Principles and Practice of Modern Chromatographic Methods

Principles and Practice of Modern Chromatographic Methods

Second Edition

Kevin Robards

Emeritus Professor, Charles Sturt University, Wagga Wagga, NSW, Australia

Danielle Ryan

Senior Lecturer in Chemistry, Charles Sturt University, Wagga Wagga, NSW, Australia

Table of Contents

Cover image

Title page

Copyright

Preface

Preface to first edition

Acknowledgements

Caution

Chapter 1: Introduction and overview

Abstract

1.1: Introduction

1.2: Coverage

1.3: Chromatographic separation simply explained

1.4: Classification of chromatography

1.5: Chromatography: Publications and equipment sales

1.6: Applications of chromatography

1.7: Comparison of chromatographic techniques

1.8: Historical aspects

1.9: Obtaining assistance

References

Chapter 2: Theoretical considerations

Abstract

2.1: Introduction

2.2: Theory of chromatography

2.3: Retention, dispersion, and distortion

2.4: Retention: Thermodynamics and molecular interactions

2.5: Retention

2.6: Measures of chromatographic retention

2.7: Dispersion

2.8: Is it resolution or separation?

2.9: Extra-column dispersion

2.10: Summary: Retention and dispersion

2.11: Column testing

References

Chapter 3: Planar chromatography

Abstract

3.1: Introduction

3.2: Why thin layer chromatography?

3.3: Theoretical considerations

3.4: Sample application in TLC

3.5: Plate development in TLC

3.6: Innovation in TLC

3.7: Thin layer plates

3.8: Stationary phases

3.9: Mobile phases

3.10: Detection: Qualitative analysis

3.11: Quantitative analysis

3.12: Separations

References

Chapter 4: Gas chromatography

Abstract

4.1: Introduction

4.2: Mobile phases and delivery systems

4.3: Sample introduction in GC

4.4: Column packings and hardware

4.5: Detectors

4.6: Column temperature and temperature programming

4.7: Derivatization

4.8: Innovation

4.9: Separations

References

Chapter 5: High performance liquid chromatography: Instrumentation and techniques

Abstract

5.1: Introduction

5.2: From HPLC to UHPLC and beyond

5.3: Solvent delivery systems

5.4: Sample introduction in HPLC

5.5: Column packings and hardware

5.6: Detectors

5.7: Post-column derivatization

5.8: Gradient elution

5.9: Multidimensional liquid chromatography

References

Chapter 6: High performance liquid chromatography: Separations

Abstract

6.1: Introduction

6.2: Separation of neutral compounds

6.3: Techniques for ionic and ionizable species

6.4: Specialty separation modes

6.5: Choosing a chromatographic method

References

Chapter 7: Supercritical fluid chromatography

Abstract

7.1: Introduction

7.2: Mobile phases and delivery systems

7.3: Sample introduction in SFC

7.4: Column packings and hardware

7.5: Detectors

7.6: Programming techniques

7.7: Separations

References

Chapter 8: Coupled systems

Abstract

8.1: Introduction

8.2: Multidimensional chromatography

8.3: Hyphenated techniques

References

Chapter 9: Analyses

Abstract

9.1: Introduction

9.2: Detectors for chromatography

9.3: Data handling

9.4: Qualitative analysis

9.5: Quantitative analysis

9.6: Metabolomics and chromatography

References

Chapter 10: Sample handling in chromatography

Abstract

10.1: Introduction

10.2: Why bother with sample handling?

10.3: Sample collection procedures

10.4: Sustainable green chemistry

10.5: Sample preparation

10.6: Pre-concentration techniques

References

Chapter 11: Preparative chromatography

Abstract

11.1: Introduction

11.2: Thin layer chromatography

11.3: Gas chromatography

11.4: Supercritical fluid chromatography

11.5: High-performance liquid chromatography

References

Index

Copyright

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Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

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Preface

My (KR) first encounter with chromatography was at the tail end of the 1960s with paper and thin layer chromatography and then with a gas chromatograph manufactured in the workshops of the University of New South Wales, Sydney, Australia. Output from the gas chromatograph was captured on a chart recorder which constantly slipped a gear or failed completely, aborting a chromatographic run. However, looking back I was fortunate as I did not have to generate my chromatograms by plotting them from spot galvanometer readings as did the very early pioneers. On the other hand, perhaps I was the unfortunate one as I am sure that the experience of those early pioneers developed an association with, and feel for, the technique of which we latecomers can only dream. It is appropriate that the second author (DR) was involved in the early days of two-dimensional comprehensive GC with Phil Marriott for it was the introduction of this modification that heralded the need for new demands on instrumentation and methods of data management.a

Our basic goal in re-writing is unchanged from the first edition and that is to provide:

•new users of chromatography with an historical context and the basic knowledge and terminology to get started in this field, and

•experienced users with a revision of the fundamentals of chromatography and its various techniques.

Why the interest in terminology and definitions? The saying that precise logical definitions of concepts are a fundamental prerequisite to true knowledge is attributed to Socrates. It is doubtful that an individual can develop a full comprehension of the science of chromatography without the appropriate language. The terminology associated with chromatography has often been used rather loosely with many texts failing to distinguish different terminologies and classification systems. Clear enunciation and differentiation of classification systems was a strength of the first edition that has been built upon in this second edition which is clearer and more logical particularly in the exposition of chromatographic theory which has been extensively revised and expanded.

As in any area, the language and associated definitions must evolve and develop with progress for, indeed, nothing in this life is constant; change is universal. The extent, nature, and rate of change have differed greatly both within and between the different areas of chromatography. For example, gas chromatography as a mature field has undergone evolutionary change in the last 30 years where revolutionary change has characterized some other areas. Columns in gas chromatography are essentially the same now as they were in 1990 although new column chemistries have been developed. One thing that has changed is the acceleration in the abuse of nomenclature. Coupled, hyphenated, multidimensional, etc. have always presented a nomenclature problem and there have been numerous attempts to resolve the situation. The historical development of the field of chromatography and the independent development and timing of developments contributed to these problems with nomenclature. However, instead of learning from past mistakes the problem has proliferated with terms such as high speed, fast, and micro gas chromatography being used with wild abandon presumably in some instances to make a paper more appealing to readers and convince editors to accept for publication. This is confusing and obfuscates the ability of a newcomer to integrate with the field.

