Metrosep a supp 4 250 4.0 column

The concentration of sulphate was measured by ion chromatography (883 Basic Ion Chromatography plus, Metrohm, Australia) following the modified method of Frankowski (2016) (link). Ground samples were extracted using Milli-Q water (1 g water to 40 g oven-dried tissue) by shaking on a horizontal shaker for 1 h followed by centrifugation at 5.292 g with a swinging-bucket rotor 11150 for 10 min at 20 °C using a Sigma 4K15 table-top centrifuge. Subsequently, 5 mL of supernatant of each extract was filtered through a 0.45-µm hydrophilic syringe filter (Sartorius, Germany) before analysis by ion chromatography. Sulphate anions were separated using a Metrosep A Supp 4–250/4.0 column (Metrohm, Australia) with a 17-min run of an isocratic elution system consisting of 1.7 mmol L⁻¹ sodium bicarbonate (NaHCO₃) and 1.8 mmol L⁻¹ sodium carbonate (Na₂CO₃) at 1 mL min⁻¹ flow rate. For quantification of sulphate, Sigma–Aldrich Multi Anion Standard 1 was used as a standard.

After the extraction processes, the anion and cation concentrations of each fraction were measured by ion chromatography (IC), using the Metrohm 930 Compact IC Flex system (Metrohm AG, Herisau, Switzerland). For cations, the samples were eluted through a Metrohm Metrosep C6-250/4.0 column with 8 mM ultrapure HNO₃ (TAMAPURE AA-100, Tama Chemical, Kawasaki, Japan) at a flow rate of 0.9 mL·min⁻¹. Anions were measured with a Metrohm Metrosep A Supp4-250/4.0 column with a chemical suppressor module. The mobile phase consisted of a mixture of 1.8 mM Na₂CO₃ and 1.7 mM NaHCO₃ (Kanto Chemical, Tokyo, Japan) at a flow rate of 0.9 mL·min⁻¹. A chemical suppressor module (Metrohm MSM) was used to decrease the background conductivity of the eluent and to transform the analytes into free anions. The column temperature was set at 35 °C throughout the analysis. Detection of cations and anions was accomplished by measuring electrical conductivity.