GEL PERMEATION CHROMATOGRAPHY

BY:- SHRADHA BASUM.PHARM-IDEPT. OF PHARM. BIOTECHNOLOGYMCOPS

CONTENTS• Introduction• Mechanism of separation• Theory of separation• Instrumentation

I. Column packingII. SolventsIII. Detectors

• Advantages & disadvantages• Applications• References

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

MECHANISM OF SEPARATION

• 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.

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

• 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 poreII. 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 columnKd = distribution coefficient

• For large molecules k d = 0, Ve = Vo,

• For molecules that can penetrate all the pores kd = 1, Ve = Vo+Vl

INSTRUMENTATION

COLUMN PACKING• Different types:-

I. Semi-rigid, cross-linked macromolecular polymersII. Rigid, controlled-pore-size glasses or silica

• Semi-rigid polymers:-I. these materials swell slightlyII. care must be taken during use III. limited to a maximum pressure of 300 psi due to bed

compressibilityEgs: 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)

• Porous glasses or silica:-I. Cover wide range of pore diameterII. Chemically resistant at pH values<10III. Used with aq. & polar organic solventsIV. 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

• Advantages of porous inorganic packingI. Column use-routine & indefinite

after calibrationII. Low possibility of sample

contamination & biodegradationIII. Bed volume-constant at high flow-

rates & pressuresIV.Thermal stability-use at elevated

temperatures

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 distortionII. Anomalous changes in elution times

• Solvent Selection Guide for Room Temp. Aqueous Soluble Polymers 

Eluent Polymer0.10M NaNo3 Neutral polymers

(PEG,PVA,Dextrans)Anionic polymers (polyalginic acid, carrageenan)

0.8M NaNo3 Cationic (polyvinylamine)

80:200.10M NaNo3/acetonitrile

Amphoteric (collagen gelatin)

SAMPLE PREPARATION

DETECTORS• Detectors used must be compatible with exclusion

columns I. 3-6m longII. Working volume-1-10mLIII. Analysis time<10 mins

• Widely used detectors :-I. Differential refractometerII. Spectrophotometric detectors

• Low-angle laser light scattering( LALLS) detectorI. Determination of absolute molecular weightsII. Provides information on variation of long chain branching

with molecular weight

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

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.

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 referencesI. http://chemcatalog.waters.com/publication/frame.php?

i=142735&p=292&pn=&ver=flexII. http://www.waters.com/waters/en_US/GPC---Gel-

Permeation-Chromatography/nav.htm?cid=10167568&locale=en_US