Searching the literature demonstrates a large change in research output since the first edition was written. Then, research into chromatographic techniques and instrumentation including consumables was relatively prolific; now it is much more sparse except in a few isolated instances. The other major difference across all areas of chromatography has been the embrace of computer technology. Computer systems now both control the instrument and process and report the results. One might expect that this would allow more time for contemplation of the system and how it functions. However, just the opposite has occurred with many chromatographers becoming operators with very little knowledge of the fundamental processes that control the separation. The result is a generation of ill-prepared analysts.b,c

It is fortunate that within all this change, some things are constant apart from death and taxes; the fundamental chemistry and basic principles of chromatography are unaltered from its inception although our understanding may have improved. The deep and concrete knowledge of basic theory behind separation science and mainly chromatographic techniques is fundamental for any analyst or lab practitioner. The users of chromatographic instruments must be aware of the theoretical aspects of their techniques in order to be able to develop and apply specific analytical methods according to their needs (unknown). This book provides this essential information to readers in a clear and concise style.

The book has been extensively revised and updated with the addition of a number of new topics plus updated schematic overviews of the content of each chapter. Topic coverage is both integrated and comprehensive based on chromatographic bibliometrics and survey reports on the relative usage of chromatographic techniques. However, the philosophical approach has not changed from the first edition. The book uses language that is clear and concise with a style and format that engages the reader and facilitates a deeper understanding – a clear, logical development of concepts and ideas that avoids the obfuscation of different terminologies and classification systems prevalent in the area. This approach makes it easier for both specialist and non-specialist readers to bridge gaps between existing knowledge and their needs (e.g. specialist to move from one technique in chromatography to another; non-specialist to obtain a comprehensive overview of chromatography).

This book will be of value and interest to both postgraduate students and undergraduate students, not only those studying chemistry, but other scientific topics as well, e.g. biology, pharmacy, forensic toxicology, etc. It will benefit all practitioners/analysts involved in all fields of applications of chromatography; not only those who lack the fundamental background in chromatography but also those who wish to refresh their knowledge with updated information. Those involved in criminal forensic law will find it a useful introduction to an area that features prominently in forensic cases.d The book will benefit users of chromatography, either those experienced in one area of chromatography that need to move to another, or new users who want to understand the background of what they do.

Preface to first edition

Chromatography is an established analytical procedure with a history of use spanning at least seven decades. It is, nevertheless, a continuously evolving technique with new variants and modified procedures. In many ways this has led to a plethora of terms that are confusing to the specialist and beginner alike. Most texts currently available are written for the specialist and concentrate on one particular form of chromatography. This is understandable, given the volume of information available on each of them but it is not of much help to the user requiring an overview of developments across all areas of chromatography. Certainly, a unified approach is essential for the novice but should also be of assistance to the specialist suddenly faced with the need to switch from one technique to another.

The authors have taught and researched extensively in both academic and industrial areas. The intention in writing this text was to appeal to as wide an audience as possible. To the non-chemist it is hoped that this material will provide an easy-to-read overview in an area that has had a profound effect in fields as diverse as clinical chemistry, geology, and food science. For many scientists engaged in these areas, their first real contact with chromatography comes when faced with an analytical problem requiring the separating power that only chromatography can provide. To the practising chromatographer involved in research or routine analyses it will provide an update in those techniques with which they are less familiar. Students will find the material suitable as an undergraduate text.

a Colin F. Poole, J. Chromatogr. A 1421 (2015) 1.

b Laura Bush, LCGC North America 30(8) (2012) 656.

c Colin F. Poole, and Salwa K. Poole, Journal of Chromatography A, 1184 (2008) 254. Colin Poole J. Chromatogr. A 1250 (2012) 157.

dhttps://www.paduiblog.com/pa-dui/a-large-problem-in-gas-chromatography-no-uniform-standard-for-gc-run-position-or-composition/.

Acknowledgements

The publication of the second edition of this book fulfils an ambition of long standing. The authors thank all who have contributed to the realization of this ambition. A special thanks to Sergio Dilli who inspired my (KR) interest in chromatography, and to KR and Phil Marriott for their exceptional mentoring (DR). Thanks are also extended to our many students over several decades who have sharpened our interest and enhanced our knowledge. We also thank the members of our families who have endured our passion and interest in this absorbing science. The contribution of Emeritus Professor Paul Haddad and Dr. Peter Jackson to the first edition is gratefully noted.

Caution

The procedures and reagents described herein can be dangerous and involve high temperatures and pressures with chemicals of potentially high or unknown toxicity. Therefore, the applicable technical instructions must be consulted in much detail before performing any of the procedures described herein.

Chapter 1: Introduction and overview

Abstract

Chromatography is simultaneously a process, a method, and a branch of science. It may be classified in different ways; one approach distinguishes planar techniques from those involving a column. It provides both qualitative and quantitative information as well as being applicable at the ultra-trace level and preparative scale. Coverage of this monograph includes all aspects of chromatography with the exception of large-scale industrial applications. This chapter serves as an introduction to chromatography and to the rest of the monograph.

Keywords:

Adsorption; Bonded phase; Development mode; Displacement; Elution; History; Ion exchange; Mechanism; Mobile phase; Stationary phase

1.1: Introduction

This is a seemingly simple and logical question. At the most basic level, chromatography is a separation technique or process. Its value depends on the ability to resolve or separate the components of mixtures with a large number of either similar (molecular size, polarity, etc.) and/or dissimilar analytes. The result is presented in a chromatogram, the nature of which is different depending on the actual process. In paper chromatography, it is a piece of chromatography paper with a series of visible spots. However, the most common form of chromatogram is now a computer-generated print-out comprising a series of peaks rising from a baseline drawn on a time axis. Each peak represents the detector response for a different compound. The time from the point of injection of sample into the chromatograph to the apex or peak maximum is referred to as the retention time of that particular compound.

