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PRINCIPLES AND PRACTICE OF MODERN
CHROMATOGRAPHIC METHODS

PRINCIPLES
ANDPRACTICE
OFMODERN
CHROMATOGRAPHIC
METHODS
SECOND EDITION
KEVINROBARDS
Emeritus Professor, Charles Sturt University, Wagga Wagga, NSW, Australia
DANIELLERYAN
Senior Lecturer in Chemistry, Charles Sturt University, Wagga Wagga, NSW, Australia

Preface
My (KR) first encounter with chromatography was at the tail end of the 1960s with paper and thin layer chroma-
tography 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 fun-
damental 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 chro-
matographic 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 30years 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, multi-
dimensional, 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 func-
tions. 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
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.
vii

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 chro-
matographic 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 sche-
matic overviews of the content of each chapter. Topic coverage is both integrated and comprehensive based on chro-
matographic 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 fundamen-
tal 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 prac-
tising 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.
d
https://www.paduiblog.com/pa-dui/a-large-problem-in-gas-chromatography-no-uniform-standard-for-gc-run-position-or-composition/.
viii Preface

CHAPTER
1
Introductionandoverview
1.1 Introduction
This is a seemingly simple and logical question. At the most basic level, chromatogra-
phy 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 chroma-
tography, it is a piece of chromatography paper with a series of visible spots. However, the most common form of
1Principles and Practice of Modern Chromatographic Methods
https://doi.org/10.1016/B978-0-12-822096-2.00006-2
Copyright©2022 Elsevier Ltd. All rights reserved.

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 ofFig. 1.1for
12 analytes in 6min. 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.
Chromatography was originally developed by the Russian botanist M. S. Tswett (1872–1919) (Fig. 1.2) as a tech-
nique 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
FIG. 1.1An example of the most common form of a chromatogram
in which thex-axis represents time and they-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.
FIG. 1.2Photograph of Mikhail Tswett (1872–1919), the son of a Russian foreign service offi-
cial 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.
2 1. Introduction and overview

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 commit-
tee 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.
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 molec-
ular 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 chroma-
tography, 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 chromatog-
raphy[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 con-
notation. In Tswett’s papers, it was coined by combining two Greek words,chroma,‘colour’andgraphein,‘
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.
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 engi-
neering, 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 mate-
rials 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
31.1 Introduction

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 62years 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 chro-
matographic 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 chroma-
tography 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 labo-
ratories, 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), cap-
illary gel electrophoresis (CGE), micellar electrokinetic capillary chromatography (MEKC), capillary electrochromato-
graphy (CEC), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP)[18].
FIG. 1.3Illustration (idealized) of classical
column chromatography showing the column
prior to addition of sample and at four different
stages of development illustrating the separa-
tion of three analytes from a mixture. Sand is
added to the top of the column to minimize dis-
turbance 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.
4 1. Introduction and overview

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 com-
pared with HPLC and CEC has failed to become a mainstream separation technique[20]. Looking at the titles of pub-
lished 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 com-
mercial 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 applica-
tions despite the lack of commercial support. Indeed, the development of rapid, effective, and selective chiral sepa-
ration 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 devel-
oped 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 inFig. 1.5where
FIG. 1.4Flow chart showing the categoriza-
tion of electrophoretic techniques.
51.3 Chromatographic separation simply explained

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, ana-
lytes 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 com-
ponents as they move through the system, as shown inFig. 1.5at times t0,t1, and t2, under ideal and real conditions.
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 time-
scale 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:
AmÐAs
wheremandsrepresent mobile and stationary phase, respectively. Under these conditions, the system can be char-
acterized 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)
:
K¼
CA
sðÞ
CA
mðÞ
(1.1)
The description ofKas 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:
H2O
1ðÞÐH2O
gðÞ
KP¼PH2O
whereK
Pis the pressure-based equilibrium constant for the evaporation process andP
H
2Ois 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 evap-
orates 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 theKvalue of a phenol is higher than that of a hydro-
carbon of corresponding chain length in a phenolic phase. The separation of two compounds on a particular chromato-
graphic 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,
FIG. 1.5Schematic representation of a chro-
matographic 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 dis-
persion of analyte bands occur during the sep-
aration process. The column is depicted at the
point of injection,t0, and at two subsequent
times,t1andt2. Although not depicted, the sol-
ute concentration profile along the column
length is Gaussian at times,t1andt2.
6 1. Introduction and overview

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 chromatog-
raphy, 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 ana-
lyte activities. However, in chromatographic systems we are normally dealing with solutions which tend towards infi-
nite 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. 1.5as 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 inFig. 1.5) which assume the normal
Gaussian distribution (provided adsorptive effects are absent—discussed in later chapters) (not depicted inFig. 1.5).
1.4 Classification of chromatography
Classification is used to simplify our understanding of the universe by dividing a subject into smaller, more man-
ageable, 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 chro-
matographic 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-2μm for UHPLC versus
5μm for HPLC with typical pump pressures
a
of 1000bar (14,500 psi) and 4000bar (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 classi-
fication is possible by specifying both the mobile and stationary phases leading, for example, to gas solid chromatog-
raphy (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 chro-
matography or GC unless a solid stationary phase is used in which case it is specifically noted by speaking of GSC.
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
a
Various pressure units are used in chromatography. Those commonly encountered and relevant conversions are
1 MPa¼10 bar¼9.869 atm¼145.038 psi¼10.197 kg cm
2
71.4 Classification of chromatography

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).
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 chromato-
graphic 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, aselution
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 inFig. 1.7, the nature
of the resulting peak profile, termed the chromatogram, differs between the three modes. IUPAC distinguishes the
three modes as follows:
FIG. 1.6Flow chart showing the classification of chromatographic systems based on mobile phase, stationary phase, configuration, and
technique.
TABLE 1.1Normal-phase and reversed-phase chromatography.
Classification Stationary phase Mobile phase Order of solute elution
Normal phase Polar Non-polar Most hydrophobic solutes first and finally most hydrophilic solutes
Reversed phase Non-polar Polar Most hydrophilic solutes first and finally most hydrophobic solutes
8 1. Introduction and overview

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.
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 sub-
stance 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 con-
centration of component coming from the column (Fig. 1.7) is seen to resemble the record obtained with frontal anal-
ysis but with an important difference. In displacement, the steps in the chromatogram represent pure components.
A + B
A + B
A + B
A + B
A + B
C + B C + A
C
C
CBA
A
B
A
A
A
FRONTAL ANALYSIS
B
C
CCC
ELUTION DEVELOPMENT
DISPLACEMENT DEVELOPMENT
FIG. 1.7Schematic 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 com-
ponents and C the displacer.
91.4 Classification of chromatography

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 com-
monly 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.
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 differ-
ences 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 inFig. 1.9.
FIG. 1.8Photograph of a laboratory set-up for liquid
chromatography-mass spectrometry.Credit: Danielle Ryan.
10 1. Introduction and overview

FIG. 1.9
Pictorial comparison of chromatographic techniques showing the various stages of the process: bed preparation, sample application, nature of the s
tationary 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 1.0mm. In GC, the internal diameters of
such columns are typically of 2–4mm and in HPLC of 4.6mm. 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 effi-
ciencies, 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.
Ingas 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
1–2m long, 2–4mm 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 1–10μg of the analyte was
relatively insignificant. However, with an improved detector and injection equivalent to 1–10μg 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 1.0mm, 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 use-
ful 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 1.0mm has been immensely successful. Open tubular
columns are also referred to as capillary columns. However, the characteristic feature of these columns is their open-
ness, which provides an unrestricted gas path through the column. Hence, open tubular column is a more apt descrip-
tion 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
12 1. Introduction and overview

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 (0.53–1.00mm 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 tem-
peratures, 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 col-
umns. 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 indica-
tion 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.
Inliquid chromatography, conventional packed columns for classical column chromatography comprised a cylin-
drical glass tube about 200–800 mm long and 20–60mm wide although larger columns have also been used in the lab-
oratory (Fig. 1.10). In almost all instances, such columns were designed for a single use.
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,
FIG. 1.10Photograph of a chemist employed by the US Food and
Drug Administration in the mid-1950s using a column chromato-
graphic apparatus to separate the constituents in a coal tar colour anal-
ysis.Credit:https://en.wikipedia.org/wiki/Column_chromatography#/
media/File:FDA_History_-_Column_Chromatography.jpg.
131.4 Classification of chromatography

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 col-
umns 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 4.6mm i.d. packed with spherical particles of between
10 and 5μm in diameter. Over this period column length gradually decreased from 300 to 150mm. Interestingly,
the original columns were either 1.0mm or 2.1mm i.d. The establishment of the 4.6mm i.d. column as standard
resulted from purely practical and pragmatic considerations rather than a rigorous theoretical study. Stainless steel
tube of 4.6mm 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 manufac-
turers 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 100mmπ4.6mm. 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 commercial-
izing columns packed with fully porous particles of decreasing sizes, 5, then 3.5, 2.5, and eventually 1.7 and 1.5μm. At
the same time, column lengths gradually decreased from 150 to 50mm. These developments witnessed the birth of
very high-pressure liquid chromatography in 2004 involving narrow bore (2.1mm i.d.) short (50mm long) columns
packed with sub-2μm 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 pel-
licular particles. The original materials, also referred to as superficially porous packings or porous layered beads, were
of 40–50μm diameter and exhibited good efficiency (a measure of separation) relative to the large (100μm) 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
1mm. Packed capillaries have a column diameter of 70μm or less and are loosely packed with particles having diam-
eters 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 adsor-
bent either coated on, or chemically bonded to, the column wall[58].Table 1.2presents 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.
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 50μm
diameter range by lengths as long as tens of centimetres. Although the flow of mobile phase can be via conventional
14 1. Introduction and overview

hydraulic means using pumping systems and nano-valves integrated onto the chip, electroosmotic flow is also
possible.
Although the situation is less clear-cut than in GC, conventional packed columns still dominate routine separations
by HPLC. A number of publications[53,55,59–64]cover the development of column technology in LC. Conventional
analytical columns in the year 2020 fall into particle-packed columns and monolithic columns of between 1 mm and
4.6mm internal diameter. The former can be subdivided into fully porous, core-shell, and non-porous particles which
are further discriminated on the basis of their chemical composition into either silica-based or polymer-based
(cross-linked organic polymers).
The development ofsupercritical fluid chromatographyoccurred in the decade from 1980 at a time when column
technology in both GC and HPLC had been well developed. Commercial column manufacturers tended to ignore SFC
until the market grew large enough to justify an investment[65]. Thus there were few SFC-specific columns and col-
umn technology for SFC was largely borrowed from HPLC for the packed column format or from GC for the open
tubular format. In the early 1980s, for a very brief period, the usual column was a standard normal-phase column
borrowed from HPLC, typically 250π4.6mm with 5μm totally porous silica packings. Capillary or open tubular
SFC was originally reported by Milton Lee and others[44,66]in 1981 and it was commercialized by 1986. It was in
this period that SFC research was dominated by gas chromatographers who transferred their knowledge of open tubu-
lar columns to their new interest and open tubular column SFC rapidly became the standard approach although there
remained a significant core employing packed column SFC[67,68]. Generally, the diameter of open tubular columns
for SFC was smaller than 100μm to maintain both reasonable analysis times and high resolution[69]. By 2000, it was
acknowledged[70]that capillary SFC had been oversold especially for the elution of polar solutes. About this same
time SFC-specific columns of both packed and open tubular varieties became progressively available from manufac-
turers. Nevertheless, SFC although considered a mature technique by age, in other ways, it is still in its infancy and
does not receive the same attention by column manufacturers as does HPLC or GC[71].
1.4.4 Separation mechanism
Molecular interactions play a fundamental role in the behaviour of the chemical and physical properties of any
physicochemical system including chromatography. The nature of this interaction between sample components
and the two phases forms a further basis for classification of chromatography. Since these molecular interactions
(Table 1.3) determine the retention behaviour of the solute, this is the most fundamental of all classifications of chro-
matography. However, in many ways it is the most difficult since, in a number of instances, it is not clear exactly what
mechanism is involved. Nevertheless, a knowledge of the mechanism is crucial to our understanding of the chromato-
graphic process, to enable predictions about the expected behaviour of a system and in choosing a stationary phase/
mobile phase combination to obtain a desired separation. In most instances it is possible to specify the predominant
mechanism operating in a particular situation even though the nominated mechanism is rarely, if ever, the sole
mechanism.
The mechanisms can be classified
b
into a number of types as follows:
TABLE 1.2Classification and column dimensions for HPLC.
Column Internal diameter (mm) Length (mm) Particle size ( μm)
Nano 0.1, 0.075 50,150 3.55, 5.0
Capillary 0.3, 0.5 35 –250 3.55, 5.0
Microbore 1.0 30 –150 3.55, 5.0
Narrow bore 2.1 15 –150 sub-2, 3.0 –3.5, 5.0
Solvent saver 3.0 150, 250 sub-2, 3.0 –3.5, 5.0
Analytical 4.6 15 –250 sub-2, 3.0 –3.5, 5.0
Semi-preparative 9.4 50 –250 5.0
Preparative 21.2, 30.0, 50.0 50 –250 5.0, 7.0, 10.0
b
The terms sorption and sorb are used in this text to denote a solute–stationary phase interaction of unspecified nature. It is used in two
circumstances; where the nature of the interaction is unknown or as a generic term to cover several types of interaction.
151.4 Classification of chromatography

TABLE 1.3Intermolecular interactions involved in different liquid chromatographic methods. Mechanism
Intermolecular interactions (interaction energy kJ mol
21
)
Van der
Waals (0.4–4) Repulsion
London
dispersion
(2–4)
Hydrophobicity
(4)
Dipole-dipole/Dipole-
induced dipole (3/1)
Charge
transfer
(4–17)
Hydrogen
bonding (5–20)
Coulomb (ion-ion,
ion-dipole)
Complex
formation
Steric
effect
Adsorption♦
Partition♦
Bonded phase♦♣♣
HILIC
Ion exchange ♣♦
Ion
interaction
♦
Size exclusion♦
Affinity ♦
Micellar♦
Complexation ♦
Ion exclusion
Chiral ♦
Important;♦Most important;♣Importance variable dependent on column packing.

adsorption
partition (including countercurrent chromatography)
bonded phase
hydrophilic interaction liquid chromatography (HILIC)
ion exchange
ion interaction
size exclusion
affinity
micellar
complexation
ion exclusion
chiral
Separations exploiting each of these mechanisms have been developed in liquid chromatography whereas gas
chromatography is restricted to separations involving one or more of the first three named mechanisms plus chiral
separations. The flexibility of SFC in terms of the mechanisms exploited approaches that of liquid chromatography.
Chromatographic retention is a very complex process involving various specific and non-specific physicochemical
interactions, reflecting the relative attraction and repulsion that the particles of the competing phases show for the
solute and for themselves through multiple molecular interactions. The three basic types of molecular interaction
or force, all of which are electrical in nature, are dispersion forces (due to charge fluctuations throughout a molecule
resulting from random electron/nuclei vibrations), polar forces (arise from electrical forces between localized charges
such as permanent or induced dipoles), and ionic forces. Many different terms are used to describe the molecular inter-
actions (e.g. hydrogen bonding,π-πinteractions, coulombic or electrostatic interactions, hydrophobic forces which
refer to dispersive interactions, etc.) but all are of one of the three basic types. The different molecular interactions that
determine solute retention comprise:
A repulsive component resulting from the Pauli exclusion principle that prevents the collapse of molecules.
Attractive or repulsive electrostatic or coulombic orientation forces between permanent charges (in the case of
molecular ions), dipoles (in the case of molecules without inversion centre), and quadrupoles (sometimes called the
Keesom interaction). Hydrogen bonding is an example of an extreme dipole-dipole interaction.
Attractive induction or polarization forces between a permanent dipole on one molecule with an induced dipole on
another (sometimes called Debye force).
Attractive (London) dispersion forces between any pair of molecules, including non-polar atoms, arising from the
interactions of instantaneous dipoles caused by random distortion of the electronic cloud of a molecule, causing a
slight electrostatic polarization. This spontaneous polarization then induces an opposite polarization in
neighbouring molecules.
The term‘van der Waals forces’is sometimes used as a collective term for the totality of forces (including repulsion)
but, more often, it is restricted to the attractive forces (Keesom, Debye, and dispersion forces).
The complex interaction between solute, stationary phase, and mobile phase due to the molecular interactions leads
to distribution of the solute between the two phases. Chromatographic behaviour is also impacted by other factors
such as steric hindrance of substituent groups within the solute molecule. The molecular forces involved in these inter-
actions are usually weaker van der Waals forces or hydrogen bonding but, in some instances, stronger ionic interac-
tions as in ion-exchange chromatography are exploited[72]and, in rare cases, specific interactions such as charge
transfer forces[73,74].
By way of illustration, dispersion forces are observed between all molecules, but they are the only forces exerted
between non-polar molecules such as hydrocarbons. Thus the separation of n-alkanes on a squalane (a branched par-
affin) stationary phase by GC involves dispersion forces alone. For these non-polar species, polarizability of the solute
molecules increases as the molecular size increases and the dispersion forces also increase allowing us to predict that
the chromatographic retention will increase as the carbon number of the n-alkane increases. As a further example,
aromatic hydrocarbons are polarizable due to theπ-electrons and exhibit induced dipole interactions as well as dis-
persive interactions. Retention and separation of these compounds can be achieved by GC on a squalane stationary
phase exploiting dispersive forces alone. Alternatively, they can be retained and separated by exploiting induced
dipole and dispersive interactions in HPLC on silica gel as a stationary phase and a dispersive mobile phase such
as n-hexane.
171.4 Classification of chromatography

In this chapter sufficient detail is given to provide an understanding of the general principles involved in each mech-
anism. This is supplemented in the relevant sections on gas chromatography, liquid chromatography[75–77](planar
and column), and supercritical fluid chromatography. It is hoped that readers will examine this and subsequent
material closely for, as Poole[71]has noted, there is a decline in understanding of separation mechanisms by those
who perform the majority of routine methods and method development. The outlook for the future development
of chromatography will be bleak if this situation is allowed to persist.
1.4.4.1 Adsorption chromatography
Adsorption was exploited by Tswett in the form of liquid-solid chromatography in columns and thus represents the
oldest of the chromatographic techniques. In separations involving adsorption[78–81], solute and mobile phase mol-
ecules compete for active sites on the surface of the solid stationary phase which is called the adsorbent. Adsorption
onto the surface of the adsorbent is distinguished from partition processes in which the solute also diffuses into the
interior of the stationary phase. A more appropriate term for the latter would probably be absorption in which case the
general process could be termed sorption. Nevertheless, the terminology used in this monograph conforms to usual
practice and refers to adsorption and partition.
Separation in adsorption chromatography is due to interaction of polar functional groups on the solute with discrete
adsorption sites on the adsorbent surface. The selectivity of the separation is dependent on the relative strength of these
polar interactions. The extent to which a solute can be accommodated on an adsorbent surface depends on its spatial
configuration and its ability to hydrogen bond with the adsorbent surface. Adsorption processes are therefore sensitive
to spatial differences in solutes and are ideally suited to separations of molecules having slight differences in shape (i.e.
geometric isomers). Adsorbents also demonstrate a unique ability to differentiate solutes possessing different numbers
of electronegative atoms such as oxygen or nitrogen, or for molecules with different functional groups. This leads to the
use of adsorption for class separations. On the other hand, partition processes depend on a competitive solubility
between two liquid phases and are quite sensitive to small differences in molecular mass. For this reason, members
of an homologous series are generally best separated by a partition system. Moreover, partition is usually more suit-
able than adsorption for highly polar substances such as amino acids and carbohydrates. In this case, the need to use
highly polar mobile phases in adsorption would negate any small differences between adsorptive properties of the
solutes and produce no separation.
Adsorption still finds use in liquid column chromatography (both classical and high performance) and is widely
exploited in thin layer chromatography, whereas applications of adsorption in GC are limited mainly to separations
where the analytes are permanent gases. Because the stationary phase in such separations is a solid, the systems are
referred to as liquid-solid chromatography and gas solid chromatography (GSC). The practical application of GSC
(based on adsorption) antedated the now more popular form of GLC (based on partition). One of the first important
accounts of GSC was published in 1946 by Claesson[82]who used displacement development for the separation of
hydrocarbons on columns packed with activated carbon. Phillips and co-workers used the same method[83]but this
approach was abandoned after 1952 in favour of partition systems as developed by Martin and James[84]. Neverthe-
less, for certain separations, namely that of permanent gases, GSC has come back into favour. Typical adsorbents for
GSC are zeolites (aluminium silicates), Porapaks (cross-linked polystyrene), and molecular sieves in addition to the
more common adsorbents such as silica gel, charcoal, and alumina encountered in liquid chromatography.
The particle size is an important characteristic of an adsorbent. To a first approximation sample retention is pro-
portional to surface area which, in turn, depends on the particle size and on the internal structure of the adsorbent
particles. The smaller the average particle size, the greater the surface area of the adsorbent and hence the number
of active sites available for adsorption. Most adsorbents are available in a range of particle sizes to suit the needs
of the various chromatographic techniques. For thin layer chromatography, particle sizes of 20 to 40μm have been
most common, whereas for classical liquid chromatography in columns the particles are larger (100 to 30μm). For
the modern counterpart (i.e. HPLC) of this particular technique they are smaller (10, 5 or 3μm) and for high perfor-
mance thin layer chromatography, particle sizes of 5μm are used. Adsorbents for GC are typically in the size range of
125–150μm up to 177–250μm.
1.4.4.2 Partition chromatography
Partition chromatography originated with the Nobel Prize winning work of Martin and Synge in 1941 and has as its
basis the partitioning of a solute between two immiscible liquids, as in solvent extraction, except that one of the liquids
is held stationary on a solid support such as silica gel, diatomaceous earth, cellulose, polytetrafluoroethylene (PTFE), or
polystyrene. The solid support is, in principle, inert and solely provides a large surface area on which the stationary
phase is retained. Partition chromatography exploits the fact that a solute in contact with two immiscible liquids (or
phases) will distribute itself between them according to its distribution constant,K(Eq.1.1). The principal
18 1. Introduction and overview

intermolecular forces involved are dispersion, induction, orientation, and donor-acceptor interactions including
hydrogen bonding. These forces provide the framework for a qualitative understanding of the separation process.
The importance of partition systems has declined in all areas of chromatography with the development of bonded
phases. Nevertheless, in GC with packed columns, partition systems are still used on rare occasions. On the other hand,
use of partition systems in liquid and supercritical fluid chromatography is restricted by instability of coated liquid
stationary phases. This is caused by the small but finite solubility of the liquid stationary phase in solvents used as
mobile phases leading to stripping of the stationary phase from the column. One application area in liquid chroma-
tography where the separation mechanism is predominantly partition is paper chromatography. Here, the water
sorbed on cellulose functions as the stationary phase.
Countercurrent chromatography[85–87]is similar to conventional liquid-liquid partition chromatography with the
distinction that the stationary liquid phase is retained in the apparatus without use of an adsorptive or porous support.
In one variant, called droplet countercurrent chromatography, the stationary phase is retained by gravitational force in
a narrow vertical tube (ca. 2mm i.d.) while droplets of an immiscible mobile phase are passed through it. The mobile
phase is either added at the top or base of the tube depending on whether it has a higher or lower density than the
stationary phase. Typically, 300 tubes are connected in series using capillary-bore polytetrafluoroethylene (PTFE) tub-
ing to provide a column of high efficiency. With the more common systems, the stationary phase is retained in more or
less segmented compartments within a coiled tubing while the mobile phase is passed through it. Coils (2–3mm i.d.)
are usually made of PTFE and range in length from a few metres to more than 100m. In contrast to conventional liquid-
liquid chromatography where the volume of stationary phase in the column is relatively small, the stationary phase in
countercurrent chromatography occupies from 40% to 90% of the total column volume. One of the problems of coun-
tercurrent chromatography is the relatively long analysis times. For example, a relatively simple separation of 10 com-
pounds may require anything from a few to several hours using centrifugally operated units up to 1–3days for units
operated in a unit gravitational field. The main role for countercurrent chromatography is for preparative scale sep-
arations in the milligram to gram range. Newer devices also function as efficient extractors to concentrate trace com-
ponents from environmental samples, such as river water and biological fluids, such as urine by replacing the
relatively long separation column with a short column.
1.4.4.3 Bonded phase chromatography
Bonded phases, in which the stationary phase is cross-linked and bonded to the internal wall of the column or chem-
ically bonded to a solid support, are popular in all forms of chromatography. The group (e.g. an octadecyl hydrocar-
bon) that is chemically bonded to the support is referred to as a ligand. The popularity of bonded phases is testimony to
their many advantages. They were originally developed for open tubular columns in GC in an attempt to stabilize the
stationary phase at elevated temperatures. Bonded phases are also available in liquid and supercritical fluid chroma-
tography where they overcome the problems of column bleed associated with physically bonded phases in which the
stationary phase is simply coated on a support material. Compared to adsorption systems in liquid chromatography,
they equilibrate faster, do not exhibit irreversible sorption, and are available with a wide range of functionalities.
GC is unique in that the mobile phase transports solutes through the chromatographic bed but it is otherwise non-
interactive. This simplifies the retention mechanism in GC as molecular interactions are essentially limited to the solute
and stationary phase. The mechanism was treated as a partition that can be approximated by a physical process of
solute vaporization and solution in the mobile and stationary phase. Thus retention is determined mostly by solute
vapour pressure and volatility. Indeed, partition is the dominant process for many solutes; however, a mixed mech-
anism of adsorption and partition is now regarded as a more accurate description although the extent of cross-linking
of bonded phases is an important factor for GC (Chapter 4).
In LC and SFC, chemically bonded phases are now common in all areas including ion exchange, hydrophilic inter-
action liquid chromatography (HILIC), etc. but the mechanism in these processes is sufficiently distinct that the reten-
tion mechanism is not regarded as bonded phase chromatography. The bonded phase mechanism is restricted to
normal-phase (liquid) chromatography (NPLC) and reversed-phase (liquid) chromatography (RPLC). With non-polar
ligands such as an octadecyl hydrocarbon, the stationary phase is always less polar than the associated mobile phase
and this constitutes RPLC. With more polar ligands, the bonded phases can be used with either more or less polar
mobile phases, and in cases involving less polar mobile phases, then it constitutes NPLC.
The mechanism of bonded phase chromatography is complex[76]but appears to involve a combination of partition
and adsorption. In a number of instances, bonded phase chromatography has been referred to as partition chromatog-
raphy because the organic surface layer is regarded as a‘bound liquid film’. However, Locke[88]concluded, as early as
1974, that bonded phases acted more like modified solids than thin liquid films. Nevertheless, the mechanism involved
with bonded phases[75]is sufficiently different from both adsorption and partition to warrant separate treatment.
191.4 Classification of chromatography

