Eluent composition protein separations

Polypeptides with MW > 10,000 often show peak broadening on the 5-or 10- m porous alkyl silicas now commonly in use for peptide separation. These supports have pore diameters typically On the order of 10 nm and the reduced efficiencies may be due to restricted diffusion of the polypeptide through the pores. The use of the larger-pore-sized silicas, e.g., 33, 50, or 100 nm, bonded withw-alkyl ligands has been found (43.44) to circumvent, to some extent, the reduced resolution and improve recovery. Because polypeptides, proteins, and some peptides show very steep dependencies of their log A s on % (see Section III,E), the issue of optimtil flow rate at any given eluent composition must, however, be taken into account with these larger-pore-size silicas. 

Amino Acid Composition. Proteins comprise 20 different kinds of amino acids. It is important to know the relative abundance of these component amino acids in a protein. Knowledge of the numbers of different amino acids is also important in determining the sequence of the amino acids in a protein molecule. To determine the amino acid composition of a protein, it is hydrolyzed in 6 M HC1 for a few hours and then separated by electrophoresis or by chromatography. The individual amino acid spots on an electrophoretogram are dyed with ninhydrin to facilitate its visualization. The number of amino acids in each spot is determined by colorimetric because the intensity of dye in each spot is related to the number of amino acids. Alternatively, the amino acids separated by chromatography as eluents are dyed with fluorescent dye, and the number of amino acid in a particular eluant is again determined spectroscopically as the number of amino acid is proportional to the amount of dye absorbed by the amino acids. The whole process is automated, and the commercially available machine called an amino acid analyzer determines the amino acid composition of a protein within a few hours. The amino acid analyzer was first developed at Rockefeller University in New York City. 

Gel filtration chromatography has been widely used to purify biomolecules due to its simplicity and efficiency (PastoreUo and TrambaioU, 2001). The technique separates proteins on the basis of their molecular size independent of the composition of the buffer used as eluent. Different media are used to perform the molecular exclusion in packed columns. The inertness and physicochemical stability of the chosen media are key requirements in column selection. Details of the technique can be found elsewhere (Amersham, 2002). Gel filtration is generally used in combination with the other chromatographic techniques described (Neyestani et al 2003). 

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