A complete separation of the twenty protein amino acids and two internal standards in 35 min has been made possible by analyzing their N-trifluoroacetyl n-butyl (TAB) derivatives simultaneously on an ethylene glycol adipate (EGA) column and on an OV-17 column using the same temperature programme. The detector signal from the EGA column is recorded up to the point just after the elution of TAB-aspartic acid; thereafter, the signal from the effluent of the OV-17 column is recorded. The TAB derivative of norleucine is poorly resolved from TAB-leucine and TAB-proline. Therefore, alpha-aminocaprylic acid has been used as the internal standard instead of noreleucine. TAB-alpha-aminocaprylic acid appears as a separate peak between TAB-serine and TAB-cysteine.