1

TABLE OF CONTENT

CHAPTER 01
Introduction to chromatography……………………………………………...3
CHAPTER 02
What is the chromatographic process?.........................................5
The critical defining properties of a chromatographic process…………...5
CHAPTER 03
Chromatography in more than one dimension…………………………...7
CHAPTER 04
Ion exchange and size exclusion chromatography………………………...9
Theory of Column Efficiency in Chromatography……………………………….…9
Theoretical plates………………………………………………………………………………….10
REFERENCE…………………………………………………………………………...………………………………………………11
 2

CHAPTER 01
INTRODUCTION TO CHROMATOGRAPHY
When we wish to measure the presence and amounts of a large number of
different compounds in a mixture, even the availability of unique spectral signatures for each
analyte may require laborious repeated measurements with different spectral techniques to
characterize the mixture fully. Living systems have evolved large protein molecules (e.g.,
receptors, enzymes, immune system antibodies) with unique 3D structures which can bind
strongly only to very specific organic compounds. Analytical procedures such as immunoassay,
enzyme mediated assays, and competitive binding assays, employ the extreme selectivity of
such protein macromolecules to measure particular biomolecules in very complex mixtures.
Microarrays of thousands of these, each with unique selectivity, can rapidly screen complex
mixtures for a list of expected components. However, precise quantitation of each detected
component is difficult to achieve this way.
Analysis of complex mixtures often requires separation and isolation of
components, or classes of components. Examples in non-instrumental analysis include
extraction, precipitation, and distillation. These procedures partition components between two
phases based on differences in the components’ physical properties. In liquid –liquid extraction
components are distributed between two immiscible liquids based on their similarity in polarity
to the two liquids (i.e., “like dissolves like”). In precipitation, the separation between solid and
liquid phases depends on relative solubility in the liquid phase.
In distillation the partition between the mixture liquid phase and its vapor (prior
to recondensation of the separated vapor) is primarily governed by the relative vapor pressures
of the components at different temperatures (i.e., differences in boiling points). When the
relevant physical properties of the two components are very similar, their distribution between
 3

the phases at equilibrium will result in slight enrichment of each in one of the phases, rather
than complete separation. To attain nearly complete separation the partition process must be
repeated multiple times, and the partially separated fractions recombined and repartitioned
multiple times in a carefully organized fashion. This is achieved in the laborious batch processes
of countercurrent liquid –liquid extraction, fractional crystallization, and fractional distillation.
The latter appears to operate continuously, as the vapors from a single equilibration chamber
are drawn off and recondensed, but the equilibration in each of the chambers or “plates” of a
fractional distillation tower represents a discrete equilibration at a characteristic temperature
A procedure called chromatography automatically and simply applies the
principles of these “fractional” separation procedures. Chromatography can separate very
complex mixtures composed of many very similar components. The various types of
chromatographic instrumentation which have been developed and made commercially
available over the past half century now comprise a majority of the analytical instruments for
measuring a wide variety of analytes in mixtures. They are indispensable for detecting and
quantitating trace contaminants in complex matrices. A single chromatographic analysis can
isolate, identify, and quantitate dozens or even hundreds of components of mixtures. Without
their ability to efficiently characterize complex mixtures of organic biochemicals at trace levels
from microscale samples, research in molecular biology as we know it today would be
impossible. We will be discussing another powerful separation technology, electrophoresis,
together with chromatography, because much of the way electrophoresis operates, and the
appearance of its separations, is analogous to chromatography, even though the underlying
separation principle is different. Chromatographic type separation is at the heart of the
instrumentation used to separate fragmented DNA molecules and to sequence the order of the
“bases” which form the genomes of all living creatures on Earth. The heroic sequencing of
around 3 billion DNA bases in the human genome, completed in the year 2001, could never
have been envisioned, let alone completed, without chromatographic-type instrumentation.
This forms the basis of the new field of genomics. Even more extensive analytical separation,
identification, and quantitation problems arise in the next new field of molecular biological
research, coming in the so-called “post-genomic” era; namely, proteomics. This requires
separation, identification, and quantitation of the thousands of proteins that may be produced
(“expressed”) by the genome of each type of living cell. The contents and proportions of this
mix will depend on what the cell is doing, and whether it is in a healthy or diseased state. The
amounts of analytes may be at the nanomolar scale or less, and unlike DNA analyses, there is
no handy polymerase chain reaction (PCR) procedure to amplify the amounts of analytes. If
genomics provides a complete parts list for an organism, then proteomics will be necessary to
understand the assembly and operating manual. Billions of dollars of chromatography-based
instrumentation, and hundreds of thousands of jobs using it, will be required for research in
these fields.
 4

Environmental monitoring and regulation have the potential of becoming another

multimillion-dollar market which can benefit from chromatography’s ability to isolate, identify,
and measure trace pollutants in complex environmental mixtures

CHAPTER 02
WHAT IS THE CHROMATOGRAPHIC PROCESS?

