Sugar pak 1

Nonstructural carbohydrates were measured using a Waters 2695 High-Performance Liquid Chromatography system (Waters, Inc., Milford, MA, USA) under the following chromatographic conditions: chromatographic column: Sugar-Pak 1 (Waters, Inc.); mobile phase: water; flow: 0.6 ml·min⁻¹; column temperature: 70°C; detector: parallax detector. For the extraction of soluble sugar [25] , 0.5 g of dried plant material was weighed and then digested for 2 h with 50 ml of distilled water. Samples were brought up to a constant volume and then filtered. For the extraction of starch [25] , 0.5 g of dried plant material was weighed and refluxed for 8 h in a 100-ml water bath of distilled water and 10 ml 2∶1 (v:v) hydrochloric acid. The samples were then neutralized with 40% sodium hydroxide, brought up to a constant volume, and filtered.

RFOs evaluation was performed in samples of 150 mg of powdered frozen material, following the HPLC method for soluble sugars described in Ramalho et al. (2013b (link)), with some modifications. To overcome the presence of non-pure peaks the separation of sugars was performed using a Sugar-Pak 1 column (300 × 6.5 mm, Waters) at 90°C, with H₂O as the eluent (containing 50 mg EDTA-Ca L⁻¹ H₂O) and a flow rate of 0.5 mL min⁻¹. Another 20 μL aliquot of each sample was injected through a DionexCarboPac PA1 analytical column (4 × 250 mm, Thermo Scientific, USA) coupled to a DionexCarboPac PA1 Guard (4 × 50 mm) at 20°C. Ultrapure water and 300 mM NaOH were used as eluents (water from 0 to 50 min; NaOH from 50 to 65 min; and water from 65 to 80 min for re-equilibration) at a 1 mL min⁻¹ flow rate. A refractive index detector (Model 2414, Waters, USA) was used for sugar detection. Sugars were quantified using specific standard curves.