In normal-phase systems, the dominant interactions between the solute and the stationary phase that cause reten-
tion and selectivity are polar in nature while dispersive interactions dominate in the non-polar mobile phase. Retention
in normal-phase bonded LC was originally regarded as similar to that in adsorption chromatography involving com-
petitive adsorption between solute and mobile phase molecules for active sites on the stationary phase surface. How-
ever, subtle nuances in retention behaviour required modifications to this simplistic retention mechanism.
In reversed-phase LC, the most extensively used of all forms of chromatography, the retention mechanism has been
studied over several decades[89]based on classical and statistical thermodynamics to describe the fundamental prin-
ciples governing separation but a unified view has not been achieved. This can be attributed to the complexity of the
retention process and the interplay of myriad molecular interactions between the analyte, mobile phase, and stationary
phase. The solvophobic theory was one of the first attempts to describe chromatographic retention using classical ther-
modynamics. The partition model was developed as an answer to the criticisms of the solvophobic theory with the
adsorption model presenting as an alternative approach. An adsorption-partition model based on solution theory
and solubility parameter models, for example, has also been presented (see Ref.[90]). Each of these and the many other
models, with a focus on the stationary phase and/or mobile phase, represents a different view of the retention mech-
anism in reversed-phase chromatography. They generally invoke either the partition or adsorption process which has
caused debate over many years about the relative role of solute partition between the phases and adsorption of the
solute to the stationary phase[91]. These two retention processes represent the extremes of a spectrum of possible
retention models. In the partition mechanism, the solute transfers from the bulk mobile phase to the stationary phase
where it is fully embedded. In the adsorption mechanism, the solute transfers from the bulk mobile phase to the inter-
face between the stationary phase and mobile phase but is not fully embedded in the stationary phase[91]. The sol-
vophobic theory was the first rigorous attempt to explain reversed-phase retention and attributed the retention process
to the mobile phase, ignoring contributions of the bonded stationary phase[75]. The theory identified the major con-
tribution to the retention process as the hydrophobic binding interaction between the solute molecule in the mobile
phase and the stationary phase. However, the actual nature of the hydrophobic interaction remains a matter of heated
debate. Indeed, it is more appropriately termed a solvophobic interaction in reversed-phase systems as the mobile
phase is rarely water but a hydroorganic mixture or a polar organic solvent. It is now clear that the stationary phase
also influences retention and that the properties of the stationary phase are influenced by the composition of the mobile
phase.
1.4.4.4 Hydrophilic interaction liquid chromatography (HILIC)
HILIC employs traditional polar stationary phases such as silica and amino or cyano bonded phases but with
mobile phases more closely associated with reversed-phase systems. It provides an alternative approach to separate
small polar compounds. The separation mechanism is based on both specific and non-specific interactions and has
been modelled as partition, adsorption, ion exchange, and size exclusion[92]. It has been reported as a variant of
normal-phase LC (NPLC) but the separation mechanism involved in HILIC is more complicated than that in NPLC
[93]. The mechanism of HILIC is not completely elucidated[94]but is thought to involve various combinations of
hydrophilic interactions, ion exchange, and reversed-phase retention by the siloxane on the silica surface of the sta-
tionary phase, which contribute to various degrees depending on the particular conditions employed[93,95,96].
1.4.4.5 Ion-exchange chromatography
Ion exchange entails a reversible, stoichiometric exchange between sample ions in the mobile phase and ions of like
charge associated with the ion-exchange surface. The stationary phase is a rigid matrix, the surface of which carries
fixed positively or negatively charged functional groups (A). Counter-ions (Y) of opposite charge are associated with
each site in the matrix and these can exchange with similarly charged ions in the mobile phase. If the matrix contains
negatively charged acidic functional groups then it is capable of exchanging cations and is called a cation exchanger; if
it bears positively charged basic groups it is an anion exchanger capable of exchanging anions. If the sample ions are
depicted as M
+
or X

the process can be represented as follows:
Cation exchange
MatrixA

Y
+
+M
+
!MatrixA

M
+
+Y
+
(1.2)
Anion exchange
MatrixA
+
Y

+X

!MatrixA
+
X

+Y

(1.3)
20 1. Introduction and overview

In order to achieve a separation, the matrix must exhibit some affinity for the sample ions. The counter-ion already on
the resin must also not be too strongly held that it cannot be displaced by sample ions. Secondary effects can arise due
to adsorption or hydrophobic interaction by the matrix itself. Due to the ionic nature of the interactions, ion exchange is
restricted to aqueous liquid chromatography.
1.4.4.6 Ion-interaction chromatography
Ion pair extraction is a valuable liquid-liquid separation technique for isolating water-soluble ionic compounds by
partitioning them between water and an immiscible liquid. The ionic solutes partition formally as ion pairs according
to the equilibrium:
nA
m+
aqðÞ
+mB
n
aqðÞ
ÐnA
m+
mB
n
ðÞ
organicðÞ
(1.4)
in whichA
m+
represents the solute andB
n
the pairing ion or vice versa. This principle was extended by Eksborg and
Schill[97]to chromatography during the 1970s and quickly gained acceptance as a versatile technique for the sepa-
ration of ionized and weakly ionized solutes. The early successes promoted general interest in ion-interaction chro-
matography which uses conventional high efficiency microparticulate, normal-phase or reversed-phase packings.
However, the forte of ion-interaction chromatography lies in its ability to simultaneously separate ionic and molecular
species. In reversed-phase chromatography, ionic species generally show little, if any, retention and are eluted as an
unresolved mixture. Ion-interaction chromatography does have some disadvantages. The ionic solutions can result in
short column life or affect metal components of the system.
The technique as described by Schill and his group became known as extraction chromatography but was dubbed
soap chromatography by Knox and co-workers[98]because of their use of the detergent cetyltrimethylammonium
bromide as the pairing ion in the mobile phase. Subsequent terms used to describe the procedure have included
ion pair, paired ion, and mobile phase ion chromatography (proposed by the Dionex Corporation), solvent generated
ion exchange, ion association chromatography, and dynamic ion exchange, although the authors prefer the use of ion-
interaction chromatography. It is unfortunate that the diversity of terms and debate over the mechanism of retention
have caused considerable confusion.
1.4.4.7 Size-exclusion chromatography
Separations in size-exclusion chromatography are based on a physical sieving process and thus differ from all other
mechanisms in the respect that neither specific nor non-specific interactions between analyte molecules and the sta-
tionary phase are involved. In fact, every effort is made to eliminate such interactions because they impair column
efficiency. Various names have been used to describe this form of chromatography including gel permeation, gel fil-
tration, and steric exclusion. Historically, gel filtration referred to separations of biopolymers, such as proteins, on dex-
tran or agarose gels using aqueous mobile phases, whereas separations of organic polymers in organic mobile phases
on a polystyrene phase were termed gel permeation.
Internal surface reversed-phase supports, or Pinkerton columns, became available in the mid-1980s[99]and involve
a dual mechanism—size exclusion and reversed-phase bonded support. These materials contain stationary phase on
the internal surface of the pores of the support with an external surface which is non-adsorptive. Thus large biomol-
ecules are eluted unretained whereas smaller molecules penetrate the pores and are separated by a conventional
reversed-phase mechanism.
1.4.4.8 Affinity chromatography
Affinity chromatography is at the opposite extreme to size exclusion in that very specific analyte-stationary phase
interactions are exploited to achieve separation. The stationary phase consists of a bioactive ligand bonded to a solid
support (e.g. cross-linked agarose or polyacrylamide). Since the latter may sterically hinder the ligand’s accessibility,
the concept of a spacer arm was introduced. This consists of a short alkyl chain inserted between the ligand and solid
support to reduce or eliminate the steric influence of the matrix. Separation relies on biospecific interactions such as
antibody-antigen interactions, chemical interactions such as the binding of cis-diol groups to boronate or other inter-
actions, whose nature is not fully understood, such as the attraction of albumin to Cibacron Blue F3G-A dye. The spec-
ificity of the ligand sets these bonded phases apart from all others. Ligands may show absolute specificity for a single
substance or may be group specific[100]. The interaction between ligand and analyte must be specific but reversible.
On adding the sample in a suitable mobile phase, the‘active’components with an affinity for the ligand are bound
and retained while the unbound material is eluted in the mobile phase. The composition or pH of the mobile phase is
then altered to weaken the specific interaction of ligand and active analyte, which is released and eluted.
211.4 Classification of chromatography

1.4.4.9 Micellar or Pseudophase liquid chromatography
The popularity of modern liquid chromatography relates partly to the unique selectivities that can be generated in
the mobile phase by the addition of modifiers. In ion-interaction chromatography, this is achieved by adding a low
concentration of modifier to the mobile phase. Here the concentration of ion-interaction reagent was intentionally
maintained below the critical micellar concentration. However, Armstrong and Henry[101]demonstrated the use
of reversed-phase mobile phases containing higher concentrations of surfactant exceeding the critical micellar concen-
tration[102]. There are a number of reports concerning the theory[103]and unique chromatographic advantages of
micellar chromatography[104]. One major advantage of micellar systems is their selectivity. Retention of solutes gen-
erally decreases with increasing micelle concentration but the rate of decrease varies considerably, producing inver-
sions in retention order.
1.4.4.10 Complexation chromatography
Complexation or chelation chromatography can be considered as a generic term to encompass all chromatographic
separations dependent on the rapid and reversible formation of a complex between a Lewis acid (e.g. metal ion) and a
Lewis base. The versatility of complexation chromatography is due, in part, to its suitability in all areas of chroma-
tography. Other reasons are the vast range of Lewis acids, Lewis bases and complexes which can be utilized, the dif-
ferent ways that they can be incorporated in the chromatographic column and the fact, that in many cases,
conventional chromatographic columns or packings (e.g. silica adsorption, reversed phase, and ion exchange) may
be utilized. Potentially important applications involve the incorporation of Lewis acid, Lewis base or complex in
the mobile or stationary phase to affect the separation of a wide range of inorganic, organic, and biochemical species.
Ligand-exchange chromatography, chelate affinity chromatography, and other terms have been used to describe
various aspects of this application.
1.4.4.11 Ion-exclusion chromatography
Ion-exclusion chromatography is defined[105]as a technique used to separate weak acids (e.g. carboxylic acids,
amino acids), weak bases (e.g. ammonia, amines), and hydrophilic molecular species such as carbohydrates and
the lower alcohols on an ion-exchange column. Because of Donnan exclusion, ionic material is excluded from the
ion-exchange resin and passes quickly through the column. Non-ionic substances are not excluded and partition
between the aqueous mobile phase and occluded water within the resin beads[106]. Because of differing partitioning
effects and van der Waals forces, non-ionic solutes are retarded by the column and separated. Thus the mixed mech-
anism that varies between solutes involves both hydrophobic adsorption and screening effect[107]. As an illustration
of an ion-exclusion separation, inorganic and organic acids may be chromatographed on a cation-exchange resin. The
strongest acids elute at the start of the chromatogram (at the column hold-up volume—Chapter 2) because they are
highly ionized and are repelled by the immobilized negative charge of the resin. Weaker acids exist largely in the
unionized molecular form and are separated by partitioning between the mobile phase and the occluded solvent
[108]. Other terms used to describe the process include ion-moderated partition chromatography[109]and ion-
exclusion partition chromatography[108].
1.4.4.12 Chiral chromatography
Chiral molecules interact with each other in a stereospecific way and are important in food chemistry, medical sci-
ences, etc. Therefore the ability to differentiate stereoisomers, specifically enantiomers, is of great importance. Indirect
approaches involve derivatization of the enantiomers of an analyte with a stereoisomerically pure reagent to form
diastereomers via covalent bonds[110]and the derivatized compounds are subsequently separated under achiral con-
ditions. In terms of the mechanism of chiral chromatography, this is a trivial process. Direct enantioseparations refer to
the separation of enantiomers in a chiral environment by way of a chiral stationary phase. Thus the mechanism
involves the reversible formation of diastereomers between the enantiomers of an analyte and the enantiomeric sta-
tionary phase. Various models have been developed to describe the specific details of the interaction between analyte
and stationary phase[110–112].
1.4.5 Other systems of classification
Other methods of classification are in current use and no doubt as new developments occur further systems will be
introduced. One method that should be mentioned is classification according to the nature of the analyte. This system
of classification gives rise to terms such as ion chromatography and fast protein liquid chromatography. Ion
22 1. Introduction and overview

chromatography originally described the particular system of ion-exchange chromatography in which a low capacity
ion-exchange column, a suppressor column, and a conductivity detector were used to measure inorganic ions. The
definition has now been broadened to include all modern chromatographic separations of ionic species. The word
‘modern’implies that the separation is high performance and that automatic, on-line detection is employed. The bulk
of ion chromatography is concerned with ion-exchange separations using low capacity ion-exchange resins; however,
ionic species are also separated by ion-interaction chromatography and ion-exclusion chromatography.
1.5 Chromatography: Publications and equipment sales
This is a convenient point at which to examine the importance and relative use that is made of the various techniques
identified in the previous sections. There are two relatively simple and convenient ways in which to perform this com-
parison. One of these is publication numbers but with the caveat expressed inSection 1.1regarding research versus
routine use. The other is the volume or monetary value of sales of chromatographic equipment.
In terms of publications, a number of bibliometric studies have been conducted looking at the entire field of chro-
matography or specific techniques such as planar chromatography[113]. The former are of more interest to the current
context. Here also there is considerable diversity in approach; some examine the literature produced by specific jour-
nals while others use broader services such as Web of Science or Scopus. Each approach has distinct advantages and
problems associated with its use and must be designed to fit the purpose of the study. When using abstracting services
in bibliometrics, a knowledge of the approach used by the service to classification of papers is essential. Whether the
analysis should be conducted on the paper title, abstract, or some other aspect must be considered carefully in the
context of the purposes of the study.
There are two aspects of bibliometrics or the more specific scientometrics. The first and simplest answers the ques-
tion, Who is doing what, and where? The second aspect is of more interest and it examines the time dependencies of the
general parameters of the academic field in an endeavour to characterize the scientific and technical evolution and
contemporary status of the field[114]. Bibliometric studies of chromatography from the 1970s are of historical interest
and few have been completed since that time[115]. A bibliometric analysis of the field of analytical chemistry for the
years 1929 to 2013 restricted to the journalAnalytical Chemistry[116]shows that the number of papers published in the
journal using the words‘gas chromato’or‘liquid chromato’in the title peaked in the years 1961–1965 and 1981–1985,
respectively. This is quite surprising in some ways but as the authors noted their approach introduced a bias towards
novel uses and the development of the particular technique. With this in mind, the data are consistent when one con-
siders the years in which GC and modern liquid chromatography were introduced.
One factor that is clear in all studies is the increasing number of publications that use chromatography or investigate
some aspect of chromatography (e.g. chemical structure-retention relationships). The other feature is the importance of
chromatography in scientific studies. For example, the number of publications in any year involving chromatography
represents a significant proportion of the total publications.
Trends in usage of individual chromatographic techniques are informative. The period from about 1940 to 1990 is
particularly significant in the development of chromatography. A CAS data search of index terms for this period[117]
shows a large decline in the number of publications involving paper chromatography which was gradually replaced
by thin layer chromatography. The analysis also shows that the period 1967–1971 is significant and it was in 1968 that
modern liquid column chromatography (or HPLC) commenced its rapid growth. A bibliometric study of gas chroma-
tography[118]showed an increase in the total number of publications involving GC viewed in 5-year blocks from 1952
to 1998 with one exception, the period 1977–1981 which showed a slight decrease from the previous block. Based on
publication numbers, SFC commenced a growth phase about 1980, peaked in 1989–1992, and decreased beginning
from 1993[118]. SFC remained a minor contributor to the overall number of chromatographic publications in 1987
when 115 papers were published[119]. This comprised approximately 0.4% of the total number of papers for the year
involving chromatography.
Data on publication numbers involving chromatography between 1980 and 2020 are presented inFig. 1.11. The
number of publications on chromatography increased during this period until 2013 and then declined until 2019 (data
are collected at September 2020). The figure provides an overview of the research being conducted in the area and an
indication of the relative importance of the various techniques. There are a number of caveats related to the data. For
example, the numbers are not mutually exclusive as a publication can have multiple chromatography terms. Evalu-
ating any increase or decrease in citation counts should be balanced with the mindset that CAS selects sources for
indexing, and the indexing itself is human applied, hence changes over time can and will be influenced by selection.
For example, a downturn in non-patent literature (NPL) could be a factor of reduced research or removal of certain
231.5 Chromatography: Publications and equipment sales

NPL sources from indexing. Alternatively, it could reflect delays in human indexing of NPL—it could lag behind in
times of increasing volume of patent literature (the volume of Chinese patents is continually increasing). Similar pub-
lication declines in other domains are not necessarily due to a decline in research. Human indexing, even with tech-
nology that enables people to do more (and better), is rate limited to the number of available scientists. A future
comparison of this data with that generated by automated indexing in other systems, such as Web of Science, would
be informative.
For several decades now, HPLC has been dominated by reversed-phase separations although other mechanisms are
finding more application in recent years. The number of publications on SFC remains small but underwent a renais-
sance commencing about the beginning of the new century[120,121]. SFC continues to occupy a particular niche
between GC and LC for some specific analyses (Chapter 7). There has been a revival of SFC in recent years, especially
in the chiral (preparative) field[122], but also more recently in the general analytical area[123].
The value of sales of chromatographic instruments was$725 million in 1990 including analytical gas, liquid, ion,
and supercritical fluid chromatographs as well as detectors employed in these instruments[124]. By 2004, the global
market for analytical instrumentation was$30 billion and this increased to$37billionin2009[125].Salesof
chromatographic and related equipment have led the field in analytical instrument sales for some years. For
instance, the separations market, essentially chromatography, accounted for 17% or$6.3 billion of the global$37
billion market in 2009. The market for chromatography instrumentation was about$8 billion in 2015 and is projected
to reach$10 billion by 2020[126]. Chromatography instrument sales haverepresented an expanding market over
this entire period from 1990. Thus there is a vast amount of chromatography being performed both in academia and
industry.
Analytical HPLC and GC collectively account for nearly three-quarters of total demand in the chromatography mar-
ket. Clinical HPLC is the fastest growing segment while the demand for thin layer chromatography continues to
decline resulting in a stagnant market for this technique. The growth of LC is being attributed to increased use of HPLC
and UHPLC techniques by academics and biopharmaceutical companies. UHPLC will continue to be in high demand
for biopharmaceutical separation analysis as it speeds up the data collection process. The growth of GC can be credited
to crude and shale oil production, growing significance of waste water treatment, increased implementation of
GC-MS, incentives to moderate pollution in the environment, an increase in food safety standards, and greater impor-
tance of GC for drug testing.
Thermo Fisher Scientific, Agilent, and Waters are dominant players in the overall chromatography market. Perkin
Elmer and Phenomenex are in the top 10 of all vendors and other important suppliers include Shimadzu, TOSOH,
VWR, and Hitachi. Numerous companies make up the remainder of the highly competitive chromatography market,
typically by capturing specialized niches in the aftermarket sales.
0
1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020
Gas chromatography
Ion exchange chromatography
Liquid chromatography
Preparative chromatography
Reversed phase chromatography
Size exclusion chromatography
Supercritical fluid chromatography
5000
Publication Year
10000
15000
Number of Publications
20000
25000
30000
35000
FIG. 1.11Number of publications indexed under high-level chromatography controlled terms in the Chemical Abstracts lexicon in the relevant
publication year. The high-level terms (e.g. gas chromatography) include all narrower terms grouped under this level such as capillary gas chro-
matography, headspace gas chromatography, etc.Credit: Matthew J. McBride, Director, Science IP, CAS, A division of the American Chemical Society.
24 1. Introduction and overview

