Reversed-phase high-performance liquid chromatography of amphoteric beta-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times.

The separation of five amino beta-lactam antibiotics by reversed-phase high-performance liquid chromatography was studied as an insight into their retention behaviour. These five amphoteric compounds are cephradine, cephalexine, cefaclor, ampicillin and amoxicillin. Both octadecylsilane-bonded silica (C18) columns and phenyl-bonded silica (phenyl) columns were used, with mobile phase pH values between 2.5 and 7.4. In the absence of ion-pairing reagents the retention times for all the five compounds were the shortest at pH 4-6. The phenyl column was found to improve the separation between cephradine and ampicillin at pH values lower than 3, when these two compounds appeared as fused peaks on the C18 on C18 columns, with mobile phases both with and without ion-pairing reagents, were compared. The addition of 0.005 or 0.02 M tetraethylammonium acetate to the mobile phase did not result in significant ion-pair formation, except at pH values higher than 5.5. A strong ion-pairing effect was obtained at pH values higher than 6 with 0.005 or 0.02 M tetrabutylammonium phosphate, and the retention was decreased at pH values lower than 4. On the other hand, 0.005 M heptanesulphonic acid exhibited an ion-pair retention effect at pH values lower than 5. The molecular structures and pK(a) values were used to account for the retention behaviour of these antibiotics in the various mobile phases.