This application displays separation of a variety of selected amino acids ranging from rather hydrophobic to more hydrophilic, including several with acidic or basic side chains. The separation employed a shallow HILIC gradient elution on an amide stationary phase, using acetonitrile and water buffered to pH 3.2 with formic acid and ammonium formate. Included amino acids were tryptophan, phenylalanine, leucine, isoleucine, glutamine, valine, cysteine, glycine, serine, glutamic acid, aspartic acid, histidine and arginine.