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.

ION EXCHANGE CHROMATOGRAPHY

Ion Exchange Chromatography:

Many biological materials for e.g. amino acids and proteins have ionizable groups and the fact is they carry net positive or negative charge that can be utilized in separating mixture of such compounds.

Ion exchange may be defined as a reversible reaction in which pre mobile ions of solid phase ion exchanger are exchanged for different ions of similar charge present in solution. In ion exchange chromatography, a reversible exchange of ion is possible between ions in liquid phase and the solid insoluble substance containing ionic site.

Ion exchange resins are porous, synthetic organic polymer containing charged group which are capable of holding positive or negative ions.

The most common properties of all ion exchange resins are

A. They are generally insoluble in water and organic solvent such as benzene, ether and carbon tetrachloride (CCl₄).

B. They are complex in nature i.e. in fact they are polymeric. The most important resins are polysterene resins formed by condensation of styrene and divinyl benzene.

Figure

Granular resin swells in water to give a gel structure due to hydration of ions. Swelling is directly proportional to the percentage of cross linking. Less the cross linking less is swelling. Ion exchange resins are mixed with cross linking reagents such as divinyl resin.

C. They have active counter ions that will exchange reversibly with other ions in a surrounding solution without any change in material.

Figure 1: Cation and Anion exchanges

There are four basic types of resins which are commonly used in ion exchanging.

1.      Strong acidic cation exchange resins

2.      Weak acidic cations exchange resins

3.      Strong basic anion exchange resins

4.      Weak basic anion exchange resins

1.  Strong acidic cation exchange resins:

It contains sulphonic acid group. Sulphonated polystyrene resins belong to this class. They are useful in pH range 1-14. They are useful mainly in fractionation of cations, inorganic separation and for separation of vitamins, peptides and amino acids.

2.  Weak acidic cations exchange resins:

It contains carboxylic acid group. Carboxylic polymetacrylate (Polymethyl methacrylate) is an example of weak acidic cation exchange resins. They are useful in pH range 5-14. They are used in biochemical separation, fractionation of cations, and separation of amino acids, antibiotics and organic bases.

3.  Strong basic anion exchange resins:

It contains quaternary ammonium groups. Quaternary ammonium polystyrene belongs to this class. And it is effective between pH 0-12. This type of resins is useful in fractionation of anion and for separation of vitamins and fatty acids.

4.  Weak basic anion exchange resins:

It contains phenol, formaldehyde or polyamines group. Phenol formaldehyde and polystyrene resins belongs to this class. They are effective in pH range 0-9. It can be useful in fractionation of anionic complexes of metals and separation of vitamins and amino acids.

Types of Resin	Functional group	Nature of Resin	Commercial Name
Cation Exchanger
Strongly acid	-CH2SO-H+	Sulphonated polystyrene	Amberlite IR-100 IR-105 IR-109
Weakly acidic	-COOH	Carboxylic polymetacrylate	Amberlite IR-6 IR-50
Anion Exchanger
Strongly basic	-CH2NR2OH	Quaternary polysteryrene	IRA-40
Weakly basic	-N(C2H5)	Polyamine Polystyrene	De-Acedite E Amberlite IR48

Techniques of Ion Exchange Chromatography:

1. Preparation of Column

The ion exchange chromatography is carried out in a chromatographic column which usually consists of a burette provided with a glass wool plug at the lower end. Generally a ratio of 10: 1 or 100:1 between height and diameter is maintained in most of the experiment. Too narrow or too wide column give uneven flow of liquid and sometimes poor separation.

2. Preparation of Ion Exchange

Ion exchange materials are first allowed to swell in buffer or in HCl or NaOH solution for 2-3 hours or sometimes overnight. Almost all ion exchange resin swells when placed in buffer or distilled water and this is due to hydration of their ions. In dry condition, the pore of resins is restricted so in order to swell the pore of resin. Resins are suspended in buffer solution or in distilled water.

3. Washing of Ion Exchangers

The ion exchange material is obtained in required ionic form by washing with appropriate solution. For e.g. the H⁺ form of cation exchange resins is obtained by washing the material with HCl then with water until the washings are neutral.

Anionic exchangers are generally supplied in the form of salt and amines. Similarly, Na⁺ form is prepared by washing the resins with NaCl or NaOH solution and then with water.

Figure 2: Ion exchange chromatography 

4. Packing of Column

This is one of the most critical factors in achieving a successful separation. The column is held in vertical position and the slurry of resins is poured into the column that has its outlet closed. The column is gently tapped to ensure that no air bubbles are trapped and that packing material settles evenly.

5. Sample Application

Sample can be loaded by using pipette or syringe. The amount of sample that can be applied to a column is dependent upon the size of the column and the capacity of resins, If the starting buffer is to be used throughout the development of column, the sample volume be 1 % to 5 % of bed volume.

6. Development an Elution of bound ions

Bound ions can be removed by changing the pH of buffer. E.g. separation of amino acid is usually achieved by using a strong acidic cation exchanger. The sample is introduced onto the column at pH of 1-2, thus ensuring complete binding of all of the amino acids.

Gradient elution used in increasing pH and ionic concentration results in the sequential elution of amino acid. Then acidic amino acid such as aspartic acid and glutamic acid are eluted first. The neutral amino acid such as glycine and valine are eluted. The basic amino acid such as lysine and arginine retain their net positive charge at pH value of 9 to 11 and are eluted at last.

