Guide to Ion-Exchange Chromatography

1 Guide to Ion-Exchange ChromatographyIntroduction ..2-4 Protocol Samples ..5 Factors Effecting Selectivity ..6-7 Components of Ion exchange Media ..8-9 Experimental Conditions & Method of exchange Chromatography is the reversible adsorption of charged molecules toimmobilized ion groups on a matrix of an opposite charge. Separation can beselectively achieved by adsorption and release of samples from the matrix. Ionexchange starts with the equilibration of the exchanger using pH, and ionicstrength. During equilibration the exchangable groups are associated with counterions. Once equilibrium is reached and the sample added the molecules undergoaddition and adsorption with an appropriate charge displace the counter ions andbind reversibly to the matrix.

2 The unbound materials will pass through thecolumn with the void volume. In the third stage, substances are removed from thecolumn by increasing the ionic strength of the eluting media is an insoluble matrix with charge groups covalently charged exchangers bind positively charged ions (cations). Exchangersbind one type of cation but, when presented with a second type of cation, it maydisplace, and/or exchange with, the first. These resins are called cation-exchangeresins. Anion- exchange resins are positively charged and bind and/or exchangenegatively charged ions (anions).Several side-chain groups of the amino acid residues in proteins are ionizable ( or glutamic acid) as are the N-terminal amino and C-terminal carboxylgroups and so proteins are charged molecules.

3 This characteristic can be used toseparate different proteins by Ion-Exchange of Exchangers Two weak exchangers can be used for protein separation are carboxymethyl-cellulose (CM-cellulose) and diethylaminoethyl-cellulose (DEAE-cellulose). Thesecovalently cross-linked agarose bead ion celluloses have been chemically has a carboxymethyl functional group -CH2 OCH2 COOH. Atneutral pH the carboxymethyl group is ionized as -CH2 OCH2 COO so that CM-cellulose is negatively charged, so it is a weak cation exchanger. DEAE-cellulosecontains an diethylaminoethyl group. It is positively charged at neutral pH and soDEAE-cellulose is a weak anion exchanger.

4 The Sepharose types are particularly useful for the separation of high molecularweight proteins. In practice, since these matrices are very similar to those used forgel filtration some molecular sieving may accompany the Ion-Exchange may either enhance or reduce the effectiveness of the fractionation comparedto using an Ion-Exchange to Ion-Exchange Chromatography3 Introduction (cont.)Types of Exchangers (cont.)Two strong exchangers are Q-Sepharose Fast Flow and SP-Sepharose Fast charged group of Q-Sepharose is a quarternary amine which carries a non-titratable positive charge. This matrix can be used at alkaline pH values at whichthe positive charge of the DEAE group would have been titrated.

5 The chargedgroup of S-Sepharose is the sulphonyl group (-SO3 ).MethodThe fractionation of proteins by Ion-Exchange Chromatography depends upondifferences in the charge of different proteins. The charge of a protein dependsupon the number and type of ionizable amino acid side chain groups. Lysineresidues, have a positively charged side chain group when ionized, whereasglutamic acid residues are negatively charged when ionized. Each ionizable sidechain group has a distinct pKa; that is, the pH at which it is half the overall number of charges on a particular protein at a particular pHwill depend on the number and type of ionizable amino acid side chain groups itcontains.

6 Since, by definition, different proteins have different amino acidcompositions, they will tend to have different charges at a given pH and so can befractionated on this protein has a isoelectric point (pI) where at a certain pH the overall numberof negative charges equals the number of positive charges and so it has no netcharge. The pI, is the proteins isoionic point. When a protein is at its pI theprotein will not bind to the Ion-Exchange resin. Below this pH the protein willhave a net positive charge and will bind to a cation exchanger, and above this pHit will have a net negative charge and bind to an anion exchanger.

7 In principle, either a cation exchanger or an anion exchanger to bind the proteinof interest. In practice proteins are stable and functionally active within a fairly narrow pHrange so the choice of ion exchanger is often dictated by the pH stability of thedesired protein. If the protein is stable at pH values below its pI, a cationexchanger should be used if it is stable at pH values above its pI, an anionexchanger would be chosen. If a protein is stable over a wide pH range, then either type of media should betried and the selection depends on the best media, usually have the ions bound to the charged groups on theresin are called counter ions.

8 For CM-cellulose the counter ion is usually Na+and for DEAE-cellulose the counter ion is normally Cl . (cont.)Method (cont.)After choice of the appropriate resin, it is mixed with buffer to form a slurrywhich is poured into a suitable Chromatography column. The pH of this startingbuffer is crucial since it will determine the charge on the proteins to be starting buffer pH should be at least one pH unit above or below the pI ofthe protein to be bound to the resin to ensure adequate binding. However, bear inmind that CM-cellulose and DEAE-cellulose are examples of weak ion weak ion exchanger is one which is ionized over only a limited pH range.

9 ThusDEAE-cellulose begins to lose its charge above pH 9 whilst CM-cellulose beginsto lose its charge below about pH 5. The term weak does not refer to thestrength of binding of ions to the resin nor to the physical strength of the resinitself. With these points in mind then, the effective starting pH range when usingDEAE-cellulose or CM-cellulose is only about pH 5 - 9. In addition to correctchoice of the pH of the starting buffer, one should take care that its ionic strengthis reasonably low since the affinity of proteins for Ion-Exchange resins decreases asionic strength increases. Indeed this property is used in one method of eluting thebound to Ion-Exchange Chromatography5 Protocol SamplesThe SpinColumns are supplied dry and need to be rehydrated, the bed of Ion-Exchange resin with starting buffer allow 10-15 minutes for rehydration.

10 Afterrehydration add a 2ml collection tube to the bottom of the SpinColumn andcentrifuge for 1 minutes at 1000rpm. After centrifugation the protein mixture isapplied. Proteins which are oppositely charged to the media at the starting pH willbind to it, so displacing the counter ions. Proteins with the same charge as theresin or with no net charge will not bind and flow straight through the different proteins bound to the column, one of which will be the protein ofinterest, will have different affinities for the ion exchanger due to differences intheir net charge. These affinities can be altered by varying the ionic strength of thecolumn buffer or alternating the pH of the elution #1A mixture of proteins are bound to the anion exchanger.