Selective determination of thiolic proteins by hydrophobic interaction chromatography coupled with on-line cold vapour atomic fluorescence spectrometry

A new analytical method is proposed for the determination and characterizat ion of thiolic proteins, based on hydrophobic interaction chromatography (H IC) coupled on-line with cold vapour atomic fluorescence spectrometry (CVAF S). Thiolic groups are derivatized pre-column by p-hydroxymercurybenzoate ( PHMB) and the derivatized proteins are separated on a TSKgel Ether-5PW colu mn. Post-column on-line reaction of derivatized proteins with bromine, gene rated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of pr otein-bound PHMB to inorganic mercury, Hg(ii), which is selectively detecte d by AFS after sodium borohydride reduction to Hg-0. Under optimized condit ions, on-line bromine treatment gives a 85 +/- 2% recovery of both free and protein-complexed PHMB in less than 2.5 s and at room temperature. Glycera ldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase, trioso phosph ate isomerase and phospho-glucose isomerase have been examined. Sensitivity and limit of detection of proteins depends on the number of -SH groups rea cting with PHMB and are in the range of 10(-8)-10(-9) mol dm(-3) with calib ration curves spanning over four decades of concentration.