Update on Undertaking Extractable and Leachable Testing

Update on Undertaking Extractable and Leachable Testing Andrew Feilden

iSmithers – A Smithers Group Company Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118 http://www.ismithers.net

First Published in 2011 by

iSmithers Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK

©2010, Smithers Rapra

All rights reserved. Except as permitted under current legislation no part of this publication may be photocopied, reproduced or distributed in any form or by any means or stored in a database or retrieval system, without the prior permission from the copyright holder.

A catalogue record for this book is available from the British Library.

Every effort has been made to contact copyright holders of any material reproduced within the text and the authors and publishers apologise if any have been overlooked.

ISBN: 978-1-84735-455-6 (Hardback) 978-1-84735-456-3 (ebook)

Typeset by Argil Services Printed and bound by Lightning Source Inc.

P

reface

The purpose of this guide is to provide an insight into the range of methods available in extraction studies and how this is linked to the analysis of the extracted species. Extraction of the species of interest from a matrix or substrate is a very important area in a wide range of fields such as pharmaceutical container closure systems (e.g., extractables and leachables), environmental (e.g., polynuclear aromatic hydrocarbons from soil) and pesticides to name but a few. This guide aims to show the possible modes of extraction, ranging from the classical Soxhlet, through pressurised solvent extraction to more esoteric techniques such as membrane-assisted solvent extraction. The key parameters of each extraction technique are discussed. Extraction is not complete without the analysis of the extracted analytes; this guide covers the basic parameters of method development for the trace analysis of volatile, semi-volatile and metal analytes (gas chromatography, high-pressure liquid chromatography and inductively coupled plasma). This is because extraction and analyses requires expert knowledge of each to enable the analysis of an extractable at trace levels. Throughout this guide, particular emphasis is made on the extraction of container closure systems used in the pharmaceutical industry (primarily orally inhaled and nasal drug products). This is an area that has the widest range of regulation, the highest diversity of analytes, and is the area of expertise of the author. The key points from the regulations and pertinent guidelines are discussed. This guide follows the process of an extractable or leachable study. Starting with sample preparation, moving onto sample extraction (extraction from solid samples (extractables) and extraction from solutions), and then onto a discussion around the key analytical techniques. These cover volatile

iii

Update on Undertaking Extractable and Leachable Testing

analytes (gas chromatography) semi-volatile (high-performance liquid chromatography) and metals (inductively coupled plasma). I would like to express huge thanks to the companies that have provided the images and information used in this guide. I also thank Dr Malcolm Kimber for his review and helpful comments. Special thanks are also given to my chief editor, Dr Caroline Feilden; her helpful comments have enabled me to produce this guide.

iv

C

ontents

1

2

Introduction ......................................................................... 1 1.1

Sample Preparation ..................................................... 5

1.2

General Extraction ................................................... 10

1.3

How Low To Go: Limit of Detection ........................ 15

1.4

Introduction to Extraction Techniques...................... 17

Extraction Techniques ........................................................ 23 2.1

Sonication ................................................................. 23

2.2

Reflux ....................................................................... 24

2.3

Soxhlet ..................................................................... 29

2.4

Supercritical Fluid Extraction ................................... 37

2.5

Sealed Vessel ............................................................. 41

2.6

Pressurised Solvent Extraction .................................. 42

2.7

Microwave Assisted Extraction ................................ 46

2.8

Shaking Extraction ................................................... 49

2.9

Direct Analysis ......................................................... 53

2.10 Headspace ................................................................ 56 2.11 Thermal Desorption ................................................. 57 2.12 Dynamic Headspace ................................................. 59 2.13 Liquid Extraction Surface Analysis ........................... 60 2.14 Extraction to Asymptotic Levels ............................... 60 3

Liquid Extraction Techniques ............................................. 69 3.1

Liquid-liquid Extraction ........................................... 69

Update on Undertaking Extractable and Leachable Testing

3.2

Solid Phase Extraction .............................................. 71

3.3

Solid Phase Microextraction ..................................... 73

3.4

Single-drop Microextraction ..................................... 75

3.5

Dispersive Liquid-liquid Microextraction ................. 75

3.6

Liquid-phase Microextraction Solidification of Floating Organic Drop ............................................. 76

3.7

Hollow-fibre Liquid-phase Microextraction ............. 76

3.8

In-tube Extraction .................................................... 77

3.9

Microextraction by Packed Sorbent .......................... 78

3.10 Stir Bar Sorptive Extraction ...................................... 80 3.11 Membrane-assisted Solvent Extraction ..................... 83 3.12 Extraction to Asymptotic Levels ............................... 84 3.13 Conclusions Regarding Extraction ............................ 84 4

5

vi

General Analysis................................................................. 89 4.1

Quantification .......................................................... 91

4.2

Response Factors ...................................................... 92

4.3

Identification ............................................................ 93

Gas Chromatography ......................................................... 97 5.1

Introduction ............................................................. 97

5.2

Instrumentation of a Gas Chromatograph ................ 98

5.3

Carrier Gas ............................................................. 100

5.4

Choice of Sample Introduction ............................... 100

5.5

Sample Injector ....................................................... 101

5.6

Information Required Before Method Development 102

5.7

Developing a Gas Chromatography Method .......... 102

5.8

Sample Preparation ................................................. 102

5.9

Effects of Changing Individual Chromatographic Parameters ................................. 103

Contents

5.10 Column Parameters ................................................ 104

5.10.1 Selection of the Stationary Phase .............105 5.10.2 Stationary Phase Film Thickness..............106 5.10.3 Column ID ..............................................106 5.10.4 Phase Ratio .............................................106 5.10.5 Column Length .......................................106 5.11 5.12 5.13 5.14 5.15 5.16 5.17 5.18 5.19 5.20

5.10.6 Conclusions Regarding Columns .............107 Oven Temperature Gradient (Temperature Programme) ...................................... 108 Injection Volume..................................................... 108 Injection Temperature ............................................. 108 Liners ..................................................................... 109 Problems with the Injection .................................... 109 Split Flow/Ratio ...................................................... 110 Column Flow Rate ................................................. 110 Cool-on-Column .................................................... 111 Programmable Temperature Vaporisation ............... 112 Choice of Detector .................................................. 113 5.20.1 Flame Ionisation Detector .......................113 5.20.2 Mass Spectrometer ..................................113 5.20.3 Electron Capture Detector .......................116 5.20.4 Thermal Conductivity Detector ...............116 5.20.5 Nitrogen Phosphorous Detectors .............117 5.20.6 Flame Photometric Detector ....................117

5.20.7 Thermal Energy Analyser, Nitrogen Chemiluminescence Detector ...................118 5.21 Derivatisation ......................................................... 118 5.22 Typical Starting Conditions for Extractable Analysis ............................................... 119

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Update on Undertaking Extractable and Leachable Testing

6

High-performance Liquid Chromatography ..................... 121 6.1

Introduction ........................................................... 121

6.2

Sample Preparation ................................................. 122

6.3

Separation .............................................................. 122

6.4 6.5 6.6 6.7 6.8 6.9

6.3.1

Reverse Phase ..........................................123

6.3.2

Normal Phase ..........................................124

6.3.3

Ion Pair Mode .........................................124

6.3.4

Ion Exchange...........................................125

6.3.5 Size-exclusion Chromatography ..............125 Developing a High-performance Liquid Chromatography Method ....................................... 125 Solvent Selection ..................................................... 126 Temperature Control .............................................. 127 Columns ................................................................. 127 pH .......................................................................... 129 Detector Selection ................................................... 129 6.9.1

Ultraviolet Detector .................................131

6.9.2 Wavelength Selection ...............................132 6.10 Fluorescence Detector ............................................. 132 6.11 Mass spectrometer .................................................. 133 6.11.1 Electrospray Ionisation ............................134 6.11.2 Atmospheric Pressure Chemical Ionisation ................................................135 6.11.3 Atmoospheric Pressure Photo Ionisation .136 6.11.4 Conclusions Regarding the Mass Spectrometer ..................................136 6.12 Evaporative Light-scattering Detector ..................... 137 6.13 Charged Aerosol Detector ...................................... 138 6.14 Nano Quantity Analyte Detector ............................ 138

viii

Contents

6.15 Typical Starting Conditions for Extractable Analysis ............................................... 139 7

Inductively Coupled Plasma - Metal Analysis ................... 143

8

Conclusion ....................................................................... 147

Abbreviations ........................................................................... 153 Index ........................................................................................ 159

ix

1

Introduction

Extraction of analytes from various matrices is very important in a wide range of industries, including food, environmental and pharmaceutical. These industries use a diverse range of analytical techniques to extract the analytes of interest from their varied matrices. This guide reviews the common extraction techniques (e.g., Soxhlet) as well as more esoteric and specialised techniques (e.g., membrane-assisted solvent extraction). Specific prominence will be on the factors that need to be considered when using each extraction technique. Particular emphasis is given to the relevance of extraction techniques to container closure systems (CCS) used in the pharmaceutical industry and the regulations for that industry. In the pharmaceutical industry, regulators are becoming increasingly aware of the need to understand whether species that can be extracted from direct contact packaging (often referred to as ‘primary packaging’) and container closures will appear as leachable species across an increasing range of dosage forms. As well as primary packaging, the potential of species to be extracted from indirect packaging also needs to be considered (e.g., foil pouches, cardboard boxes). Take for example the product recall announced by the major pharmaceutical company Johnson and Johnson of certain over-the-counter product batches because of a musty or mouldy odour, which has been linked to trace amounts of 2,4,6-tribromoanisole. The contamination is believed to stem from the breakdown of a substance applied to wooden pallets used to transport and store packaging materials. It is not just the pharmaceutical industry that has issues with leachables, the food industry also experiences similar issues. Take for example the recall in 2010 by Kellogg Company. It issued a voluntary recall of 28 million boxes of some of its most popular cereals because of

1

Update on Undertaking Extractable and Leachable Testing

an ‘uncharacteristic off-flavour and smell’ coming from packaging. The smell was attributed to a substance in the package liners that can produce an uncharacteristic waxy-like ‘off’ taste and smell. In general, regulators have always been aware of extractables and leachables for orally inhaled and nasal drug products (OINDP). This is due to their high degree of concern associated with the route of administration and the likelihood of packaging-component interaction with the dosage form, as can been seen from Table 1.1. Increasingly, regulators are becoming more concerned with other dosage forms, such as parenteral and ophthalmic drug products (PODP) as well as manufacturing equipment used in biological manufacture. This article primarily discusses the key requirements for carrying out extractable testing for pharmaceutical CCS and the factors to consider for the common analytical techniques. It will also cover the background to the common extraction techniques. Prominence will be given to the possible extraction techniques and associated common separation and quantification techniques. These approaches could quite easily be applied to any industry or application in which an analyte needs to be extracted from a matrix and analysed. It is important, however, to understand which extraction procedure would be the most applicable for the matrix involved. It is advisable to consult the guidance available before embarking on an extractable and leachable study. The extent of available information will vary for the dosage form of interest. The highest degree of concern is for OINDPs, coupled with a high likelihood of interaction, so we will focus on this area in most detail. It is also the most mature area in terms of pharmaceutical understanding and currently the most challenging on which to undertake testing.

2

Introduction

Table 1.1 Risks associated with various pack types based on degree of concern and likelihood of interaction Degree of concern associated with route

Likelihood of interaction between packaging component and dosage form High

Highest

Inhalation aerosols and solutions; injections and injectable suspensions

High

Ophthalmic solutions and suspensions; transdermal ointments and patches; nasal aerosols and sprays

Low

Topical solutions and suspensions; topical and lingual aerosols; oral solutions and suspensions

Medium

Low

Sterile powders and powders for injection; inhalation powders

Topical powders; oral powders

Oral tablets and oral (hard and soft gelatin) capsules

Adapted from Guidance for Industry: Container Closure Systems for Packaging Human Drugs and Biologics, US Department of Health and Human Services, Food and Drug Administration, Rockville, MD, May, 1999.

The level of guidance available has been increasing rapidly over the last few years. It started, initially, from a simple statement that explained that drug product is deemed adulterated ‘if its container is composed, in whole or in part, of any poisonous or deleterious substance which may render the contents injurious to health.’ [1-4].

3

Update on Undertaking Extractable and Leachable Testing

Guidance has since become progressively more comprehensive. The most recent and detailed was from the Product Quality Research Institute (PQRI) [5], (all 272 pages of it). This guidance covers general principles as well as the important questions of limit of qualification and limit of quantification. Key points from the PQRI will be discussed in later sections. In addition to the PQRI guidance on extractable and leachable testing, other types of recommended testing include pharmacopeial and International Organization for Standardization (ISO) tests, US Pharmacopeia (USP) <87> and <88> and/or ISO 10993 [6-8] may be optional. For certain applications, such as use in medical devices, these additional tests are mandatory. In addition, other countries require testing of materials to their standards (e.g., Japan: JP61). The limit of detection will be discussed further because this has an important role in the design of the extraction study and the analytical techniques used. At this point, it is worth clarifying what extractables and leachables actually are, because the uninitiated often use these terms interchangeably when discussing this research field. Extractables are chemical species that are forcibly extracted from packaging materials using appropriate solvent, temperature and time conditions. Leachables are chemical species that are found in the product under normal storage conditions that are related to the CCS. Leachables, ideally, should always be a subset of extractables. Control of leachables can be readily achieved only by controlling the level of extractables. This is under the assumptions that there is sufficient correlation between extractables and leachables, and that a sufficient extractable specification is available. Leachables take time to form, so the specification of leachables should be ‘would meet if tested’. The definition of extractables will be important when the various extraction techniques are discussed. The key part of an extractable and leachable study is the controlled extraction study (CES). This is where the critical components of a

4

Introduction

device are extracted using a range of solvents with multiple extraction techniques followed by analyses with various appropriate analytical techniques. A critical component for an OINDP is defined by the PQRI as ‘Those components that contact either the patient or the formulation, components that affect the mechanics of the overall performance of the device, or any necessary secondary protective packaging.’ The scope of the PQRI guidance is as follows: UÊ ÌÊ`œiÃʘœÌÊÃÕ}}iÃÌÊëiVˆwVÊÌiÃÌʓi̅œ`ÃÊ>˜`Ê>VVi«Ì>˜ViÊVÀˆÌiÀˆ> UÊ ÌÊ`œiÃʘœÌÊÃÕ}}iÃÌÊ>ÊVœ“«Ài…i˜ÃˆÛiʏˆÃÌʜvÊÌiÃÌà UÊ ÌÊ`œiÃʘœÌÊ}ˆÛiÊ>VVi«Ì>˜ViÊVÀˆÌiÀˆ>ÊL>Ãi`ʜ˜Ê>VÌÕ>Ê`>Ì>ÊvœÀÊ>Ê particular system UÊ /iÃÌÊ “i̅œ`ÃÊ >˜`Ê >VVi«Ì>˜ViÊ VÀˆÌiÀˆ>Ê L>Ãi`Ê œ˜Ê }œœ`Ê ÃVˆi˜ÌˆwVÊ principles for each specific system UÊ ˜ÃÕÀiÊL>ÌV…‡Ìœ‡L>ÌV…Ê՘ˆvœÀ“ˆÌÞʜvÊ«>VŽ>}ˆ˜}ÊVœ“«œ˜i˜Ìà UÊ …>À>VÌ>ÀˆÃ>̈œ˜ÊœvÊiÝÌÀ>VÌ>LiÃÊۈ>ʼ

-½ UÊ œÀÀi>̈œ˜ÊœvÊiÝÌÀ>VÌ>LiÃÊ>˜`ʏi>V…>Lià UÊ ->viÌÞʵÕ>ˆwV>̈œ˜ÊœvÊiÝÌÀ>VÌ>LiÃÊ>˜`ʏi>V…>Lià UÊ ,œṎ˜iÊVœ˜ÌÀœÊœvÊiÝÌÀ>VÌ>LiÃɏi>V…>LiÃʈ˜Ê¼VÀˆÌˆV>ÊVœ“«œ˜i˜Ìý The guidance described above has limitations, but these are to be expected when considering the very wide range of container systems.

1.1 Sample Preparation Optimal efficiency of extraction is a primary goal for any extraction process. Several approaches can be employed to optimise extraction techniques, and some techniques require this as part of the equipment design. The most common approach to optimise efficiency is to 5

Update on Undertaking Extractable and Leachable Testing

increase the sample area for a given weight of material. This can be achieved by cutting the material into smaller pieces. A more extreme approach is to mill the material to much smaller-size (millimetre or sub-millimetres) particles. Care must be taken when milling the materials because milling is a highly energetic process that can produce large quantities of heat. The heat generated can subsequently affect the material, such as promoting the loss of the more volatile species, and hence preventing the latter from being detected as extractables. In extreme cases, the material can be deformed or degraded by melting. This can potentially change the extractables chemically or prevent them from being extracted physically. To overcome the loss of the most volatile species or deformation of the material, the approach is to ‘cryomill’ the materials. Cryomilling typically uses liquid nitrogen (or solid carbon dioxide) to dissipate the build-up of heat. It also makes the materials more brittle to ease the milling and prevent heat accumulation. An example of a cryomill system is presented in Figure 1.1.

Figure 1.1 Retsch cryomill. Reproduced with permission from Retsch (UK) Limited

6

Introduction

In practice, little preparation of the sample is needed because sufficient extraction can frequently be achieved from the sample in an ‘as is’ state. The key requirement for an extraction study is to know the weight of material or the surface area to be extracted. This will be discussed further in the section addressing the required limit of detection. Another key part of sample preparation is finding out as much information as possible about the materials being extracted. This is particularly important in the pharmaceutical industry, where direct knowledge of the material composition is needed. Take for example rubber gaskets. Figure 1.2 shows the various stages of material conversion from the base rubber, through the rubber sheet, to the final cut gasket. It is not uncommon for one company to supply the base rubber (‘blob’), which will have one set of additives. Another company will then process the blob into rubber sheets, adding more additives and thus producing a complex material.

Figure 1.2 Base rubber (‘blob’), rubber sheet and cut gaskets

7

Update on Undertaking Extractable and Leachable Testing

The number of species that can be present as an extractable (and therefore possibly as a leachable) can be huge. There are a wide range of materials that can be used in CCS, and these materials are composed of a wide range of additives. An idea of the potential additives used in elastomeric and polymeric materials is given in Table 1.2 next. It aims to give a basic idea of the common types of additives and their roles; a common example is also given: UÊ œ˜œ“iÀÃÊ>ÀiÊ̅iʼLՈ`ˆ˜}ÊLœVŽÃ½ÊœvÊ̅iÊ«œÞ“iÀ°ÊÊ
˜ÊiÝ>“«iÊ could be butadiene. UÊ *>Ã̈ÈViÀÃÉ«ÀœViÃȘ}Ê>ˆ`ÃÊ>ÀiÊÕÃi`Ê̜Ê>ˆ`Ê«ÀœViÃȘ}Ê>˜`ʓœ`ˆvÞÊ the physical properties of the final product. An example could be hydrocarbon oils. UÊ ˆiÀÃÉÀiˆ˜vœÀVˆ˜}Ê>}i˜ÌÃʈ“«ÀœÛiÊ̅iÊÌi˜ÃˆiÊÃÌÀi˜}̅Ê>˜`ʈ“«>VÌÊ strength of the material. An example could be glass fibres. UÊ œÜʓœ`ˆwiÀÃʈ“«ÀœÛiÊ«ÀœViÃÃ>LˆˆÌÞÊLÞʏœÜiÀˆ˜}Ê̅iÊۈÃVœÃˆÌÞ°Ê An example could be fatty acids such as oleic acid. UÊ -ˆ«Ê>}i˜ÌÃʅi«ÊVœ“«œ˜i˜ÌÃʓœÛiÊ«>ÃÌʜ˜iÊ>˜œÌ…iÀ°Ê
˜ÊiÝ>“«iÊ would be erucamide. UÊ *ˆ}“i˜ÌÃÊ}ˆÛiÊ̅iÊVœ“«œ˜i˜ÌÊVœœÕÀÊ>˜`ʜ«>VˆÌÞ°Ê
˜ÊiÝ>“«iÊ would be titanium dioxide. UÊ œÜˆ˜}Ê>}i˜ÌÃÊ«Àœ`ÕViÊ>Êvœ>“ÊÃÌÀÕVÌÕÀiÊ܈̅ˆ˜Ê̅iÊVœ“«œ˜i˜Ì°Ê An example would be pentane. UÊ
˜ÌˆÃÌ>̈VÊ >}i˜ÌÃÊ «ÀiÛi˜ÌÊ Ì…iÊ LՈ`‡Õ«Ê œvÊ ÃÌ>̈VÊ V…>À}iÊ œ˜Ê ̅iÊ component. An example would be glycol esters. UÊ -Ì>LˆˆÃiÀÃÊ«ÀiÛi˜ÌÊLÀi>Ž`œÜ˜ÊœvÊ̅iÊ«œÞ“iÀ°Ê
˜ÊiÝ>“«iÊܜՏ`Ê be butylated hydroxytoluene. UÊ ÕÀ>̈ÛiÃÊ>ÀiÊÕÃi`Ê̜ÊVÕÀiÊÀÕLLiÀðÊ
˜ÊiÝ>“«iÊܜՏ`ÊLiÊ`ˆ‡tbutyl peroxide. The list shown above provides an idea on the diversity of species that could be extracted from a material that could be used in a CCS.

8

Introduction

Table 1.1 Advantages and disadvantages of the most common extraction techniques Equipment

Advantages

Disadvantages

Soxhlet

Standard equipment: low cost. Complete extraction possible

Slow. Typically the ‘gold standard’ extraction. High solvent volumes

Reflux

Standard equipment-low cost

Possible thermal degradation of the material

Sonication

Can be quick, equipment easily available; low cost

Possibly not the most efficient extraction

Microwave

Fast, high efficiency low solvent volumes

Choice of solvents needs to be carefully considered. Possible additional curing of elastomers

Thermal desorption

No solvent interaction

Extraction dependent upon volatility of species

Headspace

No solvent interaction

Extraction dependent upon volatility of species as well as the solid-gas partition coefficient

Supercritical fluid extraction

Novel solvent choice

Potentially difficult and expensive to set up. Equipment lifetime

Shaking

Easily automated, fast, high throughput capacity

Possibly not as efficient with low-volatility species

Pressurised assisted extraction or accelerated solvent extraction

Very fast, very efficient

Labour-intensive to prepare samples. Can be difficult to optimise

Sealed container (e.g., autoclave)

Fairly standard equipment and easy to set-up

Limited with respect to temperature

9

Update on Undertaking Extractable and Leachable Testing

1.2 General Extraction Once the sample preparation (if any) has been completed, the next stage is to carry out a CES. To carry out a CES, it is advisable to use multiple solvents of differing polarities along with multiple extraction techniques. The reason for this is twofold: (i) the guidance recommends it [5] and (ii) multiple solvents allow for the extraction of a complete range of analytes of differing polarities. This is because analytes will be soluble in solvents of similar polarity. In general, the choice of solvents is dependent upon the end-use of the materials. Consider a pressurised metered-dose inhaler (pMDI) (Figure 1.3). The CCS of a MDI comprises a wide range of materials: plastic in the mouthpiece, stem and valve, and elastomers in the valve.

Figure 1.3 Metered-dose inhaler (MDI) (schematic)

10

Introduction

As can be seen from Figure 1.3, the materials are in prolonged contact with an organic solvent: the propellant (e.g., HFA 134a (1,1,1,2-Tetrafluoroethane) or HFA 227 (1,1,1,2,3,3,3Heptafluoropropane), along with co-solvents such as ethanol). The choice of extraction solvents is typically a polar solvent (e.g., isopropanol), a non-polar solvent (e.g., iso-hexane) and something close to the actual vehicle solvent (e.g., dichloromethane). However, if the end-use is an ophthalmic bottle, then more appropriate solvents would be water, pH-adjusted water and an organic solvent (e.g., isopropanol). The choice of solvent should be representative of the end-use of the material, with a range of solvents to cover most eventualities. Ideally, the actual placebo formulation should be used as the extracting solvent. However, this has the potential of making the analysis far more challenging by increasing the complexity of the solution to be analysed (e.g., non-volatile excipients for gas chromatographic analyses). The extraction should be carried out until asymptotic levels are reached. This means that the amount of an extracted species does not increase (by typically >10%) despite extending the extraction time. However, the extraction should not be so vigorous as to deform or degrade the material because this could preclude some extractable species and prevent potential leachable species from being identified. One of the most common mistakes when carrying out an extraction study is when a solvent is chosen that dissolves the material. By dissolving the material a potential new set of species could be pulled out compared to standard extraction and it is unlikely that these dissolved species will be leachables as these dissolved species will have been extracted by a different mechanism. The generally accepted definition of general extraction techniques/ methods is by Dennis Jenke [9]: ‘Extraction techniques/methods used for CES should be vigorous, but not so aggressive as to alter the qualitative and/or quantitative nature of the extractables profile, and therefore preclude an extractables/leachables correlation. Studies must be technically justified and optimised to produce extractables profiles at least equivalent to leachables profiles obtained under worst case

11

Update on Undertaking Extractable and Leachable Testing

conditions of drug product use, allowing both qualitative and quantitative extractables/leachables correlations’. Swelling of the material would be allowed because this is often seen to a small extent in the device. Several approaches to determine asymptotic levels will be discussed later. An understanding of possible chemical reactions between potential extractables and the solvent used is also required. A classic example is the reaction between fatty acids and alcohols to form the alcohol ester. For example, using methanol as the solvent and oleic acid normally present as an extractable, the methyl ester of oleic acid could be formed depending on the extraction technique. If a different solvent is used, the ester would not be present, confirming the reaction between the extractable and the extracting solvent. This demonstrates the advantage of using multiple solvents to carry out the extraction. Another example given in the PQRI guidance is the presence of benzothiazole formed from the decomposition of mercaptobenzothiazole (Figure 1.4).

Figure 1.4 Reaction of mercaptobenzolthiazole (a special case compound) to a standard species, benzothiazole

A wide range of extraction techniques exist, with each having its own advantages and disadvantages. Studies comparing the efficiency of each technique relative to each other are not found in the

12

Introduction

literature. In general, only 2–3 techniques are compared, and these are predominately for environmental samples such as polynuclear aromatic hydrocarbons or as polyaromatic hydrocarbons in soils. If a comparison has been made, this is discussed in the first extraction section. All the extraction techniques carry out the same basic procedure in that they extract species from solid samples (‘lixiviation’) and put it into a liquid or gaseous state. Each extraction technique uses one or more of the following to optimise the extraction: heat, pressure and/or energy (sound, microwaves or mechanical). The techniques will be discussed in depth by highlighting their advantages and disadvantages but a brief summary is detailed in Table 1.3. Several industries have vast experience in the selection and optimisation of extraction techniques. These are where most of the literature in the field of extraction can be found. The food industry necessitated the development of Soxhlet extraction, but the environmental industry has also required the use of efficient extraction techniques. In addition, there are techniques that do not require extraction but instead directly analyse the sample without sample preparation. The classical and mature techniques include infrared or Raman spectroscopy. Due to the wealth of information available on these techniques, they will not be discussed in this contribution. The latest-generation techniques include direct analysis by mass spectrometry (MS) (e.g., direct analysis in real time or direct electrospray ionisation). These, along with more esoteric or specialised extraction techniques, will be discussed in more detail in subsequent sections.

