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Gel permeation chromatography
Gel permeation chromatography is a size-exclusion chromatography technique that separates analytes based on their hydrodynamic size. Molecules are separated by passing through a column containing porous gel or polymer beads. Small molecules are able to enter the pores and thus remain inside the beads longer, while larger molecules pass through more quickly. Detectors such as refractive index and UV-Vis are used to analyze the separated analyte bands exiting the column. GPC can determine molecular weight distribution and is used to analyze polymers, proteins, and other large biomolecules.
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Gel permeation chromatography
- 1. GEL PERMEATION CHROMATOGRAPHY BY:- SHRADHA BASU M.PHARM-I DEPT.OF PHARM. BIOTECHNOLOGY MCOPS
- 2. CONTENTS • Introduction • Mechanismof separation • Theory of separation • Instrumentation I. Column packing II. Solvents III. Detectors • Advantages & disadvantages • Applications • References
- 3. INTRODUCTION • Non-interactive modeof 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
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- 5. • GPC separatesmolecules 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 volumeof 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 conditionsare 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
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- 9. COLUMN PACKING • Differenttypes:- 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 glassesor 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 ofporous 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 singlesolvent 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)
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- 14. DETECTORS • Detectors usedmust 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 ofsugars 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
Editor's Notes
- #11 Silylation is the introduction of a (usually) substituted silyl group (R3Si) to a molecule.The process involves the replacement of a proton with a trialkylsilyl group, typically trimethylsilyl (-SiMe3). A general technique is to deprotonate the substrate with a suitable strong base (e.g. butyl lithium), and allow it to react with a silyl chloride (e.g. trimethylsilyl chloride). The base used in this reaction mustn't form any HCl as it will hydrolyze the silyl protecting group. The introduction of a silyl group(s) gives derivatives of enhanced volatility, making the derivatives suitable for analysis by gas chromatography and electron-impact mass spectrometry