It is not surprising that a market of this size is well served by market analyses and reports[127,128]including some
on specific market segments such as 2-Dimensional chromatography. A number of trends can be identified in these
reports. Pharmaceuticals, life sciences, and biotechnology are the largest end-user sectors for the chromatography
instruments market. However, the technique is increasingly important in the clinical, food, and environmental testing
market segments[126]. Drivers are the increased analytical testing required to comply with environmental regulations
and the regulations imposed by authorities in industries such as pharmaceuticals as consumer awareness about health
and the environment is raised. Chromatography also plays a vital role in new drug development and clinical research.
There is an expectation of increasing demand for more specialized instruments, more sensitive detection, and
enhanced data integration in the coming years.
The earlier mentioned figures do not allow for consumables and here also sales of reagents for chromatography are
increasing annually with the figures again dominated by pharmaceuticals and the food and beverage industry[129].
1.6 Applications of chromatography
Chromatography is used to solve a diverse range of problems in numerous application areas as indicated by the
following titles of articles published over a number of years in the journal‘Analytical Chemistry’.
Effects of raw material change in manufacturing process resolved[130].
Industrial analytical chemists and OSHA regulations for vinyl chloride[131].
Ion chromatography in bombing investigations[132].
Anatomy of an off-flavor investigation: The medicinal cake mix[133].
Solving mysteries using infrared spectrometry and chromatography[134].
Chemical analysis of fire debris: Was it arson?[135].
Don’t waste your breath[136].
Determination of Drugs of Abuse in Airborne Particles by Pressurized Liquid Extraction and Liquid Chromatography-
Electrospray-Tandem Mass Spectrometry[137].
Detection of Synthetic Testosterone Use by Novel Comprehensive Two-Dimensional Gas Chromatography Combustion-
Isotope Ratio Mass Spectrometry[138].
Caffeine in your drink: natural or synthetic?[139].
Authentication of Organically and Conventionally Grown Basils by Gas Chromatography/Mass Spectrometry Chemical
Profiles[140].
Universal route to polycyclic aromatic hydrocarbon analysis in foodstuff: two-dimensional heart-cut liquid chromatography-
gas chromatography-mass spectrometry. (Report)[141].
Analytical Strategies for Doping Control Purposes: Needs, Challenges, and Perspectives[142].
Despite the diversity illustrated by the earlier mentioned applications, the chromatographer is usually seeking the
answer to one or more of three questions in performing a chromatographic separation: What is present? How much is
present? or, how can a pure substance(s) be isolated from a mixture? More recently, a fourth consideration has become
important. In areas such as metabolomics, the goal may be untargeted analysis of biological samples to identify dif-
ferences between the metabolite profiles of control and test groups[143]. These different aspects of chromatography
are clearly related. For instance, measuring the amount of substance present clearly entails identification of the com-
ponent to be quantified as a first step. For convenience, however, we will look at the answers to the questions sepa-
rately and consider them as qualitative and quantitative analysis, metabolomics, and preparative chromatography.
The intent of both qualitative and quantitative analysis varies. It may be performed to provide information on the
purity of a sample, to collect physicochemical data (e.g. reaction rates), to quantify the amount of analyte in a clinical
sample to enable diagnosis and treatment, etc.
The treatment given in this section of qualitative and quantitative analysis, metabolomics, and preparative chro-
matography is expanded inChapters 9 and 11.
1.6.1 Qualitative applications
Chromatography is frequently used to confirm either the presence or absence of a compound in a sample. This is
done by comparing a chromatogram of the pure substance with that of the unknown performed under identical con-
ditions. The chromatographer must be confident that a substance identified in a sample is actually present (e.g. a
251.6 Applications of chromatography

prohibited substance in the urine of an athlete) and, equally as important, that a substance not found is indeed not
present at a stated detection limit (e.g. pesticide in a foodstuff ). One of the difficulties in the comparison is that the
chromatogram is not unique; many substances will exhibit the same chromatographic behaviour under identical con-
ditions. The magnitude of the problem is compounded by the fact that there are approximately 10 million compounds
known
c
and over 85,000 of these are registered for commercial use[144]. In short, chromatography can be used for
qualitative analysis in a limited set of circumstances, but its main use is not for screening unknowns. The best methods
of qualitative analysis are the so-called coupled or hyphenated techniques which combine the excellent separating
ability of chromatography with the capabilities of spectrometry for identification. Such techniques include gas
chromatography-mass spectrometry, gas chromatography-infrared spectrometry, and the corresponding high-
performance liquid chromatographic techniques and supercritical fluid chromatographic techniques.
Chromatography provides information on the complexity of a sample. The number of spots (TLC or PC—Chapter 3)
or peaks (GC, HPLC or SFC—Chapters 4–7) indicates the minimum number of components; conversely, the purity of a
compound can be checked and the presence of a single spot or peak is taken as an indication of purity. In other
instances, all that is required for quality control purposes is a fingerprint chromatogram showing the pattern of peaks
which can be compared with the‘normal’pattern. Patterns have been used to look for the presence or absence of
metabolites in certain diseases, for example, in phenylketonuria. One situation in which qualitative analysis is widely
used is the examination of reaction products from organic and inorganic syntheses to determine which conditions give
the cleanest products and which reactions do not occur at all (giving only reactants).
1.6.2 Quantitative applications
Chromatography is used to establish the amount of individual components in a sample by comparison with suitable
standards and calibrations. Quantitative data are widely used in industry for quality control, in clinical chemistry for
the assay of body fluids, and in environmental science for monitoring air, water, and soil samples. Chromatography
has also enabled the measurement of product distributions in reaction mixtures. Such data are useful in several areas
including physicochemical measurements (e.g. kinetic studies). Most significant in the last few decades have been the
fine chemical, biotechnology, and pharmaceutical industries. Their combined efforts have led to the production of
many high-purity chemicals and the need to characterize these new compounds has combined with the pressure
of regulatory agencies to be able to measure and quantitate them (Section 1.5—Chromatography: Publications and
equipment sales).
1.6.3 Chromatographic metabolomics
Chromatographic metabolomics is used here as a convenient title to encompass a broad range of applications
described by various names that include sample fingerprinting, profiling, untargeted and targeted metabolomics
and metabonomics. The various terms have their origin in metabolomics and are not restricted to chromatography.
This application area is not new originating from the work of Horning and Horning[145], in 1971, in relation to the
determination of metabolites using GC-MS. However, the area has expanded exponentially since the advent of high-
resolution chromatography and spectrometry coupled with the application of data management systems in
chromatography.
In one sense metabolomics is an area that has not yet matured and, as so often occurs with new fields, a controversy
arises with nomenclature[146]. Indeed, there is far from universal agreement about nomenclature but our interest is in
the application of chromatography to metabolomics rather than in metabolomics per se.
One treatment of the topic[147]distinguishes profiling or targeted metabolomics from fingerprinting or untargeted
metabolomics. Profiling involves analysis of a group of related metabolites which are typically identified and quan-
tified. Fingerprinting is based on separating as many metabolites as possible without necessarily identifying or quan-
tifying any of the separated compounds. The common feature in these applications is the extraction, in a single
chromatographic development, of as much information as possible about all peaks in the chromatogram thus provid-
ing information on the composition and comparative chemical complexity of the sample. Metabolomics has a more
ambitious goal; identification and quantification of the complete set of metabolites in a cell, tissue, or organism
[148]but this cannot yet be achieved in a single analysis.
c
Gmelindatabase (formerly theGmelins Handbuch des Anorganische Chemie), to date lists about one million and a half inorganic and organometallic
compounds, and theBeilsteindatabase (formerly theHandbuch der Organischen Chemie)lists about ten million compounds. However, the number
grows daily as chemists either synthesize or isolate from nature and identify new compounds.
26 1. Introduction and overview

Two considerations generate interest in system-wide metabolic analysis[149]. First,‘the complexity of the plant
metabolic network is such that it is not yet possible to construct predictive models of metabolic performance that allow
rational metabolic engineering of plant genomes’. Second, system-wide metabolic analysis is a useful tool for func-
tional genomics as the metabolome is the terminal downstream product of the genome. The central biochemical
and molecular biological dogma[150]holds that information flows from genomic DNA through mRNA transcripts,
which are then translated to proteins, among them enzymes. It is the enzymes that then influence the concentrations of
their substrates and products, which are integrated in tightly controlled metabolic pathways. The flux of these low
molecular mass metabolites within a cell, tissue, or organism generates the phenotype.
1.6.4 Preparative applications
The ability of chromatography to separate the components of a sample can be used preparatively to produce pure
constituents on either a laboratory or industrial scale. A single thick layer (up to 1mm) TLC plate can be used for up to
10mg of a sample. Multiple runs can be used to separate larger samples. Preparative gas chromatography and high-
performance liquid chromatography is also possible by scaling up the column dimensions to provide up to gram quan-
tities of pure components. In industry, much larger quantities ranging up to several tonnes are processed and purified
by chromatography. An example is provided by the refining of cane sugar. Operating chromatographic equipment at
this opposite extreme to that involved in analysis presents its own intriguing problems of process engineering and is
discussed in detail elsewhere[151].
1.7 Comparison of chromatographic techniques
Comparison of separation techniques in general terms is difficult because each technique has unique advantages
that suit it to particular applications while making it less suitable in other areas. However, some basis of comparison
is essential to provide guidance and reassurance for the novice chromatographer and to enable the experienced chro-
matographer to make sensible choices about new techniques and developments. The following series of tables (Tables
1.4-1.7) present an overview of various chromatographic techniques. A more detailed analysis of the various tech-
niques would address three major attributes of a chromatographic separation, namely, speed, resolution, and sensi-
tivity. However, these are constantly changing with new developments and are highly variable depending on the
specific analyte. Thus they are best addressed in relevant chapters. Cost is a fourth factor that can be crucial in many
TABLE 1.4Comparison of column techniques—gas chromatography, supercritical fluid chromatography, and high-performance liquid
chromatography.
Gas chromatography Supercritical fluid chromatography
High-performance liquid
chromatography
Sample Limited to thermally stable, volatile species although
this can be extended by derivatization
Well suited to samples not easily
handled by GC or HPLC
Few, if any sample
restrictions
Mobile
phase
Non-interactive Interactive Interactive
Resolving
power
In routine practise, open tubular column GC provides
unparalleled separations
Excellent Excellent
Detectors Very well developed and, in general, more sensitive
than HPLC detectors. Flame ionization detector gives
almost universal detection for organics
In theory, both GC and HPLC detectors
can be used. Limitation is on mobile
phase/detector compatibility
No universal, sensitive
detector as a result of the
broad applicability of HPLC
Sample
recovery
Simple Simple Less convenient
Hyphenated
techniques
GC-MS and GC-FTIR are routine procedures Well suited to coupling with
supercritical fluid extraction for on-line
sample preparation
LC-MS is a routine procedure
Capital and
running
costs
Capital costs vary greatly depending on the level of sophistication of the equipment. Running costs depend very much on local
circumstances
271.7 Comparison of chromatographic techniques

situations. It is worth noting that for any specific separation, one attribute may be absolutely critical. In this case, a
technique vastly superior in all other attributes but the critical one is useless for this application.
The choice between different chromatographic techniques is a daunting prospect for the novice chromatographer.
However, the answers to a few simple questions will often indicate the most suitable technique. The nature of the ana-
lyte and the sample matrix are the first considerations. The intent of the analysis is also important as are practical con-
siderations, such as the availability of a particular piece of equipment. For example, GC is the obvious choice for the
analysis of atmospheric gases. In other instances, the choice is less clear-cut and equipment availability and the
chromatographer’s experience and personal bias will probably determine the final choice. Nevertheless, different chro-
matographic techniques should not be considered to be competing with each other for the solution of every problem.
Each technique should be considered on its merits and used where it is most appropriate. It is only by recognizing the
complementary nature of the various techniques that maximum benefit will be achieved.Fig. 1.12illustrates this
TABLE 1.6Comparison of some liquid chromatographic techniques.
Classical column
chromatography Thin layer chromatography
High-performance thin layer
chromatography
Sample
introduction
Single, sequential Multiple, concurrent Multiple, concurrent
System Closed column Open bed Open bed
Separation
time
30 min to several hours 30–200 min 3 –20 min
Resolving
power
Low Medium High
Detection Fraction collection Mostly static, in situ, permanent record. Can be adapted to
quantitative analysis
Versatile including quantitative
analysis
TABLE 1.7Choice of liquid chromatographic technique for particular analytes.
Analyte Liquid chromatographic technique
Structural isomers of moderate polarity Liquid-solid chromatography
Members of an homologous or oligomer
series
Reversed-phase chromatography
Weak acids or bases Ion-suppression reversed-phase chromatography, ion-exchange chromatography, ion-exclusion
chromatography
Strong acids and bases Ion pair or ion-exchange chromatography
Molecular mass distribution Size-exclusion chromatography
Enantiomers Require special stationary phases containing suitable chiral centres
TABLE 1.5Comparison of liquid column chromatographic techniques.
Classical column chromatography High-performance liquid chromatography
Convenience Tedious, time consuming Rapid, easily automated
Columns Columns usually discarded after a single use Columns reusable over many months to years (if used with proper
care)
Procedure Detection and quantification achieved by fraction collection
and further off-line processing
Separation, detection, and quantification achieved on-line
Application Sample preparation, preparative scale separations Sample preparation and preparative scale possible but also
applicable to high resolution separations
Capital costs Low High
28 1. Introduction and overview

complementarity where it is seen that chromatography is a part of separation science and that the techniques that enjoy
widest use overlap to some extent. Indeed, this concept extends beyond the traditional boundaries of chromatography
as elucidated by Růžiůcka and Christian[152]who compared the synergism of flow injection and chromatographic
techniques.
The comparisons presented in this section should provide a guide to a sensible decision-making process. It is dif-
ficult to reduce the selection of a method to a simple decision tree (Fig. 1.13); either the chart becomes so complex or
contains so little information that it becomes misleading. Decision trees are examples of supervised learning but with a
significant advantage over other methods; minimal data preparation is required. FromFig. 1.13the primary deciding
factor in choosing between column methods is the volatility and thermal stability of the analyte below 350°C. How-
ever, consideration should be given to the possibility of derivatizing the sample to obtain adequate volatility for GC
particularly if detection presents a problem in LC. In those instances where liquid chromatography is the appropriate
choice, some further generalizations are possible (Table 1.7).
An understanding of the capabilities and limitations of the various techniques is a good starting point in method
selection. Experience is also a very good guide but the versatility of chromatography must always be remembered, e.g.
for many years ion exchange was limited to the separation of ionic substances largely because contemporary thought
restricted it to such separations. Nowadays, many excellent separations of non-ionic solutes are achieved by ion
exchange-type processes.
1.7.1 Analyte characteristics
In principle, the decision between GC, LC, and SFC is relatively simple. For gas chromatographic analysis, the ana-
lyte must be volatile (in general this requires a boiling point below 500°C or a vapour pressure below this temperature
of several kilopascals) and thermally stable. These limitations are not imposed on either LC or SFC. On the other hand,
the greater versatility of LC for analyte type imposes detector restrictions. As a result of volatility considerations, GC is
limited to relatively low molecular mass organic solutes for which a universal detector (the flame ionization detector,
FID;Chapter 4) exists. Strictly, the FID is not universal but it comes very close. The versatility of LC means that it is
amenable to all analyte types (providing a suitable solvent exists) including inorganic and organic species, low and
high molecular mass substances including polymers, and ionized and non-ionized materials alike. A detector capable
of sensing this broad range of analytes has not been developed and so the strength of LC also becomes its‘weakness’.
Derivatization of the analyte can be used to improve the volatility/thermal stability in GC or the detectability in LC
(and GC). The ease of detection is not a consideration in selecting SFC as detectors developed for both GC and LC can
be used.
Is either GC, LC, or SFC inherently more efficient or faster? Giddings[153]attempted to answer this question in
relation to GC and LC. He derived an expression relating column pressure and efficiency which predicts that LC would
be more efficient than GC by a factor of 10
3
if run at the same pressure. Looking at the problem the other way, GC
would be as efficient as LC if the GC were performed at a pressure 10
3
times as high as LC. The problem with the
TLC
Ion Chromatography
HILIC
Size exclusion
GC
SFC
RPLC
Chromatography Space
Separation Science Space
FIG. 1.12Schematic diagram showing the complementarity of var-
ious chromatographic techniques within a space defined by chroma-
tography and separation science.
291.7 Comparison of chromatographic techniques

first conclusion is that the time required to achieve the efficiencies in LC would be unacceptably long. The problem
with the second conclusion is that it is very difficult to perform GC at the high pressures required.
The question of the inherent speed of the three techniques was revisited in 2017[154], 5 decades after Giddings’
iconic paper. A kinetic approach was used to assess which technique produced the most theoretical plates (a measure
of efficiency—Chapter 2) for each given time, or, equivalently, requires the shortest time to achieve a given number of
plates. With no practical limitations imposed on the techniques, LC is inherently faster. Column and instrument format
for GC has been stable for many decades whereas there has been a continual stream of improvements for both LC and
SFC that continues to the present day. This shows no sign of abating. When practical considerations relating to column
dimensions and system operating pressures are considered, a fully optimized GC system (thin film coated capillary
with optimized diameter, length, and flow rate) will generate a given number of theoretical plates about 10 to 12 times
faster than a fully optimized state of-the-art LC system. Packed SFC has speed limits lying in between those of packed
bed LC and open tubular GC.
In conclusion, theory has not provided much assistance and the initial choice between GC, LC, and SFC is seldom on
the basis of differences in efficiency or time. Indeed, it is a reasonable maxim in chromatography that a technique
should not be claimed to give a faster separation than is possible with other techniques; the number of chromato-
graphic variables is sufficiently large that any such example could be demonstrated as incorrect in practice.
The choice between LC, GC, and SFC is nearly exclusively determined by the chemical nature of the analyte and
sample.
1.7.2 Characteristics of the sample matrix
In many instances, the chromatographic technique can be used to separate the analyte from its matrix and to deter-
mine its identity and concentration. In other cases, pre-fractionation is necessary to avoid accumulation of sample res-
idues in the chromatographic system. This is probably a more common occurrence in GC where accumulation and
FIG. 1.13Basic decision tree for choosing a chro-
matographic technique.
30 1. Introduction and overview

charring of non-volatile residues can lead to analyte decomposition. Nevertheless, accumulation of sample compo-
nents also occurs in LC and SFC where it can lead to various anomalies such as unstable baselines in the chromato-
gram. Pre-fractionation may involve simple filtration, solvent extraction, or some form of chromatography such as
planar or liquid column chromatography (Chapter 10). In general, classical column chromatography or one of the
many modern extensions is ideal for this purpose. It is relatively simple, inexpensive, and provides a crude separation
which is frequently all that is required at this stage.
With samples containing both volatile and non-volatile components, less sample preparation will probably be
required for LC than for GC. On the other hand, TLC is ideally suited to separations where it is desirable to observe
all the components of a sample, as any residues left at the sampling point are easily observed. For very complex matri-
ces, a high-resolution technique is clearly desirable. Although HPLC and high-performance TLC (HPTLC) provide
high resolving power, the performance of open tubular column GC is unsurpassed in this respect. For components
present at the trace level, a sensitive and/or selective detector is necessary.
1.7.3 Purpose of the analysis
For analyses where accuracy and sensitivity are not prime requirements, TLC is ideal. An example of this would be
the qualitative examination of the reaction products from an inorganic or organic synthesis. Simultaneous analysis of
several samples is also possible with TLC which is therefore well suited to routine screening of large numbers of sam-
ples as in monitoring biological fluids for the presence/absence of drugs where speed and economy are needed. Of all
chromatographic techniques, TLC is the most universal; it is suitable for all compounds (except permanent gases) and
all components of the sample are present on the plate and, with appropriate detection methods, all components can be
observed. This compares with column techniques where some sample components may be retained in the column.
However, TLC is not normally as accurate or sensitive as the column techniques. Thus analyses requiring greater selec-
tivity and quantitative accuracy can normally only be achieved with more sophisticated instrumental techniques such
as GC, HPLC, SFC, and HPTLC. Such methods are also more easily automated for process control or analysis of large
numbers of similar samples.
It is worth stating, once again, that the different techniques are complementary.
1.8 Historical aspects
There is a temptation to ignore the work of the past. However, in order to exploit fully current developments, an
awareness of past advances is desirable. A brief historical excursion at this point should place in perspective the devel-
opment of thought and activity in a technique which has had an important fundamental and applied influence in both
chemistry and other areas. Ettre has published some 50 papers that thoroughly explore the history of chromatography
in all of its forms. Specific aspects of particular note that have been examined by Ettre include:
General history and contributions of various individuals[155–158].
Invention of chromatography in the early 1900s[155,157–160].
Rebirth of chromatography in the 1930s[161].
Invention of partition and paper chromatography in the 1940s[162,163].
Preparative liquid chromatography and the Manhattan Project[164].
Development of gas chromatography, notable for some of the predictions[165].
This discussion represents an overview rather than a detailed and comprehensive account of the evolution of
thought and practice in each chromatographic technique. The reader interested in a more detailed account of the his-
tory of chromatography is referred to the various articles published since 1970[14,156–160,166–181]and to the text by
Zechmeister and Cholnoky[182]which may be difficult to access.
Credit for the introduction of chromatography is difficult to assign but is generally afforded to Tswett who was the
first to use the name‘chromatography’. Many natural processes such as the underground migration of fluids through
clays and sediments can be regarded as a chromatographic process. Various ancient texts describe procedures which
undoubtedly involve the unconscious use of chromatography. An instance is the conversion described by Aristotle of
salty and bitter water into potable water with clay. However, credit for the invention of chromatography is not attrib-
uted to the observation of these natural processes. Certain investigations in the second half of the last century may be
regarded as the precursors of chromatography. Prominent among these is the work of the American petroleum
311.8 Historical aspects

chemist D.T. Day (1859–1925). Zechmeister[182]first drew attention to Day’s activities. This was followed by a heated
exchange of ideas[175,183–185]culminating in claims that Day was the inventor of chromatography. Significant
though Day’s contributions were, his role in the development of chromatography has probably been exaggerated.
Indeed, the Editorial Board of the journal Biokhimiya[186]regarded these claims as a Western plot to‘reduce the merit
of the Russian scientist M.S. Tswett’. Zechmeister[187]stated that the work of Day,‘originating from considerations
which were quite different from Tswett’s, might well, under favourable conditions, have developed into systematic
chromatography’. It is clear then that Zechmeister recognized the potential of the work conducted by Day but did not
see him as the inventor of chromatography and rather regarded Tswett as the‘father of chromatography’.
The principles of chromatography were first outlined by Tswett on March 21, 1903 (March 8 according to the old
Russian calendar in use at that time) at a meeting of the Biological Section of the Warsaw Society of Natural Sciences.
Tswett’s biography and details of his work are covered in a number of references[159, 160, 174, 188–193]. He published
a subsequent paper in 1906 in which chromatography was used for the first time and this paper has been translated
into English and republished because of its importance[194]. The 1903 presentation represents the first report of
Tswett’s systematic investigation of the chromatographic separation of plant pigments. Tswett soon became embroiled
in a bitter controversy regarding his procedure which was either ignored or rejected outright by his contemporaries
[195]as being of little merit. Kohl, an authority on carotene pigments, cited Tswett’s failure to reference Kohl’s own
book as proof of the inaccuracy of Tswett’s results[196]. The paper by Livengood[180]provides an excellent outline of
the detailed rejection of Tswett’s work. Chromatography has also been neglected by historians of science which is
unfortunate, as Livengood notes, because chromatography affords an excellent case study for a number of
philosophico-historical questions, such as the following: what distinguishes scientific instruments from scientific tech-
niques, what is the relationship between instrumentation and theory, what is the role of explanation in the develop-
ment of instruments, and how do instruments and techniques serve as rhetorical devices? These are important
considerations but most important is the salient warning that rejection of Tswett’s work serves to all.
Tswett was so aware of the importance and scope of his discovery that he insisted—although his experiments did
not result in isolation of pure substances—against all opposition that chlorophyll was a mixture of two components.
Tswett’s involvement with chromatography had almost ceased by 1912 and there followed a 20-year period of dor-
mancy in which few researchers used the technique[167]. By 1930, the interest in natural substances and the need to
separate and purify these provided a fertile environment for the acceptance of chromatography. The rebirth occurred
in the laboratories of the Kaiser Wilhelm Institute for Medical Research at Heidelberg. Edgar Lederer
d
[197]in a careful
study of the literature found a reference to Tswett’s work and decided to apply chromatography in his own research
with Richard Kuhn on carotenoids. The work was published in a series of papers[198–200]in 1931 and the‘new’tech-
nique soon spread and became accepted as a standard laboratory procedure. An important development occurred in
1937 when Schwab and Jockers[201], at the University of Munich, adapted the technique to the analysis of
inorganic ions.
Following the rediscovery, uses of chromatography continued to expand and modifications and variants were
introduced. In the procedure as practised before 1935, a column was packed with a suitable adsorbent (e.g. calcium
carbonate, alumina) and the sample added to the top of the column. Individual components were separated by allow-
ing a solvent (termed the mobile phase or eluent) to pass through the column. The process was stopped however before
the first component emerged from the bottom of the column and the column packing was slowly removed in sections
and the‘pure’compounds recovered by extraction. In the second half of the 1930s it became the accepted practice to
‘wash’or elute the components out of the column by continued addition of mobile phase. An important achievement
was the development of a procedure[202]for continuously monitoring column effluent, by measuring its refractive
index. In response to the need for faster separations (and easier detection and recovery of sample),‘open column’chro-
matography developed[203]in the late 1930s. In this variant, solutes are separated on a thin layer of adsorbent coated
on a flat, rigid support (e.g. glass). Meinhard and Hall[204]in 1949 were the first to use starch binder to hold the adsor-
bent to the rigid support. Twenty years elapsed before the technique became widely accepted as thin layer chroma-
tography and then only following the systematic investigations of Stahl[205]and the commercial availability of
standardized adsorbents.
d
Professor Edgar Lederer was the cousin of Michael Lederer, founder and editor of the Journal of Chromatography. Michael Lederer was born in
Vienna, Austria, in 1924 but emigrated to Australia in 1938 where the first phase of his education was completed. He left Australia for Paris in late
1951 and received his Docteur es Sciences from the Sorbonne in 1954 with a thesis on paper chromatography. It was a highlight of the career of one of
the authors (KR) to meet Dr. Lederer in his laboratory in Switzerland in 1985 (a paper chromatogram was running in the background) and to be
quizzed on the education system in Australia.
32 1. Introduction and overview

