Post-column derivatization in high-performance liquid chromatography using the air segmentation principle: application to digitalis glycosides

Abstract

The possibility of using post-column derivatization techniques to improve the detection in high-performance liquid chromatography (HPLC) has been suggested before, but a serious limitation to the choice of the reactions is the kinetics, as slow reactions require longer reaction times and correspondingly larger reaction units (long spirals) with the associated problems such as a high pressure drop and loss of resolution. In this study it was attempted to overcome some of these drawbacks by applying the well known air segmentation principle used in connection with Auto-Analyzers. The studies were carried out on cardiac glycosides which, upon reaction with concentrated hydrochloric acid, yield highly fluorescent products. As this reaction is kinetically slow, with at least a 10-min reaction time for a reasonable fluorescence yield, it served ideally for the investigation. The optimal reaction conditions and the influence of solvents, reagent composition, temperature and time on the kinetics and fluorescence yield were studied. With a 10-min reaction time the band broadening was only about 15%. The reproducibility of the derivatization step is 1.2% (relative standard deviation, n = 10) and the improvement in the detection limit was at least 100-fold in comparison with UV detection. For a non-retained glycoside the detection limit is 500 pg per injection at a signal to noise ratio of 4:1. The technique was applied successfully to members of the C-group of cardiac glycosides (digoxin, digoxigenin, lanatoside C and desacetyl-lanatoside C), which were separated on a reversed phase HPLC system. Many of the concepts studied are of general validity for post-column reactions with relatively slow kinetics.