4-6- Ion Exchange Chromatography

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Chromatography

Ion Exchange Chromatography

Chapter 4

1Dr Gihan Gawish


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Definition

Dr Gihan Gawish

Ion-exchange

chromatography (orion chromatography)is a process thatallows theseparation of ionsand polar molecules

based on the chargeproperties of themolecules.

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Ion-exchange chromatography

Dr Gihan Gawish

The solution to be injected is usually called a sample,

and the individually separated components are

called analytes

It can be used for almost any kind of charged

molecule including large proteins, small nucleotides

and amino acids.

It is often used in protein purification, water analysis.

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Principle

Dr Gihan Gawish

Ion exchange chromatography retains analytemolecules based on ionic interactions.

The stationary phase surface displays ionic functionalgroups (R-X) that interact with analyte ions ofopposite charge.

This type of chromatography is further subdividedinto:

1. cation exchange chromatography

2. anion exchange chromatography.

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Ion Exchangers

Dr Gihan Gawish

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Ion exchangers Functional groups

Anion exchanger

Aminoethyl (AE-)

Diethylaminoethyl(DEAE-)

Quaternary

aminoethyl (QAE-)

Cation exchanger

Carboxymethyl (CM-)

Phospho Sulphopropyl (SP-)

Dr Gihan Gawish

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Cation exchange chromatography

Dr Gihan Gawish

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 + BR-X C +M B R-X M + C + B-- ++ ++ --

__++ ++ --

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Anion exchange chromatography

Dr Gihan Gawish

Anion exchange chromatography retains anions

using positively charged functional group:

R-X A +M B R-X B + M + AR-X A +M B R-X B + M + A++__

++ -- ++ -- ++--

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Procedure

Dr Gihan Gawish

1. A sample is introduced, either manually or with anautosampler, into a sample loop of known volume.

2. The mobile phase (buffered aqueous solution)carries the sample from the loop onto a column thatcontains some form of stationary phase material.

3. Stationary phase material is a resin or gel matrixconsisting of agarose or cellulose beads withcovalently bonded charged functional groups.

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Procedure

Dr Gihan Gawish

4. The target analytes (anions or cations) are retainedon the stationary phase but can be eluted byincreasing the concentration of a similarly charged

species that will displace the analyte ions from thestationary phase.

For example, in cation exchange

chromatography, the positively charged analytecould be displaced by the addition of positively

charged sodium ions.

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Procedure

Dr Gihan Gawish

5. The analytes of interest must then be detected

by some means, typically by conductivity or

UV/Visible light absorbance.

6. A chromatography data system (CDS) is usually

needed to control an IC.

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Procedure

Dr Gihan Gawish

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Separating proteins

Dr Gihan Gawish

Proteins have numerous functional groups that can

have both positive and negative charges.

Ion exchange chromatography separates proteins

according to their net charge, which is dependent

on the composition of the mobile phase.

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Affect of pH in the separation of proteins

Dr Gihan Gawish

By adjusting the pH or the ionic concentration of the

mobile phase, various protein molecules can be

separated.

For example, if a protein has a net positive charge

at pH 7, then it will bind to a column of negatively-

charged beads, whereas a negatively chargedprotein would not.

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Effect of pH in the separation of proteins

Dr Gihan Gawish

Proteins are charged molecules. At specific pH, it

can exist in anionic (-), cationic (+) or zwitterion

(no net charge) stage.

cationic pH =pI anionic

pH increasepH increase

*pI isoelectric point*pI isoelectric point

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Dr Gihan Gawish

Important to consider the stability of proteins in choice of

ion exchangers. Isoelectric focusing can be used to identify

suitable ion-exchanger type

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4-6- Ion Exchange Chromatography

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