Ion-exchange chromatography also developed in the late 1930s when Taylor and Urey[206]separated lithium and
potassium isotopes on zeolites. The real advance in this variant came with the application of synthetic ion-exchange
resins[207]which became commercially available in the early 1940s. Their value was demonstrated[208]in the
separation of rare earth and transuranium elements in connection with the Manhattan project of World War II.
The decade beginning in 1941 has been termed‘The golden decade of Chromatography’by Ettre[169]. Three
milestones in chromatography occurred during this period and all are associated with one person: Archer John Porter
Martin. According to the proverb, necessity is the mother of invention and, in 1941, Martin and Synge[209]developed
partition chromatography while working at the Wool Industries Research Association Laboratories in Leeds, England.
There existed a considerable need for woollen clothing for the English and Commonwealth soldiers engaged in World
War II. Wool is somewhat unique in retaining heat and the demand outstripped supply. Martin and Synge wanted to
make artificial wool and needed to determine the amino acid content of the proteins of the woollen fibres as a starting
point. They recognized that it would be very difficult to separate amino acids using the adsorption chromatographic
methods that were available and, with some lateral thinking, investigated using a liquid stationary phase (e.g. water)
retained on a solid support (e.g. silica gel) to achieve separation based on partitioning. This work titled:
A new form of chromatogram employing two liquid phases.
1. A theory of chromatography.
2. Application to the micro-determination of the higher monoamino-acids in proteins.
was sufficiently ground breaking that it was recognized with the Nobel Prize in Chemistry, 1952. The last sentence of
the Introduction in the 1941 paper predicted that a gas could function as a suitable mobile phase thus pre-empting
development of GC.
Partition chromatography (in columns) had a tremendous impact but was further strengthened with the develop-
ment of paper chromatography in 1944[210]which was originally developed for the analysis of organic compounds
but was soon extended to inorganic applications[211,212]. The thoroughness which characterizes Martin’s work is
illustrated by a problem encountered in their initial work on separating amino acids by paper chromatography using
a ninhydrin spray for detection. Following spraying, the purple amino acid spots were accompanied by‘pink fronts’
which were traced to copper salts of amino acids formed from traces of copper dust originating from an unshielded d.c.
generator[177]. Considering the advantages of simplicity and the ability to analyse several samples simultaneously, it
is not surprising that paper chromatography soon became universally accepted. Sanger’s use of paper chromatogra-
phy[213]in 1955 to separate amino acids from insulin—the first protein to be sequenced—demonstrated clearly the
immense separating power of paper chromatography. The 1944 paper by Consden et al.[210]is also notable for the
introduction of the concept of the retardation factor, R
F(SeeChapter 3).
The next major step was the development of GLC in 1952 by Martin and James while at the National Institute for
Medical Research at Mill Hill in London[84]. They demonstrated the separation of amines and carboxylic acids using a
gaseous mobile phase. Their system was very simple by modern standards but achieved a dramatic improvement in
separating ability relative to other techniques then in use such as fractional distillation. The new technique of GLC
found immediate important applications that eclipsed all other chromatographic techniques. The titrimetric detector
used in the initial work suffered serious limitations, partly overcome by adoption of the thermal conductivity detector
which was already known in the relatively inefficient technique of GSC. The most significant step in the acceptance of
GLC was the invention of the flame ionization detector in 1958 by McWilliam and Dewar in Australia[214]and Harley,
Nel, and Pretorius in South Africa[215]. This detector provided an almost universal system for detection of organic
solutes and increased the sensitivity of GLC by several orders of magnitude which, in turn, enabled the use of smaller
samples and more efficient columns. It was followed by introduction of the argon ionization and electron affinity
detectors[216], the latter being the forerunner of the selective electron capture detector.
The advent of GC contributed greatly to the development of the scientific instrument industry[173]in the decade
after World War II. This was a very important time in which analytical chemistry moved from the domination of clas-
sical, wet chemical methods based on extraction, precipitation, and titration to methods based on the measurement of a
physicochemical characteristic as in GC.
Principal developments thereafter included the introduction of open tubular or capillary columns by Marcel Golay
[42], size exclusion on cross-linked dextran gels as a result of the work of Porath and Flodin[217], and affinity chro-
matography[218]. The so-called open tubular columns dramatically increased the separating power of GC and were of
immediate interest to the petroleum industry[219]. However, their true potential was only realized with the commer-
cial availability of first glass and then fused silica open tubular columns. The widespread interest in the technique of
GLC initiated basic research on the theory of chromatography which cross-fertilized liquid chromatography.
The rekindled interest in liquid chromatography resulted in a new explosion—the development of modern liquid
column chromatography. In early work, problems of high back pressure (up to 410bar or 6000psi) resulted from use of
331.8 Historical aspects

long columns (50–100 cmπ1mm i.d.) and led to the technique being called high-pressure (later performance) liquid
chromatography[220–225]. Unlike the situation with GLC, there was no single event here which heralded the intro-
duction of the modern technique but rather there was a series of stages each representing a small advance. The major
problem with the introduction of a truly high-performance system in liquid chromatography was that theoretically
desirable small size (3–10μm) column packings[226,227]were not available. In the early stages of development, this
was overcome by the use of pellicular packings (37–44μm) with a thin porous surface layer (2μm deep) of stationary
phase over an inert core. These materials gave efficient separations but had limited sample capacity and their major
contribution was that they led to development of pumping systems and detectors[228]essential to the next stage of
development which was the introduction of truly microparticulate packings (firstly 10μm, followed by 5μm and 3μm
particles).
The development of reversed-phase chromatography[229]and gradient elution[230]paved the way for the wider
acceptance of modern column chromatography. These procedures represent variants where the mobile phase is more
polar than the stationary phase (reversed-phase chromatography) or the polarity of the mobile phase is continuously
varied throughout the analysis (gradient elution). Subsequently shorter columns (25cm, then 10 cm, and more recently,
3cm) have been the norm. The preferred name for this technique is now high-performance liquid chromatography
(HPLC) which has expanded enormously to the point where it has provided for some years the largest sales area
for scientific equipment. Related techniques have also emerged, e.g. ion chromatography, ion pair chromatography.
These techniques basically differ from HPLC only in stationary and mobile phases.
Supercritical fluids have been employed[231]as mobile phases during the past 3 decades. Nano liquid chromatog-
raphy has emerged[166]in the last decade as‘a modality of chromatography involving samples in nano litre, mobile
phase flows in nano millilitre per minute, with detection at nano grams per millilitre’. This technique is generally
performed on microchips with almost zero production of wastes. It is therefore environmentally sustainable and
compatible with the demands of green chemistry.
1.8.1 Historical developments in the theory of chromatography
Chromatography has frequently been regarded as more art than science. However, this view is far from the truth
and contributions to chromatographic theory have paralleled the development of each technique. The theoretical foun-
dations of chromatography were recognized by its discoverer. Martin and Synge, in their classic paper of 1941, pre-
sented the results of their work together with the theoretical considerations and laid the‘foundations’for the later
development of gas liquid chromatography. Furthermore, they discussed the physical factors affecting the separation
and indicated that further improvements could be achieved by using very small particles as the stationary phase and a
high-pressure difference along the column. This advice was not acted upon until 25years later when HPLC was devel-
oped. The 1944 paper by Martin and his group on paper chromatography also developed the theoretical framework for
this new technique. In 1956 van Deemter et al.[232]presented their rate theory which expressed column efficiency as a
function of the mobile phase flow velocity and the characteristics of the chromatographic system such as solute dif-
fusivity and particle diameter of the column packing.
In May 1958 at the International GC Symposium held in Amsterdam, Marcel Golay presented his findings on the
use of capillary columns in GC together with the full theory of open tubular capillary columns[170]which is still valid
today. Golay’s work was hampered by the existing detectors which had a much too large volume for the small sample
sizes of open tubular columns but, as often happens, at the same Amsterdam symposium, I.G. McWilliam and R.A.
Dewar described in detail the flame ionization detector with its much-improved specifications.
In a number of instances, theoretical considerations have been used to predict practical developments. Such was the
case when Giddings[233]pointed out that small particle size packings would be essential in order to achieve
comparable efficiencies in liquid chromatography as in GC. Small particle packings would require high column inlet
pressures to achieve reasonable mobile phase flow. This led to the development of high-performance liquid
chromatography.
1.8.2 Concluding remarks
In presenting this brief history, I have indulged a personal passion; namely, the interaction of history and specific
scientific developments. Following the initial studies of Tswett and its dormancy period, chromatography burst into
life because of a shift in thinking[176]and the fundamental changes this introduced to chemistry.
34 1. Introduction and overview

There is one thing that has not changed in the 25 years since preparing the first edition. Writing the final paragraph
in this short history is even more elusive as developments in chromatography have been characterized by exponential
growth, with one development fertilizing another in the areas of new equipment, column technologies, procedures,
and applications. Walt Jennings[234]recounted in 2008 that in 1957 he was advised by a senior professor that chro-
matography was‘a flash-in-the-pan field, and I should now find another research area’. This advice serves as a warn-
ing for all inclined to predict the future. However, it does seem that developments in chromatography and extension of
the chromatographic technique are limited only by the imagination of practitioners. Indeed, Lochmuller[235]in an
article titled‘The Future in Chromatography’said that the only thing certain about the future of chromatography
is whatever is said will undoubtedly prove incorrect. It is equally certain that any predictions will grossly underes-
timate future developments and applications in this still expanding field. As an illustration, in 1948 the resolution
of amino acids from a protein hydrolysate required 8days[236]. Ten years later the separation of the 19 common amino
acids could be achieved in 22h and, by 1982 this was further reduced to less than 30min. By 2010, the separation
required less than 6min with a detection sensitivity increased by several orders of magnitude[237].
The year 2003 marked the end of the first century of chromatography and the Journal of Chromatography published
its 1000th volume containing several reviews of recent developments including a paper on bioactive phenols by one of
the current authors. The journal also sponsored a symposium involving an invited audience of 50 held at Ermelo, Hol-
land, on June 12 and 13 of the same year.
1.9 Obtaining assistance
Many distributors of chromatographic equipment have specialist applications departments that are able to provide
assistance in choosing or developing a new method. A number publish regular newsletters and product bulletins.
Alternatively, there are many literature sources that may assist in method selection and these can be searched on-line.
Numerous books have been published on various aspects of chromatography but they all fall into one of four broad
categories as those covering:
1.Particular techniques such as gas chromatography, ion chromatography, etc.;
2.Application areas such as Chromatographic Analysis of Pharmaceuticals;
3.Theoretical aspects such as Molecular Interactions in Bioseparations; and
4.Specialized applications such as Process Chromatography: A Guide to Validation.
Books from the appropriate category should be consulted for more detailed information on a particular topic than
that provided in this book. Alternatively, a number of journals publish detailed specialist information relating to
chromatography:
Journal of Chromatography
Journal of Chromatographic Science
Chromatographia
Journal of Separation Science
LCGC Europe
LCGC North America
The latter two journals provide an excellent source of detailed practical information on the various techniques
including SFC (despite its absence from the journal title). Apart from the two sources listed here, LCGC produces other
digital and print media that are best accessed fromhttps://www.chromatographyonline.com/. Many chromatogra-
phy suppliers also provide excellent information on various aspects of chromatography and one worth mentioning is
Restek’s Chromatography Blog, ChromaBLOGraphy. Archive copies of the latter are found athttps://blog.restek.
com/. Separation Science (not to be confused with Journal of Separation Science) is another useful resource which
is freely available. American Laboratory (https://www.americanlaboratory.com/1413-Issues/) has fewer relevant
articles but is worth checking.
References
[1]M.S. Tswett, Physikalisch-chemische Studien€uber das Chlorophyll.Die Adsorption, Ber. Dtsch. Bot. Ges. 24 (1906) 316–323.
[2]M.S. Tswett, Adsorptionsanalyse und chromatographische Methode.Anwendung auf die Chemie des Chlorophylls, Ber. Dtsch. Bot. Ges.
24 (1906) 384–393.
351.9 Obtaining assistance

[3]L. Zechmeister, Progress in Chromatography 1938–1947, Chapman and Hall, London, 1950.
[4]IUPAC, Analytical Chemistry Division, Commission on analytical nomenclature, recommendations on nomenclature for chromatography,
Pure Appl. Chem. 37 (4) (1974) 445–462.
[5]International Union of Pure and Applied Chemistry (IUPAC), Nomenclature for chromatography (recommendation), Pure Appl. Chem.
65 (1993) 819.
[6]V.A. Davankov, Chromatography—basic terms—terminology, in: Commission of the Scientific Council on Chromatography, Russian Acad-
emy of Sciences National Commission, Moscow, 1997.
[7]R.L. Wixom, The beginnings of chromatography—the pioneers (1900-1960), in: C.W. Gehrke, R.L. Wixom, E. Bayer (Eds.), Chromatography-A
Century of Discovery 1900–2000. The Bridge to the Sciences, Elsevier, 2001, p. 2. 27.
[8]V.G. Berezkin, What is chromatography? Reply to a critical paper by V.A. Davankov, J. Anal. Chem. 56 (8) (2001) 778–784.
[9]J. Novák, Principles and theory of chromatography, in: Z. Deyl (Ed.), Separation Methods, Elsevier, Amsterdam, 1984, pp. 1–28.
[10]R.L. Wixom, Paradigm shifts in chromatography: Nobel awardees, in: R.L. Wixom, C.W. Gehrke (Eds.), Chromatography: A Science of Dis-
covery, John Wiley and Sons: Hoboken, New Jersey, 2010.
[11]T.I. Williams, H. Weil, Definition of chromatography, Nature (London) 170 (1952) 503.
[12]T.I. Williams, H. Weil, The phases of chromatography, Arkiv Kemi Min. Geol. 5 (1953) 283–299.
[13]H. Purnell, Gas Chromatography, Wiley, New York, 1962.
[14]G.T. Seaborg, G. Higgins, in: L.S. Ettre, A. Zlatkis (Eds.), Seaborg and Higgins. 75 Years of Chromatography—A Historical Dialogue, Elsevier,
Amsterdam, 1979.
[15] A. Moseman, This Is the mobile science lab in Ferrari’s F1 garage, 2014, [cited 2019 August 17]; Available from:https://www.
popularmechanics.com/cars/a11526/this-is-the-mobile-science-lab-in-ferraris-f1-garage-17382991/.
[16] Churyumov-Gerasimenko—hard ice and organic molecules, 2014. November 17 2014, November 21 [cited 2019 August 19]; Available from:
https://www.dlr.de/EN/Service/search/advanced-search_form.html;jsessionid¼77A757467EE30AC69E0C87AC732304F0.delivery-
replication2.
[17]P. Laszlo, On the self-image of chemists, 1950-2000, Int. J. Philos. Chem. 12 (1) (2006) 99–130.
[18] J. Precissi, Capillary Electrophoresis, 2019, [cited 2019 August 16]; Available from:https://chem.libretexts.org/Bookshelves/Analytical_
Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Instrumental_Analysis/Capillary_Electrophoresis.
[19]R. Weinberger, Capillary electrochromatography, in Practical Capillary Electrophoresis, Academic Press, 2000, pp. 293–319.
[20]C. Yan, Y. Xue, Y. Wang, in: C.F. Poole (Ed.), Capillary electrochromatography, in Capillary Electromigration Separation Methods, Elsevier,
Amsterdam, 2018, p. 210.
[21]F.M. Tarongoy Jr., P.R. Haddad, J.P. Quirino, Recent developments in open tubular capillary electrochromatography from 2016 to 2017, Elec-
trophoresis 39 (1) (2018) 34–52.
[22]F. Svec, R.E. Majors, What ever happened to capillary electrochromatography? LCGC N. Am. 27 (12) (2009) 1032–1039.
[23]Z. Mao, Z. Chen, Advances in capillary electro-chromatography, J. Pharm. Anal. 9 (4) (2019) 227–237.
[24]S. Fanali, An overview to nano-scale analytical techniques: nano-liquid chromatography and capillary electrochromatography, Electrophoresis
38 (2017) 1822–1829.
[25]Z. Liu, Y. Du, Z. Feng, Enantioseparation of drugs by capillary electrochromatography using a stationary phase covalently modified with
graphene oxide, Microchim. Acta 184 (2) (2017) 583–
593.
[26]N.H. Snow, Stationary phase selectivity: the chemistry behind the separation, LCGC Europe 31 (11) (2018) 616–623.
[27]T. Yoshida, R.E. Majors, High-speed analyses using rapid resolution liquid chromatography on 1.8-μm porous particles, J. Sep. Sci. 29 (16)
(2006) 2421–2432.
[28]D.J. Ellingson, et al., Analytical method for lactoferrin in milk-based infant formulas by signature peptide quantification with ultra-high per-
formance LC-tandem mass spectrometry. (Infant formula and adult nutritionals) (Report), J. AOAC Int. 102 (3) (2019) 915.
[29]H.H. Maurer, What is the future of (ultra) high performance liquid chromatography coupled to low and high resolution mass spectrometry for
toxicological drug screening? J. Chromatogr. A 1292 (2012) 19–24.
[30]M.-J. Motilva, A. Serra, A. Macià, Analysis of food polyphenols by ultra high-performance liquid chromatography coupled to mass spectrom-
etry: an overview, J. Chromatogr. A 1292 (2013) 66–82.
[31]H. Nishi, K. Nagamatsu, New trend in the LC separation analysis of pharmaceuticals—high performance separation by ultra high-
performance liquid chromatography (UHPLC) with core-shell particle C18 columns, Anal. Sci. 30 (2) (2014) 205–211.
[32]A. Sanches-Silva, et al., Ultra-high pressure LC for astaxanthin determination in shrimp by-products and active food packaging, Biomed. Chro-
matogr. 27 (6) (2013) 757–764.
[33]D. Guillarme, et al., New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches, Anal. Bioanal.
Chem. 397 (3) (2010) 1069–1082.
[34]A. de Villiers, P. Venter, H. Pasch, Recent advances and trends in the liquid-chromatography–mass spectrometry analysis of flavonoids, J.
Chromatogr. A 1430 (C) (2016) 16–78.
[35]O. Núñez, et al., New trends in fast liquid chromatography for food and environmental analysis, J. Chromatogr. A 1228 (C) (2012)
298–323.
[36]P. Jandera, Comparison of reversed-phase and normal-phase column liquid chromatographic techniques for the separation of low and high
molecular weight compounds, J. Liq. Chromatogr. Relat. Technol. 25 (19) (2002) 2901–2931.
[37]H. Kalász, Displacement chromatography, J. Chromatogr. Sci. 41 (6) (2003) 281–283.
[38]Nobel Prize for chemistry: Prof. Arne Tiselius, Nature, 162 (4124) (1948) 766.
[39]F. Kamarei, et al., Accurate measurements of frontal analysis for the determination of adsorption isotherms in supercritical fluid
chromatography, J. Chromatogr. A 1329 (2014) 71–77.
[40]D.C. Schriemer, Biosensor alternative: frontal affinity chromatography, Anal. Chem. 76 (23) (2004) 440A–448A.
[41]M. Srajer Gajdosik, J. Clifton, D. Josic, Sample displacement chromatography as a method for purification of proteins and peptides from com-
plex mixtures, J. Chromatogr. A 1239 (2012) 1–9.
[42]M.J.E. Golay, Vapor phase chromatography and Telegrapher’s equation, Anal. Chem. 29 (6) (1957) 928–932.
[43]M. Novotny, Recent advances in microcolumn liquid chromatography, Anal. Chem. 60 (8) (1988) 500A–510A.
36 1. Introduction and overview

