GEL FILTRATION

Gel Chromatograpphy

Gel filtration has also been called by different name: Gel permeation, Exclusion chromatography or Molecular sieve chromatography.

Gel chromatography is technique in which separation is based upon molecules, size and shape of the species in the sample.

Theory:

The chromatographic media used in this technique are porous, polymeric organic compounds with molecular sieving properties. These are cross linked polymers which swell considerably in water forming a gel of a three dimensional net work of pores. The size of pore is determined by degree of cross linking of polymeric chains. Different solutes in a mixture get separated on the basis of their molecular size and shape during their passage through a column packed with the swollen gel particles. The terms ‘exclusion chromatography’, ‘gel filtration’ and ‘molecular si’ve' chromatography are used for this separation process. The large molecules in sample are unable to penetrate through the pores into the gel beads. Obviously the volume of the solvent accessible to large molecules is very much less (V_o), whereas small molecules which can freely penetrate into the gel have access to solvent inside (V_i) as well as outside (V_o) the spherical beads.

A sample containing a mixture of large and small molecular weight substances is applied onto the column. Solvent usually a buffer is used as an eluent. As components of the sample travel down the column, the compounds whose molecular size exceeds the fractioation of range of the gel, are unable to enter into gel particles and hence remain completely excluded from beads and travel through the interstitial spaces. Molecules of smaller compounds however diffuse into the gel matrix through the pores and get distributed between the mobile phase inside as well as outside the gel particles. These thus followed a longer path than the larger molecules and hence their movement down the column is retarded. Consequently different components in the sample get separated from each other with larger molecules getting eluted first followed by smaller molecules.

For a given type of gel, the distribution coefficient, K_d which represents fraction of the liquid within the gel particles accessible to molecules of a given substance will predominately depend upon molecular size of that compound. Very large molecules, due to their exclusion, have no access to the mobile phase within the gel hence have a K_d value of 0, whereas smaller molecules for which the inner mobile phase is completely accessible have K_d value of 1. For the molecules of the intermediate size, the K_d value have varies within this range (0-1). This difference in K_d values of various compounds in the sample accounts for their separation during gel filtration. The relationship of K_d with elution volume V_e is given by:

Ve=Vo + Kd. Vi

Where:           

V_e:          is the elution volume and represents volume of the mobile phase required to elute the compound from the column.

V_o:                      is void volume or the volume of the mobile phase outside the gel particles

V_i:          is the volume of the mobile phase present inside the gel particles and can be calculated  from the relationship: Vi=a. Wr where a= dry weight of the gel and Wr = water regain. Values of Wr for different types of gels are given as:

K_d:          is the fraction of Vi accessible to a particular compound. Tranformation of Equation

Kd = ( Ve-Vo) / Vi

As can be noted, for very large molecules which are completely excluded from the gel V_e = V_o and so Kd = 0, but small molecules which can freely diffuse into and out of the gel beads V_e =V_o + V_i and so K_d = 1.

Figure 1: Elution from gel filtration

Gel Matrix

In gel chromatography, the granulated or beaded gel material is called as the packing material. The solute which are disturbuted throughout the entire gel phase is called stationary phase and the liquid flow through the bed is called as mobile phase. The solid support or gels that are used in gel chromatography are group of polymeric organic componuds that possess a three dimentional network of pore that confers the gel properties upon them. This gel have a tendency to swell in a suitable solvent and as a result of swelling of gel. The space between polymer chain increases in size. For a gel there will be a critical size of molecule that can just penetrate the interior. Molecules larger than the pores of gel are completely excluded from the region (gel). Smaller molecules can enter the gel and larger molecules can exclude the gel.

Examples of Gel

a.       one of the most widely used gel is crosslinked dextrans which is sold under the name Sephadex.

b.      Polyacrylamide cross linked dextran, called Biogel P

c.       Agarose (Sepharose or Biogel A)

d.      Polystyrene (Biobeads)

Properties of Gel

·         A good gel shoould be inert and doesn’t react with molecules to be fractionated.

·         It should be chemically stable.

·         Its particle size distribution should be controlled. For e.g. for ordinary lab work, powder gel of particle size of 70 micron in daimeter is quite effective (0.007 mm).

