Affinity chromatography- Content-Introduction Theory Instrumentation Applications Gel chromatography is a type of partition chromatography used for separating different sized molecules. Gel chromatography is also called Gel permeation chromatography or gel filtration or gel exclusion, size exclusion, molecular- sieve chromatography. The separation is based on the analyte molecular sizes since the gel behaves like a molecular sieve. In size exclusion chromatography, the stationary phase is a porous matrix made up of compounds like cross-linked polystyrene, cross-like dextrans, polyacrylamide gels, agarose gels, etc. The gel structure being used contains pores of different diameters upto maximum size. 1.The test molecules are washed through a gel column and molecules larger than the largest pores in the gel are excluded from the gel structure. 2. Smaller molecules penetrate the gel and the extent of penetration depends on the molecular size----- This delay their movement through the column This technique is used for the separation of proteins, polysaccharides, enzymes, and synthetic polymers. Instrumentation- A. Stationary phase- It is composed of semi-permeable, porous polymer gel beads with a well-defined range of pore sizes. eg. Dextran, Agarose, Acrylamide. 2. sample size and concentration- sample is applied in small volume (1-5% of the total bed volume).3. Column parameters- use long column, ratio of column diameter to column length (1:20 to :100). The method or steps used for gel preparation. 4. Choice of eluent/mobile phase- Buffers Ex- Phosphate buffer pH 7, NaCl solution, Ammonium acetate (CH3COO-NH4+ ), Ammonium bicarbonate (NH₄HCO₃) ethylenediamine acetate. 5. Effect of Flow rate- maintain with the help of pump. Elution carried out with buffer at optimal flow rate (Eg- 0.25-5ml/min) to give maximum resolution with optimal separation time.6. Separation of components from the sample- Separation of component from mixture is achieved with the help of column. The retention volume (VR).7. Detection- Using UV absorption detectors. A graph of Elution Volume (ml) Vs Molecular weight. 7. Detection- Using UV absorption detectors. A graph of Elution Volume (ml) Vs Molecular weight. For calibration of the gel in column – Calibrators - (Proteins of known molecular weight. Procedure for gel filtration technique-1. Preparation of column- 2. Washing of the column- 3. Loading of the sample-4. Elution using mobile phase (buffers)5. Detection of compounds . Applications

1. Mrs Vandana Sharma
Assistant Professor

2.  Introduction
 Theory
 Instrumentation
 Applications

4.  As a technique, size exclusion chromatography was first
developed in 1955 by Lathe and Ruthven.
 Gel chromatography is a type of partition chromatography used
for separating different sized molecules.
 Gel chromatography is also called Gel permeation chromatography
or gel filtration or gel exclusion, size exclusion, molecular- sieve
chromatography.
 The separation is based on the analyte molecular sizes since the
gel behaves like a molecular sieve.
 In size exclusion chromatography, the stationary phase is a porous
matrix made up of compounds like
cross-linked polystyrene, cross-like dextrans, polyacrylamide gels,
agarose gels, etc.
 The gel structure being used contains pores of different diameters
upto maximum size.
1.The test molecules are washed through a gel column and
molecules larger than the largest pores in the gel are excluded
from the gel structure.

5. 2. Smaller molecules penetrate the gel and the extent of
penetration depends on the molecular size----- This delay their
movement through the column
 This technique is used for the separation of proteins,
polysaccharides, enzymes, and synthetic polymers.

6.  In gel chromatography- molecules are separated based on their
size
1. A column is filled with swollen gel beads or porous glass beads-
this column is serves as a molecular sieve
2. A mixture with molecules with of different sizes is poured over
the column.
3. The larger molecules are collected first as they pass through the
spaces between the gel or glass beads. Because the pores of
beads are smaller and the large molecules cannot pass through
them.
4. The small molecules enter the beads through the pores and get
separated from the solvent
4a. Small molecules slowly travel down the beads and collected in
the separated stream means collected latter
4b. Large molecules cannot pass through the pores, and are
excluded from the beads means collected first. hence, this
technique is also called exclusion chromatography.

