Dionex ic 3000 system

In the chemical spray experiment, sugars, and sugar alcohols (sucrose, glucose, fructose, raffinose, erlose, maltose, galactose, rhamnose, sorbitol) of the leaf tissue were quantified using high performance liquid chromatography (HPLC) method as described previously (O'Rourke et al., 2015 (link)). Grounded dry tissue samples (20–50 mg) were extracted two-times with 2.5 ml of 80% ethanol by boiling the samples in glass tubes in a 60°C water bath for 30 min each. After each extraction, the tubes were centrifuged at 4500 rpm for 10 min, and the extracts were then pooled and dried in a speedvac for ~3–4 h. From this, final extract 200 μL was further dried down to remove the ethanol and rediluted with 200 μL deionized water. HPLC with a Dionex IC-3000 system including electrochemical detection cell with gold electrode and temperature controlled column compartment at 30°C (Thermo Scientific, Hemel Hempsted, UK) was used. The column used was a Dionex CarboPac PA20 3 × 150 mm analytical column (Thermo Scientific, Hemel Hempsted, UK). Ten microliters of sample was injected into the sample loop connected to the ion exchange column. The peaks were identified by comparing retention times with those of standard sugar markers with Dionex Chromeleon software.

Preliminary compositional analysis of each obtained GOS mixture was determined by glyco-profile analysis employing a High Performance Anion Exchange Chromatography and pulsed amperometric detection (HPAEC-PAD; Dionex IC-3000 system; Thermo Scientific¹). Separations were performed using a CarboPac PA1 (Thermo Scientific) analytical-anion exchange column (dimensions, 250 mm by 4 mm) with a CarboPac PA1 (Thermo Scientific) guard column (dimensions, 50 mm by 4 mm) and a detector (ED40) in the pulsed amperometric detection PAD mode (Dionex, Thermo Scientific). Qualitative analysis of the GOS Dionex profile was performed with an elution gradient according to a previously published method (Van Leeuwen et al., 2016 (link)) and the qualitative determination of the carbohydrate composition was performed by the use an elution gradient summarized in supplemental Supplementary Table 3 at a constant flow rate of 1.0 ml min^–1 at 30°C using the following eluents with programmed gradient for the analysis: (A) 100 mmol NaOH, (B) 100 mmol NaOH, 500 mmol sodium acetate (NaAC), (C) 50 mmol NaAC and (D) Milli-Q water. The obtained chromatography profiles were analyzed employing CHROMELEON software Version 7 (Dionex, Thermo Scientific).