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.

COLUMN CHROMATOGRAPHY

COLUMN CHROMATOGRAPHY

It is also called adsorption chromatography.

·         Stationary phase                      solid

·         Mobile phase                           liquid

The idea of column chromatography was first coined by American Chemist D.T. Davy in 1897 for the study of difference in chemical composition of different natural petroleum products. But it was Russian Botanist Michael Tswett in 1906 who had successfully separated green plant pigments by column techniques. It is the technique in which stationary phase e.g. silica gel, alumina, cellulose and mobile phase is either gas or liquid. The separation takes place when components of mixture are most strongly adsorbed by solid stationary phase.

Principle:

It is known that role of adsorption varies with the given adsorbent for different materials. Thus the principle of selective adsorption is used in column chromatography. In this method, mixtures to be separated are dissolved in a suitable solvent and allow passing through a column containing the adsorbent. The compound which has greater adsorbent power is adsorbed in the upper portion of the column.  The next in the lower portion of the column is compound having lesser adsorbing than other and this process is continuous as a result the compounds are separated in adsorbed in various part of the column. Then, the column may be washed with suitable solvent and each component is separated and analyzed.

A typical column chromatography system consists of the glass column, a mobile phase reservoir at the top and bottom of column contains fraction collector. The column is usually of glass of constant cross sectional area and can be of any size and length. The column is commonly made up of pyrex glass which is thermostable and chemostable. Generally long column give good resolution of components but wide columns are better for dealing with large quantities. The length: width ratios of 10:1 to 100:1 have been found most satisfactory. The glass column used should have a means of supporting the stationary phase to the base of column as possible.

Commercial column possess either a porous glass plate fused on to base of column or suitable device for supporting or replaceable nylonate which support the stationary phase. The commercial column support is to use a small plug of glass wool together with a minimum amount of quartz sand and glass beads.

Adsorbent used in column chromatography

Common adsorbent for column chromatography are sucrose, cellulose, starch, calcium carbonate, calcium phosphate, calcium oxide, silica gel, charcoal, magnesium and aluminium oxide. In order to obtain columns with good filtration properties, the adsorbent should have following properties

1.   The adsorbent must not react with substance to be analyzed i.e. chemically inert.

2.   The adsorbent should be insoluble in the solvent to be used.

3.   The color of absorbent should be such that it doesn’t interfere with chromatogram i.e. adsorbent used should be colorless. But this is not a rigid rule.

4.   The physical and chemical properties of adsorbent should not changed under experimental condition i.e. should be stable.

5.   The particle size should be uniform i.e. it should be fine enough to hold the substance and give the sharp bands. Column efficiency can be increased by smaller particles 100-200 mesh

Among the various adsorbent, alumina and magnesium have been found to give satisfactory separation with most of compound. In general, alumina seems to have higher selective power give sharper and narrow band. Sucrose and starch have power to be very successful for the separation of chlorophyll.

Classification of adsorbents:

Adsorbents have been classified into 3 groups depending on the strength according to force with which they hold solutes.

a. Week adsorbents e.g. sucrose, starch

b. Intermediate adsorbents e.g. CaCO₃, Ca-phosphate

c. Strong adsorbents e.g. alumina, charcoal

Step in Column Chromatography:

1. Preparation of column / Packing of column

The glass wool or cotton plug is used as the support for the column. It is first kept in vertical position. The chromatography column is packed with adsorbent material by filling it about 1/3^rd full solvent and slowly adding slurry of material in solvent. This is carefully poured down with glass rod to stop air buddle being trapped into the column. The suspension is allowed to settle and excess solvents are allowed to run off. This process is repeated until the column of required height is obtained (5 cm of height). Column is packed with dry power but wet packing is more common. In the dry packing, the dry powered adsorbent is introduced through the open end. Then, vaccum is created at bottom and column is tapped with light object until no more setting takes place.

2. Solvent system

In case of column chromatography, a single solvent is used. For better separation, it is found that the solution of mixture should be prepared in non polar solvent such as Petroleum ether, benzene, carbon tetrachloride, ethylacetate, acetone, cyclohexane, ether, chloroform etc.

Solvent in column chromatography functions in the following ways

a. Solvent brings about the separation of mixture into different zones/bands.

b. Solvent puts the mixture on the column

c. Solvent also elutes the components of each separated bands

For all this processes, sometimes only a single solvent is used but it is advantage to use different solvents for different functions.

