Ion exchange chromatography What is ion exchange chromatography

Ion exchange chromatography

What is ion exchange chromatography ? • IEC is a technique for separating proteins according to their charge • Its easy of use and scale up capabilities • Large volumes be applied • High resolution and high capacity method

Principle Packing material • Resin are charged molecules • Securely bound to column by covalent bonds • Negatively or positively charged groups Anion exchanger • Positively charged beads “exchange” with negatively charged counter ions • Negatively charged molecules - “anions” Cation exchangers • Negatively charged beads exchange positive counter -ions (cations)

Functional Group Counter-ion Anion Exchanger -O-CH 2 -N + Diethylaminoethyl H (CH 2 CH 3)2 (DEAE) Cation Exchanger -O-CH 2 -COO Carboxymethyl (CM) Cl Na +

Separation of protein • Proteins are charged molecules • Interaction with the packing material depends on – Overall charge – Distribution of that charge over the protein surface • They displace mobile counter ions bound to the resin • Mobile counter ions: When the packing material is suspended in buffer containing Na. Cl, the charged groups become loosely associated with Na+ and Clions of the opposite charge. These loosely bound ions are called mobile counter-ions.

• Net charge on protein will depend on – Composition of amino acids in the protein – p. H of the buffering solution. • Charge distribution will depend on – How the charges are distributed on the folded protein • • Isoelectric point ( p. I) - The p. H at which a particular protein is overall electrically neutral ie. the number of positive charges is equal to the number of negative charges. < p. I - A protein has more positively charged amino acids and therefore an overall positive charge. It will bind to cation exchangers > p. I - A a protein has more negatively charged amino acids and an overall negative charge. It will bind to anion exchangers At its p. I, a protein will not bind to either a cationic or an anionic exchanger.

Column materials - Polystyrene • • • Ion-exchangers made by co-polymerisation of styrene with divinyl benzene. Polystyrene itself is a linear polymer. Divinyl benzene, is a cross-linker Resins with low degree of cross-linking are more permeable to high molecular weight compounds, but they are less rigid and swell more when placed in buffer Sulphonation of cross-linked polystyrene results in sulphonated polystyrene resin such as Dowex 50 - strong acidic exchanger Basic exchangers are prepared by reacting cross-linked polystyrene with chlormethyl ether and then by the chlorogroup with tertiary amines – CH 2 N+ (CH 3)3 Cl- groups are ionized.

Modified cellulose and Sepharose • Modified cellulose is an alternative to polystyrene based exchangers • Cellulose is a high molecular weight i. e. carboxymethyl cellulose (CM-cellulose) where the – CH 2 OH group is converted to –CH 2 OCH 2 COOH and DEAE-cellulose [CH 2 OCH 2 N(CH 2 CH 3)2] • Commercially available in gel and bead form • Sepharose type is derived from cross-linked agarose • Sephadex and Sepharose are used for the separation of high molecular weight proteins and nucleic acids

Total exchange capacity • Defined as number of milliequivalents of exchangeable ions available either per gram of dried exchanger or per unit volume of hydrated resins – Bio Rad AG 1 -X 4 = 1. 2 meq cm-3 – DEAE-Sephadex A-25 = 0. 5 meq cm-3 – CM-Sepharose CL-6 B = 0. 2 meq cm-3 • Polystyrene exchangers are obtainable in a number of mesh size. All exchangers are supplied with counter ions i. e Na+ or Cl-.

Classification of ion exchange media Media (X = matrix) Anion exchangers X-CH 2 N+(CH 3)3 X-CH 2 NH+(CH 3)2 (CH 3 CH 2)2 X-CH 2 NH+ diethylamino-ethyl (DEAE) Cation exchangers X-SO 3 - X-COO- X-CH 2 COOcarboxymethyl (CM) Nature Applications strong intermediate weak p. H range 2 - 11 2 - 7 3 - 6 strong intermediate weak 2 - 11 6 - 10 7 - 10 amino acids peptides proteins nucleotides organic acids proteins

Buffer • The p. H of the buffer used should be one p. H unit above or below isoionic point of the compounds. • Cationic buffers are tris, pyridine and alkyl amines and they are used with anion exchangers. • Anionic buffers are acetate, barbiturate and phosphate and they are used with cationic exchangers.

Sample application • Amount dependent upon size of the column and capacity of exchanger. • For the isocratic elution, sample volume is 15% of bed volume. • For the gradient elution, sample volume is not important. • Large volumes of dilute solution can be applied as they get bound at the top of column.

Elution • Gradient elution is most common and gives better results. • With anion exchanger, p. H gradient decreases and ionic strength increases. • With cation exchangers, both p. H and ionic gradients increase.

Applications • Separation of amino acids achieved by strong acid cation exchanger – Dowex 50 -PS and SP-Sephadex-cellulose • Separation of proteins by weakly acidic and basic exchangers derived from cellulose and agarose – Proteins with isoionic point < 7 – DEAE-cellulose using low ionic strength – Proteins with isoionic point > 7 – CM-cellulose using buffer p. H 4 -5 – Proteins with isoionic point 7 – Either • Determination of base composition of nucleic acids • RNA hydrolysed by both enzymatic and base hydrolysis. DNA resisted to base hydrolysis but cleaved by DNAases.

• Separation of amino acids achieved by strong acid cation exchanger – Dowex 50 -PS and SPSephadex-cellulose – Introduction of sample at p. H 1 -2 to ensure complete binding – Gradient elution by increasing p. H and ionic concentration – Acidic amino acids, aspartic and glutamic acids elute first followed by neutral amino acids, glycine and valine. Basic amino acids retain their net negative charge up to p. H values 6 -11 and elute last.