Introduction, theory Instrumentation Applications TYPES OF GEL USED SEPARATION PROCEDURE

1. By
Mr. Vinayak R. Bodhankar
M. Pharm. (Ph.D*)

2. Content
 Introduction, theory
 Instrumentation
 Applications

3.  INTRODUCTION
 Gel chromatography is also called as Gel permeation chromatography, Size exclusion
chromatography, Molecular sieving chromatography.
 It is a chromatographic technique in which the separation of components based on the
difference of molecular weight or size.
 The stationary phase consists of beads containing pores that span a relatively narrow
size range. When the gel is packed into a column and percolated with a solvent, it
permits the large molecular weight components to pass faster without penetration of the
pores (totally excluded).
 Smaller molecules spend more time inside the beads and therefore is eluted later (after
a larger volume of mobile phase has passed through the column).
 It is one of the effective methods used to isolate and analyze the bio-macromolecular
substances.

4.  When an organic solvent is used as a mobile phase, then it is called Gel
permeation chromatography.
 When an aqueous solution is used as a mobile phase, the technique is known
as Gel filtration chromatography.
 In 1954, the scientist Mould and Sunge showed that the separation of uncharged
substances can be performed based on the size of molecules.
 However the systematic use of this principle was introduced by Porth and
Foblin in 1954 who separated the molecules of biological origin using
polysaccharide gel (Sephadex).
 Stationary phase: Polysaccharide gel (cross-linked to form pores)
 Mobile phase: Liquid solvent
 Sample: Molecules of biological origin (different sizes)

5.  PRINCIPLE
 The stationary phase in this method is an open network of polymers which are cross-
linked to each other to form the pores of consistent size. The degree of cross- linking of
polymer mainly governs the pore size.
 When the mobile phase containing mixture of solutes of various sizes is passed through
the column, the molecules which are too large to enter the pores are "excluded"
completely and get eluted with the mobile phase.
 The molecules which are smaller in size spend more time inside the pores, thus the path
travelled by them through the column is quite longer and they are eluted later.
 Large size > Intermediate size > Small size

6.  TYPES OF GEL USED
 The gels used as molecular sieves are cross linked polymers.
 They are uncharged and inert i.e., don't bind or react with the materials being
analyzed.
 Three types of gels are used:
1. Dextran (Sephadex)
2. Agarose gel
3. Acrylamide gels (synthetic gel)
Nature and properties of Gel:
 Chemically inert
 Mechanically stable
 Ideal porous structure
 Wide pore size give low resolution
 Uniform particle size

7. 1. DEXTRAN (SEPHADEX)
 Dextran is a homopolysaccharide of glucose residues.
 It is prepared with various degrees of cross-linking to control pore size.
 It is bought as dry beads, the beads swell when water is added.
 1-6 polymer of glucose is prepared by microbial fermentation of
sucrose (glucose + fructose).
 The resulting glucose provides the required a 1-6 glucosan polymer
called dextran. The resulting dextran is treated with epichlorohydrin to
give several types of cross- linked dextran.
 It is mainly used for separation of small peptides and globular proteins
with small to average molecular mass.
 Sephadex is obtained in different degrees depending on the pore size.
 High percentage of epichlorohydrin give high degree of cross linking
(small pore size).
 Lower percentage produce sephadex with large pore size.

8. Characters of Sephadex:
 Highly stable gels.
 Stable at pH 2-12.
 Their particles are free from ions.
 Insoluble in water and organic solvent.
 Swell in water and other hydrophilic solvent.
 Requires bactericidal such as Hg acetate.
2. AGAROSE GEL
 Obtained from agar and composed of alternating units of 1,3 linked Beta-D-galactose and
1,4 linked 3,6-anhydro alpha, L-galactose.
 This was subjected to epichlorohydrin to give agarose.
Characters:
 It dissolves in H2O at 50°C and on cooling form gel.
 Insoluble below 40°C.
 Freezing destroys the gel.

9. 3. ACRYLAMIDE GELS (SYNTHETIC GEL)
 It is s polymerized acrylamide or methylene-bis-acrylamide.
 The pore size is determined by the degree of cross-linking.
 They are available in wide range of pore sizes.
According to the swelling process, the gels are two types:
1. Soft gels (Xerogel i.e., gel only on swelling):
Example: Polyacrylamide gels, dextran or agarose
(used for separation of proteins in aqueous mobile phase).
2. Semirigid or rigid gels:
Polystyrene gels (separation of non-polar polymers in non-polar solvents).
Porous glass gels (separation of polar systems).

10.  INSTRUMENTATION / COMPONENTS
 Stationary Phase
 Mobile Phase
 Columns
 Pump
 Detectors
1. Stationary Phase:
Composed of semi-permeable, porous polymer gel beads with well defined range of
pore sizes.
Properties of gel beads:
 Chemically inert.
 Mechanically stable.
 Has ideal and homogeneous porous structure
 Uniform particle and pore size.
 The pore size of the gel must be carefully controlled.

12. 2. MOBILE PHASE:
 Composed of a liquid used to dissolve the bio-molecules to make the mobile
phase permitting high detection response and wet the packing surface.

13. 3. COLUMNS:
 Commercially available columns include:
 Analytical column: 7.5-8 mm diameter.
 Preparative columns: 22-25 mm diameter.
 Usual column lengths: 25, 30, 50, and 60 cm.
4. PUMP:
 These are either syringe pumps or reciprocating pumps with a highly constant
flow rate.

14. 5. DETECTORS:
Concentration Sensitive Detectors:
1. Bulk Property Detectors: Refractive Index (RI) Detector.
2. Solute Property Detectors: Ultraviolet (UV) Absorption Detector.
3. Evaporative Detectors: Evaporative Light Scattering Detector (ELSD).
Molar Mass Sensitive Detectors:
i. Light Scattering Detectors:
1. Low Angle Light Scattering (LALS) Detectors.
2. Multi Angle Light Scattering (MALS) detectors.
ii. Viscosity Detectors: Differential Viscometers.

15.  SEPARATION PROCEDURE
1. Preparation of column for gel filtration:
 Swelling of the gel: Some resin come in a powder form. These must be sonicated
first in the eluent or the desired buffer to swell.
 Packing the column: Make a slurry of gel + buffer and pour it into column
which is one third filled with the buffer.
 Washing the resin: After packing, pass several volumes of the buffer through the
column to remove any air bubbles and to test the column homogeneity.
2. Loading the sample onto the column:
The sample must enter the resin in the form of solution using a syringe.
3. Eluting the sample and detection of components:
Fractions are collected as the sample elutes from the column.

16.  APPLICATIONS
Gel chromatography is mainly used for the separation of sugars, polysaccharides,
proteins, lipids, polymers and other materials.
1. PURIFICATION:
 This technique is used for purification of biological molecules.
 Different proteins, enzymes, hormones, antibodies, polysaccharides have been
separated and purified by using appropriate gels.
 Low molecular weight dextrans can be separated from corn syrup oil.
2. DESALTING:
 Separation of monosaccharides from polysaccharides and separation of amino
acids from proteins.

17. 3. FRACTIONATION:
In this method of separation, the similar substance are eluted closer to each other.
Thus, the separation of substances which has nearly equal molecular size can be
separated.
4. PROTEIN-BINDING STUDIES
5. DETERMINATION OF MOLECULAR WEIGHT:
It is assumed that the size of molecule is proportional to the molecular weight.