Gel permeation chromatography

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Transcript of Gel permeation chromatography

  • GEL PERMEATION CHROMATOGRAPHY BY:- SHRADHA BASU M.PHARM-I DEPT. OF PHARM. BIOTECHNOLOGY MCOPS
  • CONTENTS Introduction Mechanism of separation Theory of separation Instrumentation I. Column packing II. Solvents III. 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 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
  • INSTRUMENTATION
  • 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)
  • Porous glasses or silica:- I. Cover wide range of pore diameter II. Chemically resistant at pH values