The analysis of HPLC products was carried out on a Waters 2695 coupled with a Waters 2998 Diode array detector, analysis SunFireTMC18 Column (250 mm × 4.6 mm, id 5 μm, Waters Corporation, Milford, MA, USA) and Sigma 1–14 centrifuge (Sigma, St. Louis, MO, USA) with ultramembrane filters (Microcon YM-30; Millipore, Billerica, MA, USA). A semi−preparative HPLC was performed using a 2545 Quaternary Gradient Module pump, a Fraction Collector III, a Waters 2489 UV/Vis detector, and a semi−prep SunFireTMC18 Column (100 × 19 mm, ID 5 μm, Waters Corporation, Milford, MA, USA). HSCCC was performed on a DE Spectrum HSCCC (Dynamic Extractions, Slough, UK). The kinetics of enzymatic reactions were managed using a High Performance Capillary Electrophoresis apparatus (Beckman Coulter, Krefeld, Germany).
Sunfire c18 column
Manufactured by Waters Corporation
Sourced in United States, Ireland, Germany, Japan, France, United Kingdom
The Sunfire C18 column is a reversed-phase high-performance liquid chromatography (HPLC) column used for the separation and analysis of a wide range of molecules. The column features a chemically bonded C18 stationary phase, which provides excellent retention and selectivity for non-polar and moderately polar analytes. The Sunfire C18 column is designed for reliable and reproducible performance in various HPLC applications.
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Lab products found in correlation
Hplc system, by Waters Corporation (9 mentions)
Micromass zq instrument, by Waters Corporation (8 mentions)
Empower software, by Waters Corporation (7 mentions)
Acquity uplc, by Waters Corporation (6 mentions)
Avance 3 spectrometer, by Bruker (6 mentions)
Beh c18 column, by Waters Corporation (6 mentions)
Empower 3, by Waters Corporation (6 mentions)
E2695 hplc system, by Waters Corporation (5 mentions)
Zq 4000 mass spectrometer, by Waters Corporation (5 mentions)
Alliance chromatographic system, by Waters Corporation (5 mentions)
2998 photodiode array detector, by Waters Corporation (4 mentions)
Agilent 1260, by Agilent Technologies (4 mentions)
2707 autosampler, by Waters Corporation (4 mentions)
E2695, by Waters Corporation (4 mentions)
Acquity uplc instrument, by Waters Corporation (4 mentions)
Fluorescence detector system, by Waters Corporation (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Waters 2998 photodiode array detector, by Waters Corporation (3 mentions)
E2695 separation module, by Waters Corporation (3 mentions)
Spd m20a, by Shimadzu (3 mentions)
263 protocols using sunfire c18 column
1
Comprehensive HPLC and HSCCC Analysis Protocol
2
NMR Spectroscopy and Chromatographic Procedures
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NMR spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer operating at 500.13 MHz for 1H. A 1-mm TXI microprobe with a z-gradient was used for 1H-detected experiments. Spectra were analyzed using Bruker Topspin 3.0 software. CDCl3 for NMR were purchased from Armar Chemicals. Solvents used for extraction, column chromatography and HPLC were of technical grade and were purified by distillation. Silica gel (70–230 mesh) was used for column chromatography and percolated silica gel F254 (20*20 cm) plates for TLC (both Merck). Detection was at 254 and 366 nm or by spraying with phosphor molybdic acid reagent and subsequently heating (120°C for 5 min). Analytical RP-HPLC using a SunFireTM C18 column (3.5 μM, 10 mm × 150 mm; Waters) equipped with a pre-column (3 × 10 mm) was used for analytical evaluations. Semi preparative RP-HPLC using a SunFireTM C18 column (5.0 μM, 10 mm × 150 mm; Waters) equipped with a pre-column (5.0 × 10 mm) was also used for the isolation process. Antifungal compounds were purchased from HiMedia (Mombay, India).
3
HPLC Purification and Characterization of Polyphenols
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Quercetin (12 , >98%) was purchased from Sigma-Aldrich. Gallic acid (1 , >98%), hyperoside (8 , >95%), isoquercitrin (10 , >95%), and polyamide (particle size: 0.05–0.16 mm) were from Carl Roth. HPLC-grade acetonitrile and methanol (Reuss Chemie AG), and distilled water were used for HPLC separations.
Preparative HPLC was carried out on an LC 8A preparative liquid chromatograph equipped with a SPD-M10A VP PDA detector (all Shimadzu). A SunFire C18 column (150 × 30 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used, at a flow rate of 20 mL/min. HPLC-based activity profiling was performed on an Agilent 1100 system equipped with a PDA detector. A SunFire C18 column (150 × 10 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used. The flow rate was 4 mL/min. Time-based fractions were collected with a Gilson FC204 fraction collector. ESI-MS spectra were obtained on an Esquire 3000 Plus ion trap mass spectrometer (Bruker Daltonics). NMR spectra were recorded on an Avance III 500 MHz spectrometer (Bruker BioSpin) equipped with a 1-mm TXI microprobe.
