Abstract The retention of ampicillin, amoxicillin, and penicillin G was determined on ODS and cyanopropyl (CN)-bonded to silica, on an all-organic polymeric reversed-phase column (styrene-divinylbenzene) and on bare silica. Solvents tested were 0.01 M H 3PO 4 (pH 1.6)-acetonitrile and 0.002 M KH 2PO 4 (pH 4.6)-acetonitrile in the proportions 100:0 to 10:90. Retention with 0.002 M KH 2PO 4 was weaker than with 0.01 M H 3PO 4 at all proportions of acetonitrile on all packings. With 0.01 M H 3PO 4, retention of penicillins on the polymeric column decreased rapidly from 100 to 50% aqueous phase and remained low to 10% aqueous phase. On the ODS bonded column, retention was similar, except that retention of the aminopenicillins, amoxicillin and ampicillin increased at < 30% aqueous phase. On the CN-bonded column, results were similar to those on the ODS column, except that the aminopencillins were less strongly retained when using 100% 0.01 M H 3PO 4. On the bare silica, penicillin G was poorly retained at all proportions of acetonitrile, while retention of aminopenicillins increased rapidly at < 50% 0.01 M H 3PO 4. Retention of aminopenicillins at < 50%. 0.01 M H 3PO 4 was parallel on bonded and bare silica but weaker on the bonded silica. A bonded organic layer weakened but did not prevent binding of amines to silica.