Chromatography is used to separate mixtures of substance into individual components.
These methods are applicable not only for separation, identification and quantitative analysis of amino acid mixtures but also peptides, proteins, nucleotides, nucleic acids, lipids and carbohydrates.
They are of the following types:
It is successfully used for fractionating proteins using their difference in protein charge, size, binding affinity and other properties.
It exploits differences in the sign and magnitude of the net charge of proteins at a given pH. The column matrix is a synthetic polymer (resin) containing bound anionic groups are called cation exchangers and those with bound cationic groups are called anion exchangers. By changing the pH, salt concentration separation can be optimized.
Also known as gel filtration separates proteins according to size. In this method, large protein emerges from the column sooner than the small ones. The solid phase consists of cross-linked polymer beads with engineered pores or cavities of a particular size. Large proteins cannot enter 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.
It is based on binding affinity. The beads in the column have a covalently attached chemical group called a ligand. When a protein mixture is added to the column, any protein with affinity for the particular ligand in the column binds to the beads, and its migration through the matrix is retarded.
It makes the use of high-pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can withstand the crushing force of the pressurized flow. HPLC can limit diffusional spreading of protein bands by reducing the transit time on the column, thus improving resolution.