The hydrolytic solution of proteins mainly contains free amino acids and peptides. The separation of amino acids and di- and tri-peptides is very significant and also a complicated work. This report presents a chelated metal ion affinity chromatographic (CMAC) method for the separation of alpha-amino acids and peptides. Sephadex G10 was used as the solid matrix. It was epoxy-activated by epichlorophydrin; then coupled with iminodiacetate (IDA) and chelated with copper ion to produce immobilized copper-ion affinity chromatographic packing. Some examples are given for the chromatography of model mixtures of L-Val, L-His, L-Tyr, L-Try, Tyr-Try dipeptides and protein hydrolyzing solution of fish. The separation was based on the different stabilities of copper complexes of alpha-amino acids, peptides and IDA-Sephadex G10. The components which form weak complexes with copper apparently move along with the solvent front. Alpha-amino acids-copper complexes with a stability comparable to that of copper-IDA-Sephadex G10 are retained on the matrix. Peptides form strong complexes and catch copper from the matrix. They are only slightly retained. The results showed that alpha-amino acids and peptides were completely separated under the experimental conditions.