CHROMATOGRAPHY

DEFINITION OF TERMS –
Analyte – the substance to be separated during chromatography
Analytical chromatography – this is used to determine the existence and possible also the
concentration of analyte in a sample
A bonded phase – this is a stationary or immobilised phase that is covalently bonded to the
support particles or to the inside walls of the column tubing
A chromatogram – this is the visual output of the chromatograph. In the case of an optimal
separation, different peaks or patterns on the chromatogram, correspond to different
components of the separated mixtures
A chromatograph this is the equipment that enables a sophisticated separation eg gas
chromatographic separation or liquid chromatographic separation
Chromatography – this is a physical method of separation in which the components to be
separated are distributed between two phases, one of which is stationary – stationary or
bonded phase- while the other – mobile phase moves in a definite direction.
The eluate – this is the mobile phase leaving the column
The eluent – this is the solvent that carries the analyte
Eluotropic series – this is a list of solvents ranked according to their eluting power
A preparative chromatography – this is used to purify sufficient quantities of a substance for
further use rather than analysis
The retention time – this is the characteristics time it takes for a particular analyte to pass
through the system (from the column inlet to the detector) under set conditions
The sample – the sample is the matter analysed in chromatography. It may contain a single
component or a mixture of components. When the sample is treated in the course of
analysis the phase or phases containing the analytes of interest is or are referred to as
sample where as everything out of interest separated from the sample before or in the
course of the analysis is referred to as waste
The solute – this is the sample component in partition chromatography
The mobile phase – this is the phase which moves in a definite direction. It may be a liquid
or a gas (gas chromatography) or a supercritical fluid (supercritical fluid chromatography).
The mobile phase consisting of the sample being separated or analyte and the solvent that
moves the sample through the column. In the case of HPLC the mobile phase consists of a
nonpolar solvent such as hexane in normal phase or polar solvent in reverse phase
chromatography and the sample being separated. The mobile phase moves through the
chromatographic column where the sample interacts with the stationary phase and is
separated.
Chromatography is the collective term for a set of laboratory techniques for the separation
of mixtures. It involves passing a mixture dissolved in a mobile phase through a stationary
phase which separates the analyte to be measured from other molecules in the mixture
based on different partitioning between the mobile and stationary phases. Chromatography
may be preparative or analytical. The purpose of preparative chromatography is to separate
the components of a mixture for further use and is thus a form of purification. Analytical
chromatography is done with smaller amount of material and is for measuring the relative
proportion of analyte in a mixture. The general chromatographic technique requires that a
solute undergoes distribution between two phases, one of them fixed, (stationary phase),
the other moving (mobile phase). It is the mobile phase that transfers the solutes through
the medium until eventually emerges separated from the other solutes that are eluted
earlier or later. Generally the solute is transferred through the separating medium by means
of a flowing stream of a liquid or gaseous solvent known as eluent. The stationary phase
may act through adsorption as in the case of adsorbents such as activated charcoal,
activated alumina, silica gel or ion exchange resins, or it may act by dissolving the solute,
thus partitioning the later between the stationary phase and mobile phases. In the later
process, a liquid coating held on an inert support serves as the stationary phase. Partitioning
is the predominant mechanism of separation in gas liquid chromatography, paper
chromatography, and forms of liquid chromatography. In practice, separation frequently
result from a combination of adsorption and partitioning effects.
USE OF REFERNCE SUBSTANCES IN IDENTITY TEST – In paper and TLC the ratio of the
distances (this distance being measured to the point of maximum intensity of the spot)
travelled on the medium by a given compound, to the distance travelled by the front of the
mobile phase from the point of application of the test substance is called the R f value of the
compound. The ration between the distances travelled by a given compound and a
reference substance is the RR value. If the substance to be identified, all the chromatograms
agree in colour and RF value and the mixed chromatogram yields a single spot ie R R = 1.0.
Then the test compound is same as the reference compound.
LOCATION OF COMPONENTS – The spots produced by paper or thin layer chromatography
may be located by a) direct inspection if the compounds are visible under white or visible
short wavelength or long wave length UV light; b) inspection in white or UV light after
treatment with reagents that will make the spots visible. Reagents are most conveniently
applied with an atomiser; c) use of a Geiger – Muller counter or autoradiographic
techniques in case of the presence of radioactive substance; d) evidence resulting from
stimulation or inhibition of bacterial growth by placing of removed portion of the adsorbent
and the substance on inoculate media. In open column chromatography, in pressurised
