Chromatographic techniques

1. PHARMACOGNSY-IIA (Advance) [Theory]
Course No. 513-T(Morn)
Course Incharge: Ms. Mehwish Khan
Chapter: Separation and isolation of plant
constituents
Topic: Separation mechanism in different chromatography

2. Previous lecture
Following points have been discussed in the previous lecture,
Chromatography is a separation technique in which a sample is
equilibrated between two immiscible phase (mobile and stationary
phase).
 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

3. OBJECTIVE
• In this lecture, we will study about
classification of chromatography i.e. ion
exchange, size exclusion and affinity
chromatography and also classification
according to mobile phase, polarity & shape
of chromatographic bed.

4. “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+

5. 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.

6. 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 flitration / size 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)

7. 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.

8. 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.

9. 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

10. ACCORDING TO MOBILE PHASE AND MECHANISM
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 in case of
separation by adsorption, the stationary phase is solid it is called: Gas–Solid
Chromatography (GSC).
• If separation occurs through partition, the stationary phase is liquid it is
called: Gas-Liquid Chromatography (GLC).
ACCORDING TO POLARITY OF PHASES
Liquid Chromatography (LC)
• The stationary phase is more polar and the mobile phase is less polar so it is called normal
phase chromatography
• The stationary phase is less polar and the mobile phase is more polar so it is called
reverese phase chromatography

11. 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.

12. Analytical Chromatography
• Chromatography can be used to obtain pure materials from mixtures
anddetermine the existence and also the concentration of analyte(s)
materials in a sample.
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.

13. Summary
 Ion exchange chromatography may be defined as the reversible exchange
of ions in the solution with ions electrostatically bound to stationary
phase. Bounded ions of sample can be eluted according to their binding
strength to resin by two ways.
• 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.
• Qualitative Chromatography Confirm the absence or presence of certain
constituent in the sample and Quantitative chromatography is used to
determine the concentration of analytes in a sample

14. Further reading and references
• Chromatographic Methods. A. Braithwaite and F.J. Smith.
Published by Kluwer Academic. Publisher
• The Essence of chromatography. Colin F. Poole. by Elsevier