The Russian botanist Mikhail Tswett invented the technique and coined the
name chromatography. Early in the 20th century he placed extracts containing a mixture of
plant pigments on the top of glass columns filled with particles of calcium carbonate. Continued
washing (elution) of the mixtures through the columns with additional solvent (called the
eluent) resulted in the separation of the pigments, which appeared as colored bands adsorbed
on the solid particles, moving along the column at different rates, thus appearing at different
distances down the column. He named this technique “chromatography”, from the Greek for
“color writing”. By collecting separated components as they exit the bottom of the column, one
could isolate pure material. If a detector in the exiting fluid stream (called the effluent) can
respond to some property of a separated component other than color, then neither a
transparent column nor colored analytes are necessary to separate and measure materials by
“chromatography”.
When there is a great difference in the retention of different
components on the material filling the column, a short column can
be used to separate and isolate a rapidly moving component from
highly retained material. This is the basis of a useful sample
preparation technique called solid phase extraction (SPE). Real
instrumental chromatography employs highly engineered
 5

materials for the stationary phase past which the mobile phase fluid carrying the mixture of
analytes passes, with continuous partitioning of the analytes between the two phases. The
column needs to be sufficiently long and the eluent flow sufficiently slow for the process to
approach equilibrium conditions. Then partitioning will be repeated a large number of times.
Even very slight differences among mixture components in the ratio of the amounts which
would exist in each phase at equilibrium will result in their separation into bands along the
column. These bands can be separately detected or collected as they elute off the column.

The critical defining properties of a chromatographic process are:

1. Immiscible stationary and mobile phases;
2. An arrangement whereby a mixture is deposited at one end of the stationary Phase;
3. Flow of the mobile phase toward the other end of the stationary phase;
4. Different rates or ratios of partitioning for each component of the mixture, and many cycles
of this process during elution;
5. A means of either visualizing bands of separated components on or adjacent to the
stationary phase, or of detecting the eluting bands as peaks in the mobile phase effluent.
(Note that in some applications the bands are also referred to as zones.)

The first major implementation of instrumental chromatography was based on

partition between a liquid, nonvolatile, stationary phase supported on inert solid particles
packed in metal columns, and an inert gaseous mobile phase flowing through the column from
a pressurized tank. The various partition ratios between the phases, which affected separation,
were primarily related to the components’ different volatilities (i.e., boiling points). The initial
application of this procedure, gas chromatography (GC), to petroleum hydrocarbon samples,
led to facile separation of many more components than could be achieved by the fractional
distillation process used in petroleum refining.
Instead of just petroleum “fractions”, individual specific hydrocarbons could be
resolved and isolated, if the GC column was long enough. The analogy to the theoretically well
studied multiple plate fractional distillation columns used in refining processes led to the initial
plate theory of the chromatographic process. This assumed that the separation of the bands as
they migrated down the column proceeded as a series of sequential, discrete equilibrium
partitioning, instead of the actual situation, which is a continuous, not-quite-in-equilibrium
process. Nevertheless, this plate theory proved fairly accurate for most cases. It contained
useful terms [e.g., N ¼ the number of plates in a column as a descriptor of its separation
efficiency, and the “height equivalent to a theoretical plate” (HETP)]. These remain central to
 6

the simplified theoretical characterization of the chromatographic process, despite the fact that
continuous, non-equilibrium kinetic (or rate) theory is more accurate, and there exist no actual
plates in a chromatographic column. GC was not suitable for separation and measurement of
more polar, and less volatile or less thermally stable large biomolecules. Optimized
chromatographic separations of these molecules at ambient temperatures were achieved later
using more polar, water miscible liquid mobile phases. The instruments and stationary phases
which enabled this improvement on the traditional vertical gravity column LC apparatus, form
the basis of the even more widely used HPLC

