Gel

filtratio

n

Chrom

atog

raphy

Gisha G P MSc BiotechnologyMahatma Gandhi

University, Kottayam

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 1. Gentleness of the

techniques permits separation of liable molecular species

2. 100% solute recovery3. Excellent

reproducibility4. Comparatively short

time & relatively inexpensive equipment

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

• 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.

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

Ve = Vo + KdVt

where Ve = effluent volume

Vo = void volume

Kd = distribution

coefficient

Vt = volume of solvent

inside

Vt = α Wr

α = dry wt. of gel Wr= water

regaining capacity

Kd = 0, for large molecules

Kd = 1 , for small molecules

Kd = -1 , for intermediate molecules

Types of gelCharacteristics 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)

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 α1-6 glucosan polymer called dextran

• The resulting dextran is treated with epichlorohydrin to give several types of crossed linked dextran (sephadex)

Sucrose Microbial fermentation

Specific PH

Glucose + fructose

Dertan (a-1-6 glucosan polymer)

Gl-Gl-Gl

O

CH2

CH

CH2

OH

O

Gl-Gl-Gl n

Gl-Gl-Gl-

OH

Gl-Gl-Gl-

OH

+CH2Cl

HOHC

CH2Cl

Sephadex

• 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

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.

NH

O O

NH

O

NHNH

O

NH

O

HN

O

n

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

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

Gel type Fractionation range inMolecular 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

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 inMol.wt. Units

Approximate exclusion limit in mol.wt. Units(Average porosity in A°)

60 styragel 800 1,600100 2,000 4,000400 8,000 16,0001x10 3 20,000 40,0005x10 3 100,000 200,00010x10 3 200,000 400,00030x10 3 600,000 1,200,0001x10 5 2,000,000 4,000,0003x10 5 6,000,000 12,000,0005x10 5 10,000,000 20,000,00010x10 5 20,000,000 40,000,000

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

• 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.

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

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.

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 .

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

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 .

Applications 1- separation of large

molecular weight compound as protein, carbohydrate, peptides, nucleic acids

2- desalting of colloids3- 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

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.

• Ve = Vo + KdVt

• Vt = α Wr

• Kd = Ve - Vo

α Wr

• 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)

Calibration curve

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

4 4.5 5 5.5 6

Lg Mol WtKa

v

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