2410 refractive index detector

Analysis of sugars (sucrose, fructose, glucose, glucose 6-phosphate and mannitol) was conducted by HPLC. Approximately 100 mg of leaf samples were first powdered with liquid nitrogen and then supplemented with 1 mL of water in 2 mL Eppendorf tubes. Subsequently, samples were homogenized using the Ultra-turrax homogenizer for about 2 min until complete rupture of cells. The homogenate was subjected to centrifugation at 3000× g for 5 min, the supernatants transferred to 2 mL Eppendorf tubes and then centrifuged again at 12,000× g for 5 min. Samples were filtered (0.45 µm) and about 20 µL of each extract was injected and examined using a Waters Sugar-Pak I ion exchange column (6.5 × 300 mm) at a temperature of 90 °C. The mobile phase consists of MilliQ water (pH 7) with a flow of 0.3 mL min⁻¹. The overall duration of the separation was 30 min. The elution times of sugars are as follows: glucose 6-P—about 5 min; sucrose—about 8 min; glucose—about 10 min; fructose—about 11 min; and mannitol—about 13 min. Identification of the components was obtained using a Waters 2410 refractive index detector, by comparing the retention times with those of reference standards. For each peak, the retention factor allows to identify the type of eluted molecule, while the curve area is proportional to the quantity. The protocol was repeated three times for each sample.

High Pressure Liquid Chromatography (HPLC) was used for the analysis of sugars (pectins, sucrose, fructose, and glucose) [85 (link),114 (link)]. Briefly, 100 mg of leaf samples powdered with liquid nitrogen were added to 1 mL of distilled H₂O. Samples were homogenized by Ultra-Turrax^® T-25 basic (IKA^®-Werke GmbH & Co. KG, Staufen im Breisgau, Germany), centrifuged at 3000 RCF for 5 min; the supernatants were transferred to 2 mL Eppendorf^® tubes and then centrifuged again at 12,000 RCF for 5 min (Eppendorf^® Microcentrifuge 5415D, Hamburg, Germany). Samples were filtered (0.45 µm) and 20 µL of each extract was injected into a Waters Sugar-Pak I ion exchange column (6.5 × 300 mm) at a temperature of 90 °C. The mobile phase consisted of MilliQ H₂O (pH 7) with a flow of 0.3 mL min⁻¹. The overall duration of the separation was 30 min. Identification of components was done using a Waters 2410 refractive index detector by comparing the retention times with those of reference standards. The experiment was conducted in three technical replicates for each sample. Finally, the mean and standard deviation were calculated. To verify the significance of the data obtained, the t-test (* p ≤ 0.05, ** p ≤ 0.01) were carried out.

Molecular
weights were determined by a gel permeation chromatography (GPC) system,
Waters 1515. Isocratic high performance liquid chromatography (HPLC)
pump with a Waters 2410 refractive index detector, a Waters 717 plus
autosampler, and a Rheodyne (Cotati, CA) injection valve with a 20
μL loop. The samples were eluted with CHCl₃ (HPLC
grade) through a linear Styragel HR4E column (Waters) with a molecular-weight
range of 50–100K Da at a flowrate of 1 mL/min. The molecular
weights were determined relative to a polystyrene standards (Polyscience,
Warrington, PA) calibration curve having weight-average molecular
weight (M_w) from 600 to 50 000.