Preparative HPLC was carried out on an LC 8A preparative liquid chromatograph equipped with a SPD-M10A VP PDA detector (all Shimadzu). A SunFire C18 column (150 × 30 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used, at a flow rate of 20 mL/min. HPLC-based activity profiling was performed on an Agilent 1100 system equipped with a PDA detector. A SunFire C18 column (150 × 10 mm i.d., 5 μm; Waters) connected to a pre-column (10 × 10 mm) was used. The flow rate was 4 mL/min. Time-based fractions were collected with a Gilson FC204 fraction collector. ESI-MS spectra were obtained on an Esquire 3000 Plus ion trap mass spectrometer (Bruker Daltonics). NMR spectra were recorded on an Avance III 500 MHz spectrometer (Bruker BioSpin) equipped with a 1-mm TXI microprobe.
4
Purification and Characterization of Compounds
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The NMR spectra were recorded on Bruker Avance 500 MHz and Bruker Avance III 400 MHz NMR spectrometers using acetone-d6 as an internal standard, while HRESIMS data were obtained from a Bruker MicOTOF spectrometer. Column chromatography was performed on a Sephadex LH-20 column using 100% methanol as the eluent. HPLC was performed on a Dionex-Ultimate 3000 series equipped with a binary pump, an autosampler, and diode array detector. Semi-preparative HPLC was performed on a Sunfire C18 column from Waters (5 µm, diam. 19 mm × 150 mm). Preparative HPLC was performed on a Sunfire C18 column from Waters (10 µm, diam. 19 mm × 250 mm).
5
Bioactive Fractions from Natural Extract
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All 16 fractions were tested on Micrococcus luteus ATCC 10240 (Gram positive bacterium) and Escherichia coli ATCC 35218 (Gram negative bacterium) for qualitative antimicrobial activity using the disc diffusion assay and nine of them showed encouraging (up to 18 mm diameter) bioactivity. Fraction F14 (70 mg, eluted with EtOAc-MeOH/90-10%) was the most active. The HPLC profile of F14 was recorded using a stepwise gradient of milliQ H2O and 2-100% MeCN, both with 0.1% HCOOH (flow rate: 1mL/min; Waters Sunfire C-18 column, 150 x 4.6mm, 5µm). Semi-preparative HPLC of this fraction, (Waters Sunfire C-18 column, 150 x 10mm, 5µm; flow rate 4.5 mL/min) with the same transposed gradient, gave five sub-fractions F14-1 (10 mg), F14-2 (10.7 mg), F14-3 (11.8 mg), F14-4 (17,4 mg) and F14-5 (6.8 mg). The major compound in F14-1 at retention time 12.52 min was detected and was slowly precipitating to give 3.9 mg of the pure compound 1 (noted F14-1res). Semi-preparative HPLC in the same conditions described above was performed on supernatant of F14-1 and led to 1.9 mg of the pure compound 1. Fraction F14-2 gave equally 0. These strains were obtained from the collection of the Pasteur Institute (Guadeloupe) and were cultivated on agar plates with Mueller-Hinton medium nutrient medium at 37 °C for all bioassay experiments.
6
Purification and Characterization of Organic Compounds
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Unless otherwise described, all commercial reagents and solvents were purchased from commercial suppliers and used without further purification. All reactions were performed under N2 atmosphere in flame-dried glassware. Reactions were monitored by TLC with 0.25 mm E. Merck precoated silica gel plates (60 F254). Reaction progress was monitored by TLC analysis using a UV lamp, ninhydrin, or p-anisaldehyde stain for detection purposes. All solvents were purified by standard techniques. Purification of reaction products was carried out by silica gel column chromatography using Kieselgel 60 Art. 9385 (230 − 400 mesh). The purities of all compounds were shown to be over 95% by using Waters LCMS system (Waters 2998 photodiode array detector, a Waters 3100 mass detector, a Waters SFO system fluidics organiser, a Water 2545 binary gradient module, a Waters reagent manager and a Waters 2767 sample manager) using a SunFireTM C18 column (4.6 mm × 50 mm, 5 µm particle size): solvent gradient = 60% (or 95%) A at 0 min, 1% A at 5 min. Solvent A = 0.035% TFA in H2O; solvent B = 0.035% TFA in MeOH; flow rate 3.0 (or 2.5) mL/min. 1H and 13 C NMR spectra were obtained by using a Bruker 400 MHz FT-NMR (400 MHz for 1H, and 100 MHz for 13 C) spectrometer. Standard abbreviations are used for denoting the signal multiplicities.
