This document discusses gel chromatography, which separates molecules based on size using a porous gel stationary phase. It describes the history, principle, types (gel filtration, gel permeation), instrumentation, applications, and advantages/disadvantages of gel chromatography. The key applications are separation and characterization of biomolecules and synthetic polymers.

1. © Ramaiah University of Applied Sciences
1
Faculty of Pharmacy © Ramaiah University of Applied Sciences
1
Faculty of Pharmacy © Ramaiah University of Applied Sciences
1
Faculty of Pharmacy © Ramaiah University of Applied Sciences
1
Faculty of Pharmacy
GEL CHROMATOGRAPHY
By
Burhanuddin Madriwala
M.Pharm – SEM I
Department of Pharmaceutical Chemistry
M.S Ramaiah University of Applied Sciences

2. © Ramaiah University of Applied Sciences
2
Faculty of Pharmacy © Ramaiah University of Applied Sciences
2
Faculty of Pharmacy © Ramaiah University of Applied Sciences
2
Faculty of Pharmacy © Ramaiah University of Applied Sciences
2
Faculty of Pharmacy
GEL CHROMATOGRAPHY

3. © Ramaiah University of Applied Sciences
3
Faculty of Pharmacy © Ramaiah University of Applied Sciences
3
Faculty of Pharmacy © Ramaiah University of Applied Sciences
3
Faculty of Pharmacy © Ramaiah University of Applied Sciences
3
Faculty of Pharmacy
CONTENTS
• INTRODUCTION
• HISTORICAL DEVELOPMENT
• PRINCIPLE INVOLVED
• PHASES OF GEL CHROMATOGRAPHY
• TYPES OF GEL CHROMATOGRAPHY
• INSTRUMENTATION
• CHROMATOGRAM
• MERITS & DEMERITS
• APPLICATIONS
• SUMMARY
• REFERENCES

4. © Ramaiah University of Applied Sciences
4
Faculty of Pharmacy © Ramaiah University of Applied Sciences
4
Faculty of Pharmacy © Ramaiah University of Applied Sciences
4
Faculty of Pharmacy © Ramaiah University of Applied Sciences
4
Faculty of Pharmacy
INTRODUCTION
• Gel chromatography is a technique where
components of a mixture are separated based on
their different molecular sizes on a porous gel
material used as stationary phase.
• Also knowns as molecular sieve or size exclusion
chromatography.
• Mainly used for separation of macromolecules like
proteins or synthetic polymers.

5. © Ramaiah University of Applied Sciences
5
Faculty of Pharmacy © Ramaiah University of Applied Sciences
5
Faculty of Pharmacy © Ramaiah University of Applied Sciences
5
Faculty of Pharmacy © Ramaiah University of Applied Sciences
5
Faculty of Pharmacy
HISTORY
• Discovered after 50 years from Tswett’s discovery of chromatography in 1906.
• In 1955–1956, two British biochemists, Lathe and Ruthven – separation of
polysaccharides and proteins on swollen starch granules.
• The next milestone occurred in 1959, when Per Flodin with Jerker Porath -
demonstrated size separation of peptides and oligosaccharides through a series of
cross-linked dextran packings.
• In early 1960s, scientists focused on the application of hydrophobic packings for the
Size Exclusion Chromatography of synthetic polymers.
• Concept of separation of homologues series of hydrocarbons based on molecular
size on swollen rubber granules was first demonstrated by Brewer.
• In 1962, John Moore produced a series of cross-linked polystyrene resins for the
separation of synthetic polymers.
• In 1963 – first commercial GPC equipment developed by Jim Waters.

6. © Ramaiah University of Applied Sciences
6
Faculty of Pharmacy © Ramaiah University of Applied Sciences
6
Faculty of Pharmacy © Ramaiah University of Applied Sciences
6
Faculty of Pharmacy © Ramaiah University of Applied Sciences
6
Faculty of Pharmacy
PRINCIPLE
• Two principles are involved: 1) Size separation2) Degree of penetration.
• The internal pores separates small molecules from large size molecules.
• The small molecules having size range less than internal pore size of gel beads
get stuck in the them while large molecules i.e. size greater than internal
pores passes through void volumes.
• Large molecules are separated based on the movement through void space.
• Small molecules are separated based on degree of penetration inside the
pores.