13

Update on Undertaking Extractable and Leachable Testing

Table 1.3 Advantages and disadvantages of the most common extraction techniques Equipment

Advantages

Disadvantages

Soxhlet

Standard equipment: low cost. Complete extraction possible

Slow. Typically the ‘gold standard’ extraction. High solvent volumes

Reflux

Standard equipmentlow cost

Possible thermal degradation of the material

Sonication

Can be quick, equipment easily available; low cost

Possibly not the most efficient extraction

Microwave

Fast, high efficiency low solvent volumes

Choice of solvents needs to be carefully considered. Possible additional curing of elastomers

Thermal desorption

No solvent interaction

Extraction dependent upon volatility of species

Headspace

No solvent interaction

Extraction dependent upon volatility of species as well as the solid-gas partition coefficient

Supercritical fluid extraction

Novel solvent choice

Potentially difficult and expensive to set up. Equipment lifetime

Shaking

Easily automated, fast, high throughput capacity

Possibly not as efficient with low-volatility species

Pressurised assisted extraction or accelerated solvent extraction

Very fast, very efficient

Labour-intensive to prepare samples. Can be difficult to optimise

Sealed container (e.g., autoclave)

Fairly standard equipment and easy to set-up

Limited with respect to temperature

14

Introduction

1.3 How Low to go: Limit of Detection The big question is ‘how low to go for extractables and leachables?’ Are percentage levels, trace levels, parts per million (ppm) or parts per billion (ppb) or lower required? As technology improves, ever-lower limits can be achieved. However, to achieve these low levels, very complex and expensive analytical techniques are required. Is there a safe level of extractables and leachables, one that would provide minimal risk to patients? Anyone who is exposed to a chemical species is at risk of harm. This is clarified in a quote from Paracelsus (1493-1541), the ‘father’ of toxicology: ‘All substances are poisons, there is none which is not a poison. The right dose differentiates a poison from a remedy’. The PQRI has provided definitions to help with the limit of quantification as well as identification thresholds for OINDPs, and are planning to provide limits for PODPs shortly. In addition to the PQRI guidance, the definitions given below produced by the PQRI working group will aid the reader [10, 11]. Safety concern threshold (SCT) is defined as the threshold below which a leachable would have a dose so low as to present negligible safety concerns from carcinogenic and non-carcinogenic toxic effects. Qualification threshold (QT) is defined as the threshold below which a given leachable is not considered for safety qualification (toxicological assessments) unless the leachable presents concerns regarding the structure–activity relationship (SAR). The analytical evaluation threshold (AET) is based on the SCT. It is the threshold at or above which a chemist should begin to identify a particular leachable and/or extractable and report it for potential toxicological assessment. The AET acts as a guide for the minimum sensitivity required for the leachables method(s). As low as reasonably practicable (ALARP) is used in special cases in which the SCT is exceptionally low. The SCT for extractables and leachables has been defined to be 0.15 μg/day. This differs by one order of magnitude to the threshold

15

Update on Undertaking Extractable and Leachable Testing

of toxicological concern of 1.5 μg/day quoted for potential genotoxic impurities (PGIs). A simplified explanation for the range difference is that PGIs are related to the drug substance that offers the patient a clear benefit and could be difficult to remove, whereas the container closure materials could be changed to decrease the level of extractables (and consequently leachables). See the PQRI document [5] for a more detailed explanation of the toxicological explanation for the SCT of 0.15 μg/day. The SCT is used to calculate an estimated AET. The latter is defined as the threshold at or above which an OINDP pharmaceutical development team should identify and quantify a particular extractable and/or leachable and report it for potential toxicological assessment. The calculation from the estimated AET also takes into account differing response factors of different species and gives the final AET. By working through the calculation, typical levels of AET are in the low μg/g (ppm) range. Dry-powder inhaler materials not in continuous contact with the drug product formulation should have an estimated AET of 20 μg/g. A worked calculation of final AET for a MDI is given below. For a MDI that has labelled 60 actuations, the patient takes 4 actuations per day and the amount of rubber (used in the valve) in the device is 0.5 g.

Estimated

AET

per

MDI

 

rubber

where pMDI is the pressurised metered-dose inhaler. The final AET is 2.25 μg/g (based on a 50% response (assuming a database is not available)). The use of a response factor is due

16

Introduction

to the uncertainty of an analytical response of a given analyte to a given standard. Certain analytical techniques are more applicable to database response factors. These include universal detectors such as flame ionisation detectors (FIDs) or thermal conductivity detectors (TCD) because they respond approximately equally across a range of analytes (more true with TCDs than with FIDs). One issue, however, may be that the overall sensitivity of these techniques may be inadequate. Potentially less available but more sensitive techniques can be employed, such as thermal energy analyser (TEA). Techniques such as high-performance liquid chromatography (HPLC) with ultraviolet (UV) and/or mass spectrometry detection can have huge variation in their response factors depending on the chemical nature of the analyte. These two techniques, however, may not respond to the same extent for different compounds for several reasons. For ultraviolet (UV) detection, this may be due to factors from the structure of the compound such as the presence or absence of a UV chromophore, to the extinction coefficient of the analyte, as well as the lamda maximum. For MS, the compound may not ionise, it may ionise only in negative mode compared with a standard in positive mode, and ion suppression/enhancement may be an issue if co-elution with analytes occurs. See the ‘analytical’ section for more details on the individual analytical techniques. How to determine the response factor and the development of a database will be discussed in the ‘analytical’ section.

1.4 Introduction to Extraction Techniques The various extraction techniques will now be discussed. To fully understand some of the limitations of the various techniques, their input requirements to the complete extraction process needs to be understood. Figure 1.5 shows a feedback loop required to develop a suitable extractable or leachable extractable method. As with all trace-level analytical work, the more information gained about the sample and/or the analytes, the easier the analysis will be. Some of the questions that need to be answered include: ‘how

17

Update on Undertaking Extractable and Leachable Testing

specific does the method need to be’; ‘what does the limit of detection need to be’; ‘will the matrix interfere and is any additional sample preparation required?’ Applying the above questions to a specific set of analytes, let us use nitrosamines as an example.

Figure 1.5 Feedback loop required to develop a suitable extractable or leachable analytical method

Nitrosamines are a special case because they are highly toxic and known to be carcinogens. Hence, they typically have ALARP limits of detection. Methods in the literature tend to put these limits of detection at 2-10 ng/g (or 2-10 ppb). This is therefore the driving factor in developing a suitable extractable analytical method to reach these low limits of detection. The analytes are a specific class of compounds, so should a specific detector be used? Generally this is the case because more specific and selective detectors are required to achieve these very high levels of sensitivity. For example, using a limit of 2 ng/g in the rubber used in a pMDI that has 0.5 g rubber and from which the patient takes 4 actuations per day from a 60-action

18

Introduction

product, the patient would be exposed to maximum of 70 pg/day (pg is 10–12 g). Nitrosamine analyses are typically undertaken with one of three detectors used: a TEA, a nitrogen chemiluminescence detector, or MS. The species are volatile, so these detectors are typically coupled to a gas chromatograph. Due to the low limit of detection, specific sample preparation is required. For nitrosamines, this tends to follow very closely the method devised by the American Society for Testing and Materials (ASTM) for the determination of nitrosamines in rubbers (ASTM F1313 - 90(2005) Standard Specification for Volatile N-Nitrosamine Levels in Rubber Nipples on Pacifiers). Further information on nitrosamines can be found in references [12, 13]. Factors that also need to be considered are sample concentration. If this is the case, then special care must be taken when looking for the most volatile species. Common processes for concentrating samples include simple nitrogen blowdown, automated blowdown (e.g., Zymark Turbo Vap™), a Kuderna–Danish concentrator [14], a centrifuge concentrator [15] or even a rotary evaporator. The more volatile the solvent the easier it is to remove. Hence, removing dichloromethane is a lot easier than removing water. Subsequent chapters are split between extraction techniques from solid samples, extraction from liquid samples, and analytical techniques for volatile (gas chromatography (GC)) semi-volatile (HPLC) and metals (inductively coupled plasma).

References 1.

Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Draft Guidance for Industry: Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Drug Products, Silver Spring, MD, USA, 1998.

19

Update on Undertaking Extractable and Leachable Testing

2.

US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), Guidance for Industry: Container Closure Systems for Packaging Human Drugs and Biologics, Silver Spring, MD, USA, 1999.

3.

Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Guidance for Industry: Nasal Spray and Inhalation Solution, Suspension, and Spray Drug Products – Chemistry, Manufacturing, and Controls Documentation, Silver Spring, MD, USA, 2002.

4.

US Food and Drug Administration, United States Food, Drug and Cosmetic Act; Section 501(a)(3), December 2004, Amended March 2005.

5.

Leachables and Extractables Working Group, Safety Thresholds and Best Practices for Extractables and Leachables in Orally Inhaled and Nasal Drug Products, Product Quality Research Institute, Arlington, VA, USA, 2006.

6.

Biological Reactivity Tests, In Vitro, US Pharmacopeia (USP), Chapter 87. http://www.pharmacopeia.cn/v29240/usp29nf24s0_c87.html

7.

Biological Reactivity Tests, In Vivo, US Pharmacopeia (USP), Chapter 88. http://www.pharmacopeia.cn/v29240/usp29nf24s0_c88.html

8.

TC 194, Biological Evaluation of Medical Devices, International Organization for Standardization. http://www.iso.org/iso/search.htm?qt=10993&sort=rel&type =simple&published=on

20

Introduction

9.

D. Jenke, Journal of Pharmaceutical Science and Technology, 2002, 56, 6, 332.

10. D. Ball, J. Blanchard, D. Jacobson-Kram, R. McClellan, T. McGovern, D.L. Norwood, W.M. Vogel, R. Wolff and L.M. Nagao, Toxicological Sciences, 2007, 97, 2, 226. 11. D.L. Norwood, D. Paskiet, M. Ruberto, T. Feinberg, A. Schroeder, G. Poochikian, Q. Wang, T.J. Deng, F. DeGrazio, M.K. Munos and L.M. Nagao, Pharmaceutical Research, 2008, 25, 4, 727. 12. D.L. Norwood, A.T Granger and D.M. Paskiet in, Encyclopedia of Pharmaceutical Technology, 3rd Edition, Dekker Encyclopedias, Taylor and Francis Books, New York, NY, USA, 2006, p.1693. 13. B.G. Willoughby and Scott, Nitrosamines in Rubber, Rapra Technology Limited, Shawbury, Shrewsbury, UK, 1997. 14. V. Ferreira, P. Fernández, J. Meléndez and J. Cacho, Journal of Chromatography A, 1995, 695, 1, 41. 15. Y. Kemmochi, K Tsutsumi, A. Arikawa and H. Nakazawa, Journal of Chromatography A, 2002, 943, 2, 295.

21

2

Extraction Techniques

Each of the extraction techniques has their own advantages, disadvantages and special considerations for their use. These will be discussed in the following sections as well as potential sources of the equipment. As mentioned in Chapter 1, there is no ideal universal technique. The selection is dependent upon several factors but primarily the matrix, analyte, and solvents used are the most important. It is for these reasons that multiple extraction techniques should be used.

2.1 Sonication Sonication is one of the simplest extraction techniques in terms of equipment, solvent selection and sample preparation. A known weight of material is placed in a container with a known volume of solvent. The ratio of sample-to-solvent and the overall amounts of material required are dependent upon: method requirements, analytical limits of detection, and the sample volumes required for testing. For example, standard metal analyses by inductively couple plasma will require far more solvent volume (typically 10-20 ml) compared with gas chromatography (GC) or high-performance liquid chromatography (HPLC) analyses (≤1 ml). These factors are discussed further in the ‘analytical’ section. The sample is placed in the sonic bath and sonicated for the prescribed time (or over a range of times) until asymptotic levels are reached. Potential issues can arise with sonication duration due to the relative efficiencies of sonic baths. One sonic bath can be more efficient than

23

Update on Undertaking Extractable and Leachable Testing

another in terms of energy supplied and temperature increase of the solution. The degree of temperature increase of the sample (which can have a significant influence on the degree of extraction) can vary significantly from machine to machine. The choice of solvent and the analyte being extracted can have a large impact on the efficiency of this technique [1-3]. In general, the more volatile the solvent, the greater is the efficiency of extraction when compared with other extraction techniques such as reflux. Sonication results using a lowboiling-point solvent such as dichloromethane (DCM) (boiling point 40 °C) will more closely match those achieved with an extraction technique such as reflux than if a solvent such as isopropanol (boiling point 82 °C) is used. This could be due to the kinetics involved because the reactions will be occurring at similar temperatures with DCM compared with the wide differential with isopropanol.

2.2 Reflux Reflux is another simple technique in which the sample is placed in a flask containing the solvent with a condenser fitted on top of the flask. With any setup using a condenser, the efficiency of the condenser can be important. Each condenser has a potential cooling capacity and efficiency. Different types of condenser exist, but the ‘Liebig condenser’ is the most basic water-cooled design. The inner-tube is straight, making it cheaper to manufacture (Figure 2.1). A ‘Graham condenser’ is shown in Figure 2.2. It has a spiral coil running the length of the condenser. There are two basic configurations for a Graham condenser. In the first, the spiral contains the coolant, and the condensation takes place on the outside of the spiral. This configuration maximises flow capacity because vapors can flow over and around the spiral. In the second configuration, the jacket tube contains the coolant, and condensation takes place inside the spiral. This configuration maximises collected condensate because all the vapors must flow through the entire length of the spiral, thereby having prolonged contact with the coolant. Additional

24

Extraction Techniques

cooling can be made with an additional cooling jacket on the outside of the condenser to give ultra cooling capacity.

Figure 2.1 Liebig condenser. Reproduced with permission from Ace Glass Incorporated and Sigma-Aldrich

The ‘Allihn condenser’, otherwise known as a ‘bulb condenser’ or simply a ‘reflux condenser’, consists of a long glass tube with a water jacket (Figure 2.3). There is a series of large and small constrictions on the inside tube, each increasing the surface area upon which the vapor constituents may condense. The Allihn condenser is ideally suited for laboratory-scale reflux procedures.

25

Update on Undertaking Extractable and Leachable Testing

Vapor Trail

Water Trail

Figure 2.2 Graham condenser with three possible configurations: (a) cooling with jacket, (b) cooling with spiral and (c) combined jacket and spiral cooling. Reproduced with permission from Ace Glass Incorporated and Sigma-Aldrich

A ‘Friedrich condenser’, also known as a ‘spiraled finger condenser’, consists of a large spiraled internal cold finger-type capillary tube disposed within a wide cylindrical housing (Figure 2.4). Coolant flows through the internal cold finger; accordingly, vapors rising up through the housing may condense on the cold finger as it is cooled. Compared with a standard Graham condenser of similar dimension (which also includes a spiral internal tube), the Friedrich condenser often provides more efficient condensing because the Friedrich

26

Extraction Techniques

condenser provides greater effective surface area for cooling.That is, vapors may be cooled not only by the coolant flowing through the internal cold finger, but also by the interior wall of the cylindrical housing. A Graham condenser with an additional cooling jacket as well as the internal spiral has similar cooling area and capacity.

Figure 2.3 Allihn condenser. Reproduced with permission from Ace Glass Incorporated and Sigma-Aldrich

For reflux extraction, the correct sample-to-solvent ratio should be used to obtain a suitable concentration range, as well as consideration of the amount of solution required for analyses and the required limit of detection. Heating is commenced and the material extracted at the boiling point of the solvent. The boiling point of the solvent is the key controlling factor with this technique, along with the duration

27

Update on Undertaking Extractable and Leachable Testing

of extraction. Extraction should continue until asymptotic levels are reached (unless the material being must be separated from the solution containing the analytes of interest). This may involve pouring the solution away to await a concentration step or directly for analyses. It is this separation step that makes reflux extraction less amenable to matrices comprising fine particles because losses can occur when filtering the solution. Reflux may not be suitable if the material to be extracted is particularly thermally labile because it is exposed to complete heating of the solvent. Other related techniques that also heat the sample in a solvent such as microwave-assisted extraction (MAE) or pressurised solvent extraction (PSE) [4] will be discussed in more detail in their individual sections [5-7].

Figure 2.4 Fredrich condenser. Reproduced with permission from Ace Glass Incorporated and Sigma-Aldrich

28

Extraction Techniques

2.3 Soxhlet Soxhlet extraction is probably the most common technique used for the extraction of materials from solid samples, and has been used for more than a century. It was originally used for the determination of fat in milk [8]. As such, all other extraction techniques are generally compared with Soxhlet extraction. The sample is typically placed in a thimble (usually made from cellulose) and the solvent placed in a flask below the thimble. The solvent is heated and recondenses into the thimble. Once the liquid reaches a defined level, it is siphoned back to the flask, transporting the extracted species back into the bulk liquid. The solvent is then distilled again, thereby delivering pure solvent to the material being extracted. The number of cycles (‘turnovers’) can affect the extraction. Hence, a minimum number is normally expected and this varies from material to material and species to species being extracted. The advantage of Soxhlet is the repeated delivery of fresh pure distilled solvent to the sample, allowing continual shifting of the transfer equilibrium. It is also a very simple system requiring a heater, condenser and inexpensive glassware (Figure 2.5). With its simplicity, minimal training is required. Soxhlet has the advantage over reflux in that fine-particulate matrices can be used because these are contained in the extraction thimble, resulting in only extracted analytes in solution. A standard Soxhlet apparatus can use large solvent volumes, a potential disadvantage to the technique. However, automated Soxhlet systems are available that offer additional functionality, such as warm Soxhlet, hot extraction and continuous extraction. Several solvent-extraction systems based on the Soxhlet device allow fast and safe extraction from materials. For example, FOSS have launched several types of ‘Soxtec Systems’ such as automated or semi-automated analysers which extract lipids rapidly and accurately. These instruments carry out boiling, rinsing and solvent recovery. Similarly, Soxtherm extractors from Gerhardt GmbH have been developed to reduce extraction times. The sample to be analysed is

29

Update on Undertaking Extractable and Leachable Testing

weighed into cellulose thimbles and inserted in the extraction device. All solvents except diethyl ether may be used. About 15 ml of solvent per sample is used with 75% recovery of the solvent after extraction. Extraction is completed in 30–60 min, depending on the application.

Figure 2.5 Soxhlet apparatus. Reproduced with permission from Ace Glass Incorporated and Sigma-Aldrich

30

Extraction Techniques

A device by ViscoALPHA enables the user to have a 2-, 4- or 6-place system in two versions (micro or macro). An electronic unit can control and monitor up to four extraction units individually. Compact and simple systems with 1-6 samples are sold by Behr Labor-Technic GmbH. The Büchi Extraction System B-811 is an automated system which can be used to carry out an extraction according to the original Soxhlet principle. Four different extraction methods are possible without making changes to the unit: Soxhlet standard, Soxhlet warm, hot extraction, and continuous extraction (see below). Basically, the system has an inert-gas supply to avoid oxidation during extraction and to accelerate the evaporation and drying process even with high-boiling-point solvents (up to 150 °C). An example of the Büchi Extraction System B-811 is shown in Figure 2.6, a Soxhlet Standard.

3

2 7 1

detection line

1 2 3 4 5 6 7

Solvent cup Sample tube Condenser Glass valve Optical sensor Lower heater Upper heater Sample

6

Extract

solvent level

4

Sample residue Solvent Solvent vapor

5

Figure 2.6 Büchi extraction system in standard Soxhlet mode. Reproduced with permission from Büchi UK Limited

31

Update on Undertaking Extractable and Leachable Testing

The standard Soxhlet extraction operates under the parameters listed below: UÊ /…iÊ ÃœÛi˜ÌÊ ˆ˜Ê ̅iÊ Li>ŽiÀÊ ˆÃÊ iÛ>«œÀ>Ìi`Ê LÞÊ Ì…iÊ œÜiÀÊ …i>̈˜}Ê element, condensed, and collected in the extraction chamber while the valve (4) is kept closed. The glass sample tube, placed in the extraction chamber, is covered by the condensed solvent. UÊ >V…Ê̈“iÊ̅iÊ܏Ûi˜ÌʏiÛiÊÀi>V…iÃÊ̅iʜ«ÌˆV>ÊÃi˜ÃœÀ]Ê̅iÊ̜Ì>Ê solvent containing the extracted compounds is released into the beaker while the valve remains open. This continues until the extraction chamber is completely empty. This completes one single cycle. The number of cycles and/or time defines the length of the extraction process.

detection line

solvent level

Figure 2.7 Büchi extraction system in Soxhlet warm mode. Reproduced with permission from Büchi UK Limited

32

Extraction Techniques

The Büchi extraction system in Soxhlet warm mode operates under the same basis as the standard method but under enhanced conditions, as listed below: UÊ /…iÊ ÃœÛi˜ÌÊ ˆ˜Ê ̅iÊ Li>ŽiÀÊ ˆÃÊ iÛ>«œÀ>Ìi`Ê LÞÊ Ì…iÊ œÜiÀÊ …i>̈˜}Ê element, condensed, and collected in the extraction chamber while the valve is kept closed. The glass sample tube, placed in the extraction chamber, is covered by the condensed solvent. UÊ "˜ViÊ Ì…iÊ ÃœÛi˜ÌÊ iÛiÊ ˆÃÊ `iÌiVÌi`Ê LÞÊ Ì…iÊ œ«ÌˆV>Ê Ãi˜ÃœÀ]Ê Ì…iÊ upper heating element is activated to heat up the solvent in the extraction chamber. UÊ >V…Ê̈“iÊ̅iÊ܏Ûi˜ÌʏiÛiÊÀi>V…iÃÊ̅iʜ«ÌˆV>ÊÃi˜ÃœÀ]Ê̅iÊ̜Ì>Ê solvent is released into the beaker while the valve remains open until the extraction chamber is empty.

detection line

solvent level

Figure 2.8 Büchi extraction system in hot extraction Soxhlet mode. Reproduced with permission from Büchi UK Limited

33

Update on Undertaking Extractable and Leachable Testing

The Büchi extraction system in hot extraction Soxhlet mode is often referred to as the Randall and Goldfisch extraction. The features of this system are listed below: UÊ /…iÊ ÃœÛi˜ÌÊ ˆ˜Ê ̅iÊ Li>ŽiÀÊ ˆÃÊ iÛ>«œÀ>Ìi`]Ê Vœ˜`i˜Ãi`Ê >ÌÊ Ì…iÊ condenser and collected in the extraction chamber. UÊ /…iʅœÌÊiÝÌÀ>V̈œ˜Ê«ÀœViÃÃʈÃÊV>ÀÀˆi`ʜÕÌʜ˜ViÊ̅iÊ܏Ûi˜ÌʏiÛiÊ is detected by the optical sensor for the first time and the upper heating element is activated. UÊ
ÃÊܜ˜Ê>ÃÊ̅iÊ܏Ûi˜ÌÊÀi>V…iÃÊ̅iʜ«ÌˆV>ÊÃi˜ÃœÀ]Ê̅iÊÛ>Ûiʜ«i˜ÃÊ for a few seconds and the hot solvent is drained. In comparison with the Soxhlet process, the glass sample tube remains in the hot solvent during the entire extraction time. During extraction, the solvent level in the extraction chamber remains at the detection line.

Figure 2.9 Buchi extraction system in continuous-flow Soxhlet mode. Reproduced with permission from Buchi UK Limited 34

Extraction Techniques

The main features of the Büchi extraction system in continuous-flow Soxhlet mode are listed below: UÊ /…iʓ>}˜ïVÊÛ>ÛiʈÃʜ«i˜i`ÊvÀœ“Ê̅iÊLi}ˆ˜˜ˆ˜}ʜvÊ̅iÊiÝÌÀ>V̈œ˜° UÊ /…iÊ ÃœÛi˜ÌÊ ˆ˜Ê ̅iÊ Li>ŽiÀÊ ˆÃÊ iÛ>«œÀ>Ìi`Ê LÞÊ Ì…iÊ œÜiÀÊ …i>̈˜}Ê element and condensed. During the entire process, the sample is rinsed with fresh solvent (i.e., there is ‘continuous flow’). Solvent selection is important with Soxhlet extraction. Take for example an acidified or basified solvent. Only the pure solvent would be distilled to be in contact with the sample. However, the species extracted would be in prolonged contact with the adjusted solvent (probably under exaggerated conditions). These exaggerated conditions would be produced by the distillation of water leaving an increased concentration of the modify agent (acid or base). So a more acidic or alkali solution would be present which could increase the chance of the extracted analytes reacting at this higher of lower pH. Ideal solvent systems to be used as extracting solvents are the solvent used in the device. An extreme example of this would be trying to use the solvent in a pressurised metered-dose inhaler that uses a hydrofluoroalkane (HFA) propellant. The solvent (typically HFA 227 or HFA 134a) is a hydrofluorocarbon with a boiling point of less than –15 °C for HFA 227 or –26 °C for HFA 134a. See Figure 2.10 for the chemical structures of HFA 134a and HFA 227.

Figure 2.10 Chemical structures of HFA 134a and HFA 227

35

Update on Undertaking Extractable and Leachable Testing

The HFA (134a or 227) can be added to a Soxhlet apparatus to run a sub-ambient Soxhlet in which the cooling of the solvent needs to be quite extreme because the solvent needs to be cooled to at least –30 °C (hence the term ‘sub-ambient’). The cold temperature is required to condense the propellant to allow continued extraction. This can be achieved several approaches, the simplest being the use of a dry-ice and acetone condenser (Figure 2.11).

Figure 2.11 Sub-ambient Soxhlet apparatus with a dry-ice/acetone condenser

Several challenges affect the use of sub-ambient Soxhlet apparatus. The dominant factor is the kinetics because the extraction is being carried out at –20 °C. A common rule of thumb is that for every 10 °C change in temperature the rate of reaction doubles. Hence,

36

Extraction Techniques

sub-ambient Soxhlet under the same conditions will be (very approximately) 20-times slower than Soxhlet with DCM as the solvent simply due to the kinetics of the reaction. This would require a 20-day extraction compared with a typical 24-h extraction, and for this duration it is becoming closer to a leachable study. To combat this, previously discussed factors can be employed. Firstly, the surface area of the material can be increased, typically requiring cryomilling. Secondly, by maximising the sample-to-solvent loading, the amount extracted is maximised. In addition, running the extraction for much longer than one working day is extremely challenging. A dry-ice/ acetone condenser needs to be periodically recharged with additional dry ice and acetone. A re-circulating condenser operating at –30 °C requires a specialist solvent to work (such as ethylene glycol or a fluorocarbon).

2.4 Supercritical Fluid Extraction SFE and supercritical fluid chromatography (SFC) are very closely linked and both are starting to make a re-appearance after going out of favour due to lack of equipment vendors. SFE/SFC utilises extreme conditions of temperature and pressure in such a way that the mobile phase remains as a supercritical fluid (Figure 2.12). Supercritical fluids possess unique properties, intermediate between those of gas and liquids. These are dependent upon the pressure, temperature and composition of the fluid. In particular, their viscosity is lower than that of liquids, and the diffusion coefficients are higher, allowing more efficient extractions. In addition, the density (and therefore the solvent power of the fluid) may be adjusted by varying the pressure and the temperature, affording the opportunity of theoretically undertaking highly selective extractions. The basic equipment is very similar to a standard HPLC system, with the following exceptions/additions: UÊ
ÊÜÕÀViʜvÊ "2 (commonly a tank)

37

Update on Undertaking Extractable and Leachable Testing

UÊ /…iÊ >LˆˆÌÞÊ ÌœÊ Ài}i˜iÀ>ÌiÊ Ì…iÊ "2 (i.e., remove the organic modifier before re-circulating or re-depositing in the tank) UÊ
Ê L>VŽÊ «ÀiÃÃÕÀi‡ÀiÃÌÀˆV̜ÀÊ «>Vi`Ê >vÌiÀÊ Ì…iÊ >˜>Þ̈V>Ê VœÕ“˜É extraction cell

Liquid Phase

Supercritical Area

pressure

Pc Critical Point Solid Phase Triple Point Gas Phase

temperature Tc Pressure - Temperature of a pure substance (where the subscript c denotes the critical conditions) Figure 2.12 Phase diagram showing the supercritical area. Reproduced with permission from Chromacademy

An example SFC/SFE apparatus is shown in Figure 2.13.

