1260 hplc system

The strawberry tissue was ground into powder in liquid nitrogen and weighed, and 0.5 g fine powder was placed in 1 ml extracting solution (anthocyanins: 1% hydrochloric acid-methanol solution (v:v); flavonoids: methanol), and extracted by ultrasonication in the dark for 40 min. After centrifugation at 1,2000 rpm for 10 min, the supernatant was passed through a 0.22-µm filter for further HPLC analysis. The anthocyanin compounds were analyzed on an Agilent HPLC-1260 system and separated using a ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm), with mobile phases consisting of 0.5% formic acid (A) and methanol (B). The gradient of phase B was as follows: 0-5 min 100-90%, 5-10 min 90-75%, 10-15 min 75-65%, 15-20 min 65-50%, 20-25 min 50-70%, 25-30 min 70-100%, flow rate of 1.0 mL/min and UV detection wavelength of 520 nm. Standards of cyanidin-3-glucoside and pelargonidin-3-glucoside were obtained from Sigma-Aldrich China (Shanghai). All the samples were measured in triplicate in three independent biological replicates.
The sugar content in strawberry fruit was measured using HPLC (Jia et al., 2011 (link)). Standards of sucrose glucose and fructose were acquired from Yuanye Bio-Technology (Shanghai, China). All the samples were measured in triplicate in three independent biological replicates.

HPLC analysis was performed using Agilent HPLC 1260 system, on ZORBAX Eclipse Plus Phenyl-Hexyl column (4.6 × 250 mm, 5 microns). The mobile phase consisted of A (acetonitrile) and B (0.1% H₃PO₄ in water). The elution program was isocratic at 60% (A) and 40% (B) for 20 min, at 1 mL/min flow rate. Sample injection volume was 10 μL (500 ppm) methanolic DMC solution at λ_max⁡ 330 nm. The purity of DMC was assured from chromatogram and plant extracts were analyzed for the presence of DMC and the results were calculated as % w/w.

To test the ability of DT-Bgl to degrade various substrates (Table 3), 100 μL 1 % (w/v) of these compounds was reacted with 10 μL 1 U of purified DT-Bgl. All reactions were carried out in glycine-NaOH buffer (100 mM, pH 8.5), at 60 °C for 15 min, unless specified. Reactions were stopped by boiling for 5 min, and the samples were analyzed on the HPLC 1260 system (Agilent Technologies, Santa Clara, USA), using a 7.8 × 300 mm Rezex RPM-Monosaccharide Pb⁺² column (Phenomenex Inc, Torrance, USA) fitted with a 7.8 × 50 mm LC guard column filled with the same stationary phase. An Agilent 385-Evaporative Light Scattering Detector was used, and the nebulizer and evaporator were kept at 30 °C with a nitrogen flow rate of 1.6 slm (standard liters per min). Nanopure water, filtered through a nylon membrane filter (0.45 µm pore size, Millipore, Darmstadt, Germany) and degassed, was used as the mobile phase. All the insoluble particles were filtered through a polytetrafluoroethylene syringe filter membrane (0.45 µm pore size) prior to injection into the column. All substrates and standards used were of high purity grade.