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.