An example of a 2019 state-of-the-art separation by chromatography is shown in the chromatogram of Fig. 1.1 for 12 analytes in 6 min. The chromatogram (a plot of detector response versus time) provides directly both qualitative and quantitative information. Each compound in the mixture has its own characteristic elution or retention time (the time point at which the signal appears in the chromatogram) under a given set of conditions (qualitative analysis) and the area and height of each signal are proportional to the amount of the corresponding substance (quantitative analysis). This makes chromatography a very powerful and useful technique.

Fig. 1.1 An example of the most common form of a chromatogram in which the x -axis represents time and the y -axis is the detector response. The chromatogram shows the separation of 12 analytes in 6 min. The peaks observed near 1 min are components of the sample solvent and some minor peaks are also seen throughout the chromatogram.

Chromatography was originally developed by the Russian botanist M. S. Tswett (1872–1919) (Fig. 1.2) as a technique for the separation of coloured plant pigments. A clear definition of chromatography is clearly desirable and should answer our question. Tswett gave a very pragmatic definition [1,2]. However, the first detailed definition appears to be due to Zechmeister [3] and various subsequent definitions have since been formulated. A generalized definition was provided in 1974 [4] and essentially confirmed with minor refinements in 1993 [5] by a special committee of the International Union of Pure and Applied Chemistry which regards chromatography as ‘… a method, used primarily for separation of the components of a sample, in which the components are distributed between two phases, one of which is stationary while the other moves. The stationary phase may be a solid, liquid supported on a solid, or a gel. The stationary phase may be packed in a column, spread as a layer, or distributed as a film … The mobile phase may be gaseous or liquid’. This definition neglected the possibility of using a supercritical fluid as the mobile phase which highlights the difficulties associated with providing an adequate definition.

Fig. 1.2 Photograph of Mikhail Tswett (1872–1919), the son of a Russian foreign service official and an Italian mother. He studied at the University of Geneva but then returned to Russia before working at Warsaw University and then being evacuated to Moscow in World War I. Credit: Elsevier.

While the IUPAC definition regards chromatography as a ‘method’, the Scientific Council on Chromatography, Russian Academy of Sciences [6] defined chromatography as follows:

•Science of intermolecular interactions and transport of molecules or particles in a system of mutually immiscible phases moving relative to each other;

•Process of multiple differentiated repeated distribution of chemical compounds (or particles), as a result of molecular interactions, between mutually immiscible phases (one of which is stationary) moving relative to each other leading to formation of concentration zones of individual components of original mixtures of such substances or particles; and

•Method of separation of mixtures of substances or particles based on differences in velocities of their movement in a system of mutually immiscible phases moving relative to each other.

This definition recognizes that chromatography is simultaneously a process, a method, and a branch of science. It is identified as ‘a new branch of science’ [7] and as ‘a body of knowledge that is now too large for many scientists to fully grasp?’ This reference is an excellent tribute to the pioneers and builders of chromatography and to their achievements. While the definition might appear irrelevant, in actual fact, Socrates held the view that the ‘precise logical definitions of concepts are a fundamental prerequisite to true knowledge’. Indeed, there has been considerable debate on this topic [8].

Novák approaches this problem from a different perspective and provides a phenomenological definition, a molecular kinetic definition and various working definitions [9]. What is of interest is that with each successive definition the criteria for a process to be called chromatography have generally been liberalized. This is not surprising as chromatography, like most scientific disciplines, is continuously evolving. Thus we should not allow ourselves to be distracted by the need for a clear and concise definition, but rather regard chromatography as a group of separation methods which are undergoing continuous development and refinement. Alternatively, it is also appropriate to see chromatography [10] as a unified scientific discipline: ‘the bridge—as a central science—a key foundation built on the twentieth century for major advances and discoveries yet to come across many sciences of the twenty-first century’.

The origins of the word ‘chromatography’ are no less obscure [11,12]. A 1952 paper [11] commented on the use of the word chromatography for over a century and a half prior to Tswett's use of the term although with a different connotation. In Tswett's papers, it was coined by combining two Greek words, chroma, ‘colour’ and graphein, ‘to write’ selected to indicate the individual coloured bands observed by Tswett in his separations (Fig. 1.3). At the same time Tswett emphasized that colourless substances can be separated in the same way. However, it may well be that Tswett, who was involved in a bitter controversy with his peers, gave reference to the Greek words only as an excuse for as Purnell [13] states ‘… it would be nice to think that Tswett, whose name, in Russian, means colour, took advantage of the opportunity to indulge his sense of humour’. The Germanic transcription of his Cyrillic name, Tswett is mostly used but it occasionally appears as the English transcription, Tsvet.

Fig. 1.3 Illustration (idealized) of classical column chromatography showing the column prior to addition of sample and at four different stages of development illustrating the separation of three analytes from a mixture. Sand is added to the top of the column to minimize disturbance to the sorbent as sample and mobile phase are added. This is essentially the system as used by Tswett in his original experiments. Prior to 1935 the column packing was removed from the column after use and the separated zones were extracted in order to recover the ‘pure’ components.

Irrespective of such considerations chromatography is a universal and versatile technique. While the more limited IUPAC definition justifies those involved in applications of chromatography in fields as diverse as medicine and engineering, the broader concept of a scientific discipline legitimizes basic research in the field which is an essential support for the applied aspects. It is equally applicable in all areas of chemistry and biochemistry, biology, quality control, research, analysis, preparative scale separations, and physicochemical measurements. It can be applied with equal success on the macro and micro scale. Chromatography is used industrially in the purification of such diverse materials as cane sugar, pharmaceuticals, and rare earths. On the other hand, it is widely used in the laboratory for the separation of minute quantities of substance, as in the initial chromatographic experiments leading to the discovery of element number 100 which involved only about 200 atoms. This surely represents one of the most remarkable achievements of modern science [14].

