several research groups in Italy, England, and Greece. Salimbeni et al. [156] applied LIBS for the achievement of optimum laser cleaning in the restoration of artworks. A Nd:YAG laser was adopted as an ablation source, while a diode laser (890 ran, 20 ns pulse duration) was used to detect image of the laser-induced plasma. Imaging optics and CCD camera are also used for the data recording. Burgio et al. [157] used LIBS for pigment identification in paintings in combination with Raman microscopy. Their LIBS system consists of a nanosecond Q-switched Nd:YAG laser (355 nm, 3rd harmonic), an optical fiber with a spectrograph. The detector is an OMA system with an intensified photodiode array detector. They could identify elements in the pigments layerby-layer. Bicchieri et al. [158] also used LIBS for characterization of azurite and lazurite based pigments. The LIBS setup is based on a Nd:YAG laser delivering a maximum of 0.4 J in 8 ns at a wavelength of 1064 nm and a Mechelle 7500 spectrometer coupled with an intensified CCD camera. The detection system is abe to collect, with a single laser shot, with a constant resolution l/D1 = 7500. In this study the laser energy is minimized in order to obtain the least ablation effect, with a crater diameter of approximately 10 mm, the laser power delivered on the sample is lower than l012 W/cm 2. The LIBS of pure azurite and lazurite spectra revealed the presence of the pigment and impurity elements (Mg and A1 on the azurite sample and Fe, Mg, Ti, Cu and Ca on the lazurite sample). The LIBS has been combined with the laser-induced fluorescence (LIF) technique to improve the detection sensitivity. Hilbk-Kortenbruck et al. [159] analyzed heavy metals in soils using LIBS combined with LIF. The LIBS-LIF system consists of two laser systems, a Q-switched Nd:YAG laser operating at 1064 nm with a repetition rate of 10 Hz which can deliver laser pulses with pulse energy of up to 450 mJ and a pulse width of 5-7 ns, and a dye laser

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operating at 583 nm or 574 nm pumped by a frequency doubled Q-switched Nd:YAG laser (600 mJ full power at 532 nm, 10 Hz, 6-9 ns). The second laser beam was frequency doubled by a mixing unit to generate the UV range at 291.5 nm or 276.8 nm. The radiation of the LIBS plasma as well as the laser-induced fluorescence was guided to a Paschen-Runge spectrometer via fiberoptics. The calibration curve based on the LIF signal showed significantly improved limit of detection of 0.3 and 0.5 ~tg/g for Cd and TI, respectively. Telle et al. [160] also applied LIBS-LIF technique for the analysis of metallic samples. In this study two Nd:YAG lasers, one for the laser ablation and the other for the fluorescence generation have been adopted. The ablation laser was operated at 1064 nm, while the other laser was operated at 532 nm to pump the frequency tunable Ti:Sapphire laser system. The FTIR modulator is used to gate two laser beams, because both laser beams introduce to the sample surface collinear. Romero and Lasema [161] has investigrated the diffusion of aluminum in solar cell fingers into the silicon bulk using imaging-mode LIBS. A pulsed nitrogen laser beam (337.1 nm) was used in this work. A total area of 3 x 2.5 mm 2 has been sampled with a lateral resolution of 50 mm in the minor direction axis of the elliptical crater. Spatial and depth profile distributions are provided with no previous sample treatment in a brief period of time. Knight et al. [162] investigated the feasibility of adapting LIBS technique for stand-off analysis of geological samples under Martian atmospheric conditions (5-7 torr pressure of CO2). It is demonstrated that an analytically useful laser plasma can be generated a distance up to 19 m by using only 35 mJ/pulse from a compact laser. There is a significant decrease in the analyte signas below 1 torr of ambient pressure, resulting in a higher detection limits. Multiple pulse excitation have been suggested to increase the signal strengths for soils.

5.6 Echeile spectrometer In most applications of the LIBS technology, Czemy-turner type spectrometer has been adopted. Recently more LIBS instruments are based on the echelle-type grating which is grooved or blazed such that it has relatively broad faces from which reflection occurs and narrow unused faces as developed by Florek et al. [163]. This system allows the simultaneous detection from 220 nm to 400 nm with a resolution 1/D1=15,000(4 pixels). One of the important aspects of this system is that this is a stand-alone spectrometer, therefore, it can be connected with various type of Intensified or non-intensified CCD cameras. This geometry provides highly efficient diffraction of radiation and free spectral range. Normally the echelle grating has rulings between 8 and 300 grooves/mm.

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The echelle produces many orders of diffraction within a small range of diffraction with the small range of diffraction angle and the resulting spectrum is complex. The spectrum is sorted by a prism perpendicular to the echelle grating. This creates a two dimensional array of wavelength on a focal plane. For a 79 grooves/mm echelle operating in Littrow configuration, 90 orders of diffraction are used to cover the range 200 to 800 nm (order 118 to order 28). Various types of echelle spectral system are developed and available from Multichannel Instruments adopting various CCD detectors, such as products from Apogee Instrument Inc., PCO Computer Optics, Andor Technology, Inc., etc. For Michelle 900 model from this company, the focal length is 110 mm and F-number is 4.6. The spectrograph head weighs less than 5 kg. LLA also supplies echelle spectrometer (Echelle spectra analyzer ESA 3000EV). This system can detect LIBS signal simultaneously from 200 nm to 780 nm. The linear dispersion per pixel (24mm) is 0.005 nm at 200 nm, while 0.019 nm at 780 nm. Diffraction order of the spectrometer is 30 to 120. The attached CCD camera has 1024 x 1024 pixels which can be UV sensitized or coupled to MCP by fiber optics. Detalle et al. [ 164] evaluated this system in material analysis by LIBS. The concluded that despite many disadvantages such as, change in resolution with wavelength, not equal response of the intensifier throughout the spectral range, the echelle spectrometer combined with ICCD detector can be one of the best choice in LIBS study. Uhl et al. [165] adopted the echelle spectrometer for the fast analysis of wood preservers using LIBS. The echelle grating can produce up to 100 grating orders. The simultaneous recording of the complete relevant spectral range from 200 nm to 780 nm with a linear dispersion of approximately 5-18 pm/pixel is possible with this system. The additional coupling of the CCD-chip to an image intensifier allows the detection of extremely low signals (light amplification approximately 106). 6. APPLICATIONS Laser induced breakdown spectrometry (LIBS) is a laser based analytical technique which has primarily been proposed and used for qualitative and quantitative elemental determination in a wide rage of samples such as various environmental samples, metallurgical and non-metallic solids, liquid samples, aerosols, gases samples, advanced materials as well as in some miscellaneous applications. Since around the early 1980"s there has been a steady increase in the amount of literature on the applications of LIBS. In this selection, selected applications are presented, emphasizing those that depend upon the more recent advances in LIBS. Because of the breadth of activity, the list is by no means

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exhaustive or detailed, but is given as a guide to the versatility of the LIBS technique. Among the applications of LIBS composition analysis of metallurgical samples has been most popular. Recent applications, however, are more inclined to environmental samples and liquid samples including biological samples.

6.1 Environmental applications LIBS has been applied to elemental analysis in a number of environmental samples such as air, soil, sewage sludge, and solutions. Environmental problems are among the most interesting and topical in applications and have recently attracted the most attention. Among the applications is a growing interest in the in-situ analysis, on-line monitoring, remote sensing of the pollutants, or specific elements. Fiber optic-based LIBS has the potential to be standard equipment in these applications. In addition much effort has been devoted to the development of a portable sensitive LIBS immanent for use in monitoring in the field. The detection C12 and P atoms from diisopropylmethyl phosponate in air has been performed. Results of 690 parts per million (ppm) of P were detected. The projected detection limit was 60 ppm for C12 and 15 ppm for P [ 166]. This group also used LIBS to analyze for F2 and C12 in air by monitoring atomic emission lines at 837.6 nm for C12 and 685.6 nm for F2. Minimum detectable concentrations of C12 and F2 in air were 8 and 38 ppm (w/w), respectively. Minimum detectable concentrations of C12 and F2 were, respectively, 80 ng and 200 ng in air and 3 ng for both atoms in a helium atmosphere. The relative standard deviation (RSD) or precision for replicate sample analyses was 8% [168]. The determination of Be was one of the specific applications of LIBS, became Be is known to be a significant inhalation health hazard to workers exposed to dusts of Be and its alloy. Radziemski et al. [168] demonstrated the development of a system which could routinely monitor concentrations of Be in air. They published the results of the determination for Be, Na, P, As, and Hg in air. Beryllium in atmospheric pressure in air was detected at 0.7 ~tg/m3, which is 0.6 ng/g of air (RSD=30%). The limit of detection for Na, P, As, and Hg were estimated as 0.006 mg/g, 1.2 ~tg/g, 0.5 lag/g, and 0.5 l.tg/g, respectively. The observed limit of detection for these elements were comparable with those of conventional inductively coupled plasma-atomic emission spectrometry (ICPAES). However, the ICP-AES showed a much better detection limit for other elements, particularly for Hg (0.5 I.tg/g for LIBS, 0.0006 ~tg/g for ICP-AES). Direct determination of Be on filters was also tested by this group, because a direct monitoring of air with LIBS can detect Be concentrations down to only one-third of 2 mg/m3, the U.S. standard for Be monitoring. In this study, the calibration of the detecting system was performed in several ways: (1) by

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depositing a known Be mass on the surface of the filter by a nebulizer/heatchamber apparatus, (2) by passing an aqueous suspension of the particles, which were prepared by positioning Be metal under water and ablating material off with a focused laser beam, through the filters, or (3) by passing a certain volume of well-stirred suspensions through the filters after mixing of a known mass of Be particles. The surface detection limit for particles 0.5-5 mm in diameter was 0.45 ng/cm 2, which corresponds to 3.6 ng total Be mass on the exposed area (32mm-diameter) of a 37-mm-diameter filter. The RSD for replicate sample analysis was 4 % [ 169]. They also detected Na and K in a coal gasifier product stream, airborne Be, and P, S, and C12 in various organic molecules. The detection limit for Be in air was measured as 0.5 ng/g, which is approximately 1/3 of the Occupational Safety and Health Association (OSHA) limit for the 8hour average exposure to Be [168, 170]. Approximate linear working curves were obtained over the concentration range of 5-20 gg/g. Chemical warfare agents was another area of application for environmental-related problems and the detection limit of ppm or parts per billion (ppb) was also obtained by Quigley et al. [171]. Loree et al. [172] demonstrated the capability of LIBS for remote probing into hostile environments. Potential applications included prospecting, oil shale analysis and pollution monitoring. Radziemski's group also published results on the real-time monitoring of coal gasifiers [173]. Serious attempts to real-time LIBS analyses of particulates in a combustion environmem were made by Ottesen et al. [174]. They developed an instnunent to determine the size of particles by light scattering and to measure the composition of particles by LIBS. In this study, quantities as low as 520 femtogram (fg) of Fe were determined. Overall detection limits for trace elements including Si, A1, Ti, Ca, Na in single coal particles were estimated at less than 100 ppm. Casini et al. [175] proposed a time-resolved LIBS (TRELIBS) system for the quantitative determination of small amounts of pollutants in gas. Oldenborg et al. [176] at LANL applied LIBS for measuring gaseous species involved in carbonate movemem in molten carbonate fuel cells. In this report, laser-induced breakdown spectra and photofragment fluorescence spectra of LiC1 were obtained and presented. Lancerlin et al. [ 177] applied TRELIBS for the detection and determination of CF4 in air and the detection limit ofF2 was obtained a~ 1000 ppm. Wisbum et al. [178] applied LIBS as a sensor for heavy metals in solid environmental samples. In that study, calibration plots for the analysis for heavy metals in diverse sand and soil samples were obtained. Singh et al. [179] used LIBS to characterize a large practical magneto-hydrodynamics (MHD) coal-fired flow facility (CFFF). In this study, a wavelength of 532 nm from a Nd:YAG laser was the ablation source and the characteristics of LIBS spectra at different delay times were

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described. The CFFF gas stream was a harsh environment with high turbulence and high luminosity. This group also applied LIBS to characterize the upstream region of a large MHD CFFF and a particle-loaded methane/air flame. The relative concentrations of several species were inferred by fitting the observed CFFF LIBS spectra with computer-simulated spectra [66]. In the study, they tested three different observational directions (forward, backward, perpendicular). The observed intensity was bigger in the forward direction, while backward and perpendicular observations showed comparable intensity. LIBS intensities were compared by measuring two Ca emission lines (428.94 nm and 431.87 nm). Flower et al. [180] applied LIBS technology to continuously monitor metal aerosol emissions in industrial process vents (exhaust stacks from electroplating baths), waste treatment processes (incinerators), and boilers and industrial fiamaces (coal-fired power plains). The approach used to measure total Cr concentrations in laboratory simulations of electroplating aerosols was also described in that report. Chromium concentrations less than 1 pg/cm 2 were measured. This work formed the basis for the future application to incineration and fossil-fuel power plants. Lazzari et al. [181] detected Hg in air by TRELIBS using an experimental apparatus. Cremers et al. [182] demonstrated the fiber-optic based LIBS measuremem for the remote detection of Ba and Cr in soil. Nordstrom [183] applied LIBS to evaluate the spectral characteristics of the interference from the nitrogen and oxygen componems in air using a CO2 laser. Of primary interest in the study was the atomic and molecular origin of the emission features. Comparison of the LIBS spectra with National Institutes of Science & Technology (Gaithersberg, Maryland, USA) (NIST) atomic emission data was presented in this paper. Davies et al. [184] applied fiberoptic based LIBS using a Nd:YAG laser 0~ = 1064 nm) system to detect concentrations of Cr, Cu, Mn, Mo, Ni, and V in NIST standards up to a distance of 100 m. In this investigation, a fused optical fiber with a core diameter of 550 gm was chosen to deliver a laser beam and detect the emission signal. The measured concentration of elements were approximately 200 ppm. Relative detection limit (3-6 error) was estimated as 150 + 50 ppm for Cr, 100 + 30 ppm for Cu, 210 + 70 ppm for Mn, 200 + 50 ppm for Mo, 150 + 50 ppm for Ni, 380 + 90 ppm for Si, and 200 • 50 ppm for V. In this report, the authors pointed out several sources of errors in the LIBS technique such as inhomogeneous distribution of trace elemems in the target, and variations in the alignmem of the light-collecting optics. Marquardt et al. [132] performed on-site determination of leaded paint in houses by LIBS with a doubled Nd:YAG laser (x=532nm). The measurement took less than 1 minute to perform, it required no sample preparation and it was made through overlayers of non-lead-containing paint. The limit of detection was 0.014% Pb in latex paint on a dry weight basis, with

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RSD 5-10 %. Arnold and Cremers [185] presented the results of the investigation and determination of T1 particles on filters. They detected T1 particles (< 20 jxna diameter) in less than 1 min by LIBS. Thallium was detected by forming a series of laser sparks across the filter surface. The detection limit for T1 was 40 ng/cm 2 on a filter surface. The useful dynamic range extends from 0.2 to 40 mg/filter using the strong 535.05 nm neutral T1 emission wavelength. Based on this investigation, LIBS has the potential to provide a method for rapidly analyzing multiple samples on-site at a low cost. Theriault and Liverman [186] analyzed Pb contaminated sand and soil by a fiber-optic LIBS (FOLIBS) system. Recent applications of LIBS are more inclined to practical field applications including the elemental analysis of environmental and industrial samples. Detection of metals in the environment using a portable LIBS insmnnent was performed by Yamanoto et al. [143] to monitor Pb in paint, metals in soils, and Pb and Be particles collected on filters. The authors primarily focused their research in evaluating a portable LIBS system. It was equipped with a compact laser of low pulse energies (10-20 mJ/pulse) and repetition rate (<1 Hz), for a wide rage of samples. Detection limits in ppm for elements in soils were 265 (Ba), 9.3 (Be), 298 (Pb) and 42 (Sr). The detection limit for Pb in paint was 0.8% (8000 ppm), corresponding to 0.052 mg/cm2. Direct field screening in the environment for metal contamination with a portable LIBS system can significantly reduce the time and costs associated with the sample collection, transportation, and preparation steps required by conventional laboratory methods. The authors also discuss in detail on the advantages of LIBS for environmental applications compared to X-ray Fluorescence spectrometry (XRF) which is currently the method of choice for many types of field screening measurements. Barefield et al. [187] used LIBS to analyze waste samples containing Sr. The mixed waste samples studied included vitrified waste glass and contaminated soil. A detection limit on the order of ppm for Sr was obtained by using the 407.77 nm emission wavelength. Detection limits obtained using a fiber optic cable to deliver laser pulses to soil samples containing Sr, Cr, Zr, Pb, Be, Cu, and Ni were also discussed in the report. The calibration curves for Sr were not linear in the concentration range of 50 - 1000 ppm. The detection limit for Sr, Cr, Zr, Pb, Be, Cu, and Ni were 13, 54, 65, 106, 3, 48 and 114 p p m , respectively. Schmieder [188] has applied LIBS to detect relative atmospheric abundance in turbulent CH4 [74-82-8]-air jet and in shale oil vapor. He also determined the fuel-air ratio in a CI-h-air flame with a high degree of precision. Poulain et al. [ 189] produced a laser-produced plasma (LPP) in an aerosol spray and the emission lines of Na and H ware monitored with an OMA (optical

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multichannel analyzer) to characterize the plasma spatially and temporally. The electron temperature of the plasma induced by an excimer laser was estimated to be 12,600 + 4,600 K. Calibration curve relating the Na(I) (589 nm) to H (656.3 nm) intensity ratio as a function of Na concentration, ranged from 100 to 10000 ppm. The limit of detection for Na by the current method under the experimental conditions was estimated to be approximately 165 ppm for monodisperse sprays and 925 ppm for one case involving a polydisperse spray. They also observed that the size of droplet strongly influences the observed emission intensity ratio. Taylor et al. [190] tried real-time measurements of species concentrations and temperatures of coal-gasification streams. In this study a comparison among LIBS, Coherent Anti-Stoke Raman Spectroscopy (CARS), and laser-induced fluorescence. Koskelo and Cremers [191] published detailed results on the investigation of trace element analysis on soil. Target elements for the analysis were As, Cr, Cd, Pb, Sr, Ba, Be, Cs, Hg, Se, Zr, and Sb. The measured detection limits were 1500, 28 (with 125 mJ laser energy), 141, 17, 24, 63, 2, 35, and 86 ppm for As, Cr, Cd, Pb, St, Ba, Be, Hg, and Se, respectively. Zironium and Sb were not detected with their experimental set-up and the minimum detectable amount for Cs was 500 ppm. Pakhomov et al. [192] detected Pb in concrete by LIBS and obtained a detection limit of 10 ppm. The optimum delay to obtain the best sensitivity was determined as 3 ~ts in this study. Recently, Eppler et al. [112] studied the effect of chemical speciation and matrix composition on Pb and Ba measurements in sand and soil matrices with the use of LIBS. The measured detection limits for Pb and Ba spiked in a sand matrix were 17 and 76 ppm (w/w), respectively. In spiked soil, the detection limits were 57 and 42 ppm (w/w) for Pb and Ba, respectively. Calibration curves were linear over two orders of magnitude for both elements, and measurement precision was highest (2.3% RSD) when a cylinderical lens was used. Both Ba(II) and Pb(II) emissions were dependent on analyte speciation, e.g., nitrate or oxide, in the sample. Wisburn et al. [11 l] determined selected heavy metals in soils, sand, and sewage sludge samples by TRELIBS. Various factors such as aerosol production, crater formation, size effect, timing effect, laser intensity, and humidity, affected the detection limits and the quality of the analysis were investigated. Detection limits for several elements (Zn, Cr, Pb, Cu, Ni, and Cd) were in the order of the 10 ~tg/g range, which are usually below the ecological requirements. There have been several efforts to develop a portable LIBS instrument and the test was somewhat successful. Further developments on a portable instrtanem are expected to result in a reliable and compact on-site analytical tool in the very near future. Vadillo and Lasema [193] used TRELIBS to obtain the

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spectra of minerals from different families including sulfides, vanadates and silicates and demonstrated the capability to conduct geological taxonomy and to analyze a field sample. Detection of heavy metals had been performed in different standard soil samples by using a mobile lidar LIBS system. Barbini et al. [97] and Ciucci et al. [96] had developed the semi-quantitative determination method of several metallic elements in different soils, even at low-resolution LIBS spectra, by the use of an internal standard reference method. For the most of the elements in soils, the detection limit proved to be 10-100 ppm, depending on the matrix. Xu et al. [194] developed a new data acquisition approach followed by a suitable data analysis for LIBS. It provided absolute (rather than relative) concentration of elements in particular materials, e.g., industrial dusts and soils, using a sequence of signals from single-pulse breakdown events. The detection limits of 10"12g of Zn in an aerosol (a solid or liquid particle in a gaseous medium) sample and 18 ppm of Cd in soil samples were obtained in this work. This method did not require an internal standard element of known concentration; however, standard samples are still needed to obtain calibration curves. Saggese [195] developed a field deployable LIBS system by the use of fiber optic sensor and analyzed spiked soil samples. Using the spiked and NIST standard soil samples with varying Cr concentrations, calibration curves were constructed for a variety of wavelengths. The minimum detectable limits for Cr and Pb were typically a few tenth of ppm level. The author also compared the minimum detectable limits for the fiber and free space delivery of laser beam for soil samples. It was found that there is not much degradation with the use of fibers. In recent years, the research by Hahn et al. [196] have been directed toward the development and testing of new continuous emission monitors for metals in process waste streams by LIBS. An approach based on random LIBS sampling and the conditional analysis of the resulting data was proposed as a means to enhance the LIBS sensitivity in actual waste streams. Chromium, Mn, and Fe signals were measured consistently during the test cycle by using the conditional data analysis approach. The typical LIBS "hit" rates realized in the process stack were about 2%. The resulting signal-to-noise ratio was improved by a factor of 50 with the conditional analysis approach in comparison to an ensemble average. LIBS technique has been applied to several spatially located atomic species in speleothems taken from the Nerja's Cave (Malaga, Spain) by Vadillo et al. [197]. The 532 nm output of a Nd:YAG laser was used to irradiate the samples and generate the plasma that was spectrally analyzed and detected by using an intensified CCD detector. The changes in signal intensity across the speleothems were clearly observed as a function of the growing direction. The RSD values for the CaCO3 mixed with a known concentration of Mg and

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the speleothem pellets were 9.7% and 12.7%, respectively. Lucema et al. [198] used LIBS to investigate an in-depth and 2-dimensional analysis of P, Zn, and Pb at different locations along the cental channel of a used automobile three way catalyic convertor. The authors postulated that the results showed why the lifetime of the convertor was only about 30,000-km.

