Electrochemical Reduction of 6-Benzylaminopurine at Mercury Electrodes and Its Analytical Application

Abstract

The commercial exploitation of modern, in vitro plant micropropagation methods, featuring synthetic cytokinins as essential components of the cultivation media, is rapidly increasing. Thus, development of rapid, inexpensive and less labour-intensive methods for monitoring cytokinin levels could help to optimise media consumption and reduce costs. Therefore, we studied the electrochemical behaviour of the highly active and widely used cytokinin, 6-benzylaminopurine (BAP), in aqueous solutions by DC polarography, cyclic and differential pulse voltammetry and constant potential coulometry. The BAP molecule undergoes a six-electron irreversible reduction process that starts with four-electron reduction of the protonated pyrimidine skeleton. As a result of elimination of the amine from the side chain, the N1=C6 electrochemically active bond is re-established and the last two-electron step follows. The intermediates of constant potential electrolysis were identified using mass spectrometric analysis. The dissociation constant (pKa) of BAP was found, spectrophotometrically, to be 4.16. BAP concentrations were measured using two voltammetric techniques, fast-scan differential pulse (FSDPV) and adsorptive stripping voltammetry (AdSV). The relative standard deviations for these two methods were lower than 4.5% (c < 28.7 ng ml-1) and 1.2% (c < 20 ng ml-1), while the detection limits were 7.88 and 0.80 ng ml-1, respectively. Using these techniques, BAP was determined in two types of nutrition media used for the micropropagation of plants in vitro (Gerbera and banana media). In the case of media samples containing the interfering agent adenine (i.e. Gerbera plant medium), the analyte was preconcentrated by ion-exchange chromatography and immunoaffinity chromatography. This preconcentration process gives 92% recovery. In contrast, it was possible to determine BAP levels in simple banana cultivation medium directly, without any pre-purification process. Both methods, reported here (FSDPV and AdSV), were found to be useful for rapidly monitoring BAP consumption by plants during their growth under in vitro conditions.