CHAPTER 03
CHROMATOGRAPHY IN MORE THAN ONE DIMENSION
In GC the gaseous mobile phase must be confined in a column, so that a pressure
gradient can cause it to flow past the stationary phase and eventually elute the separated
bands out the effluent end of the column. This is inherently a 1D separation, along the column,
from one end to the other. This dimensionality applies even should the column be coiled to fit
in a GC oven rather than vertically straight, like Tswett’s gravity flow liquid mobile phase
column. However, unlike gases, liquids as mobile phases do not always require confinement to
move in a desired direction or retain their volume. If they are in contact with porous beds of
small particles or fiber mats, surface forces (capillary attraction) can often induce them to flow.
Thus, it is possible to carry out the LC process on a stationary phase arrayed as a thin surface
layer, usually a planar, 2D surface. Examples include the matted cellulose fibers of a sheet of
paper, or a thin layer of silica gel or alumina particles on a planar support (e.g., a pane of glass).
An example of paper chromatography, beloved of grade-school science fairs for its simplicity, is
to place a drop of colored ink in the center of a sheet of paper. After drying, a series of drops of
organic solvent are slowly added to the spot.
Capillary attraction causes a radial eluent flow away from the spot in all directions.
The different colored dyes in the ink adhere more or less strongly to the multiple hydroxyl
groups on the cellulose molecule chains through polar or hydrogen-bonding interaction.
Therefore, the capillary eluent flow carries some further than others, resulting in a pattern of
colored circles of different radii. This recreates Tswett’s original 1D column chromatography
 7

experiment on a 2D surface using his original definition. Being on a piece of paper, it seems
even more like “color writing”. The radial separation in the earlier example is not the most
efficient way to perform surface chromatographic separations. A square planar thin layer
chromatography (TLC) plate (not to be confused with the “theoretical plates” discussed
previously!) may have a line of spots containing sample mixtures and reference standard
materials deposited just above one edge. Submerge that edge in solvent to a level just below
the line of spots, and capillary attraction will produce a unidirectional, ascending, eluent flow
which will move and separate each spot’s components vertically up the plate in separate lanes.
If they are not already colored, they may be sprayed with a reagent which will react to
“develop” a color, just as a photographic plate may be developed chemically to reveal a latent
image.
Alternatively, the separated component spots may be visualized, or even
quantified, by observing their fluorescence emission induced by exposing them to UV light, or
by their quenching of the natural fluorescence of a silica gel TLC layer. Note that an advantage
to this form of surface chromatography is that multiple 1D (i.e., in one direction)
chromatographic separations of samples or standard mixtures are carried out simultaneously in
parallel (literally). If a mixture has enough components, and some are sufficiently similar in
structure and physical properties, it is likely that chromatography in one dimension, either on a
column or a surface, will not separate all of them from each other. Many column bands or
surface spots will contain multiple unseparated components. These are said to coelute in the
chromatographic system. If we repeat the previous TLC plate separation, but this time place
just one spot containing a mixture to be separated near one corner of the plate, we will again
achieve a line of eluted spots in a lane along a vertical edge. Some of these spots may contain
still unseparated, coeluting components.
Now let us rotate the plate ninety degrees and place this line of separated
spots back in the tank just above a new eluting solvent with polarity or pH or some property
very different from the first solvent. This will elute each of the partially separated spots from
the first chromatographic separation vertically up the plate perpendicular (or orthogonal) to the
initial separation dimension. If the nature of the solvent used for the second, orthogonal elution
produces different stationary/mobile phase partition ratios for components that coeluted in
spots from the first separation, they will now separate in the dimension perpendicular to that
of the first chromatographic separation. This is an example of true 2D chromatography. It is
most easily achieved in the planar surface mode described here. A 2D column chromatography
instrument, necessary for 2D GC separations, requires more complex hardware. The effluent
from one column may be sampled one time, or in continuous segments, by a device which
collects, reconcentrates, and redirects it to a second column. The second column must have
separation selectivity different from the first one.
If the coeluting components in just a single peak isolated from the first
column are separated on the second one, the procedure is heartcutting, 2D chromatography. If
 8

segments of the effluent from the first column are continuously and very rapidly separated by
the second column at a pace matching that at which they are sampled, the resulting detector
signal may be transformed by computer to yield comprehensive 2D chromatograms, displayed
as a planar array of spots like those observed directly on the TLC plate. These examples of
multidimensional chromatography should not be confused with the use of a spectroscopic
detector (e.g., a mass spectrometer at the end of a GC column, or a UV/VIS diode array
spectrometer monitoring the effluent of a liquid chromatograph). Such hyphenated systems, so
called from their abbreviations such as GC-MS or LC-diode array, are said to add a second,
spectral, dimension to the initial chromatographic separation. If components coeluting in a
chromatographic peak have sufficiently different spectral characteristics, they may be
measured separately in the peak despite their coelution.