[44]M. Novotny, Microcolumns in liquid chromatography, Anal. Chem. 53 (12) (1981) 1294A–1297A.
[45]A. Wickers, D. Decker, R. Majors, The art and science of GC capillary column production, LCGC N. Am. 25 (7) (2007) 616–631.
[46]C.A. Cramers, J.A. Rijks, Micropacked columns in gas chromatography: an evaluation, in: J.C. Giddings, et al. (Eds.), Advances in Chroma-
tography, Marcel Dekker, New York, 1979, pp. 101–162.
[47]H. Zhao, et al., Characteristics of TGPGC on short micro packed capillary column, Anal. Sci. 18 (1) (2002) 93–95.
[48]P.Q. Tranchida, L. Mondello, Current-day employment of the micro-bore open-tubular capillary column in the gas chromatography field, J.
Chromatogr. A 1261 (2012) 23–36.
[49]R.L. Stevenson, Agilent’s GC wax column technology developments greatly advance performance of polar compound analyses, Am. Lab.
50 (8) (2018) 18–20.
[50] M.L. Duffy, Advantages and applications of wide-bore, thick-film capillary columns in GC, Am. Lab. 17 (10) (1985) 94.
[51]R.E. Majors, Anatomy of an LC column from the beginning to modern day, LCGC N. Am. 24 (8) (2006) 742–753.
[52]R.E. Majors, HPLC column technology—state of the art, LCGC N. Am. 24 (S4) (2006) 7–15.
[53]R. Majors, Historical developments in HPLC and UHPLC column technology: the past 25 years, LCGC N. Am. 33 (11) (2015) 818–840.
[54]R.E. Majors, Twenty-five years of HPLC column development: a commercial perspective, LCGC N. Am. 12 (7) (1994) 508–518.
[55]F. Gritti, G. Guiochon, The current revolution in column technology: how it began, where is it going? J. Chromatogr. A 1228 (2012) 2–19.
[56]F. Svec, Y. Lv, Advances and recent trends in the field of monolithic columns for chromatography, Anal. Chem. 87 (1) (2015) 250–273.
[57]Y. Wang, et al., Sub-2μm porous silica materials for enhanced separation performance in liquid chromatography, J. Chromatogr. A 1228 (2012)
99–109.
[58]D. Ishii, T. Takeuchi, Capillary columns in liquid chromatography, in: J.C. Giddings, et al. (Eds.), Advances in Chromatography, Marcel Dek-
ker, New York, 1983, pp. 131–164.
[59]Recent developments in LC column technology. (Special Supplement) (Column), LCGC Europe 27 (12) (2014) 620.
[60]Anatomy of an LC column: from the beginning to modern day, LCGC N. Am. 33 (2015) S33–S39.
[61]D. Bell, Recent developments in LC column technology: impact on a world of disciplines, LCGC N. Am. 34 (s4) (2016) 8.
[62]M. Dong, HPLC columns and trends, in HPLC and UHPLC for Practicing Scientists, John Wiley & Sons, 2019, pp. 45–79.
[63]F. Gritti, G. Guiochon, Perspectives on the evolution of the column efficiency in liquid chromatography, Anal. Chem. 85 (6) (2013)
3017–3035.
[64]C.E.D. Nazario, et al., Evolution in miniaturized column liquid chromatography instrumentation and applications: an overview, J. Chroma-
togr. A 1421 (2015) 18–37.
[65]T. Berger, B. Berger, A review of column developments for supercritical fluid chromatography, LCGC N. Am. 28 (5) (2010) 344–354. 356-357.
[66] M. Novotny, et al., Open-Tubular Supercritical Fluid Chromatography, 1984, Available from:https://patents.google.com/patent/
US4479380A/en.
[67]T.A. Berger, in: R.M. Smith (Ed.), Packed Column SFC. RSC Chromatography Monographs, The Royal Scoiety of Chemistry, Cambridge, 1995.
[68]
L.T. Taylor, Supercritical fluid chromatography for the 21st century, J. Supercrit. Fluids 47 (3) (2009) 566–573.
[69]R.D. Smith, B.W. Wright, C.R. Yonker, Supercritical fluid chromatography: current status and prognosis, Anal. Chem. 60 (1988) 1323A–1336A.
[70]C.F. Poole, Progress in packed column supercritical fluid chromatography: materials and methods, J. Biochem. Biophys. Methods 43 (2000)
3–23.
[71]C.F. Poole, Stationary phases for packed-column supercritical fluid chromatography, J. Chromatogr. A 1250 (2012) 157–171.
[72]F. Pacholec, C.F. Poole, Stationary phase properties of the organic molten salt ethylpyridinium bromide in gas chromatography, Chromato-
graphia 17 (7) (1983) 370–374.
[73]B.L. Karger, New developments in chemical selectivity in gas-liquid chromatography, Anal. Chem. 39 (8) (1967) 24A–50A.
[74]R.J. Laub, R.L. Pecsok, Study of charge transfer complexation by gas-liquid chromatography, J. Chromatogr. 113 (1975) 47–68.
[75]J.G. Dorsey, W.T. Cooper, Retention mechanisms of bonded-phase liquid chromatography, Anal. Chem. 66 (17) (1994) 857A–867A.
[76]S.C. Moldoveanu, V. David, Retention mechanisms in different HPLC Types, in: S.C. Moldoveanu, V. David (Eds.), Essentials in Modern
HPLC Separations, Elsevier, 2013, pp. 145–190.
[77]C.T. Santasania, D.S. Bell, Mechanisms of interaction responsible for alternative selectivity of fluorinated stationary phases (Column Watch),
LCGC Europe 29 (2) (2016) 86–92.
[78]L.R. Snyder, Principles of Adsorption Chromatography, Marcel Dekker, New York, 1968.
[79]L.R. Snyder, Mobile phase effects in liquid-solid chromatography: importance of adsorption-site geometry, adsorbate delocalization and
hydrogen bonding, J. Chromatogr. 255 (1983) 3–26.
[80]L.R. Snyder, H. Poppe, Mechanism of solute retention in liquid—solid chromatography and the role of the mobile phase in affecting separation:
competition versus“sorption”, J. Chromatogr. 184 (1980) 363–413.
[81]Y.I. Yashin, Selectivity in liquid adsorption chromatography, J. Chromatogr. 251 (1982) 269–279.
[82]S. Claesson, Arkiv foer Kemi, Mineral. Geol. 23A (1946) 1–133.
[83]C.S.G. Phillips, The chromatography of gases and vapours, Discuss. Faraday Soc. 7 (1949) 241–248.
[84]A.T. James, A.J.P. Martin, Gas-liquid partition chromatography: the separation and micro-estimation of volatile fatty acids from formic acid to
dodecanoic acid, Biochem. J. 50 (5) (1952) 679–690.
[85]A.P. Foucault, Countercurrent chromatography, Anal. Chem. 63 (10) (1991) 569A–579A.
[86]I.A. Sutherland, D. Heygood-Waddington, T.J. Peters, Toroidal coil countercurrent chromatography: a fast simple alternative to countercur-
rent distribution using aqueous two phase partition: principles, theory, and apparatus, J. Liq. Chromatogr. 7 (2) (1984) 363–384.
[87]I.A. Sutherland, D. Heygood-Waddington, T.J. Peters, Countercurrent chromatography using a toroidal coil planet-centrifuge: a comparative
study of the separation of organelles using aqueous two-phase partition, J. Liq. Chromatogr. 8 (12) (1985) 2315–2335.
[88]D.C. Locke, Selectivity in reversed-phase liquid chromatography using chemically bonded stationary phases, J. Chromatogr. Sci. 12 (1974)
433–437.
[89]C. Poole, N. Lenca, Reversed-phase liquid chromatography, in: S. Fanali, et al. (Eds.), Liquid Chromatography: Fundamentals and Instrumen-
tation, Elsevier, 2017, pp. 91–
123.
[90]P. Nikitas, A. Pappa-Louisi, Retention models for isocratic and gradient elution in reversed-phase liquid chromatography, J. Chromatogr.
A 1216 (10) (2009) 1737–1755.
37References

[91]A. Vailaya, Fundamentals of reversed phase chromatography: thermodynamic and exothermodynamic treatment, J. Liq. Chromatogr. Relat.
Technol. 28 (7–8) (2005) 965–1054.
[92]M. Jovanovic, T. Rakic, B. Jancic-Stojanovic, Theoretical and empirical models in hydrophilic interaction liquid chromatography, Instrum. Sci.
Technol. 42 (3) (2014) 230–266.
[93]B. Buszewski, S. Noga, Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique, Anal. Bioanal. Chem. 402
(1) (2012) 231–247.
[94]G. Greco, T. Letzel, Main interactions and influences of the chromatographic parameters in HILIC separations, J. Chromatogr. Sci. 51 (7) (2013)
684–693.
[95]T. Hanai, Definition of HILIC system and quantitative analysis of retention mechanisms, Curr. Chromatogr. 5 (1) (2018) 43–52.
[96]A.I. Piteni, M.G. Kouskoura, C.K. Markopoulou, HILIC chromatography—an insight on the retention mechanism, J. Chromatogr. Sep. Tech. 7
(3) (2016).
[97]S. Eksborg, G. Schill, Ion pair partition chromatography of organic ammonium compounds, Anal. Chem. 45 (12) (1973) 2092–2100.
[98]J.H. Knox, G.R. Laird, Soap chromatography—a new high-performance liquid chromatographic technique for separation of ionizable mate-
rials: dyestuff intermediates, J. Chromatogr. 122 (1976) 17–34.
[99]I.H. Hagestam, T.C. Pinkerton, Internal surface reversed-phase silica supports for liquid chromatography, Anal. Chem. 57 (8) (1985) 1757–1763.
[100]R.R. Walters, Affinity chromatography, Anal. Chem. 57 (1985) 1099A–1114A.
[101]D.W. Armstrong, S. Henry, Use of an aqueous micellar mobile phase for separation of phenols and polynuclear aromatic hydrocarbons via
HPLC, J. Liq. Chromatogr. 3 (1980) 657–662.
[102]L.J. Cline Love, J.G. Habarta, J.G. Dorsey, The micelle-analytical chemistry interface, Anal. Chem. 56 (11) (1984) 1132A–1148A.
[103]M. Arunyanart, C.L. Love, Model for micellar effects on liquid chromatography capacity factors and for determination of micelle-solute equi-
librium constants, Anal. Chem. 56 (9) (1984) 1557–1561.
[104]J.S. Landy, J.G. Dorsey, Rapid gradient capabilities of micellar liquid chromatography, J. Chromatogr. Sci. 22 (2) (1984) 68–70.
[105]D.T. Gjerde, J.S. Fritz, Ion Chromatography, second ed., Huethig, Heidelberg, 1987.
[106]J.S. Fritz, D.T. Gjerde, Ion Chromatography, fourth ed., Wiley-VCH Verlag GmbH & Co, Weinheim, 2009.
[107]B.K. Glód, Ion exclusion chromatography: parameters influencing retention, Neurochem. Res. 22 (10) (1997) 1237–1248.
[108]G.A. Harlow, D.H. Morman, Automatic ion exclusion-partition chromatography of acids, Anal. Chem. 36 (13) (1964) 2438–2442.
[109]T. Jupille, et al., Ion moderated partition HPLC, Am. Lab. 13 (8) (1981) 80–86.
[110]G.K.E. Scriba, Chiral recognition mechanisms in analytical separation sciences, Chromatographia 75 (15–16) (2012) 815–
838.
[111]A. Berthod, Chiral recognition mechanisms, Anal. Chem. 78 (7) (2006) 2093–2099.
[112]G.K.E. Scriba, Chiral recognition in separation science—an update, J. Chromatogr. A 1467 (2016) 56–78.
[113]V. Berezkin, S. Khrebtova, The development of planar chromatography in 1980-1990 and 2000-2010 (the Scientometric study), J. Planar Chro-
matogr.- -Mod. TLC 24 (2011) 454–462.
[114]V.G. Berezkin, E.N. Viktorova, Changes in the basic experimental parameters of capillary gas chromatography in the 20th century, J. Chro-
matogr. A 985 (1) (2003) 3–10.
[115]T. Braun, S. Zsindely, Some recent trends in instrumental organic analysis, TrAC Trends Anal. Chem. 9 (1990) 309–311.
[116]C.J.F. Waaijer, M. Palmblad, Bibliometric mapping: eight decades of analytical chemistry, with special focus on the use of mass spectrometry,
Anal. Chem. 87 (9) (2015) 4588–4596.
[117]K. Robards, P. Haddad, P. Jackson, Principles and Practice of Modern Chromatographic Methods, Academic Press, 1994.
[118]Y. Yashin, A. Yashin, Current trends in gas chromatography methods and instrumentation: a scientometric study, J. Anal. Chem. 56 (3) (2001)
200–213.
[119]M.D. Palmieri, An introduction to supercritical fluid chromatography, J. Chem. Educ. 65 (1988) A254.
[120]P. Schoenmakers, Editorial to "the many faces of packed-column supercritical fluid chromatography—a critical review" by Eric Lesellier and
Caroline West, J. Chromatogr. A 1382 (2015) 1.
[121]C. West, Current trends in supercritical fluid chromatography, Anal. Bioanal. Chem. 410 (25) (2018) 6441–6457.
[122]K. Kalíková, et al., Supercritical fluid chromatography as a tool for enantioselective separation; a review, Anal. Chim. Acta 821 (2014) 1–33.
[123]T. Fornstedt, R.E. Majors, Modern supercritical fluid chromatography–possibilities and pitfalls, LCGC Asia Pacific 18 (1) (2015) 27–32.
[124]Pittsburgh conference update, TrAC Trends Anal. Chem. 10 (1) (1991) v. vi.
[125]Anon, The analytical and life science instruments industry (Chromatography Market Profile), LCGC N. Am. 28 (11) (2010) 928.
[126] B. Kelly, The Chromatography Market: Outlook to 2020, 2017, [cited 2019 16 August]; Available from:https://bioinfoinc.com/
chromatography-market-outlook-2020/.
[127] Future Market Insights, Chromatography Instrumentation Market Analysis. Chromatography Instrumentation Market: Global Industry Anal-
ysis and Opportunity Assessment 2015–2025, 2019, [cited 2019 August 16]; Available from:https://www.futuremarketinsights.com/reports/
chromatography-instrumentation-market.
[128] Research and Markets, Global Chromatography Instruments Market 2019–2025—Increased Drug Tests are the Key to Market Growth, 2019,
13 May 2019 [cited 2019 August 16]; Available from:https://www.globenewswire.com/news-release/2019/05/13/1822254/0/en/Global-
Chromatography-Instruments-Market-2019-2025-Increased-Drug-Tests-are-the-Key-to-Market-Growth.html.
[129] Mordor Intelligence, Chromatography Reagents Market—Growth, Trends, and Forecast (2019–2024), [cited 2019 August 16]; Available from:
https://mordorintelligence.com:81/industry-reports/chromatography-reagents-market.
[130]J. Mitchell, Effects of raw material change in manufacturing process resolved, Anal. Chem. 46 (9) (1974) 804A–805A.
[131]S.P. Levine, et al., Industrial analytical chemists and OSHA regulations for vinyl chloride, Anal. Chem. 47 (12) (1975) 1075A–1080A.
[132]D.J. Reutter, R.C. Buechele, T.L. Randolph, Ion chromatography in bombing investigations, Anal. Chem. 55 (14) (1983) 1468A–1472A.
[133]M.R. Sevenants, R.A. Sanders, Anatomy of an off-flavor investigation, Anal. Chem. 56 (2) (1984) 293A–298A.
[134]D.J. Brown, L.F. Schneider, J.A. Howell, Solving mysteries using infrared spectrometry and chromatography, Anal. Chem. 60 (1988) 1005A–
1011A.
[135]W. Bertsch, Chemical analysis of fire debris: was it arson? Anal. Chem. 68 (17) (1996) 541A.
[136]R. Mukhopadhyay, Don’t waste your breath, Anal. Chem. 76 (15) (2004) A273–A276.
38 1. Introduction and overview

[137]C. Postigo, et al., Determination of drugs of abuse in airborne particles by pressurized liquid extraction and liquid chromatography-
electrospray-tandem mass spectrometry, Anal. Chem. 81 (11) (2009) 4382–4388.
[138]H.J. Tobias, et al., Detection of synthetic testosterone use by novel comprehensive two-dimensional gas chromatography combustion-isotope
ratio mass spectrometry, Anal. Chem. 83 (18) (2011) 7158–7165.
[139]L. Zhang, et al., Caffeine in your drink: natural or synthetic? Anal. Chem. 84 (6) (2012) 2805–2810.
[140]Z. Wang, et al., Authentication of organically and conventionally grown basils by gas chromatography/mass spectrometry chemical profiles,
Anal. Chem. 85 (5) (2013) 2945–2953.
[141]M. Nestola, et al., Universal route to polycyclic aromatic hydrocarbon analysis in foodstuff: two-dimensional heart-cut liquid chromatography-
gas chromatography-mass spectrometry.(report), Anal. Chem. 87 (12) (2015) 6195–6203.
[142]R. Nicoli, et al., Analytical strategies for doping control purposes: needs, challenges, and perspectives, Anal. Chem. 88 (1) (2016) 508–523.
[143]E. Gorrochategui, et al., Data analysis strategies for targeted and untargeted LC-MS metabolomic studies: overview and workflow, TrAC
Trends Anal. Chem. 82 (2016) 425–442.
[144]B.E. Erickson, How many chemicals are in use today? Chem. Eng. News 95 (9) (2017) 23–24.
[145]E.C. Horning, M.C. Horning, Human metabolic profiles obtained by GC and GC/MS, J. Chromatogr. Sci. 9 (1971) 129–140.
[146]G.A. Theodoridis, et al., Liquid chromatography-mass spectrometry based global metabolite profiling: a review, Anal. Chim. Acta 711 (2012)
7–16.
[147]L. Cuadros-Rodríguez, et al., Chromatographic fingerprinting: an innovative approach for food’identitation’and food authentication—a tuto-
rial, Anal. Chim. Acta 909 (2016) 9–23.
[148]A. Ribbenstedt, H. Ziarrusta, J.P. Benskin, Development, characterization and comparisons of targeted and non-targeted metabolomics
methods, PLoS One 13 (11) (2018), e0207082.
[149]P. Krishnan, N. Kruger, R. Ratcliffe, Metabolite fingerprinting and profiling in plants using NMR, J. Exp. Bot. 56 (410) (2005) 255–265.
[150]L.D. Roberts, et al., Targeted metabolomics, Curr. Protoc. Mol. Biol. 98 (1) (2012) 30.2.1–30.2.24.
[151]C.F. Poole, S.A. Schuette, Preparative-scale chromatography, in Contemporary Practice of Chromatography, Elsevier, Amsterdam, 1984, pp.
399–428.
[152]J. Růžiůcka, G.D. Christian, Flow injection analysis and chromatography: twins or siblings? Plenary lecture, Analyst 115 (5) (1990) 475–486.
[153]J.C. Giddings, Comparison of theoretical limit of separating speed in gas and liquid chromatography, Anal. Chem. 37 (1) (1965) 60–63.
[154]S. Jespers, K. Broeckhoven, G. Desmet, Comparing the separation speed of contemporary LC, SFC, and GC, LCGC Europe 30 (6) (2017)
284–291.
[155]L.S. Ettre, A. Zlatkis, Seventy-five years of chromatography—a historical dialog, J. Chromatogr. Libr. 17 (1979). Elsevier: Amsterdam.
[156]H.J. Issaq, A Century of Separation Science, Marcel Dekker, New York, 2002.
[157]L.S. Ettre, The development of chromatography, Anal. Chem. 43 (14) (1971) 20A–31A.
[158]L.S. Ettre, J.V. Hinshaw, Chapters in the Evolution of Chromatography, Imperial College Press, London, 2008.
[159]L.S. Ettre, M. S. Tswett and the 1918 Nobel prize in chemistry, Chromatographia 42 (5–6) (1996) 343–351.
[160]L.S. Ettre, M.S. Tswett, And the invention of chromatography, LCGC N. Am. 21 (5) (2003) 458–467.
[161]L. Ettre, The rebirth of chromatography 75 years ago, LCGC N. Am. 25 (2007) 640–655.
[162]L. Ettre, The predawn of paper chromatography, Chromatographia 54 (2001) 409–414.
[163]L.S. Ettre, The birth of partition chromatography, LCGC N. Am. 19 (5) (2001) 506–512.
[164]L.S. Ettre, Preparative liquid chromatography and the Manhattan project, LCGC N. Am. 17 (1999) 1104–1109.
[165]L.S. Ettre, The development of gas chromatography, J. Chromatogr. A 112 (1975) 1–26.
[166]I. Ali, H.Y. Aboul-Enein, J. Cazes, A journey from Mikhail Tswett to nano-liquid chromatography, J. Liq. Chromatogr. Relat. Technol. 33 (5)
(2010) 645–653.
[167]L.S. Ettre, C. Horvath, Foundations of modern liquid chromatography, Anal. Chem. 47 (4) (1975) 422A–446A.
[168]L. Ettre, Early evolution of gas adsorption chromatography. Part I: displacement development and the beginnings of elution-type investiga-
tions, Chromatographia 55 (7) (2002) 497–504.
[169]L.S. Ettre, 1941–1951: the golden decade of chromatography, Analyst 116 (12) (1991) 1231–1235.
[170]L.S. Ettre, Evolution of capillary columns for gas chromatography, LCGC N. Am. 19 (1) (2001) 48–59.
[171]L.S. Ettre, Fifty years of gas chromatography—the pioneers I knew, part II, LCGC N. Am. 20 (5) (2002) 452–463.
[172]L.S. Ettre, Csaba Horváth and the development of the first modern high performance liquid chromatograph, LCGC N. Am. 23 (5) (2005)
486–495.
[173]L.S. Ettre, My 50+ years in chromatography, LCGC N. Am. 27 (9) (2009) 834–842.
[174]L.S. Ettre, K.I. Sakodynskii, M. S. Tswett and the discovery of chromatography I: early work (1899–1903), Chromatographia 35 (3–4) (1993)
223–231.
[175]J. Farradane, History of chromatography, Nature (London) 167 (1951) 120.
[176]J.-F. Gal, An excursion into the history of chromatography: Mikhail Tswett, from Asti, Italy, to Tartu, Estonia, Chromatographia 82 (2) (2019)
519–521.
[177]H. Gordon, The discovery of partition chromatography. A personal view, Analyst 116 (12) (1991) 1245.
[178]J. Janák, Stepping stones in modern chromatography, Chromatographia 71 (9) (2010) 747–750.
[179]W. Lindner, History of chromatography, LCGC Europe 27 (10) (2014) 520–524.
[180]J. Livengood, Why was M. S. Tswett’s chromatographic adsorption analysis rejected? Stud. Hist. Philos. Sci. 40 (1) (2009) 57
–69.
[181]K. Usher, Phyllis Brown: a story of serendipity, perseverance, vision, and chromatography, LCGC N. Am. 30 (11) (2012) 982–985.
[182]L. Zechmeister, L. Cholnoky, Principles and Practice of Chromatography, English Ed., Chapman and Hall, London, 1941.
[183]H. Weil, T.I. Williams, History of chromatography, Nature (London) 166 (1950) 1000–1001.
[184]H. Weil, T.I. Williams, Early history of chromatography, Nature (London) 167 (1951) 906–907.
[185]L. Zechmeister, Early history of chromatography, Nature (London) 167 (1951) 405–406.
[186]Editorial Board, Questions of priority of native biochemistry, Biokhimiya 16 (1951) 478.
[187]L. Zechmeister, History, scope, and methods of chromatography, Ann. N. Y. Acad. Sci. 49 (2) (1948) 145–160.
39References