6.2 Metallurgical samples The initiation and drive for the use and development of LIBS has been due to the need for direct and rapid determination of trace elements in various types of samples. This is because no other analytical technology can do trace determination as efficiently as LIBS without complicated sample preparation. Loree and Radziemski [199] examined Cu, aluminum alloy, and steel samples by LIBS as well as time-resolved LIBS (TRELIBS) for the trace elemental determination of a sample surface. The contents of Be in beryllium-copper alloy was determined by Radziemski et al. [200] and excitation temperature within the laser-induced plasma (LIP) were determined using the Cu(I) and Cu(II) emission lines. Leis et al. [201] also applied TRELIBS with a Nd:YAG laser to find optimum conditions for laser ablation and atomization for analytical purposes. Strong temperature changes were observed depending on the sample matrix composition. Calibration curves for Cr and Si in homogeneous and low-alloyed standard steel samples were developed. The detection limits calculated on a 3-o basis were 24 gg/g for Cr at 425.2 nm, 30 gg/g for Si at 288.2 nm, and about 200 gg/g for Si at 251.4 nm. The RSD measured on the analytical wavelengths were typically 6 %. When using an appropriate Fe line as the internal standard, the RSD of the intensity ratio was only 2.4%. Nemet and Kozma [202] studied an application of TRELIBS to the direct qualitative and quantitative analysis of tertiary high-alloys (gold jewels). The investigation was carded out over a wide spectral range in the ultra-violet (UV) and visible region for the analytical line pairs of Cu-Au, Ag-Au and Cu-Ag. The RSD of the background was <4%, and the RSD of the line-pairs was < 7-8%. With most of the line-pairs, a linear correlation could be obtained, when the concentration of Cu and Ag changed between 0 - 45%. Ko et al. [203] obtained excellent analytical results almost independent of the plasma temperature and the state of evaporation of the ablated sample material for Fe and Cr by the use of internal standardization in LIBS. The Fe/Cr line pairs selected for the measurements were 300.304nm/298.865nm, 300.957nm/301.371nm, and 449.457nm/450.030nm. The standard deviation of the data from the straight line was only 3%. Belliveau et al. [204] tested NIST steel standards at ordinary conditions (atmospheric pressure and in air) and calibration for Cr, Mn, and Ni had been performed with a Nd:YAG laser. In this work the effect of laser power, critical

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alignment/focusing procedures, and number of laser pulses per analysis were also studied. The determination of Mn, Si, and Cr in solid steel was performed by Cremers and Romero [205]. They examined several factors, which effect the LIBS analysis, such as lens-to-sample distance, laser pulse energy, and position of the imaging lens. These effects are minimized by ratioing the absolute element signal to adjacent Fe-lines. Grant et al. [58, 59] studied the determination of Fe, Si, Mg, Ti, A1, and Ca in iron ore by using TRELIBS with an XeCI excimer laser and the optimum period in the plasma lifetime for spectrochemical determination was obtained. As a result, 1 to 2 E3s delay was appropriate to obtain the best signal-to-noise ratio. Observed calibration curves were linear except for A1. At higher concentration range of A1 (A1/Fe > 0.01), the curves started to level out resulting in a decrease in sensitivity. This is due to the self-absorption of the AI(I) 396.15 nm resonance line. The elements/Fe ratio detection limit was estimated from 0.001 to 0.00003 dependent on the elements concerned. They also estimated oxide concentration detection limits and the obtained results were 0.003 % for CaO, 0.15 % for SiO2, 0.023 % for MgO, 0.013 % for A1203, and 0.023 % for TiO2. This study indicated that the detection limit can be lowered by the use of resonance wavelengths, but selfabsorption may degrade the precision and accuracy of the measurement. They also pointed out if the LIBS instrument was used for the on-site analysis of iron ore, emission wavelength located in the UV region with a high cross section cannot be adopted. Aguilera et al. [206] applied fiber-optic based LIBS for the determination of C contents in steel using a Q-switched Nd:YAG laser 0~=1064 nm). The C emission line at 193.09 nm was chosen for the determination, because the 247.86 nm line suffers interference from the 247.86 nm line of Fe(II). The detection limit was estimated at 65 ppm and the RSD was 1.6 %. In this study, a CO2-free environment was necessary in order to improve the accuracy and detection limit, since atmospheric CO2 can be dissociated in air. For this purpose, nitrogen was used as a buffer gas. Matrix effects for the studied steels were identified to be small or low by comparing the calibration curves for stainless steel samples to that of non-stainless steels. This group also applied the technique to determine C content in steel samples melted in a laboratory induction furnace, and observed an RSD of 10 % in the concentration range of 150-1100 ppm [207]. The wavelengths selected for the LIBS analysis were the 193.09 nm line of C(I) and the 201.07 nm line of Fe(II). The use of the UV region allows the strong thermal emission from the molten sample to be avoided. The selection of a time window of 1 to 6 its provided high line-tobackground and signal-to-noise ratios. The detection limit for C was 250 ppm in an argon atmosphere and 65 ppm in a nitrogen atmosphere. This study

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demonstrated that LIBS could be used for direct composition analysis in molten alloys. However, the removal of the carbon content produced by the decomposition of CO2, which is produced by combustion reactions with the molten surface, is a major obstacle in applying LIBS to on-line analysis of molten metals. Owens and Majidi [61] used a Nd:YAG laser (~ = 1064 nm) based TRELIBS for the investigation of National Bureau of Standards (NBS, now called NIST) powder sample containing 0.02 % A1, by placing the sample between two layers of tape attached to a Plexiglas plate. In this study, one of the main interests was to develop a good signal-to-noise ratio with a single laser pulse, because multiple laser firings generate the need for an increased amount of samples, which results in a large consumption of material. The composition of the sample changes after each plasma sampling for solid samples of alloys. They adopted TRELIBS and monitored the emission only during for a small portion of the plasma lifetime to increase signal-to-noise ratio. They also adopted an alternative calibration technique using imbedded aluminum in a nonconductive resin. Advantages of resin standards include uniformity of an analyte distribution and shot-to-shot reproducibility. The lowest detectable concentration of A1 in the resin was 16.9 ~tg/g. Rasberry et al. [208] performed an experiment on iron-steel samples containing 0.1-1% Cr using LIBS. Their analytical curve showed a slope of 0.75 in a log-log plot representation of the intensity ratio of Cr at 359.3 nm and Fe at 296.6 nm versus concentration with a RSD of about 10 %. Kagawa and Yokoi [209] also performed the determination of Cr in iron-steel samples with a nitrogen laser which resulted in an improved precision over that of the work by Rasberry et al. [208]. The minimum detectable concentration of Cu in iron, and Mg in aluminum were not lower than 0.05 % due to the lack in performance of the monochromator, as the authors claimed. This group also investigated Zn plasma intensively by observing several emission wavelength of Zn with an XeC1 excimer laser as well as a transversely excited atmospheric pressure (TEA) CO2 laser. A calibration curve was obtained for Cr in carbon steel by observing the 425.4 nm emission wavelength and the detection limit was estimated as 20 ppm with background equivalent concentration (BEC) of 12 ppm [62]. Thiem et al. [210] studied a simultaneous quantitative elemental analysis of NIST transition metal alloy samples by using a Nd:YAG laser. Linear calibration curves were obtained for elements with the composition of A1 (0.21.2 %), Cu (0.021-0.49 %), Fe (4.5-51.0 %), Ni (30.8-80.3 %) and Zn (6-12.8 %) using non-resonant lines. The detection limit varied with sample composition complexity from 0.0001% for Ni in a Cu alloy, Standard Reference Material (SRM 111) to 0.16 % for A1 in a complex granular sample (SRM 349a). The spectral wavelengths used in the determination were 309.3 nm for

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A1, 357.9 nm for Cr, 351.8 nm for Co, 324.7 nm for Cu, 372.0 nm for Fe, 403.3 nm for Mn, 352.5 nm for Ni, 390.5 nm for Si, 399.9 nm for Ti, and 334.5 nm for Zn. Thiem and Wolf [211] performed the analysis of trace elements (A1, Ca, Cu, Fe, K, Mg, Mn, Si, Ti) in aluminum and manganese ore and U.S. Geological Survey (Boulder, Colorado, USA) manganese modules using both LIBS and ICP-AES. In this work, the two methods were compared and contrasted as to sample preparation, difficulty of analysis, cost of insmunentation, and values obtained for the determinations of the standard reference materials (SRMs). Lee and Sneddon [212] used an ArF excimer laser (z=193 nm) for ablation to perform the quantitative analysis of elements in solid steel samples. A calibration curve was developed which related the Cr concentration in a solid steel matrix to the intensity ratio of Cr(I) 520.84 nm to Fe(I) 516.75 nm. The Cr concentration, as determined ranged 0.062 % to 1.31%. A detection limit of 20 mg/g (approximately 0.002 %) was estimated. Sabsabi and Cielo [213] obtained a calibration curve for Mg, Mn, Cu, and Si in aluminum alloy samples. The calibration curves for Cu and Mg exhibit a curvature for higher concentration due to the self-absorption of the Cu(I) 327.4 nm and Mg(I) 285.2 nm resonance wavelength. The strong resonance wavelengths can be used, however, for lower concentrations with improvement of the detection limit. The detection limits for each element were estimated as 0.5 ppm for Mg, 10 ppm for Cu, 14 ppm for Si, and 2 ppm for Mn. The RSD for 50 consecutive measurements of Mg, Mn, Cu, and Si was 4% for strong (resonance) wavelength at high concentration and 6 % at low concentration. Sabsabi and Cielo [214] also performed the quantitative analysis of copper alloys by LIBS using a Nd:YAG laser (~ = 1064 nm). Calibration curves for Fe, Ni, and Ag were produced. The RSD ranged from 2 to 10 % of the analyte concentration. The detection limits were 20, 10, and 1 mg/g for Fe, Ni, and Ag, respectively. These values are comparable to conventional AES methods. This group also performed the research on A1 and Mg in aluminum-alloy [215]. Nemet and Kozma [202] applied a Q-switched Nd:YAG laser based TRELIBS for the quantitative analysis of elements in high alloys (gold jewels). For this purpose, several line pairs of Cu-Au, Ag-Au, and Cu-Ag in the wavelength range of 295-525 nm were applied for the determination of the components in high alloys. The deviations between this study and the certified values were less than 3 %. The detection limit for Cu in low-alloys was estimated at 15 ppm using 324.5 nm wavelength and 160 ppm using 521.83 nm wavelength. The RSD was 4-8 %. This study also proposed a possible application of LIBS for quantitative analysis of several other tertiary and binary high-alloys (Fe-Cu-Ni, Cu-Zn-Ni, Cu-Zn, Cu-Sn and Cu-Pb). Noll et al. [216] performed quantitative elemental determinations in an iron-matrix. This study

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was to improve the detection limits of LIBS for multi-element analysis. The influence of the laser pulse structure on the emission of the LIP was also studied. Sattmann and Noll [217] studied the effect of single and double pulses from Qswitched Nd:YAG laser on the intensity of emission signal for trace elements. Line intensities were increased by a factor of about two using double pulses. Quantitative microchemical analysis of low-alloy steel was also performed with single and double pulses. Cremers [218] published the results of the analysis of trace elements (Si, V, Cu, Mn, Cr) in metal samples at a distance from 0.5 m to 2.4 m by using a fiber-optic cable. This study motivated work on the elemental analysis by LIBS at a remote distance up to few hundred meters. Observed concentrations of elements were comparable to the predicted concentrations for the concentration range of 0.05 to 2.95%. RSD of the measurement were estimated from 9 % for Si to 28 % for Cu. The simultaneous detection of Mg, Mn, Fe, and Pb in aluminum was performed by Bescos et al. [219]. In that study, linear calibration curves for those elements over 0.01-1 % concentration range were obtained. They suggested the potential application of this method to online industrial analysis. Gonzalez et al. [220] studied the determination of S content in steel by LIBS using a Q-switched Nd:YAG laser. They measured the emission intensity ratio of S(I)/Fe(II) in the wavelength region of 550 nm (543.23, 545.38, and 547.36nm lines) and 180 nm (180.73, 182.03 nm, and 182.62 nm). A detection limit of 70 ppm and a RSD of 7 % was obtained. Calibration curves are linear over the range of 0.008 to 0.28 % of S concentration. No noticeable matrix effects were observed in this study. Lorenzen et al. [221] applied LIBS to the in-process quality assurance and process control in different industrial branches such as steel production and plant making. Their instrument, which was called LIESA (laser-induced emission spectral analysis) was equipped with a high power laser source (irradiance 9 1 x 1 0 8 - 5 x 109 W/cm 2) and optical fiber bundle for the detection of emission signals. Relative detection limits of between 10 to 100 ppm was demonstrated for most of the detectable elements in various matrixes ~ (steel, rubber, rock and glass). They also developed procedures to convert relative measurements with RSDs of between 1 and 2 % into absolute concentration values with a relative accuracy of about 3 %. Bender and Sattler [222] have performed the determination of Pb, Zn, Ca, Si, and Fe in cold leadzinc slag samples. They also performed the experiments to verify the results with hot slag samples in a laboratory oven, but it was unsuccessful. Sattler [223] applied LIBS to control a sorting application for nonferrous metal recovery from scrap. The electronics of the LIBS-AES detector sends a signal to an evaluation computer which controls the sorting application. By this method, 3-5 metric tons of nonferrous metals can be sorted out of a shredder in 1

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hour. Oh et al. [224] performed the quantitative analysis of Zn, A1, Pb, Cu, and Fe in zinc-base alloy and obtained an accuracy of 5 % for the A1 contents in the composition range of 0.1-5 %. Talmi et al. [225] used a LIBS system with a ruby laser (x - 694 nm, 0.6-1.2 J, repetition rate of 4 pulses/min.) to monitor both the surface and depth profiles of the elemental content of a variety of sample types including a ruby rod and ceramic material with blemishes, electrical capacitors, integrated circuits, and surface-coated electrical conductors. Detection limits obtained were in the range of 2-500 ppm depending on the element, the wavelength used, and the matrix. Ernst et al. [226] applied LIBS as a means to assess radiation embrittlement by the detection and quantification of Cu in A553b steel. The LIBS results were comparable to those from atomic absorption spectrometry (AAS) in precision and accuracy. Copper content <0.02% was not resolvable for AAS, whereas LIBS was able to detect and quantify Cu down to 0.01%. The average error for levels <0.50% was 0.010 wt % for LIBS and 0.026 wt % for AAS. Anderson et al. [227] carried out the depth profile measurements of coatings on steel using LIBS. Linear calibrations against coating thickness for Zn/Ni (2.7 to 7.2 ~tm) and Sn (0.38 to 1.48 pro) on steel were achieved with good precision (RSD of 3.5%). An ultra-thin coating of Cr (20 nm) on steel was also detected by LIBS. Song et al. [99] applied the TRELIBS technique to determine various trace elements including Mg, Cu, and Cr for zinc-based alloys, and Si and Ca for rock samples. The analytical signals for trace elements were integrated within 20 ms after an optimum gate delay time (200 ns). The detection limit (S/N ratio - 3) was element-dependent and varied with complexity in sample composition but was in the order of 5-100 ppm. Precision was typically 5 to 10 %. St-Onge et al. [100] determined several elements (A1, Cu, Fe, Pb, and Sn) in solid zinc alloys using LIBS. In this experiment, optimal conditions for the determination were evaluated with the variation of time gating parameters and distance from focusing lens to target. Detection limits lower than about 60 ppm were achieved for all elements except Cu, for which a 544 ppm 3 s-limit was found for the line used. Kim et al. [228] used the time-resolved LIBS technique for the determination of A1 impurities in zinc-alloy. Calibration curves for A1 were developed by the use of the relative intensities of two lines; AI(I) 308.215 nm and Zn(I) 307.590 nm under air and argon atmosphere. Davies et al. [229] used a fiber optic system to solve the problem of transporting the laser beam from a remote laser to the sample and of collecting the irradiation output from the laser produced plasma. Various elemental determinations including Cr, Ni, Mn, Mo, Si, and V at concentrations smaller than 5 x 10.4 g/g were carried out with the use of a reference line for an internal standard. The authors applied this analytical system for the study of steel components in an operating nuclear

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reactor with significant savings compared with conventional analytical methods since the measurements did not require reactor shut-down. The measuring system may therefore be placed remote from the analyte which may be situated in a hostile environment such as an operating nuclear reactor. We can conclude that almost all of the metal and particularly transition metal elements in different matrices have been target elements in trace determination by LIBS, and detection limits in the ppm to ppb range have been obtained. Further work in this direction will be the application to on-site analysis of elemems, improvement of precision in measurement, development of accurate calibration procedures, and of course improvement of detection limits.

