Oxidative chemiluminescence assay of 2,4-dinitrophenylhydrazine

Abstract

This paper describes the development of an assay for 2,4-dinitrophenylhydrazine suitable for application to the determination of carbonyl compounds. Complete factorial design was used to investigate the effects of solvents and carrier on the oxidative chemiluminescence of phenylhydrazines in flow injection analysis. 2,4-Dinitrophenylhydrazine gave a better analyte:blank signal ratio than other phenylhydrazines. Aqueous propan-2-ol was found to be the preferred solvent and aqueous formic acid containing no sensitiser the preferred carrier solution. Rhodamine B sensitiser enhanced all signals but, by its effect on blank signals, reduced the signal to blank ratio. Simplex optimisation was carried out on six variables. The criterion was (A – B)/B, where A and B are the analyte and blank signals, respectively; conditions giving analyte signals of relative standard deviation >10% were eliminated whatever the value of (A – B)/B. The optimum conditions were fine-tuned by univariate exploration and emerged as 22.0% propan-2-ol in the sample, 0.75 M formic acid carrier, 5.0 × 10^–5M permanganate and 0.041 M sulfuric acid as oxidant, 3.25 ml min^–1 total flow-rate and the water-bath at 40 °C. These conditions can be explained in terms of a combination of flow-rate and factors known to increase the rate of oxidation of 2,4-dinitrophenylhydrazine. The effects of formic acid carrier and propan-2-ol solvent are best understood in terms of their effects of enhancing the signal by energy transfer and diminishing it by competition for permanganate. The log–log calibration for 2,4-dinitrophenylhydrazine in optimum conditions was linear (r = 0.9972, n = 10) from 1 × 10^–7 to 2 × 10^–5M; the detectivity calculated at three standard deviations above blank was 1.1 × 10^–7M (1.1 pmol analyte). The method was found to be subject to interference from common metal ions from V to Zn in the periodic table, especially Fe²⁺, though EDTA largely corrected this latter effect.