Reversed-Phase High-Performance Liquid Chromatography of Peptides

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Abstract
A reversed-phase high performance liquid chromatographic methd for the determination of some peptides including growth-hormone releasing factor was studied. For the column packing, the endcapping of residual silanol groups of silica gel was important to obtain symmetric elution peak with high resolution, especially for basic peptides. Other parameters were optimized as follows: particle size, 5 .mu.m; pore size, 100.ANG.; stationary phase, C18. For the mobile phase, a mixture of acetonitrile and 0.1% trifluoroacetic acid containing 0.1 M sodium chloride was selected. Species and concentration of the salt and the counter ion, and pH of the mobile phase had a great influence on selectivities among peptides. The addition of the salt improved column efficiency and peak symmetry, and was effective to establish the analytical conditions in isocratic elution. Under these chromatographic conditions, coefficient of variations in repetitive injections of each peptide were below 0.65%.