38

Extraction Techniques

Organic Modifiers

Figure 2.13 Example of SFC/SFE apparatus. Reproduced with permission from Chromacademy

SFE may be carried out in static or dynamic mode. The pressure in the system is maintained by means of a restrictor (fixed or variable, the latter making the pressure independent of the flow rate). At the end of the restrictor, the fluid is depressurised and the extracted analytes trapped in an organic solvent or on a solid phase-filled cartridge (from which the analytes are later eluted with a small volume of organic

39

Update on Undertaking Extractable and Leachable Testing

solvent). Due to the numerous parameters affecting the extraction efficiencies, SFE affords a high degree of selectivity. However, this makes the optimisation quite tedious and difficult in practice. The parameters to consider are linked to the extraction parameters inside the cell, to the nature of the solutes, or to the nature of the matrix. The important parameters in SFE are the pressure and temperature inside the cell. A pressure increase leads to a higher fluid density, thus increasing the solubility. The inverse is observed with the temperature; however, increasing the temperature may enhance the solubility of volatile analytes. In addition, higher temperatures may be required to overcome solute–matrix interactions, as observed for the extraction of polycyclic aromatic hydrocarbons or polynuclear aromatic hydrocarbons (PAH) and polychlorinated dibenzo-p-dioxins from environmental matrices. The polarity of compounds is the most significant factor to be considered when working with SFE. Pure CO2 efficiently extracts non-polar to low-polarity compounds. For polar solutes, a modifier is added to enhance the extraction. For very polar and ionic compounds, the modifier may be a complexing agent, an ion-pair reagent or a derivatisation reagent. As an example, the addition of tetrabutylammonium enables the extraction of anionic surfactants from sewage sludge to be carried out. The addition of the modifier directly to the matrix (before the extraction) may help in disrupting the analyte–matrix interactions; however, it requires that a static extraction be done first to avoid sweeping the modifier out of the cell. In cases where the analytes do not readily derivatise, addition of a derivatisation reagent may still be useful because it can react with the active sites of the matrix, thus enhancing the extraction, as has been observed during the extraction of PAHs from urban dust [9]. The users of SFE must be aware that addition of a modifier to CO2 presents severe drawbacks due to technical factors. Hence, it should be avoided or minimised whenever possible.The presence of the modifier changes the values of the critical pressure and temperature, so that too high a modifier content may result in a temperature lower

40

Extraction Techniques

than the critical value. This results in a subcritical state, with higher viscosity and lower diffusion coefficients than the supercritical state. In this case, the technique is commonly called ‘enhanced-fluidity liquid extraction’. In addition, as the modifier enhances the solvating power of the fluid, it reduces the extraction selectivity as more matrix materials or non-target analytes are co-extracted. Finally, the modifier condenses upon depressurisation, which may result in elution of the retained compounds when a solid trap is used as the collection device because then it may act like a chromatographic device. The nature of the matrix (water content, percentage of organic carbon, humic/fulvic materials) and its physical characteristics (e.g., porosity or particle size) are of prime importance for the success of an extraction [9], as with other extraction techniques. Milling the matrix is recommended to limit the diffusion step inside the matrix and to increase the surface area. This increases the rate of extraction when it is limited by matrix effects. Also, addition of a drying agent (e.g., sodium sulfate) may prevent plugging of the restrictor by ice in the presence of humid matrices. Caution must also be taken when filling the vessel to ensure a homogeneous bed of material (to prevent channelling) and to take into account possible swelling of the matrix (such as polymers) upon introduction of supercritical CO2. In addition, very fine particles may be swept out of the cell by the fluid and result in plugging and mechanical-transfer problems. Finally, a sorbent may be added in the cell to retain matrix material and increase the selectivity of the extraction.

2.5 Sealed Vessel Sealed vessels are another very simple technique. The sample is placed in the solvent with appropriate levels of sample and solvent to achieve appropriate limits of detection for the method. A sealed vessel can be thought of as a simple version of MAE or pressurised solvent extraction. These will be discussed in more detail in later sections. The sample is heated for a set period of time. Typically the sample is heated with reduced pressure so the temperature of the solvent is above its normal boiling point, thereby helping to

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Update on Undertaking Extractable and Leachable Testing

speed up the extraction. Sealed-vessel or autoclave extraction is predominately used with aqueous media, whether with pure water or pH-adjusted water. Typical conditions involve temperatures at 121 °C with various extraction times [10]. The conditions in the United States Pharmacopeia <381> Elastomeric Closures for Injections is as follows: ‘Heat in an autoclave so that a temperature of 121 ±2 °C is reached within 20 to 30 minutes, and maintain this temperature for 30 minutes. Cool to room temperature over a period of about 30 minutes’. The use of organic solvents is much less common, but examples exist with the use of alcohols [11] and these tend to be very specialist applications. If an organic solvent is to be used then the general conditions of heating with the solvent at 10 °C below the boiling point of the solvent are recommended.

2.6 Pressurised Solvent Extraction Pressurised Solvent Extraction (PSE), accelerated solvent extraction (ASE) [trademark of Dionex] or pressurised fluid extraction (PFE) and pressurised liquid extraction were introduced in the mid-1990s as an alternative to other extraction methods such as Soxhlet or sonication for solid samples. With PSE, the sample is placed in a stainless-steel cell through which a solvent is passed, utilising high temperatures and pressures. Two forms of the PSE apparatus exist. One from Dionex is shown in Figure 2.14, with variations in cellextraction size and solvent selection. Another PSE system, the speed extractor, is available from Büchi (see Figure 2.15 for the apparatus) The elevated temperature and pressure typically leads to a significantly higher capacity of the extraction solvent to dissolve the target analytes, improves the rate of mass transport and the effectiveness of sample wetting and matrix penetration. This improves the desorption of analytes from active sites on, and within, the sample particles.

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Extraction Techniques

Figure 2.14 Accelerated solvent extraction apparatus. Reproduced with permission from Dionex UK Limited

ASE may be conducted in two ways: UÊ Dynamic ASE, where the solvent is continuously pumped through the extraction cell. UÊ Static ASE, where the extraction cell is filled with solvent, pressurised for a specified time, and then drained to the collection vial. It is also possible to combine the two or to undertake multiple extraction cycles. Most ASE applications (~75%) have been carried 43

Update on Undertaking Extractable and Leachable Testing

out in the static extraction mode. Such systems can reach temperatures of up to 200 °C and pressures up to 20 MPa, and can accommodate cells of various volumes. ASE has a significant advantage over several extraction techniques in that binary solvent mixtures can be used using one weak solvent to carry out pre-extraction to remove interfering (less strongly sorbed) compounds before the extraction of the compounds of interest [12, 13]. The extract complexity may also be reduced by using a sequential extraction procedure with solvents of increasing solvent strength [14]. Such procedures may also be used to assess the strength of matrix–analyte interactions [15].

Figure 2.15 Speed Extractor E916 from Büchi. Reproduced with permission from Büchi UK Limited

The selectivity of the extraction or leaching process may be further enhanced through the addition of a matrix retainer to the extraction cell. Use of alumina as a fat retainer was suggested in 1996 in a Dionex application note [16]. Since then, many other adsorbents have been used for the same purpose, for example, Florisil, silica gel and diol- and cyanopropyl-silica [17].

44

Extraction Techniques

The Büchi apparatus differs from the ASE apparatus in that it is a parallel system in which up to 6 samples can be analysed simultaneously rather than in series. One has higher throughput, the other higher flexibility. The ASE system can be left running for extended periods with changes to the solvents used, whereas the Büchi system processes up to 6 samples at a time. The Büchi Speed Extractor analysis is shown in Figure 2.16. The solvent mixture is transferred into separate extraction cells by a pump at high pressure (<15 MPa) and heated (<200 °C). The extracts are collected in vials to be concentrated or used directly for analyses.

Figure 2.16 Extraction approach for the Büchi Speed Extractor. Reproduced with permission from Büchi UK Ltd

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Update on Undertaking Extractable and Leachable Testing

2.7 Microwave Assisted Extraction Microwave extraction typically occurs in one of two systems: focused microwave-assisted extraction (FMAE), where the extraction occurs in an open vessel under atmospheric pressure, or pressurised microwave-assisted extraction (PMAE), which is a closed pressurised system. FMAE is less common but has been applied to certain examples as well as specific extractions such as PAHs, [18-21], polychlorinated biphenyls (PCBs), pesticides [22, 23], organometallic compounds [24, 25], and dioxins or furans [26] from spiked, real, and reference-material samples. PMAE is far more common but suffers from several disadvantages in terms of analysis time because it takes time to ensure that the extraction vessels can cope with the extraction pressure and are suitably sealed. It can also take a considerable amount of time for the vessels to cool to room temperature before the pressure is reduced and they can be opened. Examples exist of its use in the extraction of environmental pollutants such as hydrocarbons [27], PAH [28, 29], organochlorine pesticides [30, 31], PCBs [32], dioxins or furans, triazines and alkyl or aryl phosphates; and in soil, sediment and sludge sample matrices. The primary concern with microwave heating is the ability of the solvent to absorb the microwaves. That is, dielectric polarisation of the solvent must be possible. This heating efficiency is dependent upon the dielectric constant (g) of the solvent and, in most cases, is proportional to the polarity of the solvent. Examples of the dielectric constant for common extractable solvents are shown in Table 2.1.

Table 2.1 Dielectic constant of common solvents Solvent

Dielectric constant (g)

Hexane

1.89 at 20 °C

Dichloromethane

8.93 at 25 °C

Methanol

33.0 at 20 °C

Water

80.1 at 20 °C

46

Extraction Techniques

In addition to the ability of the solvent to absorb microwave energy, its ability to convert the energy to heat is important. The efficiency of the conversion of microwave energy to heat is given by the dielectric loss factor or loss tangent. Loss tangents of common solvents are shown in Table 2.2.

Table 2.2 Microwave loss tangents of common extraction solvents Solvent

Loss tangent (2.45 GHz at 20 oC)

Hexane

0.020

DCM

0.042

Methanol

0.659

Water

0.123

Therefore, the overall efficiency of heating using microwave energy is usually expressed by the dissipation factor, which is the ratio of the dielectric loss factor and the dielectric constant of the involved matrix [33]. With respect to the overall efficiency, these data show a large difference between various solvents and, moreover, methanol has been shown to be more favourable than water despite its lower dielectric constant [34]. Standard setups suffer from those in which only one solvent mix can be used at a time because the rate of heating can and does vary quite dramatically. Other possible issues are that the material to be extracted can be altered. It can be termed as ‘accelerations of chemical transformations in a microwave field that cannot be achieved or duplicated by conventional heating’ [35]. A typical example would be the additional curing of rubber with microwaves. Whether this happens in standard extraction conditions has not been proved but will be dependent upon several factors, including temperature, as well as possibly solvent and extraction

47

Update on Undertaking Extractable and Leachable Testing

duration. Another factor to consider with microwave heating is that for some solvents the average temperature of the solvent can be considerably higher than the atmospheric boiling point because the microwave power is dissipated over the whole volume of the solvent, potentially degrading or altering the material. An example of a commercial system is shown in Figure 2.17. It has the safety systems required of a microwave, which are: UÊ

œÊ…>˜`ˆ˜}ʜvÊ«ÀiÃÃÕÀˆÃi`ÊÛiÃÃiÃ

UÊ Closed rotor with protection lid UÊ Stable oven cavity with resealing safety door UÊ Control of pressure increase UÊ Controlled pressure limits for each type of vessel UÊ 7 MPa safety disc (overpressure tolerance)

Figure 2.17 A commercial microwave system. Reproduced with permission from Anton Parr

48

Extraction Techniques

2.8 Shaking Extraction Shaking in its simplest form involves placing a sample in a solvent and applying mechanical energy to the system manually or with a mechanical shaker. In certain circumstances, this may be sufficient but other systems are available that can provide mechanical shaking at elevated temperatures. Two systems exist that can carry out simultaneous extraction and analysis for volatile and semi-volatile species. This simultaneous extraction and analysis can be a tremendous time-saver and hence can also maximise equipment utilisation and minimise analyst involvement. As with all extraction techniques, the principle is reliant on the use of appropriate temperature, time and solvent conditions. For the volatile species, the automated extraction and analysis is as follows. A vial is loaded with an appropriate amount of material (weight) and the vial capped and crimped. The vial is then loaded onto the equipment. The remainder of the process can be fully automated, as shown in the following example. A known volume of solvent is added by the instrument, containing an appropriate internal standard. The vial is automatically transferred to the heating agitator, where it is shaken at elevated temperatures for a set time. See Figure 2.18 for the apparatus. After the set time, a sample is taken automatically and injected directly into the GC with mass spectrometry (MS) detection or flame ionisation detection or both (see a subsequent section for more detailed description of the analysis). The apparatus can also add a range of solvents during the analysis, allowing for prolonged unattended operation. This standard analysis can allow for the usual identification and quantification to take place. The time and the temperature in the agitator can be varied to produce asymptotic extraction levels. The asymptotic levels can be readily plotted in graphs. The equipment shown in Figure 2.18 is an Agilent 7890GC, 5975B MSD and Gerstel MPS 2 dual-rail system supplied and configured by Anatune Limited. The MPS 2 contains a solvent-handling syringe (1 ml), a Solvent Filling Station and Agitator on the Prep Rail,

49

Update on Undertaking Extractable and Leachable Testing

and a 10-μl injection syringe with wash facilities on the other rail. Both rails can access a 10-ml or 20-ml vial capacity of up to 240 vials for long unattended experiments. The system is controlled with Gerstel Maestro software with full integration into an Agilent MSD Chemstation producing a single sequence table. For a more simplified system, a single rail can be used but, in this case, the solvent containing an internal standard would have to be added manually.

Figure 2.18 Shaking apparatus Agilent 7890GC, 5975B MSD and Gerstel MPS 2 dual-rail system. Reproduced with permission from Anatune Limited

Example data generated using the shaking apparatus is shown in Figure 2.19. This was generated using the prescribed extraction duration with the agitator set to 10 °C below the boiling point of the

50

Extraction Techniques

solvent. It shows that DCM is the most effective solvent at extracting compounds, and that asymptotic levels are reached after ~30 min. In this case, the optimum extraction time for DCM is very close to the GC analysis time. This allows maximum analysis to be undertaken in the minimum time if the extraction is carried out while the analysis of the previous samples is carried out.

Figure 2.19 Asymptotic plot showing the levels of a given species with each solvent against time

The shaking system described above could also be connected to a HPLC inlet system. For such a system, careful considerations must be made when deciding whether an extraction solvent could be used with a reverse-phase (aqueous-based) chromatographic setup. For example, hexane is not water-soluble. In addition, if the system were to be used for HPLC sample preparation, then it would take up a considerable amount of laboratory space.

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Update on Undertaking Extractable and Leachable Testing

For the semi-volatile-to-non-volatile species, a similar mechanical extraction system exists: the I-Chem explorer (Figure 2.20). The equipment fits into a standard Agilent 1100 HPLC stack, thereby maximising laboratory space.

Figure 2.20 The I-Chem explorer. Reproduced with permission from Reaction Analytics Incorporated

The sample, with solvent, is heated together with agitation for appropriate times, similar to the previous system. By sampling at prescribed intervals, asymptotic extraction conditions can be determined. Sample agitation is achieved by the use of a magnetic flea. The vial can be heated up to 150 ºC and with stirring up to 1500 rpm. After heating, the sample is automatically analysed by liquid chromatography with suitable detectors, such as ultraviolet and/or MS. This technique does suffer from several drawbacks. As discussed

52

Extraction Techniques

previously, solvents such as DCM and hexane cannot be easily used with this system because they are not miscible with typical HPLC mobile phases. The solvent also has to be added before the analysis/ heating being started, so accurate timings of asymptotic levels may not be feasible. However, as long as these can be produced reproducibly, this should not be an issue.

2.9 Direct Analysis If we define direct analysis as an approach in which the extraction of an analyte from a material and/or a solution cannot be separated from the actual analysis, then there are an ever-increasing number of analytical techniques that fit this description. The classical techniques for direct analysis would be infrared and Raman spectroscopy. However, these techniques, whilst very useful, do not alter the material being tested and as such are not carrying out an extraction. These will not be discussed further save for that they could be used non-destructively to identify and quantify extraction solutions. Another classical example would be thermal desorption/headspace, in which the sample is placed in a vial and heated. As such, they will be discussed in more detail in a subsequent section. Even so, there are a wide range of direct analysis techniques. The most common direct analysis techniques involve ‘direct analysis in real time’ (DART) [36] and desorption electrospray ionisation (DESI) [37]. These techniques may seem to be solventless systems, but there is a degree of ablation/ excitation of the surface using a solvent or an excited gas to allow for the analysis of species on or near the surface of a material. In general, they can be seen as a three-stage process. They wet the surface, the analytes are dissolved into the solvent, and finally the ions are injected from the liquid surface into the gas phase and analysed. A summary of ambient ionisation techniques and their year of introduction is given in Table 2.3. There is a wide and very varied list of techniques, with some showing more relevance to the analysis of materials. Only two of the most commonly used techniques will be discussed in more detail: DESI and DART.

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Update on Undertaking Extractable and Leachable Testing

Table 2.3 Direct analysis techniques with acronym and year of introduction Technique

Reference

Year of Introduction

DESI

DESI

2004

Surface sampling probe

[38]

2004

DART

[39]

2005

Atmospheric solids analysis probe

[40]

2005

Electrospray laser desorption ionisation

[41]

2005

Fused droplet electrospray ionisation

[42]

2005

Direct atmospheric pressure chemical ionisation

[43]

2005

Matrix-assisted laser desorption electrospray ionisation

[44]

2006

Jet desorption electrospray ionisation

[45]

2006

Extractive electrospray ionisation

[46]

2006

Desorption sonic spray ionisation

[47]

2006

Atmospheric pressure thermal desorption ionisation

[48]

2006

Helium atmospheric pressure glow discharge ionisation

[49]

2006

Plasma-assisted desorption ionisation

[50]

2007

Dielectric barrier desorption ionisation

[51]

2007

iÕÌÀ>Ê`iÜÀ«Ìˆœ˜ÊiÝÌÀ>V̈ÛiÊ electrospray ionisation

[52]

2007

Laser diode thermal desorption

[53]

2007

Laser ablation electrospray ionisation

[54]

2007

Desorption atmospheric pressure photo-ionisation

[55]

2007

54

Extraction Techniques

Infra red laser ablation electrospray ionisation

[56]

2008

Flowing atmospheric-pressure afterglow

[57]

2008

Easy ambient sonic spray ionisation

[58]

2008

Remote analyte sampling transport and ionisation relay

[59]

2008

Laser ablation flowing atmosphericpressure afterglow

[60]

2008

Low temperature plasma

[61]

2008

Desorption electrospray metastableinduced ionisation

[62]

2009

Liquid micro-junction surface sampling probe/electrospray ionisation

[63]

2009

Surface activated chemical ionisation

[64]

2009

Single-particle aerosol mass spectrometry

[65]

2009

Reproduced with permission from D.J. Weston, Analyst, 2010, 135, 661. ©2010, The Royal Society of Chemistry [66]

DESI involves the spray of charged micro-droplets from a pneumatically assisted electrospray needle, as per standard electrospray ionisation (ESI) MS. The spray is directed towards the surface of the object, where it impacts the surface, desorbing the analytes into the gas phase where it is ionised and subsequently sampled by the mass spectrometer. Several factors can affect the response and selectivity of analytes, such as capillary tip-to-sample and sample-to-collector distances, as well as angles of incidence. DART relies on the formation of a plasma discharge in a heated helium gas stream to give chemical ionisation at atmospheric pressure. The helium atoms react with water molecules via chemical ionisation processes, and subsequent downstream ionisation of the sample

55

Update on Undertaking Extractable and Leachable Testing

occurs by thermal desorption into the hot gas stream and then into the mass spectrometer. For more details on the ionisation techniques, see ‘Ambient ionisation MS: current understanding of mechanistic theory; analytical performance and application areas’ [66]. In general, DART is used in fit-for-purpose applications because it is more geometrically independent when compared with a technique such as DESI. DART has been used to identify common stabilisers used in polypropylenes [67]. Other surface-analytical techniques such as energy dispersive X-ray or time-of-flight secondary ion MS will not be discussed but they too have their potential niche area of analysis.

2.10 Headspace Headspace is the classical technique that can extract the sample without the need for solvent. As such, only volatile-to-semi-volatile species are extracted. Headspace is a means of introducing the volatile components from a liquid or solid sample into a gas chromatograph for analysis when it is difficult or impossible to inject the actual sample. Also, if very volatile species are present and an extracting solvent would interfere with the analysis, headspace offers an analytical methodology. The basic principle is well documented [68] and is as follows. The solid, liquid or gaseous sample is placed in a vial and sealed. The vial is heated, allowing the volatile components to escape out of the sample to form a gaseous headspace. After a set time, the headspace gas is extracted from the vial and injected into a gas chromatograph for analysis. Headspace theory is based round the tendency of a material to go into the gas phase, producing the partition coefficient, K. K is defined as CL divided by CG (where CL is the concentration of the analyte in the condensed phase and CG is the concentration of the analyte in the gas phase). This is shown schematically in Figure 2.21. Reducing K will increase the sensitivity of the Headspace analysis. Methods to reduce K are listed below: UÊ
``ˆÌˆœ˜Ê œvÊ “ˆ˜iÀ>Ê Ã>ÌÃÊ ÌœÊ Ì…iÊ “>ÌÀˆÝÊ ­Ãœ`ˆÕ“Ê ÃՏv>ÌiÊ ˆÃÊ common)

56

Extraction Techniques

UÊ
``ˆÌˆœ˜ÊœvÊ>˜œÌ…iÀʏˆµÕˆ`Ê̜Ê̅iʓ>ÌÀˆÝ UÊ ˜VÀi>Ș}Ê̅iÊÌi“«iÀ>ÌÕÀi UÊ
}ˆÌ>̈œ˜ÊœvÊ̅iÊÃ>“«i

Figure 2.21 Headspace extraction (schematic)

In general, headspace is a simple, reliable and easily automated technique which allows for the analysis of very volatile species without potential interference from an extracting solvent. It does not, however, provide as-low detection limits as other headspace techniques such as solid-phase microextraction (SPME), in-tube extraction, or dynamic headspace (DHS). The advantages of these advanced headspace techniques will be discussed in subsequent sections.

2.11 Thermal Desorption Thermal desorption techniques are widely used in GC for the measurement of trace impurities in air samples. In this technique, a known volume of sample air is passed through a trap comprising a tube containing a powdered adsorbent such as charcoal. Contaminants in the air are trapped in the adsorbent bed. After

57

Update on Undertaking Extractable and Leachable Testing

collection of the sample, the trap is then inserted into the carrier gas path at the inlet to the chromatograph, and rapidly heated to release the analytes into the column. Figure 2.22 shows the thermal desorption unit along with a selection of desorption tubes. However, for the analysis of solid samples, the material to be analysed is placed in the tube over which a gas is passed while the tube is heated. In thermal desorption, the partition is a dynamic process in which the analyte goes from the gas state and is then moved to the inlet of a gas chromatograph where they are cryofocused (by Peltier cooling, liquid CO2ʜÀʏˆµÕˆ`Ê 2 [in increasing order of cooling efficiency]). Once the analyte is moved away from the solid sample, the partition coefficient favours the transfer of the analyte away from the solid sample into the gaseous state [69]. With Headspace, the process is typically static, where once the equilibrium coefficient is reached no further analyte is extracted.

Figure 2.22 Thermal desorption apparatus along with a selection of desorption tubes. Reproduced with permission from Marks International Limited

58

Extraction Techniques

DHS is a combination of headspace and thermal desorption and will be discussed in the following section.

2.12 Dynamic Headspace Dynamic headspace (DHS) is a combination of headspace and thermal desorption. The principle involves the extraction as per standard headspace but then subsequent concentration of the volatile components. DHS involves the agitation of the sample while the headspace is purged with an inert gas (which is then passed through an absorbent tube to be concentrated). The absorbent tube is then transferred to a thermal desorption unit, where standard thermal desorption takes place (Figure 2.23).

TDU-Liner

Adsorbent

Trapping 20 - 70ºC

Thermal desorption 20 - 350ºC

Gas

Sample Storage 4 - 200ºC

Extraction 10 - 200ºC

selectable dry purge

Sample introduction

Figure 2.23 Gerstel DHS apparatus. Reproduced with permission from GERSTEL GmbH & Co. KG

59

Update on Undertaking Extractable and Leachable Testing

In general, DHS offers lower detection limits than static headspace and solid-phase micro extraction (SPME; see subsequent section).

2.13 Liquid Extraction Surface Analysis The liquid extraction surface analysis (LESA) process brings the extraction solvent from a pipette tip into contact with the surface of >ÊÃ>“«iʅi`ʈ˜Ê̅iÊÃ>“«iÊ«>ÌiʜvÊ>˜Ê
`ۈœ˜Ê/Àˆ6iÀÃ>Ê >˜œ>Ìi°Ê Analyte is extracted from the surface [70]. The solvent is then retracted back into the pipette tip and sprayed through the ESI chip in the normal manner (Figure 2.24).

2.14 Extraction to Asymptotic Levels There are two basic approaches to determine asymptotic levels. The most common is to test the sample by the analytical methods to be used at various time-points and record the result until a constant response level is achieved. Care must be taken to maintain the sampleto-solvent ratio. This requires the addition of extra solvent replacing the solvent that had been removed or multiple extraction setups. The extraction must take care not to deform or degrade the material [71]. Another less common approach is to use an orthogonal technique to check the total level of extractables. Examples include total organic carbon (TOC) or ion mobility. TOC is applicable only if aqueous extraction solvents have been used. Ion mobility is dependent upon the ability of an analyte to be ionised [72].

60

Extraction Techniques

(a)

Sampling tip

ESI Chip

Mass spectrometer Well plate, side view front plate Sample on surface of tissue slice/MALDI plate, and so on.

Solvent

(b)

Solvent drawn from reservoir into pipette tip (c)

Solvent eluted from tip and held on sample surface (d)

Analytes extracted directly from surface into solvant (e)

Sample analysed by chip-based nanoelectrospray mass spectrometry

Figure 2.24 The LESA process (schematic). Reproduced with permission from Advion BioSystems, Incorporated

61

Update on Undertaking Extractable and Leachable Testing

References 1.

°Ê->ˆ“]Ê°,°Ê i>˜]Ê*°Ê
L`Տ>…Ê>˜`Ê<°Ê<>Ž>Àˆ>]ÊJournal of Chromatography A, 1997, 791, 361.

2.

°,°Ê >˜œœÊ>˜`Ê*°°Ê iÃœ˜]ÊJournal of Chromatography A, 2005, 1066, 9.

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T.F. Guerin, Journal of Environmental Monitoring, 1999, 1, 1, 63.

4.

ii“Ži˜]Ê °Ê/…iœL>`Ê>˜`Ê °7°Ê7i˜V>܈>Ž]ÊAnalytical Chemistry, 1997, 69, 2171.

5.

K. Ganzler, A. Salgo and K. Valko, Journal of Chromatography, 1986, 371, 299.

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K. Ganzler and I. Szinai, Journal of Chromatography, 1990, 520, 257.

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F. Soxhlet, Die Gewichtsanalytische Bestimmung des Milchfettes, Polytechnisches J. (Dingler‘s), 1879, 232, 461.

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B.A. Benner, Jr., Analytical Chemistry, 1998, 70, 21, 4594.

10. D.R. Jenke, E.K. Chess and G. Jakubowski International Journal of Pharmaceutics, 1994, 108, 1, 11. C.B. Muchmore, J.W. Chen, A.C. Kent and K.E. Tempelmeyer, American Chemical Society, Division, Fuel Chemistry, Preprints, 1985, 30, 2, 24. 12. J. McKiernan, Journal of Analytical Atomic Spectrometry, 1999, 14, 4, 607. 13. M. Papagiannopoulos and A. Mellenthin, Journal of Chromatography A, 2002, 976, 1/2, 345.

