Cation h guard column

A 2 mL aliquot of the 18 mL ethanol extractives supernatant derived from the AIR preparation protocol was dried down under nitrogen (N) gas in a Techne Sample Concentrator (Techne, Charleston, SC, USA). The extractives precipitate was resuspended in 200µL of Milli-Q^® water (18.2 Ω), filtered through a 0.45 µm filter plate, and then loaded into the high-performance liquid chromatography (HPLC) system. The samples were run on an Agilent 1260 Infinity™ LC series system (Agilent, Santa Clara, CA, USA), with a Bio-Rad 300 × 7.8 mm Aminex 87 H column (Bio-Rad, Hercules, CA, USA) and a Bio-Rad cation H guard column. The Agilent 1260 refractive index detector (RID) was held at 35 °C. The samples were run using an isocratic 4 mM sulphuric acid eluent at 0.6 mL/min⁻¹ and 60 °C for 16 min. For the quantification of sucrose, glucose, and fructose, the conditions using a temperature of 18 °C and 10 mM sulphuric acid eluent at a flow rate of 0.3 mL/min⁻¹ for 22 min. The extractive precipitate sample volume was 3µL (at RT). A combined sucrose, fructose and glucose standard from Sigma-Aldrich (St Louis, MI, USA) at concentrations of 5, 10, and 20 g/L was run as a signature reference, which displayed satisfactory accuracy of the HPLC instrument. For further details of the methods and instruments utilised, see [69 ].

The extracts in the volumetric flasks were filtered through a cellulose acetate syringe filter with a pore size of 0.45 μm (dismic‐25cs, Advantec, CA, USA) into 1 ml glass vials. The samples were analyzed with a high‐performance liquid chromatography (HPLC) system, using a 300 mm × 7.8 mm Rezex ion exclusion column (Phenomenex Inc., Torrance, CA, USA) attached to a Cation‐H guard column (Bio‐Rad, Richmond, CA, USA) held at 25°C. Analysis was performed by injecting 20 μl of sample or standard onto the column using an aqueous solution of 25 mM sulfuric acid (HPLC grade Baker Chemicals, Phillipsburg, NJ, USA) as the mobile phase, pumped isocratically at 0.6 ml/min, with peaks detected at 210 nm. The HPLC equipment consisted of a Shimadzu LC‐10AD pump, CTO‐10A column oven, SPD‐10Avp UV–vis detector (Shimadzu, Kyoto, Japan) and a Waters 717 plus auto‐sampler (Waters, Milford, MA, USA). Data acquisition and processing were undertaken using a PeakSimple Chromatography Data System (model 203) and PeakSimple software version 4.37 (SRI Instruments, Torrance, CA, USA). The oxalic acid peak was identified by comparing the retention time to a standard solution, and by spiking an already‐filtered sample containing a known amount of oxalic acid standard. The insoluble oxalate content of each sample was calculated by difference between the total and the soluble oxalate contents.