7. © Ramaiah University of Applied Sciences
7
Faculty of Pharmacy © Ramaiah University of Applied Sciences
7
Faculty of Pharmacy © Ramaiah University of Applied Sciences
7
Faculty of Pharmacy © Ramaiah University of Applied Sciences
7
Faculty of Pharmacy
conti….

8. © Ramaiah University of Applied Sciences
8
Faculty of Pharmacy © Ramaiah University of Applied Sciences
8
Faculty of Pharmacy © Ramaiah University of Applied Sciences
8
Faculty of Pharmacy © Ramaiah University of Applied Sciences
8
Faculty of Pharmacy
PHASES OF GEL CHROMATOGRAPHY
STATIONARY PHASE
• on which separation takes place.
• Microporous gel material having bead like structure with internal pores.
• Rigid, semi rigid and soft gels used.
• Size of gel beads range from 3-20 µm with internal pore size of 4-200 nm.
• Examples include dextran(sephadex ) , agarose(sepharose) , polyacrylamide gel
,porous silica gels etc.
• Type of gel to be used depends on size range of molecules to be separated.

9. © Ramaiah University of Applied Sciences
9
Faculty of Pharmacy © Ramaiah University of Applied Sciences
9
Faculty of Pharmacy © Ramaiah University of Applied Sciences
9
Faculty of Pharmacy © Ramaiah University of Applied Sciences
9
Faculty of Pharmacy

10. © Ramaiah University of Applied Sciences
10
Faculty of Pharmacy © Ramaiah University of Applied Sciences
10
Faculty of Pharmacy © Ramaiah University of Applied Sciences
10
Faculty of Pharmacy © Ramaiah University of Applied Sciences
10
Faculty of Pharmacy
Conti…..
Properties of gel
• Chemically inert.
• Mechanically stable.
• Should withstand pressure of mobile phase.
• Should be stable at temperature of up to 100°C.
• Should have uniform porosity.
• Should have larger size of beads to allow proper separation of higher molecules.
Mobile phase
• Aqueous &non-polar solvents both are used.
• Examples of non-polar solvents include tetrahydrofuran, trichloromethane , hot
trichlorobenzene etc.

11. © Ramaiah University of Applied Sciences
11
Faculty of Pharmacy © Ramaiah University of Applied Sciences
11
Faculty of Pharmacy © Ramaiah University of Applied Sciences
11
Faculty of Pharmacy © Ramaiah University of Applied Sciences
11
Faculty of Pharmacy
• Examples of aqueous solvents include water or buffer solutions. Buffer solutions
used are: -
1. Tris buffer (tris(hydroxymethyl)aminomethane) – pH:7.5-9.0
2. Sodium phosphate buffer mostly used – pH: 5.8-7.4
3. MOPS buffer (3-(N-morpholino)propane sulfonic acid) – pH: 6.5-7.9
4. MES buffer (2-(N-morpholino)ethane sulfonic acid) - pH: 5.5-6.7
5. BES buffer (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) - pH: 6.4-7.8

12. © Ramaiah University of Applied Sciences
12
Faculty of Pharmacy © Ramaiah University of Applied Sciences
12
Faculty of Pharmacy © Ramaiah University of Applied Sciences
12
Faculty of Pharmacy © Ramaiah University of Applied Sciences
12
Faculty of Pharmacy
TYPES OF GEL CHROMATOGRAPHY
Gel filtration chromatography
• Stationary phase is hydrophilic.
• Aqueous mobile phase used.
• Polyvinyl alcohols used as gel.
• Copolymers of polyglycerometh-
acrylates or vinyl polyacetates are also
used as gels.
• Water or buffer solutions are used as
mobile phase.
• Soft gels are used.
• Biomolecules are separated.
Gel permeation chromatography
• Stationary phase is hydrophobic.
• Organic solvents used as mobile phase.
• Styrene-divinylbenzene copolymer gel
is mostly used.
• Tetrahydrofuran used as mobile phase.
• Semi-rigid & rigid gels are used.
• Synthetic polymers are separated.