Polar solvents like water, alcohol etc hold the solute fast and separation of the solute from the solvent is more difficult. The more polar is adsorbent solute, the more polar the mobile phase should be used. This is reason why adsorption is generally not useful for polar compounds. Thus, the transfer of mixture to the adsorbent column is usually carried out by making use of non polar solvents whereas elution of solute from the column can be carried out by using strongly polar solvents.

3. Application of Samples

Several methods are available for the application of sample to the top of the prepared column.

1.   Simple way is to remove most of the mobile phase from above the column by substrate and just to drain remainder solvent into the column bed. Then, sample is carefully applied by pipette and allowed just to run in the column. A small volume of mobile phase is applied to wash final traces of sample into column bed. Mobile phase is then carefully added to column to the height of 2-5 cm. Then, column is connected to a suitable reservoir that contains more mobile phase so that the height of mobile phase can be maintained at 2-5 cm.

2.   An alternative procedure which avoids the necessity to drain the column to the surface of the bed is to increase the density of the sample by the addition of sucrose to a concentration of about 1 % when the solution is layered into the liquid above the column bed, it will automatically sink to the surface of column and hence can be quickly passed into the column.

3.   A third method involves the use of capillary tubing or use of roller pumps to pass the sample directly to the column surface.

In all cases, care must be taken to avoid overloading the column with sample, otherwise irregular separation will occur.

4. Column development and sample elution

In column chromatography, the term development implies the irrigation of the adsorbed mixture and resolution of component into distinct bands. After sample application, the next step is to remove the material from the column with appropriate solvent. Development may be carried out in several ways.

A. Frontal development: A large volume of mixture to be separated is passed through a column of adsorbent. Pure solvent appears first followed by least strongly held substance then a mixture of least and then least strongly held and so on. Only the strongly held substances appear in pure form.

B. Displacement development: A solvent is used which have the higher affinity for the stationary phase than any of the components of mixture to be separated. All components are thus displaced down of the column. In displacement development, the solvent interact most strongly with chromatographic material (adsorbent) then the compound to be separated down the column, thus displacing the bound molecule.

C. Elution development: The components of applied sample are separated by continuous flow of mobile phase through the column and this is called elution development. In this method the solvent interact with the column material more weakly than the solute molecules and drags the bound molecule gradually eluting them from the column.

5. Collection and analysis of fraction

The fraction from the column is collected into the series of test tubes either manually or with fraction collector. Each fraction is then analyzed for the presence of compound. For colored compounds, detection can be achieved simply by visual observation. But for colorless compound detection can be done by Spectrophotometric method or by Colorimetry method or by UV adsorption method. E.g. Protein can be detected and quantified at 190-220 nm and at 260-280 nm. For amino acids, quantization is done at 570 nm by reacting with ninhydrin.

Application of Column Chromatography

i.    Adsorption chromatography has been used primarily for the separation of organic compounds and separation based on adsorption chromatography depends upon the equilibrium that governs the distribution of various solute species between the solvent and the surface of solid. Large difference exists in the tendencies of compounds to be adsorbed. The tendencies to be adsorbed decreases in following manner;

Acid > Alcohol > Carbonyl compounds > Ester > Hydrocarbon

ii.   It is useful for separating non polar or fairly polar organic molecules.

iii. Commonly used adsorbents: Silica, Alumina, Carbon etc are commonly used adsorbents. Silica adsorbents are acidic and good for the separation of basic materials whereas the alumina is basic and better suited for the separation of acidic materials.

iv. Adsorption chromatography is most commonly used to separate non-ionic, water insoluble compounds such as triglycerides, vitamins and many drugs.

v.   It has widely been used for the chromatography of polycyclic compounds, phenols and amines.

vi. It is used to determine amino acid content of protein hydrolysate.

vii. It is used in the separation of urinary ketosteroids, separation of plasma cortisol and separation of mixtures containing stereoisomers or related compounds.

viii. It is used in testing the homogeneity of colored compounds.

ix. It is used in removing small impurities whose structures differ widely from that of major components. The impurities get adsorbed.

x.   In establishing the identity and non identity of two substances: E.g. two substances whose identity is to be determined are mixed and the solution is allowed to pass through a vertical adsorbent column. On elution, bonds are developed corresponding to substances. If only one bond produced it shows that the two substances are different.

NOTE: It should be noted that success of column chromatography depends upon the following factors.

·         Nature of adsorbents

·         Nature of solvents

·         Rate of flow of solvent through column

·         Dimension of column

·         Particle size of adsorbents

Temperature of column during separation