Post on 10-May-2015
description
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