liquid chromatography and in gas chromatography, the retention time, t, defined as the
time elapsed between the sample injection and appearance of the peak concentration of
the eluted sample zone may be used as a parameter of identification. The ratio of the
retention of the test substance, the reference compound and a mixture of these, to the
retention time of an internal standard is called the relative retention time R R and is also used
frequently as a parameter for identification.
TYPES OF CHROMATOGRAPHIC TECHNIQUES
1. Technique by chromatographic bed eg paper chromatography, thin layer
chromatography, column chromatography
2. Displacement chromatography – gas chromatography, liquid chromatography
3. Affinity chromatography – supercritical fluid chromatography
4. Technique by separation mechanism – ion exchange chromatography, size exclusion
chromatography
5. Special techniques – reversed phase chromatography, two dimensional
chromatography, simulated moving bed chromatography, pyrolysis gas
chromatography, fast protein liquid chromatography
GAS CHROMATOGRAPHY
The distinguishing features of gas chromatography are a gaseous mobile phase and a solid
or immobilised liquid stationary phase. Gas chromatography also known as gas – liquid
chromatography – GLC – is a separation technique in which the mobile phase is a gas. Gas
chromatography is always carried out in a column which is typically packed or in capillary.
Gas chromatography is based on a partition equilibrium of analyte between a solid
stationary phase(often a liquid silicone based material) and a mobile phase(most often
Helium). The stationary phase is adhered to the inside of a small diameter glass tube (a
capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely
used in analytical chemistry, though the high temperature used in GC make it unsuitable for
high molecular weight biopolymers or proteins frequently encountered in biochemistry. It is
well suited for use in petrochemical industries, environmental monitoring and industrial
chemical fields. It is also used extensively in chemistry research. Liquid stationary phases are
available in packed or capillary columns. In the packed column, the liquid phase is deposited
on a divided inert solid support such as diatomaceous earth, porous polymers or graphitised
carbon which is packed into a column that is typically 2-4mm in internal diameter and 1 –
3M in length. In capillary column which contain no packing , the liquid phase is on the inner
surface of the column and may be chemically bonded to it. In gas chromatography, the solid
phase is an active adsorbent such as alumina, silica gel or carbon packed into a column.
Polyaromatic porous resin, which are sometimes used in packed columns, are not coated
with a liquid phase. When a vaporized compound is introduced into the carrier gas and is
carried into the column, it is partitioned between the gas and the stationary phase by a
dynamic counter current distribution process. The compound is carried down the column by
the carrier gas, retarded to a greater or lesser extent by sorption and desorption on the
stationary phase.
LIQUID CHROMATOGRAPHY
Liquid chromatography is a separation technique in which the mobile phase is a liquid.
Liquid chromatography can be carried out either in a column or a plane. Present day liquid
chromatography that generally utilises very small packing particles and a relatively high
pressure is called high performance liquid chromatography or high pressured liquid
chromatography – HPLC. In this technique, the sample is forced through a column that is
packed with irregular or spherically shaped particles or a porous monolithic layer (stationary
phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two
different sub classes based on the polarity of the mobile and stationary phases. Technique in
which the stationary phase is more polar than the mobile phase eg toluene as the mobile
phase and silica as the stationary phase, is called normal phase liquid chromatography. And
the opposite eg water methanol mixture as the mobile phase and octadecylsilyl as the
stationary phase is called reverse phase liquid chromatography. The normal phase has fewer
application and reverse phase is used considerably more. Separation by HPLC are achieved
by partition, adsorption or ion exchange processes depending upon the type of stationary
phase used. HPLC has distinct advantages over gas chromatography for the analysis of
organic compounds. Compounds to be analysed are dissolved in a suitable solvent and most
separation take place at room temperature thus most drugs being volatile or thermally
unstable compounds can be chromatographed without decomposition. Most
pharmaceutical analysis are based on partition chromatography and are completed within
30mins.
Size-exclusion chromatography
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a
chromatographic method in which molecules in solution are separated by their size, and in
some cases molecular weight. It is usually applied to large molecules or macromolecular
complexes such as proteins and industrial polymers. Typically, when an aqueous solution is
used to transport the sample through the column, the technique is known as gel-filtration
chromatography, versus the name gel permeation chromatography, which is used when an
organic solvent is used as a mobile phase. The chromatography column is packed with fine,
porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or
polyacrylamide (Sephacryl or BioGel P). The pore sizes of these beads are used to estimate
the dimensions of macromolecules. SEC is a widely used polymer characterization method
because of its ability to provide good molar mass distribution (Mw) results for polymers. …