The achievement of such separations demonstrates the role of chromatography not only in chemistry but also in science and medicine where the importance of chromatography is indisputable. Two Nobel Prizes in Chemistry (to A. Tiselius of Sweden in 1948 and to A.J.P. Martin and R.L.M. Synge of Great Britain in 1952) have been awarded for work directly in the field of chromatography. In addition, chromatography played a vital role in work leading to the award of a further 25 Nobel Prizes in the 62 years between 1937 and 1999, and 19 between 2000 and 2007 [10].

The use and importance of chromatography to society can be illustrated in many ways. The value of sales of chromatographic equipment demonstrates a direct economic importance to the community. However, there are many other economic impacts that are difficult to assess. For example, the use of chromatography in ensuring the health of the environment, quality of our food, water and air supply, and clinical monitoring, among others, are less tangible and more difficult to assign a dollar value. Equipment sales also provide an indirect measure of how widely chromatography is used. Another measure is the use of scientific publications but this reflects research output rather than routine daily use occurring in government organizations and departments, food and pharmaceutical industry laboratories, hospitals, racing clubs, sporting organizations, the mobile science lab in Ferrari's F1 garage (the oven with a 30-m coil inside is a gas chromatograph) [15], the Philae lander on Comet 67P located over 500 million kilometres from Earth [16], or any one of the many laboratories worldwide using chromatography.

The authors of a paper on the self-image of chemists between the years 1950 and 2000 [17] identified the 1960s as the period of ‘chromatographic takeover.’ They noted the existence at that time of two worlds, ‘that of traditional chemistry, basically unchanged for two or three centuries, and that of modern chemistry, with a plethora of new and powerful physical methods’ with chemists seeing themselves with a foot in each of the two worlds. Hopefully, there are at least some chemists still straddling both of these worlds.

1.2: Coverage

In this book we examine all techniques that fit the definition of chromatography provided earlier in this chapter. The order of presentation of topics has changed from the first edition to better reflect the distinction between planar and column techniques. New chapters have been added on hyphenated techniques and preparative chromatography while the chapter on theory has been extended and re-written.

References have been updated and extended and partly for this reason but also reflecting the greater ease of literature searching, the bibliography with each chapter has been deleted.

Electrophoretic techniques bear some similarity to chromatography in that a mobile and stationary phase are involved and, on this basis, it is easy to argue for the inclusion of modern electrophoretic techniques in the current book. Six types of capillary electroseparation (Fig. 1.4) can be identified as: capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic capillary chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP) [18].

Fig. 1.4 Flow chart showing the categorization of electrophoretic techniques.

The means of driving the mobile phase in liquid chromatographic separations has evolved from gravity in classical column chromatography through capillary action in paper and thin layer chromatography to hydrodynamic pressure in high-performance liquid chromatography. However, in the case of electrophoresis, solute migration is also under the influence of an external force in the form of an electric field.

Of the six techniques, CEC is a true hybrid of electrophoresis and chromatography and a stronger argument could be made for its inclusion. The CEC process takes place in a capillary column, containing a selected stationary phase, where the mobile phase is delivered by an electro-osmotic flow controlled by the application of a relatively high electric field. CEC presents a number of advantages relative to high-performance liquid chromatography (HPLC) [19] but the most significant consideration is the improved chromatographic efficiency. The flow rate in CEC is independent of particle diameter and column length and there is no pressure dependency unlike HPLC, where the column pressure is inversely proportional to the particle diameter squared and directly proportional to column length, So, CEC has the potential to generate higher plate counts than HPLC. The plug-like flow velocity profile in the electro-driven system further reduces band dispersion and thus achieves a higher efficiency [20] to the parabolic or Gaussian profile associated with hydraulic-driven flow in HPLC.

There are divergent views on the utility of CEC. According to one source published in 2018 [21], interest in open-tubular CEC (OT-CEC) continues to thrive whereas a 2009 paper posed the question ‘what ever happened to capillary electrochromatography?’ [22]. Can these views be reconciled or has the technique experienced a resurgence between 2009 and 2017? Between 1998 and 2009 there was an average of around 150 published papers per year. The technique does appear to have had a renaissance since 2009 based on publication numbers but these are still relatively few compared with HPLC and CEC has failed to become a mainstream separation technique [20]. Looking at the titles of published papers gives a feeling that it is a technique seeking application areas. The development of novel stationary phases [23] has always been the research focus. The theoretical basis of CEC has been firmly established so that aspect is not an impedance. Rather the major issues in the lack of widespread acceptance are the absence of dedicated commercial instruments [24] with the features needed to compete with HPLC and CE, and the lack of applications where established methods fail. However, scientists are still investigating the parameters for CEC and developing applications despite the lack of commercial support. Indeed, the development of rapid, effective, and selective chiral separation methods is getting increasingly important for drug quality control, pharmacodynamic and pharmacokinetic studies, and toxicological investigations [25] and this is one area in which CEC may compete.

We have excluded treatment of CEC in this book for two reasons. Firstly, the means of driving the mobile phase in electrophoresis is not encapsulated by the definition we have given of chromatography. Secondly, CEC has not developed into a routine technique or established particular niche markets. The authors acknowledge the potential CEC offers and recognize that its inclusion in the next edition of this book may be warranted to the exclusion or, more likely, reduction in treatment of another technique(s).

1.3: Chromatographic separation simply explained

The definition of chromatography implies that the separation of the components of a sample occurs by distribution of the components between two phases, one of which is stationary (a solid or liquid) and the other moving or mobile (a liquid, gas or supercritical fluid). Consider a two-component mixture which is introduced at time, to, into a moving phase which is in contact with a second phase, the stationary phase. A continuous supply of fresh mobile phase is then provided to transport the sample components through the stationary phase. As the analytes come into contact with the stationary phase, they distribute or partition between the two phases depending on their relative affinities for the phases as determined by molecular structures and intermolecular forces. This process is depicted in Fig. 1.5 where analyte A has a higher affinity than analyte B for the stationary phase and thus spends a greater proportion of the available time in the stationary phase. When an analyte is present in the mobile phase, it will pass through the system with the same velocity as the mobile phase but when it is in the stationary phase, its velocity will be zero. Hence, analytes with a high affinity for the stationary phase will move through the system very slowly whereas analytes with a lower affinity will migrate more rapidly. This differential migration rate of analytes results in separation of the components as they move through the system, as shown in Fig. 1.5 at times t0, t1, and t2, under ideal and real conditions.

