Eclipse xdb c18 column

Optical rotations were acquired on a JASCOP-1020 polarimeter. ECD data were measured on a Chirascan spectropolarimeter. IR spectra were measured on PerkinElmer infrared spectrophotometer. 1D and 2D NMR spectra were recorded on Bruker Avance 400 or 600 DRX spectrometers in acetone-d₆, methanol-d₄, DMSO-d₆ and chloroform-d. Column chromatography (CC) was performed on silica gel (200–300 mesh; Qingdao Marine Chemical Plant Branch., China), RP-C18 (ODS-A, 50 μm, YMC, Kyoto, Japan), or Sephadex LH-20 (100–200 mesh; Beijing Solarbio Technology Co., Ltd., China). Plates precoated with silica gel GF254 (Rushan, Shandong Sun Desiccant Co., Ltd.) were used for thin layer chromatography (TLC). An Agilent HPLC series 1260 and Shimadzu LC-20AR were used for analysis and isolation. For analysis, an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) was used. The isolation was achieved on an Agilent semi-preparative Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm). HPLC-MS data were acquired on an Agilent 1260 Series system coupled with an Agilent Accurate-Mass-Q-TOF MS 6520 system equipped with an Electrospray ionization (ESI) source.

The concentration of DON in compound feed and feed ingredient samples was measured using HPLC. In analysis, Agilent 1100 series (Santa Clara, CA, USA) including a degasser, auto sampler, a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm), and a guard column C18 (4.6 × 10 mm, 5 μm) were used at 30 °C. DON was separated using HPLC for 20 min at a flow rate of 1 mL/min and detected with a diode array detector at 220 nm. The mobile phase was composed of HPLC grade water and acetonitrile, which was used in the gradient mode. The retention time was 4.4 min after injection of 20-μL samples.

Leaf tissue (0.1 g fresh mass) was sampled and added to 1 ml extract (acetonitrile:water, 1:1). The supernatants were extracted on ice for 4 h and centrifuged at 4°C for 12,000 × g for 10 min. An aliquot (800 μl) of the supernatant was purified by solid-phase extraction. The solid-phase extraction cartridges were washed using 1 ml of methanol and equilibrated with 1 ml 50% ACN/H₂O (v/v). The samples were loaded, and then the flow-through fraction was discarded. The cartridge was then rinsed using 1 ml of 60% ACN/H₂O (v/v). Then, the samples were evaporated to dryness under a gentle stream of nitrogen and reconstituted in 100 μl of 10% ACN/H₂O (v/v). All the samples were vortexed for 30 s, sonicated in an ice-water bath for 5 min, and then, centrifuged at 4°C for 15 min at 12,000 × g. The ZORBAX Eclipse XDB C18 column (4.6 mm × 280 mm; 5.0 μm) was used to analyze samples. After the crude extract was purified by reverse-phase solid-phase extraction, ether extraction, and derivatization, the endogenous hormone concentration of mung bean leaf was measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS, Agilent Technologies, Ltd., Waldbronn, Germany) with Chromosep C18 column (C18 Sep-Pak Cartridge, Waters Corp., Milford, MA, United States) (Ma et al., 2008 (link); Teng et al., 2010 (link)).

The viscosity of polysaccharide was determined using an Ubbelohde viscometer. Protein content of the polysaccharide fraction was measured by the Bradford method as previously described.^{22 (link)} The total carbohydrate and sulfate content in PHSP_(hp) and GLSP_(hp) were determined as previously described.^{20 (link)} The molecular weight distribution of polysaccharides was determined by high performance gel-permeation chromatography (HPGPC) (Agilent-1100, Santa Clara, U.S.A.) equipped with a ZORBAX Eclipse XDB-C18 column (250 × 4.6 mm², column temperature 30 °C). The Fourier transform infrared spectra of polysaccharide was analyzed with a Fourier transformed infrared spectrometer (FT-IR) (Vector-22, Bruker, Switzerland) in the wave number range of 4000–400 cm⁻¹ using the KBr-disk method.

SGPL-1 activity measurement was performed by the quantification of (2E)-hexadecenal following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) as described before [18 (link)]. In brief, cells were extracted by a cold methanol-chloroform 0.9% NaCl mixture on ice. The organic phase was dried and solved in acetonitrile. Derivatization was performed with a mixture containing 0.6 mg/ml DAIH in acetonitrile and 7% of 2 M HCl at 4°C. The analysis of the aldehyde was conducted with an Agilent 1200 liquid chromatography system coupled to an Agilent 6530 quadrupole/time-of-flight mass spectrometer (both from Waldbronn, Germany). Chromatographic separation was performed on a ZORBAX Eclipse XDB-C18 column.