Application - The main application of gel-filtration chromatography is the fractionation of

proteins and other water-soluble polymers, while gel permeation chromatography is used to
analyze the molecular weight distribution of organic-soluble polymers. Either technique
should not be confused with gel electrophoresis, where an electric field is used to "pull" or
"push" molecules through the gel depending on their electrical charges. The amount of time
a solute remains within a pore is dependent on the size of the pore. Larger solutes will have
access to a smaller volume and vice versa. Therefore, a smaller solute will remain within the
pore for a longer period of time compared to a larger solute.
The advantages of this method include good separation of large molecules from the small
molecules with a minimal volume of eluate, and that various solutions can be applied
without interfering with the filtration process, all while preserving the biological activity of
the particles to separate. The technique is generally combined with others that further
separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for
certain compounds. With size exclusion chromatography, there are short and well-defined
separation times and narrow bands, which lead to good sensitivity. There is also no sample
loss because solutes do not interact with the stationary phase.
The other advantage to this experimental method is that in certain cases, it is feasible to
determine the approximate molecular weight of a compound. The shape and size of the
compound (eluent) determine how the compound interacts with the gel (stationary phase).
…

Method - SEC is used primarily for the analysis of large molecules such as proteins or
polymers. SEC works by trapping smaller molecules in the pores of the adsorbent
("stationary phase"). This process is usually performed within a column, which typically
consists of a hollow tube tightly packed with micron-scale polymer beads containing pores
of different sizes. These pores may be depressions on the surface or channels through the
bead. As the solution travels down the column some particles enter into the pores. Larger
particles cannot enter into as many pores. The larger the particles, the faster the elution.
The larger molecules simply pass by the pores because those molecules are too large to
enter the pores. Larger molecules therefore flow through the column more quickly than
smaller molecules, that is, the smaller the molecule, the longer the retention time.
One requirement for SEC is that the analyte does not interact with the surface of the
stationary phases, with differences in elution time between analytes ideally being based
solely on the solute volume the analytes can enter, rather than chemical or electrostatic
interactions with the stationary phases. Thus, a small molecule that can penetrate every
region of the stationary phase pore system can enter a total volume equal to the sum of the
entire pore volume and the interparticle volume. This small molecule elutes late (after the
molecule has penetrated all of the pore- and interparticle volume—approximately 80% of
the column volume). At the other extreme, a very large molecule that cannot penetrate any
the smaller pores can enter only the interparticle volume (~35% of the column volume) and
elutes earlier when this volume of mobile phase has passed through the column. The
underlying principle of SEC is that particles of different sizes elute (filter) through a stationary phase
at different rates. This results in the separation of a solution of particles based on size.
Provided that all the particles are loaded simultaneously or near-simultaneously, particles of
the same size should elute together.
Each size exclusion column has a range of molecular weights that can be separated. The
exclusion limit defines the molecular weight at the upper end of the column 'working' range
and is where molecules are too large to get trapped in the stationary phase. The lower end
of the range is defined by the permeation limit, which defines the molecular weight of a
molecule that is small enough to penetrate all pores of the stationary phase. All molecules
below this molecular mass are so small that they elute as a single band.

