Zorbax eclipse xdb c18 column

To compare the performance of the proposed method with that of other analytical methods, certain OAs and PAs in the white and red wines (see preparation of different wines in Section 2.1.2.) were analyzed. Glycerol and tartaric acid were measured enzymatically and with a colorimetric method, respectively, on a Y15 enzymatic autoanalyzer (BioSystems S.A., Barcelona, Spain) using kits from BioSystems [33 (link)]. For PAs, p-coumaric acid and ferulic acid were determined with HPLC with an Agilent 1200 series liquid chromatograph (HPLC-diode array detection) using an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 250 mm, 5-μm; Agilent Technologies) according to a modification of the method of [20 (link)].

The crude extracts were dissolved in ACN-H₂O (8:2) and assessed by HPLC-DAD and UPLC-DAD-QTOF using modified chromatographic conditions from literature^{28 (link)}. For UPLC-DAD-QTOF, samples were ionized by ESI with a MicrOTOF Bruker Daltonics mass spectrometer (Milford MA, USA). Mass spectra were obtained in positive ionization mode and the following parameters were applied: capillary energy of 3.5 kV, nitrogen was used as nebulization, drying gas (5.5 bar and 10 L/h), and temperature drying time of 220 °C. UPLC conditions were carried out using the isocratic system: 0–18′ (60:40; A:B), 18–32′ (50:50; A:B), and 32–40′ (100% of B %), 0.3 mL.min⁻¹ flow rate and detection at 245 nm (A-triethylammonium acetate buffer 35 mM, 0.2% v/v, pH 6.9; B-ACN); and an Agilent Zorbax Eclipse XDB-C18 column: 3.5 µ, 4.6 × 150 mm. For HPLC-DAD, the samples were analyzed with the same chromatographic conditions described above, except for the flow rate, which was 0.8 mL.min⁻¹.

Root ground powder (0.1 g) was weighed accurately and extracted with 15 mL of 67% methanol in an ultrasonic bath (250 W, 40 kHz) for 45 min. Extract solutions were cooled down and filtered through a 0.45 μm filter before detection. Glycyrrhizin and liquiritin were separated and detected by high-performance liquid chromatography (HPLC; Agilent-1200, Santa Clara, CA, USA) through an Agilent ZORBAX-Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm). Standard materials of glycyrrhizin (Batch No.110731) and liquiritin (Batch No. 11610) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products, China, to generate the calibration curve [30 (link)].

The protein, moisture, and ash content of the SJP, SJP-A, and SJP-C sample were measured in accordance with the AOAC standard methods (AOAC International, 2005 ). The total carbohydrate content was determined using the phenol–sulfuric acid method (Dubois et al., 1956 (link)). The uronic acid and sulfate group contents were determined using the m-hydroxydiphenyl (Blumenkrantz and Asboe-Hansen, 1973 (link)) and barium sulfate turbidimetric methods (Dodgson, 1961 (link)), respectively. The monosaccharide component was analyzed on an Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system coupled to a ZORBAX Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 µm; Agilent Technologies, Santa Clara, CA, USA). Samples were acid-hydrolyzed using 2 mol/L of trifluoroacetic acid at 110°C for 6 h. Further derivatization and HPLC evaluation were conducted in accordance with methods proposed in a previous study (Fu et al., 2019 (link)). The MWs were measured using a TSKgel G4000PW_XL column (7.8 mm × 300 mm × 10 μm, Tosoh Co., Ltd., Japan) equipped with a refractive index detector (G1362A; Agilent Technologies). Dextran standards of 1, 3.65, 5, 12, 21, and 80 kDa were used for calibration. The following experimental parameters were used: column temperature, 35°C; flow rate, 0.3 ml/min; and mobile phase, 10 mM of NaH₂PO₄ and 200 mM of NaNO₃ dissolved in ultrapure water.

Structural determination of the antimicrobial compound was performed by spectroscopic techniques and literature comparison. The antimicrobial compound was analyzed by thin-layer chromatography on a silica 60 plates (Merck, Germany) with chloroform-acetic acid-ethanol at 95:5:2.5 (v/v) as the mobile phase, followed by spraying of an iron reagent (0.1 M FeCl₃ in 0.1 M HCl). LC-MS was performed with an Agilent 1290 Infinity LC system coupled to Agilent 6520 Accurate-Mass Q-TOF mass spectrometer (dual ESI source) equipped with an Agilent Zorbax Eclipse XDB-C18 column. The ultraviolet/visible absorption spectrum was recorded with the photodiode array detector equipped with the above-mentioned HPLC. The mobile phase was composed of water (A, 0.5% formic acid) and acetonitrile (B, 0.5% formic acid), the gradient program of which was 0–12.00 min 90% A and 10% B and 12.00–15.00 min 100% B. The follow rate of the mobile phase was 0.3 ml/min, and the column temperature was set to 25°C. The injection volume was 10 μl.