·         It should be high mechanical rigidity

Table 1: Molecular weight fractionating range of Sephadex and Sepharose

Gel type	Fractionation range (MW)		Bed Vol. (ml/gm dry material)	Dry bead diameter (mm)
	Peptides and globular proteins	Dextrans
Sephadex G-10	-700	-700	2-3	40-120
Sephadex G-5	-1500	-1500	2.5-3.5	40-120
Sephadex G-25 (Fine)	1000-5000	100-5000	4-6	20-80
Sephadex G-50 (Fine)	1500-30000	500-10000	9-11	20-80
Sephadex G-75	3000-80000	1000-50000	12-15	40-120
Sephadex G-100	4000-150000	1000-100000	15-20	40-120
Sephadex G-150	5000-150000	1000-150000	20-30	40-120
Sephadex G-200 (Super fine)	5000-250000	1000-200000		10-40
Sephacryl S-200	5000-2.5X105	1X103-8X104		40-105*
Sephacryl S-300	1X104-1.5X106	1X103-7.5X105		40-105*
Sepharose 2 B	7X104-40X106	1X105-20X106		60-200*
Sepharose 4 B	6X104-20X106	3X104-5X104		60-140*
Sepharose 6 B	1X104-4X106	1X104-1X106		45-165*

Steps involved in Gel Chromatography

1. Selection of a column

The column consists of a straight glass tube with a bed support at the bottom. The bed support allows only the liquid to pass through without disturbing the bed material. Column of 100 cm length and 10-20 cm in height  are sufficient for lab work in many cases.

2. Gel Preparation

There are two main methods of preparation of the gel.

       I.            First, the powder gel is mixed with excess of solvent to be used as eluent. It is then allowed to swell and left as such till the equilibrium condition is achieved. This procedure takes longer time.

    II.            Second, t.he powder gel is mixed with excess of solvent and the slurry so obtained is warmed to about 100°C for about 30 mins in a water bath. By warming, bacteria and fungus if present in suspension is also killed and dissolved air is also removed. As a result, the gel swells in few hours. The slurry is cooled before packing.

3 . Packing of the column

The method for packing of the column with gel depends upon type of gel to be used. Soft gel like sephadex are packed carefully while hard gel donot require much precaution.

There are generally  2 methods

1.      The column is first filled with eluent and then the slurry of the powder gel is poured  into the column through a funnel attached to the top of the column. the entire amount of slurry should be added on one step. Packing in many steps should be avoided as it gives uneven packing.

2.      It is most employed method. The gel is allowed to swell in the solvent. It is prepared by warming the slurry and allowed to cool before packing. After swelling , the gel is allowed to settle and the supernatant liquid is off to about half the volume of sedimented gel. This is again mixed with the solvent with constant stirring to make a slurry of the gel. The slurry is then carefully poured into the column in one step with the help of glass rod.

Figure 2: Gel filtration of different cellular inclusions

4. Application of sample

Sample can be loaded at the top of gel surface with the help of the pipette or syringe. The sample of 1-2% of total bed volume is sufficient. In group separation, sample of 25 -30 % of the total bed volume is requried.

5. Elution method

Then the sample solution is allowed to pass down through gel bed. Small volume of the eluent is added by means of pipette having a bent tip. And the last traces of sample is washed with eluent. Single solvent is used for elution. E.g.: sodium chloride, H₂O, organic solvents are used as elution buffer.

6. Collection and analysis of eluate

Each fraction of eluate is collected by keeping the flow rate 1 ml/min.

7. Analysis

It can be done by spectrophotometric methods or colorimetric methods. For the separation of polysaccharide, each fraction collected can be identified by paper chromatography.

Application

•     It is used in separation of sugars, salts, polypeptides, amino acids, proteins, lipids,  polystyene and silicon polymers.
•       The main appliction of gel chromatography is in purification of biological macromolecules, viruses, proteins, enzymes, hormones, antibodies, nucleic acids and polysaccharides by the of appropriate gel. Sephadex G75 is used for purifying macromolecules such as various species of RNA viruses. Sephadex G15 have been used in separation of maltose and glucose.
•      It is used for solution concentration. Solution of high relative molecular mass substances can be concentrated by the use of sephadex G-25. Water and low relative molecular mass substances are adsorbed by the swelling gel whereas high relative molecualr mass substances remain in the solution. Then the gel is removed by centrifugation leaving high molecular mass substances in the solution increasing the concentration.
•           Plasma protein fractions can be quantitatively determined in the diagnosis of certain human disease such as Hyperglobulinema.