Determination of p-Hydroxyphenoxyacetic Acid in Phenoxymethyl Penicillin Fermentations

Abstract

A method based on TLC separation and subsequent colorimetric determination was developed for the determination of p-hydroxyphenoxyacetic acid in phenoxymethyl penicillin (penicillin V) fermentation samples. The p-hydroxyphenoxyacetic acid is extracted from penicillin culture fluid into amyl acetate. A portion of the extract is dried and then dissolved in acetone. An aliquot is chromatographed on a silica gel thin-layer plate by ethyl acetate-chloroform-acetic acid (9:1:0.15 v/v). The zones are located by spraying with an aqueous mixture of equal volumes of 1% ferric chloride and 1% potassium ferricyanide. The zones are transferred into glass tubes, treated by sodium nitrite in dilute sulfuric acid, and neutralized by ammonia. The absorbance of the orange supernate, which is proportional to the concentration of p-hydroxyphenoxyacetic acid, is measured spectrophotometrically at 478 nm.