6.3 Applications to liquids and solutions A variety of LIBS techniques have achieved limited success in liquids and solutions. Application of LIBS to trace metal deterrtfination within liquids offers considerable potential if the attributes of minimal sample preparation and high detection sensitivity can be realized. While pre-breakdown events can be minimized, LIBS analysis of liquids in-situ possess an inherent difficulty that stems from the high local density within the liquid. The high collision rate within a plasma confined by liquids significantly broadens spectral transitions relative to those for a rarefied gas [230, 231]. Boiron et al. [232] applied LIBS for the determination mono-atomic ions in individual fluid inclusions. They analyzed major cations (Ca 2+, Mg 2+, Na +, and K +) contained in macroscopic electrolyte solutions. For Ca + and Mg 2+, calibration curves were obtained in the concentration range of 0.1 to 1.2 M using synthetic solutions. The detection limits were estimated as 0.005 M (200 ppm Ca) for Ca + at 393.336 nm and 396.847 nm emission wavelengths and 0.001 M (25 ppm Mg) for Mg 2+ at 279.553 nm and 280.270 nm emission wavelengths. Wachter and Cremers [233] used LIBS to detect U in solution for possible application to process control in nuclear fuel reprocessing facilities. A pulsed Nd:YAG laser were focused on the surface of the liquid in order to generate sparks. The detection limit for U in 4M nitric acid was 0.1 g/L. Measurement RSD was 1-2 % for a 4.2 g/L solution with the use of 1600 laser sparks, corresponding to a measurement time of about three minutes. A calibration curve was prepared that covered U concentrations from 0.1 to 300 g/L. The effect of some experimental parameters on the analysis were also discussed in this paper. Archontaki and Crouch [234] used an isolated droplet generator (IDG) as a novel sample introduction system for LIBS. The IDG converts liquid samples to equally-spaced, and uniform-sized droplets. Calibration curves were linear over three orders of magnitude for several elements. The detection limits for solutions with the FIA (flow injection analysis)/IDG/LIBS system were in the low ppm range. Cremers et al. [235]

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used the LIBS technique with a Nd:YAG laser (15 ns duration, 45 mJ/pulse) to analyze liquids spectroscopically for element constituents. Emission from onceionized and neutral atoms and simple molecules were observed. Detection limits for Li, Na, K, Rb, Cs, Be, Mg, Ca, B, and A1 in aqueous solutions were established. Most of these elements were only detectable at levels above 1 ppm, although the detection limit for Li was 0.006 ppm. The RSD for replicate sample analysis was 4-8%. Ito et al. [236] used a Q-switched Nd:YAG laser for the LIBS measurement to detect FeO(OH) in water and the concentration of the turbid solution down to a few ppm. In this study, a cell-less measurement was achieved using a coaxial nozzle flow system which allowed the effect of ambient gas on the emission intensity and decay lifetime of the breakdown plasma to be studied. This group also published a result of quantitative detection for colloidal and particulate Fe in water [149]. Using sequential laser pulses from the Qswitched Nd:YAG laser as an excitation source, the determination of FeO(OH) concentration in the tens of ppb range was demonstrated. With sequential pulse excitation, the detection limit was decreased to 16 ppb compared to the 0.6 ppm observed with single pulse excitation. Knopp et al. [230] performed the quantitative detection of ions of Li, Na, Ca, Ba, Pb, Cd, Hg, Er, in aqueous solution by using an excimer pumped dye laser based LIBS. The detection limit was dependent on the elements and was estimated as 500 ~tg/L for Cd 2+, 12.5 ~tg/L for Pb 2+, 7.5 ~g/L for Na +, 13 ~tg/L for Li+, 6.8 ~tg/L for Ba 2+, and 0.13 ~tg/L for Ca 2+. No Hg 2+ and Er 2+ emission could be detected even at concentrations of up to the ~tg/L levels. On the other side, for Er 3+ in suspensions of ErBazCu3Ox particles, a better than 103 times higher sensitivity was found than for dissolved Er 3+. This result shows the potential to analyze colloid-borne metal ions with an increased sensitivity. Solution analysis continues to be a relevant part of metal analysis. Pichahchy et al. [237] evaluated LIBS for the detection of elements in metals located under water. Repetitive laser spark (RSS) and repetitive spark pair (RSP) were performed on the metals by focused pulses from Q-switched Nd:YAG laser (z = 1064 nm). They found that the plasma on the second pulse was useful to monitor the element composition of the metals. Calibration curves for Cr, Cu, Mn, and Si in steel were prepared by analyzing a set of certified steel reference standards positioned under water and the detection limits of 367, 520, 1200, and 1190 ppm were obtained for Cr, Cu, Mn, and Si, respectively. Arca et al. [238] checked the feasibility of quantitative determination of trace element concentration in water by LIBS. The Nd:YAG laser beam was focused on the flee surface of the water sample and then collected the plasma emission by an optical system coupled with an optical fiber to 1-meter spectrometer. The concentrations for the main components of water (Mg, Ca, CI, Na, Si, K) determined by LIBS were

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in very good agreement with those from standard chemical analysis, thus confirming the reliability of the LIBS method for precise quantitative analysis of traces in water, up to a few parts per million. Considerable fundamental research on the effect of laser wavelength on liquid plasma properties by the use of two different laser system (Nd:YAG and ArF excimer) was carded out by Ho et al. [239, 240]. An ArF excimer laser irradiation (~ = 193 nm) produced the plasma plumes at much lower (<1 eV) temperature but at a comparable electron density to that of Nd:YAG laser irradiation (~ =532 nm). The authors suggested that this cool plasma was ideal for analyzing the biologically important elements, such as Na, K, and Ca. In 1997, Haisch et al. [241] used the LIBS technique for elemental analysis of heavy metal colloids with particle diameters between 0.1 and 1 mm to study the nature and abundance of colloids in aquifer systems. The fourth harmonic ( ~ - 266 nm) of a Nd:YAG laser beam with a pulse energy of 70 mJ was employed for better coupling to the target surface. The plasma emission was optimized by a time-resolved LIBS system with a gate-delay time of 0.75 gs and an integration time of 300 ns. The authors utilized a miniaturized ultrafilteration system with 0. l mm membrane filter to accumulate the colloidal particles on the membrane surface and ft~her analyzed it. Berman and Wolf [23 l] analyzed aqueous solutions containing Ni or the chlorinated hydrocarbons (CHCs) with the use of LIBS technique. Two different laser wavelengths (1064 nm and 355 nm) were used for the production of the laser-induced plasma and compared their analytical performance. The detection limits for Ni in water were 36.4 _+ 5.4 gg/L and 18.0 _+ 3.8 pg/L for the irradiation at 1.064 gm and 355 nm, respectively. However, the detection limits for various CHCs; CCl4, CHCI3, C2C14, and C2HCI3, determined in this work were several tens of ppm. This was higher than the values needed for environmental many applications. Van der Wal et al. [242] presented an alternative approach to trace metal determination in liquids in which 1.0 mL of liquid was deposited onto a carbon planchet and then evaporated, thus transforming the liquid analysis to a solid surface analysis. Using optimized excitation and detection conditions, 15 metals (Mg, A1, Si, Ca, Ti, Cr, Fe, Co, Ni, Cu, Zn, As, Cd, Hg, Pb) were determined. The limit of detection (LOD) ranged from 10 ppb to 10 ppm for these elements. A 100 ppb LOD represents detection of 130 picograms (pg) of metal (approximately 2 picomoles) in a single measurement calculated from the laser spot size on the sample. Pardede et al. [243] applied LIBS to determine metals deposited from water samples by means of electrolysis. Detection limits in the parts per billion (ppb) were obtained for several elements. Schmidt and Goode [244] determined a selected number of elements from

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solutions by either filtering the solution through an ion exchange membrane with suction or placing the membrane in the solution and allowing the ions to equilibrate with the membrane. LIBS was then used to determine the metal concentration. 6.4 Applications to aerosols and gases The analysis of small particles (i.e., sub-micrometer to several micrometers in diameter) using in-situ, real-time techniques has a wide range of applications. Areas of interest include atmospheric sciences, process monitoring and control and effluent waste stream monitoring. Cheng et al. [245] applied LIBS for the detection of trace concentrations of Group II and V hydrides, such as Phosphine (PH3), Arsine (ASH3), and Diborane (BEH6). These three gases are widely used in the semi-conductor industry and the toxicity of each has impeded the development of diagnostic procedures. In metal organic chemical vapor deposition (MOCVD), a Ge impurity was introduced to the reactor in the form of germane (GeH4) as a contaminant in AsH3. The detection limit was estimated as 1 ppm. Essien et al. [246] determined the concentration of Cd, Pb and Zn in aerosols by LIBS. Samples were generated in aerosol form by a nebulizer-heat chamber arrangement. Calibration graphs for these elements in dry aerosols were developed using the ratio of the intensity of the analyte line to the adjacent background. Detection limits for Cd, Pb and Zn in aerosols were 0.019, 0.21, 0.24 ktg/g, respectively at a signal to noise ratio of 3. Recently, Williamson et al. [137] evaluated LIBS for the detection of candidate replacement compounds (CF4, CF3H, CF2H2, and C2FsH) to halogenated hydrocarbons (halons). Certain halons, such as CF3Br (Halon1301) and CF2CIBr are among the most effective fire suppressants. The fundamental Nd:YAG laser beam (n = 1.064 nm) was focused into an air flow containing 0.0005-5% of the analyte halocarbon compounds. The emission of F(I) was clearly resolved with the maximum intensity at 685.6 nm. The detection limit of 40 ppm for fluorine (CaFsH) in air was obtained with a laser energy of 120 mJ/pulse. Where, the detection limit for fluorine (CF3H) in air was 60 ppm and the noise for five consecutive measurements of shot-to-shot spectra was 8%. Subsequent research by Lancaster et al. [247] evaluated LIBS as a means of detecting the fire suppressants CF3Br, C3F7H, and CF4 and the refrigerant C2F4H2 using time- and space-resolved measurements. Limits of detection for these halocarbons are somewhat greater (less sensitive) than those for flurocarbons. A two-part calibration scheme response to (I) a known mass

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concentration, (II) and a known discrete particle mass for real-time sizing and elemental analysis of single particles, based on LIBS was demonstrated by Hahn [248]. This calibration technique yielded excellent results in laboratory tests, and proved to be both robust and highly sensitive during initial field experiments. Tran et al. [249] used LIBS to determine gaseous and particulate fluorides in air and other gaseous mixtures. The limit of detection for fluorine with LIBS in a pure He atmosphere for collection on a filter was 5 pg/m 3 for a sampling time of 10-min at a flow rate of 10 L/min. 6.5 Applications to non-metallic solids Lee and Sneddon [55] analyzed K(I) in solid glass by observing the emission wavelength at 764.6 nm in the laser-produced plasma. The detection limit for K in solid glass was estimated as 0.13 pg/g and the RSD was estimated as + 10 %. Uebbing et al. [250] applied a Nd:YAG laser (x = 1064 nm, 13 mJ) as an atomizer coupled with the another Nd:YAG laser (x = 1064 nm, 115 mJ) for reheating the LIP to increase the analyte line intensities and to improve the detection limit. Calibration curves for A1 and Mn in glass and steel samples and for Mg and Mn in glass, copper, and aluminum were obtained by internal standardization. Generally, detection limits of these elements were in the higher ktg/g range. However, detection limits were four to ten times better than those from experiments without re-excitation. Kagakawa et al. [209] detected B and Si in borosilicate glass and Cr in iron-steel samples using N2 laser based LIBS. Kumiawan et al. [25] used an XeCI excimer laser and TEA CO2 laser for the determination of Li, Be, Na, Mg, AI, K, Ca, Ti, Zn, Zr, and Ba in glass. The detection limit for Li in glass was estimated as 10 ppm by using the emission wavelength observed at 670.7 nm. Detection limits for other elements were dependent on the buffer-gas pressure. Detection limits at one torr buffer gas pressure were 30 ppm for B(I), 14 ppm for Na(I), 130 ppm for Mg(I), 54 ppm for Al(I), 190 ppm for K(I), 85 ppm for Ca(I), 410 ppm for Ti(I), 160 ppm for Zn(I), 290 ppm for Zr(I), and 180 ppm for Ba(I). The detection limit required for the ordinary glass analysis were 100 ppm and the results obtained were adequate for real sample applications. Franzke et al. [251] investigated minor components contained in the naturally occurring minerals by using a UV laser pulse as a ablation source (370 nm). The minerals investigated in this study included FeS2, ZnS, PbS, ZrO2, PbCrO4, K2S, Sb2S3, and Mo2S3. For the more sensitive elements, such as Fe and Pb, the detection limit was estimated as approximately 10 ppm or 35 pg/cm 2. Stoffels et al. [252] detected time-resolved emission fi'om laser-ablated U by using aQ-switched Nd:YAG laser (z = 1064 nm). They found that the time dependence of the

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emission intensity was strongly affected by the nature and pressure of the buffer gas. Jensen et al. [253] performed mechanistic studies of LIBS to model environmental samples to detect the C content of a physical mixture of Eu203 and K2Cr207 with granular, crystalline SiO2. Excitation of the powdered specimens was achieved with a KrF excimer laser light (x = 248 nm, 30 ns pulse width, fluence/pulse 0.3-30 J/cm 2, irradiance 10-100 Mw/cm 2) in one atmosphere of air. The detection limits with a quartz matrix for the detection system were 100 ppb for Eu and 2 ppb for Cr. They claimed these limits could be easily lowered by factor of ten or more through more efficient light collection alone. Singh et al. [66] detected Sn and As in metal halides of these elements. Blacic et al. [254] at Los Alamos National Laboratory (Los Alamos, New Mexico) applied LIBS for elemental analysis of the planetary surface. They intended to deploy a portable LIBS on a surface-rover vehicles on large bodies such as Mars and the Moon and to use the system from a spacecratt in a close rendezvous with small bodies such as comets and asteroids. They used a Nd:YAG laser with the energy of several hundred mJs and CCD array or diode array for the detection of the emission signal. Absorption spectral analysis of the emission signal will generate mineralogical information that provides a remote geochemical characterization of the rock samples. They demonstrated remote LIBS analysis of terrestrial rock surfaces at ranges over 25 m, and the ability of the system to operate on a six-wheeled Russian robotic rover vehicle. They estimated an absolute accuracy of 10-15 percent for most of minor elements on the planetary surfaces. Elemental analysis of solid samples has been popular among the applications of LIBS. Marquardt et al. [132] determined the lead concentration in paint in homes using the fiber-optic probe LIBS technique. The measurement took less than 1 minute to perform and the limit of detection was 0.014% in latex paint, on a dry weight basis, with RSD of 5-10%. Yoon et al. [255] applied the LIBS technique to the analysis of elemental distribution mapping for Ba, Cu, Fe, Mn, Pb, Si and Sr, in polished rock sections by the use of a frequency-doubled 532 nm Nd:YAG laser beam under atmospheric conditions. For each element, mapping of 50 x 50 mm samples could be made in 30 minutes. The detection limits of the elements were in the range of a few ppm to several thousands ppm depending on the elemental species. The RSD for 50 consecutive measurements of Cu, Fe and Si was 5%, and for Ba, Mn, and Sr was 7 % for the selected wavelengths. The Winefordner group [256] developed a LIBS/Laser excited atomic fluorescence spectrometry (LIBS/LEAFS) combination system for the determination of Co in three solid sample matrices: graphite, soil, and steel. The optimum gate delay time between the ablating and exciting pulses

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was 16 ms. Relative standard deviations were within 15-30% for the low concentration range (0.5-100 ppm) and less than 10% for larger concentration (> 100 ppm). A detection limit of 0.2 ppm was obtained for the determination of Co in the graphite matrix, whereas those for Co in soil and steel matrices were somewhat higher: l ppm and 20 ppm, respectively. On an absolute basis, the LODs were 30 fg for Co in graphite, 190 fg for Co in soil, and 500 fg for Co in steel. LIBS has been evaluated for depth profiling of P doping in silicon by Milan et al. [257]. The depth resolution was about 1.2 mm pulse l in this work which is sufficient for many applications. However, it should be kept in mind that its value changes even with small variations in the experimental conditions. Marquardt et al. [258] developed a fiber-optic probe LIBS system to determine the concentration of Pb in samples of dry paint. They used one optical fiber for laser excitation and a separate fiber for light collection. Both fibers were 1 ~m core diameter, 0.48 NA and the fibers were approximately 4 m long. A Nd:YAG laser operating at 1064 nm was used to produce a laserinduced plasma. Typical laser power at the probe tip for the Pb analysis was 19.0 mJ/pulse. The detection limit for Pb in dry paint were 0.014 % (w/w dry weight) and the precision of the measurements was 5 to 10%. Lucema et al. [259] presented the capability of LIBS for V determination in a xV-2TiO2-SiO3 catalyst. The microplasma was generated onto the sample surface using a pulsed Nd:YAG laser beam operating in the second harmonics (532 nm). The focusing of the laser beam on the surface was optimized to improve the signal-to-noise ratio and consequently the detection limit for the analysis. The detection limit for V was estimated to be 38 ~tg/g in 2TiO2-SiO2. The precision for measurements was better than 6% RSD in the concentration range 200-1000

g/g. Fichet et al. [260] used LIBS to investigate impurities at concentrations around 100 ppm, rapidly and in-situ at atmospheric pressure using a specially designed glove box, in two nuclear solid materials, UO2 and PuO2. The 18 elements; Ag, Al, B, Ba, Bi, Ca, Cr, Cu, Fe, Ga, In, Li, Mg, Mn, Na, Pb, Sr, and T1, have been observed in UO2 at concentrations of around 500 ppm and 12 elements; Ag, Ba, Ca, Cr, Cu, Ga, In, Li, Mn, Na, Sr, and V, in PuO2 at concentrations around 100 ppm. Detection limits of about 100 ppm were found in both matrices for the impurities studied. 6.6 Applications for advanced materials A better knowledge of the behavior of the main impurities which are usually introduced in advanced materials during production helps to reduce additional contamination. Silicon is the most extensively studied semiconductor

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mainly owing to its wide application in the microelectronics device manufacture and in the photovoltaic solar cell industry. Ciocan et al. [261] applied a laserablated microwave-induced plasma atomic emission spectrometry (LA-MIPAES) system to the direct determination of trace elemems (Mg, A1, Si, and Fe) in a high-temperature superconducting ceramic (Yba2Cu307) and the determination of a low Na concentration in high-purity natural and synthetic quartz used for the production of optical fibers. They used internal standardization for the normalization of the emission signal and calibrated with relevant spectra of standard reference samples (A1, Cu, borax glass). The measured concentrations for elements (Si, Fe, A1, Mg) by LA-MIP-AES and ICP-AES were compared to that of LIBS. Both methods revealed very similar result, e.g., for Si, 2670 + 330 ~tg/g by LA-MIP-AES and 3300 + 300 ~tg/g by LIBS. The concentrations for other elements in YbaECU307 were 114 + 40 ~tg/g for Fe, 114 + 8800 ~tg/g for A1, and 26 + 5 ktg/g for Mg. For A1 a strong inhomogeneity was found in differem spots of the target. In this study, internal standards such as a known concentration of Cu were used for the determination. The concentration of Na on natural and synthetic quartz was 0.28 to 2.0 ~tg/g. The detection limit for Na was estimated as 40 ng/g by adopting the square root of 3-6. The precision of the measurement was not as good as ICP-AES, but was comparable to that of LA-MIP-AES. Ottesen [262] applied the LIBS technique to analyze the elememal composition of contaminants found on electronic microcircuits fabricated on alumina substrates and obtained spatially resolved data with some degree of depth profiling information. One of the potential uses of LIBS in the field involves depth profiling of surface coatings. The composition of each individual layer, particularly at the interface, was much more informative than the composition of the average over a range of depths. Hidalgo et al. [263] investigated the emission spectra of a laser-generated plasma from titanium dioxide anti-reflection coatings in solar cells. A method for measuring time TiO2 films between 40 and 400 nm within the typical values used in solar cells, based on the LIBS technique has been developed. A pulsed nitrogen laser at 337.1 nm was used with a pulse width of 10 ns and a laser fluence of 8.6 J/cm 2 on the sample. The capability of LIBS to resolve complex depth profiles was also demonstrated by Vadillo and Lasema [264] using electrolytically deposited brass samples. Ablation depths of 6.5 ng per pulse were obtained, which imply absolute detection limits of the order of fg per pulse for an element present in the sample at a concentration of a few ppm. Vadillo et al. [265] studied the lateral and depth resolution for surface analysis using LIBS. A pulsed nitrogen laser at 337.1 nm (3.65 J/cm2) was used to irradiate solar cells employed for photovoltaic energy production. The emission lines for Ag(I) at 545.9 mm,

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C(II) at 588.9 nm and Si(II) at 634.7 nm were detected clearly by a single-shot LIBS spectrum of a solar cell. Further research by Romero and Laserna [266] was performed to generate selective chemical images for Ag, Ti, and C from silicon photovoltaic cells with a multichannel LIBS system. Both surface and depth distributions were amenable with this approach. Lateral resolution of 80 mm and depth resolution of better than 13 nm for TiO2 coatings were achieved in this work.

