An improved reversed-phase liquid chromatographic method for the analysis of low-molecular mass organic acids in plant root exudates

Abstract

Reversed-phase column liquid chromatography (RPLC) was used for the separation and quantification of 10 low-molecular-mass organic acids (malic, malonic, lactic, acetic, maleic, citric, cis-aconitic, succinic, fumaric, and trans-aconitic) in plant root exudates. A mobile phase of 93% 25 m M KH 2PO 4 at pH 2.5 and 7% methanol at a flow-rate of 1 ml min −1 resolved all 10 acids in 15 min on a C 18 column. Experiments demonstrated a significant ( P<0.05) effect of sample pH on detector response, with peak heights being significantly lower at pH 6.0 compared with pH 2.5, but peak area showed no significant difference. At pH 8.0 and above, both peak height and area differed significantly from injections made at pH 2.5. Limits of detection (LOD) for the 10 acids ranged from 0.05 to 24 μ M. Finally, the improved method was applied for the analysis of root exudates from soil cultured field pea, Banksia attenuata, white lupin, and chickpea.