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Extraction Techniques

14. M. Bergknut, Environmental Toxicological: Chemistry, 2004, 23, 8, 1861. 15. H. Schroder, Journal of Chromatography A, 2003, 1020, 1, 131. 16. Dionex Application Note ASE 322, Dionex Corp., Sunnyvale, California, USA, 1996. 17. C. Huie, Analytical Bioanalytical Chemistry, 2002, 373, 1/2, 23. 18. Y.Y. Shu and T.L. Lai, Journal of Chromatography A, 2001, 927, 131. 19. S. Dupeyron, P.M. Dudermel and D. Couturier, Analysis, 1997, 25, 286. 20. M. Letellier, H. Budzinski, P. Garrigues and S. Wise, Spectroscopy, 1996/1997, 13, 71. 21. L.E. Garcia-Ayuso, J.L. Luque-Garcia and M.D.L. de Castro, Analytical Chemistry, 2000, 72, 3627. 22. A. <Տœ>}>]Ê °Ê ÌÝiL>ÀÀˆ>]Ê°
°ÊiÀ˜>˜`iÃÊ>˜`Ê°°Ê Madariaga, Journal of High Resolution Chromatography, 2000, 23, 681 23. °°Ê >œ]Ê<°Ê7>˜}]Ê°Ê1ÀÀÕÌÞ]Ê°°Ê*œ““ˆiÀÊ>˜`Ê°Êœ˜ÌÕÀÞ]Ê Journal of Agricultural and Food Chemistry, 2001, 49, 5092 24. J. Szpunar, V.O. Schmitt, R. Lobinski and J.L. Monod, Journal Analytical Atomic Spectrometry, 1996, 11, 193. 25. I.R. Pereiro, V.O. Schmitt, J. Szpunar, O.F.X. Donard and R. Lobinski, Analytical Chemistry, 1996, 68, 4135. 26. E. Eljarrat, J. Caixach and J. Rivera, Chemosphere, 1998, 36, 2359.

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27. A. Pastor, E. Vazquez, R. Ciscar and M. de la Guardia, Analytica Chimica Acta, 1997, 344, 241. 28. R.C. Lao, Y.Y. Shu, J. Holmes and C. Chiu, Microchemical Journal, 1996, 53, 99. 29. V. Lopez-Avila, R. Young and W.F. Beckert, Analytical Chemistry, 1994, 66, 1097. 30. K. Li, J.M.R. Belanger, M.P. Llompart, R.D. Turpin, R. Singhvi and J.R.J. Pare, Spectroscopy, 1996/1997, 13, 1. 31. Silgoner, R. Krska, E. Lombas, O. Gans, E. Rosenberg and M. Grasserbauer, Journal of Analytical Chemistry, 1998, 362, 120. 32. G. Dupont, C. Delteil, V. Camel and A. Bermond, Analyst, 1999, 124, 453. 33.
°Ê<œÌœÀâޘΈ, Critical Reviews in Analytical Chemistry, 1995, 25, 43. 34. A. Molins, E.A. Hogendoorn, H.A.G. Heusinkveld, D.C. van >ÀÌi˜]Ê,ÊÛ>˜Ê<œœ˜i˜Ê>˜`Ê,°
°Ê >Փ>˜˜ÊChromatographia, 1996, 43, 527. 35. C.O. Kappe, D. Dallinger and S.S. Murphree in Practical Microwave Synthesis for Organic Chemists, John Wiley & -œ˜Ã]ÊœLœŽi˜]Ê ]Ê1-
] 2008. 36. R.B. Cody, J.A. Laramee and H.D. Durst, Analytical Chemistry, 2005, 77, 2297. 37. <°Ê/>Ž>ÌÃ]Ê°°Ê7ˆÃi“>˜]Ê °Êœœ}>˜Ê>˜`Ê,°°Ê œœŽÃ]Ê Science, 2004, 306, 471. 38. M.J. Ford and G.J. Van Berkel, Rapid Communication in Mass Spectrometry, 2004, 18, 1303.

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39. R.B. Cody, J.A. Laramee and H.D. Durst, Analytical Chemistry, 2005, 77, 2297. 40. ° °ÊV Üi˜]Ê,°°ÊV>ÞÊ>˜`Ê °-°Ê>ÀÃi˜]ÊAnalytical Chemistry, 2005, 77, 7826. 41. °<°ÊÕ>˜}]Ê°°ÊÃÕ]Ê °°Ê7Õ]Ê-°9°Êˆ˜]Ê9°°Ê>]Ê/°°Ê Cheng and J. Shiea, Rapid Communication in Mass Spectrometry, 2007, 21, 1767. 42. I.F. Shieh, C.Y. Lee and J. Shiea, Journal of Proteomic Research, 2005, 4, 606. 43. <°Ê/>Ž>ÌÃ]Ê°Ê œÌÌi‡,œ`Àˆ}Õiâ]Ê °Ê/>>ÌÞ]Ê°Ê …i˜Ê>˜`Ê,°°Ê Cooks, Chemical Communications, 2005, 15, 1950. 44. J.S. Sampson, A.M. Hawkridge and D.C. Muddiman, Journal of the American Society for Mass Spectrometry, 2006, 17, 1712. 45. <°Ê/>Ž>ÌÃ]Ê °Ê âÕVâÞ]Ê°Ê>̜˜>Ê>˜`Ê,°Ê-ŽœÕ“>Êˆ˜Ê the Proceedings of the 54th ASMS Conference on Mass Spectrometry and Allied Topics, Seattle, WA, USA, 2006. 46. H. Chen, A. Venter and R.G. Cooks, Chemical Communications, 2006, 19, 2042. 47. ,°Ê>``>`]Ê,°Ê-«>ÀÀ>«>˜Ê>˜`Ê° °Ê LiÀˆ˜]ÊRapid Communications in Mass Spectrometry, 2006, 20, 2901. 48. °Ê …i˜]Ê<°Ê"ÕÞ>˜}Ê>˜`Ê,°°Ê œœŽÃ]ÊAngewandte Chemie International Edition, 2006, 45, 3656. 49. W.C. Wetzel, F.J. Andrade, J.A.C. Broekaert and G.M. Hieftje, Journal of Analytical Atomic Spectrometry, 2006, 21, 750. 50. L.V. Ratcliffe, F.J. Rutten, D.A. Barrett, T. Whitmore, D. Seymour, C. Greenwood, Y. Aranda-Gonzalvo, S. Robinson and M. McCoustra, Analytical Chemistry, 2007, 79, 6094. 65

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51.

°Ê >]Ê°Ê<…>œ]Ê-°Ê<…>˜}]Ê °Ê9>˜}Ê>˜`Ê8°Ê<…>˜}]ÊJournal of the American Society for Mass Spectrometry, 2007, 18, 1859.

52. °Ê …i˜]Ê
°Ê7œÀ̓>˜˜Ê>˜`Ê,°Ê˜`Ê°*°Ê >Ìi“>˜]ÊAnalytical Chemistry, 2007, 79, 4657. 54. *°Ê i“iÃÊ>˜`Ê
°Ê6iÀÌiÃ]ÊAnalytical Chemistry, 2007, 79, 8098. 55. M. Haapala, J. Pol, V. Saarela, V. Arvola, T. Kotiaho, R.A. Ketola, S. Franssila, T.J. Kauppila and R. Kostiainen, Analytical Chemistry, 2007, 79, 7867. 56. Y.H. Rezenom, J. Dong and K.K. Murray, Analyst, 2008, 133, 226. 57. F. Andrade, J. Shelley, W. Wetzel, M. Webb, G. Gamez, S. Ray and G. Hieftje, Analytical Chemistry, 2008, 80, 2654. 58. ,°Ê>``>`]Ê,°Ê-«>ÀÀ>«>˜]Ê/°ÊœÌˆ>…œÊ>˜`Ê° °Ê LiÀˆ˜]Ê Analytical Chemistry, 2008, 80, 898. 59. R.B. Dixon, J.S. Sampson, A.M. Hawkridge and D.C. Muddiman, Analytical Chemistry, 2008, 80, 5266. 60. J.T. Shelley, S.J. Ray and G.M. Hieftje, Analytical Chemistry, 2008, 80, 8308. 61. ° °Ê>À«iÀ]Ê °
°Ê …>Àˆ«>À]Ê ° °ÊՏˆ}>˜]Ê8°Ê<…>˜}]Ê,°°Ê

œœŽÃÊ>˜`Ê<°Ê"ÕÞ>˜}]ÊAnalytical Chemistry, 2008, 80, 9097. 62. °Ê Þ>`œ˜}]Ê
°-°Ê>…i˜>Ê>˜`Ê°°ÊiÀ˜>˜`iâ]ÊAnalytical Chemistry, 2009, 81, 7788.

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63. G.J. Van Berkel, V. Kertesz and R.C. King, Analytical Chemistry, 2009, 81, 7096. 64. S. Crotti and P. Traldi, Combinatorial Chemistry & High Throughput Screening, 2009, 12, 125. 65.
° °Ê>À̈˜]Ê°,°Ê>ÀµÕ>À]Ê*°/°Ê-Ìiii]Ê
° °Êœ˜iÃÊ>˜`Ê°Ê Frank, Analytical Chemistry, 2009, 81, 9336. 66. D.J. Weston, Analyst, 2010, 135, 661. 67. M. Haunschmidt, Analyst, 2010, 135, 80. 68. B. Kolb and L.S. Ettre, Static Headspace Gas Chromatography]ÊӘ`Ê `ˆÌˆœ˜]Ê7ˆiއ6 ]ʘV°]Ê iÜÊ9œÀŽ]Ê 9]Ê1-
]Ê£™™Ç° 69. °Ê<ÜiˆLi˜Ê>˜`Ê
°°Ê-…>Ü]ÊPDA Journal of Pharmaceutical Science and Technology, 2009, 63, 353. 70. V. Kertesz and G.J. Van Berkel, Journal of Mass Spectrometry, 2010, 45, 3, 252. 71. D. Jenke, Journal of Pharmaceutical Science and Technology, 2002, 56, 6, 332. 72. 9°
°Ê<œœÌœÛÊJournal of Analytical Chemistry, 2006, 61, 6, 519.

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3

Liquid Extraction Techniques

Once an extraction solution has been produced there may be a need to concentrate the sample, make the sample more amenable to certain analytical techniques, or to separate the analytes of interest from interfering species. These situations can arise when extractable testing has taken place as well as the need for the extraction of leachables. For leachables, sample preparation can be a key step in their identification and quantification because it is, in some cases, the slowest and most complicated step overall. Sample preparation is necessary to extract, isolate, and concentrate the analytes of interest from complex matrices into a suitable solvent because most analytical instruments cannot handle the sample matrix directly. Therefore, sample preparation has a direct impact on accuracy, precision, and quantification limits, and is often the rate-determining step of the analytical process (especially when trace determination has been the purpose). The examples in Table 3.1 are not a complete list of possible techniques but a selection to give an idea of what is available to extract and concentrate analytes from liquids.

3.1 Liquid-liquid Extraction Liquid-liquid extraction (LLE) is the simplest form of the extraction and purification of analytes from liquid samples. The basic principle is the tendency of an analyte to prefer one solvent over another immiscible solvent. The often-quoted partitioning coefficient of a species is Log P. This is defined as the ratio of the concentration in octanol divided by the concentration in water. The Log-P values of a vast number of species have been calculated, and there are computer

69

Update on Undertaking Extractable and Leachable Testing

programs that will calculate Log P and Log D simply from the chemical structure of the species. The use of high-purity solvents can make this technique expensive, especially if large volumes are required and then there is the disposal of these solvents. Carrying out LLE requires the use of two immiscible liquids and soluble samples. It is very useful for separating analytes from interferences by partitioning the sample between these two immiscible liquids or phases. Typically, with LLE one phase will be aqueous (often the denser or heavier phase) and the second phase is an organic solvent (usually the lighter phase). Hydrophilic compounds prefer the polar aqueous phase, whereas hydrophobic compounds will prefer the organic solvent. Analytes extracted into the organic phase are recovered relatively easily by evaporation of the solvent, whereas analytes extracted into the aqueous phase are often amenable to direct analysis by highperformance liquid chromatography (HPLC). Extraction efficiency can be improved by solvent selection, solvent volume, pH, and by using the salting-out effect.

Table 3.1 Liquid purification techniques Technique

Acronym

Liquid-liquid extraction

LLE

Solid-phase extraction

SPE

Solid-phase microextraction

SPME

Stir-bar sorptive extraction

SBME

Dispersive liquid–liquid microextraction

DLLME

Single-drop microextraction

SDME

Liquid-phase microextraction solidification of floating organic drop

LPMESFOD

In-tube extraction

ITEX

Micro-extraction by packed sorbent

MEPS

Hollow-fibre liquid-phase microextraction

HFLPME

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Liquid Extraction Techniques

The salting-out effect involves the addition of an inorganic salt and has often been used to enhance the activity coefficients of volatile components in aqueous solutions, thereby increasing the concentration in the organic layer. The salting-out effect is dependent upon the analyte and type of salt applied. The higher is the number of carbon atoms in a compound the greater is the salting-out effect. This results from the lower solubility of the high-molecular-weight compounds in water. However, with improvements in analytical techniques (especially in detector sensitivity and specificity), even classical LLE is coming back in favour now that smaller samples can be used. This implies that extraction solvent volume can be dramatically reduced, sometimes to only a few millilitres. Some of these micro liquid-liquid extraction techniques are discussed in subsequent sections.

3.2 Solid-Phase Extraction Solid-phase extraction solves several problems associated with LLE, such as incomplete phase separations, poor quantitative recoveries and disposal of large quantities of organic solvents. SPE is generally more efficient than LLE, giving quantitative extractions that are easy to carry out, rapid, and which allow automation. Solvent use and laboratory time are reduced. SPE is used most often to prepare liquid samples and extract semi-volatile or non-volatile analytes, but it can also be used with solids that are pre-extracted into solvents. SPE products are excellent for sample extraction, concentration, and clean-up. It is used in four very wide classes of applications: UÊ ˆœœ}ˆV>ÊÃ>“«iÃÊ>˜`ʘ>ÌÕÀ>ÊVœ“«œÕ˜`ÃÊ UÊ *…>À“>ViṎV>ÃÊ>˜`Ê`ÀÕ}ÃÊ UÊ œœ`Ê>˜`ÊLiÛiÀ>}iÃÊ UÊ ˜ÛˆÀœ˜“i˜Ì>ÊÃ>“«iÃÊ>˜`Ê«œÕÌ>˜ÌÃ

71

Update on Undertaking Extractable and Leachable Testing

There is a wealth of information available on this technique, ranging from books to application papers from SPE suppliers. As such, only a very basic introduction to SPE will be presented here. A literature search on Scifinder TM produced 17574 references for SPE (with 1359 references for the year 2009 alone). If there is a need for SPE then a literature search on a specific application or class of application is strongly recommended. There are two very basic approaches to SPE: 1) The analyte of interest can be retained and the matrix interferences are washed through. 2) The analyte of interest is washed through and the matrix interferences are retained. SPE cartridges are available in a wide variety of chemistries, adsorbents, and sizes. Selecting the most suitable product for each application and sample can be very important. As a rough guide, the phases are categorised by the primary interaction mechanism with the analyte of interest: UÊ Reversed phase (extraction of hydrophobic analytes from aqueous matrix) UÊ Normal phase (extraction of polar analytes from non-polar organic solvents) UÊ Ion exchange (extraction of charged analytes from aqueous or non-polar organic samples) UÊ *…>ÃiÃÊ܈̅ÊVœ“Lˆ˜i`ʓՏ̈«iʈ˜ÌiÀ>V̈œ˜Ê“iV…>˜ˆÃ“ÃÊ>ÀiÊV>i`Ê mixed-mode phases.

3.3 Solid Phase Microextraction (SPME) SPME was the first successful modern microextraction technique [1, 2]. It is now commonly used for the analysis of trace components

72

Liquid Extraction Techniques

in a wide variety of matrices. The SPME technique uses a small polymer-coated fibre that is placed in solution or in the headspace of a sample for a period of time. Analytes are diffused to the surface of the polymer coating until equilibrium is achieved. The types of compounds which can be analysed by this technique range from non-polar to semi-polar materials due to the different fibre materials available. Most applications favour headspace over immersion sampling, therefore the analytes must be sufficiently volatile to partition into the headspace of the vial being sampled. This extraction technique has become a standard technique, with fully automated systems available. Other techniques are available that carry out a similar function to SPME, and some of these other techniques will be discussed in subsequent sections. In a similar way to SPE, there are two basic types of sorption process for the fibres: adsorbent-type fibres or absorbent-type fibres. A summary of the mode of sorption of fibres are given in Table 3.2, and a selection of types of fibres given in Table 3.3. Once withdrawn from the sample source, in the case of gas chromatography (GC), the fibre is placed into the hot injection port and sorbed analytes are thermally desorbed into the GC column to be separated and detected. This is the most common approach. Alternatively, HPLC can be employed. In this case, the analytes need to be solvent-extracted (or stripped) from the polymer coating, which can prove to be a much slower process than thermal desorption. A variation of the SPME technique, the stir-bar sorptive extraction technique (SBSE) is discussed in a later section. One of the main drawbacks of SPME is attaining accuracy and reproducibility. The following factors can affect recovery: vial size and selection, headspace versus direct sampling, salt addition, pH adjustment [3], stirring or agitation, fibre selection, extraction time and desorption conditions [4].

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Update on Undertaking Extractable and Leachable Testing

Table 3.2 Summary of the mode of sorption of SPME fibres Adsorbent-type fibres

Absorbent-type fibres

Physically traps or chemically reacts bonds with analytes: Porous material High surface area Chemical derivatising agent

Analytes are extracted by partitioning the liquid phase: Retained by thickness of coating

Analytes may compete for sites

Analytes do not compete for sites

Fibres have limited capacity for extracted analtyes

Fibres can have high capacity

Table 3.3 Types of SPME fibres Material

Type

Polarity

7-μm PDMS

Absorbent

Nonpolar

100-μm PDMS

Absorbent

Nonpolar

30-μm PDMS

Absorbent

Nonpolar

85-μm polyacrylate

Absorbent

Polar

65-μm PDMS-divinylbenzene (DVB), StableFlex™

Adsorbent

Bipolar

65-μm CW-DVB, StableFlex

Adsorbent

Polar

85-μm carboxen-PDMS, StableFlex

Adsorbent

Bipolar

55-μm/30-μm DVB/Carboxen™-PDMS, StableFlex

Adsorbent

Bipolar

However, automation of SPME fibre addition to the sample and other steps can help with sample throughput, precision and accuracy. Figure 3.1 shows an automated SPME system. Past failings of this technique has been the robustness of the fibre, but automation can circumvent this problem. 74

Liquid Extraction Techniques

Figure 3.1 Automated GC SPME analysis with expansion of the SPME injection. Reproduced with permission from GERSTEL GmbH & Co. KG

3.4 Single-drop Microextraction Single-drop microextraction (SDME) [5, 6], is a reduced-volume version of LLE. It uses a single drop of an immiscible organic solvent (typically 1-10 μl) suspended from the end of a microsyringe needle in an aqueous solution (donor phase). With SDME, once equilibrium has been achieved, the drop is retracted into the syringe barrel and injected directly into the analytical instrument. Standard LLE parameters will affect the extraction efficiency.

3.5 Dispersive Liquid–liquid Microextraction Dispersive liquid-liquid microextraction (DLLME) [7, 8] involves the use of a three-component solvent system. The analytes are typically in the aqueous solvent. To this is rapidly injected a mixture of a very small volume of extracting solvent and a disperser solvent. The 75

Update on Undertaking Extractable and Leachable Testing

extracting solvent is at a higher density than water and the disperser solvent is miscible with the extracting solvent and the aqueous sample solvent. This forms a cloudy solution because of the formation of fine droplets of extraction solvent, which has been dispersed among the sample solution. The analytes in the aqueous sample solution are extracted into the organic solvent very quickly due to dispersion of fine droplets in sample solution. Once injected, a cloudy mixture is formed due to the tiny droplets of immiscible extracting solvent in the aqueous environment. Due to the high surface area of these extracting solvent droplets, analyte extraction is quite efficient and extremely rapid. The final step involves centrifugation with the sedimented layer collected to await further analysis.

3.6 Liquid-phase Microextraction Solidification of Floating Organic Drop Liquid-phase microextraction solidification of floating organic drop is a technique similar to DLLME in that a small volume of immiscible organic solvent with a melting point near room temperature (e.g., undecanol) is added to an aqueous sample containing the analyte of interest [9]. The phases are stirred using a magnetic flea for a period of time and the mixture transferred to an ice bath. This causes the low-melting-point solvent to solidify, enabling it to be removed from the solution. Once removed from the vial, the solvent can melt, releasing the analyte for analysis.

3.7 Hollow-Fibre Liquid-Phase Microextraction Hollow-fibre liquid-phase microextraction (HFLPME) is closely related to SDME but, by using the hollow fibre, the drop can be protected. Analytes of interest are extracted from aqueous samples through the thin layer of organic solvent, which is immobilised within the pores of a porous hollow fibre into the acceptor solution inside the lumen of the hollow fibre. The fibres are typically disposable, this aids in the elimination of sample carryover. The pores in the walls of

76

Liquid Extraction Techniques

the hollow fibre cause it to display some selectivity by preventing the extraction of macromolecules such as proteins, as well as particles from the sample matrix. Consequently, HFLPME can provide sample cleanup and pre-concentration. A typical hollow fibre is made from hydrophobic polypropylene with the following dimensions: 600-μm id, a 200-μm wall thickness and a 0.2 -μm pore size. It has a capacity of just 25 μL solvent.

3.8 In-Tube Extraction The principle of in-tube extraction (ITEX) is very similar to that of SPME. The sample is heated and/or agitated in a sealed sample vial until equilibrium is achieved. The analysis is carried out with the steps shown in Figure 3.2. The ITEX Trap needle pierces the sealed sample vial. A heated gas-tight syringe pumps headspace gas through ITEX Trap. The loaded ITEX Trap flash is heated up to 350 °C and desorbed into a hot GC Injector. Finally, after thermal desorption, the hot ITEX Trap is cleaned with inert flush gas [10]. A range of traps are available to aid the extraction. The types of traps and their use is presented in Table 3.4.

Table 3.4 Range of traps used in in-tube extraction Material

Use

Tenax TA

Volatiles and semi-volatiles

Carbotrap/carbopack

Hydrophobic character minimises analyte displacement by water

Carbosieve/carboxen

For very volatile analytes (e.g., vinyl chloride)

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Update on Undertaking Extractable and Leachable Testing

1

2

Conditioning Adsorption

3

Desorption

4

Trap cleaning

Figure 3.2 ITEX extraction process (schematic). Reproduced with permission of CTC Analytics AG

3.9 Microextraction by Packed Sorbent Microextraction by packed sorbent (MEPS) is related to SPE. In MEPS, ~1–2 mg of solid packing material is inserted into the barrel of a syringe as a plug with polyethylene filters on both sides, or between the syringe barrel and the injection needle as a cartridge. Possible packing materials include C18 end-capped; C8; C2; C8/SCX; C8/SAX; and silica. The dimensions of the packing material are scaled from a conventional SPE bed and, in this way, MEPS can be adapted to most existing SPE methods by scaling the reagents and sample volumes from the conventional device to the MEPS. The basic approach is as follows (Figure 3.3):

78

Liquid Extraction Techniques

UÊ ->“«iʫՓ«i`Ê̅ÀœÕ}…ÊÃÞÀˆ˜}iÊ­œ˜ViʜÀʓՏ̈«iÊ̈“iî UÊ 7>ÅÊ܈̅ÊÜ>ÌiÀÊ̜ÊÀi“œÛiÊ՘Ü>˜Ìi`ʓ>ÌÀˆÝÊ­£{‡Óxäʖ® UÊ ÕÌiÊ܈̅Ê܏Ûi˜ÌÊ`ˆÀiV̏Þʈ˜ÌœÊ̅iʈ˜iV̜ÀʭLJÓxäʖ®ÊQ11, 12] Common examples of its use are in the extraction of drugs and metabolites from biological samples [13].

PHASE 1

PHASE 2

PHASE 3

PHASE 4

Sampling (1-n times)

Washing

Elution solvent

Injecting in test equipment

Figure 3.3 MEPS analyses (schematic). Reproduced with permission from SGE Analytical Science

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Update on Undertaking Extractable and Leachable Testing

3.10 Stir Bar Sorptive Extraction Stir bar sorptive extraction (SBSE) has the same basic principle as LLE and SPME. SBSE involves a magnetic flea coated in glass which is then in turn coated with polydimethyl siloxane (PDMS). The apolar analytes are sorbed by PDMS. The bar is placed in a vial and left to stir for a suitable time (typically 30–240 min) (Figure 3.5).Controlled kinetically, the extraction time is determined by the sample volume, the stirring speed and the stir-bar dimensions. Each needs to be optimised for each application. Revisions to the application of SBSE in environmental situations have been carried out by Kawaguchi and co-workers [14] and for biomedical analysis by David and Sandra [15]. The available surface area of the coated stir bar is substantially higher than the surface area of an SPME-coated fibre (50–250-times greater). Hence, larger amounts of analyte can be extracted and the overall sensitivity is improved. A special thermal desorption unit is used to transfer sorbed analytes into the GC. Once extraction has been completed, the bar is removed and wiped to remove water droplets. It is then placed in the thermal desorption unit for further analysis. Depending upon the octanol–water partition coefficients of the analytes, sample volume, stir-bar size, and analytical system being used (usually GC–mass spectrometry), detection limits of 1 ng/l have been obtained.

Figure 3.4 Twister bar. A magnetic flea coated in glass with a PDMS coating

80

Liquid Extraction Techniques

Figure 3.5 Vial with the twister bar stirring. Reproduced with permission from GERSTEL GmbH & Co. KG

It has been suggested that SBSE has major advantages over liquid and SPME [16]. These are listed below: UÊ

œÊ`ˆÃ«>Vi“i˜ÌÊivviVÌÃ

UÊ >V…ÊÃ̈À‡L>Àʅ>ÃÊ>ʏ>À}iÊ>“œÕ˜ÌʜvÊ«…>ÃiÊ܅i˜ÊVœ“«>Ài`ÊÜˆÌ…Ê SPME (which has only a small amount of phase). Consequently with SPME, if a sample contains some major components then often these will be the only things seen because the major components displace minor components from the fibre. This is not the case with SBSE UÊ

œÊ܏Ûi˜ÌÊ«i>ŽÊ̜ʓ>Îʈ“«œÀÌ>˜ÌÊVœ“«œ˜i˜ÌÃÊ

UÊ ˆ}…ÊÀiVœÛiÀˆiÃÊ>ˆ`ˆ˜}Ê̅iÊ`iÌiÀ“ˆ˜>̈œ˜ÊœvÊÌÀ>ViÊVœ“«œ˜i˜ÌÃÊ UÊ -̈ÀÊL>ÀÃÊV>˜ÊLiÊÀiÕÃi`ʓ>˜ÞÊ̈“iÃÊ In liquid phase or headspace, the extraction process is simple. Once compounds have been recovered into the PDMS coating of the stir bar, there are sufficiently stable to be stored or transported without losses. In general, for volatile compounds, the twister may be thermally desorbed to the GC column [17].

81

Update on Undertaking Extractable and Leachable Testing

Figure 3.6 Twister bar being placed into the thermal desorption unit. Reproduced with permission from GERSTEL GmbH & Co.KG

For less volatile compounds, the twister can be solvent back-extracted and injected into an liquid chromatography (LC) or LC-MS, but the efficiency of this step is typically far less than the thermal desorption approach [18]. The PDMS phase is a non-polar liquid phase, so it should preferably extract low-polarity analytes. High-polarity compounds are not well recovered. Therefore, the possibility of in situ derivatisation (in which derivatisation and SBSE are done simultaneously) has been described for fatty acids, phenols [19], barbiturates and benzodiazepines [20].

