Determination of organic acids and their role in nickel transport within cucumber plants

Abstract

Reversed-phase (RP) and ion-pair (IP) high performance liquid chromatographic (HPLC) methods were developed for the direct analysis of the organic acids (citric, malic and fumaric) present in xylem sap samples of cucumber plants grown in ‘nickel free’ and nickel contaminated nutrient solutions. Both methods were developed using Nucleosil C18 RP columns and phosphate buffers with organic additives (2.5 v/v% acetonitrile for the RP and 10 v/v% methanol for the IP method) as mobile phases. The main advantage of the RP method over the IP method is in its easiness and rapidity of separation. The IP method that uses tetrabutylammonium hydrogen sulfate (TBAHSO 4) in a concentration of 5 mmol/dm 3 dissolved in the eluent, ensured better separation for malic acid. For the elucidation of the role of organic acids in nickel transport in xylem vessels, size exclusion chromatography (SEC) methods — using a silica Macrosphere column and acetate buffer – were elaborated. Analyzing the model solutions of citric or malic acids containing nickel(II) sulfate with the aid of an ‘off-line’ SEC-graphite-furnace atomic absorption spectrometer (SEC-GF-AAS) system, the implication of citric acid in the transport of nickel may be justified. However, neither the separation of the acids nor their identification in the real samples could be carried out with this method because of the following reasons: their similar molecular size and the presence of small inorganic ions in the saps in high concentrations like nitrate ions.