Development of an Impregnated Reagent and Automation of Solid-Phase Analytical Derivatization for Carbonyls: Proof of Principle

This study undertakes reduction of scale and automation of a solid-phase analytical derivatization of carbonyls with 2,4-dinitrophenylhyrazine on a styrene-divinylbenzene resin (XAD-2). Three processes are tested. In the batch process, an aqueous phase consisting of 50 microL of sample and 150 microL of reagent solution is contacted with 6 mg XAD-2 by shaking. An impregnated reagent consisting of 2,4-dinitrophenylhydrazine hydrochloride (DNPH) deposited on XAD-2 enables two additional processes. In-vial derivatization with an impregnated reagent requires shaking 50 microL of sample with 6 mg of the impregnated reagent and reduced the reaction time from 10 to 5 min. The third process involves packing impregnated reagent a mini-column and flowing 50 microL of sample through under positive pressure supplied by a Harvard Pump. This reduces sample preparation time to 1 min. Studies are conducted with worst-case model analytes: butanone, 2-pentanone, and malonyldialdehyde. The carbonyl of the two ketones is hindered, and, thus, these two compounds react very slowly with DNPH in aqueous solution. Malonyldialdehyde is highly water soluble, and it does not react in aqueous phase but also would not sorb onto XAD-2 for reaction. Nevertheless, derivatization/extraction of all model compounds any of the three procedures result in reproducible and high yields.