Abstract
Seven isocratic liquid chromatography (LC) methods have been examined for the separation of benzylpenicillin and its related substances on C18 or C8, phenyl and poly(styrene–divinylbenzene) columns. The method proposed by a manufacturer, using a C8 column and a mobile phase consisting of 0.05 M phosphate buffer, pH 3.5–methanol (62:38, v/v) gave the better selectivity. The amount of organic modifier was slightly adapted when it was used with different columns. Similar selectivity was obtained not only on C8 materials but also on C18 packings. The selectivity on poly(styrene–divinylbenzene) was worse. A resolution test with phenylacetic acid was used as the method performance test. The robustness of this method was examined by applying a full factorial design to test the influence of content of organic modifier in the mobile phase, the pH of the mobile phase, the concentration of buffer in the mobile phase and the column temperature. The results show that the method is robust.
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