7. Analysis of eluate

Equal fraction of each elute are collected at different test tube keeping the flow rate at 1 ml per minute. The eluate collected in each fraction is mixed with ninhydrin color reagent. The mixture is then heated to 105°C to develop the color and intensity of color is determined by colorimeter method or spectrophotometer method at 540 to 570 nm.

The protocol for amino acid separation (purification) by ion exchange chromatography

Figure 3: Separation of amino acids by ion exchange chromatography

Requirements:

                                                        i.            Strongly acidic resin (e.g. amberlite IR -120)

                                                      ii.            HCl (4 mol / L)

                                                    iii.            HCl (0.1 mol/L)

                                                    iv.            Tris-HCl buffer (0.2 mol/L)

                                                      v.            NaOH (0.1 mol /L)

                                                    vi.            Amino acid mixture (Dissolve aspartic acid, histidine and lysine in 0.2N HCl)

                                                  vii.            Acetate buffer (4 mol/L)

                                                viii.            Ninhydrine reagent (store in brown bottle)

Procedure:

1. Preparation of ion exchanger

·         Gently stir the resin With HCl (4 mol/L) until fully swollen.

2. Washing

·         Allow the resin to settle, then decant the acid and repeat the wasting with 0.1N HCl, and resuspend it in the solution.

3. Preparation and packing of column

·         Clamp the column vertical position.

·         Fill the column with resin suspended with 0.1 N HCl and allow to settle down and height of which is made 11 cm.

4. Smaple application                  

·         Add 0.2 ml of amino acid mixture to the top of column wihtout disturbing ion exchange resins.

·         Add 0.2 ml of 0.1 N HCl, allow it to float into the column and repeat this process twice.

5. Development of Chromatogram

·         Finely apply 2 ml of 0.1 N HCl to the top of resin and connect the column to a reservoir containing 500 ml of 0.1 N HCl.

6. Elution of Amino acid

·         Collect 2 ml of each eluted sample in 40 test tubes keeping flow rate at 1 ml per minute.

·         Test five of the tubes at a time for the presence of amino acid by spotting a sample from each tube onto a filter paper. Dip this in acetone solution of ninhydrin and heat in an oven at 105°C. If amino acids are present they will show as blue spot in the filter paper.

7. Detection of Amino acids

·         Adjust the pH of each tube to five by addition of few drops of acid or alkali. Add 2 ml of ninhydrin reagent and heat in a boiling water bath for 15 minutes.

·         Cool the tube to room temperature and add 3 ml of 50 % ethanol and read the absorbance at 570 nm in spectrophotometer.

Figure 4: Elution of amino acids on the basis of charge behavior in different pH medium

Application of IEC

·         It is used for separation of similar ions from one another because different ion undergo exchange reaction to different extent.

·         IEC can be used for removal of inferring radicals. E.g. PO₄^3- ion interferes in the estimation of calcium or barium ions by oxalate method. Therefore its removal is achieved by passing a solution of calcium and barium ions through a sulphonic acid cation exchanger. The Ca²⁺ or Ba²⁺ ions held by the resin will be removed by using suitable eluent. Ca²⁺ or Ba²⁺ ions will get exchanged with H⁺ ion while PO₄^3- will pass as such through the column.

·         IEC is also used for the softening of hard water. The hardness of water is due to presence of Ca²⁺ or Mg²⁺ or other divalent ions and these ions can be removed by passing hard water through cation exchangers. Ca²⁺ and Mg²⁺ or any divalent ions are retained in clumn, Na⁺ ions pass into solution. These Na ions are harmless for washing purposes.

ResSO₃^2-(Na⁺)₂    +  2 Ca²⁺    ®      ResSO₃^2- Ca²⁺   +  2Na⁺

·         IEC is used for separation of amino acids and protein.

·         It can be used for demineralization of water which requires removal of cations as well as anions. Water is first passed through an acidic cation exchange where cation like Na⁺, Ca²⁺, Mg²⁺ are exchanged by H⁺ ions. It is then passed through basic anion exchanger, where anions like Cl are exchanged by OH^- ions of exchanges. Then, H⁺ and OH^- which pass into the solution combined to form unionized water.

Protein Purification Protocol

Requirements:

1.      DEAE Cellulose (Diethyl amino ethyl cellulose)

2.      Eluting buffer

i. Sodium Phosphate (0.1 mol/L)

ii. Sodium Phosphate (0.2 mol/L)

iii. Sodium Phosphate (0.3 mol/L)

3.      Human Serum

4.      UV Spectrophotometer

Procedure:

                              1.      Column prepared from Cellulose (0.1 mol/L, swelling buffer)

2.      Washing (0.2 mol/L)

3.      Column preparation vertically clamp

4.      Resin solution add 1 L

5.      Sample Application

6.      Carefully pipette 0.5 ml of serum sample on the top of the column. Allow the serum to pass into DEAE cellulose.

7.      Development and Elution.

8.      Then , elute slowly with 3 ml of eluting buffer.

9.      Fraction collection.

10.  Analysis of Amino acid

11.  Calculate the recovery of protein by determining the protein content of serum and then take the absornbance at 280 nm in spectrophotometer.