Using activated charcoal to remove substances interfering with the colorimetric assay of inorganic phosphate in plant extracts

Abstract

AimOrganic substances in leaves of several southwest Australian native species interfere with sensitive colorimetric assays and prevent quantification of inorganic phosphate concentration ([Pi]). We aimed to develop a reproducible routine procedure for treating leaf extracts with activated charcoal (AC) to remove interfering substances, allowing the determination of [Pi] by the malachite green spectrophotometric assay.MethodsLeaf extracts of native plants from southwest Australia in 1% (v/v) acetic acid were treated with 10 mg mL−1 acid-washed AC for removal of interfering substances. Standard solutions (0 to 18 μM Pi) with and without AC treatment were compared to quantify Pi loss. A spiking and recovery test was performed to validate the AC treatment.ResultsLeaf extracts treated with AC exhibited distinguishable absorbance peaks for the malachite green-orthophosphate complex between 630 and 650 nm, as opposed to untreated samples. The Pi-adsorption by AC represented a relatively larger fraction of [Pi] in solutions at 0–4 μM Pi range and stabilised at higher [Pi] when maximum adsorption capacity of AC reached at 11.7 μg Pi g−1AC. The Pi recovery after AC treatment in spiked samples ranged between 100 and 111%.ConclusionThe AC treatment successfully removed interfering substances from samples but caused Pi loss. Thus, quantification of [Pi] in AC-treated extracts requires sample [Pi] ≥ 6 μM Pi and the use of AC-treated standards. The error of the AC treatment was minor compared with environmental variability of leaf [Pi]. The AC treatment was a reproducible time- and cost-effective method to remove interfering substances from leaf extracts.