6.7 Miscellaneous applications Experimental studies on laser-ablated ZrC were performed by Wantuck et al. [267] for the investigation of fuel corrosion diagnosis in nuclear fuel. Monitoring of the fuel corrosion products is important not only for understanding corrosion characteristics, but to assess the performance of an actual, operating nuclear propulsion system. Anglos et al. [268] employed the LIBS technique for the in-situ analysis of pigments used in painting. Appropriate emission lines for the identification of the metallic elements in the pigments examined were proposed. Furthermore, a test of an 18th century oil painting was examined by LIBS and the different pigments used in the original and in the restored part of the work were clearly identified. Anglos [269] reviewed the use and potential of LIBS in art and archaeology. The LIBS technique has also been applied as a real-time diagnostic technique for the laser cleaning of natural marble surface by Maravelaki et al. [270]. They demonstrated that LIBS can be used for the on-line control to the extent of laser cleaning at each spot of the surface by analyzing the spectra of the plasma emission. The LIBS technique has been successfully applied as an on-line diagnostic technique in the laser cleaning process of polluted limestone by Gobemado-Mitre et al. [271]. Monitoring of the relative intensities of selected emission lines such as Ti, Fe, and Si, can be used as an indicator of the extent of the cleaning process. Therefore, an efficient on-line control of the laser cleaning process of limestone has been achieved by monitoring the consecutive LIBS spectra while the encrustations are being removed. Kim et al. [272] applied LIBS for compositional mapping of a commercial printed circuit board. A Nd:YAG laser beam with second harmonic module was focused on the solid surface with the optimum energy of 5 mJ. The authors successfully accomplished their study for a few millimeters size of samples. This is not the sample size for the scanning electron microscope (SEM)/energydispersive X-ray (EDX) technique. Sattmann et al. [273] studied the LIP emission of the visible and near-UV spectral region of polyethylene (PE), polypropylene (PP), polyethylene (PET) and polyvinyl chloride (PVC) polymer samples to evaluate the feasibility of LIBS for the identification of different

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polymer materials. Spectral features were measured with the use of the 725.7 nm chlorine wavelength, the 486.13 nm lib line, and the 247.86 nm carbon line. The evaluation with neural networks of the spectral data measured with clean samples enables accurate identification in the range of about 93 to 96% for PE and PP and of >99% for PET and PVC. The application of LIBS for certain cases, such as a high-speed separation of washed PET and PVC bottle. 7. CONCLUSION This chapter has shown the potential and versatility of L1BS as an analytical technique to determine elements or metals in a wide variety of samples, including solids, liquids (solutions) and gases. It has not achieved widespread use in the biological and clinical area, in part, due to the lack of commercial instrumentation, its somewhat reduced sensitivity compared to conventional and competitive analytical techniques such as those described in the other chapters of this volume. It lack of ability to determine the chemical form or species is also a drawback in the biological and clinical area. The greatest and most cutting edge determination in biological or clinical samples is in this area. However, LIBS continues to attract the interest of scientists and researchers throughout the world. It will become more widespread in the cbiolical and clinical area in the near future. It is not uncommon to find sessions devoted to this technique at scintific meetings and more recently a complete conference in this area. A special issue of Applied Optics devoted to LIBS is currently underway and should be published late in 2002/early 2003. LIBS continues to thrive. REFERENCES 0

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246. M. Essien, L.J. Radziemski, L.J. and J. Sneddon, J. Anal. Atom. Spectrom., 3 (1998) 985. 247. E.D. Lancaster, K.L. McNesby, R.G. Daniel and A.W. Miziolek, Appl. Optics, 38 (1999) 1476. 248. D.W. Hahn, Appl. Phys. Lea., 72 (1988) 2960. 249. M. Tran, B.W. Smith, D.W. Hahn and J.D. Winefordner, Appl. Spectrosc., 55(11) (2001) 1455. 250. J. Uebbing, J. Brust, W. Sdorra, F. Leis and K. Niemax. Appl. Spectrosc., 45 (1999) 1419. 251. D. Franzke, H. Klos and A. Wokaun, Appl. Spectrosc., 46 (1992) 587. 252. E. Stoffels, P.V.D. Weijer and J.V.D. Mullen, Spectrochim. Acta, 46B (1991) 1459. 253. L.C. Jensen, S.C., Langford, J.T. Dickinson and R.S. Addleman, Spectrochim. Acta, 50B (1995) 1501. 254. J. Blacic, D. Pettit, D.A. Cremers, and N. Roessler, N. In Lunar and Planatary Inst., Workshop on advanced technologies for planatary instruments, (1993) Part 1 and 2. 255. Y.Y. Yoon, T.S. Kim, K.S. Chung, KY. Lee and G.H. Lee, Analyst, 122 (1997) 1223. 256. I.B. Gomushkin, J.E. Kim, B.W. Smith, S.A. Baker and J.D. Winefordner, Appl. Spectrosc., 51 (1997) 105. 257. M. Milan, P. Lucena, L.M. Cabalin and J.J. Lasema, Appl. Spectrosc.,52 (1998) 444. 258. B.J. Marquardt, B.M. Cullum, J.J. Shaw and S.M. Angel, Proc. SPIE-Int. Soc. Opt. Eng., 3105 (1997) 203. 259. P. Lucema, L.M. Cabalin, E. Pardo, F. Martin, L.J. Alemany and J.J. Lasema, Talanta, 47 (1998) 143. 260. P. Fichet, P. Mauchien and C. Moulin, Appl. Spectrosc., 53 (1999) 1111. 261. A. Ciocan, L. Hiddemann, J. Uebbing and K. Niemax, J. Anal. Atom. Spectrom., 8 (1993) 273. 262. D.K. Ottesen, Appl. Spectrosc., 46 (1992) 593. 263. M. Hidalgo, F. Martin and J.J. Laserna, Anal. Chem., 68 (1996) 1095. 264. J.M. Vadillo and J.J. Lasema, Talanta, 43 (1996) 1149. 265. J.M. Vadillo, S. Palanco, M.D. Romero and J.J. Lasema, Fresenius J. Anal. Chem., 355 (1996) 909. 266. M.D. Romero and J.J. Lasema, Anal. Chem., 69 (1997) 2871. 267. P.J. Wantuck, D.P. Butt and A.D. Sappy, Los Alamos National Laboratory Report No. LA-UR, 92 (1992) 1557. 268. D. Anglos, S. Couris and C. Fotakis, Appl. Spectrosc.,51 (1997) 1025.

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269. D. Anglos, Appl. Spectrosc., 55(6)(2001 186A. 270. P.V. Maravelaki, V. Zafiropulos, V. Kilikoglou, M. Kalaitzaki and C. Fotakis, Spectrochim. Acta, 52B (1997)41. 271. I. Gobemado-Mitre, A.C. Prieto, V. Zafiropulos, Y. Spetsidou and C. Fotakis, Appl. Spectrosc., 1998, 51, 1125e. 272. T. Kim, C.T. Lin and Y. Yoon, J. Phys. Chem. B, 102 (1998) 4284. 273. R. Sattmann, I. Monch, H. Krause, R. Noll, S. Couris, A. Hatziapostolou, A. Mavromanolakis, C. Fotakisi, E. Larrauri and R. Miguel, Appl. Spectrosc.,52 (1998) 456.

Chapter 7

Application of graphite furnace atomic absorption spectrometry in biological and clinical samples Joseph Sneddon I and David J. Butcher 2 1-Department of Chemistry, McNeese State University, Lake Charles, Louisiana 70609, USA and 2-Department of Chemistry and Physics, Western Carolina University, Cullowhee, North Carolina 28723, USA I. INTRODUCTION Atomic absorption involves the measurement of the reduction of intensity of optical electromagnetic radiation, from a light source, following its passage through a cell containing gaseous atoms (the atom cell). Atomic absorption spectroscopy generally refers to the study of fundamental principles of this phenomenon, whereas atomic absorption spectrometry (AAS) refers to its use for the quantitative determination of metals in a wide variety of samples, although these terms are often used interchangeably. AAS is applicable for the determination of most metals (almost all metals and metalloids and some non-metals, approximately 70 elements in the Periodic Table), in a wide variety of samples including biological, clinical, environmental, food, and geological, and hence is one of the most commonly used analytical techniques for elemental or metal determination. Two types of atom cells have been commonly used for AAS. The flame is widely used became of its ease of use for elemental analysis at the parts per million (ppm) (ktg/mL) level. However, the use of a graphite furnace as the atomizer is used when a limited sample volume is available or lower analyte concentrations (parts per billion) (ppb) (ng/mL) level are present. In this case, the technique is commonly referred to as graphite furnace atomic absorption spectrometry (GFAAS) or electrothermal atomization atomic absorption spectrometry (ETAAS). An earlier name of carbon furnace atomic absorption spectrometry (CFASS) is not widely used. The purpose of this chapter is to provide a brief and general overview of the technique including the basic principles and theory, and insmunentation. This will show the reader the potential of GFAAS in the determination of metals in clinical and biological samples. Selected and recent applications are highlighted.

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1.2 S P E C T R O S C O P Y Spectroscopy is defined as the interaction of electromagnetic radiation (light) with matter. Electromagnetic radiation is described as having both wave and particle properties. Wave properties include frequency (v, Hz), wavelength (~,, meters), velocity, and amplitude. Light is also considered to be composed of particles called photons and have a characteristic energy (E, Joules). The relationships between energy, frequency, and wavelength are given by: E = hv = h--c-c

(1)

2 where c is the speed of light in a vacuum (2.99792 x 108 m/s) and h is Planck's constant (6.62608 x 10.34 J s). The electromagnetic spectrum covers a wavelength range of over 14 orders of magnitude, including the gamma ray, X-ray, ultraviolet, visible, infrared, microwave, and radio frequency regions. For atomic absorption spectrometry, we will focus on a relatively limited region of the spectrum between 180 and 900 nm (ultraviolet, visible, and near infrared). These wavelengths are involved in electronic transitions of valence electrons. 1.2.1. I n t r o d u c t i o n to a t o m i c s p e c t r o s c o p y

It is beyond the scope of this chapter to provide a detailed description of the process of atomic spectroscopy. These details are available elsewhere [1, 2, 3] but a short review is given for completeness. Atomic spectroscopy involves the interaction of light with gaseous atoms. A device converts a sample (usually a solution) into gaseous atoms and is called an atom cell. Typical atom cells include flames, plasmas, and graphite furnaces. There are three basic types of atomic spectroscopy: atomic emission, atomic absorption, and atomic fluorescence. While the three processes are related they do offer three unique analytical techniques. In order to introduce these phenomena, we will initially consider an atom with only two electronic energy states, in which the ground (lowest energy) state is designated 0 and the excited state as 1. It can generally be assumed that under normal conditions the majority of atoms are in the ground state. Atomic emission (AE) involves the transfer of energy, usually as heat, from the atom cell to the atom to promote a valence electron in the atom from the ground state to the excited state. The atom then may emit a photon, and deactivate to the ground state (emission). The energy of the photon is equal to the difference in energy between the states. This process is called an electronic transition.

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Atomic absorption (AA) involves the transfer of the energy of a photon to an atom (absorption) to promote a valence electron in the atom from the ground to the excited state. In order for absorption to occur, the energy of the photon must be identical to the difference in energy between the lower and higher energy levels of the atom. Atomic fluorescence (AF) involves the excitation of atoms from a lower energy state (usually the ground state) to a higher energy state by light, followed by the emission (fluorescence) of a photon to deactivate the atoms. AFS can be considered to be a combination of atomic absorption and emission because it involves radiative excitation and de-excitation. Atomic spectra are characterized by their relative simplicity, typically consisting of narrow lines, which correspond to the limited number of possible energy levels. Each element has a unique set of energy levels and hence a unique spectnnn. At the temperatures of the atom cells used for atomic absorption (15003000~ the vast majority of atoms are present in the lowest energy level, called the ground state, and consequently the most sensitive lines involve transitions from the ground state, which are called resonance transitions. A transition from the lowest energy state is called a first resonance transition. The full width at half maximum (half-width) of most atomic lines is typically 0.01 - 0.05 nm, which is much narrower than liquid-phase molecular bands, whose widths are typically 10100 nm. The absence of vibrational or rotational levels in atoms results in the narrow widths of atomic lines. 1.3 GFAAS A N A L Y T I C A L SIGNAL: ABSORBANCE The fundamentals of a quantitative atomic absorption measurement are illustrated in Figure 1. In the ideal case, a monochromatic light beam from the source of intensity (Io) enters a cell containing the gaseous analyte. The transmitted beam (I) then passes into a detection system and converts the light beam into an electrical signal. If analyte is present in the cell, then the transmitted beam is less intense than the incident beam. On the other hand, if no analyte is present in the cell, then the incident and transmitted beams are equal. The ratio of the transmitted beam to the incident beam is defined as the transmittance, T (unitless):

T= •

Io

(2)

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Figure 1. Schematic diagram of atomic absorption process, where b is the pathlength of the atom cell, Io is the incident intensity, and I is the transition intensity which represents the fraction of light transmitted through the cell. Alternatively, the percent transmittance, % T, is defined as % T = I x 100% Io

(3)

Transmittance values range from 0, in which case no light passes through the cell, meaning a very high concentration of analyte atoms are present in the cell, to 1, in which no atoms are present in the cell. There is not a linear relationship between transmittance and concentration, and hence quantitative measurements are usually made using absorbance, A (unitless): A

=

-logT

-

I=log -lOglo

~

(4)

Notice that the absorbance increases as transmittance decreases, indicating as more atoms are present in the cell, the absorbance increases. Absorbance is a unitless number, typically, 0 to 2, with optimum precision between 0.1 and 0.5. The quantitative relationship between absorbance and concentration (c, g/L solution) is described by the Beer-Lambert Law:

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A : abc

(5)

where a is a constant called the absorptivity (L g-I cm-l) and b is the pathlength of the cell (cm). The magnitude and units of absorptivity are determined by the units for the pathlength and concentration. The absorptivity and concentration are related to the absorption coefficient k (cm l ) by a : 0.434 k

(6)

c

Atom cells for AAS typically employ a relatively long illuminated volume because of the direct proportionality between absorbance and pathlength. The GFAAS absorption signal differs from that of flame AAS because of the temporal variation of the atom population, producing a transient signal. A discrete amount of sample (10-50 laL) is introduced into the graphite furnace, which is heated to a series of temperatures for specified times. This process is called an atomization cycle. During the atomization step, the tube is heated to a sufficiently high temperature to convert the sample into gaseous atoms. A plot of absorbance versus time shows no signal at relatively low temperatures, before the atoms have been produced, an increase in absorbance as atoms are formed, and a decrease in absorbance as atoms are swept out of the atom cell. 1.4 THE NATURE OF THE TRANSIENT GFAAS SIGNAL: M E C H A N I S M OF ATOM F O R M A T I O N IN A GRAPHITE FURNACE

Within a few years after the development of GFAAS as an analytical technique in the late 1950s and 1960s, questions were raised regarding the chemical and physical processes involved in the conversion of the (commonly) aqueous sample into gaseous atoms, followed by their removal from the furnace. Although a considerable amount of information has been obtained regarding these processes, a commercial graphite furnace is a sufficiently complex system that uncertainty exists regarding the mechanism of atom formation for many elements. A very general mechanism for atom formation is Metal Salt (aq)

('), Metal Salt (s)

Metal (s).-(4)~ Metal (g)

(2), Metal Oxide (s)

(3),

(7)

A graphite tube is typically 20-30 mm in length and 3-6 mm in diameter. The tube is surrounded by argon to prevent combustion in air at elevated

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temperatures. The sample is introduced into the furnace through a dosing hole (1-3 mm in diameter). In many cases, chemical compounds called chemical modifiers are also added to improve the sensitivity or accuracy for a given analyte. The temperature of the tube can be controlled from ambient up to approximately 2700~ with heating rates up 1500~ It has been shown to be beneficial for many elements to insert a platform into the tube onto which the sample is placed. In our general mechanism (Equation 7), we have chosen to show vaporization of the metal as a starting point for the mechanisms, although some metals may vaporize as a compound. The first step involves the relatively straightforward removal of solvent (usually water) from the sample. The remaining steps include chemical/physical surface processes, such as homogeneous or heterogeneous solid-solid interactions, solid-phase nucleation, and diffusion into graphite; heterogeneous gas-solid interactions, i.e., adsorption/desorption and reaction of molecules with the wall to form atoms; homogeneous gas phase reactions; and processes by which analyte leaves the furnace. Steps (2) and (3) involve the chemical and physical processes which occur on the surface of the graphite tube. These processes are probably the least well understood because of the unavailability of a technique to monitor species on the surface of a commercial tube during a heating cycle. In fact, many of the techniques employ graphite substrates different from those used in GFAAS, or involve measurements made at room temperature. In spite of their limitations, these methods have revealed significant information regarding the mechanism of atom formation. 1.5 INSTRUMENTATION A block diagram for a graphite furnace atomic absorption instrument is given in Figure 2. The graphite tin,ace serves as the atom cell, which converts liquid or solid samples into gaseous atoms. A power supply provides current to control the temperature of a graphite tube between ambient and approximately 3000~ A light source is used to radiatively excite analyte atoms in the tube. The quantity of light absorbed is recorded by a detection system to do quantitative analysis. The detection system is composed of a wavelength selector, which is used to separate the analytical wavelength from other wavelengths emitted by the source, and a detector, which converts electromagnetic radiation into an electric signal. A signal processor amplifies the signal and sends it to a readout device, which in modem instrumentation is a microcomputer. In addition, the microcomputer serves to control other components of the instrument. A background correction system is required to correct for attenuation of the source by molecular absorption or scatter and perform accurate quantitative analysis. A discussion and description of the complete instrumentation for GFAAS is beyond the scope of this chapter and is

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described elsewhere [1, 2, 3]. This chapter will concentrate on the graphite furnace or electrothermal atomizer part of GFAAS instrumentation.

i" _ '1 ~engtht " ..=[~etector ~ [Signal Gra Light acete~.' I "I~Ve~i lPr~176 [[Readout ~1 Device ! Fun~hi Furnace Power

Supply

Figure 2. Block digram of the instrumentation for graphite furnace AAS. reference [ 1] for details

See

1.5.1 Graphite Furnace Since the initial use of a graphite fiamace with atomic absorption spectrometry by L'vov in 1959, considerable development in graphite furnace materials and design, called modem fitmace technology, has occurred that has increased the sensitivity and accuracy of the atomizer for practical analysis. This section focuses upon a description of successful furnace design. The graphite furnace serves as the atom cell, whose function is to convert the analyte in a sample (solid, liquid, or gas) into gaseous atoms that can be monitored spectroscopically. A schematic diagram of this device, which is also called an electrothermal atomizer (ETA), is given in Figure 3. A graphite tube, typically 3-6 mm in inner diameter and 20-40 mm in length, is heated resistively by a high current (up to several hundred amps), low voltage (6-12 V) ac power supply. Sample introduction into the tube is performed through the dosing hole in the center of the fiamace length. The source beam passes through the tube in the axial direction and on to the detection system. The temperature of the tube can be controlled from ambient to 2700~ with a precision of + 10~ up to 200~ and ~:

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50 ~ at the higher end of the range. The tube is mounted in graphite inserts and are held in place by brass or stainless steel water-cooled electrodes that are electrically connected to the power supply. The internal and external walls of the tube are bathed in a purge gas, usually argon, to exclude oxygen and prevent combustion of the graphite surfaces. Argon is preferable to nitrogen because the latter forms compounds with several elements (e.g., aluminum) and toxic CN gas. Removable quartz windows serve to prevent intrusion of oxygen into the fiu'nace through the optical path. The internal flow of gas is usually turned off ("gas-stop") when the analytical measurement is performed to maximize the time that the analyte atoms are present in the tube ("residence time") and the sensitivity. The internal gas flow generally may be specified by the analyst during each step of the furnace heating cycle. In addition, it is usually possible to switch to an alternative gas (e.g., air or oxygen for oxygen ashing) at specified times. The temperature of the tube is controlled two ways. During the majority of the fm'nace cycle, the tube temperature is regulated by the amount of voltage (current) applied. However, in order to obtain the best sensitivity and accuracy, the ~ce is usually heated with the maximum possible heating rate (> 1000~ to a temperature just above the minimum required to completely atomize the analyte. An optical temperature sensor is used to monitor the tube wall temperature in most modem commercial designs during maximum power heating. When the tube temperature exceeds the selected atomization temperature, the optical pyrometer ttmas off the maximum power heating and temperature control is retraced to the fia'nace power supply. Alternatively, some instruments use the optical temperature sensor to control the temperature of the tube during the pyrolysis and atomization steps.

