Course Title

Pharmacognosy-IIA (Advanced) [Theory]

Topic : Chromatography Classification and mechanism of
separation of molecules

Course Incharge
Dr. Mehwish Khan
(PHARM-D,MPHIL, Ph. D)
 Learning Objectives

 Student will understand separation mechanism in different types of

chromatography
 In Adsorption chromatography mixture of gas or liquid gets separated
when it passes over the adsorbent bed
 Partition chromatography, separation of the components of the mixture
get distributed into two liquid phases
 Ion exchange chromatography, reversible exchange of ions in the solution
with ions electrostatically bound to stationary phase
• Size-exculsion chromatography uses porous particles to separate
molecules of different sizes
• Affinity chromatography is a method of separating biochemical mixtures
based on a highly specific interaction
• Chromatography can be classified according to mobile phase and the
shape of chromatographic beds on which separation occur
 Chromatography
Introduction
• In 1906, Mikhail Tswett, the Russian botanist discovered
chromatography.
• Chromatography is a separation technique in which a
sample is equilibrated between a mobile and a stationary
phase.
• The stationary phase may be a solid, or a liquid supported
on a solid or gel, the mobile phase may be either a gas or
a liquid.
The chromatographic method of separation, in general,
involves following steps
• Adsorption or retention of substances on the stationary
phase
• Separation of the adsorption of substances by the
mobile phase
• Recovery of the separated substances by a continuous
flow of the mobile phase; the method being called
elution
• Qualitative and Qantitative analysis of the eluted
substances
CHROMATOGRAPHY TERM
• The analyte is the substance to be separated during chromatography.
The term Analytical chromatography is used to determine the existence and possibly
the concentration of analyte(s) in a sample .

A bonded phase is a stationary phase that is covalently bonded to the support particles
or to the inside wall of the column tubing.

A chromatogram 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 mixture.

A chromatograph is a sophisticated equipment that enables separation, e.g. gas

chromatographic (GC) or liquid chromatographic separation (LC).

Chromatography is a physical method of separation that distributes components to

separate between two phases, one stationary (stationary phase), the other (the mobile
phase) moving in a definite direction. Differences in rates of movement through the
medium are calculated to different retention times of the sample.
• The eluate is the mobile phase leaving the column.

• The eluent is the solvent that carries the analyte.

• An immobilized phase is a stationary phase that is immobilized on the support particles,

or on the inner wall of the column tubing.

• The mobile phase is the phase that moves in a definite direction. It may be a liquid (LC
and Capillary Electrochromatography (CEC)), a gas (GC), or a supercritical fluid
(supercritical-fluid chromatography, SFC). The mobile phase consists of the sample
being separated/ analyzed and the solvent that moves the sample through the column. In
the case of HPLC the mobile phase consists of a non-polar solvent(s) such as hexane in
normal phase or polar solvents in reverse phase chromatography and the sample being
separated. The mobile phase moves through the chromatography column (the stationary
phase) where the sample interacts with the stationary phase and is separated.

• Preparative chromatography is a method by which sufficient quantity of a pure

substance is obtained for further use, rather than analysis.
The retention time is the characteristic time it takes for a particular analyte to pass through the system
(from the column inlet to the detector) under set conditions.

The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a
mixture of components. When the sample is treated in the course of an analysis, the phase or the phases
containing the analytes of interest is/are referred to as the sample whereas everything out of interest
separated from the sample before or in the course of the analysis is referred to as waste.

The solute refers to the sample components in partition chromatography.

The solvent refers to any substance capable of solubilizing another substance, and especially the liquid
mobile phase in liquid chromatography.

The stationary phase is the substance fixed in place for the chromatography procedure.
Examples include the silica layer in thin layer chromatography
TECHNIQUES BY CHROMATOGRAPHIC BED SHAPE:

• Column chromatography

• Flash chromatography

• Phosphocellulose chromatography

• Planar chromatography

• Paper chromatography

• TLC chromatography

• Displacement chromatography
 Classification of chromatography
Based on mechanism of separation
The mechanism of separation depends mainly on the
nature of the stationary phase. Based on separation
mechanisms chromatography can be classified into:
• adsorption chromatography
• Partition chromatography
• Ion Exchange Chromatography
• Molecular Exclusion
• Affinity Chromatography
 Adsorption Chromatography
• Adsorption chromatography is a process of separation of components in a
mixture introduced into chromatography system based on the relative
differences in adsorption of components to the stationary phase present in the
chromatography column.
• This adsorption chromatography applies to only solid-liquid or solid-gas
chromatography. Because the adsorption phenomenon is inherent property of
solids and hence it is used with only solid stationary phase chromatographies.
Adsorbent A substance which is generally porous in nature with a high surface
area to adsorb substances on its surface by intermolecular forces is called
adsorbent.
• Silica gel is the most common stationary phase in adsorption chromatography.
• Mobile phase which is either in liquid or gaseous form.
• The mixture of gas or liquid gets separated when it passes over the adsorbent
bed that adsorbs different compounds at different rates.
 Column chromatography
• It involves the separation of a mixture over a column of adsorbent
(stationary phase) packed in a glass tube.
• The mixture adsorbed on adsorbent is placed on the top of the adsorbent
column packed in a glass tube.
• An appropriate eluant which is a liquid is allowed to flow down the column
slowly
• The most readily adsorbed substance are retained near the top and the
others come down to various distance in the column

The substance loosely adsorbed to the

stationary medium comes in the
earlier fraction
 Thin Layer Chromatography
• This is separation of substances of a mixture over a thin layer of an
adsorbent coated on glass plate.
• A thin layer about 0.2mm thick of an adsorbent (silica gel or
alumina) is spread over a glass plate of suitable size.
• The plate is known as thin layer chromatography plate
• The solution of the mixture to be separated is applied as a small
spot about 2cm above one end of the TLC plate.
• Mobile phase travel with the sample in upward direction. Polar
molecule do not move faster because of polarity of silica gel. Less
polar molecule faster with mobile phase
• Calculate Rf values of separated molecules of mixture
 Gas-Solid chromatography
• The principle of separation in GSC is adsorption.
• Gas chromatography employs an inert gas as the mobile phase (nitrogen,
helium, hydrogen as "carrier gas“)
• The mixture of component to be separated is converted to vapours and
mixed with gaseous mobile phase.
• The components which are adsorbed strongly to stationary phase travel
slowly and eluted later.
• Gas-solid chromatography is relatively rare, but it is used to separate
atmospheric gases
 Partition Chromatography
• This form of chromatography is based on a thin film formed on the
surface of a solid support by a liquid stationary phase.
• Separation of components of a sample mixture occurs because of
differential partitioning between stationary and mobile phase.
• Partition chromatography is process of separation whereby the
components of the mixture get distributed into two liquid phases
due to differences in partition coefficients during the flow of mobile
phase in the chromatography column
 Liquid-Liquid Chromatography (LLC)

• The stationary phase immobilizes the liquid surface layer.