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Liquid Extraction Techniques

3.11 Membrane-assisted Solvent Extraction The basic approach involves LLE through a polymer membrane. Using this extraction technique, even dirty aqueous matrices can be studied chromatographically without numerous manual sample preparation steps. The principle of membrane extraction is based on a simple physical mechanism: the distribution between two membrane-separated phases is controlled by an exchange through the permeable membrane. To carry out good membrane extractions, extraction time must be carefully controlled [21].

Figure 3.7 Process flow of membrane-assisted solvent extraction of aqueous samples in 20-ml headspace vials. Reproduced with permission from GERSTEL GmbH & Co. KG

This method requires the steps listed below: 1) Sample is transferred to a 20-ml headspace vial and sealed with a cap containing an integral membrane

83

Update on Undertaking Extractable and Leachable Testing

2) The membrane is filled with extraction solution (this can done manually or with an auto sampler) 3) The sample is heated and agitated for a specific time 4) Extracted sample is withdrawn from the vial and injected into the GC or HPLC When this type of extraction is undertaken, the organic components are enriched in the extraction phase depending on their distribution coefficients. Suspended material or very polar or ionic components cannot pass through the membrane and therefore do not enter the organic phase. This techniques has been used in several areas, including environmental [22,23] and biomedical [24].

3.12 Extraction to Asymptotic Levels There are two basic approaches to determine asymptotic levels. The most common is to test the sample by the analytical methods to be used at various time-points and record the result until a constant level is achieved. Care must be taken to maintain the sample-to-solvent ratio. This requires the addition of extra solvent replacing the solvent that had been removed or multiple extraction setups. The extraction must take care not to deform or degrade the material [25]. Another less common approach is to use an orthogonal technique to check the total level of extractables. Examples include total organic carbon (TOC) or ion mobility. TOC is applicable only if aqueous extraction solvents have been used. Ion mobility is dependent upon the ability of an analyte to be ionised.

3.13 Conclusions Regarding Extraction No extraction technique is perfect. Each technique has its own disadvantages but also advantages, whether they be speed, ease of use, cost of the equipment, sample preparation or extraction efficiency. The relative advantages of each technique can vary depending on

84

Liquid Extraction Techniques

the solvent used as well as the analyte(s) to be extracted. The main factor that has the biggest influence is the matrix. This assessment of multiple extraction techniques has been done for several analytes but never a complete set. Examples exist for explosives in soil [26], and polynuclear aromatic hydrocarbons in soil, but this has not been assessed for all the techniques and for all solvents. One of the most comprehensive extraction studies exploring the widest range of extraction techniques and solvents is being undertaken by the Extractable and Leachable Safety Information Exchange (ELSIE) group [27]. However, this will partly change once the ELSIE group completes a controlled extraction study using the most common extraction techniques and a wide range of solvents. The variability of each technique is one of the main reasons why guidance exists to specify which multiple extraction techniques are used to carry out controlled extraction studies.

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C.L. Arthur and J. Pawliszyn, Analytical Chemistry, 1990, 66, 2145.

2.

C.L. Arthur, D.W. Potter, K.D. Buchholz, S. Motlagh and J. Pawliszyn, LCGC North America, 1992, 10, 9, 656.

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C.L. Arthur, L.M. Killam, K.D. Buchholz and J. Pawliszyn, Analytical Chemistry, 1992, 64, 1960.

4.

H. Lord and J. Pawliszyn, Journal of Chromatography A, 2000, 885, 153.

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H. Liu and P.K. Dasgupta, Analytical Chemistry, 1996, 68, 1817.

6.

M.A. Jeanott and F.F. Cantwell, Analytical Chemistry, 1996, 68, 2236.

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7.

D.M. Razaee, Y. Assadi, M.R. Milani Hosseini, E. Aghaee, F. Ahmadi and S. Berijani, Journal of Chromatography A, 2006, 1116, 1.

8.

R.E. Majors, LCGC North America, 2008, 26, 12, 1158.

9.

E.M.R. Khalili Zanjani, Y. Yamini, S. Shariati and J.A. Jonsson, Analytica Chimica Acta, 2007, 585, 286.

10. M.A. Jochmann, X. Yuan, B. Schilling and T.C. Schmidt, Journal of Chromatography A, 2008, 1179, 96. 11. A. El-Beqqali, A. Kussak and M. Abdel-Rehim, Journal of Chromatography A, 2006, 1114, 234. 12. H. Miyaguchi, Journal of Chromatography A, 2009, 1216, 4063. 13. Z. Altun and M. Abdel-Rehim, Analytica Chimica Acta, 2008, 630, 116. 14. M. Kawaguchi, R. Ito, K. Saito and H. Nakazawa, Journal of Pharmaceutical and Biomedical Analysis, 2006, 40, 500. 15. F. David and P. Sandra, Journal of Chromatography A, 2007, 1152, 54. 16. L.S. De Jager, G.A. Perfetti and G.W. Diachenko, Analytica Chimica Acta I, 2009, 635, 162. 17. E. Baltussen, P. Sandra, F. David and C.A. Gramers, Journal of Microcolumn Separations, 1999, 11, 737. 18. P. Popp, C. Bauer and L. Heinrich, Analytica Chimica Acta, 2001, 436, 1. 19. B. Tienpont, F. David, K. Desmet and P. Sandra, Analytical and Bioanalytical Chemistry, 2002, 373, 46.

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20. B. Tienpont, F. David, T. Benijts and P. Sandra, Journal of Pharmaceutical and Biomedical Analysis, 2003, 32, 569. 21. B. Hauser, M. Schellin and P. Popp, Analytical Chemistry, 2004, 76, 20, 6029. 22. N. Carro, I. Garcia, M. Ignacio and A. Mouteira, International Journal of Environmental Analytical Chemistry, 2009, 89, 8-12, 759. 23. R. Rodil, M. Schellinand and P. Popp, Journal of Chromatography A, 2007, 1163, 1/2, 288. 24. C. Vincelet, J-M. Roussel and D. Benanou, Analytical and Bioanalytical Chemistry, 2010, 396, 6, 2285. 25. D. Jenke, Journal of Pharmaceutical Science and Technology, 2002, 56, 6, 332. 26. T.F. Jenkins and C.L. Grant, Analytical Chemistry, 1987, 59, 9, 1326.

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4

General Analysis

Once the material has been extracted, what is the next stage? How are the species which are extracted identified and quantified? How is this achieved? The following section discusses the key factors on developing the analytical methods which addresses these questions. Extractable analysis requires detailed understanding of trace level (parts per million) or ultra trace level (parts per billion (ppb)) analysis. A wide range of techniques are required to analyse all the potential extractables. However, this chapter cannot go into depth on all the possible techniques, so only the pertinent points for the key analytical techniques are discussed. This encompasses the three main classes of extractables based on their analytical techniques: volatile, semivolatile and metal analytes. Special-case analysis such as nitrosamines, polycyclic aromatic hydrocarbons (PAH) or mercaptobenzothiazole analysis is not discussed. The selection of techniques is dependent upon several factors, as listed below: UÊ -œÕÌiÊV…>À>VÌiÀˆÃ̈VÃʭ܅>ÌʈÃÊ̅iÊÌ>À}iÌÊVœ“«œÕ˜`® UÊ 7…>ÌʈÃÊ̅iÊÃ>“«iʓ>ÌÀˆÝÊ­ÛiÀÞʈ“«œÀÌ>˜ÌÊvœÀʏi>V…>Liî UÊ œ˜Vi˜ÌÀ>̈œ˜ÊiÛiÊœvÊ܏ÕÌiÊ­¯ÊœÀÊ««L® UÊ œiVՏ>ÀÊÜiˆ}…ÌÊ­ˆ“«>VÌʜ˜Êۜ>̈ˆÌÞ® UÊ *œ>ÀˆÌÞÊ­`iÌiÀ“ˆ˜iÃÊۜ>̈ˆÌÞ]Ê܏ÕLˆˆÌÞÊ>`ÜÀ«Ìˆœ˜® UÊ œ}Ê*Ê­œ}Ê ®

89

Update on Undertaking Extractable and Leachable Testing

UÊ ՘V̈œ˜>Ê}ÀœÕ«Ã UÊ /…iÀ“>ÊÃÌ>LˆˆÌÞÊ>˜`ÊÀi>V̈ۈÌÞ UÊ iÌiVÌ>LˆˆÌÞÊ ­i°}°]Ê «ÀiÃi˜ViÊ œÀÊ >LÃi˜ViÊ œvÊ >˜Ê ՏÌÀ>ۈœiÌÊ ­16®Ê chromophore) Figure 4.1 shows the potential analytical techniques depending on the polarity and the molecular weight of the extractable. The use of derivatisation can make an analyte more amenable to another technique. Examples of derivatisation could include the methylation of fatty acids to increase their volatility and make them more amenable ̜Ê}>ÃÊV…Àœ“>̜}À>«…ÞÊ­ ®]ʜÀÊ>``ˆÌˆœ˜ÊœvÊ>ÊV…Àœ“œ«…œÀiÊ̜ʓ>ŽiÊ a molecule amenable to high-performance liquid chromatography ­* ®‡ÕÌÀ>ۈœiÌÊ `iÌiV̈œ˜°Ê *ÞÀœÞÈÃÊ V>˜Ê LÀi>Ž‡`œÜ˜Ê >Ê >À}iÊ non-volatile molecule to a much smaller entity with more volatility, Vœ˜ÃiµÕi˜ÌÞÊ>œÜˆ˜}ʈÌÊ̜ÊLiÊ>˜>ÞÃi`ÊLÞÊ ° ÕVi>Àʓ>}˜ïVÊÀiܘ>˜ViÊ­ ,®ÊëiVÌÀœÃVœ«ÞʈÃʘœÌÊ>˜Ê>˜>Þ̈V>Ê technique but it can be used in certain circumstances, typically if the analyte is known, interference is absent and sufficient sensitivity V>˜Ê LiÊ >V…ˆiÛi`Ê ÜˆÌ…Ê ÃՈÌ>LiÊ >˜>ÞÈÃÊ Ìˆ“iÃ°Ê ,Ê …>ÃÊ ÃiÛiÀ>Ê advantages, the main one being its general intrinsic quantification. œÀÊ>˜ÞÊ}ˆÛi˜Ê«ÀœÌœ˜ÊœÀʜ̅iÀÊ ,‡ÃՈÌ>LiÊ>̜“Ê­i°}°]Ê19ʜÀÊ14N), the response is equal irrespective of the compound.

ʅ>ÃÊŜܘʫÀœ“ˆÃiÊvœÀÊ>Ê܈`iÊÀ>˜}iʜvÊ>˜>ÞÌiÃ]ÊLÕÌÊ`œiÃÊÃÕvviÀÊ from loadability issues. However, due to technological improvements ˆ˜Êˆ˜ÃÌÀՓi˜Ì>̈œ˜]Ê

ʈÃÊÃÌ>À̈˜}Ê̜ÊÀi‡i“iÀ}iÊ>ÃÊ>ÊÀœLÕÃÌÊ>˜>Þ̈V>Ê technique.

œ˜ÃiµÕi˜ÌÞ]Ê̅iÊL>ÈVÃʜvÊ`iÛiœ«ˆ˜}Ê>ÊÃՈÌ>LiÊ>˜>Þ̈V>Ê“i̅œ`Ê after extraction by any or all of the extraction techniques discussed in ̅iÊ«ÀiۈœÕÃÊÃiV̈œ˜Ê>ÀiÊ`ˆÃVÕÃÃi`ÊvœÀÊ ]Ê Ê>˜`ʈ˜`ÕV̈ÛiÞÊVœÕ«i`Ê plasma. This is because they are the most appropriate primary techniques used for the analysis of samples after extraction studies.

90

General Analysis

ˆ}ÕÀiÊ{°1 Possible analytical techniques based on molecular Üiˆ}…ÌÊ>˜`Ê«œ>ÀˆÌÞ°Ê­ˆµÕˆ`ÊV…Àœ“>̜}À>«…ÞÊ­ ®]ÊV>«ˆ>ÀÞÊ iiVÌÀœ«…œÀiÈÃÊ­

®]ÊÈâi‡iÝVÕȜ˜ÊV…Àœ“>̜}À>«…ÞÊ­- ®]ʈœ˜Ê V…Àœ“>̜}À>«…ÞÊ­ ®Ê>˜`Ê}iÊwÌÀ>̈œ˜ÊV…Àœ“>̜}À>«…ÞÊ­ ®®

4.1 Quantification A key part of extractable testing is quantification of the extractable species. This is typically achieved by the use of internal standards. Key factors of an internal standard are listed below: UÊ ÌÊŜՏ`ÊLiÊ܏ÕLiʈ˜Ê̅iÊiÝÌÀ>V̈œ˜Ê܏Ûi˜Ì UÊ ÌÊŜՏ`ÊLiÊVœ“«>̈LiÊ܈̅Ê̅iÊ«>À̈VՏ>ÀÊ>˜>Þ̈V>ÊÌiV…˜ˆµÕi UÊ ÌÊŜՏ`ÊLiʼÜi‡Li…>Ûi`½Êˆ˜Ê̅iÊ«>À̈VՏ>ÀÊ>˜>Þ̈V>Ê“i̅œ`°Ê
ʼÜi‡Li…>Ûi`½Êˆ˜ÌiÀ˜>ÊÃÌ>˜`>À`ʈ˜Ê>Ê ʓi̅œ`]ÊvœÀʈ˜ÃÌ>˜Vi]Ê will not have a significant tailing factor, and will not irreversibly adsorb onto the column

91

Update on Undertaking Extractable and Leachable Testing

UÊ ÌÊŜՏ`ÊLiÊÃÌ>Liʈ˜Ê̅iÊ>˜>Þ̈V>Ê“>ÌÀˆÝ UÊ ÌÊ LiÊ V…i“ˆV>ÞÊ ˆ˜iÀÌÊ vÀœ“Ê ̅iÊ œÌ…iÀÊ >˜>ÞÌiÃ]Ê >˜`Ê ˜œÌÊ LiÊ interfered with by other analytes or components in the analytical matrix UÊ ÌÊŜՏ`Ê«œÃÃiÃÃÊ>ÊÀi뜘ÃiÊȓˆ>ÀÊ̜Ê̅œÃiʜvʜ̅iÀÊ>˜>ÞÌiÃʈ˜Ê the particular analytical technique/method

œ““œ˜Êˆ˜ÌiÀ˜>ÊÃÌ>˜`>À`ÃÊ>ÀiʜvÌi˜Ê`iÕÌiÀ>Ìi`ÊLiV>ÕÃiÊ̅iÃiʓiiÌÊ all the criteria listed above. In extractable testing, it is quite common to use two internal standards. One standard would be at the final analytical evaluation threshold (AET) level and the other at typical levels for quantification.

4.2 Response Factors To accurately quantify a given analyte, the relative response factors ­,,Ã®Ê œÀÊ Ì…iÊ Ài뜘ÃiÊ v>V̜ÀÃÊ ­,®Ê œvÊ >˜Ê >˜>ÞÌiÊ ÌœÊ >˜Ê ˆ˜ÌiÀ˜>Ê standard is required. The uncertainty of each analytical method used for definitive extractables testing should be determined. The use of authentic standards (if available) can be used to develop a ,Ê`>Ì>L>Ãi°ÊÊ
Ê,ʈÃÊ`iw˜i`Ê>Ã\ Ê

,ÊrÊ
aÉ a

where Aa is the response of an individual analyte (e.g., chromatographic «i>ŽÊ>Ài>®Ê>˜`Ê a is the concentration (or mass) of the individual analyte. A more precise uncertainty evaluation can be obtained through the ÕÃiʜvÊ,,]Ê܅ˆV…ʈÃÊ`iw˜i`Ê>ÃÊvœœÜÃ\ Ê

,,ÊrÊ isAa/Ais a

7…iÀi\Ê isÊrÊ œ˜Vi˜ÌÀ>̈œ˜Ê­œÀʓ>ÃîʜvÊ>˜Êˆ˜ÌiÀ˜>ÊÃÌ>˜`>À`

92

General Analysis

AisÊrÊ,i뜘ÃiʜvÊ̅iʈ˜ÌiÀ˜>ÊÃÌ>˜`>À` AarÊ,i뜘ÃiʜvÊ>˜Êˆ˜`ˆÛˆ`Õ>Ê>˜>ÞÌi

arÊ œ˜Vi˜ÌÀ>̈œ˜ÊœvÊ̅iʈ˜`ˆÛˆ`Õ>Ê>˜>ÞÌi /…iÊ ,,Ê ˜œÀ“>ˆÃiÃÊ Ì…iÊ ˆ˜`ˆÛˆ`Õ>Ê ,Ê ÌœÊ Ì…iÊ ,Ê œvÊ >˜Ê ˆ˜ÌiÀ˜>Ê standard. This estimated uncertainty, for the selected analysis, should be applied to the estimated analytical evaluation threshold (AET) to calculate the final AET. /…iÀiÊ>Àiʏˆ“ˆÌ>̈œ˜ÃÊ̜ÊÕȘ}Ê,ðÊ/…iÞÊܜÀŽÊÛiÀÞÊÜiÊvœÀÊ>˜>Þ̈V>Ê ÌiV…˜ˆµÕiÃÊ̅>ÌÊÕÃiʼ՘ˆÛiÀÃ>Ê`iÌiV̜Àýʭi°}°]ʜ˜iÃÊ̅>ÌÊÀi뜘`Ê̜Ê>Ê degree irrespective of the chemical nature of the analyte). The classic iÝ>“«iʈÃÊ>Êy>“iʈœ˜ˆÃ>̈œ˜Ê`iÌiV̜Àʈ˜Ê ʭ̅iÊܜÀŽˆ˜}ÃʜvÊ̅ˆÃÊ detector will be discussed in a subsequent chapter). An ultraviolet ­16®Ê `iÌiV̜ÀÊ ˆ˜Ê * Ê ˆÃÊ `i«i˜`i˜ÌÊ Õ«œ˜Ê ̅iÊ >˜>ÞÌiÊ …>ۈ˜}Ê >Ê chromophore and a maximum absorbance at the same wavelength. ˜Ê}i˜iÀ>]ʈÌʈÃÊ«œÃÈLiÊ̜Ê}i˜iÀ>ÌiÊ>Ê,Ê`>Ì>L>ÃiÊvœÀÊ ÊLÕÌÊ̅ˆÃÊ ˆÃÊÛiÀÞÊ`ˆvwVՏÌÊvœÀÊ* °

4.3 Identification In addition to the quantification of extractables, they also need to be identified. This is primarily achieved by mass spectrometry ­-®Ê܅i̅iÀʈÌÊLiÊVœ“Lˆ˜i`Ê܈̅Ê ]Ê ʜÀÊ

°Ê-iiV̈œ˜ÊœvÊ̅iÊ separation technique and the ionisation technique is dependent upon the molecular weight and polarity of the analyte. These factors `iÌiÀ“ˆ˜iÊ ˆvÊ Ì…iÊ >˜>ÞÈÃÊ ˆÃÊ  ‡-Ê œÀÊ * Ê ÜˆÌ…Ê >̓œÃ«…iÀˆVÊ pressure photo-ionisation (APPI), atmospheric pressure chemical ˆœ˜ˆÃ>̈œ˜Ê­
* ®]ʜÀÊiiVÌÀœÃ«À>Þʈœ˜ˆÃ>̈œ˜Ê­ -®Ê­Figure 4.2). The guidance offers a range of identification reports: confirmed, confident or tentative. This is similar to the identification of trace-level organic compounds in environmental samples. This identification is dependent upon the criteria summarised in Table 4.1.

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Update on Undertaking Extractable and Leachable Testing

ESI

Molecular weight

10,000 APPI

1,000

APCI 100 GCMS 10 Non polar

Polarity

Very Polar

ˆ}ÕÀiÊ{°2 Selection of the separation technique and mode of ionisation (schematic)

Table 4.1 Identification Categories for Structure Elucidation of Extractables Category

94

Identification data

A

>ÃÃÊëiVÌÀœÃVœ«ÞÊvÀ>}“i˜Ì>̈œ˜ÊLi…>ۈœÕÀ

B

œ˜wÀ“>̈œ˜ÊœvʓœiVՏ>ÀÊÜiˆ}…Ì



œ˜wÀ“>̈œ˜ÊœvÊii“i˜Ì>ÊVœ“«œÃˆÌˆœ˜



>ÃÃÊëiVÌÀœÃVœ«Þʓ>ÌV…iÃÊ>Õ̜“>Ìi`ʏˆLÀ>ÀÞʜÀʏˆÌiÀ>ÌÕÀiÊ spectrum

E

>ÃÃÊëiVÌÀœÃVœ«ÞÊ>˜`ÊV…Àœ“>̜}À>«…ˆVÊÀiÌi˜Ìˆœ˜Êˆ˜`iÝÊ matches authentic specimen

General Analysis

Confirmed identification means that identification categories A, B ­œÀÊ ®]Ê>˜`Ê Ê­œÀÊ ®Ê…>ÛiÊLii˜ÊvՏwi`ÊQ£R° Confident identification means that sufficient data to preclude all but the most closely related structures have been obtained. Tentative identification means that data have been obtained that are consistent with a class of molecule only. A challenging area for complete identification of an analyte can be sourcing a suitable reference standard. This can be costly in terms of the price of the material but also the time taken to source this reference standard. In some cases, this will not be possible and, if this is the case, a similar class of compound should be sourced to }ˆÛiÊ̅iÊLiÃÌÊ>««ÀœÝˆ“>̈œ˜ÊvœÀÊ>Ê,°Ê

Reference 1.

,°°Ê …ÀˆÃ̓>˜]ÊEnvironmental Science and Technology, 1982, 16, 3, 143A.

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5

Gas Chromatography

5.1 Introduction The following section covers the basic requirements for developing a method on a gas chromatograph, which is a highly efficient and robust technique to analyse the species from an extractable or leachable study. This provides an insight into the important factors associated with gas chromatography (GC), and the key parameters to change and develop a method rapidly and easily. This chapter covers the basic conditions of the method but does not cover the fundamentals of GC and/or GC-mass spectrometry (MS). This is left to reference sources such as CHROMacademy [1] and others [2, 3]. In GC, a sample is injected onto a column that separates the individual components and generates a signal that is expressed as a chromatogram. This is based on the different partitioning of each component between two phases, the mobile gas phase and the stationary liquid phase coated on a column. The compounds must have sufficient volatility and be thermally stable to be analysed by GC. The main challenge to method development in GC is sample introduction; once this has been solved, 80-85% of the method development is complete. Separation of the analytes is normally very straightforward because GC has plenty of resolving power with very efficient separations. Detection is relatively straightforward, with GC having a wide variety of detectors but also ‘universal detectors’ which can detect any species. The only restrictions on using GC are the lack of thermal stability or volatility, but these can be partly overcome by the use of derivatisation

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Update on Undertaking Extractable and Leachable Testing

or pyrolysis. In general, most common solvents used in the extraction process are very amenable to GC analysis with the only exception being water. This is primarily because of the expansion volume when the liquid is injected and volatilised into the gas chromatograph. The expansion volumes of common extraction solvents are shown in Table 5.1.

Table 5.1 Expansion volumes of common solvents (inlet 280 oC, injection volume 1 μl and an outlet pressure of 14.6 psi) Solvent

Expansion Volume μl

Percentage of typical liner capacity (900 μl)

Isopropanol

297

33

Dichloromethane

355

39

Hexane

174

19

Water

1261

140

Data taken from Agilent Technologies Flow calculator Freeware

Ideally, the injection volume should not be more than 50% of the liner capacity to prevent overload and consequently irreproducible injection due to liner contamination.

5.2 Instrumentation of a Gas Chromatograph A gas chromatograph comprises seven components: gases, sample introduction, column, detector, electronics, output device, and temperature control (Figure 5.1).

98

Gas Chromatography

Figure 5.1 Gas chromatograph (schematic)

Mobile phases are generally inert gases such as helium, or hydrogen (nitrogen has been used in the past). The most common type of sample introduction injection port consists of a septum through which a syringe needle is inserted to inject the sample. The injection port is maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture to ensure that the entire sample will be vaporised. The partitioning behaviour is dependent upon temperature, as well as the different interaction of each component with the stationary phase coated on the column. Hence, the column is usually contained in a thermostat-controlled oven. The separation of components with a wide range of boiling points is accomplished by starting at a low oven temperature and increasing the temperature over time to elute the high-boiling-point components. Most columns contain a liquid stationary phase on a solid support. As the components leave the column, they pass to the detector, resulting in a signal that gets registered as a deflection in the baseline. As a rule of thumb, there should be a temperature gradient between the sample introduction (coldest), the final column temperature (after it has completed its temperature programme) and the detector (hottest).

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Update on Undertaking Extractable and Leachable Testing

5.3 Carrier Gas The carrier gas is chemically inert and transports the sample components through the column to the detector. The gas supply is regulated to restrict the gas pressure to the instrument and is monitored with pressure gauges and flow meters. In addition, the gas supply system contains clean-up cartridges to remove unwanted impurities. The gases are provided ‘oxygen free’ to prevent reactions with the column liquid phase and the sample components. The carrier gases frequently used are helium and hydrogen. Helium is generally preferred for capillary columns due to potential safety issues but this may well shift to hydrogen due to cost implications and more efficient separations.

5.4 Choice of Sample Introduction Solid, liquid and gaseous samples can be analysed by various introduction techniques for GC but, independent of sample type, sample introduction must comply with the following: UÊ Ìʅ>ÃÊ̜ʈ˜ÌÀœ`ÕViÊ̅iÊÃ>“«iʈ˜ÌœÊ̅iÊVœÕ“˜Ê­i°}°]Ê>˜>ÞÌiÃʘii`Ê to be volatalised) UÊ Ìʅ>ÃÊ̜ÊLiÊÀi«Àœ`ÕVˆLi UÊ Ìʅ>ÃÊ̜ʅ>ÛiʘœÊivwVˆi˜VÞʏœÃÃià UÊ Ìʅ>ÃÊ̜ÊLiÊÀi«ÀiÃi˜Ì>̈ÛiʜvÊ̅iÊÃ>“«i For solids, samples can be introduced as a headspace sample, by thermal desorption by pyrolysis, or on a heated probe. Liquid injection is the most common form for samples. In addition to the solid-sample introduction techniques, there are several other introduction methods, including split, splitless, cool-on-column, and programme temperature vaporisation (PTV). These types can be

100

Gas Chromatography

subdivided into even more specialised sample introduction techniques such as pulsed splitless and large-volume injection. These are not be discussed in this chapter.

5.5 Sample Injector The injector provides a means of introducing a sample solution into a hot environment. This in turn causes the sample to vaporise, thereby allowing it to be transferred to the column by the carrier gas. The most common type of injector encountered with GC is the split/splitless (S/S) injector used with a capillary column. The size of the capillary column limits the amount of the analyte that can be injected, otherwise chromatographic overloading occurs, potentially invalidating the subsequent data. The solution is the S/S capillary GC injector. As the name suggests, this inlet can be used in a split or splitless mode. The manufacturers of these systems design them so that the carrier gas flow onto the column is constant to maintain the chromatographic requirements of the column, and yield reproducible retention times for the analytes. However, there is also a certain amount of gas that goes out via a split vent, and this controls the amount of sample that enters the column. If the split vent is closed (usually via computer control) then the entire sample introduced into the injector goes on the column. This is typically used for trace-level analysis, where there is a large difference in boiling point between the analytes and the solute. If the split vent is open then, depending on the ratio of gas flow going onto the column or out through the split, a proportion of the vaporised sample is lost to waste via the split vent, and only a portion of the sample is introduced to the column. The inlet temperature, to allow for complete transfer of the analyte in question, should be set to the following temperature: Inlet temperature = 0.0008* (boiling point of analyte) 2 + 0.2274*(boiling point of analyte) – 33.799

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Update on Undertaking Extractable and Leachable Testing

(For more details, see Anatune application note 18A) For splitless analysis, the oven temperature is set at least 5 °C cooler (typically 10 °C if possible with the solvent, e.g., dichloromethane boils at 40 °C and cooling a gas chromatograph to 30 °C can be very difficult) than the boiling point of the solvent to produce a solventfocusing effect. For split analysis, the oven temperature is to at least 10 °C above the boiling point of the solvent.

