Download - Gel permeation chromatography
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