Investigation of perchlorate, phosphate and ion-pairing eluent modifiers for the separation of cephalosporin epimers

The retention behavior of several pairs of 7 alpha- and 7 beta-cephalosporin epimers was investigated using perchlorate, phosphate and ion-pairing eluent modifiers. At pH 2.5, sodium perchlorate, sodium phosphate and sodium pentanesulfonate all provided separation of epimers with free 7-amino groups. When the 7-amino group was blocked, as in cephalexin and cefaclor, sodium perchlorate gave the best separation at pH 2.5. A tetrabutylammonium ion-pairing system at pH 7.0 provided separation of all epimer pairs containing a free carboxylic acid at the 3 position. Hydrophobic, residual silanol and ionic interactions were factors in the retention mechanism of the cephalosporins under the conditions investigated. An ionic interaction of perchlorate with the protonated amine of the cephalosporin was postulated as an explanation of the retention and selectivity effects observed with perchlorate as an eluent modifier.