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An improved reversed-phase liquid chromatographic method for the analysis of low-molecular mass organic acids in
1School of Plant Biology (M090), Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. gcawthra@agric.uwa.edu.au
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 mM KH2PO4 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 C18 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 microM. Finally, the improved method was applied for the analysis of root exudates from soil cultured field pea, Banksia attenuata, white lupin, and chickpea.
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