1. GEL PERMEATION
CHROMATOGRAPHY
BY:-
SHRADHA BASU
M.PHARM-I
DEPT. OF PHARM. BIOTECHNOLOGY
MCOPS

2. CONTENTS
• Introduction
• Mechanism of separation
• Theory of separation
• Instrumentation
I. Column packing
II. Solvents
III. Detectors
• Advantages & disadvantages
• Applications
• References

3. INTRODUCTION
• Non-interactive mode of separation
• Particles of column-range of pore size & pore
networks
• Solute molecules separated on the basis of size &
shape
• Also called gel permeation chromatography,
exclusion chromatography and molecular sieve
chromatography
• Molecular sieve chromatography-separation carried
out on natural or synthetic zeolites
• General formula of a typical zeolite-M2/n. Al2O3 x SiO2
. y H2O
• Separation not based on any distribution ratio
• Not strictly chromatographic

4. MECHANISM OF SEPARATION

5. • GPC separates molecules in solution by their “effective
size in solution.”
• To prepare a sample for GPC analysis the resin is first
dissolved in an appropriate solvent.
• Inside the gel permeation chromatograph, the dissolved
resin is injected into a continually flowing stream of
solvent (mobile phase).
• The mobile phase flows through millions of highly porous,
rigid particles (stationary phase) tightly packed together in
a column.
• The pore sizes of these particles are controlled and
available in a range of sizes.

6. THEORY
• Total volume of column packed with a gel that has been
swelled by water or other solvent is given by
• V t = Vg + Vl + Vo
where,
Vt = total bed volume
Vg = vol. occupied by solid matrix of gel
Vl = vol. of solvent held in pores or interstices
Vo = free vol. outside the gel particles

7. • If conditions are assumed such that
I. time taken for solute molecules to diffuse into pore is less
as compared to time spent by molecule near pore
II. separation process independent of diffusion process
• Under these conditions
Ve = Vo+K d . Vl
Ve =vol. of effluent flowing through column between point of sample
injection & sample emergence from column
Kd = distribution coefficient
• For large molecules
k d = 0, Ve = Vo,
• For molecules that can penetrate all the pores
kd = 1, Ve = Vo+Vl

8. INSTRUMENTATION

9. COLUMN PACKING
• Different types:-
I. Semi-rigid, cross-linked macromolecular polymers
II. Rigid, controlled-pore-size glasses or silica
• Semi-rigid polymers:-
I. these materials swell slightly
II. care must be taken during use
III. limited to a maximum pressure of 300 psi due to bed
compressibility
Egs: styrene divinylbenzene polymers (for compounds of MW
range of 100-500 million) & suspension polymerization of 2-
hydroxyethyl methacrylate with ethylene dimethacrylate (can
withstand pressure upto 3000 psi)

10. • Porous glasses or silica:-
I. Cover wide range of pore diameter
II. Chemically resistant at pH values<10
III. Used with aq. & polar organic solvents
IV. Non-polar solvents-deactivate surface with silylation &
avoid irreversible retention by polar solutes
Silylation:-
• introduction of substituted silyl group (R3Si) to a molecule
• process involves replacement of proton with trialkylsilyl group such as
trimethylsilyl(-SiMe3)
• deprotonate the substrate with a suitable strong base (e.g. butyl
lithium)
• allow it to react with a silyl chloride (e.g. trimethylsilyl chloride)
• base used in this reaction must not form HCl- hydrolyze the silyl
protecting group
• introduction of a silyl group(s) gives derivatives of enhanced volatility-
making the derivatives suitable for analysis by GC

11. • Advantages of porous inorganic
packing
I. Column use-routine & indefinite
after calibration
II. Low possibility of sample
contamination & biodegradation
III. Bed volume-constant at high flow-
rates & pressures
IV.Thermal stability-use at elevated
temperatures

12. SOLVENTS
• Requires single solvent to dissolve & chromatograph
sample
• Issues caused by high viscosity of high MW samples
• Viscosity difference between injected sample & MP is
high-
I. Peak distortion
II. Anomalous changes in elution times
• Solvent Selection Guide for Room Temp. Aqueous Soluble
Polymers
Eluent Polymer
0.10M NaNo3 Neutral polymers
(PEG,PVA,Dextrans)
Anionic polymers (polyalginic acid,
carrageenan)
0.8M NaNo3 Cationic (polyvinylamine)
80:20
0.10M NaNo3/acetonitrile
Amphoteric (collagen gelatin)

13. SAMPLE PREPARATION

14. DETECTORS
• Detectors used must be compatible with exclusion
columns
I. 3-6m long
II. Working volume-1-10mL
III. Analysis time<10 mins
• Widely used detectors :-
I. Differential refractometer
II. Spectrophotometric detectors
• Low-angle laser light scattering( LALLS) detector
I. Determination of absolute molecular weights
II. Provides information on variation of long chain branching
with molecular weight

15. ADVANTAGES & DISADVANTAGES
• Has well defined
separation time
• Can provide
narrow bands
• Low chance for
analyte loss
• Determination of
MW of polymers
• Less time of
analysis
• Requires at least
10% difference
in MW for
reasonable
resolution of
peaks
• Pre-filtration of
sample

16. APPLICATIONS
• Separation of sugars polypeptides, proteins, liquids, butyl
rubbers, polystyrenes, silicon polymers.
• Sephadex G-25 : for separation of salts & amino acids
from proteins .
• Sephadex G-75 : fractionation & purification of proteins
polysaccharides & nucleic acids.
• Polymers can be characterized for number average
mol.wt. (Mn), weight average mol. wt. (Mw), size average
mol. wt. (Mz), polydispersity index.

17. REFERENCE
• Book reference
I. Willard H H, Merritt l l, Dean J A, Settle F A. Instrumental
Methods of Analysis.2012:7:644-648
II. Sharma B K. Instrumental Methods of Chemical
Analysis.2004:pg161-170
• Web references
I. http://chemcatalog.waters.com/publication/frame.php?i=1
42735&p=292&pn=&ver=flex
II. http://www.waters.com/waters/en_US/GPC---Gel-
Permeation-
Chromatography/nav.htm?cid=10167568&locale=en_US