5.6 Information Required Before Method Development The only information that needs to be gathered initially is whether or not the analyte(s) in question have sufficient volatility and thermal stability to be amenable to GC. If not, this can be overcome by derivatisation or pyrolysis. Similarly, if an analyte does not have a UV chromophore then, assuming sufficient volatility and thermal stability, GC should be the method of choice. As an approximation, a molecule with a molecular weight of <600 amu has a good chance of being able to be analysed by GC and, as the weight decreases, the amenability to GC increases. The applicability of GC is presented in Figure 5.2.

5.7 Developing a Gas Chromatography Method The following sections highlight the starting conditions for any analyte and the parameters that need to be adjusted to provide a suitable method. A suitable method will be dependent upon many factors ranging from the matrix, the sensitivity required, and the amount of material available.

5.8 Sample Preparation For liquid injection, the sample is dissolved in a suitable organic solvent (remembering water should be avoided if possible due to liner

102

Gas Chromatography

capacities). Ideally, for clean samples ‘cool-on-column’ is the injection of choice, but typically S/S is used with a split, depending on analyte concentration, of ~20-50, with a flame ionisation detector (FID) or MS detector. For trace analysis, splitless injection is commonly employed.

No

Volatile? Yes Semi Volatile?

Yes

Liquid Chromatography

If LC Fails

Derivatisation (GC or LC)

No Thermally Stable?

No

Yes Gas Chromatography

Figure 5.2 When to use GC

5.9 Effects of Changing Individual Chromatographic Parameters The following sections provide guidance on what each of the chromatographic parameters can be used to affect. Each section describes only the effect of changing that particular parameter in

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Update on Undertaking Extractable and Leachable Testing

isolation and not combinations of changes that are often used. It is possible to alter more than one parameter to change the chromatography and, in many cases, this will be required. However, during the development of the method, only one parameter should be changed at a time. The changing of some parameters requires others to be altered in conjunction. For example, if the column flow rate is changed and you wish to retain the same split ratio, then the split flow would also need to be altered accordingly.

5.10 Column Parameters The following section covers the factors associated with changing column variables. The choice of column to be used in GC tends to be a capillary column rather than a packed column. The separating efficiency of a capillary column is much greater than that of a packed column, and the capillary column offers improved detection limits because of the sharp peaks obtained and by minimising the effect on stability by vaporisation of the stationary phase (‘bleed’) into the detector. Capillary columns are manufactured from fused silica tubing, and are typically 10-60 m in length with an internal diameter (id) in the range 0.1-0.53 mm. A polyimide coating is applied to the outside of the silica tubing to improve its mechanical robustness. A film of the stationary phase, typically 0.1-5 μm-thick, is coated on the inside wall of the column. Narrow-bore columns with thin stationary-phase coatings provide fast analysis times, but the sample capacity (loading) of such columns is very small. Overloading of the stationary phase, resulting in a reduction in column efficiency, occurs if too large a sample amount is injected. Larger-diameter columns contain more stationary phase, and give longer analysis times but are less prone to sample overload problems. The stationary phase must be chemically inert, non-volatile, and thermally stable at the column temperature employed for the analysis. A wide range

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of capillary columns is available, mainly using high-boiling-point organic polymers as the stationary phase. As a general rule, non-polar sample materials such as saturated hydrocarbons are best analysed using non-polar stationary phases of the 100% dimethyl polysiloxane type. These separate on the basis of the differences in the boiling point of the analytes. Polar sample materials such as alcohols and ketones require a polar phase such as polyethylene glycol that separates on the basis of hydrogenbonding interactions. In-between these extremes there are many other stationary phases with different polarities suitable for a wide range of applications. Common definitions for columns include fused silica open tubular, wall-coated open tubular and porous-layer open tubular columns.

5.10.1 Selection of the Stationary Phase Stationary phases are selected based on three interaction types: dispersive interactions, dipole interactions, and hydrogen-bonding interactions. Separation of a non-polar analyte such as an aliphatic species would undergo predominantly dispersive types of interactions. Therefore, for optimum retention, a dispersive-type stationary phase (e.g., 100% polydimethylsiloxane (methyl) (e.g., DB-1, ZB1))-type columns are used. The most common stationary-phase phenyl dimethylpolysiloxane (phenyl) has dispersive and induceddipole interactions. The degree of each is dependent upon the ratio of monomers. A 5% phenyl to 95% dimethyl (e.g., DB-5, ZB-5) is the column of choice for general method development because it has the highest temperature range with dispersive and induceddipole interactions. This makes it an ideal choice for a wide range of unknown analytes. (As a rule of thumb, the number after a manufacturer’s code indicates the approximate ratio of monomers, so a DB-5 manufactured by J&W has a 5% phenyl phase.)

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5.10.2 Stationary Phase Film Thickness Thicker film columns (decreasing phase ratio (see later)) are more retentive. Conversely, thinner films (higher phase ratio) are less retentive. These are therefore options for gaining or reducing retention time. Typically, the thinnest film that can achieve the desired separation will be used because this will have the lowest column bleed.

5.10.3 Column Internal Diameter Columns with smaller ids are generally more efficient that the largerid columns. This results in sharper peaks, and they can be used with faster temperature gradients, therefore reducing run times. However, smaller id columns have less stationary phase and therefore the amount of sample that can be introduced onto the column before overloading occurs is much lower. The typical analyte capacity for a 530-μm id column is 1000-2000 ng. However, for a 250-μm id column, the analyte capacity is 50-100 ng and for a 100-μm id column the analyte capacity is <10 ng. Reducing the column id by half doubles the column efficiency.

5.10.4 Phase Ratio The phase ratio is a measure of the ratio of stationary-to-mobile phase. It is useful for determining the relative retentiveness of columns with different ids. This can be useful if the application dictates a change of column id.

5.10.5 Column Length Particularly when using a temperature programme method, the column length has little effect on resolution or analysis time because the temperature has a much more significant effect. There is usually little point in changing the column length to try and achieve

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significantly improved resolutions (although it may be used as a last resort to achieve a final refinement in separation because it also significantly increases retention time). Typically, columns >30 m in length are used with very complex mixtures such as fragrances or in the petrochemical industry. Theoretical improvement of the resolution of an isothermal analysis after doubling the column length would be by a factor of only 1.4.

5.10.6 Conclusions Regarding Columns 1. Typically, if no information about which stationary phase to use is available, a 5% phenylmethyl polysiloxane phase is selected because this has a wide temperature range and a mixed mode of interaction with analytes. In addition, columns with low bleed characteristics should be used, so MS-type columns are recommended. These are identified by the ‘MS’ prefix at the end of the column name (e.g., DB-5MS) 2. Use the least polar stationary phase that provides satisfactory resolution and analysis times. Non-polar stationary phases have superior lifetimes to polar phases. 3. Use a stationary phase with a polarity similar to that of the solutes. This approach works more often than not; however, the best stationary phase is not always found using this technique. 4. If poorly separated solutes possess different dipoles or hydrogenbonding strengths, change to a stationary phase with a different amount (not necessarily more) of the dipole or hydrogen-bonding interaction. Other co-elutions may occur upon changing the stationary phase, therefore the new stationary phase may not provide better overall resolution. 5. If possible, avoid using a stationary phase that contains a functionality that generates a large response with a selective detector (e.g., cyanopropyl containing stationary phases with nitrogen phosphorus detectors (NPD)). This will increase the

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detector background and hence may reduce detection levels for analytes. 6. In general, a 250-μm column offers the best compromise in terms of sample capacity and resolution for initial method development.

5.11 Oven Temperature Gradient (Temperature Programme) The oven temperature gradient is one of the most effective parameters for affecting the chromatography. Gradients can be run at different ramp rates to optimise separations. It is also not uncommon for temperature programmes to have more than one ‘step’. Software simulation programmes such as HIPAC and Dry-Lab can be used to predict what affect the temperature programme will have when using these steps. However, in some cases only a few of the chromatographic parameters may be covered by the software and all of the parameters should be considered holistically. For example, the temperature programme can be used to make peaks elute later, but an increased film thickness would also have the same (but potentially larger) effect.

5.12 Injection Volume The injection volume can be increased or decreased to alter the amount of sample being introduced. It is important that the vapour volume does not exceed the liner capacity; the flow calculation programme should be used to determine this (see Section 6.5). Typically, 0.5-1.0 μl is the normal injection volume.

5.13 Injection Temperature One should ensure that the temperature of the injection is suitable for the sample in question. Degradation can occur if the analyte is thermally unstable at the temperature used. Roughly speaking, a reduction of 10 °C will halve the amount of degradation occurring.

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Discrimination can occur if the injection-port temperature is too high or too low, and a good balance should be chosen. It is essential that this is tested as part of the method development process. Equations can be used to determine if a given species will be volatalised and analysed depending on the boiling point of the analytes [4].

5.14 Liners A wide range of inlet liners are available and, though many organisations and individuals assertively recommend particular types and configurations, there are few rules that are valid for all applications. A high-quality simple straight-glass liner that has been packed with deactivated glass wool (ideally deactivation should have been undertaken after the wool was introduced into the liner) is generally the best all-round starting point. For headspace, a simple narrow-bore glass liner is the optimum liner. If the method development indicates that this configuration of liner is contributing to thermal degradation, then other liners such as the carbofrit and Jennings cup liner could be tried. One should consult with the individual instrument manufacturer’s catalogue for a comprehensive list of liners and their key applications.

5.15 Problems with the Injection The injector has to introduce the sample fast enough in a reproducible manner so that there are no efficiency losses and the sample is representative. Problems with the injection can be discrimination, injector degradation, and vaporisation outside the liner. Discrimination can occur if the injector temperature is set up at a level that does not vaporise all the components of the mixture, and therefore only the most volatile components go onto the column. This means that we are not injecting a representative sample. Discrimination can also occur if vaporisation occurs outside the liner. Every given sample has a certain temperature of vaporisation.

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If the sample enters the injector, it vaporises instantly, and the vapour expands inside the liner. If the injection volume is too large, the vapour can expand outside the liner, and the sample going onto the column will not be representative of the entire sample, hence the importance of injecting the right volume. In addition, this can cause serious contamination of the carrier gas feed through, resulting in a time-consuming cleaning operation. This is why it is rare to use water as the solvent for GC because, under typical conditions, 1 μl of water would expand to >1 m of vapour overloading the liner, whose capacity is typically 900 μl. Table 5.1 details common solventexpansion volumes. Injector degradation: some compounds degrade at high temperatures. If the injector temperature is set up at or above the degradation temperature of the compound, then it will degrade as it enters the injector. This can be avoided by reducing the injector temperature (although there has to be a balance between reducing the injector temperature and ensuring that the entire sample is vaporised instantaneously).

5.16 Split Flow Ratio Increasing the split flow can be used to decrease the quantity of analyte that is introduced on to the column. Conversely, decreasing the split flow will increase it. The split ratio is the ratio of the split flow to the column flow, and indicates the proportion of the sample that is introduced onto the column. For example, a split flow of 50 ml/ min and a column flow of 1.2 ml/min, gives a split ratio of 42:1. That is, 1/42 of the sample injected will be introduced onto the column.

5.17 Column Flow Rate The gas (mobile phase) flow through the column is a very important parameter. It can be expressed in ml/min or cm/s. Maximum efficiencies for helium carrier gas are 20-40 cm/s and for hydrogen it is 30-55 cm/s. 110

Gas Chromatography

However, because the viscosity of fluids change with temperature, and for most GC methods temperature gradients are used, the flow rate will change during the actual run. The flow rate at the start of the run (50 °C) is 40 cm/s. At the end of the run (300 °C) it has changed to 26.4 cm/s. Some modern gas chromatographs have options to run in constant flow mode (i.e., the column head pressure will adjust throughout the run so that the flow rate remains constant). Methods run in this mode are becoming increasingly popular because they can offer maximum column efficiency throughout the whole temperature gradient.

5.18 Cool-on-Column Cool-on-column works by injecting the sample directly into the top of the column. There is no inlet liner between the needle and the column as in the S/S inlet. Instead, the needle passes through a septum and a metal insert, and goes directly into the top of the column, which is pushed up against the insert. The fact that the needle expels the sample straight into the column has sveral advantages over S/S operation, as listed below: UÊ
Ê«ÀœLi“ÊœvÌi˜Ê«Àœ`ÕVi`ÊLÞÊVœ˜Ûi˜Ìˆœ˜>Êˆ˜iV̈œ˜ÊˆÃÊÃ>“«iÊ discrimination. This is caused by volatilising sample components and/or solvent in the hot needle as it injects. By using cool-oncolumn, the sample remains homogeneous in the needle and leaves it, negating this effect. UÊ >˜`ÊLÀœ>`i˜ˆ˜}ʈÃÊ>˜œÌ…iÀÊ«ÀœLi“ÊœvÊVœ˜Ûi˜Ìˆœ˜>Êˆ˜iV̈œ˜Ã°Ê This can occur if a sample or its components do not wet the column very well. As it deposits onto the column, it may separate into small beads, leading to broadened peaks. As the sample does not re-condense with cool-on-column, it always wets the column equally. UÊ /œÊˆ˜iVÌÊÃ>“«iÃʈ˜Ê>Ê-É-Ê̅iÞʓÕÃÌÊwÀÃÌÊLiÊۜ>̈ˆÃi`°Ê/…ˆÃʓ>ÞÊ cause the sample to degrade or react to form byproducts, which

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may affect the accuracy of the results. Using cool-on-column, the sample is injected directly onto the column in its liquid form, avoiding the problem. UÊ >ÅL>VŽÊ˜iÛiÀʜVVÕÀð UÊ œiÃʅ>ÛiʈÃÃÕiÃʈvʼ`ˆÀÌÞ½ÊÃ>“«iÃʜÀÊÃ>“«iÃÊVœ˜Ì>ˆ˜ˆ˜}ÊÃi“ˆ‡ volatile materials are used because contamination can occur.

5.19 Programmable Temperature Vaporisation When using Programmable temperature vaporisation (PTV), the sample is introduced into a cold injector, which is then heated at a controlled rate so as to vaporise the analytes and transfer them to the column. This technique ensures that the sample components are transferred onto the column at the minimum possible temperature, and is particularly suitable for the analysis of samples containing thermally unstable compounds. The PTV injector can be used in split and splitless modes. In addition, it can also be used for largevolume sample injections. In many applications of GC, the analysis sample consists of a dilute solution of analytes in a volatile solvent. Using a conventional S/S column injector, it is not possible to inject sample volumes of >5 μl without causing a reduction in the separating efficiency of the column. However, a PTV injector can handle comparatively large volumes of such samples, with dramatic benefits in terms of the limits of detection. If operated in the large-volume injection mode, the PTV is maintained at a low temperature, with the split vent open during the sample introduction phase. The sample is injected relatively slowly so as not to flood the injector and to allow the volatile solvent to vaporise and exit to atmosphere via the split vent. The vent is then closed and the injector temperature programmed so as to transfer the analytes into the column. Sample volumes >100 μl can be injected using this technique.

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5.20 Choice of Detector GC has a very wide choice of detectors that have universal appeal or can be very selective and specific for a class of analytes. The list of detectors shown below is not exhaustive but covers the most common detectors. The detectors are listed in an approximate order of the most important/widely used. The two most commonly used detectors are the flame ionisation detector (FID) or the mass spectrometer.

5.20.1 FID The FID is the most widely used GC detector because of its high sensitivity (detection limit ~1×10-12 g/s), wide linear response range (106), low cost, and ease of use. The FID responds to all organic substances with the exception of formic acid, formamide and formaldehyde, but does not respond to He, Ne, Ar, Kr, Xe, H2, O2, N2, H2S, CO, CO2, COS, SO2, NH3, NO, NO2, N2O or water. For an excellent description of the principles of an FID, see [5].

5.20.2 Mass Spectrometer The mass spectrometer has become the most important detector used in GC because the detector offers very high sensitivity but, most importantly, it enables positive identification of the sample constituents. In a mass spectrometer detector, the column effluent is passed into an ionisation chamber and bombarded with highenergy electrons from a hot tungsten/rhenium filament. Any organic substances present in the carrier gas are fragmented by collision with these electrons. There are two basic modes of ionisation for GC-MS: electron impact (EI) and chemical ionisation (CI). There is a third, rarer mode, field ionisation (FI), for the softest mode of ionisation. EI is the most common mode of ionisation and has several benefits.

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Figure 5.3 How EI works

The ion species resulting from this process are accelerated into a mass/ion separation device such as a magnetic sector, quadrupole spectrometer, a time-of-flight or an ion trap where they are characterised on the basis of their mass-to-charge ratio. Figure 5.4 shows the ‘decision tree’ showing when to use a particular mass spectrometer. The mass spectra resulting from this process are generally characteristic of the sample component and can be used for identification purposes. In some applications, the electron bombardment from this type of source may be too energetic, resulting in complete disintegration of the sample molecule and producing a mass spectrum that provides little useful information. In such cases, an alternative technique, CI, may be used. In this method, a gas such as methane (or butane or ammonia) is fed into the mass spectrometer inlet where subsequent electron bombardment results in the creation of low-energy ions from the gas. These low-energy ions then interact with the sample components to produce less fragmented mass spectra, producing a pseudo molecular ion.

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Identification by GC-MS required

Problem solved

Identification by Library match or by expected theoretical fragmentation

Yes

Attempt Identification by EI

Molecular ion present

Insufficient certainty from Library match Molecular ion present

Problem solved

Excessive or no fragmentation Molecular ion not present

Try “softer” ionisation

Low energy EI

Chemical Ionisation

Positive

Problem solved

Negative

Choice of gases Methane Ammonia Butane

MS/MS

Molecular ion present

Problem solved

Problem solved

Molecular ion not present

Accurate Mass

Problem solved

FI

Figure 5.4 Selection of the type of mass spectrometer analyser depending on the challenge faced

The main advantages of MS is that the EI mass spectra produced are independent of instrument used and are fully library-searchable

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with typically >100,000 spectra in a library. Very rarely do species not ionise in EI because EI is the most universal mode of ionisation. One disadvantage is excessive fragmentation of the parent molecule, which provides no categorical evidence to enable identification.

5.20.3 Electron Capture Detector The electron capture detector (ECD) uses a radioactive beta emitter (Ni63) to ionise some of the carrier gas and produce a current between a biased pair of electrodes. If organic molecules that contain electronegative functional groups such as halogens, phosphorus, and nitro groups pass by the detector, they capture some of the electrons and reduce the current measured between the electrodes. The ECD responds strongly to many substances containing halogens, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, lead and tin organic compounds. Among the applications of the ECD is the detection of trace amounts of pesticides in crops and water samples [6]. The typical detection limit of the ECD is 2 ×10–14 g/s with a linearity range (pulse modulated system) of 104.

5.20.4 Thermal Conductivity Detector A typical design for a thermal conductivity detector system includes four tungsten filaments mounted in a steel block. Two of the filaments are in a chamber in the block through which the carrier gas flows after emerging from the end of the column. The other two are in a second chamber through which a reference stream of carrier gas passes. The filaments are electrically connected in the arms of a Wheatstone Bridge network. Electrical power is applied to the bridge and the filaments become hot due to current flow. The actual temperature achieved by the filaments is a function of the power applied to the bridge and the thermal conductivity of the gas passing through the detector. Under stable conditions, the same gas is flowing through both chambers of the detector and the bridge attains equilibrium, resulting in a steady output. The performance of the TCD can be

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improved by use of a system in which the filaments are maintained at constant temperature during elution of the sample components by variation of the applied power. The power required to maintain the filaments at constant temperature is outputted to the data system and produces a chromatogram. Compared with the variable temperaturetype of TCD, the constant-filament-temperature system gives greater sensitivity and improved linear range. The TCD responds to any sample component that has a different thermal conductivity to that of the carrier gas. Thus, the detector will respond to organic and inorganic materials, and its main application is for the detection of substances (mainly permanent gases and water) that cannot be detected with the FID.

5.20.5 Nitrogen Phosphorus Detectors The NPD is a modified FID in which an alkali metal halide is continuously vaporised into the flame. In the presence of the alkali metal halide vapour in the flame, organic phosphorus- or nitrogencontaining substances burn to produce ionic species which are unusually stable. In a normal FID, the ions produced by combustion of the sample rapidly decrease in concentration in the area above the flame. If the collector electrode is moved away from the immediate vicinity of the flame, little or no response is obtained. In contrast, in the NPD, the ions formed from organophosphorus or nitrogen substances persist much longer and, if the collector electrode is placed in a suitable position, a selective response to phosphorus or nitrogen compounds can be obtained. Applications of the NPD include measurement of trace amounts of pesticides in crops and water samples and of drugs in body fluids.

5.20.6 Flame Photometric Detector The flame photometric detector (FPD) is based on the fact that, when burnt in a hydrogen-rich flame, phosphorus- and sulfurcontaining substances give a characteristic light emission. Phosphorus

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compounds emit light at 526 nm and sulfur compounds at 394 nm due to the formation in the flame of the hydrogen phosphorus and oxygen PO and S2 species, respectively. In the FPD, an optical filter designed to block light other than at 526 nm or 394 nm is placed between the flame and a photomultiplier detector. In this way, a selective response to phosphorus and/or sulfur compounds is obtained. The response of the FPD to phosphorus compounds is linearly proportional to sample concentration but, in the case of sulfur, because the response is due to formation of the S2 species, it follows an approximately square law relationship. The main applications of the FPD include the detection of traces of organophosphorus and sulfur pesticides, and for the determination of H2S, SO2, and mercaptans.

5.20.7 Thermal Energy Analyser, Nitrogen Chemiluminescence Detector Thermal energy analysers or nitrogen chemiluminescence detectors work on the same basic principle. They are highly specific and sensitive detectors primarily used for the analysis and quantification of nitrosamines at ppb levels. When nitrosamines are analysed, they are injected as normal on a GC system to separate the nitrosamines. Once eluted, they are pyrolysed (at ~500 °C) and form a nitrosyl radical. These nitrosyl radicals are then reacted with ozone under a vacuum to form the energetically excited state of NO2. The NO2 relaxes to the ground state and emits photons in the red and infrared regions (chemiluminescence) which are detected by the instrument.

5.21 Derivatisation Properties for a good derivitisation process are listed below: UÊ -iiV̈ÛiÊÀi>V̈œ˜Ê܈̅ÊÜi‡`iw˜i`Ê}ÀœÕ«Ã

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Gas Chromatography

UÊ œœ`ÊÀi>V̈œ˜ÊŽˆ˜ïVÃ]Êv>ÃÌÊÀi>V̈œ˜ UÊ ˆ}…ÊÀi>V̈œ˜Êވi`à UÊ ˆ`ÊÀi>V̈œ˜ÊVœ˜`ˆÌˆœ˜Ã UÊ -Ì>LiÊÀi>V̈œ˜Ê«Àœ`ÕVÌÃ]ʘœÌÊ̜œÊۜ>̈i UÊ ÝViÃÃÊÀi>}i˜ÌÊi>ÃÞÊ̜ÊÀi“œÛiÊ­˜œÌÊ`ˆÃÌÕÀLˆ˜}Ê̅iÊ>˜>ÞÈî UÊ ,i>}i˜ÌÃÊ>ÀiʘœÌÊ̜݈V UÊ œœ`Ê`iÌiV̜ÀÊÀi뜘Ãi UÊ /iÃ̇ÌÕLiÊV…i“ˆÃÌÀÞ The benefit or necessity of derivatising an analyte has to significantly outweigh the time and effort to carry out the derivatisation.

5.22 Typical Starting Conditions for Extractable Analysis Very generic conditions should be employed if you do not have any information on the analytes being extracted. This will involve a generic column with intermediate dimensions, optimal flow, with a very generic temperature gradient starting below the boiling point of the solvent to the column maximum. Ideally a cool-on-column injector should be used but a split-splitless injector in split mode can be used. This enables the low detection limits required. FID and mass spectrometer detectors should be used. Good starting conditions are presented in Table 5.2. Typically, leachable analysis will employ exactly the same conditions as extractable testing. These conditions will not be perfect, but they make a good start until more information on the analytes can be found and the conditions optimised.

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Table 5.2 Starting GC analytical conditions for extractable or leachable analysis Parameter

Condition

Injection mode:

Splitless injection or cool-on-column

Injection volume:

1 μl

Injector temperature/ program:

Cool-on-column oven +3 °C, oven track ON 280 °C for splitless injection

Column:

DB-5MS, 30 m × 0.25 mm (0.25-μm film) or equivalent

Oven temperature:

35 °C for 2 min, heated at 10 °C/min to 325 °C and hold for 10 min

Carrier gas flow

Constant flow (helium) at 35 cm/s

Scan mode:

Scanning: m/z 30-500 amu

References 1.

LC-GC’s ChromAcademy, http://www.chromacademy.com

2.

H. Hubschmann, Handbook of GC/MS, Wiley-VCH, Weinheim, Germany, 2001.

3.

W. Jennings, E. Mittlefehldt and P. Stremple, Analytical Gas Chromatography, 2nd Edition, Academic Press, New York, NY, USA, 1997.

4.

Procedure for Using the Optic 2 for Selective Exclusion, ANATUNE Application Note 18A. http://www.anatune.co.uk

5.

J.V. Hinshaw, The Flame Ionisation Detector, 2006, 19, pp.4. http://www.lcgceurope.com

6.

C. Bicchi, C. Cordero, C. Iori, P. Rubiolo, P. Sandra, J.H. Yariwake and V.G. Zuin Journal of Agricultural Food Chemistry, 2003, 51, 1, 27.

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6

High-performance Liquid Chromatography

6.1 Introduction The following section covers the basic requirements for developing a method on a high-performance liquid chromatography (HPLC) system. The same basic factors concerning analysis with HPLC apply to ultra-performance liquid chromatography (UPLC) or ultra-highperformance chromatography because the mode of separation and detection is common to all three. UPLC is simply HPLC undertaken at high flow rates using sub-2-μm porous particles (i.e., 1.5-1.9 μm) to achieve high efficiency and/or short cycle times. Often an elevated temperature (40–60 °C) is used to reduce the back pressure and/or allow higher flow rates. The back pressure is typically twice as high as in conventional liquid chromatography (LC), often 800 bar. A more extensive treatise can be found in the literature [1]. Typically, UPLC is undertaken with 30-150 mm × 2.1 mm i.d. columns at flow rates of 0.2-1.0 ml/min. This section provides a basic insight into the important factors associated with HPLC and the key parameters to change, rapidly and easily develop a suitable method. This section gives a basic introduction but [2] and [3] cover this area in much more detail. In HPLC, a sample is injected onto a column that separates the individual components and generates a signal that is expressed as a chromatogram. This is based on the different partitioning of each component between two phases, the liquid mobile phase and the solid stationary phase of the column. The main challenges to method development in HPLC differ quite dramatically to those for gas chromatography (GC). In HPLC, sample introduction is

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straightforward because a liquid is being introduced into a liquid. It is the separation and detection that are the challenges in HPLC. The detection issue is due to the lack of a true ‘universal’ detector, and the separation is due to the less efficient columns when compared with GC. Some detectors in HPLC are non-destructive, so they can be used in series. The most commonly used is an ultraviolet (UV) detector, followed by a mass spectrometer.

6.2 Sample Preparation Sample preparation for HPLC is very straightforward. The sample needs to be filtered to prevent blocking of the needle and/or column, which is more important with UPLC than with HPLC due to the packing particle size. The sample diluent needs to be miscible with the mobile phase. This can be an issue with reverse-phase and extraction solvents such as hexane and dichloromethane (e.g., nonwater miscible solvents).

