Introduction to Chromatography

By: Pharma Tips | Views: 6145 | Date: 01-Apr-2011

Chromatography is the science, which includes the studies of separation of molecules based on differences in their structure and/or composition. Chromatographic separations can be carried out using a ` of supports, includingo Immobilized silica on glass plates (thin layer chromatography)o Volatile gases (gas chromatography), paper (paper chromatography)o Liquids, which may incorporate hydrophilic, insoluble molecules (liquid chromatography).

1.1   WHAT IS CHROMATOGRAPHY?

Chromatography is the science, which includes the studies of separation of molecules based on differences in their structure and/or composition. Chromatographic separations can be carried out using a ` of supports, including
o     Immobilized silica on glass plates (thin layer chromatography)
o    Volatile gases (gas chromatography), paper (paper chromatography)
o    Liquids, which may incorporate hydrophilic, insoluble molecules (liquid chromatography).

1.2   TYPES OF LIQUID CHROMATOGRAPHY

1.2.1 Ion-Exchange Chromatography

Proteins are made up of twenty common amino acids. Some of these amino acids possess side groups ("R" groups), which are either positively or negatively charged. A comparison of the overall number of positive and negative charges will give a clue as to the nature of the protein. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic. When the protein contains a predominance of ionic charges, it can be bound to a support that carries the opposite charge. A basic protein, which is positively charged, will bind to a support, which is negatively charged. An acidic protein, which is negatively charged, will bind to a positive support. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge. Families of molecules (acidic, basics and neutrals) can be easily separated by this technique. This is perhaps the most frequently used chromatographic technique used for protein purification.

1.2.2 Hydrophobic Interaction Chromatography ("HIC")

Not all of the common amino acids found in proteins are charged molecules. There are some amino acids that contain hydrocarbon side-chains, which are not charged and therefore cannot be purified by the same principles involved in ion-exchange chromatography. These hydrophobic ("water-hating") amino acids are usually buried away in the inside of the protein as it folds into its biologically active conformation. However, there is usually some distribution of these hydrophobic residues on the surface of the molecule. Since most of the hydrophobic groups are not on the surface, the use of HIC allows a much greater selectivity than is observed for ion-exchange chromatography. These hydrophobic amino acids can bind on a support, which contains immobilized hydrophobic groups. It should be noted that these HIC supports work by a "clustering" effect; no covalent or ionic bonds are formed or shared when these molecules associate.
 
1.2.3 Gel-Filtration Chromatography

This technique separates proteins based on size and shape. The support for gel-filtration chromatography is beads, which contain holes, called "pores," of given sizes. Larger molecules, which can't penetrate the pores, move around the beads and migrate through the spaces, which separate the beads faster than the smaller molecules, which may penetrate the pores. This is the only chromatographic technique, which does not involve binding of the protein to a support.

1.2.4 Affinity Chromatography

This is the most powerful technique available to the chromatographer. It is the only technique, which can potentially allow a one-step purification of the target molecule. In order to work, a specific ligand (a molecule which recognizes the target protein) must be immobilized on a support in such a way that allows it to bind to the target molecule. A classic example of this would be the use of an

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