Sugar pak 1 column

Soluble sugars were extracted from flour samples (400 mg DW), following the method of [43 (link)]. Samples were homogenized in 20 mL of cold H₂O, stirred for 30 min on ice, sonicated for 5 min and centrifuged (15,000 g, 20 min, 4 °C). The supernatant was collected, and extraction procedure was repeated with the pellet. Both supernatants were joined and cleared with nylon syringe filters (0.45 µm) before injection. Sugars separation was performed with an HPLC system coupled to a refractive index detector (Model 2414, Waters, MA, USA), using a SugarPak 1 column (300 mm length × 6.5 mm diameter, Waters) at 90 °C, with H₂O as eluent (containing 50 mg EDTA-Ca L⁻¹ H₂O) and a flow rate of 0.5 mL min⁻¹. Standard curves were used for the quantification of each sugar.

Soluble sugars (sucrose, fructose, glucose, and sorbitol) were determined in approximately 400 mg of powdered frozen leaf material, based on the method previously described [63 (link)] with alterations as in [64 (link)]. Briefly, the samples were homogenized in 4 mL of cold H₂O with 50 mg of polyvinylpolypyrrolidone, left to extract for 20 min on ice at 100 rpm and centrifuged (12,000× g, 5 min, 4 °C). The supernatant was boiled to denature the proteins (3 min), placed on ice (6 min) and centrifuged again. The obtained clear solution was then filtered (0.45 µm, nylon) before the injection of a 50 μL aliquot into an HPLC system equipped with a refractive index detector (Model 2414, Waters, Milford, MA, USA). The separation of sugars was performed using a SugarPak 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 for 22 min. Standard curves were used for the quantification of each sugar.

Sugar extraction was performed in 40 g of pulp (removed from several grape berries at harvest) per sample (n = 3), according to [37 (link)], with minor modifications. Pulp weighted was liquified with 150 mL of cold ultrapure water and then adjusted to 200 mL. Samples were kept in ice, then transferred to ultrasounds (for 5 min), and later centrifuged (15,000× g, 15 min, 4 °C). Thereafter, the supernatant was removed to glass tubes and kept in ice. The pellet was then resuspended in ultrapure water and centrifuged in the same conditions. The supernatant, after the 2nd centrifuge, was transferred to glass tubes (ca. 20 mL) and immersed in a boiling bath (for 4 min). The tubes were then removed and put into ice (for 6 min), and, once cold, the samples were centrifuged (15,000× g, 20 min, 4 °C). Samples were injected in an HPLC (Waters, Milford, MA, USA) system, coupled to a refractometric detector (Waters 2414), equipped with a SugarPak 1 column (Waters 6.5 × 300 mm) and pre-column (Wat 088141) with SugarPak II inserts (Wat 015209), after being filtered (nylon 0.45 mm) and stored. Ultrapure water containing 50 ppm calcium EDTA was used as the mobile phase, with a flow of 0.5 mL min⁻¹, and an injection volume of 40 µL for each sample. Data were later analyzed with Breeze software, and quantification was performed based on the calibration curves of sucrose, glucose, and fructose.