The filtered solution that is collected at the end is known as the eluate. The void volume
includes any particles too large to enter the medium, and the solvent volume is known as
the column volume.
Following are the materials which are commonly used for porous gel beads in size exclusion
chromatography
Material and Fractionation range
Sr. Trade name (Molecular mass in Da)
No

1 Sephadex G-10 0 to 700

2 Sephadex G-25 1000 to 5000

3 Sephadex G-50 1500 to 30000

4 Sephadex G-75 3000 to 70000

5 Sephadex G-100 4000 to 150000

6 Sephadex G-150 5000 to 300000

7 Sephadex G-200 5000 to 800000

8 Bio-gel P-2 100 to 1800

9 Bio-gel P-6 1000 to 6000

10 Bio-gel P-60 3000 to 60000

11 Bio-gel P-150 15000 to 150000

12 Bio-gel P-300 16000 to 400000

13 Sepharose 2B 2 x 106 to 25 x 106

14 Sepharose 4B 3 x 105 to 3 x 106

15 Sepharose 6B 104 to 20 x 106

 …

ION EXCHANGE CHROMATOGRAPHY

ion-exchange chromatography separates ions and polar molecules based on their affinity
to the ion exchanger. It works on almost any kind of charged molecule— including large
proteins, small nucleotides, and amino acids. However, ion chromatography must be done
in conditions that are one unit away from the isoelectric point of a protein. The two types of
ion chromatography are anion-exchange and cation-exchange. Cation exchange
chromatography is used when the molecule of interest is positively charged. In this type of
chromatography, the stationary phase is negatively charged and positively charged
molecules are loaded to be attracted to it. Anion-exchange chromatography is when the
stationary phase is positively charged and negatively charged molecules are loaded to be
attracted to it. It is often used in protein purification, water analysis, and quality control. The
water-soluble and charged molecules such as proteins, amino acids, and peptides bind to
moieties which are oppositely charged by forming ionic bonds to the insoluble stationary
phase. The equilibrated stationary phase consists of an ionizable functional group where the
targeted molecules of a mixture to be separated and quantified can bind while passing
through the column—a cationic stationary phase is used to separate anions and an anionic
stationary phase is used to separate cations. Cation exchange chromatography is used when
the desired molecules to separate are cations and anion exchange chromatography is used
to separate anions. The bound molecules then can be eluted and collected using an eluant
which contains anions and cations by running higher concentration of ions through the
column or changing pH of the column.
One of the primary advantages for the use of ion exchange chromatography is only one
interaction involved during the separation as opposed to other separation techniques;
therefore, ion chromatography may have higher matrix tolerance. Another advantage of ion
exchange is the predictability of elution patterns (based on the presence of the ionizable
group). For example, when cation exchange chromatography is used, cations will elute out
last. Meanwhile, the negative charged molecules will elute out first. However, there are also
disadvantages involved when performing ion-exchange chromatography, such as constant
evolution with the technique which leads to the inconsistency from column to column. A
major limitation to this purification technique is that it is limited to ionizable group.
PRINCIPLE
Ion-exchange chromatography separates molecules based on their respective charged
groups. Ion-exchange chromatography retains analyte molecules on the column based ionic
interactions. The ion exchange chromatography matrix consists of positively and negatively
charged ions. Essentially, molecules undergo electrostatic interactions with opposite
charges on the stationary phase matrix. The stationary phase consists of an immobile matrix
that contains charged ionizable functional groups or ligands. The stationary phase surface
displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. To
achieve electroneutrality, these inert charges couple with exchangeable counterions in the
solution. Ionizable molecules that are to be purified compete with these exchangeable
counterions for binding to the immobilized charges on the stationary phase. These ionizable
molecules are retained or eluted based on their charge. Initially, molecules that do not bind
or bind weakly to the stationary phase are first to wash away. Altered conditions are needed
for the elution of the molecules that bind to the stationary phase. The concentration of the
exchangeable counterions, which competes with the molecules for binding, can be
increased or the pH can be changed. A change in pH affects the charge on the particular
molecules and, therefore, alters binding. The molecules then start eluting out based on the
changes in their charges from the adjustments. Further such adjustments can be used to
release the protein of interest. Additionally, concentration of counterions can be gradually
varied to separate ionized molecules. This type of elution is called gradient elution. On the
other hand, step elution can be used in which the concentration of counterions are varied in
one step. This type of chromatography is further subdivided into cation exchange
chromatography and anion exchange chromatography. Positively charged molecules bind to
cation exchange resins while negatively charged molecules bind to anion exchange resins. ]
The ionic compound consisting of the cationic species M+ and the anionic species B- can be
retained by the stationary phase.
Cation exchange chromatography retains positively charged cations because the stationary
phase displays a negatively charged functional group: Anion exchange chromatography
retains anions using positively charged functional group: Note that the ion strength of either
C+ or A- in the mobile phase can be adjusted to shift the equilibrium position, thus retention
time.