Fig. 1.5 Schematic representation of a chromatographic development on a packed column showing the separation of a mixture of two dyes of equimolar concentrations under ideal conditions and in the situation pertaining in real systems, where both separation and dispersion of analyte bands occur during the separation process. The column is depicted at the point of injection, t 0 , and at two subsequent times, t 1 and t 2 . Although not depicted, the solute concentration profile along the column length is Gaussian at times, t 1 and t 2 .

Even though the system is dynamic, it must be operated as close to equilibrium conditions as possible by optimizing the mobile phase velocity and designing the stationary phase to allow rapid equilibration to be achieved; i.e. the timescale for distribution of solute molecules between phases must be rapid compared to the velocity of the mobile phase. We can write for any solute, A:

where m and s represent mobile and stationary phase, respectively. Under these conditions, the system can be characterized by a thermodynamic distribution constant, K, which is usually expressed as the ratio of analyte concentration in the stationary phase, CA(s) to that in the mobile phase, CA(m):

si2_e    (1.1)

The description of K as a distribution constant is consistent with IUPAC nomenclature but older terms such as partition coefficient and distribution coefficient are also still in use.

An analogy might improve understanding [26] of Eq. (1.1). If we assume that there has been rain which has left a puddle of water. It is well known that the puddle disappears over time due to evaporation even though the ambient temperature never reaches the boiling point of water. This can be described by the following equilibrium:

where KP is the pressure-based equilibrium constant for the evaporation process and PH2O is the vapour pressure of the water. On a cool dry day, the air above the puddle becomes saturated (100% relative humidity). The liquid and gaseous water molecules are in or close to equilibrium and no further evaporation occurs. On a windy day the puddle evaporates more rapidly because the gaseous water molecules are being continuously removed so that more molecules enter the gas phase to restore the equilibrium.

The distribution constant of Eq. (1.1) is a characteristic physical property of an analyte which depends only on the structure of the analyte, the nature of the two phases, and the temperature. Phenolic solutes, for example, would be expected to form intermolecular attractions with phenolic stationary phases to a much higher degree than would hydrocarbon solutes exposed to the same stationary phase. Thus the K value of a phenol is higher than that of a hydrocarbon of corresponding chain length in a phenolic phase. The separation of two compounds on a particular chromatographic system requires that they have different distribution constants. Conversely, two compounds with the same distribution constant will not be separated. In this case, the separation can be improved by varying the mobile phase, the stationary phase, or the temperature of the system. In practice, it is often difficult to predict the effects of changing the mobile phase or stationary phase and the only method is to make the change experimentally. In gas chromatography, the partition properties of the gases used as mobile phase are similar and the mobile phase is described as non-interactive, so that only the stationary phase and temperature can be varied to improve separation. The greater versatility of liquid and supercritical fluid chromatography is possible because the mobile phase is interactive and all three variables can be altered, although temperature changes are very restricted.

Eq. (1.1) is an oversimplification, since K, like any thermodynamic equilibrium constant, is really a quotient of analyte activities. However, in chromatographic systems we are normally dealing with solutions which tend towards infinite dilution and therefore the activity coefficient is one. This equation also assumes that the analyte is present as only one molecular structure or ion and that the analyte does not interact with other analyte molecules at infinite dilution. Considering the low levels of analytes involved, this is a reasonable assumption. The concentration profiles depicted in Fig. 1.5 as ideal are never achieved in practice. At the molecular level, various solute diffusional effects and random statistical motion of molecules cause spreading of the analyte bands (as shown in Fig. 1.5) which assume the normal Gaussian distribution (provided adsorptive effects are absent—discussed in later chapters) (not depicted in Fig. 1.5).

1.4: Classification of chromatography

Classification is used to simplify our understanding of the universe by dividing a subject into smaller, more manageable, and more specific parts. Books can be classified according to many criteria including colour of the cover, height, or subject matter. The latter is the most useful system for a librarian to use but height is more useful for me because I can then fit books on my limited shelf space. Similarly, any one of several factors (stationary or mobile phase, separation process, or even the type of solute) can serve as a basis for classification of chromatography. Thus chromatographic separations can be classified in a number of ways depending on the interests of the chromatographer. While the ultimate goal of any classification system is simplification, unfortunately this multiplicity of overlapping systems, together with the diversity of chromatography as now practised, has led to a proliferation of terminology which can be confusing for both the novice and specialist chromatographer alike.

There are other aspects of nomenclature that are potentially confusing to the novice. For instance, as we will see, a chromatographic system in which a column is connected to a pump and spectrophotometer is called an HPLC, but if we take the same pump and column and connect it to a mass spectrometer, we have a hyphenated technique or, more specifically, in this instance, an LC-MS. One might say that this distinction is illogical but it is understandable in terms of the historical development of the field of chromatography. In other examples, the terms rapid resolution LC [27], ultra-high-performance liquid chromatography (UHPLC) [28–31], and ultra-high pressure LC [32] describe essentially the same procedure [33] but emphasize different aspects reflecting the personal bias of the speaker. For example, UHPLC [34,35] is distinguished from HPLC by the size of stationary phase particles: sub-2 μm for UHPLC versus 5 μm for HPLC with typical pump pressuresa of 1000 bar (14,500 psi) and 4000 bar (5800 psi), respectively.

A grasp of the fundamentals presented here and in what follows will help to reduce such confusion.