6.3 Separation In HPLC, separation can occur in five basic modes: normal-phase, reversed-phase, ion-exchange, ion-pairing and size-exclusion. The basic separation principle is presented schematically in Figure 6.1. In summary, sample molecules can distribute between the stationary phase (typically the silica particles in the column) and the mobile phase. A sample analyte is carried forward only if it is in the mobile phase. Analyte C that spends more time in the stationary phase compared with the mobile phase is eluted later than analyte A, whose distribution is predominately in the mobile phase and passes through a column very quickly. These distributions of analytes between the two phases are affected primarily by the chemical composition of the sample, column packing, solvent system and temperature. These factors will be discussed in later sections.

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Figure 6.1 Principles of HPLC separation (schematic)

The various modes of separation are described in more detail below.

6.3.1 Reverse Phase Reverse phase is the most common mode of separation, with ~75% of all HPLC separations carried out by this mode. With reverse-phase HPLC, the stationary phase is non-polar and the mobile phase is polar. It is a good choice for mixtures with a different number of types or location of alkyl or polar functions and, as such, is the primary mode of separation for extraction studies. This mode of separation can separate weak acids and bases as long as the pH of the eluent

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is buffered to keep them non-dissociated. This can be challenging if the identity of the analytes is unknown. There is a very wide variety of reverse-phase packing materials, with C18 (C18H37 bonded to the porous silica surface) being the most common. Stationary phases will be discussed in subsequent sections. The most common solvents used are either weak e.g. water or stronger solvents e.g., methanol (MeOH), acetonitrile and tetrahydrofuran (THF)

6.3.2 Normal Phase In normal-phase HPLC, the stationary phase is polar and the mobile phase is non-polar. Normal-phase HPLC is most commonly used for separating geometric and positional isomers. Normal-phase HPLC has little affinity for alkyl groups, therefore it is not very successful for separating compounds differing only by alkyl groups. The most common solvents used are the iso-hexane (weak solvent) and then stronger solvents such as chloroform and dichloromethane.

6.3.3 Ion Pair Mode Separation of ionisable organic compounds on reverse-phase columns is preferred over ion-exchange (stationary phase with fixed ionic sites) because it offers higher efficiency and greater control over selectivity. It differs from reverse phase in that the eluent contains a hydrophobic counter ion called an ion-pairing agent. It is widely believed that ion-pairing agents adsorb on the stationary phase to form the equivalent of an ion-exchange stationary phase. In addition to ion-pairing agents, eluents usually contain a buffer to keep the sample components ionised. Retention is controlled by the concentration and type of the ion-pairing agent, the ionic strength, the pH and organic solvent composition. MeOH is preferred to

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acetonitrile (MeCN) or THF because it provides better solubility for buffers (0.02-0.2 M) and ion-pairing agents (0.005-0.5 M).

6.3.4 Ion Exchange Ion exchange is less likely to be used for extractable testing because the species typically found in extractable or leachable testing do not have the chemical characteristic required. It is more likely to be used for the separation of inorganic ions, proteins, peptides, nucleotides and amino acids. Retention in ion-exchange chromatography is controlled by pH and ionic strength of the eluent rather than its organic solvent content. Elevated temperatures often improve efficiency. MeOH and MeCN may be added to modify retention (added because of hydrophobic interactions with the backbone of the stationary phase).

6.3.5 Size-exclusion Chromatography Size-exclusion chromatography is used for samples that contain high-molecular-weight compounds and is less likely to be used in extractable testing in which compounds are significantly different in molecular weight.

6.4 Developing a High-performance Liquid Chromatography Method The following sections highlight the key factors that need to be considered to develop a suitable method. The objective of a method is for the adequate resolution of the components of interest in a reasonable time. Analysis is dependent upon many factors ranging from the matrix, the sensitivity required, and the amount of material available. The key parameters associated with development of a HPLC method are solvent strength, temperature, solvent type,

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additives, column type, and pH. These parameters can be split into three categories. The ‘continuous variables’ which can take any value and allow interpolation between selectivity extremes are solvent strength, temperature, solvent type, additives. The ‘discontinuous variable’, column type, does not allow for an interpolation between selectivity extremes. The final variable, pH, is dependent upon the analyte in question so it can be a continuous or discontinuous variable.

6.5 Solvent Selection Solvent selection is dependent upon the four main types of interactions: dispersion, dipole, hydrogen-bonding and dielectric interaction. These interactions have been summarised by Synder and are shown in Figure 6.2. Solvents are chosen near the apex of the triangle to obtain the widest selectivity differences. For reversephase HPLC these are MeOH, THF or MeCN as the strong solvent and water as the weak solvent. By adjusting the concentration of the weak solvent and strong solvent, the required selectivity could be achieved. In more complex separations, quaternary solvent mixtures can be used (but this is rare).

Proton acceptor II III VI

MeOH THF MeCN

II IV VIII

Proton donor

I III VI VII

V Dipole Interaction

Figure 6.2 Solvent selectivity: the Synder triangle

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High-performance Liquid Chromatography

6.6 Temperature Control Increasing the column temperature has several advantages: UÊ ˜VÀi>Ș}ÊivwVˆi˜VÞʭۈ>ʈ˜VÀi>Ãi`ʓ>ÃÃÊÌÀ>˜ÃviÀ® UÊ iVÀi>ÃiÃÊÀiÌi˜Ìˆœ˜Ê̈“i UÊ iVÀi>ÃiÃÊVœÕ“˜Ê«ÀiÃÃÕÀiÊ`Àœ« UÊ “«ÀœÛiÃÊÃ>“«iÊ܏ÕLˆˆÌÞ Elevated temperatures may also cause a change in selectivity. Even if an elevated temperature is not needed, it is worthwhile to thermostat the column because variations in room temperature may cause a drift in retention.

6.7 Columns Selecting a particular column when the analytes are unknown can be very challenging. However, the following basic criteria should be applied: UÊ œœ`ÊÃÌ>LˆˆÌÞÊvœÀÊ̅iÊÃÕL‡Ó‡–“ÊÛiÀȜ˜Ê>Ìʅˆ}…Ê«ÀiÃÃÕÀiÊ­nää‡ 1000 bar) UÊ œœ`ÊL>ÌV…‡Ìœ‡L>ÌV…ÊÀi«Àœ`ÕVˆLˆˆÌÞ UÊ ˆvviÀi˜ÌÊÃiiV̈ۈÌÞ UÊ œœ`Ê«i>ŽÊÅ>«i UÊ ˆ}…ÊivwVˆi˜VÞ UÊ œÜÊLii`ʈ˜Ê16Ê>˜`ʓ>ÃÃÊëiVÌÀœ“iÌÀÞÊ­-® A key factor with HPLC when compared with GC is that a type of column such as a C18-type column from one supplier can vary in selectivity to a C18 column from another supplier. This is due to

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differences in the bonding chemistry involved in joining the packing material to the silica stationary support. A schematic of the stationary phase is shown in Figure 6.3.

R1 Silica Stationary Phase Support

Si

O

Monofunctional bonding chemistry

Si

R

R1 R1 = typically is a methyl group R = typically C18, C8, Phenyl, CH2CH2CN Figure 6.3 A stationary phase used in HPLC (schematic)

The factors affecting the column selectivity are listed below: UÊ *œÀœÕÃʜÀʘœ˜‡«œÀœÕÃÊȏˆV> UÊ -ÕÀv>ViÊV…i“ˆÃÌÀÞ\ÊÌÞ«iʜvÊȏ>˜œÊ}ÀœÕ«ÃÊ UÊ >ÀLœ˜Êœ>`\ÊÊ«iÀVi˜Ì>}iʜvÊV>ÀLœ˜ÊLœ˜`i`Ê̜Ê̅iÊȏˆV>Ê«>À̈Vi UÊ /Þ«iʜvÊLœ˜`ˆ˜}\ʓœ˜œ“iÀˆVʜÀÊ«œÞ“iÀˆV UÊ i}ÀiiÊ œvÊ i˜`V>««ˆ˜}\Ê À>˜}iÊ vÀœ“Ê ˜œ˜iÊ ÌœÊ i݅>ÕÃ̈ÛiÞÊ i˜`‡ capped

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High-performance Liquid Chromatography

UÊ iÌ>ÊVœ˜Ìi˜Ì\Ê`i«i˜`Ãʜ˜Ê̅iʓi̅œ`ʜvÊȏˆV>Ê«Ài«>À>̈œ˜ The characterisation differences in column chemistry were first presented by Tanaka [4]. The phase’s hydrophobicity, shape selectivity, hydrogen-bonding capacity and ion-exchange capacity at pH 2.7 and 7.6 were characterised and these were progressed further [5]. Characterisation of the stationary phase by M. Euerby and P. Petersson has been very extensive because they have characterised 135 stationary phases [6]. By selecting differences between stationary phases, a step change in the selectivity of a column can be achieved.

6.8 pH In typical HPLC analysis, to produce robust methodology, it is recommended that a pH is selected which is at least two pH units above or below the dissociation constant (pKa) of the analyte(s). In this way, analyte(s) are present in a completely neutral or ionised state and, consequently, small variations in pH have a minimal effect on the resultant chromatography. However, this can be problematic if the analytes are unknown. Hence, typically a LC-MS-friendly acid buffer is added. LC-MS-friendly buffers are volatile buffers that do not crystallise out and contaminate the ion source. Potential buffers are shown in Table 6.1, along with the corresponding pKa and buffer range. Addition of an acid can provide an extra source of protons to help promote/aid ionisation and maintains consistency in whether or not an analyte is fully ionised.

6.9 Detector Selection The choice of a detector is dependent upon the chemical structure of the components and the requirements of the method. With unknown analytes, a range of detectors may need to be used and some of these detectors, being non-destructive, can be used in series. An HPLC detector should have the features listed below: UÊ ˆ}…ÊÃi˜ÃˆÌˆÛˆÌÞÊ>˜`ÊÀi«Àœ`ÕVˆLi]Ê«Ài`ˆVÌ>LiÊÀi뜘Ãi

129

Update on Undertaking Extractable and Leachable Testing

UÊ ,i뜘`Ê̜Ê>Ê܏ÕÌiÃ]ʜÀʅ>ÛiÊ«Ài`ˆVÌ>LiÊëiVˆwVˆÌÞ UÊ 7ˆ`iʏˆ˜i>ÀÊ`ޘ>“ˆVÊÀ>˜}iÆÊÀi뜘ÃiÊ̅>Ìʈ˜VÀi>ÃiÃʏˆ˜i>ÀÞÊÜˆÌ…Ê the amount of solute UÊ ,i뜘ÃiÊ՘>vviVÌi`ÊLÞÊV…>˜}iÃʈ˜ÊÌi“«iÀ>ÌÕÀiÊ>˜`ʓœLˆiÊ«…>ÃiÊ flow UÊ ,i뜘`ʈ˜`i«i˜`i˜ÌÞʜvÊ̅iʓœLˆiÊ«…>Ãi UÊ

œÌÊVœ˜ÌÀˆLÕÌiÊ̜ÊiÝÌÀ>‡VœÕ“˜ÊL>˜`ÊLÀœ>`i˜ˆ˜}

UÊ ,iˆ>LiÊ>˜`ÊVœ˜Ûi˜ˆi˜ÌÊ̜ÊÕÃi UÊ *ÀœÛˆ`iʵÕ>ˆÌ>̈ÛiÊ>˜`ʵÕ>˜ÌˆÌ>̈Ûiʈ˜vœÀ“>̈œ˜Êœ˜Ê̅iÊ`iÌiVÌi`Ê peak UÊ >ÃÌÊÀi뜘Ãi

Table 6.1 Potential LCMS-friendly buffers with pKa and buffer range Buffer

pKa

Buffer range

Trifluoracetic acid (TFA)a

0.5

Formic acid

3.8

Ammonium formate

3.8

Acetic acid

4.8

Ammonium acetate

4.8

3.8-5.8

Triethylamine

11.0

10-12

a

2.6-4.8

TFA should be avoided if at all possible due to ion suppression

iÌiV̜ÀÃÊ vœÀÊ * Ê >ÀiÊ `iÈ}˜i`Ê ÌœÊ Ì>ŽiÊ >`Û>˜Ì>}iÊ œvÊ ViÀÌ>ˆ˜Ê physical or chemical attributes of the solute or mobile phase in the chromatographic process in one of four ways:

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High-performance Liquid Chromatography

UÊ
ÊLՏŽÊ«Àœ«iÀÌÞʜÀÊ`ˆvviÀi˜Ìˆ>Ê“i>ÃÕÀi“i˜Ì UÊ
˜>ÞÌi‡Ã«iVˆwVÊ«Àœ«iÀ̈ià UÊ œLˆi‡«…>Ãiʓœ`ˆwV>̈œ˜ UÊ Þ«…i˜>Ìi`Ê ÌiV…˜ˆµÕiÃ]Ê i°}°]Ê Ê * Ê VœÕ«i`Ê ÜˆÌ…Ê “>ÃÃÊ spectrometry

6.9.1 Ultraviolet (UV) Detector UV detection is the most widespread detection methods for LC, especially in the pharmaceutical industry. This is due to its high sensitivity, broad linear range and ease of operation, as well as its compatibility with most mobile-phase solvents. However, this assumes that the analytes of interest possess a UV-absorbing chromophore. Other detectors such as refractive index (RI) detection have been employed for the detection of UV-unresponsive compounds. However, RI detection has the disadvantages of low sensitivity and incompatibility with gradient elution, making it less suitable for complex mixtures at trace levels. On this basis, RI detectors will not be discussed further. Analytes will absorb different amounts of light in the UV and visible regions. A beam of UV light is passed through the analyte after it is eluted from the column. A detector is positioned on the opposite side which can measure how much light is absorbed and transmitted. The amount of light absorbed will be dependent upon the amount of the compound that is passing through the beam. This is based on the Beer-Lambert law:

A= εcd where A is the absorbance, ε is the molar absorbtivity coefficient, c is the concentration and d is the path length.

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Update on Undertaking Extractable and Leachable Testing

UV detectors are split into two types. One is a variable wavelength detector, which measures one wavelength at a time, but can detect over a wide range of wavelengths. The other is a diode array `iÌiV̜ÀÊ­
®]Ê܅ˆV…Ê“i>ÃÕÀiÃÊ̅iÊ܅œiÊ16ÉۈÈLiÊëiVÌÀՓʜvÊ Ü>Ûii˜}̅ÃÊȓՏÌ>˜iœÕÃÞ°Ê ÕiÊ̜Ê̅iÊÀ>˜}iʜvÊÜ>Ûii˜}̅ÃÊ̅>ÌÊ >Ê
ÊV>˜Ê“i>ÃÕÀiÊȓՏÌ>˜iœÕÏÞ]ʈÌʈÃÊ̅iÊLiÃÌÊ16Ê`iÌiV̜ÀÊvœÀÊ analysing unknown analytes.Ê /…iÊ
Ê >ÃœÊ ṎˆÃiÃÊ >Ê `iÕÌiÀˆÕ“Ê or xenon lamp that emits light over the UV spectrum range. Light from the lamp is focused by means of an achromatic lens through the sample cell and onto a holographic grating. The dispersed light from the grating is arranged to fall on a linear diode array. The resolution of the detector will depend on the number of diodes in the array, and also on the range of wavelengths covered. Consequently, ̅iÊՏ̈“>ÌiÊÀi܏ۈ˜}Ê«œÜiÀʜvÊ̅iÊ
Ê܈Ê`i«i˜`ʜ˜Ê̅iÊÃi“ˆ‡ conductor manufacturer and on how narrow the individual photo cells can be commercially fabricated.

6.9.2 Wavelength Selection ÕÀˆ˜}ʓi̅œ`Ê`iÛiœ«“i˜Ì]ʈÌʈÃÊÀiVœ““i˜`i`Ê̜ÊÕÃiÊ>Ê
Ê>˜`Ê record the signal for one channel at 220 nm that can be considered to be a relatively ‘general’ wavelength. It is also recommended to use another channel with a broad bandwidth (e.g., 220-400 nm) to get as general UV detection as possible (typically 254 nm). Selection of the optimum wavelength requires knowledge of the UV spectra vœÀÊ>˜ÞÊ>˜>ÞÌiÆÊvœÀÊ̅iÊ}Ài>ÌiÃÌÊÃi˜ÃˆÌˆÛˆÌÞ]ÊÕÃiÊ̅iÊλ max. However, normally it is a compromise wavelength for detecting all the sample components because they will all have their own maximum.

6.10 Fluorescence Detector A fluorescence detector excites the sample with excitation light and breaks up the emitted fluorescence light with a fluorescence monochromator. It extracts the required fluorescence wavelengths and measures the intensity with a photomultiplier.

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High-performance Liquid Chromatography

When light is emitted by molecules that are excited by electromagnetic radiation, the phenomenon is termed ‘photoluminescence’. If the release of electromagnetic energy is immediate, or stops on the removal of the excitation radiation, the substance is said to be ‘fluorescent’. If, however, the release of energy is delayed, or persists after the removal of the exciting radiation, then the substance is said to be ‘phosphorescent’. Fluorescence has been shown to be extremely useful as a detection process, and detectors based on fluorescent measurement have provided some of the highest sensitivities >Û>ˆ>LiÊ ˆ˜Ê * °Ê iÌiV̈œ˜Ê ÌiV…˜ˆµÕiÃÊ L>Ãi`Ê œ˜Ê y՜ÀiÃVi˜ViÊ afford greater sensitivity to sample concentration, but less sensitivity to instrument instability, (e.g., sensor temperature and pressure). This is due to the fluorescent light being measured against a very low light background (i.e., against a very low noise level). This is opposite to light absorption measurements, where the signal is superimposed on a strong background signal carrying a high noise level. Unfortunately, relatively few compounds fluoresce in a practical range of wavelengths. However, some compounds (including products from foods, drugs, and dye intermediates) exhibit fluorescence and can be monitored by fluorescent means. In addition, many substances can be made to fluoresce by forming appropriate derivatives.

6.11 Mass spectrometer The mass spectrometer has become the most important detector used in HPLC. A schematic of the basic factors to consider for an HPLC mass spectrometer is shown in Figure 6.4. There are also a wide range of mass spectrometers that are available, from the simplest and cheapest single quadrupole mass spectrometer (it gives molecular ion information with limited fragmentation), through to triple quadrupoles, ion traps, time-of-flight, magnetic sector instruments to the most expensive Fourier-transform ion cyclotron resonance mass spectrometers. Each mass spectrometer gives mass spectra based on the mass-to-charge ratio of the analyte, but can give additional information such as accurate mass, enabling ascertainment of the elemental composition of unknowns. For each mass spectrometer

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there are three basic modes of ionisation for HPLC use. These are electrospray ionisation (ESI), atmospheric pressure chemical ionisation (APCI) and atmospheric pressure photo ionisation (APPI). The method can have a significant impact on the nature of the ionisation, so these modes are discussed in more detail.

Figure 6.4 HPLC mass spectrometer (schematic)

6.11.1 Electrospray Ionisation In ESI, the solvent and sample are nebulised by the use of a gas stream in the presence of a large voltage (≤6 kV) that is applied between the end of a capillary carrying the HPLC mobile phase and the mass spectrometer entrance. As part of this process, the mobilephase solvent evaporates from the droplets (‘desolvates’). The charge density in the droplets increases until the Raleigh limit is reached, and coulomb explosions take place. Under the influence of the electrostatic potentials in the spray chamber, the analyte ion is desorbed from the droplet. This process is affected by the solution chemistry, the flow rate, the sample pKa, the solution pH, and solution conductivity. i˜Vi]ʈÌÃÊ>`Û>˜Ì>}iÃÊ>Ài\ʈÌʈÃÊ̅iÊÜvÌiÃÌʈœ˜ˆÃ>̈œ˜Ê>Û>ˆ>LiÆÊ* Ê -ʈ˜ÌiÀv>ViÊ܈̅Ê̅iÊLiÃÌÊÃi˜ÃˆÌˆÛˆÌÞÆÊ̅iʓ>ÃÃÊÀ>˜}iÊvœÀʓՏ̈«ÞÊ V…>À}i`Ê>˜>ÞÌiÃʈÃÊiÝÌi˜`i`ÆÊ>˜`ʈÌÊܜÀŽÃÊ܈̅Ê>Ê܈`iÊÀ>˜}iʜvʓi`ˆ>Ê 134

High-performance Liquid Chromatography

to highly polar analytes. Its disadvantages are: the solution chemistry ˆ˜yÕi˜ViÃÊ Ì…iÊ ˆœ˜ˆÃ>̈œ˜Ê «ÀœViÃÃÆÊ ˆÌÊ ÜœÀŽÃÊ œ˜ÞÊ vœÀÊ “œ`iÀ>Ìi‡Ìœ‡ …ˆ}…Þ‡«œ>ÀÊ>˜>ÞÌiÃÆÊ>``ÕVÌʈœ˜ÃÊ­œÌ…iÀÊ̅>˜Ê³Ê­i°}°]ʳ >®®Ê >ÀiÊ«œÃÈLiÊ܈̅ÊܓiÊ>˜>ÞÌiÃÆÊ>˜`ʈÌʈÃÊÃi˜ÃˆÌˆÛiÊ̜ʅˆ}…ÊyœÜÊÀ>Ìið

6.11.2 Atmospheric Pressure Chemical Ionisation In APCI, the mobile phase and analyte are nebulised and the droplets are vaporised. Vaporised sample and solvent enter the corona region between the needle and capillary. Nitrogen and solvent molecules are ionised in the corona discharge region, becoming reagent gas ions. The reagent gas ions react with sample molecules, ionising them (Figure 6.5). This process is affected by two main factors: solution chemistry and spray chamber setup. These can then be split further to flow rate, sample pKa, solution pH, solution conductivity, and choice of mobile phase for the solution chemistry. The spray chamber setup comprises: inner needle position, nebuliser pressure, capillary and chamber voltage settings, and vaporiser tempera

e – + N2

Gas Phase

N2+ + solvent

e – N2+ + 2e –

Gas Phase

[solvent+H]+ + N2

[solvent+H]+ + Analyte

Gas Phase

[Analyte+H]+

Figure 6.5 Ionisation theory for APCI

Its advantages are that it is complementary to ESI for less polar analytes and has good sensitivity for compounds of intermediate

135

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molecular weight. It is less sensitive to solution-chemistry effects than ESI and can tolerate higher flow rates without a decrease in sensitivity. The disadvantages are that it requires some compound volatility and is less useful for thermally labile compounds

6.11.3 Atmoospheric Pressure Photo Ionisation APPI is very similar to the APCI source except photons with 10.2 eV are used to ionise the molecules (M) directly:

Ê

ÊÊʳʅv = M³°Ê³Êi

ʜÀʈ˜`ˆÀiV̏ÞÊ܈̅Ê̅iÊÕÃiʜvÊ>Ê`œ«>˜ÌÊ­ ®\

Ê

Ê ÊÊÊʳÊʅνÊÊÊrÊÊ ³°ÊÊʳÊÊi

Ê Ê Ê Ê Ê Ê Ê ³°ÊʳÊÊÊÊÊrÊʳ°ÊʳÊÊ Then M³°ÊʳÊÊ-ÊÊÊrÊÊʳÊʳÊÊ­-‡® So M³°ÊÊcould be the major species very atypical for HPLC MS. The same factors affecting APCI also affect APPI except the mass and polarity range is less. APPI is used only if APCI and ESI modes have failed.

6.11.4 Conclusions Regarding the Mass Spectrometer In general, for extraction analysis, the analytes are of lower molecular weight and are typically more on the non-polar range than polar

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High-performance Liquid Chromatography

range. Hence, the preferred ionisation technique is APCI. This is unless specific information on the analyte is known. However, multimode sources such as ESI/APCI are available but there is a degree of compromise with these multimode sources. They may successfully ionise more species but the overall sensitivity is lower. Another critical point is that HPLC MS is not a panacea. Not everything ionises and, unless suitable chromatography can be achieved, the technique can suffer from ion suppression or ion enhancement that can have a major effect on possible quantification.

6.12 Evaporative Light-Scattering Detector ˜Ê>˜ÊiÛ>«œÀ>̈Ûiʏˆ}…̇ÃV>ÌÌiÀˆ˜}Ê`iÌiV̜ÀÊ­ - ®]Ê̅iÀiÊ>ÀiÊ̅ÀiiÊ parts to the analysis of analytes. In the first part (step 1), the eluent is nebulised by passing through a needle, and mixed with nitrogen gas, forming a dispersion of droplets. These small droplets are selected to minimise background noise. The selection of droplets is a function of their size, preventing larger droplets from entering the evaporation or ‘drift tube’. Large droplets cause increased background noise because they are more difficult to evaporate. This selection of droplets makes detection at low evaporation temperatures possible, resulting in low baseline noise and excellent sensitivity (even for semi-volatile solutes). In step 2 (evaporation), the droplets pass through a heated drift tube where the mobile phase evaporates, leaving a fine mist of dried sample particles in solvent vapour. The small droplets are evaporated at low temperatures to avoid loss of compounds. Solute molecules are obtained from the vapour using low-temperature technology in the heated evaporation or drift tube. The low temperatures used in the standard operating mode minimise the potential of thermal decomposition of the compound of interest. The final step (detection), the sample particles pass through a flow cell where they are hit with a laser light beam. Light scattered by the sample particles is detected, generating an electrical signal. This

137

Update on Undertaking Extractable and Leachable Testing

՘ˆµÕiÊ `iÌiV̈œ˜Ê “i̅œ`Ê ˆÃÊ Ì…iÊ ŽiÞÊ ÌœÊ Ì…iÊ - Ê ÛiÀÃ>̈ˆÌÞÊ >˜`Ê performance. All particles scatter light, so all samples are detected with high sensitivity and accuracy, regardless of their functional groups or optical properties. In theory, all samples are detected with nearly equivalent response factors, making concentration determination easier. However, unsatisfactory quantification, reproducibility, sensitivity and dynamic range have been reported [7, 8].

6.13 Charged Aerosol Detector
Ê V…>À}i`Ê >iÀœÃœÊ `iÌiV̜ÀÊ ­
®Ê ˆ˜ÛœÛiÃÊ ˜iLՏˆÃ>̈œ˜Ê œvÊ Ì…iÊ column eluent with nitrogen. The resulting mobile-phase droplets are dried to remove the mobile phase, which produces analyte particles. A secondary stream of nitrogen becomes positively charged as it passes a high-voltage, platinum corona charger. This charge is mixed with the analyte particles, and charge is transferred to the analyte particles through an absorptive, collision/diffusion-based mechanism that allows for detection of any non-volatile molecule. The charge from the analyte particles is then transferred to a collector, where it is measured via an electrical aerosol analyser, the signal being in direct proportion to the mass of the analyte particles [9, 10].
Ê
ÊÅ>ÀiÃÊ̅iʏˆ“ˆÌ>̈œ˜ÃʜvÊ - ʈ˜Ê̅>ÌÊ̅iÊÀi뜘ÃiÊÛ>ÀˆiÃÊ with the composition of the mobile phase, and peak areas can be increased with the increase of organic additives in the mobile phase ܅i˜Ê}À>`ˆi˜ÌÊiṎœ˜ÊˆÃÊ>««ˆi`°Ê/…iÀiʈÃÊiۈ`i˜ViÊvœÀÊ̅iÊ
ÊÌœÊ œÕÌ«iÀvœÀ“Ê>˜Ê - ÊQ££R°

6.14 Nano Quantity Analyte Detector Another aerosol/nebulisation-based HPLC detector is the nano µÕ>˜ÌˆÌÞÊ>˜>ÞÌiÊ`iÌiV̜ÀÊ­ +
TM®°Ê/…iÊ«Àˆ˜Vˆ«iʜvÊ̅iÊ +
Ê is based on nebulisation and evaporation of the mobile phase at elevated temperature and consequent analyte condensation with supersaturated auxiliary water vapour. This results in the formation

138

High-performance Liquid Chromatography

of relatively large droplets which are later detected using a lightscattering detector. It is this increase in particle size that tremendously increases the light-scattering signal and thus increases the sensitivity. Only particles above a critical size can act as condensation nucleation sites that increase the signal–noise ratio due to discrimination of small droplets from the mobile phase [12]. Unlike other aerosol-based detectors, there is no interference from sensor noise or drift that can interfere with resolution and sensitivity [13].

