The effect of ion pairing reagents in the retention profile of zwitterionic cephalosporins

The retention of three zwitterionic cephalosporins, Cefepime, Cefpirome, and Ceftazidime, in a broad pH range was studied by means of reversed phase high performance liquid chromatography (RP-HPLC) and reversed phase ion-pair liquid chromatography (RP-IPC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexane-, heptane-, and octane-sulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion pair chromatography. The presence of the permanently charged quaternary nitrogen in the cephalosporin molecules leads to zwitterionic species over a broad pH range, masking the carboxylic acid function and stabilizing the retention. The effect of the counter ion depends on the ionization state of the compounds. Formation of ion pairs with the opposite charged center of the molecules led to significant increase in retention. At pH values favoring the zwitterionic species a decrease in retention was observed as a result of the disruption of the intramolecular interactions and the deliberation of the free charge. The extent of ion pair formation and the competition with the zwitterionic species, depended on the counter ion concentration in the mobile phase and was reflected in retention. The effect of the size of the counter anion alkyl chain on retention was investigated at different pH as well.