[188]V.G. Berezkin, Biography of Mikhail Semenovich Tswett and translation of Tswett’s preliminary communication on a new category of adsorp-
tion phenomena, Chem. Rev. 89 (1989) 279–285.
[189]K.I. Sakodynskii, M. S. Tswett—his life and work, Chromatographia 3 (1970) 92–94.
[190]K.I. Sakodynskii, M.S. Tswett—his life, J. Chromatogr. 49 (1970) 2–17.
[191]K.I. Sakodynskii, The life and scientific works of Michael Tswett, J. Chromatogr. 73 (1972) 303–360.
[192]K.I. Sakodynskii, New data on M.S. Tswett’s life and work, J. Chromatogr. 220 (1981) 1–28.
[193]K.I. Sakodynskii, K. Chmutov, M. S. Tswett chromatography, Chromatographia 5 (1972) 471–476.
[194]H.H. Strain, J. Sherma, M. Tswett, Adsorption analysis and chromatographic methods: application to the chemistry of chlorophylls, J. Chem.
Educ. 44 (4) (1967) 238.
[195]J.E. Meinhard, Chromatography: a perspective, Science 110 (2859) (1949) 387–392.
[196]F.G. Kohl, F.G. Kohl, Kohlens€aure-assimilation und chlorophyll-funktion, Ber. Deut. Bot. Ges. 24 (1906) 124–134.
[197]E. Lederer, La renaissance de la mπethode chromatographique de M. Tswett en 1931, J. Chromatogr. 73 (1972) 361–366.
[198]R. Kuhn, E. Lederer, Fraktionierung und isomerisierung des carotins, Naturwissenschaften 19 (1931) 306.
[199]R. Kuhn, E. Lederer, III. Gewinnung eines aktiven trocknen Pr€aparats aus den Bl€attern der Futterr€ube, Ber. Dtsch. Chem. Ges. 64 (1931)
1349–1357.
[200]R. Kuhn, A. Winterstein, E. Lederer, Zur kenntnis der xanthophylle, Z. Physiol. Chem. 197 (1931) 141–160.
[201]G.-M. Schwab, K. Jockers, Anorganische chromatographie (I. Mitteilung), Angew. Chem. 50 (1937) 546–553.
[202]A. Tiselius, Adsorption analysis of amino-acids and proteins, Arkiv Kemi Min. Geol. 15 (6) (1941) 1–5.
[203]M.S. Shraiber, The beginnings of thin-layer chromatography, J. Chromatogr. 73 (1972) 367–370.
[204]J.E. Meinhard, N.F. Hall, Surface chromatography, Anal. Chem. 21 (1949) 185–188.
[205]E. Stahl, Thin Layer Chromatography (English translation), second ed., Academic Press, New York, 1969.
[206]T.I. Taylor, H.C. Urey, Fractionation of the Lithium and potassium isotopes by chemical exchange with zeolites, J. Chem. Phys. 6 (1938) 429.
[207]O. Samuelson, Fresenius’Zeitschrift f€ur Analytische, Chem. Aust. 116 (1939) 328.
[208]F.H. Spedding, et al., The separation of rare earths by ion exchange. III. Pilot plant scale separations, J. Am. Chem. Soc. 69 (11) (1947) 2812–2818.
[209]A.J.P. Martin, R.L.M. Synge, A new form of chromatogram employing two liquid phases: a theory of chromatography. 2. Application to the
micro-determination of the higher monoamino-acids in proteins, Biochem. J. 35 (12) (1941) 1358–1368.
[210]R. Consden, A.H. Gordon, A.J.P. Martin, Qualitative analysis of proteins: a partition chromatographic method using paper, Biochem. J. 38 (3)
(1944) 224–232.
[211]M. Lederer, The chromatographic separation of antimony, Anal. Chim. Acta 2 (1948) 261–262.
[212]F.H. Pollard, J.F.W. McOmie, I.I.M. Elbeih, Inorganic paper chromatography and detection of cations by fluorescence, Nature (London) 163
(4138) (1949) 292.
[213]J.S. Jeffers, Frederick Sanger Two-Time Nobel Laureate in Chemistry, Springer Nature, 2017.
[214]I.G. McWilliam, The origin of the flame ionization detector, Chromatographia 17 (5) (1983) 241–243.
[215]J. Harley, W. Nel, V. Pretorius, Flame ionization detector for gas chromatography, Nature (London) 181 (4603) (1958) 177–178.
[216]J.E. Lovelock, S.R. Lipsky, Electron affinity spectroscopy—a new method for the identification of functional groups in chemical compounds
separated by gas chromatography, J. Am. Chem. Soc. 82 (2) (1960) 431–433.
[217]J. Porath, P. Flodin, Gel filtration: a method for desalting and group separation, Nature (London) 183 (4676) (1959) 1657–1659.
[218]R. Axπen, J. Porath, S. Ernback, Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature (London)
214 (5095) (1967) 1302–1304.
[219]D.H. Desty, A. Goldup, B.H.F. Whyman, The potentialities of coated capillary columns for gas chromatography in the petroleum industry, J.
Inst. Pet. 45 (1959) 287–298.
[220]I. Halász, P. Walkling, Different types of packed columns in liquid-solid chromatography, J. Chromatogr. Sci. 7 (3) (1969) 129–136.
[221]C. Horvath, S.R. Lipsky, Column design in high pressure liquid chromatography, J. Chromatogr. Sci. 7 (1969) 109–116.
[222]J.F.K. Huber, Evaluation of detectors for liquid chromatography in columns, J. Chromatogr. Sci. 7 (3) (1969) 172–176.
[223]J.J. Kirkland, Controlled surface porosity supports for high-speed gas and liquid chromatography, Anal. Chem. 41 (1) (1969) 218–220.
[224]R.P.W. Scott, J.G. Lawrence, The effect of temperature and moderator concentration on the efficiency and resolution of liquid chromatography
columns, J. Chromatogr. Sci. 7 (2) (1969) 65–71.
[225]L.R. Snyder, Column efficiency in liquid-solid adsorption chromatography. H.E.T.P. [height equivalent to a theoretical plate] values as a func-
tion of separation conditions, Anal. Chem. 39 (7) (1967) 698–704.
[226]J.H. Knox, M. Saleem, Kinetic conditions for optimum speed and resolution in column chromatography, J. Chromatogr. Sci. 7 (10) (1969)
614–622.
[227]L.R. Snyder, Column efficiencies in liquid adsorption chromatography: past, present and future, J. Chromatogr. Sci. 7 (6) (1969) 352–360.
[228]H. Felton, Performance of components of a high pressure liquid chromatography system, J. Chromatogr. Sci. 7 (1) (1969) 13–16.
[229]G.A. Howard, A.J.P. Martin, The separation of the C12-C18 fatty acids by reversed-phase partition chromatography, Biochem. J. 46 (5) (1950)
532–538.
[230]R.S. Alm, R.J.P. Williams, A. Tiselius, Gradient elution analysis. I. A general treatment, Acta Chem. Scand. 6 (1952) 826–836.
[231]M. Saito, History of supercritical fluid chromatography—
instrumental development, J. Biosci. Bioeng. 115 (6) (2013) 590–599.
[232]J.J. van Deemter, F.J. Zuiderweg, A. Klinkenberg, Longitudinal diffusion and resistance to mass transfer as causes of nonideality in chroma-
tography, Chem. Eng. Sci. 5 (6) (1956) 271–289.
[233]J.C. Giddings, Liquid chromatography with operating conditions analogous to those of gas chromatography, Anal. Chem. 35 (13) (1963)
2215–2216.
[234]W. Jennings, From academician to entrepreneur—a convoluted trek (the history of chromatography), LCGC N. Am. 26 (7) (2008) 626–631.
[235]C.H. Lochmuller, The future in chromatography, J. Chromatogr. Sci. 25 (12) (1987) 583–586.
[236]L.R. Snyder, The future of chromatography: optimizing the separation, Analyst 116 (1991) 1237–1244.
[237] C. Woodward, J.W. Henderson, T. Wielgos, High-Speed Amino Acid Analysis (AAA) on 1.8μm Reversed-Phase (RP) Columns, 2019, [cited
August 16]; Available from:https://www.agilent.com/cs/library/applications/5989-6297EN.pdf.
40 1. Introduction and overview

CHAPTER
2
Theoreticalconsiderations
2.1 Introduction
At the most fundamental level chromatography is a process used for the separation of compounds occurring in a
mixture. The chromatographer must consider a number of factors in establishing and optimizing a chromatographic
system. First, the resolution of the compound(s) of interest from each other and from other sample components must be
considered. Second, an important practical consideration is the time taken for the analysis. Third, a means must be
available to detect the analyte(s).
a
The latter is often a function of the detector selectivity and the presence of interfer-
ences. The remaining factors of resolution and analysis time are interrelated and can often be improved significantly by
the chromatographer, based on an understanding of the separation process.
In chromatography, a mobile phase percolates or passes through or over the chromatographic bed.
b
A sample is
introduced to the stationary phase bed and the mobile phase transports the components of the sample that interact
to different degrees with the stationary phase. These components undergo molecular interactions with the stationary
phase and are eventually swept out of the column. Fluid dynamics, mass transfer phenomena, and equilibrium ther-
modynamics play an important role in the outcome of the separation[1]. The relative importance of the thermody-
namics of phase equilibria and of the kinetics of mass transfer depends on experimental conditions. What does this
mean when it is well known that thermodynamics always trumps kinetics? A reaction will proceed if the Gibbs free
a
Analyte or solute is used interchangeably in this book although the former is often used to emphasize that we are dealing with a solute that is being
analysed.
b
This is defined by IUPAC as the physical form in which the stationary phase is used.
41Principles and Practice of Modern Chromatographic Methods
https://doi.org/10.1016/B978-0-12-822096-2.00002-5
Copyright©2022 Elsevier Ltd. All rights reserved.

energy for the reaction is favourable; conversely, it will not proceed if the Gibbs free energy change for the reaction is
unfavourable. Consider the reaction:
C
(s;diamondÞ !C
(s;graphiteÞ
If this reaction proceeds, then the expensive diamond you have sitting at home needs an insurance policy. The Gibbs
free energy for this reaction is indeed favourable and so your lovely diamond will absolutely convert to a pile of graph-
ite but should you rush out and purchase an insurance policy? Fortunately, you do not require insurance against this
reaction as the reaction converting diamond to graphite is extremely slow because an enormous activation energy of
370kJmol
1
is a barrier to the reaction. As the saying goes, diamonds are forever on the timescale that is of interest to
us. Thus the conversion is extremely slow requiring billions of years and we can say that on the timescale in which we
are interested that the reaction is under kinetic control. In the same manner, chromatographic processes occur on a
timescale where some are under thermodynamic control (as expected) but kinetics is important in others. (Note that
diamond forms in the first place because it is the stable form at high pressure.)
Two concurrent phenomena occur during chromatographyas the sample components traverse the stationary
phase bed, namely, retention of analyte particles
c
(which leads to separation if experimental conditions are chosen
appropriately) and dispersion of the analyte particles; the latter leading to spreading or broadening (sometimes incor-
rectly labelled as zone distortion) of the analyte bands. The first of these involves purely thermodynamic processes
accounting for the interactions between analyte and chromatographic phases leading to differential sorption or reten-
tion and hence separation. The second is kinetic in origin and results in broadening of the analyte bands as they pro-
gress through the chromatographic system. The latter, which opposes separation and is clearly undesirable, is a
consequence of the finite rate of mass transfer kinetics of the solute between the phases. It involves non-
thermodynamic contributions due to molecular diffusion, eddy diffusion, mass transfer resistance, and the finite rate
of sorption-desorption kinetics. As was shown in Fig. 1.5, it interferes with the separation process relative to the ideal
situation. In order to optimize the chromatographic process, we need to understand theprocesses of sample retention and
zone dispersion (seen as zone broadening)and to be able to describe them quantitatively using mathematical expressions.
Since the inception of chromatography, chromatographers have attempted to describe the processes occurring dur-
ing a chromatographic separation by mathematical equations. The original theory can be traced to the Nobel Prize
winning paper (Fig. 2.1) of Martin
d
and Synge[2]. This was further developed by Craig[3]and became known as
the Plate Theory. Subsequently, Glueckauf[4]published a paper titled‘The plate concept in column separations’.
The so-called rate theory also came into prominence about the same time[5]and this was followed by a paper by
Giddings[6]which established rate theory as the backbone of chromatographic theory, the plate theory slipping into
relative obscurity. The contribution of these and other theories to an understanding of the practice of chromatography
is examined in this chapter. Discussion is concentrated on those aspects which can be manipulated by the chromatog-
rapher to improve the separation process. The basic concepts and theories underlying all chromatographic techniques
are effectively the same regardless of the nature of the stationary or mobile phases.
A number of definitions, symbols, and terms will be introduced in this chapter. A clear understanding of the terms
elution, development, band, zone, and peak is required. As commonly used in chromatography, the last three terms
describe the concentration or mass profile of an analyte in space. Band represents this distribution while the analyte is
still in or on the system whereas peak describes the distribution of analyte once it has left the system and been mon-
itored by some sort of detector. Zone is a generic term covering both bands and peaks used when further distinction is
unnecessary. The process by which mobile phase is caused to flow over the stationary phase to effect the chromato-
graphic separation is known as development (Section 1.4.2). When the analytes are actually‘washed out’of the chro-
matographic system, they are said to have been eluted or elution has taken place. In elution chromatography, the
mobile phase is frequently referred to as the eluent (also spelt eluant) and the eluate is the combination of eluent
and analyte exiting the chromatographic system.
Recommendations were made on definitions very early in the expansion phase of chromatography by a number of
groups including ASTM[7,8]and IUPAC[9]but because chromatography was still evolving, the recommended ter-
minology was often contrary to current usage and the style adopted in the principal chromatography journals. Ettre
c
The nature of the particles, molecules or ions, will depend on the chromatographic system.
d
Martin and Synge, two young chemists (Martin was 31 and Synge was 26), originally presented their work at the 214th Meeting of the (British)
Biochemical Society held in London, at the National Institute for Medical Research in June 1941. They met originally at Cambridge where Martin
completed his doctoral studies. A.T. James in a personal memoire (originally published inINFORM, 6 (1995) 820) states,“Cambridge did not think a
great deal of A.J.P. Martin—he didn’t get a terribly good degree”. For another account, see Leslie S. Ettre, LCGC, 19 (2001) 506.
42 2. Theoretical considerations

[10]summarized the situation as it applied in 1980 to liquid[11]and gas chromatography[12]. Current recommended
nomenclature can be found by searching Commission on Analytical Nomenclature of IUPAC or ASTM and chroma-
tography. Unfortunately, universal agreement still has not been reached on all of these definitions and, in some cases,
the terminology varies between the different chromatographic techniques as a consequence of the historical develop-
ment of certain of the techniques in relative isolation. Nevertheless, the disparity between current usage[13]and
recommended terminology[14]has decreased in recent years.
2.2 Theory of chromatography
A comprehensive theory of chromatography must account for and explain the two processes occurring during chro-
matographic development (Fig. 2.2), namely:
1.Retention, and
2.Dispersion.
It must also allow prediction of retention data. However, this is considered the‘Mt. Everest of separation science’
according to Analytical Chemistry[15]due to the complexity of the underlying phenomena and principles that
FIG. 2.1Title and extract of the Nobel Prize win-
ning paper by Martin and Synge.Extract from Bio-
chemical Journal: A.J.P. Martin, R.L.M. Synge, A new
form of chromatogram employing two liquid phases.
A theory of chromatography. 2. Application to the micro-
determination of the higher monoamino-acids in proteins,
Biochem. J. 35(12) (1941) 1358–1368.
FIG. 2.2Schematic outline of the processes
occurring during a chromatographic develop-
ment and leading to solute resolution.
432.2 Theory of chromatography

determine retention. This makes direct modelling difficult and accounts for the large number of both mathematical and
empirical relationships that have been used to describe and characterize chromatographic columns and the separation
of peaks in chromatograms. A third phenomenon, that of peak distortion, occurs in real chromatograms.
The original theories, plate and rate theory, explain first-order chromatographic phenomena by providing mathe-
matical expressions that include both retention and dispersion. A number of sources make the bold assertion that‘there
are two theories to explain chromatography, plate theory and rate theory’. This statement is a very fitting testimony to
the impact of both plate and the original rate theory and is still substantially true in the correct context. However, it will
probably leave the novice chromatographer very confused[16]as an on-line search of a journal such as the Journal of
Chromatography for the terms‘chromatography’and‘theory’or‘model’will almost certainly produce at least one
relevant article per issue of the journal with titles seemingly unrelated to either plate or rate theory. A selection of such
papers shows the diversity and the potential for confusion:
Simulation and theory of open-tube dispersion in short and long capillaries with slip boundaries and retention[17].
Correlation and prediction of partition coefficient using non-random two-liquid segment activity coefficient model
for solvent system selection in countercurrent chromatography separation[18].
Determination and evaluation of gas holdup time with the quadratic equation model and comparison with non-
linear models for isothermal gas chromatography[19].
Retention models and interaction mechanisms of acetone and other carbonyl-containing molecules with amylose
tris[(S)-α-methylbenzylcarbamate] sorbent[20].
The statistical overlap theory of chromatography using power law (fractal) statistics[21].
Dependence on saturation of average minimum resolution in two-dimensional statistical-overlap theory: Peak
overlap in saturated two-dimensional separations[22].
Closer inspection of these articles will reveal terms such as Laplace domain, Fourier transform, statistical overlap
theory, microscopic or statistical theory, and stochastic dispersive model and that is just in the first paragraph. This is
likely to deter most, if not all readers, other than the most mathematically minded from reading beyond that point. The
treatment in this chapter is not intended as a mathematical treatise but rather to place the various theories and models
in a conceptual framework that facilitates understanding and eliminates the confusion. It was stated[23]as early as
1972 that the chemical literature contained an apparently unlimited collection of chromatographic theories. Another
interesting point is the seemingly arbitrary use of the terms‘theory’and‘model’in the titles (vide infra).
In what context is the claim that there are two theories still correct? The plate theory developed by Martin and Synge
[2]and the classical rate theory of van Deemter and colleagues are of historical significance and have had a profound
impact on the development of chromatography. The third contribution that has probably been largely unrecognized is
that of Wilson[24]. Together these three groups laid the framework of contemporary separation theories and models.
Wilson focused on the thermodynamic influence of a non-linear isotherm (Section 2.3) neglecting the influence of mass
transfer kinetics and axial dispersion (band broadening contributions) on the band profile whereas Martin and Synge
did the opposite and assumed a linear isotherm in order to study non-ideal effects.
Plate theory and rate theory were well developed and experimentally confirmed by 1960. These covered the basic
principles and first-order effects involved in a chromatographic separation. What then are all of these‘new’theories
and models? The intervening period from 1960 to 2019 has witnessed a number of very significant contributions to the
theory of chromatography but these are largely extensions and confirmation of the existing theories plus a closer look
at second-order effects. These second-order effects or secondary interactions (e.g. residual adsorptive effects due to
adsorbent reactive sites in a liquid-phase coated adsorbent) were considered a problem in earlier separations. It is only
more recently that these secondary effects have been exploited in mixed mode chromatography by the connection of
two columns with different modes (two-dimensional chromatography), the combination of two or more types of sta-
tionary phases in a column[25,26], the packing of one mixed-mode stationary phase into a column, and the chemical
synthesis of new stationary phases with different functional groups[27].
The interested reader is referred to Liang and Lin[28]for a challenging and somewhat different approach to the
treatment of the many theories of chromatography.
2.2.1 Why bother with theory?
The study of theory in chromatography has been essential as all of the major developments in the technique have
been predicted from theoretical concepts. Moreover, a thorough and detailed knowledge and understanding of the
principles of chromatography allows appropriate choices about the best system to use and how to optimize conditions.
44 2. Theoretical considerations

Without the insight provided by theoretical studies, the choice of a chromatographic system and its optimum use
would become art or serendipity rather than science.
The theory of chromatography provides a number of outputs that can be identified as follows:
explaining and enhancing the understanding of the mechanisms controlling retention;
modelling and explaining the processes causing dispersion and hence zone broadening and thus predicting zone
shape and position as it moves through the chromatographic column; and
providing retention data at the point the zone exits the column
e
providing a basis for understanding and modifying the effects of the operating variables of the system (e.g. phase
composition, temperature, mobile phase pH) on both retention and dispersion implying that the system can be
optimized in terms of these variables;
characterizing the selectivity of the stationary phase (and mobile phase). This may be to select a new stationary
phase from a list of nominally equivalent phases or, to select an orthogonal phase[29,30]that provides totally
different selectivity; and
enabling us to make predictions about solute retention behaviour (both peak shape and retention time).
The first three outputs provide an answer to our two fundamental questions; what is the shape/size of the zone (i.e.
how much zone broadening has occurred) and what is the retention time. It is important to realize that a model which
is good in terms of one of these outcomes may be poor in achieving another. More specifically, one may have a useful
model for retention that is simultaneously a poor model for characterizing stationary phase selectivity[31].
2.2.2 Model or theory, which is it?
In commenting on the results of our on-line search of the Journal of Chromatography, we commented that both
terms, theory and model, are used although closer examination of the journal reveals that model is used more exten-
sively than theory. Is there a distinction between a theory and a model? According to the Stanford Encyclopedia of
Philosophy[32]the distinction between the two, while perplexing, is a very hazy one and it is often difficult, if not
impossible, to draw a line.
The semantic view of theories asserts that we should view a theory as a family of models whereas the syntactic view
regards models as superfluous additions that are at best of pedagogical, aesthetical, or psychological value. In the for-
mer case, models are viewed as complements of theories, as substitutes for theories (when the latter are too complex to
manipulate), or as preliminary theories. So, the question is: is there a distinction between models and theories and, if so,
how do they relate to one another? In many instances in chromatography, but not all, the two terms have been used
almost interchangeably.
The Stanford Encyclopedia of Philosophy also notes the proliferation of model types and, of the long list quoted,
those that are relevant to chromatography including mathematical models, conceptual models, computational models,
developmental models, and explanatory models. This perhaps also accounts for the number and diversity of chro-
matographic models and theories that differ in the aspects of the chromatographic process covered and in their com-
plexity. Thus some models have general applicability across the range of chromatographic processes while others are
limited to one or more of the processes (e.g. ion-exchange or reversed-phase chromatography). The complexity of the
model will depend on the number and type of assumptions (e.g. of non-ideal, linear behaviour, or of local equilibrium
in rate theory—Section 2.7) that are a compromise between simplicity of the model and its relevance to the actual
process.
2.2.3 Taxonomy of models
This section provides a systematic framework for the classification of the various models and theories that have
been developed to explain chromatographic behaviour. It is hoped that the structure this provides will facilitate a bet-
ter understanding of the various approaches. At the same time, it is informative to have a means of classifying the vast
array of theories that are applied to modelling solute behaviour. Bringing order and structure to the maze of theories
and models is not easy. G.W. Poole, a physicist, in a lecture delivered in the early 1940s at the University of London, as
e
Note that there are two types of concentration or band profile, the spatial profile and the historical profile. Spatial profiles give the distribution of the
concentrations of a band along the column at a given time. Historical profiles give the variation of the concentration during the migration of the band
past a given point of the column. These two profiles differ because axial diffusion occurs constantly, and hence disperses more the rear of the band
than its front which passes first at any given point[1].
452.2 Theory of chromatography

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C. L. S. C. NOTES ON REQUIRED
READINGS FOR MARCH.
PREPARATORY LATIN COURSE IN ENGLISH.
P. 11.—“Matriculate.” The roll or register book in which the
Romans recorded names was called matricula, from this we have the
verb to matriculate, to admit to a membership in an institution or
society, and the noun matriculate, the one admitted.
P. 17.—“Latium,” lāˈshe-ŭm. One of the principal divisions of
ancient Italy, lying south of the Tiber. Its boundaries varied at
different periods.
P. 18.—The Greeks called themselves Hellenes, their language the
Hellenic.
“Æneas,” æ-nēˈas. See the Æneid of Virgil, page 251 of
“Preparatory Latin Course.”
“Mars.” For the story of Mars and Romulus, see page 73 of
“Preparatory Latin Course.” The date of the founding of the city is
given as 753 B. C., and the line of legendary rulers numbered seven.
P. 19.—“Pyrrhus.” For his history see Timayenis, vol. ii.
“Cineas,” cinˈe-as. The friend and prime minister of Pyrrhus. So
eloquent was he that Pyrrhus is said to have declared that “the
words of Cineas had won him more cities than his own arms.” He
went twice to Rome on important embassies for the king, and
probably died in Sicily while Pyrrhus was there.