13. © Ramaiah University of Applied Sciences
13
Faculty of Pharmacy © Ramaiah University of Applied Sciences
13
Faculty of Pharmacy © Ramaiah University of Applied Sciences
13
Faculty of Pharmacy © Ramaiah University of Applied Sciences
13
Faculty of Pharmacy
INSTRUMENTATION
• Solvent delivery system
• Pumps
• Autosampler
• Sample injector
• Column(30 cm with internal
diameter of 7.5 mm)
• Detector( refractive index ,
light scattering ,uv
absorption detectors etc.)
• Recorder

14. © Ramaiah University of Applied Sciences
14
Faculty of Pharmacy © Ramaiah University of Applied Sciences
14
Faculty of Pharmacy © Ramaiah University of Applied Sciences
14
Faculty of Pharmacy © Ramaiah University of Applied Sciences
14
Faculty of Pharmacy
CHROMATOGRAM

15. © Ramaiah University of Applied Sciences
15
Faculty of Pharmacy © Ramaiah University of Applied Sciences
15
Faculty of Pharmacy © Ramaiah University of Applied Sciences
15
Faculty of Pharmacy © Ramaiah University of Applied Sciences
15
Faculty of Pharmacy
MERITS
Short
analysis
time
Well
defined
separation
Less
mobile
phase
consumed Good
sensitivity
Less
chance of
sample
loss
Narrow
bands
formed

16. © Ramaiah University of Applied Sciences
16
Faculty of Pharmacy © Ramaiah University of Applied Sciences
16
Faculty of Pharmacy © Ramaiah University of Applied Sciences
16
Faculty of Pharmacy © Ramaiah University of Applied Sciences
16
Faculty of Pharmacy
DEMERITS
Low
resolution
Proteolysis
problem in
protein
separation
Pre-
filtration of
sample
required
Less
efficient
than other
techniques Limited
number of
peaks
Difference(>10%)
in size of different
molecules
required

17. © Ramaiah University of Applied Sciences
17
Faculty of Pharmacy © Ramaiah University of Applied Sciences
17
Faculty of Pharmacy © Ramaiah University of Applied Sciences
17
Faculty of Pharmacy © Ramaiah University of Applied Sciences
17
Faculty of Pharmacy
APPLICATIONS
• Determination of molecular weight of different compounds using hyphenated
techniques like LC-MS or molar mass sensitive detectors.
• Used for separation of biomolecules like proteins, polysaccharides, nucleic acids,
hormones, enzymes etc.
• Estimating molecular weights of synthetic polymers, elastomers, polyisoprene,
rubbers etc.
• Characterisation of properties of different macromolecules.
• Study of quaternary structure of proteins.

18. © Ramaiah University of Applied Sciences
18
Faculty of Pharmacy © Ramaiah University of Applied Sciences
18
Faculty of Pharmacy © Ramaiah University of Applied Sciences
18
Faculty of Pharmacy © Ramaiah University of Applied Sciences
18
Faculty of Pharmacy
SUMMARY
• Gel chromatography separates molecules based on their different sizes using gel as a
stationary phase.
• No distribution or chemical interaction involved in the technique.
• It is divided into gel permeation & gel filtration chromatography based on type of
stationary & mobile phase used.
• Stationary & mobile phase can be hydrophilic & hydrophobic in nature.
• Mostly used for separation of macromolecules.
• Used for separation of wide range of polymers, biomolecules etc.

19. © Ramaiah University of Applied Sciences
19
Faculty of Pharmacy © Ramaiah University of Applied Sciences
19
Faculty of Pharmacy © Ramaiah University of Applied Sciences
19
Faculty of Pharmacy © Ramaiah University of Applied Sciences
19
Faculty of Pharmacy
REFERENCES
• Rouessac, F. and Rouessac A. (2007) Chemical Analysis - Modern Instrumentation
Methods and Techniques. 2nd Edition. England, John Wiley & Sons Ltd Publishing,
pp.160-167.
• https://www.chromatographyonline.com/view/early-development-size-exclusion-
chromatography-historical-perspective.