Reaction flow chromatography for rapid post column derivatisations: The analysis of antioxidants in natural products

The analysis of antioxidants from complex samples is conveniently achieved using liquid chromatography, which provides sample fraction, coupled with an on-line antioxidant assay, which provides detection. One particularly useful on-line antioxidant assay that has routinely been coupled with HPLC involves the diphenylpicrylhydrazyl radical (DPPH), which provides a positive test for phenolic antioxidants through a decolorisation of the DPPH reagent. A limitation of this assay, however, is the need to employ a reaction coil, which is often large with respect to the peak volume, consequently adding substantial band broadening to the separation. In this study we introduce a new concept that can be employed for systems requiring post column derivatisations, such as the DPPH assay. We have termed this ‘reaction flow’ chromatography, whereby, the derivatisation reagent can be added directly into one of the outlet ports of a parallel segmented flow column. Subsequently, the mixing between the derivatising reagent and the solute is very efficient removing the need to employ reaction coils. The concept is tested here using the DPPH assay for the analysis of antioxidants in samples derived from natural origin.