“Cavour,” käˈvoorˌ. (1810-1861.) An Italian statesman. After a
varied experience in war and politics, Cavour was called in 1850 to
the cabinet of Victor Immanuel, king of Sardinia. Italy was then
divided into several states, some under Austria, others under papal
rule. Cavour turned all his ability to defeating the Austrian powers
and breaking the pope’s authority, in order to unite Italy. In all the
struggles he was one of the chief advisers. In 1861 the states were
united. It has been said of him, “he was one of the most
enlightened, versatile and energetic statesmen of the age.… It is
now conceded on all hands that to him more than any other man is
owing the achievement of the unity of Italy.”
“Victor Immanuel.” (1820-1878.) Became king of Sardinia in 1849
by his father’s abdication. He took part in the Crimean war with
France and England, and was joined by France in the war for Italian
independence. In 1861 he assumed the title of King of Italy, having
united many of the northern provinces. In 1866 he annexed Venetia,
and in 1870 the last of the papal states. In 1871 he transferred his
seat of power to Rome.
“Carthage,” carˈthage. The city was situated in the middle and
northernmost part of the north coast of Africa. It was founded about
one hundred years before Rome, and so rapidly its conquests and
influence advanced that it soon became evident that the rulership of
the western world lay between these two cities. Jealousy kept each
on the alert, and B. C. 264 a dispute about matters in Sicily brought
about the first Punic war, which lasted until B. C. 241. The second
Punic war (B. C. 218-201) resulted in a complete relinquishment of
all power by Carthage. The third (B. C. 149-146) was ended by the
complete destruction of Carthage.
P. 20.—“Hamilcar.” A famous leader in the latter part of the first
Punic war; the father-in-law of Hasdrubal, and father of Hannibal.
After this war and a campaign in Africa, Hamilcar undertook to
establish an empire for Carthage in Spain. After nine years he fell in
battle there and was succeeded by Hasdrubal, who finished the work
and formed a treaty with Rome, regulating the boundaries. After

Hasdrubal’s death Hannibal took his place, but breaking the treaty,
brought about the second Punic war, where he won several brilliant
victories, though finally defeated by Scipio Africanus.
“Regulus.” A Roman leader captured by the Carthaginians in the
first Punic war, and held five years. The Carthaginians desiring peace
sent him to Rome with an embassy to help negotiate, but he
dissuaded his countrymen from accepting the terms. Before leaving
Carthage he had given his word to return if peace was not made,
and in spite of the protest of Rome, he kept the promise. He is said
to have been tortured to death on his return. This story, however, is
suspected to be an invention of the Romans.
“Fabius.” Was five times Roman consul. After the first victories of
Hannibal in the second Punic war, Fabius was appointed dictator.
Here he earned the title of “Master of Delay.” Merivale says: “His
tactics were to throw garrisons into the strong places, to carry off
the supplies of all the country around the enemy’s camp, wherever
he should pitch it, to harass him by constant movement, but to
refuse an engagement.”
P. 21.—“Gracchus.” The family name of two brothers, Tiberius and
Caius, who soon after the destruction of Carthage (146) tried to
relieve the sufferings of the Roman poor. The former was made
tribune in 133, and immediately tried to arrange for a fair division of
public lands, so that the poor citizens might each obtain a small
farm. The opposition was so great that in the attempt to reëlect
Tiberius a riot occurred and he was slain. Ten years afterward Caius
became tribune; he succeeded in carrying several measures to
better the condition of the poor, but through the jealousy of the
senate, his power with the people was broken, and finally during a
disastrous fight between his party and his opponents he fled and
caused a slave to kill him.
“Jugurtha.” See page 82 of “Preparatory Latin Course.”
“Marius.” See page 87 of “Preparatory Latin Course.”

P. 27.—“King William.” See ThÉ Chautauèuan for February, page
252.
P. 28.—“Mommsen,” mŭmˈzen. A German historian, born in 1817.
He has held professorships in jurisprudence or archæology at various
universities, and has published several books. His “History of Rome”
is the most important. It has run through five editions, and been
translated into French and English.
P. 29.—“Curtius.” According to this legend the earth in the Roman
forum gave way B. C. 362. The soothsayers declared that the chasm
could only be filled by throwing into it Rome’s greatest treasure.
Curtius, a young nobleman, declared that Rome possessed no
greater treasure than the citizen willing to die for her, and mounting
his steed leaped into the abyss, which closed upon him.
P. 31.—“Medusa.” One of the Gorgons, frightful beings, whose
heads were covered with hissing serpents; they had wings, brazen
claws and enormous teeth. Medusa was fabled to have been a
beautiful maiden of whom Athena was jealous, and in consequence
turned her into a gorgon. Her head was so fearful that every one
who looked at it was changed into stone. See illustration, page 115.
P. 33.—“Roman Mile.” A thousand paces, or 1600 yards.
P. 34.—“Cretan.” From the island of Crete, one of the largest of
the Mediterranean Sea. It became a Roman province B. C. 66. The
people were celebrated as archers, and were frequently employed as
mercenaries by other nations.
“Balearic.” The Balearic Islands, a group east of Spain, were
known to both Greeks and Romans by this name, derived from the
Greek verb to throw, because of the skill of the inhabitants as
slingers. The Romans subdued the islands 123 B. C.
P. 37.—“Longwood.” The largest of the plains on the island of St.
Helena.
P. 38.—“Trajectory.” The curve which a body describes.

“Cineas.” It is said that when Cineas (see note above) returned
from an embassy at Rome, he told the king that there was no people
like that; their city was a temple, their senate an assembly of kings.
P. 45.—“Montesquieu,” mŏnˈ-tĕs-kūˌ. French jurist and philosopher
(1689-1755).
P. 46.—“Marcus Aurelius.” Roman Emperor from 161-180, called
“The Philosopher.” Smith says of him: “The leading feature in the
character of Aurelius was his devotion to literature. We still possess
a work by him written in the Greek and entitled ‘Meditations,’ in
twelve books. No remains of antiquity present a nobler view of
philosophical heathenism.”
“Bœthius.” A Roman statesman and philosopher, said to be “the
last Roman of any note who understood the language and studied
the literature of Greece.” His most celebrated work was “On the
Consolation of Philosophy.”
P. 48.—“Ennius.” (B. C. 239-169.) Called Father Ennius.
“Plautus.” (B. C. 254-184.) “Terence.” (B. C. 195-159.)
“Menander.” (B. C. 342-291.) A distinguished poet at Athens, in
what was called the “New Comedy.”
P. 50.—“Cato.” (B. C. 234-149.) Cato was famous in military affairs
in early life; after that he entered on a civil career. In 184 he was
elected to the censorship, the great event of his life. Here he tried to
turn public opinion against luxury and extravagance. Cato wrote
several works; only fragments of his greatest, “A History of Rome,”
have been saved.
P. 51.—“Boileau,” bwâˈlō. (1636-1711.) A French poet and critic.
P. 52.—“Æschines.” See Greek history.
“Hortensius,” hor-tenˈsi-us. (B. C. 114-50.) Hortensius was the
chief orator of Rome until the time of Cicero, by whom, in the

prosecution of Verres, he was completely defeated. He held many
civil offices, but in old age retired from public life.
P. 53.—“Livy.” (B. C. 59-A. D. 17.) Livy spent the greater part of
his life in Rome, where he was greatly honored by the emperors. His
reputation is said to have been very great in all countries. His best
known work was a history of Rome, in one hundred and forty-two
books, only thirty-five of which are in existence.
“Tacitus,” “Suetonius.” See page 61 of this volume of ThÉ
Chautauèuan.
“Nepos.” A contemporary of Cicero, of whose life nothing is
known. The chief works of Nepos were biographies, of which we
have only fragments.
“Georgics.” See page 236 of “Preparatory Latin Course.”
P. 54.—“Horace.” (B. C. 65-8.) Horace was the son of a freedman
who attempted to educate his son, sending him to Rome and then to
Athens. While in the latter place Brutus came to Athens, and Horace
joined his army. Returning to Rome he found his father’s estate
gone. He lived in poverty until some of his poems were noticed by
Virgil. Mæcenas became his patron, and afterward Augustus. His
works are The Odes, Satires, Epistles, and The Art of Poetry.
“Ovid.” See page 100 of “Preparatory Latin Course.”
P. 63.—“Historia Sacra.” Sacred history.
P. 65.—“Æsop.” A writer of fables who lived about B. C. 570. He is
said to have been born a slave, but was freed. He was thrown from
a precipice by the Delphians because of a refusal to pay them
money which Crœsus had sent to them. It is uncertain whether
Æsop left any written fables, but many bearing his name have been
popular for ages.
“Putative,” pūˈta-tive. Reputed; supposed.

P. 66.—“Viri Romæ.” Men of Rome.
“Valerius.” A historian of the time of the Emperor Tiberius. The
circumstances of his life are unknown. His work remaining to us is
on miscellaneous subjects, sacred rites, civil institutions, social
virtues, etc.
P. 69.—“Fra Angelico,” frä-än-gelˈe-cō. At the age of twenty he
entered a monastery, where he spent the rest of his life. His
paintings of angels were so beautiful that he won the name of Fra
Angelico—the Brother Angelic. He was called to Rome to decorate
the papal chapel, and offered the position of Archbishop of Florence,
but refused it. He painted only sacred subjects, and would never
accept money for his pictures.
P. 70.—“Repertories,” rĕpˈer-to-ries. A book or index in which
things are so arranged as to be easily found.
“Metellus Pius.” A prominent Roman of the first century B. C. He
held various civil offices, was a commander in the Social war, and
carried on war against the Samnites, in 87. Afterward he was in
arms in Africa, and in 79 went as proconsul to Spain. He died about
60 B. C.
P. 71.—“Dolabella,” dŏl-a-bĕlˈla.
P. 72.—“Caninius,” ca-nĭnˈi-ŭs. One of Cæsar’s legates in Gaul and
in the civil war.
“Drusus.” He won successes in the provinces after the death of
Augustus, and was pointed out as the successor of Tiberius.
Sejanus, the favorite of Tiberius, aspired to the empire. He won the
wife of Drusus to his plans, and persuaded her to administer a slow
poison to her husband, which finally caused his death.
P. 75.—“Egeria.” She had been worshiped by the people of Latium
from the earliest times, as a prophetic divinity. Numa consecrated to
her a grove in the environs of the city, where it is said that he used
to meet her. The grotto and fountain of Egeria are still pointed out to

travelers. It is said that on the death of Numa, Egeria was so
inconsolable that she was changed into a fountain.
“Aurora.” In Grecian mythology the goddess of the morning, who
sets out before the rising of the sun and heralds his coming.
“Nympholepsy,” nĭm-pho-lĕpˈsy. The state of being caught by the
nymphs; ecstasy.
P. 77.—“Numidia,” nu-midˈi-a. A country of Northern Africa, now
Algiers.
P. 78.—“Bohn.” An English publisher who has republished in the
English language, and in cheap form, most of the rare standard
works of the different literatures of Europe. His library now numbers
between 600 and 700 volumes.
P. 80.—“Numantine.” This war was waged by the Numantians, a
little people of Spain, not numbering more than 8,000 fighting men,
against Rome. Their city, Numantia, was taken B. C. 133, after a
long siege.
P. 82.—“Cato.” (B. C. 95-46.) Great-grandson of Cato the Censor.
His character was stern and stoical, and in his public and military life
he was famous for his rigid justice and sternness against abuses.
Cato opposed Cæsar throughout his life. When Cæsar entered Africa
he tried to persuade Utica to stand a siege, but failing, committed
suicide.
P. 103.—“Clymene,” clymˈe-nē. The mother of Phæton.
“Styx.” The chief river of the infernal world, according to Grecian
mythology, around which it flows seven times. The name comes
from the Greek word to hate. Milton calls it “Abhorred Styx, the flood
of burning hate.”
“Hours.” The Hours were the goddesses who presided over the
order of nature and over the seasons. They gave fertility to the
earth, and furnished various kinds of weather. The course of the

season is described as the dance of the Hours. In art they are
represented as beautiful maidens, carrying fruits and flowers.
P. 194.—“Tethys,” tĕˈthys. The goddess of the sea. The wife of
Oceanus, and mother of the river gods.
P. 105.—“Seven Stars.” By these seven stars are meant the sun,
moon, Mars, Mercury, Saturn, Jupiter and Venus.
“Serpent.” The constellation of Draco, which, stretching between
Ursa Major and Ursa Minor, nearly encircles the latter.
“Boötes,” bo-oˈtes. The constellation commonly known as Charles’
Wain, or the Wagoner. Boötes is said to have been the inventor of
the plow, to which he yoked two oxen. At his death he was taken to
heaven and set among the stars.
“Libya.” A name for the continent of Africa, applied here to the
Sahara Desert.
“Dirce.” It is fabled that a king of Thebes drove away his wife into
the mountains of Bœotia, where she died, leaving two sons. When
the boys grew up they returned to Thebes and killed both their
father and his wife, Dirce, who had been an assistant in his crime.
Dirce was dragged to death by a bull, and her body thrown into a
well, which was from that time called the “Well of Dirce.” The
celebrated statue of the Farnese bull represents the death of Dirce.
“Pyrene,” pyrˈe-ne.
“Amymone,” amˈy-moˌne. The daughter of Danaus, who had fled
with his family from Egypt to Argos. The country was suffering from
drought, and he sent out Amymone to bring water. She was attacked
by a Satyr but rescued by Neptune, who bade her draw his trident
from a rock. Thereupon a threefold spring gushed forth, which was
called the river and well of Amymone.
“Tanais,” tanˈa-is. The river Don.

“Caicus,” ca-īˈcus. A river of Asia Minor.
“Lycormas,” ly-corˈmas.
“Xanthus,” zanˈthus. The chief river of Lycia, in Asia Minor.
“Mæander,” mæ-anˈder. A stream of Asia Minor. The greater part
of its course is through a wide plain, where it flows in the numerous
windings which have made of its name the verb to meander.
“Ismenos,” is-mēˈnos. A small river in Bœotia.
“Phasis,” phāˈsis. A river flowing through Colchis, into the Black
Sea.
“Tagus.” One of the chief rivers in Spain.
P. 106.—“Cayster” or “Caystrus,” ca-ysˈter. A river of Lydia and
Ionia, in Asia Minor. It is said that it still abounds in swans, as it did
in Homer’s time.
“Pluto.” The god of the infernal world.
“Cyclades,” cycˈlă-des. A group of islands in the Ægean Sea, so
called because they lay in a circle around Delos.
“Phocæ,” phōˈcæ. Sea calves, or sea monsters of any description.
“Doris.” The daughter of Oceanus, and wife of her brother
Nereus; sometimes her name is given to the sea itself.
P. 107.—“Presto,” prĕsˈtō. Quickly; at once.
P. 108. “Burke,” Edmund. (1730-1797.) An English statesman,
writer and orator.
“Lucian,” lūˈci-an. (A. D. 120-200.) A Greek author.
“Molossian,” mo-losˈsian. The Molossi were a people in Epirus,
inhabiting a country called Molossis. They were the most powerful
tribe in Epirus.

P. 109.—“Daphne,” dăphˈne.
P. 110.—“Peneus,” pe-neˈus. The name of the chief river of
Thessaly. As a god Peneus was the son of Oceanus.
“Claros,” claˈros. A small town on the Ionian coast, with a
celebrated temple and oracle of Apollo.
“Tenedos,” tĕnˈe-dŏs. A small island of the Ægean, off the coast of
Troas, also sacred to Apollo.
“Patarian,” pa-taˈri-an. From Patara, one of the chief cities of Asia
Minor, in Lycia. Apollo had an oracle here, and a celebrated temple.
P. 114.—“Narcissus.” A youth who was fabled to be so hard of
heart that he never loved. The nymph Echo died of grief because of
him. Nemesis caused him to fall in love with his own image as he
saw it in a fountain, and Narcissus died because he could not
approach the shadow. His corpse was metamorphosed into the
flower which has his name.
“Dædalus.” A character of Grecian mythology, fabled to be the
inventor of many contrivances, as well as a sculptor and architect.
Having incurred the displeasure of the king of Crete, he was obliged
to flee from the island. Accordingly he made wings for himself and
his son Icarus. Dædalus flew safely to shore, but Icarus went so
near the sun that the wax by which his wings were fastened melted,
and he was drowned in that part of the Ægean called the Icarian
Sea.
“Baucis.” Baucis and Philemon were an aged couple living in
Phrygia. Jupiter and Mercury having occasion to visit this part of the
world, went in the disguise of flesh and blood. Nobody would receive
them until Baucis and Philemon took them into their hut. Jupiter
took the couple to a hill near by, while he punished the inhospitable
by an inundation; he then rewarded them by making them guardians
of his temple, allowing them to die at the same moment, and
changing them into trees.

“Lycidas,” lĭsˈi-das. A poetical name under which Milton laments
the death of his friend Edward King, who had been drowned.
“Comus.” In the later age of Rome, a god of festive joy and mirth.
In Milton’s poem entitled “Comus, a Masque,” he is represented as a
base enchanter who endeavors, but in vain, to beguile and entrap
the innocent by means of his “brewed enchantments.”—Webster.
P. 123.—“Rhodes.” An island of the Eastern Ægean. It was long
celebrated for its schools of Greek art and oratory.
“Pontifex,” ponˈtĭ-fex. A Roman high priest, a pontiff. The
pontifices constituted a college of priests, superintended the public
worship, and gave information on sacred matters. Their leader was
called pontifex maximus.
“Quæstor.” The title of a class of Roman officials, some of whom
had charge of the pecuniary affairs of the state, while others
superintended certain criminal trials.
“Ædile.” A magistrate of Rome who superintended public
buildings, such as temples, theaters, baths, aqueducts, sewers, etc.,
as well as markets, weights, measures, and the expenses of
funerals.
P. 125.—“Proconsul.” The title given to those who, after holding
the office of consul, were sent to some province as governor.
P. 126.—“Ascham.” (1515-1568.) The foremost scholar of his time,
celebrated for his superior knowledge of Greek and Latin.
P. 127.—“Æduans,” ædˈu-ans. Their country lay between the Loire
and the Saone.
P. 126.—“Lingones.” A people living to the east of the source of
the Mosa river. (See map.)
P. 137.—“Sequani.” A tribe of Gallia Belgica (see map), taking their
name from the river Sequana, near the source of which they lived.

P. 139.—“Soissons,” swäˌsōnˈ, almost swīˌsōnˈ. About fifty miles
northeast of Paris.
P. 112.—“Bellovaci.” They dwelt in the north of Gallia, beyond the
Sequana river. (See map.)
P. 143.—“Ambian.” These people, with the Nervii and the Aduatuci
(p. 147) were all tribes of Gallia Belgica.

NOTES ON REQUIRED READINGS IN
“THE CHAUTAUQUAN.”
READINGS FROM FRENCH HISTORY.
P. 215, c. 1.—“Gallia.” For Gallia and the tribes Aquitani, Celtæ
and Belgæ, see Professor Wilkinson on Cæsar in “Preparatory Latin
Course.”
“Burgundians.” A race of early Germans who in 407 A. D. crossed
the Rhine and settled between the Rhone and Saone. In 534
Burgundy was taken possession of by the Franks.
“Franks.” See page 63 of the present volume of ThÉ Chautauèuan.
“Clovis.” See page 129 of the present volume of ThÉ Chautauèuan.
“Salian Franks.” There were two tribes of the Franks, one called
Salian, from the river Sala or Yssel, upon which they dwelt, the other
Ripuarian, from the Latin ripa, bank, the name showing their location
on the banks of the Rhine.
“Merovingians.” See notes, page 185 of present volume of ThÉ
Chautauèuan.
“Childeric,” or Hilderik. The race had become so weak that the
rulers have been well described as the “shadow kings.” This last
ruler of the Merovingians was thrust into a convent, where he soon
died.
“Pepin,” pēpˈin. The son of Charles Martel. See page 129 of ThÉ
Chautauèuan. His wars were successful. The most interesting was

against the Lombards, who were threatening Rome. He compelled
them to give up to the Church of Rome a considerable territory
which was, says a writer, “The foundation of that temporal power of
the papacy, the end of which we have seen with our own eyes.”
“Charlemagne,” sharˈle-mānˌ. See page 131 of fourth volume of ThÉ
Chautauèuan.
“Hugues.” Hugh, in English; “Capet,” cāˈpet or căpˈet.
“Louis le Gros.” Louis the Great.
“Feudal system.” That system where land is held of superiors, on
condition of military service.
P. 215, c. 2.—“Oriflamme.” From the Latin auriflamma, or flame of
gold. A flag or banner of red or flame colored cloth, cut into long
points at the end and mounted on a gilded lance. It originated in a
certain abbey of France, where it was used in religious services.
“Touraine,” tô-rān; “Poitou,” pwä-tôˈ. These provinces had come to
England on the accession of Henry II. (1154), to whom they
belonged.
“Gallican Church.” The Catholic Church of France, which holds
certain doctrines differing from those of the church at large. This
church claims that the pope is limited as far as France is concerned,
by the decisions of the Gallican Church, that kings and princes are
not subject to him, and that he is not infallible. This pragmatic
sanction of St. Louis in 1269 was the most important outbreak
against Rome that ever took place in the Gallican Church.
“Le Bel.” The Beautiful.
“Navarre,” nă-varˈ. A province of France on the northern slope of
the Pyrenees.
“Champagne,” shŏnˌpäñˈ. See map.