1.5.2 Graphite tube material and design Early GFAAS work in the 1970s employed relatively large, longitudinallyheated tubes (50 mm in length, 8 mm in diameter) which had the advantages of being able to accommodate large sample volumes and relatively easy alignment of the source through the tube. Longitudinally heated furnaces are commonly referred to as Massmann furnaces, who first described this design in the late 1960"s. However, large fia'naces cannot be heated rapidly and tend to heat unevenly, which allows condensation of material in the cool areas. During the early period, open graphite atomizers were also employed for AAS. These atom cells provide very high sensitivity for simple aqueous solutions, but are unsuitable for analysis of complex sample matrices. Atomization occurs into a relatively cool environment that induces molecule formation between the analyte and its matrix. The most commonly used material for early graphite tubes was polycrystalline graphite (electrographite), which is porous, allowing diffusion of

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analyte molecules into the material, and is relatively reactive towards metals, which may cause interferences. The porosity and reactivity of electrographite may induce tailing, memory effects, or incomplete atomization. This material is particularly unsuitable for the determination of several elements (e.g., vanadium, titanium, molybdenum) that form stable, involatile carbides.

i

GraphiteFumace I

;I

DO~eng l, power supp,, Water-cooled

\ I

Io0t,ca, I I I

I Temp.

Ix ISensor ]

Electrode\

I

Quartz W~nd~

Source ......... ...~ .."~.1..................... [~....'~.. DeTOction / System raphite~ '

-

--I

,be

I

~ Cooling Internal ~ Cooling External Water Gas Water Gas Exit Flow Inlet Flow

(i~ Argon Supply

Figure 3. Schematic diagram of graphite furnace atomizer Modem graphite furnace systems employ graphite tubes (20-30 mm in length; 3-6 mm in diameter) that can be rapidly heated (> 1000~ to reduce interferences. A high heating rate also allows vaporization with a lower final atomization temperature, which is particularly advantageous for involatile elements. The problems with electrographite can be greatly reduced by the formation of a layer of pyrolytic graphite (50 lam) on the surface of a polycrystalline tube. Pyrolytically coated graphite tubes are produced by heating an electrographite tube in 5 % methane/argon. The dense layer of pyrolytic graphite serves to reduce diffusion of analyte into the graphite and chemical

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J. SNEDDON and D.J. BUTCHER

reactivity. Carbide-forming elemems should only be determined with pyrolytically coated tubes to obtain acceptable sensitivity and accuracy. A variety of other materials have been employed as tube substrates for graphite finnace AAS which include total pyrolytic carbon, glassy carbon, metal furnaces (e.g., tantalum, tungsten), metal liners (thin metal sheets), and metal impregnated into graphite. In general, these approaches have not been proven to superior to pyrolytically coated tubes for a wide variety of applications, and hence they have been employed in a relatively small number of laboratories. Up until recently, all commercial graphite furnaces were heated by a longitudinal flow of current through the furnace. Recently it has been shown that the temperature of the gas phase in a longitudinally heated graphite furnace may vary as much as 1200~ fi'om the center to the ends. The relatively cool temperatures at the ends of the furnace, caused by contact with the water cooled electrodes, may allow analyte atoms to condense, or analyte-containing molecules to form, in the colder areas. Condensation has been shown to induce chemical interferences. One approach to minimize these effects is the use of a transversely-heated graphite atomizer (THGA) with integrated contacts. The temperature gradients are reduced with these atomizers compared to longitudinally heated fiamaces, resulting in reduced interferences compared to the Massmann design. However, it has been postulated that the analyte and matrix may condense in cool regions in a THGA. In addition, commercially available THGAs (Perkin-Elmer Corporation, Norwalk, CT) are employed in a longitudinal magnetic field for ac Zeeman effect background correction that has dictated the use of a relatively short tube length (18 mm) that resulted in a reduction of the peak area sensitivity by a factor of two. In order to reduce interferences and improve the sensitivity, pyrolytically coated carbon disks with small (3.2 mm) apertures, called end caps, have been inserted in the end of the tube. The use of end caps reduces the rate of diffusion of the analyte out of the tube, and provides comparable peak area sensitivity to longitudinally heated fin'naces. In addition, they increase the temperature of the gas phase at the ends of the tube, reducing condensation and the potential for interferences.

1.5.3 Furnace Heating Cycle In order to make an analytical measurement by GFAAS, it is necessary to set up a program to control the temperature of the fiu'nace following introduction of the sample. An optimized temperature program allows vaporization of non-analyte (matrix) species, such as solvent and organic matter, before atomization of the analyte, in order to reduce potential interferences. It is also essential that all analyte

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vaporize in a temporally reproducible way, and that all is removed by the end of the fumace program. A typical fiwnace program consists of the following steps: (1) sample introduction; (2) a dry step, to remove solvent (usually water) from the sample; (3) a pyrolysis step (also called char or ash) to remove organic and other volatile materials in the sample before the analyte is vaporized; (4) a cool-down step to allow the fitmace to reach ambient temperature; (5) an atomization step in which the analyte is atomized and the integrated absorbance recorded; and (6) a clean step to remove any residual material from the graphite tube. With modem instrumentation, sample introduction is normally done with an autosampler. For the introduction of liquid samples, which are used in the vast majority of GFAAS work, the autosampler consists of a mechanical arm attached to a piece of plastic tubing and a series of standard and sample solutions in cups positioned in a sample tray. One end of the tubing is connected to a pump and a deionized water supply, while the other end is connected to a mechanical arm that may be inserted into the solutions. The sample tray rotates, and moves laterally, to allow sampling of each of the solutions in the tray. The tubing is inserted into a sample, and the pump draws a user-specified volume into the tubing, and the mechanical arm moves the tubing through the dosing hole into the graphite tube to deliver the sample. Generally sample volumes of 5-50 ~tL are employed; most modem programs use 10-25 laL. Most autosamplers allow introduction of up to three solutions into the furnace (e.g., sample, standard, chemical modifier). The autosampler then rinses the tubing with deionized water to clean it, and the heating cycle of the furnace begins. It is usually necessary to observe the sample introduction process the first few samples of the day to ensure the solution is precisely and accurately delivered into the furnace. The end of the tubing normally should pass through the center of the dosing hole and be positioned 2-3 mm above the graphite substrate during sample introduction. Normally the position is set manually by the user, before a graphite furnace cycle is initiated. A dentist's mirror, or a solid state charge coupled device (CCD) camera available on some instruments, is used to observe the sample introduction process. The CCD camera has been used to optimize set-up and alignment of the graphite tube and to ascertain that all of the liquid has been deposited correctly in the tube. Phase transitions of the injected samples may be observed during the dry and pyrolysis steps. A second type of autosampler employs deposition of the sample as an aerosol spray. This autosampler has the advantage that it can also be used for flame AAS, and is well-suited for use with a graphite tube that has been preheated, which decreases the time required for the dry step and hence the analysis time. However, aerosol deposition requires several milliliters of sample (although only 20-50 ~tL

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are introduced into the furnace), and is problematic with samples that are viscous or contain undissolved material. The use of an autosampler has three principal advantages for GFAAS. Precision is usually degraded with manual pipetting because it is difficult to reproducibly dispense the sample in the same location. Manual pipetting may cause introduction of material on the edge of the dosing hole of the tube. Typical precision values of several percent are obtained with manual sample introduction, as compared to one percent or less with an autosampler. Second, each heating cycle requires 2-3 minutes, and hence manual operation is unproductive for the operator. Third, pipette tips employed in manual pipetting may introduce contamination. The dry step serves to remove liquid present in the sample. With aqueous samples, the furnace is heated to 110-150~ lower temperatures are used with wall operation and higher temperatures with a probe or platform. The tin,ace is heated relatively slowly (2 - 20~ to a temperature just high enough to completely remove the solvent without spattering. Improper drying can lead to poor precision. It is normally prudent to observe the drying step for the first few furnace cycles of the day, using a mirror or camera system described above. The pyrolysis step serves to remove non-analyte (matrix) components of the sample. A moderate heating rate of 50-200~ is generally used during this process. It is usually desirable to use as high a pyrolysis temperature as possible without vaporization of the analyte, and determination of the optimum temperature is generally required for each element and type of sample. Pyrolysis temperature optimization involves measuring the absorbance from a standard or sample at a fixed atomization temperature with a variety of pyrolysis temperatures. Usually it is necessary to do temperature optimization for standards and samples to ensure the pyrolysis temperature is optimum for both. It is usually desirable to use a pyrolysis temperature of at least 1000~ to minimize interferences. The use of chemical modifiers allows the use of relatively high pyrolysis temperatta'es, even for volatile elements (e.g., lead). The introduction of air or oxygen into the fimmce during this step allows ashing in the graphite tube (oxygen ashing). Oxygen ashing allows more complete vaporization of some organic matrices, such as blood, and prevent formation of carbonaceous residue which cause interferences. Oxygen ashing is performed at temperatures below 800~ to prevent combustion of the furnace. Following the pyrolysis step, the furnace temperature is returned to ambient using a cool down step. The use of a cool-down step before atomization helps ensure atomization of the sample into a hot environment, which has been shown to reduce interferences.

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In the atomization step, the temperature of the graphite tube is rapidly increased with maximum power heating (> 1000~ to just above the temperature required to atomize the analyte (1600-2700~ The analytical measurement is performed during the atomization step, a transiem absorption signal is obtained. Optimization of the atomization temperature is generally required for standard solutions and samples, and involves the measurement of absorbance of a standard or sample with a fixed pyrolysis temperature and a series of atomization temperatures. The clean step serves to remove residual material from the sample in the fin'nace. The tube is usually heated at a few hundred degrees per second up to 2500-3000~ After the clean step, the tube is allowed to cool to ambient temperature, and the cycle is initiated again. Typical fim~ce programs require 2-3 minutes per analysis. Fast fimmce programs have been developed in order to approximately double the sample throughput of GFAAS. Typically the sample is introduced into a hot (150-200~ furnace, and short (5 s) pyrolysis steps are used at relatively high temperatures (> 1000~ Good results have been obtained for a number of elements in relatively easy matrices. More work needs to be performed to determine whether fast programs can be widely used for routine analysis. 1.5.4 Methods of Atomization

Early commercial atomic absorption atomizers heated at relatively slow rates (500-800~ with sample introduced on the wall of the tube, and hence atomization occurred as the fiwnace was heating to its final temperature. Under these conditions, atomization occurred into a tube whose temperature varied fi'om fiwnace cycle to furnace cycle, and was not isothermal along the length of the tube, with the center several hundred degrees hotter than the ends. Wall atomization was shown to be less suitable for real sample analysis with volatile elemems because these temperature variations were shown to degrade precision and induce the formation of analyte-containing molecules that cause chemical interferences, although wall atomization is preferable for extremely involatile elements. Several approaches have been investigated to ensure atomization occurs into a relatively high temperature environment. The most commonly used approach is the L'vov platform or simpley called platfoma, in which a sample is introduced onto a small graphite shelf (usually pyrolytically coated polycrystalline graphite or totally pyrolytic graphite) inside the tube. Currem does not pass directly through the platform, and hence it is primarily heated radiatively by the tube walls. Consequently, the use of a platform with a rapidly heated furnace ensures that atomization occurs after the tube, and the gas inside it, have reached a relatively constant temperature after maximum power

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heating. This means that atomization occurs into a hot environment, minimizing interferences. Today most manufacturers offer graphite tubes with integrated platforms. Transversely heated graphite tubes include a pyrolytically coated platform machined from the same piece of graphite. Contact between the tube and platform is minimized to reduce heat transfer through direct contact. A second approach is the delayed atomization cuvette (DAC), in which a graphite tube is modified so that the outer diameter at the middle is greater than at the ends, with a constant inner diameter. In a delayed atomization cuvette, sample is introduced into the middle, thicker region. The thinner ends of the fin'nace are heated more rapidly than the center, allowing vaporization into a relatively hot environment. In general this approach does not seem to be as effective at reducing interferences as the L'vov platform. Probe atomization involves the use of a graphite probe that is inserted into and removed from the tube by a stepper motor. Sample is deposited onto the probe with the probe inside the fiamace, and the sample is dried and ashed. The probe is then withdrawn from the finaaace which is subsequently heated to the atomization temperature. The probe is rapidly reinserted into the furnace, allowing atomization into a hot environment. Probe atomization has not been as widely employed as platform atomization, probably because of the added complexity of the instrumentation, and because the insertion of the cool probe into a hot tube cools the vapor, which prevents isothermal vaporization. In addition, the probe hole provides an additional avenue for loss of analyte. A two-step fiimace employs two power supplies, one to heat the graphite tube transversely, and the other to heat a graphite cup, just below an aperttwe in the tube, into which sample is introduced. The tube is heated to the atomization temperature, and subsequently the cup is heated to vaporize the analyte into the isothermal tube. Imerestingly, this design is very similar to the first graphite fumace instrument described by L'vov. The design has not been available in commercial instrumentation, probably because of the additional cost of two power supplies, and has relatively small advantages for most analytical applications compared to conventional atomization with a transversely heated furnace, although two-step fia'naces have been employed for ftmdamental studies.

1.6 Sample Preparation and Sample Introduction Sample preparation involves the conversion of a sample into a form that is suitable for analysis, which in general, is into a solution, although methods have been developed that allow the use of solids. In general, the quality and rate of GFAAS analysis is dependent upon the success of sample preparation procedures. Sample introduction involves the transfer of a standard or prepared sample into the graphite furnace, and the method employed depends on the state of the sample after

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sample preparation. Due to problems with this process, Browner and Boom [4] has referred to sample introduction as the "Achilles' Heel of Atomic Spectroscopy." These two processes are closely related and hence will be considered together in this section. A book edited by Sneddon [5] discusses various methods of sample introduction in atomic spectroscopy. Liquid, gaseous, and solid samples are all determined by graphite ftwnace atomic absorption, and here a general overview of sample preparation/introduction will be provided, along with representative applications. A detailed discussion on sample preparation as it relates to clinical and biological samples is presented later. Conventional dissolution methods (acid digestion, combustion, fusion) as well as methods to analyze solids with minimal sample preparation (slurry and solid sampling) are discussed. Methods of preconcentration/isolation of analyte, such as extraction, chromatography, and flow injection, allow the removal of the analyte from its matrix and a reduction in the detection limit. A variety of applications of these methods have been employed with GFAAS. GFAAS has also been used to obtain quantitative information on the chemical form of metal present in samples, which is called metal speciation.

1.6.1 Liquids Aqueous samples (e.g., fiver water, sea water, etc.) can be introduced directly into the graphite ~ a c e with an autosampler. If the sample is viscous, such as blood, or colloidal (milk), then it is necessary or advisable to dilute the sample with an appropriate solvent. Usually deionized water or dilute nitric acid are employed for this purpose. Surfactants, such as Triton X-100, are added to some samples to lower surface tension and promote thorough mixing of the diluted sample. The use of a digestion procedure has been shown by some analysts to improve the detection limit and remove some interferences. The determination of lead in blood has been widely investigated due to the toxicity of the element, the relatively low concentration levels (typically 1 ng/mL in "normal" blood), and severe matrix effects. Deval and Sneddon [6] described a method for the direct, simultaneous determination of cadmium and lead in blood reference samples with self-reversal background correction. The use of an ammonium dihydrogen phosphate chemical modifier allowed the use of an elevated pyrolysis temperature that removed the blood matrix. A detection limit of 1.06 ng/mL was reported for lead, which allowed its direct determination at concentration levels between 4.8 and 17 ng/mL. Good accuracy was obtained by this method.

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1.6.2 Solids The majority of samples for analysis by GFAAS are solids, which are generally converted to solutions, and introduced in that form into the graphite tube for analysis. Some solids may be dissolved by simple dissolution in water (e.g., heroin) but most require a digestion procedure. Most procedures involve the dissolution of 0.1-1 g of solid per 100 mL solution. The primary types of dissolution procedures are wet decomposition (acid digestion), combustion (dry ashing), and alkali fusion. The direct analysis of solids is also possible, and two basic techniques have been employed: slurry sampling, in which a powdered material is suspended in a solution that is aspirated into the atom cell (see chapter 6 by Matusiwiecz), and solid sampling, in which a solid is directly inserted into the graphite furnace. Sample preparation methods are generally considered to be critical to quantitative analysis became significant errors may occur due to loss of analyte due to volatilization or precipitation The use of standardized methods of sample preparation would facilitate meaningful comparison of detection limits, linear dynamic ranges, and other analytical figures of merit between various spectrometers. 1.6.3 Wet Decomposition Wet decomposition, or acid digestion, involves the use of mineral acids and oxidizing agents (hydrogen peroxide) to affect dissolution of a sample. Acid digestion is employed for a variety of organic and inorganic solids. Wet digestion procedures may be used to dissolve the entire sample (total decomposition), dissolve a fraction of the entire sample (strong attack), or simulate the transfer of elements in the environment, such as the assimilation of elemems from soil by plants (moderate attack). Acids commonly used in these procedures include nitric, sulfi~c, perchlofic, hydrochloric, and hydrofluoric. Hydrochloric acid is usually not recommended for ftLmace analysis to avoid chloride interferences Nitric acid generally serves as the primary oxidizing acid, and sulfuric acid is a dehydrating agem and has a high boiling point (300~ which increases the rate of decomposition of some samples. The combination of hydrogen peroxide with sulftndc acid produces permono sulfuric acid in-situ. Perchloric acid, although a potential explosion hazard, is a very strong oxidizing agent, and hence is typically mixed with nitric acid to reduce its reactivity. Hydrofluoric acid is required for the dissolution of silicates. Total decomposition of most samples requires hydrofluoric acid combined with other acids. Strong attacks, which are performed with strong acids without hydrofluoric, are easier to use, but will not dissolve silicate residues. This selectivity may be an advantage if the goal is to evaluate levels of pollution.