1.4.1: Mobile phase

One system of classification recognizes the importance of the mobile phase and divides chromatography into three broad areas of liquid chromatography (LC), gas chromatography (GC), and supercritical fluid chromatography (SFC) (Fig. 1.6), depending on whether the mobile phase is a liquid, gas, or supercritical fluid, respectively. Further classification is possible by specifying both the mobile and stationary phases leading, for example, to gas solid chromatography (GSC) and gas liquid chromatography (GLC) in which the mobile phase is a gas but the stationary phase is a solid (GSC) or a liquid (GLC). As GSC is little used except in very specific applications one normally refers to gas chromatography or GC unless a solid stationary phase is used in which case it is specifically noted by speaking of GSC.

Fig. 1.6 Flow chart showing the classification of chromatographic systems based on mobile phase, stationary phase, configuration, and technique.

More recently, supercritical fluids have been employed as mobile phases and these techniques are at present termed supercritical fluid chromatography (SFC) irrespective of the state of the stationary phase. The situation with liquid chromatography is intermediate in that distinction between liquid-solid chromatography (LSC) and liquid-liquid chromatography (LLC) is sometimes made but, in most cases, it is just referred to as liquid chromatography (LC) regardless of the state of the stationary phase.

1.4.1.1: Reversed-phase and normal-phase chromatography

In liquid and supercritical fluid chromatography, systems involving a polar stationary phase and a non-polar mobile phase are termed normal-phase systems. With this combination of phases, solute retention generally increases with solute polarity. On the other hand, if the stationary phase is less polar than the mobile phase, the system is described as reversed phase and polar molecules have a lower affinity for the stationary phase and elute faster (Table 1.1).

Table 1.1

The choice of these terms is purely historical and no special significance (beyond that indicated) is attached to the use of the term ‘normal’. The original separations of plant extracts performed by Tswett used a polar stationary phase (chalk in a glass column) with a much less polar (indeed, non-polar) mobile phase and this combination became known as normal phase. However, reversed-phase systems because of their broad applicability and better reproducibility are now far more common than normal-phase systems in liquid chromatography.

With normal-phase systems, increasing the mobile phase polarity makes it more like the stationary phase so that the mobile phase competes more effectively with the stationary phase for solute molecules. The solute molecules therefore spend less time in the stationary phase and elute faster. Using a similar argument, we predict slower elution as mobile phase polarity is increased in reversed-phase chromatography [36].

1.4.2: Development mode

The chromatographic bed describes the configuration of the stationary phase. In the simplest terms, the bed is either planar or a column. The process whereby a sample comprising a mixture of solutes progresses through the chromatographic bed, while in the mobile phase, is called chromatographic development. Three modes of chromatographic development were identified in 1943 [37] by the 1948 Chemistry Nobel Laureate [38], Arne Tiselius, as elution development, displacement development, and frontal analysis. The development mode refers to the manner in which the sample and mobile phase are applied to the stationary phase bed (column or plane) and as shown in Fig. 1.7, the nature of the resulting peak profile, termed the chromatogram, differs between the three modes. IUPAC distinguishes the three modes as follows:

•Frontal Chromatography. A procedure in which the sample (solid, liquid, or gas) dissolved in mobile phase is fed continuously into the chromatographic bed. In frontal chromatography no additional mobile phase is used.

•Displacement Chromatography. A procedure in which the mobile phase contains a compound (the Displacer) more strongly retained than the components of the sample under examination. The sample is fed into the system as a finite slug.

•Elution Chromatography. A procedure in which the mobile phase is continuously passed through or along the chromatographic bed and the sample is fed into the system as a finite slug. Elution development is now virtually the only development technique employed for analytical separations.

Fig. 1.7 Schematic representation of the different chromatographic development modes showing the effect on migration of sample components and the resulting zone profile. A and B represent sample components and C the displacer.

In frontal chromatography the sample is swept continuously onto the column by the mobile phase during the entire course of the process. When the column becomes saturated with respect to a particular component, that component is then eluted from the column. When the zone of pure component has completely eluted, it is followed by a mixture with the next component, and so on. A complete separation cannot be achieved and the method has limited application for quantitative measurements. A typical application would be the estimation of a trace impurity in a high purity substance where the impurity can be concentrated in front of the main constituent, provided that it was the less strongly retained. Frontal analysis is now probably of academic interest only although it is useful for obtaining thermodynamic data from chromatographic measurements [39,40]. It is quite inappropriate for most practical analytical applications and has been completely superseded by elution development.

The advent of displacement chromatography can be attributed [37] to Tiselius. With this mode, the sample is applied to the system as a discrete plug as in elution, but unlike elution, the mobile phase has a higher affinity for the stationary phase than any sample component. Alternatively, a substance more strongly retained than any of the components of the sample is added continuously to the mobile phase. This substance is known as the displacer and it ‘pushes’ the sample components down the column. The mixture resolves itself into zones of pure components in order of the strength of retention on the stationary phase. Each pure component displaces the component ahead of it with the last and most strongly retained component being forced along by the displacer. The record depicting the concentration of component coming from the column (Fig. 1.7) is seen to resemble the record obtained with frontal analysis but with an important difference. In displacement, the steps in the chromatogram represent pure components. Historically it was used for preparative separations of amino acids and rare earth elements. More recently, it has found many applications in the realm of biological macromolecule purifications [41].

In this book we are referring to elution development in all cases unless specifically mentioned otherwise.

1.4.3: Technique

Chromatography can be performed using very simple apparatus as in Tswett's original experiments or, as commonly practised nowadays, with much more sophisticated equipment (Fig. 1.8) costing into the million-dollar mark for the most sophisticated system. However, all of these diverse techniques can be classified into one of two broad categories; namely, column and planar chromatography, but these encompass a number of variants.

Fig. 1.8 Photograph of a laboratory set-up for liquid chromatography-mass spectrometry. Credit: Danielle Ryan.

1.4.3.1: Planar and column chromatography

There are two techniques in which the stationary phase is supported on a planar surface: paper chromatography (PC) and thin layer chromatography (TLC) which collectively are termed planar chromatography. With PC, a sheet of paper or substrate-impregnated paper comprises the plane (sorbed water is the stationary phase) whereas in TLC, the plane is a flat sheet of glass, plastic or aluminium coated uniformly with a thin layer of solid comprising the stationary phase. Alternatively, the stationary phase may be packed in a closed column and the technique is referred to as column chromatography.