7
HPLC Analysis of Phenolic Compounds
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The HPLC system was comprised of a Waters e2695 HPLC system equipped with a Waters SunFireTM C18 column (250 mm × 4.6 mm, 5 μm, Waters, Milford, MA, USA) as well as a Diode Array Detector (DAD, Waters 2998, Milford, MA, USA). The mobile phase consisted of 0.1% formic acid aqueous solution (v/v, solution A) and acetonitrile solution (solution B) in the gradient elution at 0.8 mL/min with time-course increasing of solution B to 15% B for 0–5 min, 15–20% B for 5–10 min, 20–25% B for 10–20 min, 25–35% B for 20–30 min, 35–50% B for 30–40 min, 80% B for 40–50 min, and 15% B for 50–55 min. The column temperature was set at 30 °C. The detected wavelength was set at 280 nm. Before HPLC analysis, the samples were filtered through a 0.25-μm membrane filter (Millipore, Billerica, MA, USA). Accurate amounts of standard phenolics were added to GL extracts after enzymatic hydrolysis, and they were extracted as described in Section 4.3 , Section 4.4 and Section 4.5 . As calculated according to the amount found and amount added, the recovery rate of these phenolics ranged from 95.31% to 101.07% (Table 3 ). The contents of individual phenolics were expressed as milligram per 100 g DM of GL samples.
8
HPLC Analysis of Compounds
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High-Performance Liquid Chromatography (HPLC) analysis was performed using an Alliance e2695 system (Waters, Milford, MA, United States) equipped with an autosampler and photodiode array detector (DAD, Waters 2998) and a SunfireTM C18 column, 5 μm (4.6 × 150 mm) (Waters®) attached to a guard column (SunfireTM C18, 5 μm, 4.6 × 20 mm, Waters®). The mobile phase consisted of a gradient of H2O (A):CH3CN (B) with a flux rate of 2 mL/min for 60 min (5%–100% B) and a UV detection wavelength of 210–600 nm.
9
HPLC Analysis of Metabolites
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A 900 µL aliquot from the elicited culture medium was extracted with an equal amount of ethyl acetate in a 2 mL microcentrifuge tube by vortexing for 30 s. Then, 500 µL of the upper organic phase was removed, transferred to an amber HPLC vial, and dried under a nitrogen stream using a Reacti-Vap III apparatus (Thermo Fisher Scientific, Waltham, MA, USA). HPLC analyses were performed in an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The extract was resuspended in 500 µL of MeOH and analyzed by reversed-phase HPLC. Briefly, the chromatography was performed in a SunFireTM C18 column (5 µm, 4.6 × 250 mm, UV detection at 340 nm) (Waters, Milford, MA, USA) at 40 °C and a flow rate of 1.0 mL/min. The mobile phase was composed of MeOH (A) and 0.5% HCO2H (v/v) (B). The column was initially calibrated with B for 1 min. Then, a linear gradient was performed using the following program: 60% A to 65% A for 1–20 min, 65% A and 35% B to 100% B for 20–25 min, and 100% B for 25–30 min. Calibration curves for reference compounds were established previously [36 (link)]. Similarly, re-elicited culture medium was extracted and analyzed as described above.
10
Quantitative Analysis of Nucleotides
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Fresh-frozen tissues were washed once with PBS, and homogenized in 10% Trichloroacetic Acid (T6399, Sigma-Aldrich, St. Louis, MO, USA). After incubating on ice for 10 min, the mixtures were centrifuged at 12,000 g for 15 min at 4 °C, and the nucleotide containing supernatant was washed 4 times with water-saturated diethyl ether. Then the supernatant was froze at −80 °C, and freeze-dried at −20 °C overnight use FreeZone Freeze Dry Systems (LABCONCO, Inc., MO, USA). The extracted nucleotides were dissolved in 100 μL ultrapure water and centrifuged at 8,000 rpm for 5 min. Supernatant was filtered with 0.45 μm filter and then analyzed by HPLC on a Agilent 1260 (Agilent Technologies, Inc., CA, USA) equipped with a Waters SunFireTM C18 column (250 mm × 4.6 mm i.d., 5 μm, Waters, Milford, MA, USA) and detected by UV-VIS absorbance at 260 nm. The nucleotides were eluted with isocratic elution (flow rate: 0.75 ml/min, Column temperature: 25 °C) using a mixture of 10% acetonitrile and 90% water (0. 01 mol/L Tetrabutylammonium bromide, 0.05 mol/L Na2HPO4-NaH2PO4, pH6.4). The amount of each nucleotide was determined by external standard method. The following standards were used: ATP (A2383), ADP (A2754), AMP (01930), IMP (57510), GMP (G8377), GDP(G7127), GTP(G8877) (Sigma-Aldrich, St. Louis, MO, USA).
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