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Moderate attacks typically involve treating samples with dilute acids or other salts to evaluate the bioavailability of elements. In other applications, it is desirable to monitor the concentration of a metal that is exchanged by a cation of an added salt solution (e.g., ammonium acetate, potassium chloride), which is called the exchangeable concentration. Wet decomposition can be performed with either open or closed systems. Open systems may include teflon beakers, or test tubes in a shallow aluminum block, on a hot plate. Open acid digestion is suitable for relatively "easy" samples (e.g., food and agricultural samples) and is relatively inexpensive, but is unsuitable for some samples, relatively time-consuming (1-24 hours or more), and may allow evaporative loss of volatile elements. Closed digestion systems allow pressures above atmospheric to be developed in the vessel. Higher pressures allow the acids to boil at higher temperatures, and facilitate complete oxidation of the sample. The efficiency of digestion is commonly evaluated by the residual carbon content, which is a convenient, quantitative measure. In addition, loss of volatile elements is eliminated and the rate of digestion is increased. Examples of closed digestion systems include a decomposition bomb, high pressure asher, or a microwave digestion vessel. The former consists of a teflon container surrounded by a stainless steel body. After introduction of the sample and reagems, the emire bomb is heated in a muffle fiamace at temperatures up to 200~ Higher temperatures may be achieved with a high pressure asher (HPA) system, which is composed of a quartz digestion vessel mounted in an autoclave. Unlike the decomposition bomb, this system allows simultaneous monitoring of the temperature and pressure of the sample during the decomposition procedure. Several sizes of vessels are available (2-70 mL); the smallest fits directly on a Perkin-Elmer GFAAS autosampler, allowing analysis from the digestion vial. Microwave digestion involves the use of 2450 MHz electromagnetic radiation to dissolve samples in a teflon or quartz container. Microwaves interact with polar molecules and induce alignment of the molecular dipole momem with the microwave electric field. The field changes constantly, causing rotation of the molecules and intermolecular collisions, producing heat. Consequently, the rate of microwave digestion is dependent upon the coupling efficiency of microwaves with mineral acids. Nitric acid has the highest efficiency, with a value nearly as high as water, followed by hydrofluoric and sulfuric acids. Microwave ovens specific for chemical digestions are recommended for safety considerations. Both open and closed systems have been used with microwave digestion. Most dissolutions are performed with teflon vessels because it is inert with respect to metals, although the maximum temperature to which they may be heated is 200~ This property prevems the use of large quantities of

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sulfuric acid (boiling point 300~ which may deform the vessels. To obtain higher temperatures, quartz vessels are employed. Closed systems allow faster digestions (< 30 minutes), the digestion of more difficult samples (e.g., polymers, geochemical), and a reduced risk of analyte volatilization, but are relatively expensive. For example, a commercial microwave oven is $15,000-$20,000, and digestion vessels are approximately $100 each. A particular advantage of microwave dissolution procedures is the ease of automation. A relatively new development is the combination of microwave digestion with on-line sample and reagent flow transport. These commercial instruments (CEM, Matthews, North Carolina and Questron, Mercerville, New Jersey) offer the potential for rapid, automated sample preparation. Solid samples are converted to 0.1-1% slurries by the addition of suitable acids. As with slurry sampling techniques discussed briefly below (see Chapter 6 for greater details), it is usually necessary to produce samples with a homogeneous, small particle size to ensure a uniform slurry. Some samples may require a "pre-digestion" step in order to prevent plugging of the instrument. Agitation of the slurries is performed by a paddle or ultrasound in order to produce a homogeneous slurry. An aliquot of slurry is obtained in a sampling loop, and then pumped through the microwave system. An output autosampler is used to control the volume of digest delivered. Mineral acid solutions are placed in an acid-rinse reservoir to facilitate selfcleaning of the instrument. In our opinion, the relatively high cost and moderate performance of these instruments makes their value questionable for GFAAS. In addition, it is necessary to homogenize and size-fractionate samples, as required with slurry sampling GFAAS. Slurry sampling accessories are more economical than continuous-flow digestion systems, and analysis can be performed directly after slurry formation. 1.6.4 Combustion

Combustion (dry ashing) procedures involve heating a sample to a sufficiently high temperature (400~176 to remove the organic constituents. The traditional method involves placing the sample in a crucible (platinum or ceramic) or a test tube, followed by insertion in a muffle fiarnace for 1-6 hours to induces quantitative decomposition and removal of organic material. The residue is composed of carbonates and oxides. The analyte is then extracted from the ash with a mineral acid (usually nitric acid for furnace work). Dry ashing has the advantage of relative ease of use and allows decomposition of relatively large sample sizes followed by concentration in a relatively small volume of acid. However, it is a relatively slow process and is limited to relatively "easy" samples. Losses of volatile elements (Hg, As, Se, Cd,

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Pd, T1) may occur. Losses of analyte may also occur due to retention of analyte in the ash. For example, nitric acid will not dissolve silica present in the ash. In general, combustion methods have been replaced by wet decomposition procedures for most analyses by GFAAS.

1.6.5 Fusion Fusion procedures are well suited for the dissolution of samples that cannot be dissolved by other procedures (e.g., geological samples). The sample is mixed with a four-to-ten-fold excess of a fusion reagent, which are usually alkali metal hydroxides, carbonates, or borates (e.g., lithium metaborate), and placed in a platinum or graphite crucible. The crucible is then inserted into a muffle fin'nace at 800-1000~ for 15 minutes - 6 hours to form a molten salt. The melt is then poured in a dilute acid solution (usually nitric acid for GFAAS). The principal advantage of fusion is its applicability to nearly all samples. On the other hand, the large quantities of flux reagents may increase the blank level, making the technique unsuitable for many GFAAS analyses. In addition, volatile elements may be lost in the fusion step.

1.6.6 Solids analysis with slurry sampling (see Chapter 5) An alternative to the dissolution of powdered samples is a technique called slurry sampling, in which the material is suspended in a liquid diluent. The liquid depends on the nature of the sample. For example, for most biological and agricultural samples, the diluent is usually dilute (5 %) nitric acid with a surfactant (e.g., Triton X-100) to ensure good wetting of the sample and to prevent the formation of clumps. The elimination of a dissolution step has the advantages of reducing analysis time and the probability of analyte loss during sample preparation. In addition, quantities of reagents are frequently decreased, which reduces the risk of contamination, and less sample dilution isrequired, which may lower the determinable mass of analyte. GFAAS is well suited to slurry sampling compared to flame and plasma methods because of the relatively long time that the sample remains in the atomizer (long residence times) which usually induces complete atomization of particles. In order to obtain precise and accurate results by slurry techniques, it is necessary to produce a homogeneous slurry. This usually requires the use of a mill to convert the sample into a powder with a small (< 10 ~tm), homogeneous sample size. Typically 1-15 mg of powder are added per 5 mL of diluent. An effective method of agitating the slurry is also required. Methods of agitation include the stabilization of the slurry with a thickening agent (e.g., glycerol) or homogenization of the slun'y. Stabilization can prevent accurate pipetting by the autosampler and hence is not recommended.

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Homogenization has been performed by use of a magnetic stir bar, an vortex mixer, the introduction of gas bubbles, high pressure homogenization, or an ultrasonic probe. Magnetic stirring has the disadvantage that magnetic particles may adhere to the bar. Vortex mixing has been shown to be ineffective at producing homogeneous slurries of dense materials (e.g., sediments) and cannot be easily automated. The use of manual pipetting is inconvenient and also results in a degradation in precision. Gas bubbling was shown to ineffective at forming homogeneous slurries of samples in which the analyte was associated with larger particles. Ultrasonic agitation appears to be the method of choice for slurry preparation. Ultrasound induces disaggregation, wetting, and dispersal of solid particles in a liquid. In addition, it has been shown to enhance extraction of analyte into the diluent. This commercially-available device (Perkin-Elmer, Norwalk, Connecticut) consists of a titanium ultrasonic probe mounted above the autosampler that effectively agitates powdered samples. A gas-actuated cylinder is employed to control the vertical position of the probe, and its operation is synchronized with the autosampler. After the sample has moved directly below the autosampler arm, the probe moves into the sample cup, and the ultrasound is activated to produce a homogeneous sample. The probe is then lifted out of the sample and the ultrasound turned off. The autosampler arm then enters the cup, removes an aliquot, and dispenses it to the fiwnace. Slurry sampling has the potential for rapid analysis compared to dissolution procedures, but with some limitations. First, the measurement of small masses of sample (2 - 50 mg) is required, which is time-consuming, and may not be representative of the bulk sample. Second, it is usually necessary to characterize the particle size and homogeneity of the sample, as well as the distribution of the analyte between the solid and liquid phases of the slurry. If a significant fraction of the analyte is extracted into the diluent, the analytical performance will be similar to a digestion. However, if the analyte remains associated with the solid, then the precision will probably be reduced compared to digestion procedures. Third, careful optimization must be performed to obtain good results. GFAAS parameters to be considered include pyrolysis and atomization temperatures, amount and type of chemical modifier, and oxygen ashing. In addition, it is also necessary to characterize the sample in terms of homogeneity, density, and particle size. It is generally assumed that at least 50 particles should be introduced in each 20 laL injection into the graphite tube. For a material with a density of 1 g/mL, 20 mg of sample is required per milliliter of diluent. Although accurate analyses have been performed with particle sizes exceeding 100 ~tm, it may be necessary to use a nonstandard autosampler capillary. The precision may be degraded as well.

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1.6.7 Direct solid sampling Direct introduction of solid samples (direct solid sampling) eliminates sample preparation procedures, which reduces analysis time and prevents contamination by reagents. In addition, there is no dilution of the sample, which allows measurement of lower levels of analyte than dissolution procedures. Typically a few milligrams of solid material are introduced into the furnace. 1.6.8 Laser ablation Laser ablation (LA) involves the use of a laser beam to ablate or vaporize a solid sample. Spectroscopy may be done in the plasma generated by the laser beam (see Chapter 7 on Laser Induced Breakdown Spectrometry (LIBS)), or the vaporized sample may transported to a conventional atom cell. The majority of work in this area involves the use of an inductively coupled plasma (ICP) as the atom cell, with detection by either optical emission spectrometry (OES) or mass spectrometry (MS). The popularity of LA-ICP methods is due to the low transport efficiency (-~ 1 % ) of conventional nebulization methods of sample introduction. Laser ablation allows a significant improvement in efficiency. Of course, the efficiency of GFAAS is 100 %, and hence no gain in efficiency will be achieved by LA. ICP-OES and ICP-MS also have the advantage of being multielemental techniques, compared to GFAAS, which until the early 1990s was almost exclusively single-elemental. However, laser ablation has the ability to vaporize microparticles (e.g., individual crystal grains in minerals), which cannot be achieved with other solid sampling techniques. LA-GFAAS has considerable potential for microsampling, although more development is required to develop standard accessories and methods. Commercial ablation cells are available for ICP techniques which can be used for GFAAS. One problem remains the relatively low collection efficiency of metals by impaction methods. The use of electrostatic precipitation may be useful in this regard. 1.6.9 Pre-concentration/separation methods The levels of elements in some samples (e.g., semiconductor industry, environmental samples) are below the detection limits attainable by graphite fim~ce atomic absorption. Some matrices (e.g., silicates) cause significant degradation of detection limits, and hence separation of the analyte from the matrix is required. Pre-concentration/separation techniques are used to increase low levels of analyte and remove the sample matrix from the analyte. The enrichment factor (E) is a quantitative measure of the degree of pre-concentration. It is defined as the concentration of analyte after the pre-concentration step divided the analyte concentration in the original solution.

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Commonly used methods for pre-concentration/separation include extraction and chromatography. Major disadvantages of these pre-concentration techniques include their labor-intensive nature and unsuitablity to automation. Recently, flow injection (FI) has been employed with graphite furnace AAS, which has allowed rapid, automated pre-concentration/separation procedures. Other methods include the use of biological organisms for pre-concentration, co-precipitation, solid sorption, and a liquid supported membrane. 1.6.9.1 Extraction

Extraction procedures involve the transfer of the analyte from a solvent (usually water) to a second solvent (usually organic). In order to obtain quantitative extraction, it is necessary to ensure that the analyte is all in the same chemical form, usually as its most common cation, and to control the pH of the aqueous phase. The limit of detection is generally improved by a factor of 10-20 by extraction methods, with a maximum enrichment of approximately one hundred. The analyte must be converted to an uncharged compound or to an ion-association complex in order to increase its solubility in an organic solvent. The extraction process is evaluated in terms of the distribution ratio D : D = C~o,~

(8)

CA,W

where CA,Org and CA,w are the concentrations of analyte in the organic and aqueous phase, respectively. The mass of analyte remaining in the aqueous phase atier n extractions (mA,w,n) is given by

(

tn

Vw mA,w,n -- DVo~g+ Vw mA,w,o

(9)

is the initial mass of analyte in the aqueous phase and Vw and Vo~g are the volumes of the aqueous and organic phase, respectively. In general, it can be assumed that quantitative transfer of analyte may be achieved in one step for D values exceeding 100. Metal chelating agents, such as 8-hydroxyquinoline (oxine) or ammonium pyrollidine dithiocarbamate (APDC), are one class of compounds used to remove analytes from a sample matrix. These chelating agents are commonly weak acids designated by HR. They can be used for a wide variety of metals (Mn+). The equilibrium for the extraction process for a chelate may be expressed as w h e r e mA, w,0

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Mn+ (aq) + nHR (org) ~ MRn (org) + ~

(aq)

383

(10)

and the distribution ratio for a metal-chelate system is given by I!

DM = BnI~x [HRo~g aM [H +]~

(11)

where Kex is the overall extraction constant; g. is the formation constant of the metal chelate; and t3/,M is the fraction of the total metal concentration presem as the uncomplexed metal. Equation (11) demonstrates that the distribution ratio decreases as the pH decreases. Variation of the pH can therefore be used to control the metal ions extracted. The pH,~ value, which is defined as the pH at which 50 % of a metal is extracted, is used to evaluate the selectivity of an extraction. In general, a difference of three units in pH~ value is required to quantitatively separate two metal ions using a single batch extraction. For metals which cannot be separated on the basis of pH, additional complexing agents called masking agents may be employed. Masking agents, which include EDTA and ammonia, serve to tie up one of the metals and prevent its extraction into the organic phase. Ion association complexes involve the formation of a soluble ionic compound containing the analyte and a suitable counterion. In order to be suitable for extraction, these complexes should have no net charge or include sufficient nonpolar functional groups to allow high solubility in nonpolar solvents. Examples of ion-association complexes include Fe(o-phen)32+ / 2CIO4 (where o-phen = orthophenanthroline) and [(C2Hs)20]3IT(H20), / FeCl4. Extraction methods are relatively simple, and may allow extraction of several elemems or only one depending upon the analytical requirements. However, these procedures are difficult to automate, relatively labor-intensive, and have interferences that reduce the extraction efficiency. 1.6.9.2 Chromatography A variety of chromatographic procedures have been employed for preconcentration/separation. Although typical enrichment factors of 100 are obtained, concentration factors up to 2000 have been reported. In order to obtain good accuracy, it is necessary to convert the analyte into one chemical form. A prescribed volume of sample is loaded onto a column using a mobile phase that does not elute the analyte, but (ideally) allows removal of the matrix. A second mobile phase is then added that serves to quantitatively and rapidly remove the analyte from the colmlm, resulting in a relatively concentrated solution.

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Ion-exchange techniques have been employed with cationic resins, which contain acidic functional groups (e.g., -SO3H, -CO2H) and anionic resins, which contain basic functional groups (-NR3H). The exchange processes for cationic resin with a metal Mn+and anionic resin with an anion A n are nRSO3 -H+ (s) + M "+ (a,0 ~ (RSO3")n Mn+ (s) + nil+ (aq)

(12)

and nRYm3 +x" (s) + An" (at0 -'~ (RNR3+)n An" (s) + n X (aq)

(13)

The distribution coefficient for ion exchange (D~) is given by De = concentrationof analyte in the resin (amount / kg dry resin) concentration of analyte in solution (amount / L solution)

(14)

Cationic resins can be used to preconcentrate metal cations, while anionic resins allow the removal of negatively charged interferents and separation of the analyte if an anionic complex of the metal is formed (e.g., ZnCI42). Chelating ion exchange resins, such as Chelex-100, include a functional group that forms chelates with metal cations and have the advantage of forming stronger complexes with most transition metal cations. Chelex-100 and other chelating ion-exchange resins are well suited to sea water samples because they do not interact significantly with alkali metal cations (Na +) which may interfere with conventional ion-exchange resins. A more recent development is the use of preconcentration with a reversed-phase liquid chromatography procedure. A conventional reversed-phase column is used to separate metal ions that have been treated with a chelating agent.

1.6.9.3 Flow injection analysis Flow injection (FI) analysis involves the introduction of a sample (typically 50 btL) in a flowing stream of liquid (~ 1 mL/min) in narrow-bore (0.5 mm), nonwetting tubing for quantitative analysis. A peristaltic pump is generally used to transport the liquid in a laminar flow pattern. A detection system, which is used to measure the analyte concentration, may be virtually any instrument. An autosampler is often used to inject the samples into the flow stream. A variety of types of chemical processes may be automated by the systems. For example, a column, extraction module, or dialysis module may be used to separate the analyte from other sample constituents to minimize interferences in the detection system. Alternatively, reagents may be injected into the system to react with the analyte. It

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is desirable to ensure good mixing by coiling the tubing tightly or packing the tubing with beads to produce a packed bed reactor. The degree of mixing of the sample with the flow stream is referred to as dispersion (D). The dispersion of an FI system is usually quantified by the ratio of the analyte concentration injected (Co) to the analyte concentration at the peak maximum (Cp)

o = Co

(15)

Cp The dispersion of an FI system may be controlled experimentally by variation of several design parameters. For example, dispersion increases with tubing length, tubing diameter, and the flow rate, and decreases with volume injected, tight coiling, and packing with glass beads. As discussed above, the ability of a preconcentration system to increase the analyte concentration may be expressed by the enrichment factor E. However, in some cases a FI system is operated under different experimental conditions than a batch system which may lead to an increase in sensitivity. Consequently, if experimental conditions are not identical between a batch and an FI system, the increase in sensitivity should be referred to as an enhancement factor. Compared to conventional batch procedures, in which each sample is located in a separate vessel (e.g., extraction), FI is a continuous flow technique in which a series of samples are injected into a length of tubing separated by solvent. The basic processes of loading and removal from a column are similar to those employed in chromatography, but FI is distinguished from chromatography because it is designed for rapid quantitative analysis of a limited number of analytes instead of the separation of any number of compounds. FI has been widely employed with flame AAS as a method of preconcentration since the early 1980s became of its compatibility with a continuous flow system. The combination of FI with GFAAS did not occur until the late 1980s, but since then a number of applications have appeared in recent years. The interest in FI- GFAAS may be related to the ability to do automated preconcentration steps and to the availability of a commercially available FI system for use with atomic absorption instnunents. In general, the combination of GFAAS with FI for preconcentration requires specific features in terms of the immanent design First, GFAAS operates in a batch mode, and consequently preconcentration of the analyte is performed in parallel and must be synchronized with the atomization cycle in a discontinuous manner. Second, the maximum volume that can be accommodated in a graphite tube is less than 100 laL; this value is reduced

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with a platform and organic solvems that are commonly used for elution. When preconcentration is achieved by chromatography, it is therefore necessary to use microcolumns (15 laL), and it may not be possible to collect all of the analyte. Third, GFAAS is relatively sensitive to high concentrations of matrix elemems, and hence it is usually necessary to incorporate a column washing step to remove residual sample matrix before elution of the sample. Fourth, the combination of these specifications results in a relatively complicated elution sequence that generally must be computer controlled. Several methods of separation have been employed with GFAAS, including sorption, ion exchange, extraction, coprecipitation, supported liquid membrane, and electrochemistry. Flow injection provides a convenient method for automated sample preconcentration, with typical enrichmem factors of 20-50. We expect this technique to replace batch methods of preconcentration. FI has also been used with GFAAS as an interface for sample introduction into the furnace One interesting application of FI involves its coupling with in situ trapping of volatile hydrides in a graphite tube. The hydride generation (HG) technique involves the conversion of the analyte to a volatile hydride with a chemical reagent (usually sodium borohydride) which is then swept into an atom cell (generally a heated quartz tube) where the molecule dissociates in gaseous atoms. Elements which form volatile hydrides include antimony, arsenic, bismuth, germanium, lead, selenium, tellurium, and tin. Other volatile molecules have been used for sample introduction by similar procedures, including chlorides, fluorides, B-diketonates, and dithiocarbamates. In addition, aqueous mercury may be reduced into the metal which is volatile enough to be determined in a quartz tube maintained at room temperature (cold vapor mercury determination. Disadvantages of these conventional HG procedures include dilution of the analyte by cartier gas and hydrogen and low atomization efficiency in quartz tubes due to their relatively low maximum temperatures. The in situ trapping technique involves flow of hydride into a heated graphite tube which serves to decompose the hydride and condense the analyte on the tube. In general, absolute detection limits are degraded by HG-GFAAS compared to conventional GFAAS, since the efficiency of the HG procedure is not 100 %. The use of flow injection with HG-GFAAS provides a convenient approach to automate sample introduction procedures and a reduction in interferences compared to a batch system. However, in general these methods are relatively difficult to implemem, and the sensitivity for most elements is comparable to that obtained by conventional GFAAS. We consequently do not recommend these procedures for routine analysis.