If the stationary phase is a liquid, it must be immobilized on the thin layer or in the column and this is conveniently achieved by coating or chemically bonding the liquid stationary phase to an inert solid support (e.g. silica) which is then packed in the column or spread in a thin layer over a flat plate. Planar procedures have been restricted to liquid chromatography because of the technical difficulties associated with confining a gas or supercritical fluid to a planar surface. In contrast to planar procedures, column chromatography is used in LC, GC, and SFC. In the case of liquid chromatography, there are two variants which because of their chronological development may be termed classical column chromatography and modern column chromatography. The latter is referred to as high-performance liquid chromatography, HPLC. The term liquid chromatography is often used to denote liquid chromatography in columns and particularly (ultra)high-performance liquid chromatography, (U)HPLC because of its extensive use.

Methods involving gaseous, liquid, and supercritical fluid mobile phases will be treated individually in later chapters. Although there are few fundamental reasons for separate treatment, it is nonetheless warranted by differences in equipment and operational procedures. For now, it is sufficient to make a comparison of the different techniques on the basis of operational procedures and results as shown in Fig. 1.9.

Fig. 1.9 Pictorial comparison of chromatographic techniques showing the various stages of the process: bed preparation, sample application, nature of the stationary phase, method of mobile phase addition, method of detection, and format for presentation of results.

Column types

It is often stated that the column is the heart of the chromatograph emphasizing its importance to the separation. Column procedures may be further classified according to the nature and dimensions of the column. Conventional column procedures both in gas and liquid chromatography and, more recently, supercritical fluid chromatography, exploit ‘wide’ bore packed columns with internal diameters (i.d.) exceeding 1.0 mm. In GC, the internal diameters of such columns are typically of 2–4 mm and in HPLC of 4.6 mm. These packed columns generally contain a stationary phase consisting of either a solid or a liquid coated or bonded to an inert solid support. Specific details relating to stationary phases will be presented in the relevant chapter. This section is concerned with the physical construction of the actual column.

There are many benefits associated with column miniaturization and the first step in this direction was made in 1957 with the development of capillary columns for GC [42]. It is now realized that miniaturized separation columns, whether used in various forms of gas, liquid, or supercritical fluid chromatography, share similar technologies and instrumental requirements [43]. In 1981 Novotny [44] identified advantages of microcolumns as higher column efficiencies, improved detection performance, various benefits of drastically reduced flow rates, and the ability to work with smaller samples. Priorities have changed over the years as different applications have varied the emphasis of these unique capabilities of miniaturized systems. Miniaturization is not without problems however, and the chief disadvantages of capillary columns are that they are more demanding of instrument performance, less forgiving of poor operator technique, and possess a lower sample capacity than packed columns.

In gas chromatography, columns can be classified [45] as follows:

Conventional packed columns were originally constructed of stainless steel or other metal such as copper of about 1–2 m long, 2–4 mm i.d., and formed into a coil. As the performance of detectors improved, less sample was applied to the system and these metal columns were too reactive for the smaller quantities of analytes. For example, with a sample injection corresponding to 1 mg analyte, decomposition catalysed by the metal tubing of 1–10 μg of the analyte was relatively insignificant. However, with an improved detector and injection equivalent to 1–10 μg of analyte, catalytic decomposition of this same amount of analyte represented a very significant loss. Thus columns constructed of glass which was more inert were introduced but with similar dimensions to the previously used metal columns.

Miniaturization of the columns (diameter) proceeded in two directions. Micropacked or packed capillary columns [46,47], characterized by small internal diameters, usually less than 1.0 mm, are miniaturized versions of conventional packed columns. They are typically constructed of stainless steel and are 1–2 m long. Their use has been limited by practical problems, particularly with injection at high back pressures but they are commercially available and are useful for separation of gas mixtures including sulphur compounds or light hydrocarbons. In contrast, the development of open tubular columns [48] with an internal diameter less than 1.0 mm has been immensely successful. Open tubular columns are also referred to as capillary columns. However, the characteristic feature of these columns is their openness, which provides an unrestricted gas path through the column. Hence, open tubular column is a more apt description although both terms will undoubtedly continue to be used and can be considered interchangeable (but with a caution to check the context). The columns were originally constructed of glass but the commercial availability in the 1980s of fused silica open tubular columns coated on the outside with a protective polyimide layer revolutionized the industry. They rapidly became widely accepted and used as the standard against which other columns were judged. Highly inert metal open tubular columns were a more recent addition.

If the stationary phase is applied directly as a thin layer to the internal wall of the open tubular column, then it is known as a wall coated open tubular (WCOT) column. While non-bonded phases are simply coated on the internal wall of the column, the stationary phase can also be chemically bonded to the wall. In most cases, the polymer chains of the stationary phase are also cross-linked. Generally, a bonded and cross-linked phase is preferable because it can be used to higher temperatures and has less column bleed (Chapter 4) during use. Moreover, these phases can be rinsed with solvents to remove any accumulated non-volatile materials. Low polarity stationary phases are typically bonded and cross-linked but, in some instances, such as highly polar phases, options are more restricted [49].

The sample capacity of WCOT columns can be adjusted by varying the column diameter and the film thickness of the stationary phase. Alternatives to the WCOT column are the porous layer open tubular (PLOT) column and surface coated open tubular (SCOT) column. The inner wall of the column is extended in PLOT columns by addition of a porous layer such as fused silica. In SCOT columns, the stationary phase is applied to a solid support which is coated on the internal wall of the column. SCOT columns were popular because of higher sample capacity although wide bore or mega bore (0.53–1.00 mm i.d.) WCOT columns are competitive in this respect and are easier to use and more stable. SCOT column technology allows access to many stationary phases that are not compatible with conventional WCOT column manufacturing technology. PLOT columns are ideal for separating compounds that are gases at room temperatures, low molecular mass hydrocarbon isomers, and reactive analytes such as hydrides, amines, and sulphur gases. The various column types have been compared by Duffy [50].

