Ion-exchange chromatography

1. Genaral information
- Ion Exchange Chromatography (IEC) is a powerful liquid chromatographic technique
used for bioseparation. The separation is done by a reversible interaction between
charged molecules of the sample with charged ligands attached to a column.
- Ion-exchange chromatography is a type of chromatography that separates analytes
based on charge. A column is used that is filled with a charged stationary phase on a
solid support, called an ion-exchange resin. Strong cation-exchange chromatography
preferentially separates out cations by using a negatively-charged resin while strong
anion-exchange chromatography preferentially selects out anions by using a positively-
charged resin.

- Classification: Ion-exchange chromatography is divided 2 type: Cation-exchange

chromatography is used for the positively charged molecules. In this type of
chromatography, the stationary phase is negatively charged, which attracts the
positively charged molecules of interest. Whereas, in anion-exchange chromatography,
the stationary phase is positively charged which attracts the negatively charged
molecules.
+ Strong-cation exchange resins typically feature sulfonic acid functional groups
while weak cation-exchange resins have carboxylic acids.
+ Strong anionic-exchange resins contain quaternary amines while weak anion-
exchange resins feature secondary or tertiary amines
2. The elements affecting
- The resin: Some of the factors affecting resin choice are anion or cation exchanger,
flow rate, weak or strong ion exchanger, particle size of the resin, and binding
capacity.
- The pH of buffet: The pH needs to be maintained so that the analyte of interest is
charged and will bind to the resin. In cation-exchange chromatography, raising the pH
will cause the analyte to be less positively charged and less likely to interact with the
 negatively-charged resin. Similarly, in anion-exchange chromatography, lowering the
pH will cause the analyte to be less negatively-charged and less likely to interact with
the positively-charged resin. Thus pH adjustments can also be used to selectively elute
analytes from the column.
3. Tools and equipment
 In laboratory:
- Ion- exchange column is Cylindrical glass column. Column is often stuffed the
cationit: R-H or R-Na, the anionit: R-OH or R- Cl.

- Injector, conduits, valves, detector and recorder or data system.

 In industry:
- Centrifugal filter equipment

- Ion- exchange column: Cylindrical columns, several meters high, made of metal.

- Injector, conduits, valves, detector and recorder or data system.

- The solvent reservoirs are made of metal or plastic. The solvent reservoirs are attached
with a solvent selection valve and high-pressure pump.
4. The applycation
The method offers a sizeable sample-handling capacity, powerful resolving ability,
broad applicability, moderate cost, and the ease of handling. These characteristics have
made the ion-exchange chromatography one of the most versatile and widely used
liquid chromatography techniques.
- Ion-exchange chromatography is widely used in biochemistry to isolate and purify
protein samples
- Some type of ion-exchange chromatography is also used in water softeners.
- Ion-exchange chromatography is also useful in metal purification. It can be used to
purify actanides, such as plutonium, and remove it from spent nuclear reactor fuel
rods.
 - This type of chromatography is popular for sample preparation, for example in the
cleanup of proteins or nucleic acid samples.

EX1: purify protein samples

Process 1: anion-exchange process

the buffer
CH3COONa pH= 5.8

whey feed
(protein) column

Eluent:
centrifugal CH3COONa anion-
pH= 5.0 exchange
process

Column after
protein pI > 5.0 (β- process 1
lactoglobulin, albumin)

Eluent:
centrifugal CH3COONa
pH= 4

Column after
protein pI >4 (α- process 2
lactalbumin)
Process 2: cation-exchange process

Column after
process 2

The buffer:
CH3COONa centrifugal cation-
pH= 5.5 exchange
Eluent: process
CH3COONa
pH= 8.5

protein pI >8.3
(immunoglobulin G và
lactose)

EX2: Water softeners

Hard water
( Mg2+, Ca2+)

ion exchange column

( Na+, RCOO-)

Anion links with

Mg2+, Ca2+
ion exchange column
(RCOO)2Mg, (RCOO)2Ca
…
Desorp by
Soft water NaCl through