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1.6.9.4 Other pre-concentration/separation methods Several methods of pre-concentration are available that have been used for a limited number of applications, which include liquid membrane devices, electrochemical cells, co-precipitation, and the use of organisms for preconcentration. 1.6.9.5 Metal speciation Metal speciation is the quantitative determination of each of the chemical forms of a metal presem in a sample. Considerable interest has developed in speciation over the past twenty years because the toxicity and mobility of metals in the environmem and organisms is dependent upon their chemical form. Metal compounds may be classified as inorganic, complexed ions, or organometallic. A variation in the toxicity of differem oxidation states exists for some metals. For example, chromium(VI) is considerably more toxic than chromium(III). In general, the organometallic compounds are more toxic than inorganic compounds because the former have greater permeability through biomembranes and may accumulate in fatty tissues. Mercury is an example of this type of element, where alkylated mercury compounds (e.g., methyl mercury) are more toxic than inorganic mercury (although these species are also regarded as toxic). Tin compounds (e.g., tributyltin) have been of interest because of their use as algicides, fungicides, and molluscicides. These compounds may accumulate to toxic levels in shellfish and fish, although inorganic tin is an essential trace elemem. Arsenic is an exception to the general rule because some organometallic forms, such as arsenobetaine, arsenocholine, and some arsenosugars, are relatively nontoxic, but inorganic arsenic(III) (arsenite) and arsenic(V) (arsenate) are toxic. A considerable body of literature is available on metal speciation. Here we discuss some general aspects of speciation with an emphasis on some recem GFAAS applications. The various chemical forms of a metal must be separated by a method which does not change the chemical structure of the analytes prior to detection by GFAAS or another method. Perhaps the most commonly used separation technique is extraction, either with acids or organic solvents. It is necessary to verify the recovery of the procedure by measurement of the extraction recovery for each analyte. This procedure involves spiking a sample with each analyte and measuring the concentration after extraction. An alternative procedure is derivatization of analytes to achieve preconcentration of the analytes. For example, hydride generation can be employed to preconcentrate hydride-forming elements. Alternatively Grignard reactions may be employed to induce pentylation of alkyllead and alkyltin species and produce compounds that can be separated easily by gas chromatography. Derivatization methods may lead to errors because of

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incompleteness of reactions (e.g., arsenobetaine is not converted to a volatile hydride by sodium borohydride), and probably should be avoided when possible. Some examples of metal speciation with GFAAS include separation of the analytes has been achieved by a number of procedures, including gas chromatography (GC), liquid chromatography (LC), extraction, and coprecipitation A disadvantage of GFAAS with many conventional separation procedures is its incompatibility with a flowing system. These problems may be alleviated by performing the separations in parallel by flow injection methods. Although clearly more work needs to be done in this area, quantitative direct speciation by GFAAS without any separation steps would certainly reduce analysis time and complexity. In conclusion, although traditional methods of speciation may be difficult to interface with GFAAS, flow injection provides a convenient way to determine various forms of elements in an on-line, automated fashion. We expect a number of new methods to be developed in this area. 1.7 Determination of elements by GFAAS One of the goals of this chapter is to provide some guidelines for quantitative analysis by GFAAS. First, the criteria that are used to evaluate whether GFAAS can be used to do a particular analysis are outlined. The second section discusses sampling, storage of samples, and sample preparation. The emphasis is on contamination, which is a common source of error in trace analysis. The use of quality control procedures is discussed to evaluate analytical procedures, including the use of standard reference materials and recovery checks. The instrument optimization protocols required to do quantitative analysis by GFAAS are discussed, such as pyrolysis temperature optimization, atomization temperature optimization, and the type and quantity of chemical modifier. 1.7.1 Applicability The applicability of an elemental analysis technique involves consideration of the analyte, the amount of available sample, and the concentration levels of the analyte. The first criterion involves consideration of the applicability of GFAAS to determine a particular elemem. In general, GFAAS is applicable to the determination of most metals and metalloids, with the exception of a few refractory elements (e.g., tantalum). The atomic absorption cookbook given by all commercial instrtnnents provides a list of determinable elements. The amount of sample must also be considered. An advantage of GFAAS compared to other techniques is the small amount of sample required. Each graphite fiu~ace cycle employs approximately 10-20 pL of solution (dissolved sample or liquid), and since determinations are normally performed in triplicate, approximately 30-60 pL are required. If a smaller volume is available, but the

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analyte levels are relatively high, it is possible to dilute the sample before analysis to increase the volume. Slurry and solid sampling methods may employ as little as 1-4 mg of solid per cycle, allowing analysis of a few milligrams of sample, although such small masses may not be representative of the bulk of the material. Assuming the analyte is determinable by GFAAS, and sufficient sample is available, the third criterion to consider is the concentration levels in the sample following sample preparation procedures (See 1.6). Generally 0.1 - 1 g of solid sample are dissolved and diluted per 100 mL volume. The useful (linear) range of the calibration graph is usually assumed to be between the limit of quantitation (approximately five times the detection limit) to the level of linearity, typically two to three orders of magnitude. It may be possible to detect values closer to the detection limit, but degraded precision and accuracy should be expected. In addition, some sample matrices may degrade the detection limit, increasing the limit of quantitation. Data from the atomic absorption cookbook and the approximate concentration of analyte in the sample should be used to determine whether the levels fall within the useful range of the graph. Obviously, if this condition is met, the analyst may continue to the next step. However, if the concentration levels are too low, the analyst has two options. The easiest option, if available, is the use of a more sensitive technique. This may not be possible became GFAAS is one of the most sensitive techniques. Possible options include inductively coupled plasma- mass spectrometry (ICP-MS) and neutron activation analysis. The second option is to use one of the preconcentration techniques (e.g., extraction, chromatography, flow injection). These techniques also offer the advantage of separating the analyte from the matrix, which may reduce interferences. Their primary disadvantages are their time-consuming nature and inconvenience. If the concentration levels are above the useful range of the calibration curve, several options are available. First, if the concentration levels are sufficiently high, the use of a less sensitive technique, such as flame AAS or inductively coupled plasma optical emission spectrometry, is appropriate. These techniques are faster and usually easier than GFAAS. A second option is dilution of the sample by deionized-distilled water, which has the advantage of diluting potential interferences. Third, many elements have less-sensitive alternative wavelengths, listed in the cookbooks, that may be employed to determine relatively high concentration levels. A final option is the use of a low internal flow of gas through the graphite tube during the atomization step, which serves to more quickly remove the atoms from the atomizer, and reduce the sensitivity. This option should probably be used as a last resort because a gas flow may reduce the temperature during the atomization step and cause chemical interferences.

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The concentrations of metals or elements in many biological and clinical samples are low, and ot~en very little sample is available. The electrothermal atomizer or graphite fiamace also allows in-situ treatment of the sample such as removal of potential interfering and often complex matrix. Hence, the attractiveness of GFAAS in the biological and clinical area.

1.7.2 Sampling, sample storage, and sample preparation The use of well-designed sample collection and storage procedures is required to ensure collection of representative samples with good precision and accuracy. In order to obtain a representative sample, it is first necessary to consider the size of the gross sample required that is truly representative of the entire sample. It is then necessary to reduce the gross sample to laboratory samples, which are employed for analysis, with maintenance of the chemical integrity of the analytes. Ideally, in addition to ensuring that the laboratory sample is representative of the entire sample, it is necessary to ensure that no addition or deletion of analyte has occurred prior to analysis. Reduction of the sample size involves homogenization of the sample by thorough mixing. Some samples, such as soils and fertilizer blends, are heterogeneous. In these cases the particle size should be reduced as much as possible in order to obtain representative portions for analysis. Particle size reduction of hard materials may be performed with laboratory mills and grinders. Sot~ samples, such as foods and tissue, may be homogenized with mixers or blenders. It should be pointed out that in some cases it may not be desirable to homogenize the entire sample. For example, consider fruits with non-edible skins The levels of metals may be higher in the skins than in the fruit due to pollution. However, is the concentration in the skin of interest? It may be more appropriate to analyze the edible portion. Loss of analyte may occur during transport, storage, and sample preparation. For example, the analyte may coprecipitate with other salts (e.g., urine samples). These losses may be eliminated by complete digestion of the sample. The analyte may absorb on to the wall of a container. Absorption losses can be minimized by the use of thoroughly cleaned Teflon or polyethylene containers, acidification of the sample to pH < 1, and minimization of the contact time. Although volatilization of elements is normally associated with sample preparation procedures, losses of volatile compounds (e.g., mercury compounds) may occur at room temperature. Contamination is a particularly significant factor in GFAAS because of the relatively low (pg/mL - ng/mL) concentration levels determined. Air particulate matter (dust) may be a major source of contamination, both at the sampling location and in the laboratory. When collecting plant samples, it is ot~en desirable to

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monitor the concentration of metals in tissues independent of the air particulate matter. In this case it is necessary to wash the samples in order to remove the deposition, which may lead to additional contamination/losses. The prevention of contamination by air particulate matter may be achieved by the use of clean environments. Class 100 clean rooms appear to be adequate at removing most particulates. Ideally, the air entering the clean room should be purified with high efficiency particulate air (HEPA) filters, which serve to remove at least 99.99 % of 0.3 l~m particles. The analyst may also be a source of contamination. Human skin is a source of sodium and other elements. Significant contamination may also be produced by hair and clothing. Ii is recommended limiting access to the clean room and requiring special dust-~ee clothes, shoes, and hats. Equipment and chemicals are potential sources of contamination. Collection and storage should be performed using clean containers (washed in detergent followed by soaking in 1 M nitric acid) made of a high-purity material in a relatively clean area with controlled temperature conditions appropriate for the samples. For trace element analysis, recommended materials include polyethylene, teflon, and synthetic quartz. Sample vessels should be permanently labelled to allow random assigmnent to prevent bias from particular containers. Colorless pipette tips should be used for solution preparation because the color of some tips is due to the presence of certain metals. Homogenization of samples by grinding or blending induces considerable physical contact between the sample and this equipment. Considerable contamination may be induced during this step in the sample preparation procedure. The choice of material to be used for grinding is dependent upon the analytes. For example, steel is a durable, relatively inexpensive material, but may induce contamination of iron, chromium, and manganese, while tungsten carbide is brittle and expensive but elemental contamination is limited to tungsten, cobalt, and a few rare earth elements. High quality deionized water is essential for trace element analysis. A number of water purification systems are available commercially. Further purification of water using a sub-boiling distillation unit may be necessary for extremely low concentration levels. The use of high purity chemical reagents is obvious for trace analysis. Contamination may be a problem even with the use of high quality reagents. The levels of copper and zinc in blood samples are typically in the ~tg/mL range, and contamination from acids is not a problem. However, the other elements are present at the pg/mL- ng/mL levels, and concentrations in the reagents are the same as or greater than those in the samples. These data indicate that it may be necessary for the laboratory to purify acids for trace analysis. Quartz sub-boiling

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distillation units may be used to reduce metal concentrations in all acids except hydrofluoric, which must be purified in all teflon units. Details on sample preparation procedures are given in section 1.6. The significance of contamination control is given in this example, where the determination of manganese in serum by neutron activation analysis is reported in the absence and presence of contamination controls. Contamination controls for this work involved the use of care in sample handling, carefully cleaned glassware and plasticware for the collection and storage of samples, purified reagents, and a cleanroom. The concentration levels reported here are of the same order of magnitude as would be expected by GFAAS, and similar results would be expected with this technique. The manganese levels are a factor of ten lower in the absence of contamination, and the relative standard deviation (RSD) was reduced from approximately 100 % to 15 %. These data illustrate the concept of "garbage in, garbage out" for elememal analysis. Reliable data cannot be obtained during the analysis step if errors are introduced in the collection and preparation steps. This trend has been confirmed in several analyses in the environmental (e.g., sea water) and clinical chemistry (e.g., blood serum) literature, in which ambient levels of metals have "fallen" over the past 30 years because of the elimination of contamination in more recent work.

1.7.3 Quality control procedures Quality in analytical procedures is characterized by the magnitude of errors and the extent to which the errors affect the final results. Accredited laboratories are required to document the accuracy and precision of methods and results as described by international organizations such as the International Standards Organization (ISO), International Union of Pure and Applied Chemistry (IUPAC), and Association of Official Analytical Chemists (AOAC). All samples and procedures must be carefully documented during collection, storage, and analysis. Careful attention must be paid to blanks and calibration standards. Calibration and reagent blanks should be prepared and analyzed to establish a zero baseline and a background value, respectively. Generally two independent sets of high quality calibration standards should be employed. Calibration standards are generally prepared by serial dilution of concentrated stock solutions (1000 ~tg/mL). Directions for preparation of stock solutions are provided with the list of standard methods provided with the instrumem, or alternatively commercial standards may be purchased. Stock and calibration standards are usually stored in acid solution in plasticware to increase their stability. Analytical standards for GFAAS should be prepared daily in plasticware (rather than glassware) in dilute nitric acid (0.2 %) by serial dilution techniques. Generally dilutions should be performed with mechanical pipettes with volumes between 0.1 and 5 mL.

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Standard reference materials (SRMs), which are samples that have been analyzed by at least two independent methods, should be analyzed with the "real" samples to assess accuracy. A wide variety of standard reference materials are available, such as manufactured matrix (e.g., plant process quality control for metals, cement, glass, paint, and automobile catalysis), agricultural products, environmental samples (soil, sediment, sludge, and water), botany, marine science, geology, coal, and medicine. Some government agencies that supply SRMs are listed in Table 1. A number of private companies also produce reference materials. Recovery checks involve the addition of an aliquot of analyte to a "real" sample to evaluate the recovery efficiency of the method. Recovery checks should be incorporated randomly in a sequence of analyzed samples. The level of spiked analyte may be equal to the expected level of analyte, in order to evaluate differences between analyte from the sample or from the spike. On the other hand, minimal error in the recovery factor is obtained when the added analyte is several times larger than the native analyte. It is necessary to ensure that the total concentration of analyte is on the linear portion of the calibration graph. Samples should be analyzed using strict quality control procedures. Blind samples are standards that are submitted for analysis as "real" samples. Analysis of replicates involves repeated analysis of a sample during a series of measurements in order to evaluate the precision of the analytical system. Typically at least one sample should be replicated in every ten analyses. In addition to quality control within a laboratory, it is also necessary to verify comparability between laboratories. External quality assurance systems are employed to assess the reliability of results from more than one laboratory. 1.7.4 Development of GFAAS methods The successful use of GFAAS for real sample analysis involves the use of modem fia'nace technology [1, 2]. Method development should be initiated by consultation with an atomic absorption "cookbook" of experimental conditions for the determination of a particular element, provided by most manufacturers. Methods are usually outlined for the determination of elements in particular samples which may include the concentration levels that correspond to the linear region of the calibration graph; sample preparation procedures; immanent conditions, such as available wavelengths, with their relative sensitivities, slitwidth, and temperature programs; and type and amount of chemical modifiers. However, it is the opinion of the authors that the analyst should use these conditions as general guidelines and develop their own sample preparation methods and instrument conditions. Additional information may be obtained by careful examination of the atomic spectrometry literature before attempting an analysis to avoid "reinventing the wheel." Methods of sample preparation are discussed in

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section 1.6. It is recommended that the reader focus on references published since the mid-1980s, subsequent to the development of modem furnace technology, for the most relevant information. Table 1 Representative government suppliers of standard reference materials

Agency Community Bureau of Reference (BCR), Brussels, Belgium (numerous standards in many areas) National Institute of Standards & Technology (NIST), Gaithersberg, Maryland, USA (numerous standards in many areas) National Research Council (NRC), Montreal Road, Ottawa, Canada (marine standards) National Institute for Environmental Studies (NIES), Ibaraki, Japan (environmental standards) Geological Survey of Japan, Ibaraki, Japan (geological standards) United States Geological Survey (USGS), Denver, Colorado, USA (geological standards) Laboratory of the Government Chemist (LGC), Middlesex, United Kingdom (numerous standards in many areas) Canadian Certified Reference Materials Program (CCRMP), Ottawa, Ontario, Canada (geological standards) Agricultural Research Center (ARC), Jokionen, Finland (agricultural standards) National Research Center fro certified Material (NRCCRM), Beijing, China (numerous standards in many areas) ,

,

A number of GFAAS instrumental conditions need to be selected or optimized. For example, a method of background correction should be selected, if more than one is available on an instrumem. Many modem instrumems have selfreversal or Zeeman and the continuum source method. In general, self-reversal or Zeeman is preferable to the cominuum source method, although there are exceptions to this statemem.

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The type of graphite must be selected as well. A pyrolytically coated graphite tube with platform atomization gives optimum performance for most elements. However, if cost of graphite is an important consideration, some volatile elements may be accurately determined with less expensive uncoated graphite tubes, and some involatile elements may be accurately determined without a platform. The cookbook should be consulted for the type of graphite recommended for a particular application. The use of chemical modifiers has been shown to reduce chemical interferences for GFAAS. The atomic absorption cookbook and literature should be investigated for the most appropriate choice of chemical modifer for a given analyte. For example, palladium has been commonly used to determine a variety of volatile elements because of its ability to stabilize them sufficiently to allow pyrolysis temperatures above 1000~ and remove much of the matrix. Frequently several modifiers have been employed for a given element, and it is desirable to experimentally evaluate the most suitable. After the modifier has been selected, it is necessary to optimize the amount of reagent employed. Chemical interferences caused by organic matrices have been effectively removed by the use of oxygen ashing at temperatures below 800~ to prevent oxidation of the tube. Lastly, the atomization cycle of the graphite tube needs to optimized for the particular analysis. Most cookbooks provide temperature programs that can be used as a starting point. In general, conditions for the dry cycle are determined by the graphite employed (e.g., whether a platform is present or not), and hence they usually do not need to optimized for each analysis. The pyrolysis and atomization temperatures are optimized for aqueous standards and the samples. Ideally, no difference would be observed in the optimum temperatures for standards and samples. The characteristic mass or limit of detection should be determined and compared to values specified in the cookbook to evaluate the analytical performance of the system. Quality control procedures should then be employed to evaluate the precision and accuracy of the analysis. These procedures include the sample collection methods, including contamination control, the use of high quality standards, and the use of standard reference materials and recovery checks. From the discussion above, it should be apparent that a number of experiments must be performed in order to optimize conditions and perform a determination for elements or metals by GFAAS. Normally one might expect to spend a few hours to several days performing optimizations for the determination of an element in a "new" sample.

