Ionpac as11 hc

For reproducibility, only well-developed fruits at nodes 8–16 of the plant were selected for the experiment. The fruits were juiced and the extracted paste underwent centrifugation (12,000 rpm, 8 min). The supernatant was then passed through an activated CNWBOND LC-C18 solid phase extraction column. Activation involved a sequential process: 2 mL of methanol, followed by 2 mL of a 1:1 methanol–water solution, then 2 mL of water. After 2 min, 2 mL of water was added, and the activation was allowed to proceed for 30 min, followed by discarding 1 mL. The remaining portion was passed through a 0.22 μm membrane, and the filtrate was used for detection. The content was determined using ion chromatography. The ion chromatograph used was a Thermo Scientific Diongx ICS-5000+. For anion analysis, the analytical column employed was a Dionex IonPAC AS11-HC (250 mm × 4 mm), with a guard column of IonPac AG11-HC (4 mm × 50 mm). The column temperature was maintained at 30 °C, and the eluent solution consisted of KOH solution at a flow rate of 1 mL/min. The standard curve was generated based on standard samples.

All mineral content analyses were conducted on dried samples, which were processed using a model MF10.1 grinding mill (IKA-Werke GmbH & Co. KG, Staufen, Germany). Leaf mineral (NO₃, P, K, Ca, S, Mg) and organic acid (citrate and malate) composition was determined using ICS-3000 ion chromatography equipment (Dionex, Sunnyvale, CA, USA). Anionic and cationic separations were obtained via the IonPac AS11-HC and IonPac CS12A analytical columns, and quantified against chromatography standards using electrical conductivity detectors (Dionex, Sunnyvale, CA, USA) as mentioned in detail by Rouphael and collaborators [19 (link)]. All mineral contents are quantified as mg 100 g⁻¹ fw.

Penicillium oxalicum F9 culture medium (1 ml) was placed into a centrifuge tube and centrifuged at 8,000 r·min⁻¹ for 5 min. The supernatant was filtered through a 0.22-μm filter membrane and injected by a 1 ml syringe into an ICS-2000 ion chromatograph (DIONEX, United States) for detection using a DIONEX IonPac®AS11-HC anion-exchange column (aqueous; sample flow rate: 1.20 ml·min⁻¹, column temperature: 30°C, injection volume: 0.50 ml). The chromatographic data were analyzed according to the instructions using a Chromeleon chromatographic working station.

Pulp concentrations of cations (K+, Ca²⁺, Mg²⁺ and Na⁺) and anions (NO₃⁻, PO₄³⁻and SO₄²⁻) were analyzed as previously described in detail by Rouphael et al. (2017) [21 (link)]. The cations were separated on an ion chromatograph (ICS-3000, Dionex, Sunnyvale, CA, USA) using a Dionex IonPac CS12A (4 × 250 mm) analytical column connected to a Dionex IonPac CG12A (4 × 50 mm) guard column. Anions were separated using a Dionex IonPac AS11-HC analytical column (4 × 250 mm) connected to a Dionex IonPac AG11-HC (4 × 50 mm) guard column. The concentrations of all minerals were expressed in g kg⁻¹ dry weight (dw). The total protein content was assessed based on the nitrogen content according to the Kjeldahl method, using a nitrogen-to-protein conversion factor of 6.25 [22 ].

The total concentration of Al in organs (Al/total; air-dried at 60°C and stainless-steel milled) was determined by ICP-OES after microwave assisted high pressure acid-digestion (65% HNO₃:30% H₂O₂ 4:1). Certified reference material (CTA-OTL-1 oriental tobacco leaves) was mineralised under the same conditions for quality assurance. The concentration of Al in organ infusions (Al/infusion) was determined by ICP-OES after leaching (15 min) and filtering of the suspension of organ biomass and boiled deionised water (1/50, w/v) [41 (link)] through filtration paper ‘Filtrak 390’ (Niederschlag, Germany) with porosity 3–5 μm and flow rate 0.1 ml/s [DIN 53137]. After filtration through a 0.45-μm nylon membrane filter, concentrations of low-molecular-weight organic acid (LMWOAs; acetate, citrate, formate, lactate, malate, maleate, propionate, tartrate, and oxalate) anions in the same organ infusions were determined by means of ion-exchange chromatography with suppressed conductivity. An ion chromatograph ICS 1600 (Dionex, USA) equipped with IonPac AS11-HC (Dionex, USA) guard and analytical columns was used. The eluent composition was 1–37.5 mM KOH with a gradient of 1–50 min; and flow rate was set to 1 mL/min. To suppress eluent conductivity an ASRS 300–4 mm suppressor (Dionex, USA) and Carbonate Removal Device 200 (Dionex, USA) were used.

3.0 g petals of each sample were ground with liquid nitrogen and centrifuged at 12,000 rpm for 20 min, and the supernatant was transferred to 1.5 mL centrifuge tube. Based on this, 200 μL supernatant and 800 μL ultrapure water were transferred to 2 mL centrifuge tube together, which was passed through 0.22 μm membrane filters (Shanghai ANPEL Scientific Instrument Co., Ltd., China). This experiment was performed three times for each sample, and the extracts were used for analysis of organic acids. Qualitative and quantitative analysis of organic acids was performed using Ion Chromatograph System (ICS-2100, Thermo, USA). The anion exchange column was Ion Pac AS11-HC (2.0 mm × 50 mm) and AG11-HC (2.0 mm × 250 mm) (Dionex, USA). The column temperature was 30 °C, injected volume was 25 μL, flow rate was 0.3 mL/min and the electric current of suppressor was 50 mA. In addition, the eluent was KOH with 1.0 mmol/L at 0 min, 1.0 mmol/L at 8 min, 30.0 mmol/L at 28 min, 50.0 mmol/L at 35 min, 50.0 mmol/L at 40 min, 1.0 mmol/L at 41 min, and then returned to 1.0 mmol/L at 55 min.

The freeze-dried samples (0.05 g) were digested with HNO₃-HCL-HF (volume ratio of 1:3:1) by Microwave Digestion System (Milestone, BG, Italy), and the filtrate was analyzed by ICP-MS (Thermo Fisher Scientific, OH, USA) to determine As concentration. The same procedure used to generate the plants was used to prepare blanks and samples of the standard reference material (wheat, GBW07603, IGGE, Langfang, China) as a quality control measure. The check recovery was within 90–110%. Four forms (As(III), As(V), MMA, DMA) were separated using an inductively coupled plasma liquid chromatograph from Perkin Elmer Instruments Ltd. (AAS, PinAAcle 900T, Perkin Elmer, MA, USA) for the separation. Chromatographic column type: Dionex IonPac AS11-HC, size: 250 × 4, equilibration time: 20 min. Mobile phase composition: (A): 0.6 mL sulfuric acid, 0.3 mL nitric acid, and 3 mL ammonia (pH = 10); (B): ultrapure water. Mobile phase flow rate: 0.5 mL min⁻¹. The chromatographic peaks of the samples are shown in the Supplementary Materials (Figure S1).