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Types of Chromatography: Affinity, Gel, Ion exchange and Reverse phase

BY: Muniba Safdar | Category: Genetics | Submitted: 2010-07-25 14:50:59
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Article Summary: "Chromatography is any separation technique that distributes the components of a mixture between two phases. Chromatography has various types but in Proteomics 'Liquid Chromatography' is mostly used..."


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Chromatography is any separation technique that distributes the components of a mixture between two phases.

1. Fixed stationary phase
2. Free moving mobile phase

Chromatography has various types but in Proteomics "Liquid Chromatography" is mostly used. For the representation of chromatography in liquid chromatography beads are used in stationary phase packed in column while mobile phase is dissolved sample. Other types of chromatography are:

i. Affinity chromatography
ii. Gel filtration chromatography or size exclusion
iii. Ion exchange chromatography
iv. Reverse phase chromatography

i. Affinity Chromatography

It is a selective (specific to study function and interaction of protein) technique in which binding affinity is exploited. We attach any component with the beads, those components which specifically bind with the desired protein. For example, concavalin A (specifically binds with glucose).

Three step process:

Step 1: Covalently attach X-molecule e.g. glucose to the beads or to the column.
Step 2: Adding a mixture of protein to the column then wash the column with buffer to remove unbound proteins except concavalin.
Step 3: Eluting the desired protein by adding a high concentration of a soluble form of X-molecule or altering the conditions to decrease binding affinity (changing of temperature and pH).

ii. Gel filtration chromatography or size exclusion

This technique separates protein on the basis of size. In gel filtration, beads are made of two gels Agarose and Polyacrylamide.

• The sample is applied to the top of a column consisting of porous bead made of an insoluble but highly hydrated polymer such as Dextran or agarose.
• Smaller molecules can enter these beads but large one cannot.
• The result is that smaller molecules are dissolved in the aqueous solution both inside the beads and between them.
• Large molecule flow more rapidly through this column and emerge first.
• Molecules that are of a size to occasionally enter a bead will flow from the column at an intermediate position.

iii. Ion exchange chromatography

This technique separates proteins on the basis of charge. There are two methods of ion exchange chromatography.

a. Anion exchange chromatography

• In anion exchange chromatography our stationary phase is highly positive charge.
• Beads are made of Diethylaminoethyl cellulose (DEAE-cellulose).
• Negatively charged proteins are bound with the beads but positively charge will elute out.
• Add buffer to remove unbound proteins.
• We can unbind the bound protein by changing its pH or buffer.

b. Cation exchange chromatography

• In cation exchange chromatography beads are made of Carboxymethyl cellulose.
• Positively charged proteins are bound with the beads but negatively charge will elute out.
• We can add sodium chloride, it will compete with the protein and will attach with beads.
• We can also change the pH.

iv. Reverse phase chromatography

• In this technique stationary phase is made of non-polar material such as silicon or alumina.
• Polar proteins will bound with the beads and remaining protein will elute out. Because silicon has a high affinity to attach with polar molecule.
• If we add alkyl chain with the silicon beads non-polar protein will be attached with the proteins.

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