“Brie,” bre. A former province of France, lying between the Seine
and the Marne.
“Valois,” väl-wäˈ.
“Salic Law.” According to this, “no woman could succeed to Salian
soil.” The only descendant of Charles IV. was his infant daughter, and
when the lords met to decide on the succession after his death, they
followed this law; for as Froissart says, “The twelve peers of France
said and say that the crown of France is of such noble estate, that
by no succession can it come to a woman nor a woman’s son.”
P. 216, c. 1.—“Le Sage,” the wise; “Crécy,” krĕsˈe; “Poiters,” pwä-
terzˈ; “Le Bien Aime,” the Beloved; “Agincourt,” ă-zhan-koor; “Le
Victorieux,” the Victor; “Le père du peuple,” the father of his people.
“Valois-Orleans.” Louis XII. was the representative of the line
nearest to the Valois family, that is, he was a son of the Duke of
Orleans, and a grandson of the younger brother of Charles VI., thus
representing both families.
“Valois Angoulême,” ŏnˌgooˌlāmeˈ. Louis XII. dying without heirs,
the kingdom fell to the heirs of his uncle, the Count of Angoulême.
Francis became a competitor with Charles I., of Spain, for the throne
of Spain, but the latter was successful. This led to the war which
was ended by Francis being made a prisoner at Pavia.
“St. Bartholomew.” There had been a struggle for many years
between the Protestants and Catholics, which finally took the form of
a conflict between the houses of Guise and Condé. Henry of Navarre
was the successor to the throne—a marriage was arranged between
him and the sister of the king, and August 18, 1572, was to be the
wedding day. Many of the leading Huguenots were in Paris. It has
been said that this wedding was but a scheme to bring them
together; at any rate Coligni, a leading Huguenot, was fired upon by
an assassin. The Huguenots became excited and threatened
revenge. Catherine persuaded her son that they intended
massacring the Catholics, and Charles gave an order for a general

slaughter of the Protestants. The order was executed in nearly every
city and town of France, and nearly 100,000 persons were put to
death.
“Confederation of the League.” This holy league, or “Catholic
Union,” as it was called, was supported by the pope and Philip II., of
Spain. Its head was Duke Henry of Guise, who aimed at the French
throne.
“Guise,” gheez.
“Bourbon,” boorˈbon. A French ducal and royal family, different
branches of which have ruled Spain, France, Naples and Parma. The
civil wars which were carried on between these houses were no less
than eight in number.
“Richelieu,” reshˈeh-loo.
“Mazarin,” măz-a-reenˈ.
“Fronde.” A faction which opposed putting all the power of France
into the hands of the government, as Richelieu and Mazarin both
attempted. The name of frondeurs (slingers) was applied to them
because in their sneering and flippant attacks upon Mazarin they
were said to resemble boys throwing stones from slings.
“Tiers état.” Third estate. Before the reign of Philip the Fair, the
people had had no voice in the government; but in his struggle with
the papacy, as he desired to have the whole body of citizens on his
side, he convened an assembly of the middle class of citizens, beside
the clergy and nobility. The third body was called the third estate.
P. 216, c. 2.—“États Généraux,” States general. An assembly of
the nation, which consisted of representatives of the clergy, nobility,
and the third estate.
“National Assembly.” Upon the meeting of the states general, the
nobles and the clergy insisted that the meetings of the body and its
deliberations should be conducted according to class distinctions;

this met with the opposition of the third estate, who finally declared
themselves the only body having a right to act as the legislature of
France, and summoned the clergy and nobles to attend their
deliberations. They called themselves the National Assembly.
“Bastille,” bas-teelˈ. The state prison and citadel of Paris. It was
begun in 1366; destroyed in 1789.
“Marie Antoinette,” mäˈrēˌ ŏnˌtwäˈnĕtˈ.
“Dauphin.” The title given to the eldest son of the king of France,
under the Valois and Bourbon lines. It corresponds to “Prince of
Wales” in England. It originally belonged to the counts of Dauphiny.
“Cis-Alpine,” sis-alˈpin. On this side of the Alps, that is, on the
south or Roman side.
“Marengo,” ma-rĕnˈgō; “Prestige,” prĕs-tijˈ.
P. 317.—“D’Artois,” darˌtwäˈ; “Louis Phillippe,” loo-ē fe-leep; “Coup
d’état,” a stroke of policy in state affairs; “Sedan,” se-dänˈ, a town of
France, 130 miles northeast of Paris; “Bordeaux,” bor-dō; “Thiers,”
te-êrˈ; “Grèvy,” grā-vē.
P. 317, c. 2.—“Champs-de-Mars,” shân-duh-marce. An extensive
parade ground of Paris, on the left bank of the Seine. It has been
the scene of many very remarkable historic events, and is now used
for great reviews, etc. The buildings of the exposition of 1867 were
erected upon it.
“Friesland,” freeceˈland. A province of Holland.
“Teignmouth,” tinˈmuth.
“Hengesdown,” henˈges-down.
“Narbonnese,” narˌbonˌnesˈ. One of the four provinces into which
Augustus divided Gaul was named from Narbonne, a city near the
Mediterranean, Gallia Narbonensis or Narbonnese Gaul.

P. 318, c. 1.—“Montfort.” The wife of the duke of Brittany, who
had succeeded his brother, Jean III. It seems that the latter had left
the duchy to his nephew, Charles of Blois, but Montfort took
possession. War was declared, and the king of France aided Blois,
the king of England, Montfort. The latter was taken prisoner and his
wife took the field.
“Blois,” blwä; “Penthièvre,” pĕnˈtĕvrˌ.
“Van Artevelde,” vän arˈta-velt. A citizen and popular leader of
Ghent, who for a long time was almost ruler of Flanders. In this war
the people, under Artevelde, supported the English, while the
nobility were in sympathy with the French.
“Froissart,” froisˈärt. (1337-1410.) A French history writer.
“D’Harcourt,” därˈkōrtˌ.
“Harfleur,” har-flurˈ; “Cherbourg,” sherˈburg; “Valognes,” väˌloñˈ (n
like ni in minion). “Carentan,” käˈrŏnˌtŏnˌ; “Caen,” kŏn; “Louviers,” loo
ˌve-āˈ; “Vernon,” vĕrˌnōnˈ; “Verneuil,” vĕrˈnuhl; “Mantes,” mants;
“Meulan,” moi-lăn; “Poissy,” pwâ-sē; “Ruel,” roo-äl; “Neuilly,” nuhˌyēˈ;
“Boulogne,” bou-lōnˈ; “Bourg-la-reine,” boor-la-rain.
“Béthune,” bā-tün; “Ponthieu,” pŏn-te-ŭh.
P. 318, c. 2.—“Hainault,” ā-nōl; “De Vienne,” deh ve-enˈ; “De
Manny,” deh mănˌneˈ.
P. 319, c. 1.—“Eustace de St. Pierre,” eūsˈtace deh sănˌpe-êrˈ;
“D’Aire,” d’air; “Domremy,” dôn-rŭh-me; “Neufchâtel,” nushˌäˌtelˈ;
“Vancouleurs,” vŏnˌkooˈluhrˌ; “Baudricourt,” bōˈdrēˌkoorˌ; “Chinon,” she-
nōng.
“Cap-a-pie,” kăpˌa-peeˈ. From head to foot.
P. 319, c. 2.—“La pucelle,” the maid; “Trémoille,” trāˌ-mooyˈ;
“Boussac,” booˈsäkˌ; “Xaintrailles,” zanˈträlˌyeˌ; “La Hire,” läˌērˈ; “Dunois,”

düˈnwâˌ; “Jargeau,” zharˌghōˈ; “Meung,” mŭng; “Beaugency,” bōˈgán-cēˌ;
“Patay,” pa-tāyˈ.
P. 320, c. 1.—“Compiègne,” kŏmˌpe-ānˈ; “Ligny,” lē-nyē;
“Vendôme,” vŏnˌdōmˈ.
P. 320, c. 2.—“Épernon,” āˈpĕrˌnōnˌ; “Angoumois,” ŏnˈgooˌmwäˈ;
“Saintonge,” săn-tōnzhˈ.
P. 321.—“Sancy,” sanˈcē; “Ile de France,” eel-deh-frŏnss; “Picardy,”
picˈar-dee; “Auvergne,” ō-vĕrnˈ; “Gaetano,” gā-ā-täˈno, usually written
Cajetan.
“Sorbonne,” sor-bŭn. The principal school of theology in the
ancient university of Paris. Its influence was powerful in many of the
civil and religious controversies of the country.
“Arques,” ark; “Dreux,” druh; “Evreux,” ĕvˈruhˌ; “Ivry,” ēvˈrēˌ; “Eure,”
yoor.
P. 321, c. 2.—“Reiters,” rīˈters; “Mayenne,” mäˌyenˈ; “Meaux,” mō;
“Senlis,” sŏnˌlēsˈ.
P. 322, c. 1.—“Brisson,” brēˌsōnˈ; “Grève,” grāv.
“Sully.” A French statesman, the chief adviser of Henry IV.
P. 322, c. 2.—“Bèarnese,” bāˈarˌnēseˌ. Bèarn, a former southwest
province of France, belonged to the kings of Navarre. From this
possession Henry IV. received the title of the Bèarnese.
“Eustache,” uhsˌtäshˈ; “Merri,” mā-rē; “Guincestre,” ghinˈcestrˌ;
“Villeroi,” vēlˈrwä; “Vervins,” vĕr-vănˈ.
“Escurial,” ĕs-koo-re-älˈ. A palace and mausoleum of the kings of
Spain.
P. 323, c. 1.—“Saluzzo,” sâ-lootˈso; “Rosny,” ro-ne; “Gontaut de
Biron,” gŏnˈ-toˌ deh beˌ-rōnˈ; “Malherbe,” mälˌêrbˈ.

P. 323, c. 2. “Praslin,” präˌlănˈ; “Montbazon,” mōnˌbäˌzŏnˈ; “Crèqui,”
krā-keˈ; “Mirabeau,” meˌräˌbōˈ.
“Equerry,” e-quĕrˈry. An officer of nobles, charged with the care of
their horses.
“Cœur Couronné,” etc. The crowned heart pierced with an arrow.
“Curzon en Quercy,” kür-sōnˈ ĕng kwerˈcēˌ.
P. 324, c. 1.—“Bruyère,” brü-eˌyêrˈ. (1646?-1696.) French author.
“Fouquet,” fooˌkāˈ. (1615-1680.) A French financier, convicted of
dishonesty and treason under Louis XIV.
“De la Vallière,” deh lä väˌle-êrˈ; “Montespan,” mŏnˌtes-pănˈ.
“Bossuet,” boˌsü-āˈ (almost bosˌswāˈ). (1627-1704.) French bishop
and orator.
“Lauzun,” lōˌzŭnˈ. (1633?-1723.) A French adventurer.
“Pignerol,” pē-nyŭh-rŭl. A city of Piedmont, Italy.
“Iron Mask.” The man in the iron mask was a prisoner who died in
the Bastile in 1703. He was brought there in 1698, from the state
prison of Marguerite, by the governor who had been changed to the
Bastile. His face was covered with a black velvet mask, fastened with
steel springs. He was never allowed to remove this, nor to speak to
any one except his governor. After his death everything he
possessed was burned. There have been many theories as to his
identity, but no one has been thoroughly proven.
P. 324, c. 2.—“Marcillac,” mär-ceelˌlakˈ; “Rochefoucauld,” roshˌ-foo
ˌkōˈ; “Marèchal,” mäˌrāˌshalˈ; “Fontanges,” fōnˌtanzhˈ.
“Scarron,” skărˌrōnˈ. She had been the wife of Paul Scarron, a
French author, who died in 1660. “Maintenon,” mănˈtŭhˌnōn.

P. 325, c. 2.—“Della Guidice,” dĕlˈlä gweeˈde-cā; “Alberoni,” ăl-bä-ro
ˈnee.
P. 326, c. 1.—“Lettres de Fénelon,” etc. Letters of Fénelon to the
duke of Chevreuse.
P. 326, c. 2.—“Nunc et in,” etc. Now and in the hour of death.
READINGS IN ART.
P. 331, c. 1.—“Transept.” Any part of a church which projects at
right angles with the body and is of equal or nearly equal height to
this. Transepts are in pairs, that is, the projection southward is
accompanied by a corresponding projection northward.
“Nave.” The central portion of a cathedral, distinguished from the
choir.
“Arcade.” Ranges of arches supported on piers or columns.
“Triforium,” tri-fōˈri-um.
P. 331, c. 2.—“Apse,” ăpse; “Apsidal,” ăpˈsi-dal.
“Chapter-house.” The house where the chapter or assembly of the
clergymen, and their dean, belonging to a cathedral, meet.
“Hospitium,” hos-pĭshˈi-ŭm.
“Castellated.” Adorned with turrets and battlements, like a castle.
“Dais,” dāˈis. A raised floor at the upper end of a dining hall.
“Lancet.” High, narrow, and sharp pointed.
“Piers.” A mass of stonework used in supporting an arch; also the
part of the wall of a house between the windows or doors.
P. 332, c. 1.—“Cuspated,” cuspˈāt-ed. Ending in a cusp, that is, the
projecting point thrown out from foliations in the heads of Gothic

windows.
“La Sainte Chapelle.” The holy chapel.
“Chartres,” shartˈr; “Bourges,” boorzh; “Corbel,” a projecting stone
or timber supporting, or seeming to support, some weight.
P. 332, c. 2.—“Tudor,” tūˈder. So called from the house on the
English throne at the time of the growth of the style.
“Elizabethan,” elĭzˌa-bēthˈan.
“Newel-post.” The stout post at the foot of the staircase, on the
top of which the rail rests.
“Wren.” (1632-1723.) An English architect, the designer of St.
Paul’s, in London. After the London fire of 1666, he drew the plans
for over fifty churches and many important public buildings of the
city.
“Mural,” belonging to a wall.
“Beaumanti,” bĕ-ä-mänˈte.
SELECTIONS FROM AMERICAN LITERATURE.
P. 333, c. 2.—“Ichthyophagi,” ĭchˌthy-ŏphˈa-gi. A compound word of
Greek origin, meaning fish eaters.
“Dunes.” Same as downs, little sand hills piled up near the sea.
“Badahuenna,” bad-a-huenˈna.
“Hercynian,” her-cynˈi-an.
P. 334, c. 1.—“Bouillon,” booˌyŭnˈ.
“Brabantine,” braˈbran-tīne.
P. 335, c. 2.—“Cortés,” kôrˈtez.

P. 336, c. 1.—“Narvaez,” nar-väˈĕth; “Chiapa,” che-āˈpä.

CHAUTAUQUA NORMAL
GRADUATES,
Class of 1883.
John Aiken, Washington, Pennsylvania.
Mrs. W. C. Armor, Bradford, Pennsylvania.
Addie M. Benedict, Jamestown, New York.
Vinola A. Brown, Morning Sun, Ohio.
Clara J. Brown, Morning Sun, Ohio.
Martha Buck, Carbondale, Illinois.
Anna C. Cobb, New York City.
Kittie E. Carter, Randolph, New York.
Mary E. Coles, Philadelphia, Pennsylvania.
Mrs. Hattie E. Chambers, Bradford, Pennsylvania.
Sarah I. Dale, Franklin, Pennsylvania.
Miss H. M. Dawson, Tidioute, Warren Co., Pennsylvania.
Harriet E. Elder, South Bend, Indiana.
Will T. Edds, Gerry, New York.
Rev. W. H. Groves, Fayetteville, Tennessee.
Mrs. H. M. Graham, Garrettsville, Portage Co., Ohio.
Ida E. Goodrich, Geneva, Ashtabula Co., Ohio.
Myrtie C. Hudson, Ann Arbor, Michigan.
Maria R. Jones, Meriden, Connecticut.
Eleanor M. Matthews, Gerry, New York.
Sarah A. Mee, Buffalo, New York.
Mrs. Rosetta Page, Frewsburgh, New York.
Mary J. Perrine, Rochester, New York.
Lucie A. Pooley, Bridgeville, Allegheny Co., Pennsylvania.
Mrs. P. P. Pinney, Clarion, Clarion Co., Ohio.

Nellie H. Skidmore, Fredonia, New York.
Rev. Orange H. Spoor, Charlotte, Eaton Co., Michigan.
Mary A. Sowers, Carbondale, Illinois.
Mary Stevenson, Leech’s Corners, Mercer Co., Pennsylvania.
Will B. Stevenson, Leech’s Corners, Mercer Co., Pennsylvania.
Kate M. Thorp, Napoli, Cattaraugus Co., New York.
Mattie R. Weaver, Latrobe, Westmoreland Co., Pennsylvania.
OTTAWA ASSEMBLY.
Mrs. N. S. Zartman, Kansas City, Missouri.
Mrs. M. E. Wharton, Ottawa, Kansas.
Mrs. A. C. Hodge, Ottawa, Kansas.
B. F. Thayer, Wamego, Kansas.
N. W. Beauchamp, Kansas, Illinois.
Cornelia C. Adams, Ottawa, Kansas.
Mrs. D. Holaday, Ottawa, Kansas.
Mrs. H. E. M. Pattee, Williamsburg, Kansas.
Robert Bruce, Ottawa, Kansas.
L. Ettie Lester, Ottawa, Kansas.
Jennie Gott, Ottawa, Kansas.
Emma W. Parker, Ottawa, Kansas.
Rev. F. L. Walker, Grenola, Kansas.
Alberlina Wickard, Ottawa, Kansas.
Mrs. J. F. Drake, Emporia, Kansas.
Miss Emma J. Short, Ottawa, Kansas.
Mrs. J. P. Stephenson, Ottawa, Kansas.
J. K. Mitchell, Osborne, Kansas.
Emma E. Page, Ottawa, Kansas.
Rev. C. R. Pattee, Williamsburg, Kansas.
R. Henry Stone, Kansas City, Missouri.
Rev. P. P. Wesley, Great Bend, Kansas.
Mrs. C. W. Holmes, Ottawa, Kansas.
May L. Parker, Olathe, Kansas.

SUNDAY-SCHOOL PARLIAMENT.
T. Harry Farrell, Kingston, Ontario.
Mrs. Sarah W. Hopkins, Madison, New York.
Nellie Lavelle, Kingston, Ontario.
Florence E. Kinney, Syracuse, New York.
Minnie Lavelle, Kingston, Ontario.
Mrs. Effie Williams, Plainfield, New Jersey.
James Farrell, Kingston, Ontario.
Harry A. Lavelle, Kingston, Ontario.
Mrs. T. W. Skinner, Mexico, New York.
Avery W. Skinner, Mexico, New York.
Fannie S. Jaques, Merrickville, Ontario.
FRAMINGHAM CHILDREN’S CLASS.
Bessie M. Adams, Northboro, Massachusetts.
James A. Babbitt, Swanton, Vermont.
Winfield H. Babbitt, Swanton, Vermont.
Harry R. Barber, Worcester, Massachusetts.
Laura M. Batchelder, West Medway, Massachusetts.
Arthur T. Belknap, Framingham, Massachusetts.
Jesse H. Bourne, Foxboro, Massachusetts.
Albert C. Comey, South Framingham, Massachusetts.
Bernia Comey, South Framingham, Massachusetts.
Willie Desmond, West Medway, Massachusetts.
Bertha Elliott, Revere, Massachusetts.
Annie T. Francis, Fitchburg, Massachusetts.
M. Gracie Full, South Framingham, Massachusetts.
Maud Grumelle [No address].
George Hancock, Milford, Massachusetts.
Lewis K. Hanson, Natick, Massachusetts.

Lillian R. Hemenway, Framingham, Massachusetts.
Bertha J. Hopkins, Worcester, Massachusetts.
Kate E. Lawrence, South Framingham, Massachusetts.
Stella Mann, Boston Highlands, Massachusetts.
C. L. Reynolds, Framingham Center, Massachusetts.
Florence M. Sears, Worcester, Massachusetts.
Cora E. Thayer, Allston, Massachusetts.
Fred P. Wheeler, Allston, Massachusetts.
Ellen M. Works, Southboro, Massachusetts.
Frank S. Wright, Natick, Massachusetts.
FRAMINGHAM CHILDREN’S CLASS—
ADVANCED GRADE.
Phillips P. Bourne, Foxboro, Massachusetts.
Mattie P. Cushing, Hudson, Massachusetts.
William O. Cutler, Natick, Massachusetts.
Joseph H. Hall, Natick, Massachusetts.
Mary A. Harriman, Framingham, Massachusetts.
Lewis K. Hanson, Natick, Massachusetts.
Howard Mason, Natick, Massachusetts.
Harry D. Neary, Framingham, Massachusetts.
Ida M. Neary, Framingham, Massachusetts.
Edward O. Parker, East Holliston, Massachusetts.
Bertie M. Stetson, Holliston, Massachusetts.
G. Adelbert Watkins, South Framingham, Massachusetts.
Theodore S. Bacon, Natick, Massachusetts.
Millie S. Bruce, Southville, Massachusetts.
Harry R. Barber, Worcester, Massachusetts.
Geo. F. Beard, South Framingham, Massachusetts.
Albert Comey, South Framingham, Massachusetts.
John Connelly, Cochituate, Massachusetts.
Bertha May Cushing, Hudson, Massachusetts.
Fred L. Francis, Fitchburg, Massachusetts.

Emeline Hancock, Milford, Massachusetts.
Emma L. Huse, Somerville, Massachusetts.
Stella Mann, Boston Highlands, Massachusetts.
Florence B. Moultrop, Framingham, Massachusetts.
Ida M. Neary, Framingham, Massachusetts.
Emma J. Parker, East Somerville, Massachusetts.
Charles H. Phipps, South Framingham, Massachusetts.
Cora E. Thayer, Allston, Massachusetts.
Hattie Stratton, South Framingham, Massachusetts.
Fred R. Woodward, Natick, Massachusetts.
Frank S. Wright, Natick, Massachusetts.
FRAMINGHAM PRIMARY TEACHER’S UNION.
Mrs. Emma D. Daniels, Framingham, Massachusetts.
Minnie E. Gaskins, Mattapan, Massachusetts.
Georgie A. Goodnow, Sudbury, Massachusetts.
Jessie E. Guernsey, Framingham, Massachusetts.
Minnie L. Jackson, South Gardner, Massachusetts.
Addie M. Knight, Magnolia, Massachusetts.
Helen Virginia Ross, Charleston Station, Massachusetts.
Ellen Letitia Ruggles, Milton, Massachusetts.
Josie Bell Stuart, Lowell, Massachusetts.
Mrs. M. D. Thayer, Allston, Massachusetts.
Mrs. S. Isabella Valentine, Hopkinton, Massachusetts.
Mrs. I. G. Wheeler, Allston, Massachusetts.
FRAMINGHAM NORMAL UNION.
S. Addie Alexander, Marlboro, Massachusetts.
Willis N. Bailey, Buckingham, Connecticut.
Elsie L. Ball, Milford, Massachusetts.
Alice Bertha Besse, Lowell, Massachusetts.

Mrs. Harriet E. Bates, Boston, Massachusetts.
Mary Amittai Bradford, Mystic Bridge, Connecticut.
Hannah K. Bradford, Mystic Bridge, Connecticut.
Mrs. Lizzie E. Bird, Boston, Massachusetts.
Mrs. L. S. Brooks, Fitchburg, Massachusetts.
Nellie M. Brown, Lowell, Massachusetts.
Nellie E. Canfield, South Britain, Connecticut.
Hattie D. Fuller, Hudson, Massachusetts.
Rev. A. Gardner, Buckingham, Connecticut.
Miss M. E. Harrington, North Amherst, Massachusetts.
F. M. Harrington, Northboro, Massachusetts.
O. A. Heminway, Framingham, Massachusetts.
Clara D. Jones, North Abington, Massachusetts.
Miss Ida A. E. Kenney, Worcester, Massachusetts.
Addie M. Knight, Magnolia, Massachusetts.
Caroline M. Lee, Wayland, Massachusetts.
J. H. O. Lovell, Oakham, Massachusetts.
Helen M. Locke, Magnolia, Massachusetts.
Mrs. S. T. McMaster, Watertown, Massachusetts.
Sarah M. Potter, Providence, Rhode Island.
Delia Pinney, Ludlow, Vermont.
Margaret S. Rolfe, Newburyport, Massachusetts.
Julia A. Robinson, North Cambridge, Massachusetts.
Luella H. Simonds, Lowell, Massachusetts.
Mrs. Harriet B. Steele, Reading, Massachusetts.
Rachel Steere, Greenville, Rhode Island.
Clara E. Stevens, Newburyport, Massachusetts.
Ellen K. Stone, Framingham, Massachusetts.
Anna A. Ware, West Medway, Massachusetts.
Mrs. William L. Woodcock, Winchendon, Massachusetts.
L. D. Younkin, Boston, Massachusetts.

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Principles And Practice Of Modern Chromatographics Methods Second Edition Robarts

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Principles And Practice Of Modern Chromatographics Methods Second Edition Robarts

About This Presentation

Slide Content

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