Chapter 4

Ion Exchange Chromatography

Dr Gihan Gawish


Dr Gihan Gawish .Definition 2 Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules.

water analysis. small nucleotides and amino acids. It is often used in protein purification. and the individually separated components are called analytes It can be used for almost any kind of charged molecule including large proteins. Dr Gihan Gawish .Ion-exchange chromatography 3 The solution to be injected is usually called a sample.

. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into: cation exchange chromatography anion exchange chromatography. Dr Gihan Gawish 1.Principle 4 Ion exchange chromatography retains analyte molecules based on ionic interactions. 2.

Ion Exchangers 5 Dr Gihan Gawish .

Ion exchangers ± Functional groups 6 Anion exchanger Aminoethyl (AE-) Diethylaminoethyl (DEAE-) Quaternary aminoethyl (QAE-) Cation exchanger Carboxymethyl (CM-) Phospho Sulphopropyl (SP-) Dr Gihan Gawish .

Cation exchange chromatography 7 Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group .+ + _ R-X C +M B R-X M + C + B + + - Dr Gihan Gawish .

Anion exchange chromatography 8 Anion exchange chromatography retains anions using positively charged functional group: R-X A +M B + _ + - + R-X B + M + A + Dr Gihan Gawish .

Dr Gihan Gawish 2. The mobile phase (buffered aqueous solution) carries the sample from the loop onto a column that contains some form of stationary phase material. 3.Procedure 9 1. into a sample loop of known volume. . A sample is introduced. Stationary phase material is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. either manually or with an autosampler.

Procedure 10 4. the positively charged analyte could be displaced by the addition of positively charged sodium ions. in cation exchange chromatography. The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. Dr Gihan Gawish . For example.

Procedure 11 5. 6. Dr Gihan Gawish . The analytes of interest must then be detected by some means. A chromatography data system (CDS) is usually needed to control an IC. typically by conductivity or UV/Visible light absorbance.

Procedure 12 Dr Gihan Gawish .

Separating proteins 13 Proteins have numerous functional groups that can have both positive and negative charges. Dr Gihan Gawish . Ion exchange chromatography separates proteins according to their net charge. which is dependent on the composition of the mobile phase.

For example. whereas a negatively charged protein would not. various protein molecules can be separated.Affect of pH in the separation of proteins 14 By adjusting the pH or the ionic concentration of the mobile phase. then it will bind to a column of negativelycharged beads. Dr Gihan Gawish . if a protein has a net positive charge at pH 7.

cationic pH =pI pH increase anionic *pI isoelectric point Dr Gihan Gawish .Effect of pH in the separation of proteins 15 Proteins are charged molecules. cationic (+) or zwitterion (no net charge) stage. it can exist in anionic (-). At specific pH.

Stability of proteins stable below pI value. use matrix of small pore size 10. use anion-exchanger Molecular size of proteins <10. Dr Gihan Gawish .000 mw. use Sepharose equivalent grade 2. use cation-exchanger stable above pI value.000-100.000 mw.Choosing your ion-exchanger: know your proteins 16 1.

Important to consider the stability of proteins in choice of ion exchangers. Isoelectric focusing can be used to identify suitable ion-exchanger type 17 Dr Gihan Gawish .

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