7. Swollen
gel
beads
Large
molecules
Small
molecules
Large
molecules
Small
molecules

8. In short

9. Only for explanation (not compulsory to write in exam paper
Add with
principle in
last if asked
about Theory

10. A. Stationary phase
 It is composed of semi-permeable, porous polymer gel beads
with a well-defined range of pore sizes.
 It has the following properties:
1. Chemically inert
2. Mechanically stable
3. With ideal and homogeneous porous structure (wide pore size
give low resolution).
4. A uniform particle and pore size.
Examples of gel:
 Dextran (Sephadex) gel: An α 1-6-polymer of glucose natural gel
 Agarose gel: A 1,3 linked β-D-galactose and 1,4 linked 3,6-
anhydro-α, L-galactose natural gel
 Acrylamide gel: A polymerized acrylamide, a synthetic gel

11. 1. Matrix choice-
 The gel filtration matrices compromise of porous beads
containing cross linked polyacrylamide, agarose, dextran or
combination of these.
 These matrices are supplied in
 suspended form or
 as dry powders.
 Desirable properties of matrix
1. They should be compatible with the molecules to be separated
and should be stable in organic solvents, pH, and temperature
2. They should be inert when molecules undergoing separation so
that the molecule don't get partially adsorbed to the matrix – if
this will happens the migration of molecules through the column
will be retarded.
Eg Sephadex-
 Original medium based on dextran (a linear polymer)- that has
been modified to offer varying degree of cross linking to
determine the material pore size

12.  sephadex is a strongly hydrophilic polymer and swell in aqueous
solutions, like dextran
Polyacrylamide beads-
 Like dextran- hydrophilic and swell in aqueous solution.
Chemically more stable than dextran gels.
Agarose gels-
 Hydrophilic and used when very large pore sizes is needed
Polystyrene gels-
 hydrophobic in nature, used with non-aqueous solvents for
organic chemical applications
2. sample size and concentration- sample is applied in small
volume (1-5% of the total bed volume)
 The sample viscosity -(increase with the sample concentration)
 A high viscosity- irregular sample migration through the column
and reduce the column flow rate= loss of resolution
3. Column parameters- use long column, ratio of column diameter
to column length (1:20 to :100)

13. Dry gel powder (A weighted amount)- Mixed with solvent (to be
used as eluent ) and made to swell
The resultant mixture is kept aside till equilibrium is attained
Then the column slurry warmed at 100
o
C in a water bath
The gel swells in a few days.
The slurry is cooled and packed in column
The method or steps used for gel preparation
Type of column packing
1. Porous glasses or silica and
2. Porous cross-linked organic gels eg- dextrans, methylate acrylic
based gels, polyvinyl alcohal based gels, Hydroxyethyl cellulose
gels
Detectors- based on
–UV fluorescence,
-UV absorption or
-Change in refractive index

14. 4. Choice of eluent/mobile phase- In gel chromatography, the
molecules are separated based on their relative sizes.
 Due to this reason, the technique is independent of the of type of
eluent used.
 The elution conditions (pH, essential ions, cofactors, protease
inhibitors etc) that will fulfill the requirements of desired molecule
should be selected
 Mobile phase: Buffers Ex- Phosphate buffer pH 7, NaCl solution,
Ammonium acetate (CH3COO
-
NH4
+
), Ammonium bicarbonate
(NH₄HCO₃) ethylenediamine acetate
5. Effect of Flow rate- maintain with the help of pump
 Maximum separation in gel chromatography- can be achieved by low
flow rate indicate longer separation time
 Higher flow rates can be used with rigid material such as Sephacryl
HR range
 Elution carried out with buffer at optimal flow rate (Eg- 0.25-
5ml/min) to give maximum resolution with optimal separation time

