Separation of phenolic compounds and corresponding glucuronides by coupled-column micellar reversed-phase liquid chromatography

Abstract

SummaryAn isocratic HPLC technique for separation of phenolic compounds and corresponding glucuronides in urine is developed. Sample pre-treatment, often a tedious and rate-limiting factor, was eliminated by use of a coupled column system. Spiked urine samples were injected directly into a C4 precolumn and a selected fraction was transferred on-line from the precolumn to a silanized C18 analytical column in the backflush mode. Analyte peak enrichment was attained by employing mobile phases of different elution strengths. The weaker mobile phase (7 % v/v acetonitrile) was used to strongly retain the analytes, on the precolumn while most of the polar endogenous compounds were washed to waste. Elution and transfer of the trapped analytes from the precolumn to the analytical column was achieved by introducing a stronger mobile phase (20 % v/v acetonitrile) with the aid of a switching valve. The use of cetyltrimethylammonium bromide as counter ion and micellar agent in the mobile phase involved a high selectivity for the analytes relative to the urine matrix components and allowed simultaneous analysis of the glucuronides and parent compounds without the need of gradient elution. The system demonstrated a good repeatability on spiked urine samples.t Who passed away July 21, 1996

Optimization Strategy for Isocratic Separation of Halogenated Phenols by Reversed Phase Liquid Chromatography with Methanol-Water Mobile Phase

“…After HPLC separations, target phenols can be identified by means of a diode-array detector 23,24 . Isocratic elution from C18 columns with binary eluents of methanol-water is commonly used 15,25 .…”