Gel permeation chromatography

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 Column packing Solvents 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-M 2/n. Al 2 O 3 x SiO 2 . y H 2 O 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 + V l + V o where , V t = total bed volume Vg = vol. occupied by solid matrix of gel V l = vol. of solvent held in pores or interstices Vo = free vol. outside the gel particles

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

8. instrumentation

9. Column packing Different types :- Semi-rigid, cross-linked macromolecular polymers Rigid, controlled-pore-size glasses or silica Semi-rigid polymers:- these materials swell slightly care must be taken during use 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:- Cover wide range of pore diameter Chemically resistant at pH values<10 Used with aq. & polar organic solvents Non-polar solvents-deactivate surface with silylation & avoid irreversible retention by polar solutes Silylation :- introduction of substituted silyl group (R 3 Si) to a molecule process involves replacement of proton with trialkylsilyl group such as trimethylsilyl(-SiMe 3 ) 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 Column use-routine & indefinite after calibration Low possibility of sample contamination & biodegradation Bed volume-constant at high flow-rates & pressures 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- Peak distortion Anomalous changes in elution times Solvent Selection Guide for Room Temp. Aqueous Soluble Polymers  Eluent Polymer 0.10M NaNo 3 Neutral polymers (PEG,PVA,Dextrans) Anionic polymers (polyalginic acid, carrageenan) 0.8 M NaNo 3 Cationic (polyvinylamine) 80:20 0.10M NaNo 3 /acetonitrile Amphoteric (collagen gelatin)

13. Sample preparation

14. detectors Detectors used must be compatible with exclusion columns 3-6m long Working volume-1-10mL Analysis time<10 mins Widely used detectors :- Differential refractometer Spectrophotometric detectors Low-angle laser light scattering( LALLS) detector Determination of absolute molecular weights 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. (M n ), weight average mol. wt. ( M w ), size average mol. w t. (M z ), polydispersity index.

17. reference Book reference Willard H H, Merritt l l, Dean J A, Settle F A. Instrumental Methods of Analysis.2012:7:644-648 Sharma B K. Instrumental Methods of Chemical Analysis.2004:pg161-170 Web references http ://chemcatalog.waters.com/publication/frame.php?i=142735&p=292&pn=& ver=flex http ://www.waters.com/waters/en_US/GPC---Gel-Permeation-Chromatography/nav.htm?cid=10167568&locale=en_US