15. 6. Separation of components from the sample-
Separation of component from mixture is achieved with the help of
column
Separation mechanism- In gel chromatography, the polymer
molecules are separated based on their molecular size.
 Separation is achieved when the polymer molecules elute through
the column(s) packed with a porous material
 Smaller molecules are retained in the pores while the larger ones
are excluded.
 Thus, first the largest molecule (having the greatest
hydrodynamic volume) elutes from the column and then the
smaller molecules.
 The retention volume (VR) - The volume of liquid at which a
solute elutes from a column or the volume of liquid
corresponding to the retention of a solute on a column is the
retention volume (VR), which is related to the void volume (Vo)
and internal pore volume of the column

16.  7. Detection- Using UV absorption detectors
 A graph of Elution Volume (ml) Vs Molecular weight
 For calibration of the gel in column –
 Calibrators - (Proteins of known molecular weight)
 like- Yeast alcohal dehydrogenase (1,50,000 Daltons), Bovine serum
albumin (66,000 Daltons), carbonic anhydrase (29,000 Daltons) and
Cytochrome C (12,400 daltons) are used
 A graph of MW vs elution volume – plotted
 For unknown compounds – separation is carried out using the same
column of gel, elution volume is determined – and FROM THE GRAPH-
the molecular weight of each compound is determined
10,00,000
1,00,000
10,000
1,000
10 15 20
M.W. in
Daltons
Elution volume (ml)
Calibration curve

17. Easy Diagram

18. A generic size exclusion chromatography
Explained Diagram of
Gel Chromatography

20. Steps
1. Preparation of column- slurry of stationary phase with the help
of mobile phase – allow to become gel
 After sufficient time – in column (made of glass or inert material)
is filled to get packed bed of gel.
 Stationary phase- porous beads containg cross linked poly
acrylamide, agarose, dextran or combination of these
2. Washing of the column- The packed bed is equilibrated with
buffer, which fills the pores in the beads as well as the space
between the beads.
 Now the packed bed of column is ready for separation.
 NOTE- The buffer is not required to improve resolution- but
buffer is used at the end of separations to remove any molecules
which are present in the column to facilitate next separation.

21. 3. Loading of the sample- The compounds to be separated, in the
form of solution is charged into the column, followed by flow of
mobile phase with a specific flow rate.
4. Elution using mobile phase (buffers)- Elution is carried out with
buffer at optimum flow rate (eg- 0.25-5ml/min) to give
maximum resolution of peaks
and
small volume fractions are collected for detection of individual
component.
 Depending upon separation – a pressure upto 350 psi (Pounds
per square inch0 are used
 5. Detection of compounds – Detection of compound is carried
out by connecting outlet of column to detector.
 Usually UV detector monitored at 214nm or 280nm and a
chromatogram is obtained

23.  Separation of protein in mixtures-
 Molecular weight of each protein is different – gels for required
specification is used to separate – mixture of proteins into pure
component
 This technique is frequently used for –
1. The separation of small proteins, large proteins
2. Polynucleotides
3. DNA fragments
4. Small virus particles
5. Peptides
6. Globular proteins
7. Peptide hormones
8. Monoclonal antibiotics etc
 Determination of approximate molecular weight of unknown
 sample-
 The gel column is calibrated, followed by separation of
unknown compounds in the same column

24.  From the elution volume, and calibration graph- the
approximate MW of given unknown sample is determined
Purification of macromolecules, proteins, enzymes, amino acids,
polysaccharides
 When impurities are present, they can also be separated as the
molecular sizes of impurities are different when compared to
components of interest.
 Each fraction is collected separately and detected using suitable
technique
Desalting of proteins-
 When salts are used in the separation of proteins in to fractions-
to remove these salts for purification of proteins- gel filtration is
used
 Salts travel slower in the gel ( smaller size) and proteins
excluded from the column faster ( due to larger size)