6.15 Typical Starting Conditions for Extractable Analysis Without any information on the analytes being extracted, very generic conditions should be employed. This will involve a generic column with an estimated pH to provide some degree of buffering capacity, >œ˜}Ê܈̅Ê>Ê܈`iÊ}À>`ˆi˜ÌÊiṎœ˜Ê>˜`ʓՏ̈«iÊ`iÌiV̜ÀÃ°Ê iÌiV̜ÀÃÊ would typically be a UV detector followed by a mass spectrometer in series. Good starting conditions are presented in Table 6.2. Typically, leachable analysis will employ exactly the same conditions as those used in extractable testing. These conditions will not be perfect. However, they will make a good start until more information on the analytes can be found and conditions optimised.

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Update on Undertaking Extractable and Leachable Testing

Table 6.2 Starting HPLC analytical conditions for extractable or leachable analysis Parameter

Condition

Flow rate:

1 ml/min

Injection volume:

20 μl

UV wavelength:

210-480 nm

Column:

C18

Temperature

50 °C

Mobile phase:

A 5 mM ammonium acetate in H2O B MeOH

Gradient

Mass spectrometer

Time (min)

%B

0

20

20

98

30

98

31

20

APCI positive and negative mode of ionisation from m/z 150-1500 amu

References 1.

°,°Ê>ââiœ]Ê1° °Ê iÕi]Ê°ÊiiÊ>˜`Ê,°-°Ê*Õ“L]ÊAnalytical Chemistry, 2005, 77, 460A.

2.

°,°Ê-˜Þ`iÀ]Ê°°ÊˆÀŽ>˜`Ê>˜`Ê°°Ê>V…ʈ˜ÊPractical HPLC Method Development, 2nd Edition, John Wiley & Sons, Inc., New York, NY, 1997.

3.

°7°Ê œ˜}ʈ˜ÊModern HPLC for Practicing Scientists, Wiley-Interscience, USA, 2006.

140

High-performance Liquid Chromatography

4.

°Êˆ“>Ì>]Ê°ÊÜ>}ÕV…ˆ]Ê-°Ê"˜ˆÃ…ˆ]ʰʈ˜˜œ]Ê,°Ê ŽÃÌii˜]Ê °ÊœÃœÞ>]Ê°Ê
À>ŽˆÊ>˜`Ê °Ê/>˜>Ž>]ÊJournal of Chromatographic Science, 1989, 27, 721.

5.

M.R. Euerby, P. Petersson, W. Campbell and W. Roe, Journal of Chromatography A, 2007, 1154, 138.

6.

M.R. Euerby and P. Petersson, Journal of Chromatography A, 2003, 994, 13.

7.

°9°Ê<…œÕ]Ê*°Ê …>“ˆ˜>`i]Ê°Ê>Õ`ˆ˜]Ê*°Ê*Àœ}˜œ˜]Ê
°Ê >ˆiÌÊ >˜`Ê °ÊiÀÀˆiÀ]ÊJournal of Chromatography A, 1999, 859, 99.

8.

°Ê>Õ`ˆ˜]Ê
°Ê >ˆiÌÊ>˜`Ê*°Ê …>“ˆ˜>`i]ÊJournal of Chromatography A, 2004, 1051, 43.

9.

T. Górecki, F. Lynen, R. Szucs and P. Sandra, Analytical Chemistry, 2006, 78, 3186.

10. ,°7°Ê ˆÝœ˜Ê>˜`Ê °-°Ê*iÌiÀܘ]ÊAnalytical Chemistry, 2002, 74, 2930. 11. °Ê-…>œ`œ˜}]Ê7°Êii]Ê°Ê i]Ê°Ê*>ÀŽ]Ê-°Êܜ˜Ê>˜`Ê°Êii]Ê Journal of Pharmaceutical and Biomedical Analysis, 2010, 51, 973. 12. °Ê"ÃœÛÎ>ÆÊ<°Ê>“i˜ˆŽÊ>˜`Ê/°Ê >Ì…>“]ÊJournal of Chromatography A, 2009, 1216, 30, 5774. 13.
°°ÊœÀœ«V…>Ž]ÊJournal of Analytical Chemistry, 1993, 65, 841.

141

7

Inductively Coupled Plasma Metal Analysis

For the analysis of metals, inductively coupled plasma (ICP)-optical emission spectrometry and/or ICP–mass spectrometry (MS) are the most common instrument types for elemental determinations. These have replaced the older atomic absorption spectrophotometry. ICP is formed by coupling the energy from a radiofrequency (1–3 kW or 27–50 MHz) magnetic field to free electrons in a suitable gas, typically argon. The magnetic field is produced by a water-cooled coil, and the electrons are accelerated in circular paths around the magnetic field lines that run axially through the coil. Neutral particles are heated indirectly by collisions with the charged particles upon which the field acts. The field does not penetrate the conductor uniformly and therefore the largest current flow is at the periphery of the plasma. This is the so-called ‘skin’ effect and, coupled with suitable gasflow geometry, it produces an annular or doughnut-shaped plasma. The present generation of ICP emission spectrometers provides: limits of detection in the range 0.1–500 μg/l of metal in solution; a substantial degree of freedom from interferences; and a capability for simultaneous multi-element determination facilitated by a directly proportional response between the signal and the concentration of the analyte over a range of about five orders of magnitude. The most common method of introducing liquid samples into the ICP is by using pneumatic nebulisation, in which the liquid is dispensed into a fine aerosol by a high-velocity gas stream. The fine gas jets and liquid capillaries used in ICP nebulisers may cause inconsistent operation and even blockage if solutions containing high levels of dissolved solids, or particular matter, are used. These problems have been overcome by using nebulisers based on the following design: the liquid is pumped from a wide-bore tube and then to the nebulising

143

Update on Undertaking Extractable and Leachable Testing

orifice by a V-shaped groove [1] or by the divergent wall of an overexpanded nozzle [2]. Such devices are able to handle most liquids and even slurries without difficulty. ICP-MS is the method of choice for the determination of a very wide range of trace elements, with detection limits down to and below 1 part per trillion. The basic principle of ICP-MS is as follows. Samples are decomposed to neutral elements in high-temperature argon plasma and analysed based on their mass-to-charge ratios. An ICPMS can be thought of as four main processes with the first two being the same as basic ICP (including sample introduction and aerosol generation and ionisation by an argon plasma source) followed by mass separation and the detection system. ICP-MS does not depend on indirect measurements of the physical properties of the sample. The elemental concentrations are measured directly; individual atoms are counted, giving the key attribute of high sensitivity. The technique has the additional benefit of unambiguous spectra and the ability to directly measure isotopes of the same element. Typical limits of detection in the pharmaceutical industry are presented in Table 7.1. These limits are derived from the permitted daily exposure limits provided in the European Medicines Agency Guideline [3] as well as the limits proposed in the stimulus article published in Pharmacopeial Forum, which recommends revisions to the United States Pharmacopeia General Chapter on Inorganic Impurities [4].

144

Inductively Coupled Plasma - Metal Analysis

Table 7.1 European Medicines Agency (EMEA) and United States Pharmacopeia (USP) limits for metals Metal Al Sb As Be B Cd Cr Co Cu Fe In Ir Pb Li Hg Mg Mn Mo Ni Os Pd Pt Rb Rh Ru Se Sr Tl Sn V W Zn

EMEA limit (ppm) NAa NA NA NA NA NA 2.5b NA 25 25 NA 1 NA NA NA NA 25 2.5 2.5c 1 1 1d NA 1 1 NA NA NA NA 2.5 NA 130

USP limit (μg/g) 500 0.2 0.15 1 100 0.25 1.5 10 5 150 1 1 0.1 6 0.15 Under deliberation 70 2.5 10 1 1 1 Under deliberation 1 1 2.5 300 0.04 300 NA 3.8 15

a

NA = Not Applicable The limit for Cr (VI) via inhalation is 10 ng/day c The limit for Ni via inhalation is 100 ng/day b

145

Update on Undertaking Extractable and Leachable Testing

References 1.

R.F. Suddendorf and K.W. Boyer, Analytical Chemistry, 1978, 50, 13, 1769.

2.

B.L. Sharp, inventor; no assignee; GB 8,432,338, 1984.

3.

EMEA Guideline on the Specification Limits for Residual Metal Catalysts or Metal Reagents, EMEA/CHMP/ SWP/4446/2000.

4.

Anon, Pharmacopeial Forum, 2008, 34, 5, 1345.

146

8

Conclusion

Extraction and analysis of extractables and leachables is a very challenging area. It requires a high degree of knowledge and skill to quantifiably extract and then identify a wide range of analytes from a range of matrices at trace or ultra-trace levels. No single extraction technique can extract all species without deforming/degrading the base material. Pushing the extraction to the point just before the material is deformed/degraded can be quite a challenge, one that sometimes inexperienced analysts fail. No extraction technique is perfect and each technique has its own disadvantages but also advantages, whether they be speed, ease of use, cost of the equipment, sample preparation or extraction efficiency. The relative advantages of each technique can vary depending on the solvent used, the analyte(s) to be extracted. However, the factor that has the largest influence is the matrix. This assessment of multiple extraction techniques has been done for several analytes but never a complete set. Examples exist for explosives in soil [1], and polynuclear aromatic hydrocarbons in soil, but this has not been assessed for all the techniques and for all solvents. One of the most comprehensive extraction studies exploring the widest range of extraction techniques and solvents is being undertaken by the (Extractable and Leachable Safety Information Exchange) ELSIE group. However, this will partly change once the ELSIE group completes a controlled extraction study using most of the common extraction techniques and a wide range of solvents. The variability of each technique is one of the main reasons why guidance exists to specify that multiple extraction techniques are used to carry out controlled extraction studies.

147

Update on Undertaking Extractable and Leachable Testing

In addition, no single analytical technique can identify and quantify all analytes. This can also be have the added challenge of not knowing what the analytes may be. As with all analysis, the more information that is available on the analytes the easier the analysis can be. It can be thought of as finding a needle in a haystack and at least knowing what the needle looks like. Also, the better the sample preparation in terms of the purity of the solution and the optimisation of the concentration of the analytes the easier the analysis can be. Information from the material supplier and along the supply chain can be critical in making the extraction study easier. To give an idea of the scale of the number of species that could be present, a database by Toxicon [2] has shown that they have 1600 volatile, semi-volatile or non-volatile organic compounds. All this adds to the complexity of the study, and that is before the latest paradigm from the regulators of quality by design. The basic premise of this is that quality cannot be tested into a product it has to be designed in. To explore this, multiple batches of materials must be tested, covering the input materials but also the design space of the processing conditions. Figure 8.1 shows a material processing design space covering moulding temperature and time. Hence, extractable testing could comprise, for a very simple pressurised metered-dose inhaler (pMDI) with five different components, a total of 270 analyses (assuming three different batches tested, with three solvents, two extraction techniques and three analytical techniques). This is the smallest number of analyses that can be undertaken because it is assumed that everything will be quantified and identified in a single analysis and that asymptotic levels are achieved in a single analysis. Also, this number assumes that no special case species are identified as possible extractables.

148

Minimum processing time

Max. vendor recommended processing time

C

D

Temp

“WindowB.” for material extractables evaluation

B

“Window A” for material extractables evaluation

Max. vendor recommended processing temperature

Required Processing window for dimension control of component

Minimum processing temp

A Processing Time

149

Conclusion

Figure 8.1 Material-processing design space

Update on Undertaking Extractable and Leachable Testing

Once the controlled extraction is completed, the species need to be assessed toxicologically: this can be a very time-consuming process. An estimated time to toxicologically assess butylated hydroxytoluene (a species for which a plethora of information was available) was 30 h. This is probably an extreme value, but puts into scale the effort required to assess maybe 30-40 extracted species. Finally comes leachable testing, which will require testing at accelerated conditions over the shelf-life of the product (maybe 6 time-points), with suitably validated methods. These methods require the following parameters to be investigated (see [3] for more details): UÊ ˆ˜i>ÀˆÌÞ Ê

UÊ

,>˜}i

UÊ
VVÕÀ>VÞ UÊ -ÞÃÌi“Ê«ÀiVˆÃˆœ˜Ê­ˆ˜ÃÌÀՓi˜ÌÊ«ÀiVˆÃˆœ˜® UÊ i̅œ`Ê«ÀiVˆÃˆœ˜ Ê

UÊ

,i«i>Ì>LˆˆÌÞ

Ê

UÊ

˜ÌiÀ“i`ˆ>ÌiÊ«ÀiVˆÃˆœ˜

UÊ -«iVˆwVˆÌÞ UÊ iÌiV̈œ˜Êˆ“ˆÌɵÕ>˜ÌˆÌ>̈œ˜Êˆ“ˆÌ UÊ -Ì>˜`>À`Ê>˜`ÊÃ>“«iÊÃÌ>LˆˆÌÞ UÊ ,œLÕÃ̘iÃà UÊ -ÞÃÌi“ÊÃՈÌ>LˆˆÌÞ Three methods would need to be validated covering the volatile, semi-volatile and metal species. Validating methods with multiple analytes (for pMDI this could be >30 species) over potentially wide ranges (some species may be present at μg/container closure system to mg/container closure, a three-order of magnitude range). Overall,

150

Conclusion

this is a very time-consuming and challenging undertaking. In the pharmaceutical industry, there are several groups (ELSIE and the Polymer Forum) that are trying to simplify the extraction and analysis of container closure systems. Nevertheless, this simplification requires an understanding of the principles of sample preparation, the extraction process, subsequent analysis and how they are all interlinked.

References 1.

T.F. Jenkins and C.L. Grant, Analytical Chemistry, 1987, 59, 9, 1326.

2.

P. Christiaens, J-M. Beusen, F. De Smedt, A. Cerstiaens, L. Habel, P. Geys, I. Janssen, D. Verbiest and B. Palmaers, The Toxikon Europe Screener Database: a powerful tool for the identification of organic compounds during Controlled Extraction Studies for OINDPs, http://www.toxikon.be/documents/pdf/DDL%202010%20 Abstract%20for%20presentation%20-%20Toxikon%20 Europe.pdf

3.

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedures,  Ê+ÓÊ­,£®]Ê 1996.

151

A

bbreviations

AET

Analytical evaluation threshold

ALARP

As low as reasonably practicable

APCI

Atmospheric pressure chemical ionisation

APPI

Atmospheric pressure photo-ionisation

ASE

Accelerated solvent extraction

ASTM

American Society for Testing and Materials

CAD

Charged aerosol detector

CCS

Container closure system(s)

CE

Capillary electrophoresis

CES

Controlled extraction study

CI

Chemical ionisation

DAD

Diode array detector

DART

Direct analysis in real time

DCM

Dichloromethane

DESI

Direct electrospray ionisation

DHS

Dynamic headspace

153

Update on Undertaking Extractable and Leachable Testing

DLLME

Dispersive liquid–liquid microextraction

DVB

Divinylbenzene

ECD

Electron capture detector

EI

Electron impact

ELSD

Evaporative light scattering detector(s)

ELSIE

Extractable and Leachable Safety Information Exchange

EMEA

European Medicines Agency

ESI

Electrospray ionisation

FI

Field ionisation

FID

Flame ionisation detectors

FMAE

Focused microwave assisted extraction

FPD

Flame photometric detector

GC

Gas chromatography

GC-MS

Gas chromatography-mass spectrometry

GFC

Gel filtration chromatography

HFA

Hydrofluoroalkane

HFLPME

Hollow fibre liquid phase microextraction

HPLC

High-performance liquid chromatography

IC

Ion chromatography

ICP

Inductively coupled plasma

ICP-MS

Inductively couple plasma–mass spectrometry

154

Abbreviations

id

Internal diameter

ISO

International Organization for Standardization

ITEX

In tube extraction

LC

Liquid chromatography

LESA

Liquid extraction surface analysis

LLE

Liquid-liquid extraction

LPMESFOD Liquid-phase microextraction solidification of floating organic drop MAE

Microwave-assisted extraction

MDI

Metered-dose inhaler

MeCN

Acrylonitrile

MeOH

Methanol

MEPS

Microextraction by packed sorbent

MS

Mass spectrometry

NMR

Nuclear magnetic resonance

NPD

Nitrogen phosphorus detector

NQAD™

Nano quantity analyte detector

OINDP

Orally inhaled and nasal drug products

PAH

Polycyclic aromatic hydrocarbon(s) or polynuclear aromatic hydrocarbons

PCB

Polychlorinated biphenyls

PDMS

Polydimethyl siloxane

155

Update on Undertaking Extractable and Leachable Testing

PFE

Pressurised fluid extraction

PGI

Potential genotoxic impurities

PMAE

Pressurised microwave-assisted extraction

pMDI

Pressurised metered-dose inhaler

PODP

Parenteral and Ophthalmic drug products

ppb

Parts per billion

ppm

Parts per million

PQRI

Product Quality Research Institute

PSE

Pressurised solvent extraction

PTV

Programmable temperature vaporisation

QT

Qualification threshold

RF

Response factor

RI

Refractive index

RRF

Relative response factor

SBSE

Stir-bar sorptive extraction

SCT

Safety concern threshold

SEC

Size exclusion chromatography

SDME

Single-drop microextraction

SFC

Supercritical fluid chromatography

SFE

Supercritical fluid extraction

SPE

Solid-phase extraction

156

Abbreviations

SPME

Solid-phase microextraction

S/S

Split/splitless

TCD

Thermal conductivity detectors

TEA

Thermal energy analyser

TFA

Trifluoroacetic acid

THF

Tetrahydrofuran

TOC

Total organic carbon

TOF

Time-of-flight

UPLC

Ultra-performance liquid chromatography

USP

United States Pharmacopeia

UV

Ultraviolet

157

INDEX

Index Terms

Links

A Accelerated solvent extraction

9

dynamic

43

speed extractor

44

static

43

Acetonitrile

124

Allihn condenser

25

14

42

44

27

American Society for Testing and Materials Analysis, low temperature plasma Analyte capacity

19 55 106

Analytical evaluation threshold

15

Analytical methods, feedback loop

17

Antistatic agents

8

Analysis probe, atmospheric solids

54

Analysis, trace-level

89

Atomic absorption spectrophotometry

92

101

143

B Beer-Lambert Law

131

Base rubber, blob

7

Blowing agents

8

Büchi apparatus

45

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Büchi autoclave extraction

42

Büchi extraction system

31

continuous-flow Soxhlet mode

34

speed extractor

45

Bulb condenser

25

C Capillary column

104

Carrier gas

100

Centrifuge concentrator

19

Changed aerosol detector

138

Chemical ionisation

113

atmospheric pressure

93

direct atmospheric pressure

54

surface activated

55

Column flow rate

134

110 104

Column length

106

Columns, narrow-bore

104

Columns, porous-layer open tubular

105

110

Complexing agent

40

Compounds, hydrophilic

70

Compounds, hydrophobic

70

Condenser, spiraled finger

26

Container closure systems

1

8

Controlled extraction study

4

10

Cool-on-column

100

103

Cool-on-cool injection

119

111

120

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Cryomill

6

Curatives

8

37

D Desorption ionisation, plasma-assisted

54

Derivatisation

118

in situ

82

reagent

40

Desorption ionisation, dielectric barrier Detector, magnetic sector

54 114

Dichloromethane

24

Dielectric constant

46

interaction

126

loss factor

47

Diode array detector

132

Dipole interactions

105

37

Direct analysis

53

in real time

13

53

105

126

Dispersive liquid-liquid microextraction

70

75

Dissipation factor

47

Dry powder inhaler

16

Dry-ice/acetone condenser

36

Dispersive interactions

Dry-lab software Dynamic headspace

51

53

108 57

59

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

E Electrical aerosol analyser

138

Electron capture detector

116

Electron impact mass spectra

115

Electrophoresis, capillary

90

93

Electrospray ionisation

93

134

chip

60

desorption

53

extractive

54

fused droplet

54

infrared laser ablation

55

jet desorption

54

laser ablation

54

laser desorption

54

mass spectrometry

55

neutral desorption extractive

54

Raleigh limit

136

134

Electrospray metastable-induced ionisation, desorption 555 Endcapping, degree of

128

Energy dispersive X-ray

56

Enhanced-fluidity liquid extraction

41

European Medicines Agency

145

Evaporative light-scattering detector

137

Evaporative light-scattering, drift tube

137

Extractable analysis, ultra trace level

89

Extractable and leachable safety information exchange

85

This page has been reformatted by Knovel to provide easier navigation.

Index Terms Extraction, headspace Extraction, in-tube Extraction, pressurised assisted

Links 9

14

56

60

73

81

83

109

57

70

77

9

14

Extraction, pressurised fluid

42

Extraction, pressurised microwave-assisted

46

Extraction, pressurised solvent

28

41

Extraction, restrictor

38

41

Extraction, shaking

9

14

49

70

72

78

103

113

Extraction, solid-phase cartridges

72

F Fibres, absorbent-type

73

Fibres, adsorbent-type

73

Field ionisation

113

Fillers/reinforcing agents

8

Flame ionisation detector

17

117

119 Flame photometric detector Flow modifiers Flowing atmospheric-pressure afterglow

117 8 55

Fluorescence detector

132

photomultiplier

132

Focused microwave-assisted extraction

46

Fourier-transform ion cyclotron resonance mass spectrometers Friedrich condenser

133 26

28

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

G Gas chromatography

19

23

49

51

57

73

80

84

90

93

97

100

102

104

110

121

127 band broadening

111

injection temperature

108

injection volume

108

liquid injection

100

sample injector

101

injector degradation

110

inlet liners

109

111

mass-to-charge ratio

114

144

oven temperature gradients

108

temperature

101

Gas chromatography - mass spectrometry

80

93

97

Gas chromatography – mass spectrometry - electron impact

113

Gas chromatography - solid-phase microextraction

75

Gel filtration chromatography

91

Graham condenser

24

26

H Helium atmospheric pressure glow discharge ionisation Helium carrier gas

54 110

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Hydrofluoroalkane 134a

11

35

Hydrofluoroalkane 227

11

35

High-performance liquid chromatography

17

19

23

30

37

70

73

84

93

121

125

133

140 capacity

129

chromatography

125

detector

129

inlet system

51

ion-exchange

122

ion pairing

122

mobile phase

134

liquid

121

normal-phase

124

mass spectrometry

137

125

shape selectivity of stationary phase 129 ultraviolet detection HIPAC software Hollow-fibre liquid-phase microextraction Holographic grating

90 108 71

76

132

Hydrofluoroalkane propellant

35

Hydrogen bonding interactions

105

Hydrophobicity

129

107

126

I Identification report, confident

95

Identification report, confirmed

95

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Identification report, tentative

95

Identification, tentative

95

Infrared spectra

13

53

Inhaler, metered-dose

10

16

Inlet liner, Carbofrit

109

International Organisation for Standardisation Tests

4

Ion chromatography

91

Ionisation, direct electrospray

13

Ion trap Ion-pair reagent Isothermal analysis

114

133

40

124

107

L Laser ablation flowing atmosphericpressure afterglow Laser diode thermal desorption

55 54

Leachable analysis

119

140

Leachable testing

125

150

Limit of detection, qualification threshold

15

Limit of detection, safety concern threshold

15

Liquid chromatography

52

82

121

131

90

93

Liquid chromatography – mass spectrometry

82

Liquid extraction, salting-out effect

70

Liquid extraction, surface analysis

60

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Liquid extraction techniques, ion mobility

60

84

Liquid micro-junction surface sampling probe/electrospray ionisation Liquid-liquid extraction

55 69

80

70

76

83

Liquid-phase microextraction solidification of floating organic drop Lixiviation

13

M Mass spectrometer

detectors

13

17

19

52

93

113

122

127

133

136

140

49

119

Matrix-assisted laser desorption electrospray Mercaptobenzothiazole, analysis of Methanol

54 12

89

124

126

Microextraction by packed sorbent

79

Microextraction, single-drop

70

75

Microextraction, solid phase

57

60

70

77

80

81

Micro-extraction by packed sorbent

70

78

Micro-liquid-liquid extraction

71

Microwave assisted extraction

28

41

73

46

Microwave-assisted extraction Microwave energy Microwave extraction

47 9

14

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Mobile phase

30

99

122

138

N Nano quantity analyte detector Nitrogen chemiluminescence detector Nitrogen phosphorus detectors

138 19

118

107

117

Nitrosamines

18

Nuclear magetic resonance spectroscopy

90

O Optical emission spectrometry, inductively coupled plasma

143

mass spectrometry

144

Orally inhaled and nasal drug products

2

5

15

P Packaging, direct contact

1

Packaging, indirect

1

Packaging, primary

1

Paracelsus 15 Parenteral and ophthalmic drug products

2

Partitioning coefficient

69

Peltier cooling

58

Phenylmethyl polysiloxane phase

107

Phosphorescence

133

Photo-ionisation, atmospheric pressure

136

Photo-ionisation, atmospheric pressure

93

134

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Photo-ionisation, desorption atmospheric pressure Photoluminescence

54 133

Polyaromatic hydrocarbons

13

Polychlorinated dibenzo-p-dioxins

40

Polycyclic aromatic hydrocarbons

40

Polydimethyl siloxane

80

Polynuclear aromatic hydrocarbons

13

Potential genotoxic impurities

16

Pressurised metered-dose inhaler

10

16

148

150

4

15

Product Quality Research Institute document

16

guidance

12

Programmable temperature vaporisation injector Pseudo molecular ion

100

89

18

35

24

27

112

112 114

Q Quadrupole spectrometer

114

R Raman spectroscopy Reflux condenser Refractive index detection

13

53

9

14

25 131

Remote analyte sampling transport and ionisation relay

55

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Response factor

16

relative

92

92

95

122

125

S Single-particle aerosol mass spectrometry

55

Size-exclusion chromatography

91

Slip agents

8

Solid phase extraction, mixed-mode

72

Solid phase extraction, ion exchange

72

Solid phase extraction, reversed phase

72

Solvent extraction, membrane-assisted

1

Solvent extraction, pressurised Sonication

42 9

14

23

42

1

9

13

29

34

37

42

29

32

35

122

128

Sonic spray ionisation, desorption

54

Sonic spray ionisation, easy ambient

55

Soxhlet extraction apparatus sub-ambient

36

warm mode

31

Soxtherm extractors

29

Split flow ratio

110

Split/splitless injector

101

Stabilisers Stationary phase

83

8 105

hydrogen bonding capacity

129

non-polar

107

polar

107

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Stationary phase (Cont.) solid

121

Stir bar sorptive extraction

70

73

Stirring, magnetic flea

76

80

Supercritical fluid chromatography

37

Supercritical fluid extraction Surface sampling probe Solvent selectivity, Synder triangle

9

14

80

37

54 126

T Temperature system, constant-filament

117

Tetrahydroduran

124

Thermal conductivity detectors Thermal desorption ionisation atmospheric pressure Thermal energy analyser

17

116

9

14

53

80

82

100

19

118

56

54 17

Time-of-flight secondary ion mass spectrometry

56

Total organic carbon

60

Twister bar

80

84

U Ultra-performance liquid chromatography

121

Ultraviolet detection

17

Ultraviolet detector

93

Universal detectors

93

US Pharmacopeia

4

122

131

139

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

V Vaporisation

109

Variable wavelength detector

132

Viscosity

37

41

W Wheatstone bridge network

116

This page has been reformatted by Knovel to provide easier navigation.