This technique is used for separating any mixtures of substance into individual components . These techniques are applicable in many processes such as for separation, for identification and for quantitative analysis of amino acid mixtures , peptides, proteins, nucleotides, lipids and carbohydrates . Here some types are explaining :
a) Column chromatography- This process is successfully used for differentiates proteins using their difference in protein charge , size, binding energy , affinity and other properties .
b) Ion-exchange chromatography- This technique utilize differences in the sign and magnitude of the net charge of proteins at a available pH. The column matrix is a synthetic polymer such as resin which containing bound anionic groups are called cation exchangers and those with bound cationic groups are called anion exchangers. By changing their pH , salt concentration separation will be maximized.
c) Size exclusion chromatography- This technique is also known as gel filteration which separates proteins according to size . In this method , large protein emerges from the column faster than the small ones. The solid phase having cross-linked polymer beads with engineered pores or cavities of a particular size . Large proteins cannot enter into the cavities and so they take a short path through the column , whereas small proteins enter the cavities and are slowed down by the more labyrinthine path through the column.
d) Affinity chromatography- This technique is based on binding affinity and energy . The beads which is present in the column have a easily attached to the chemical group which is ligands . When any protein mixture is added into the column , but here any protein have special affinity for the particular ligand in the column binds with beads , and its migration through the matrix is delayed.
e) High-performance liquid chromatography- This techniques is use always high-pressure pumps which increases the movement of the protein molecules down into the column , as well as higher quality of chromatographic materials which can withstand the compressing force of the pressurized flow .
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