Carbopac ma 1 column

WSCs were separated and quantified from plant extracts and enzyme assays by HPLC-PAD as previously described (Bachmann et al., 1994 (link); Peters et al., 2007 (link); Peters and Keller, 2009 (link)). Briefly, The BC-100 HPLC system comprised a Ca²⁺/Na⁺ moderated ion partitioning carbohydrate column (Benson BC-100 column, 7.8 × 300 mm; Benson Polymeric, Reno, Nevada, USA) operated at 90°C and isocratically eluted with 0.005% (w/v) Ca/Na₂-EDTA at a flow rate of 0.6 ml min⁻¹. To confirm the identities of certain carbohydrates, samples were also analyzed by anion exchange chromatography using a CarboPac MA1 column (4 × 250 mm; Dionex, Sunnyvale, CA, USA) operated at 30°C and isocratically eluted with 0.6 N NaOH at a flow rate of 0.4 ml min⁻¹. WSCs on both systems were quantified in silico, using the Chromeleon v. 6.4 software package, against a series of 5 nmol of standard sugars. The quantity of standard sugars used corresponds to the linear response range of the both chromatographic systems.

For the determination of mannitol content, mycelia were freeze-dried and ground into powder by a TissueLyser (Qiagen, Hilden, Germany). The method for determining mannitol levels in V. volvacea mycelia by high-performance anion chromatography-pulsed amperometric detection (HAPEC-PAD) has already been described [20 , 21 (link)].
The samples (0.1 g) were ultrasonically extracted in 10 mL of ultrapure water for 30 min and centrifuged at 3600 × g for 20 min at temperature of less than 25°C. Then, the supernatant was filtered with a 0.45-μm filter (Millipore, Bedford, MA, USA) and diluted 5 times for analysis by an ICS2500 HPAEC-PAD system comprising a GP50 quaternary gradient pump, an EG50 automatic eluent generator, an LC30 column oven, a Dionex CarboPac MA1 column, and an ED50 electrochemical detector (Dionex, Sunnyvale, CA, USA). The temperature of the column was 30°C, the mobile phase was 480 mM NaOH solution, and the flow rate was 6.67 μL s⁻¹. The adopted external standard mixture included mannitol (Sigma, USA). Each standard was dissolved in deionized water and diluted to several standard solutions to generate a calibration curve [22 (link)].

Monosaccharide contents were obtained by the hydrolysis of the polysaccharides in 2 M H₂SO₄ at 110 °C for 60 min, followed by neutralisation with 1 M NaOH; the monosaccharides were quantified by high-performance anion-exchange chromatography (HPAEC-PAD) [20 ], using the Shimadzu Prominence HPLC system (Shimadzu, Japan), equipped with an Antec II Decade electrochemical detector (ANTEC Leyden, The Netherlands). The analysis was carried out on a Dionex CarboPac MA-1 column, thermostated at 35 °C. Elution was performed using 450 mM NaOH at a flow rate of 0.4 ml min⁻¹, and the sample injection size was 10 μl.
The sulphate content was quantified using the BaCl₂-gelatin turbidity method [21 (link)] after hydrolysing the samples in 1 M HCl at 115 °C for 5 h.
The pH of 1% (w/v) polysaccharide sols was determined using a Mettler Toledo SevenEasy pH meter.

The concentrations of simple carbohydrates (i.e., fructose, glucose, and sucrose) and sugar alcohols (i.e., glycerol and mannitol) were quantified in the aqueous extracts in triplicate by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), using ICS 3000 chromatograph systems equipped with a CarboPac PA-20 and CarboPac MA-1 column, respectively (Thermo Fisher Scientific), as described previously (De Bruyn et al., 2017 ). Quantification was performed by external calibration, including an internal standard [IS; solution of 0.02 g of rhamnose (Merck) per liter of acetonitrile (Thermo Fisher Scientific)].

Xylose and glucose present in the S. cerevisiae culture supernatants were quantified by high-pressure liquid chromatography (HPLC) analysis. The samples were centrifuged at maximum speed in a benchtop centrifuge for 10 min and analyzed on a Dionex ICS-5000+ instrument (Thermo Scientific), equipped with a CarboPac MA1 column. Separation was performed by isocratic elution with 480 mM NaOH, at a flow rate of 0.4 mL min⁻¹ for 35 min.

After extension growth had ceased (250 DABB), final architectural measurements were made of six ‘RG’/‘M9’ and six ‘RG’/‘RG’ grafted trees. A segment of stem tissue was collected from 20 cm above the graft junction (scion), 5 cm below the graft junction (rootstock stem) and roots. Tissue was snap-frozen in liquid nitrogen, dried in a freeze-dryer, and then ground to a fine powder. A 0.05 g subsample was extracted with 80% ethanol with Adonitol added as the internal standard and then incubated for 1 h at 60 °C. Extracted samples were centrifuged and the supernatant decanted off. The residue was re-suspended in 80% ethanol re-spun and supernatants combined. The insoluble residue was transferred into Erlenmeyer flasks and analysed for starch as per Smith et al.⁴⁶ A subsample of the supernatant was taken and dried using a centrifugal concentrator; samples were then re-dissolved in ultrapure water. The sugars were analysed using DIONEX ICS-5000 Reagent-Free IC (RFIC; Thermo Fisher Scientific, Waltham, MA, USA) system with a CarboPac MA1 column with electrochemical detection.