Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography: I. Development and Assessment of Chromatographic Conditions *1

Methods for determining nine low molecular weight organic acids in root exudates were developed by using reversed phase high performance liquid chromatography with UV (ultraviolet) detection at 214 nm. The mobile phase was 18 mmol L -1 kH 2PO 4 adjusted to pH 2.25 with phosphoric acid and the flow rate was 0.3 mL min -1 . The analytical column was a reversed phase silica based C 18 column (Shim pack CLC ODS). The root exudates were collected through submerging the whole root system into aerated deionized water for 2 hours. The filtered exudate solutions were concentrated to dryness by rotary evaporation at 40 °C, dissolved in 10 mL mobile phase. The chromatographic conditions of organic acid determination were analyzed. The results showed that there was a high selectivity and sensitivity in the organic acid determination by reversed phase high performance liquid chromatography. Coefficients of variation for organic acid determination were lower than 10% except lactic acid. The recoveries were consistently between 80.1% to 108.3%. Detection limits were approximately 0.05 to 4.5 mg L -1 for organic acids except succinic acid with the detection limit of 7.0 mg L -1 . Phosphorus deficiency may contribute to the release of organic acids in soybean root exudates especially malic, lactic and citric acids.

Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography:Ⅱ.Influence of Several Testing Conditions

Effects of column temperature and flow rate on separation of organic acids were studied by determining nine low-molecular-weight organic acids on reversed- phase C18 column, using high performance liquid chromatography (HPLC) with a wavelength of UV (ultraviolet) 214 urn and a mobile phase of 18 mmol L-1 KH2PO4 buffer solution (pH 2.1). The thermal stability of organic acids was determined by comparing the recoveries of organic acids in different temperature treatments. The relationships between column temperature, flow rate or solvent pH and retention time were analyzed. At low solvent pH, separation efficiency of organic acids was increased by raising the flow rate of the solvent because of lowering the retention time of organic acids. High column temperature was unfavorable for the separation of organic acids. The separating effect can be enhanced through reducing column temperature in organic acid determination due to increasing retention time. High thermal stability of organic acids with low concentrations was observed at temperature of 40 ℃-45℃. Sensitivity and separation effect of organic acid determination by HPLC were clearly improved by a combination of raising flow rate and lowering column temperature at low solvent pH.

Assessment of Cation Exchange Membrane Method and Estimation of Potassium Critical Values

Soil and tissue analyses are usually used in identifying potassium(K) deficiencies and predicting K fertilizer requirements of crops.The critical levels of both soil and fresh leaf tissue at seventh leaf stage were developed and assessed for canola,chickpea and sunflower grown on two Saskatchewan,soils,with six rates of K fertilizer supply, in a growth chamber experiment.The available potassium in soils was determined by two methods:1)resin strip extraction, and 2) NH4OAC extraction. The potassium in fresh leaves was determined at seventh leaf stage by a simple procedure using a common garlic press and injector to extract the plant sap and testing the sap with a handheld ion selection electrode meter. The results shoWed significant relationships between the resin strip extractable K and NH4OAC extractable K, and between the plant uptake of total K and the supply of available K in the soils determined by the two methods. Good relationships were also found between the potassium in fresh leaves and the plant uptake of total K for canola, chickpea and sunflower.The resin strip extraction for K was calibrated using common NH4OAC extraction, and recommended for routine analyses because of its simplicity and sensitivity.

Determination of Organic Acids in Root Exudates byHigh Performance Liquid Chromatography: III. Effectsof Interfering Factors

A solution culture experiment was conducted to investigate the effects of collection time and interferingions on separation and determination of low-molecular-weight organic acids in root exudates of soybeanusing the method for directly collecting root exudates. The suitable collection time of root exudates andthe interfering ions affecting organic acid determination were determined. The method for removing theinterfering ions was established and analyzed. The release amount of root exudates increased with theincrease of collection time from 0 to 120 min but decreased with increasing of collection time from 120 to 240min. The maximum exuding amounts of organic acids were observed in root exudates at the collection time of120 min. There was a significant difference of organic acid components between the treatments of collectiontime of 120 min and 240 min. Citric acid was found only in the treatment of 120 min collection time. NO3-was the main interfering ion in organic acid determination and had the same retention time as oxalic acid.Anion exchangs resin (SAX) properly treated by HPLC (high performance liquid chromatography) solventcould remove NO3- anion in sample solution of root exudates, thus enhancing the recoveries of organic acidsin root exudates. There was no significant effect of the chemicals added into sample solution such as H3PO4,SAX and KNO3 on the retention time of organic acids.