Paper Chromatography

This is a technique that involves placing a small dot or line of sample solution onto a strip of
chromatographic paper. The paper is placed in a jar containing a shallow layer of solvent
and sealed. As the solvent rises through the paper, it meets the sample mixture which start
to travel up the paper with the solvent. This paper is made up of cellulose, a polar substance
and the compounds within the mixture travel further if they are non polar. More polar
substances bond with the cellulose more quickly and therefore do not travel thus far. The
paper or adsorbent is of suitable texture and thickness.

Thin layer chromatography – TLC

The adsorbent in a TLC is a relatively thin, uniform layer of dry, finely divided material
applied to a glass, plastic or metal sheet or plates, that is instead of using a stationary phase
of paper. It involves a stationary phase of a thin layer of silica gel, alumina or cellulose. It
has an advantage of faster runs, better separations and the choice between different
adsorbents. For even better resolutions, and to allow for quantification, high performance
TLC can be used. The coated plate can be considered an open chromatographic column. The
separation achieved may be based on adsorption, partition or combination of both effects.

Column Chromatography

Column chromatography is used for the separation and isolation of different constituents of
extracts. Column chromatographic grade adsorbents of choice are used for packing the
columns and various organic solvents are used as eluting solvents. A glass column of
suitable size is selected depending upon the quantity of the extract and number of
components present in it. The column of smaller size for laboratory scale fractionation and
very large rocket columns with built-in sintered glass filter in the neck, for large scale
industrial use are available for the purpose. If such a filter is not is not present it becomes
necessary first to plug the neck of the column with a wad of glass or cotton wool. Silica gel is
either made into slurry with the solvents or as such introduced from the top of the column.
The next step is to add the sample solution to the top of the column in such a way that a
narrow band is formed, for further elution. To this end the sample is dissolved in minimum
volumes of solvents. The solvent used should be one of those selected for later elution and
preferably the one with the lowest polarity. The extract is sometimes converted to dry
slurry by using little quantity of silica gel and introduced at the top of silica gel column and
cotton plug is placed on it. In order to fractionate the components of varying solubility, the
gradient elution technique is followed. The column is subsequently eluted with various
proportions of solvents in the order of increasing polarity. The fractions collected from the
bottom are distilled to recover the solvents, while the concentrated residue of the fraction
thus obtained is stored in a clean and dry glass vials and monitored by TLC studies. The
mixture of component obtained by such fractionation can be subjected to re-fractionation
so as to get the pure components. Column chromatography therefore becomes a
purification process. Most of the extracts obtained from the crude drugs are
multicomponent mixtures of several constituents. The potent component may be present in
major or minor amount or even in traces. Chromatographic fractionation if skilfully used
guarantees the purification of diverse types of natural products, biochemical vitamins,
hormones etc
Vaccume liquid chromatography – VLC – is same as column chromatography described
above. The only difference is that a vacume or pump is input at the bottom of the column to
add pressure to the column and greatly reduce time spent.