Downstream processing (purification by chromatography)

Question 1

Describe chromatography

Answer 1

Chromatography usually consists of a stationary phase and mobile phase.
* The stationary phase is the porous solid matrix packed in a column (equilibrated with a suitable solvent) on to which the mixture of compounds to be separated is loaded. The compounds are eluted by a mobile phase.

Question 2

State some examples of different types of chromatography

Answer 2

1. Ion-exchange chromatography (net charge)
2. Hydrophobic interactions chromatography (polarity)
3. size exclusion chromatography
4. Affinity chromatography (biological affinity)

Question 3

Describe gel-filtration chromatography

Answer 3

In this technique, the separation of
molecules is based on the size, shape and molecular weight.
The sponge-like gel beads with pores serve as molecular sieves for separation of smaller and bigger molecules. A solution mixture containing molecules of different sizes (e.g. different proteins) is applied to the column and eluted.
The smaller molecules enter the gel beads through their pores and get trapped.
On the other hand, the larger molecules cannot pass through the pores and therefore come out first with
the mobile liquid.

Question 4

What is gel-filtration chromatography used for in the industrial scale?

Answer 4

At the industrial scale, gel-filtration is particularly useful to
remove salts and low molecular weight compounds from high molecular weight products.

Question 5

What are the 2 types of ion-exchangers in ion-exchange chromatography?

Answer 5

cation- exchangers which have negatively charged groups like carboxymethyl and sulfonate, and

anion- exchangers with positively charged groups like diethylaminoethyl (DEAE)

Question 6

Name some cation exchangers and anion exchangers

Answer 6

cation-exchangers:
Dowex HCR and Amberlite IR,

anion-exchangers:
Dowex SAR and Amberlite IRA

Question 7

What is affinity chromatography?

Answer 7

Affinity chromatography is based on an interaction of a protein with an immobilized ligand. The ligand
can be a specific antibody, substrate, substrate analogue or an inhibitor. The immobilized ligand on a solid matrix can be effectively used to fish out complementary structures

Question 8

For affinity chromatography, list some examples of ligand-type of protein

Answer 8

1. Antibody - antigen
2. cofactor - enzyme
3. receptor - hormone
4. hapten - antibody
5. inhibitor - enzyme
6. lectins - glycoproteins
7. heparin - coagulation factors
8. metal ion - metal ion binding proteins

Question 9

Name some matrices that are used for LC purification of proteins

Answer 9

1. Agarose
2. porous silica
3. Cellulose
4. Polyacrylamide
5. cross-linked dextran
6. polystyrene