Gel filtration copy

chromatography, separation of proteins

1. Gel filtration Chromatography Gisha G P MSc Biotechnology Mahatma Gandhi University, Kottayam

2. Introduction Gel filtration (chromatography), is also known as molecular sieve / molecular exclusion chromatography. Gel filtration chromatography separates molecules according to their size and shape. Advantages Gentleness of the techniques permits separation of liable molecular species 100% solute recovery Excellent reproducibility Comparatively short time & relatively inexpensive equipment

3. Principle The stationary phase consists of gel beads containing pores that span a relatively narrow size range. Separation is achieved by differential distribution of sample component b/w the stationary solvent within the pores of a gel & the mobile eluting solvent out side the pores. Mixture of molecules placed on top of equilibrated column , Large molecules are “excluded” from the pores and travel through the column faster but small molecules are “included” – can diffuse into the pores and elute later

4. Degree of retardation of molecules is a function of the molecules size and pore diameter. Exclusion limit – molecular weight of the smallest molecule incapable of entering the gel pores.

5. Column Parameters Vs= volume of solvent held in the pores. This is normally approximated to Vt-Vo = volume of beads Vo = Elution volume of a large “totally excluded” molecule such as blue dextran Vt = Physical volume of column

6. V e = V o + K d V t where Ve = effluent volume Vo = void volume Kd = distribution coefficient Vt = volume of solvent inside V t = α W r α = dry wt. of gel Wr= water regaining capacity K d = 0, for large molecules K d = 1 , for small molecules K d = -1 , for intermediate molecules

7. Types of gel Characteristics of gel material Chemically inert Wide choice of pore and particle size Uniform particle and pore size Mechanically rigid Types Sephadex (cross linked dextran) Sepharose /Bio-Gel A ( agarose ) Bio-Gel P ( polyacrylamide) Bio Glass /Porasil ( Porous glass& silica granules ) Styragel / BioBeads S (polystyrene)

8. Sephadex Popular gel for biomolecule separation . A 1-6-polymer of glucose is prepared by microbial fermentation of sucrose (glucose + fructose) The resulting glucose provides the required α 1-6 glucosan polymer called dextran The resulting dextran is treated with epichlorohydrin to give several types of crossed linked dextran (sephadex)

9. Pore size controlled by molecular wt. of the dextran & amount of epichlorohydrin used. By controlling cross linking reaction , various class of gel beads with exclusion limits b/w 1 – 200,000 Da can be produced. Characters of sephadex 1- highly stable gels 2- stable at PH 2-12 3- their particles are free from ions 4- insoluble in water and organic solvent 5- they swell in water and other hydrophillic solvent 6- they require bactericidal such as Hg acetate

10. Polyacrylamide Can become compressed in the column, cause slow flow rates. Insoluble in water and common organic solvents , pH 2-11. Polymerized acrylamide into bead form. Numbered like P-10 , P-100. Number multiplied by factor of 1000 indicate exclusion limit in Da. Used to separate molecules up to 300,000 Da . Large pored gels lack mechanical rigidity.

11. Gel type Fractionation range in Molecular wt units Hydrated bed volume ml/g of dry gel Water regain ml/g of dry gel Bio Gel P-2 200-2,000 3.8 1.6 P-4 500- 4,000 6.1 2.6 P-6 1,000 – 5,000 7.4 3.2 P-10 5,000- 17,000 12 5.1 P-60 30,000 – 70,000 18 6.8 P-100 40,000 – 100,000 22 7.5 P-200 80,000 – 300,000 47 13.5

12. Agarose Use for study of viruses , nucleic acids and polysaccharides. Stable at pH 4-10 Freezing temperature and temperature above 30°C cause alterations in gel structure Chromatography performed b/w 0°C & 30°C Produced from agar. Linear polysaccharides of alternating residues of D- galactose & 3,6-anhydro-L- galactose. Hydrophilic , free of charged groups, completely inert. High porosity, use to separate biomolecules of several million Da

13. Gel type Fractionation range in Molecular weight units Agarose 0.5m (10%) 10,000 to 250,000 1.0m(8%) 25,000 to 700,000 2.0m(6%) 50,000 to 2,000,000 15.0m(4%) 200,000 to 15,000,000 50.0m 100,000 to 50,000,000 150m 500,000 to 150,000,000