CHAPTER 04
ION EXCHANGE AND SIZE EXCLUSION CHROMATOGRAPHY
Ion exchange chromatography uses supports with ion exchange functionalities as
the stationary phase. The mechanism of separation is based on ion exchange equilibria.
Hydrophobic interactions play a strong role in most ion exchange separations nevertheless,
particularly in anion exchange chromatography. In size exclusion chromatography, solvated
molecules are separated according to their size by their ability to penetrate into porous pockets
and passages in the stationary phase. Some types of chromatography are considered together
as a separate technique, such as gas chromatography for gas–solid and gas–liquid
chromatography. In every case, successive equilibria determine to what extent the analyte
stays behind in the stationary phase or moves along with the eluent (mobile phase). In column
chromatography, the column may be packed with small particles that act as the stationary
phase (adsorption chromatography) or are coated with a thin layer of liquid phase (partition
chromatography). In gas chromatography, the most common form today is a capillary column in
which a virtual liquid phase, often a polymer, is coated or bonded on the wall of the capillary
tube. We will see that this results in greatly increased separation efficiency. In addition to these
terms, we will use a number of others throughout the properties of gas and liquid
chromatography. These are summarized here for easy reference.
 9

Theory of Column Efficiency in Chromatography

Band broadening in chromatography is the result of several factors, which
influence the efficiency of separations. Mathematically, column chromatography is the easiest
to treat; we can quantitatively describe the efficiency of a column and evaluate the factors that
contribute to it

THEORETICAL PLATES
The separation efficiency of a column can be expressed in terms of the number of
theoretical plates in the column. A theoretical plate is a concept derived from distillation
theory, whereby each theoretical plate in chromatography can be thought of as representing a
single equilibrium step, such as in our Excel simulations. They are a measure of the efficiency or
resolving power of a column; the more the number of plates, the more efficient is the column.
The plate height, H, is the length of a column, L, divided by the number of theoretical plates,

To avoid a long column, H should be as small as possible. These concepts apply to all forms of
column chromatography. Experimentally, the plate height is a function of the variance, σ2, of
the chromatographic band and the distance, x, it has traveled through the column, and is given
by σ2/x; σ is the standard deviation of the Gaussian chromatographic peak. The width at half-
height, w1/2, corresponds to 2.355 σ, and the base width, wb, corresponds to 4 σ. The number
of plates, N, for a solute eluting from a column is given by:
 10

The narrower the peak, the greater the number of plates. Analyte, tR is the retention time, and
w1/2 is the peak width at half-height in the same units as tR. Retention volume VR may be used
in place of tR. It should be noted that wb is not the base width of the peak but the width
obtained from the intersection of the baseline with tangents drawn through the inflection
points at each side of the peak. N can also be expressed in terms of w b

REFERENCES
A. Berthod, Countercurrent Chromatography, The Support-Free Liquid Stationary Phase. Comprehensive Analytical Chemistry (D. Barcelo, Ed.),
Volume ´ XXXVIII, Elsevier, 2002,

J. C. Giddings, Unified Separation Science. New York: Wiley-Interscience, 1991.

C. E. Meloan, Chemical Separations: Principles, Techniques, and Experiments. New York, Wiley, 1999.

J. Cazes, ed., Encyclopedia of Chromatography. New York: Marcel Dekker, 2001.

R. E. Majors and P. W. Carr, “Glossary of Liquid-Phase Separation Terms,” LC.GC, 19(2) (2001) 124.

L. S. Ettre, “Nomenclature for Chromatography,” Pure Appl. Chem., 65 (1993) 819

H. J. Isaaq, ed., A Century of Separation Science, New York: Marcel Dekker, 2002.

L. S. Ettre, “The Rebirth of Chromatography 75 Years Ago,” LC.GC North America, 25(7), July 2007, p. 640. An excellent tracing of the birth and
evolution of chromatography. www.chromatographyonline.com.

W. Jennings, The History of Chromatography. “From Academician to Entrepreneur—A Convoluted Trek,” LC.GC North America, 26(7), July
2008, 626. An interesting account of the life of capillary chromatography pioneer Walt Jennings and the establishment of his column
preparation company. www.chromatographyonline.com.

R. G. Wolcott, J.W. Dolan, and L. R. Snyder, “Computer Simulation for the Convenient Optimization of Isocratic Reversed-Phase Liquid
Chromatography Separations by Varying Temperature and Mobile Phase Strength,” J. Chromatogr. A, 869 (2000) 3.
 11

J. W. Dolan, L. R. Snyder, R. G. Wolcott, P. Haber, T. Baczek, R. Kaliszan, and L. C. Sander, “Reversed-phase liquid chromatographic separation of
complex samples by optimizing temperature and gradient time: III. Improving the accuracy of computer simulation,” J. Chromatogr. A, 857
(1999) 41