It is difficult to establish accurately the proportion of routine separations that use open tubular versus packed columns. The vast majority of published papers (probably well in excess of 90%) involve the former. However, papers represent research activity rather than routine applications. On the other hand, the relative amount of space devoted to the different column types in column manufacturers’ literature and the volume of sales probably give a good indication of the relative importance across all uses. On this basis, we can state that separations on open tubular columns dominate GC except in a very few specialty areas.

In liquid chromatography, conventional packed columns for classical column chromatography comprised a cylindrical glass tube about 200–800 mm long and 20–60 mm wide although larger columns have also been used in the laboratory (Fig. 1.10). In almost all instances, such columns were designed for a single use.

Fig. 1.10 Photograph of a chemist employed by the US Food and Drug Administration in the mid-1950s using a column chromatographic apparatus to separate the constituents in a coal tar colour analysis. Credit: https://en.wikipedia.org/wiki/Column_chromatography#/media/File:FDA_History_-_Column_Chromatography.jpg.

The design of a column for HPLC is very simple in theory. All that is needed is an inert tube that will retain the stationary phase and allow movement of the mobile phase. However, the practical requirements are very demanding as noted by Majors [51,52]: ‘The tube itself must be able to withstand the pressure generated by the packed bed and should not expand or change its dimensions if the pressure or temperature is high or contract if the tube is cooled. Next, one needs a device to contain the packing and not permit any minute particles (from a distribution of particles) to exit the packed column. Even with this restriction, this device must be able to provide adequate flow and be inert’. In columns used at high pressures for HPLC a sintered metal frit is used for this purpose and a means of sealing the column is required so that it holds this pressure without leaking fluid. ‘Sealing is accomplished by compression endfittings or special modified endfittings that can consistently withstand these high pressures. These fittings must allow flow to enter and exit the column at these high pressures without leaking. The fittings also should provide narrow, well-swept flow channels so that flowing solvent can pass through them at high and low linear velocities without the creation of flow disturbances, flow eddys, or uneven flow. For modern high efficiency columns, the flow design should allow the injected sample to pass through these channels in a narrow plug without spreading this band. Other column design issues that are important are the smoothness of the inside wall, which is in contact with the packing, the chemical resistance, and inertness of the column components in contact with the mobile phase and the sample, the cost of the materials used to construct the hardware, and the sturdiness of the assembled column hardware’.

Column technology in HPLC [51–54] advanced rapidly in the late 1960s and early 1970s. Between 1975 and 2000, the conventional column for HPLC was a stainless steel tube of 4.6 mm i.d. packed with spherical particles of between 10 and 5 μm in diameter. Over this period column length gradually decreased from 300 to 150 mm. Interestingly, the original columns were either 1.0 mm or 2.1 mm i.d. The establishment of the 4.6 mm i.d. column as standard resulted from purely practical and pragmatic considerations rather than a rigorous theoretical study. Stainless steel tube of 4.6 mm was readily available at a reasonable price, was compatible with compression fittings borrowed from packed column gas chromatographs, and was of sufficient wall thickness to withstand operating pressures. Thus it was adopted as the standard material for column construction. More recently, glass-lined or Teflon-lined stainless steel has been introduced for column construction to enhance inertness and reduce surface interactions. Many manufacturers supply columns for biochromatography with polyetheretherketone (PEEK) construction.

The next major development was the commercialization of monolithic silica columns by Merck in 1999. These new columns were made of a single block of silica, with a bimodal pore size distribution, encapsulated in a PEEK tube with dimensions of 100 mm × 4.6 mm. These columns offered faster analyses than the conventional packed columns at equivalent resolution and looked a very promising alternative in the early 2000s. Polymeric monolith columns were also developed consisting of a continuous cross-linked, porous monolithic polymer usually polymethacrylates or methacrylate copolymerizates. Interest in monolithic columns faded and they seemed to be on their way to oblivion [55] but they experienced a renaissance [56] and are enjoying renewed popularity.

Manufacturers of conventional packings responded to the potential threat of monolithic columns by commercializing columns packed with fully porous particles of decreasing sizes, 5, then 3.5, 2.5, and eventually 1.7 and 1.5 μm. At the same time, column lengths gradually decreased from 150 to 50 mm. These developments witnessed the birth of very high-pressure liquid chromatography in 2004 involving narrow bore (2.1 mm i.d.) short (50 mm long) columns packed with sub-2 μm particles [57]. These columns offered superior performance to the monolithic columns due to the radial heterogeneity of the silica rods in the latter and this probably explained the sudden decline in interest in the monolithic columns.

A second and very unexpected development in column technology occurred a few years later with a re-visit to pellicular particles. The original materials, also referred to as superficially porous packings or porous layered beads, were of 40–50 μm diameter and exhibited good efficiency (a measure of separation) relative to the large (100 μm) porous particles then in vogue but they had poor sample capacity. Thus they were rejected in the early 1970s in favour of packings based on smaller porous particles. The pellicular packings that have emerged in the last few years have much smaller particle sizes than their 1960s forerunners and potentially can deliver comparable separations to the very fine fully porous particles.

There are three types of microcolumn for liquid chromatography although their use is not widespread. Microbore columns are similar in construction to conventional packed columns except that the column diameter is reduced to 1 mm. Packed capillaries have a column diameter of 70 μm or less and are loosely packed with particles having diameters of from 5 to 30 micrometres. Nano or open microtubular columns are the equivalent of the capillary or open tubular column in GC. Ideally, they have diameters of 10–30 micrometres and contain a stationary phase or an adsorbent either coated on, or chemically bonded to, the column wall [58]. Table 1.2 presents a nomenclature applied to LC columns by Majors [51]. Nano columns are most commonly constructed from fused silica while stainless steel, lined stainless steel, and PEEK-clad fused silica tubing have been used to make capillary LC columns.

Table 1.2

Nano columns can be replaced potentially by chip-based LC systems [52] in which the chromatography column is fabricated on glass or plastic chips. The narrow channels can be etched or laser ablated with dimensions in the