• Mobile phases passes over the coated surface with spotted sample and
depending upon relative solubility in the coated liquid and mobile
phase, separation of mixture occurs.
Paper Chromatography is example of partition Chromatography. Special
paper is used, trapped water in it which act as stationary phase.
• Mixture of components spotted on the base of paper and rises up with
the suitable solvent system which act as mobile phase
• The component of sample mixture appear separated because of
differences in their partition coefficient.
• The molecule dissolve greater in the coating of paper will elute last as
compare to those molecules do not dissolve in the coating of stationary
phase
 Gas-liquid Chromatography (GLC)
• A form of chromatography in which stationary phase is a liquid, Layer of liquid
on inert solid support and packed in a long column
• Mobile phase is a gas (Inert carrier gas usually He or N) and the principle of
separation in GC is differential partition of components.
• The mixture of components to be separated is converted to vapour and mixed
with gaseous mobile phase
• Due to interaction of vaporized samples elute at different times. The
component which is more soluble in stationary phase travel slower and eluted
later. The component which is less soluble in stationary phase travels faster
and eluted out first
“Ion exchange chromatography may be defined
as the reversible exchange of ions in the solution with
ions electrostatically bound to stationary phase.”
The stationary phase is an ion exchange resin to which a cationic or anionic
groups are covalently bonded.
Ions of opposite charges (counter ions) in the mobile phase will be attracted to
the resin and compete with the components of the mixture for the charged
group on the resin.
Consider column having E - Y+ cation exchanger in which E - is negative charged
exchanger and Y+ is the mobile counter ion. X+ be the cation in the sample
having charge greater than Y+. The X+ ion can exchange sites with the counter
ion Y+
 Ion – Exchange chromatography
Bounded interest of ion (X+ ) can now
be eluted by either of the two ways;
1. By adding a component M+ having
magnitude of charge more than
that of X+ so that M+ will replace
X+ and X+ will be eluting out.
2. By changing pH of the solvent
(mobile phase) to neutralize the
component X+ have no charge and is
then unbounded from the matrix and
can be eluted out.
 Molecular Exclusion or Gel filtration chromatography
• Size-exculsion chromatography (SEC), also called gel filtration or gel-
permeation chromatography (GPC), uses porous particles to separate
molecules of different sizes.
Stationary phase used for gel exclusion chromatography macromolecular
complexes include dextran (Sephadex), polyacrylamide and
dextranpolyacrylamide (Sephacryl).
• Each is available with a variety of different ranges of pore size in the
beads, permitting separation of macromolecules of different size
• A mixture of molecules dissolved in liquid (the mobile phase) is applied
to a chromatography column which contains a solid support in the form
of microscopic spheres, or “beads” (the stationary phase).
• Size of pores in beads determines the exclusion limit (what goes
through the beads and what goes around the beads)
Working of gel-permeation chromatography Larger molecules pass around
or are “excluded” from the beads .
• Large sample molecules cannot or can only partially penetrate the pores,
whereas smaller molecules can access most or all pores.
• Thus, large molecules elute first, smaller molecules elute later, while
molecules that can access all the pores elute last from the column.
• Particles of different sizes will elute (filter) through a stationary phase at
different rates.
 Affinity Chromatography
• Affinity chromatography is a method of separating biochemical
mixtures based on a highly specific interaction such as that between
antigen and antibody, enzyme and substrate, or receptor and ligand
• It is a method of separating a mixture of proteins or nucleic acids
(molecules) by specific interactions of those molecules with a
component known as a ligand, which is immobilized on a support.
• If a solution or mixture of proteins is passed over (through) the
column, one of the proteins binds to the ligand on the basis of
specificity and high affinity (they fit together like a lock and key).
• The other proteins in the solution wash through the column because
they were not able to bind to the ligand.
• The ligand/molecule complex dissociates by changing the pH.
 Affinity Chromatography
APPLICATIONS
• Production of Vaccines -
antibody purification from
blood serum
• Used in Genetic Engineering -
nucleic acid purification
• Basic Metabolic Research -
protein or enzyme purification
from cell free extracts
 ACCORDING TO MOBILE PHASE
Liquid Chromatography (LC)
• The mobile phase is liquid. In case of separation by adsorption the
stationary phase is solid so it is called: Liquid-Solid Chromatography
(LSC).
• If separation occurs through partition the stationary phase is liquid
so it is called: Liquid –Liquid Chromatography (LLC).
Gas Chromatography (GC)
• Where the mobile phase is inert gas nitrogen or helium. Again if the
stationary phase is solid it is called: Gas–Solid Chromatography
(GSC).
• When stationary phase is liquid it is called: Gas-Liquid
Chromatography (GLC).
 According to the technique
(methods of holding the stationary phase)

Planar or Plane Chromatography:

• In this type of chromatography the stationary phase is used in the form of
layer. Plane chromatography is further classified into:
a- Thin Layer Chromatography (TLC):
• A technique used to separate non-volatile mixtures. It is performed on a
sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of
adsorbent material, usually silica gel, aluminium oxide (alumina), or
cellulose.
b- Paper Chromatography (PC):
• A specific type of papers is used as stationary phase in the form of sheets.
Columnar or Column Chromatography (CC):
• The stationary phase is held in to a tube made of glass or metal.
 Analytical Chromatography

• Chromatography can be used to obtain pure materials from mixtures.

Qualitative Chromatography
• Confirm the absence or presence of certain constituent in the sample
• Thin-layer chromatography (TLC) is a widely used method for qualitative
analysis to determine the number of components in a mixture, to
determine the identity of substances in sample
Quantitative Chromatography
• Quantitative chromatography is used to determine the concentration of
analytes in a sample.
• The components is identified by its retention time and concentration
calculated from intensity of detector signal.
• HPLC/ GC can be used for these applications.
 Summary
 Separation mechanism in different types of chromatography depends mainly on the
nature of the stationary phase
 Adsorption chromatography, mixture of gas or liquid gets separated when it passes
over the adsorbent bed that adsorbs different compounds at different rates
 Partition chromatography is process of separation whereby the components of the
mixture get distributed into two liquid phases due to differences in partition
coefficients
 Ion exchange chromatography may be defined as the reversible exchange of ions in
the solution with ions electrostatically bound to stationary phase
• Size-exculsion chromatography (SEC), also called gel filtration or gel-permeation
chromatography (GPC), uses porous particles to separate molecules of different sizes
• Affinity chromatography is a method of separating biochemical mixtures based on a
highly specific interaction
• Chromatography cab be classified according to mobile phase and shape of
chromatographic bed on which separation of molecules occurred.
 Further reading and references
• Chromatographic Methods. A. Braithwaite and F.J. Smith.
Published by Kluwer Academic. Pulisher
• The Essence of chromatography. Colin F. Poole. Elsevier