14. Styragel Hydrophobic gel used for complete aqueous separation. Polymerized styrene , cross linked by divinyl benzene. Swells in organic solvent , rigid than hydrophilic gels. Unaffected by high temperatures up to 150°C Solvents – tetrahydrofuran , cyclohexanone , carbon tetrachloride Type Fractionation range in Mol.wt. Units Approximate exclusion limit in mol.wt. Units (Average porosity in A°) 60 styragel 800 1,600 100 2,000 4,000 400 8,000 16,000 1x10 3 20,000 40,000 5x10 3 100,000 200,000 10x10 3 200,000 400,000 30x10 3 600,000 1,200,000 1x10 5 2,000,000 4,000,000 3x10 5 6,000,000 12,000,000 5x10 5 10,000,000 20,000,000 10x10 5 20,000,000 40,000,000

15. Controlled pore glass beads Fine glass spheres of porosilicate glass Large no. of narrow sized pores. High flow rate , high rigidity. Adsorb significant amount of protein on their surface To avoid this –treat with hexamethyldisilazane. Exclusion limit 3000 to 9 million Da

16. Sephacryl HR: Sephacryl High Resolution (HR) is a composite gel prepared by covalently cross-linking dextran with N, N'-methylene bisacrylamide to form a hydrophilic matrix of high mechanical strength. Superdex: It is based on highly cross-linked porous agarose beads to which dextran has been covalently bonded.

17. Column preparation Gel must be swollen , attain equilibrium. Greater porosity much time for equilibration. Previously swollen gel is added in form of slurry & allowed to settle. Air bubble should not be formed. Equilibrate the column with 1-2 column volumes of buffer before starting a separation

18. Sample application Considerable care must be taken to avoid disturbing the bed surface. 1) Close the outlet and remove most of the buffer above the gel surface by suction. 2) Layer the sample on top of the bed. 3) Open the column outlet and allow the sample to drain into the bed 4) Wash the sample which remains on the bed surface and on the column wall into the bed with a small amount of eluent. 5) Refill the column with eluent and reconnect to a Mariotte flask or pump.

19. Elution & flow rates Samples are eluted from column using a single buffer . Resolution decreases as flow rate increases Allow time for molecules to diffuse in & out of matrix b/w mobile phase & stationary phase in order to achieve a good separation .

20. Precautions Preparing the gel from too thin a suspension or packing the column in stages, often results in a badly packed bed. Avoid disturbing the bed surface , an uneven bed surface leads to uneven separated bands and poor resolution. Do not allow the bed to run dry Damaging of matrix affect separation process , since the fractionation is based on pore size. Buffer and matrix should be degassed , air bubbles entering the column can lead to poor resolution. Experimental set up should be maintained at same temperature

21. Thin layer gel chromatography First done by Determann , Johanson , & Rymo. Used for clinical studies. Small sample volume. Hydrated gel is applied to the plate , placed on an air tight container at an angle of 20°. Plate is connected to reservoirs at both ends by means of filter paper bridges. Equilibration carried out for at- least 12 hrs. Sample applied either as spot or as a band. The plate is then developed and separated components detected by suitable methods .

22. Applications 1- separation of large molecular weight compound as protein, carbohydrate, peptides, nucleic acids 2- desalting of colloids 3- molecular weight determination (A linear relationship exists between the logarithm of the molecular weight and the elution volume) Separation of large molecular weight compounds Chief use of gel filtration Ultimate purification Protein , enzymes , hormones , antibodies, nucleic acids , polysaccharides and even viruses can be separated Low molecular weight compounds such as amino acids , small peptides and oligonucleotides can also be separated Useful in separation of 4S & 5S tRNA

23. Desalting of colloids Removal of salt& small molecules from macromolecules. Easily performed in gel filtration , distribution coefficient of salt molecules differ from macromolecules Sephadex G -25 columns are used Molecular weight determination Distribution coefficient of a given macromolecule is a function of size & shape. V e = V o + K d V t V t = α W r K d = V e - V o α W r

24. Distribution coefficients of standard proteins of known molecular wt. are plotted against log of their mol.wt. shape of proteins vary – error in mol.wt. determination Solvent confers identical shapes – guanidium chloride.(6M , pH 6) Gels – 4% agarose (10,000 to 30,000 Da) & 6% agarose (1,000 to 80,000 Da)

25. References Gel filtration ,principles and methods , Amersham Biosciences . Biophysics , Vasudeva Pattabi, N. Gautham High Resolution Chromatography , A Practical Approach , Edited By P.A Millner Separation and Purification Techniques In Biotechnology, Frederick J. Dechow www. edvoteck/ Principles of Gel filtration Chromatography/ www.sigmaaldrich.com Gel filtration chromatography , Lave Fischer , Elsevier / north –Holland Biochemical press 1980