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1.8. APPLICATIONS The final part of this chapter describes recent developments in instnnnentation, methodology, and selected applications for GFAAS. It is not designed to be a comprehensive review, but to describe some of the more interesting developments in these techniques. Recently, Butcher [7] has reviewed some of the more interesting and innovative areas of GFAAS. Some recent reviews on the application of GFAAS to the biological and clinical area should be consulted [8, 9, 10, l l] 1.8.1 M u l t i e l e m e n t continuum source G F A A S

One of the traditional weaknesses of ETA-AAS is its low sample throughput because of single-element detection. In recent years, several instruments have been developed that involve the use of multiple hollow cathode lamps to allow simultaneous determination of 2-6 elements [12]. However, these instruments require lamp changes and optical recalibration for a different set of elements. An alternative instrument design to HCL excited ETA-AAS, using a continuum source (xenon short-arc lamp) using an echelle spectrometer with a two-dimensional charge-injection device (CID) array detector has been developed [13]. This system provides greater simplicity with the high spectral resolution required for continuum source ETA-AAS. The analytical capabilities of the instrmnent were evaluated by the determination of cobalt, nickel, copper, zinc, and lead. Table 2 summarizes detection limits of this instrument compared to the detection limits of manufacturer's HCL-excited system using the electrothermal atomizer. In all cases, the continuum source system had higher values than the commercial system. These data also demonstrate that the detection limits degrade as a function of wavelength. This was attributed to a reduction in the intensity of the continuum source below 280 nm. In addition, the detection limit for copper degraded by a factor of three in the multi-element mode compared to the single-element mode. This degradation was attributed to an increase in integration time and background scatter in multi-element analysis. The ability of the system to perform real-sample analysis was verified by the accurate determination of lead in drinking water. In summary, this prototype insmunent has considerable potential for multielemental analysis, although the system is limited by relatively poor detection limits because of the low intensity of the continuum source in the ultraviolet. The authors suggested the use of a pulsed continuum source would provide better intensity in this region of the specmnn, resulting in better detection limits for many elements.

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Table 2 Comparison of detection limits for Echelle/CID system to the manufacturer's detection limit Element

Wavelength nm

Cu

324.75

Pb Co Ni Zn

283.31 240.73 232.00 213.86

Echelle/CID Detection Limit, pg 3 9 8 90 60 30

Elements Manufacturer" s Simultaneously Detection Limit Determined HCL-AAS, pg 1 0.5 3 3 4 3 8 3 5 3 1

1.8.2 Determination of lead in blood by tungsten-coil AAS The determination of lead in blood is a very common analysis because of the toxicity of this metal, particularly to children [ 1]. Although a variety of techniques have been proposed for this analysis, ETA-AAS has been widely used because of its excellent detection limits in the low picogram range and its use of small sample volumes. The use of a chemical modifier, such as palladium, allows the use of a sufficiently high pyrolysis temperature to remove the matrix and allow accurate analysis [6]. Salido et al. [14] reported the determination of lead in blood using tungsten coil AAS. A tungsten coil, obtained from a slide projector bulb, fit into a ceramic bulb mount in a quartz cell. Nylon bushings, which contain quartz windows, screwed into both ends of the cell. The cell was purged continuously with 10 %HJAr to minimize oxidation of the coil. A solid state power supply provided 0 15 A at 120 ACV to the coil for temperature control. Non-absorbing lead lines at 280.0 nm and 287.0 nm on either side of the analytical wavelength (283.3 nm) were used to provide background correction. A CCD spectrometer served as the detector. A commercial ETA-AAS spectrometer was used to evaluate the performance of the W-coil AAS instrumentation. Aqueous standards and blood samples were treated by an extraction procedure. A lead complex was formed with ammonium pyrrolidine dithiocarbamate (APDC). This hydrophobic chelate was subsequently extracted into methyl iso-butyl ketone (MIBK). The resulting MIBK solution was then introduced into the AAS instruments. Under optimized pyrolysis conditions with the tungsten coil (2.3 A), acceptable absorbance profiles were obtained. The

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presence of double-humped profiles observed at low pyrolysis temperatures were attributed to incomplete decomposition of the lead chelate. Analytical figures of merit for ETA-AAS and tungsten coil-AAS are shown in Table 3. The detection limits and characteristic masses for the systems were within a factor of two of each other, and the linear dynamic ranges were identical. The method detection limits for both techniques were well below the Centers for Disease Control (CDC) target value of 10 - 20 pg/L. The systems were also compared for the determination of lead in NIST bovine blood SRMs and painters' blood samples. For the SRMs, the tungsten coil instrument results were with 8 % of the certified values with an RSD below 10 %. For the painters' blood samples, the tungsten coil results were 91.7 % of the ETA-AAS values. The tungsten coil system was also shown to meet the CDC required accuracy limits of • 40 pg/L, demonstrating the suitability of this tungsten-coil instrument for the determination of lead in blood. Table 3 Analytical figures of merit for ETA-AAS and ttmgsten coil-AAS

Figure of merit D etecti0n limit Method detection limit Characteristic mass (pg) Linear dynamic range (orders of magnitude)

ETAAS 8 (0.4) 16 (0.8) 13 2

Tungsten Coil-AAS 12 (0.6) 24 (1.2) 28 2

1.8.3 Determination of arsenic and tin Hydride generation [15] (HG) atomic spectrometry involves the chemical conversion of analytes to volatile hydrides which are decomposed to atoms into a suitable atom cell. The atom cell is usually a quartz tube which is heated electrically or inside a flame. For AAS, this technique is particularly sensitive for elements whose absorption lines are below 200 nm, such as selenium and arsenic. A detailed description of HG techniques including GFAAS is provided in Chapter 2 of this book. Pacquette et al. [16] reported the first coupling of HG with laser excited atomic fluorescence spectrometry (LEAFS) for the determination of arsenic and selenium. Laser light from a XeF excimer laser (351 nm) was used to pump a dye laser to produce ~460 nm radiation. A second harmonic generation crystal was used to fi'equency double the visible light to produce ultraviolet light between 230

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and 235 nrrL The ultraviolet light was converted to arsenic and selenium absorption wavelengths (193 - 197 nm) using stimulated Raman scattering for excitation of arsenic and selenium. A laboratory constructed system was used for HG, consisting of a reaction vessel, a glass U-tube water trap (maintained in dry ice-isopropanol to remove water vapor), and a glass U-tube hydride trap (immersed in liquid nitrogen). A commercial ETA served as both the trapping cell and atomizer. Palladium was added to the tube and dried. The graphite tube was heated to 200~ as the hydrides were introduced through a quartz tube in the dosing hole to trap the analyte on the tube. Atomization was performed with a conventional ETA heating program. A comparison of limits of detection of various hydride generation techniques is available in Table 4. Table 4 Limits of detection for arsenic and selenium by selected hydride generation techniques i

HG Technique HG-ICP-LEAFS HG-ETA-LEAFS HG-ICP-OES HG-ICP-MS HG-AAS HG-ETA

Arsenic pg pg/mL 5000 1000 200 40 100 1000 0.6 12 16 80 49 7

i

Selenium pg pg/mL 300 60 800 160 200 20 18 90 29 140 49 7

The HG-ETA-LEAFS detection limits were 200 pg and 800 pg for arsenic and selenium, respectively. These values were approximately 40 times and 400 times worse than the most sensitive HG method, HG-inductively coupled p l a s m a mass spectrometry (ICP-MS), and 1000 times less sensitive than previous ETALEAFS detection limits. The relatively poor detection limits were attributed to low transport/trapping efficiency caused by the laboratory constructed HG system. It was concluded that high efficiencies would lower detection limits to the low picogram or high femtogram mass range.

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1.8.4 Determination of cadmium and zinc by double resonance laser excited atomic fluorescence in an electrothermal atomizer Double resonance (DR) laser-excited atomic fluorescence spectrometry (LEAFS) involves the use of two wavelengths of laser light to promote the analyte atoms to relatively high energy levels with subsequent fluorescence. DR-LEAFS provides high sensitivity, with typically detection limits below 100 fg. In addition, it provides very high spectral selectivity, including excellent discrimination against scattered laser light. Ezer et al. [17] reported the use of DR-LEAFS for the determination of zinc and cadmium. A XeF excimer laser was used to pump two dye lasers at a repetition rate of 30 Hz. For cadmium, the output of one dye laser was frequency doubled to produce 228.802 nm (maximum energy, 540 ~tJ) and the other was used directly (643.847 nm). Cadmium fluorescence was detected at 361.1 or 346.7 nm. For zinc, the first dye laser output was converted to 213.856 by stimulated Raman scattering, and the second used directly at 636.235 nm. The fluorescence was detected at 334.5 nm. Power dependence studies were performed to evaluate whether the transitions were saturated. The cadmium UV transition was saturated at less than 1 ~tJ, but the visible transition was not saturated. The best detection limits were obtained with 361.1 nm detection: 70 fg (7 pg/mL) with the laser tuned on the analytical wavelength (contamination limited) and 40 fg (4 pg/mL) with the laser tuned off the analytical wavelength. In the case of zinc, high background levels of zinc contamination were reported to effect the detection limit. It was also suggested that the contamination could be induced by molecular species, such as NO. The zinc detection limits were 6 pg (600 pg/mL) (on-wavelength) and 700 fg (off-wavelength). The capability of the system to do practical analysis was examined by the analysis of a bovine serum SRM. The sample was diluted in water and analyzed without a matrix modifier. For zinc, a measured value of 940 + 60 ng/g was in good agreement with the certified value of 890 • 60 ng/g. Cadmium could not be determined in the SRM because the levels were below the DR-LEAFS limit of detection. In summary, DR-LEAFS was shown to be a highly sensitive method capable of accurate real-sample analysis. 1.8.5 Copper determination in biological materials by ETAAS using W-Rh permanent modifier A recent study by Lima et al. [18] involves a tungsten-rhodium treatment on the integrated platform of a transversely heated graphite fiimace atomizer was used as a permanent chemical modifier for the determination of copper in biological materials such as copepod homogenate, fish flesh homogenate, tuna homogenate,

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pig kidney, rye grass, brown bread, plankton, and mussel tissue. The samples were all certified by agencies similar to that described in Table 1. The W-Rh permanent modifier was stable up to 250-300 atomization cycles when using volume of 20 OL of a digested samples and increased the lifetime of the graphite tube by over 1000 atomization cycles. Detection limits were in the sub E]g/g range for copper. Accuracy was at least as good as standard GFAAS methods. 1.8.6 Determination of urinary lead, cadmium and nickel in steel production workers by GFAAS The following is an example of the use and value of GFAAS in a clinical situation. A recent paper by Homg et al. [19] describes a GFAAS method for the determination of urinary lead, cadmium and nickel in steel workers. The objective was the screening of workers under routine clinical laboratory conditions. After pre-treatment with acid, the samples were digested via a microwave oven and determined by GFAAS. The analytical reliability (accuracy and precision) of the GFAAS method was ascertained through the certified standards as well as comparison to two electrochemical methods (differential pulse stripping voltammetry and hanging mercury drop electrode) and found to be excellent. This was also confirmed using National Institute Standards & Technology (NIST) (Gaithersberg, Maryland-Standard Reference material (SRM) 2670- freeze dried urine. Typical urine concentrations for lead, cadmium and nickel in steel production workers is shown in Table 5. Also shown in Table 5 are results for quality control (QC) and control concentrations. 1.8.7 Determination of platinum in clinical samples Platinum has been proposed as an anti-cancer drug and its determination in a wide variety of body fluids and tissues is frequently required. In too large a presence or concentration in the body it can be toxic. A recent review by Yang et al. [20] describes various methods, including GFAAS to determine platinum in clinical samples. The high sensitivity of GFAAS makes it attractive for determination of platinum, particularly for patients treated with platinum containing drugs, although a drawback is the complex matrix associated with clinical samples. However, recent pharmo-kinetic studies with oxaplatin suggest that the sensitivity of GFAAS may not be adequate for the accurate determination of the "free" platinum in plasma ultrafiltrate beyond a 24-hr period [21 ]. A GFAAS method was developed for the determination of platinum in human plasma, plasma ultrafiltrate and urine from cancer patients orally receiving a platinum based drug. The sensitivity was enhanced by using a volume of 150-1xL [22]. GFAAS has been used in the determination of platinum in high protein solutions as plasma-protein

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bound cisplatin [23], multiple tumour samples [22], neurologic tissue [24], and human plasma. Table 5 Urine concentrations a for lead, cadmium and nickel for steel workers, QC-workers and control groups i

i

lead, ~tg/L 52.3 +19.1 (20.26-89.60)

i

cadmium, ~tg/L i

Production Workers

i

nickel, i

~tg/L i

9.55 + 5.33 (3.08-22.61)

36.6 + 16.5 (17.0-79.5) 29.8 + 13.1 _ (4.45-51.0)

QC-workers

48.0 + 7.9 (37.98-68.61)

7.96 + 2.21 (3.18-10.32)

Controls

31.1 + 16.2 (3.88-58.12)

3.45 + 2.07 (0.60-6.81)

4.39 9 2.35 (1.73-6.82)

Each value represents the mean standard deviation. The number in parenthesis shows the range in each case.

a

1.9. CONCLUSION This chapter has given an overview of GFAAS with selected applications in the biological and clinical area. GFAAS as an analytical technique developed from the late 1960"s until the late 1980"s/early 1990"s by significant improvements in instrumentation (background correction, sample introduction techniques, atomisation techniques (platform, probe or surface)), innovative engineering (constant temperature atomisation, multielement determination, etc.) and a greater understanding of the mechanism of atomization. However, since the early 1990"s it is probably true to say that there have been no significant improvements in GFAAS. This is due, in part, to the fact that the technique has evolved into a standard and widely accepted technique for trace element or metal determination, particularly at the ppb or lower concentrations where there is limited sample available. The ability to treat the sample in-situ is attractive, particularly when there is a complex matrix. The ability to determine a few (two to six elements simultaneously) can be useful although a compromise in experimental conditions often leads to a degradation in detection limit in the simultaneous mode compared

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to the single element determination. It will continue to be used to provide necessary and reliable information in many fields, including the clinical and biological fields. REFERENCES 1 D

2e

0

.

5. .

7. 8.

0

10.

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INDEX Immobilized borohydride on ion exchange column and moveable reduction bed, 62-64 In-situ trapping/ETAAS, 71-73 Vesicle-assisted, 64-65 Hyphenated techniques, 80 CE/HG, 84-87 HPLC/HG, 80-83

Absorbance, 363-365 Atom formation, 365-366 Atomic absorption spectrometry Applicability, 388-395 Development of method, 393-394 Sampling and storage, 390-392 Quality control procedures, 392-393 Applications, Arsenic and tin, 398-399 Cadmium and zinc by double resonance laser excited AFS in an electrothermal atomizer, 400 Copper in biological materials, 401-402 Lead in blood by tungsten coil AAS, 397398 Lead, cadmium and nickel in urine of steel workers, 401 Platinum in clinical samples, 401-402 Multielement continuum source, 396-397 Flame, 7, 243-244 Furnace, graphite furnace or electrothermal atomization, 7-8, 81-84, 244-246, 361-404 Furnace design and materials, 368-369 Furnace heating cycle, 370-373 Instrumentation, 366-367 Atomic emission spectrometry, 247 Flame, 247 Atomic fluorescence spectrometry, 249, 400 Atomization (methods of), 373-374

Inductively coupled plasma-atomic emission spectrometry (ICP-AES), 8-9, 24-27, 11-13, 8487,247-248 Application, 18-20, Inductively coupled plasma-mass spectrometry (ICP-MS), 27-30, 84-87, 117-120, 248 Applications, 141-149, 201-204, 210-214 Double focusing, 119-120, 125-128 Electrostatic mass analysers, 123 Magnetic mass analysers, 121-123 Nier-Johnson geometries, 124-125 Field -flow fractionation, 179-186 Comparison with SEC, 204-205 Electrical FFF (E1FFF), 192-194, 203 FFF modes, 186-187 FFF Sub techniques, 187-188 Flow FFF (FIFFF), 194-197,203-204 Frit outlet, 216-217 Instrumentation, 198-199 On-channel, 215-216, 221-223 Opposed-flow sample concentration, 217221 Optimization, 199-200 Sedimentation FFF (SdFFF), 188-190, 201-202 Thermal FFF (ThFFF), 190-192, 202-203 Interferences, Atomization in gas phase for HG, 68-71 Chemical in liquid phase and pre-reduction in HG, 74-78 Matrix, 139, Non-spectral, 18 Spectral, 16-18, 129-136 Isotopes, 20-29, 150-159 Accuracy, 150 Blanks, 152-153 Isobaric interferences, 153 Isotope dilution, 157-158 Paleoanthropological, 157 Precision, 153-155 Resolution, 155 Tracers, 20-23, 156-157 Copper, Nickel, 24-26 Calcium, 25-26 Iron, 26-27 Selenium, 27-28

Body fluids or biological or clinical samples (human serum, urine, plasma protein and tissues), 5-7, 15, Direct current plasma, 247 Electron densities, 306-308 Excitation temperature, 304-306 Flow-injection techniques, 246 Hydride generation Applications, 87-114 Arsenic, 87-92 Antimony, 99-102 Bismuth, 107-109 Germanium, 102 Lead, 102 Miscellaneous, 103 Selenium, 92-99 Tin, 102 Electrochemical, 54-55 Fast gas-liquid separation, 60-62

405

Others, 28-29 Laser-induced breakdown spectrometry, 287-290 Analytical characteristics, 293-297 Applications, 309-330 Advanced materials, 328-329 Aerosols and gases, 324-325 Environmental, 309-315 Liquids and solutions, 322-324 Metallurgical, 315-321 Miscellaneous, 329-330 Non-metallic solids, 325-328 Basic principles, 292-293 Factors influencing plasma production, 295306 Ambient conditions, 300-302 Electric and magnetic fields, 302-303 Irradiation energy, 297-298 Physical properties, 298-300 Plasma shielding, 303-304 Sampling geometry, 304 Wavelength, 295-296 Fundamental studies, 290-304 Instrumentation, 314-326 Echelle spectometer, 325-326 Excimer laser, 316 Field instrumentation, 319-322 Fiber based, 316-319 New approaches, 322-325 Nd-YAG laser, 316 Laser-induced plasma production, 293-295

Slurry sampling or slurry sample introduction, 237-284 Calibration, 241 Chemical modification, 241 Nomenclature, 242 Particle size, 240 Precision and accuracy, 242 Slurry concentration, 241 Slurry preparation, 239-240 Speciation, 29-30, 158-159, 205-209, 387-388 Capillary electrophoresis, 169-170 DNA adducts quantification, 165 Gas chromatography, 167-169 HPLC, 159-160 Ion-exchange, 162-164 Off-line strategies, 170 Organics solvents-induced interferences, 167 Selenium, 161-165 Size exclusion, 159 Spectroscopy, 361 Thermal vaporization techniques, 249-250

Microwave-induced plasma-atomic emission spectrometry, 248-249 Reference materials, 30, 394 Reference methods, 30 Sample introduction, 13-14, 374-375 Combustion, 378-379 Chromatography, 383-384 Flow injection analysis, 383-386 Extraction, 382-383 Fusion, 379 Laser ablation, 381 Liquids, 375 Metal speciation, 387-388 Pre-concentration/separation, 381-382 Other, 387 Solids, 357-358,376 Solids with slurry, 379-380 Wet decomposition, 376-378 Sample preparation, 31,378-379 Sensitivity (and limit of detection or detection limit), 139-141,247-251

406