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GHORMADE SIR GEL CHROMATOGRAPHY GEL CHROMATOGRAPHY : GEL CHROMATOGRAPHY Gel chromatography (GC) is a chromatographic method in which molecules are separated based on their size Gel chromatography also known as- Size exclusion chromatography Gel permeation chromatography Gel filteration chromatography molecular sieve chromatography Slide 3: When an organic solvent is used as a mobile phase, then it is tend to called as Gel permeation chromatography when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography ADVANTAGES:- : ADVANTAGES:- Very simple to perform Not sensitive to eluant composition and its temperature Range of separation can be varied by varying the contents of gel matrices Gels are very stable and can be reused Principle- : Principle- Slide 6: Smaller molecules penetrate the particles to varying extents depending upon their shape & size, There is thus a partition of the molecules between the liquid inside the gel particles & that outside, The smaller the molecules, the larger the percentage of liquid within the particles that is available to them Molecules therefore leave the column in the order of decreasing molecular size, the larger size will leave the column first followed by the smaller sizes depending on their partition (shape & size) ranges Principle:- : Principle:- Size exclusion chromatography or Molecular sieve chromatography. How does it work? If we assume proteins are spherical… size Molecular mass (daltons) 10,000 30,000 100,000 Gel filtration chromatography : Gel filtration chromatography flow Gel filtration chromatography : Gel filtration chromatography flow Gel filtration chromatography : Gel filtration chromatography flow Gel filtration chromatography : Gel filtration chromatography flow Gel filtration chromatography : Gel filtration chromatography flow Slide 13: Elution volume (Ve) is the volume of a solvent required to elute a given solute from the column after it has first contacted the gel. Elution volume (Ve) = Vo +Kd Vi For two substances of diff mol. Wt. the difference in their eluant value Vs is Vs=Ve-Ve” Vs=(Kd-Kd”)Vi Thus for complete separation of two subs , the sample volume must not be larger than Vs. To separate a protein sample by gel filtration chromatography : To separate a protein sample by gel filtration chromatography PHM2213 2009/10 14 INSTRUMENTATION : INSTRUMENTATION Colum Gel Packing 0f Colum Preparation of sample Application of the sample Solvents Detector INSTRUMENTATION : INSTRUMENTATION Slide 17: A]Column- Straight glass tube with a bed support at the bottom Diameter and length should be larger Gel- Requirements of gel Matrix of gel should be inert chemically Gel should be stable chemically Gel should have low content of ionic group Matrix of gel should have the uniform porosity Degree of swell should be small A) CLASSIFICATION OF GEL : A) CLASSIFICATION OF GEL B] CHOICE OF GEL : In GC two types of separations are done:- DESALTING:- separation of high molecule substance from low molecular weight subs FRACTIONATION:-similar sub are eluted closer to one another B] CHOICE OF GEL C]PARTICLE SIZE : The material with particle diameter lower than 40 micro can be used D]GEL PREPARATION Dry powder is allowed to swell in liquid n then is used as eluant or Warm the gel slurry in boiling water at 100 degree Celsius C]PARTICLE SIZE D]DRYING OF GEL : Gel can be stored very well in wet state and there is no need of drying Ex:-dextran gel 3] PACKING OF COLOUM General method Process of packing depend upon nature of gel A}soft gel B}hard gel 0 D]DRYING OF GEL 4] PREPARATION OF SAMPLE : 5] APPLICATION OF SAMPLE a} pipette b}valve loop c}commercial plunger 4] PREPARATION OF SAMPLE 6] SOLVENTS : The choice is based on the solubility characteristics' of sample and detector It must swell the soft gel The solvent must be compatible with the detector 7] DETECTOR Differential refractometer UV photometer 6] SOLVENTS 8] APPLICATIONS : Purification Molecular wt. determination Solution concentration Desalting Protein-binding studies 8] APPLICATIONS REFERENCES:- : SEPARATION METHODS :-BY M .N. SHASTRI HIMALAYA PUBLICATIONS PAGE NO-154,155,156 ANALYTICAL CHROMATOGRAPHY:-BY G.R.CHATWAL ,HIMALAYA PUBLISHING HOUSE PAGE NO:-57-64 PHARMACEUTICAL ANALYSIS VOL 2:-BY KASTURE,MAHADIK,WADODKAR,MORE NIRALI PRAKASHAN PAGE NO-80-85 REFERENCES:- Slide 26: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.