The dried extract was redissolved with 200 μl of buffer containing 5% methanol and 0.05% TFA in water for HPLC analysis. The chromatographic separation was performed using a Waters e2695 HPLC system equipped with a reversed phase C18 column (Agilent ZORBAX Eclipse XDB‐C18, 9.4 mm I.D. × 250 mm, 5μm) and a Waters 2998 photodiode array detector. The column was equilibrated with 95% solvent A (0.05% TFA in water) and 5% solvent B (0.05% TFA in methanol) at a flow rate of 1 ml min−1. The gradient elution steps were as follows: 0–1 min, 5% solvent B; 1–30 min, a linear gradient to 50% solvent B; 30–40 min, to 99% solvent B, and the UV absorption was 254 nm.
E2695 hplc system
Manufactured by Agilent Technologies
The E2695 HPLC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical separation and quantification of chemical compounds. It features a solvent delivery system, a sample injection unit, a column compartment, and a detector, all integrated into a compact and robust design. The E2695 HPLC system is capable of performing various chromatographic techniques to analyze a wide range of samples.
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4 protocols using e2695 hplc system
1
HPLC Analysis of Plant Extracts
2
Comprehensive Analytical Characterization of ONZ
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The concentration of ONZ was measured in a Waters e2695 HPLC system equipped with an Agilent C18 column (4.6 × 250 mm, 5 μm). The mobile phase was composed of methanol and water (20:80, v/v) with a flow rate of 1.0 mL/min. The ONZ was measured at a wavelength of 318 nm, and the column temperature was 30 °C. An ultraviolet–visible light (UV–Vis) spectrophotometer (D6000, Hach, USA) was used to obtain the UV spectra of the ONZ solution. The chemical oxygen demand (COD) and total organic carbon (TOC) were measured using a COD tester (DRB200, Hach, USA) and a TOC analyzer (TOC-VCPH, Shimadzu Corporation, Japan), respectively. The pH of the solution was measured using a Mettler-Toledo pH meter. An ion chromatograph (DX600, Dionex, USA) was used to detect the inorganic ions. The carbon content was determined by element analysis (Vario EL Ⅲ, Elementar, Germany). The iron content was determined with inductively coupled plasma atomic emission spectrometry (ICP-OES, ICPOES730, Agilent, USA) after HNO3 digestion.
3
Microbial Population and Metabolite Analysis
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The brine was centrifuged at 2,054 g and 4°C for 10 min to harvest microorganisms. The separated supernatants and pellets were stored at −80°C for metabolite analyses and population analyses of three LABs, respectively.
Mannitol was measured using a Waters e2695 HPLC system equipped with an SPELC‐NH column (Agilent) at 35°C. The mobile phase was 78% acetonitrile and 22% water, and the flow rate was 1 ml/min. Lactic acid was analyzed using HPLC on an CAPECELL PAK MGS5 C18 column (4.6 mm × 250 mm; 5 μm) at 40°C with 0.1% phosphoric acid and methyl alcohol (97.5:2.5 volume ratio) as eluent. Acetic acid was analyzed using HPLC on an CAPECELL PAK MGS5 C18 column (4.6 mm × 250 μm; 5 μm) at 25°C with diammonium phosphate (pH adjusted to 2.7–3.5 with 1 mol/L phosphoric acid) as eluent. All samples were detected at 210 nm, except for acetic acid, which was detected at 214 nm. Amino acid content was determined using an amino acid automatic analyzer HitachI L‐8500 (Hitachi Ind., Tokyo, Japan).
Mannitol was measured using a Waters e2695 HPLC system equipped with an SPELC‐NH column (Agilent) at 35°C. The mobile phase was 78% acetonitrile and 22% water, and the flow rate was 1 ml/min. Lactic acid was analyzed using HPLC on an CAPECELL PAK MGS5 C18 column (4.6 mm × 250 mm; 5 μm) at 40°C with 0.1% phosphoric acid and methyl alcohol (97.5:2.5 volume ratio) as eluent. Acetic acid was analyzed using HPLC on an CAPECELL PAK MGS5 C18 column (4.6 mm × 250 μm; 5 μm) at 25°C with diammonium phosphate (pH adjusted to 2.7–3.5 with 1 mol/L phosphoric acid) as eluent. All samples were detected at 210 nm, except for acetic acid, which was detected at 214 nm. Amino acid content was determined using an amino acid automatic analyzer HitachI L‐8500 (Hitachi Ind., Tokyo, Japan).
4
Amino Acid Profile Analysis Protocol
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CP content was determined by rapid N cube (NY/T 2007-2011) using a N-to-protein conversion factor of 6•25. The AA contents were determined in triplicate 5-mg samples following hydrolysis in 500 µl of constant-boiling HCl (6 mol/l) for 24 h at 110 ± 1°C in a hydrolysis tube (24) . The liberated AA were derived with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and α-aminobutyric acid was used as the internal standard. The derivatives were separated on a Waters E2695 HPLC system equipped with a C18 column (150 mm × 4•6 mm, 5•0 µm; Agilent) and quantified using Waters 2475 fluorescence detector at 395 nm emission and 250 nm excitation. To determine cysteine and methionine, we used performic acid oxidation at 0°C for 16 h, followed by neutralisation with HBr; then, we applied hydrolysis as described above. The concentration of titanium in the diets and ileal samples was determined through the method described by Short et al. (25) . The samples were ashed, then digested in 60 % (v/v) sulfuric acid and finally added to 30 % hydrogen peroxide. Absorbance at 410 nm was measured. Tryptophan (Trp) was determined using the method described by Rutherfurd & Gilani (24) . Free AA molecular weights were used for the calculation of the weight of each AA.
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