Column chromatography was carried out on flash silica gel (Sorbent 230–400 mesh). TLC analysis was conducted on silica gel plates (Sorbent Silica G UV254). Reverse phase high performance liquid chromatography (RP-HPLC) was carried out with Waters 600. Analytical HPLC was performed on a SunfireTM C18 Column (5 μm, 4.6 × 150 mm) at a flow rate of 1 ml/min measured by Waters 2996 Diode Array Detector. Preparative HPLC was performed with a COSMOSIL 5C18-AR-300 Column (5 μm, 20 × 250 mm) at a flow rate of 8 ml/min measured by Waters 2996 Diode Array Detector. For all HPLC, solvent A (water with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA) were utilized. All UV/vis spectra and kinetics experiments were recorded on an Agilent 8453 UV/vis Spectrophotometer. Pseudo first order rate constants from all kinetics experiments were calculated using the Agilent UV/vis Chemstation software package Rev. A.10.01. Fluorescence measurements were obtained using a Varian Cary Eclipse Fluorescence Spectrophotometer. 1H (300 MHz) and 13C nuclear magnetic resonance (NMR) (100 MHz) spectra were collected on a Bruker Spectrometer at the ambient temperature in DMSO. High-resolution electrospray ionization (ESI) mass spectra were obtained on a Bruker Daltonics Esquire 6000 with ESQ6K Operator. BL-420E+, Taimeng Technology Corporation of Chengdu, China.
2996 diode array detector
Manufactured by Waters Corporation
Sourced in United States
The 2996 Diode Array Detector is a high-performance liquid chromatography (HPLC) detector designed by Waters Corporation. The device is capable of analyzing a wide range of compounds by detecting their absorbance across a spectrum of wavelengths. It provides reliable and accurate data for various analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Hplc system, by Waters Corporation (2 mentions)
Alliance 2695, by Waters Corporation (2 mentions)
1290 infinity hplc system, by Agilent Technologies (1 mentions)
Sunfire c18 ods column, by Waters Corporation (1 mentions)
Accq reagent, by Waters Corporation (1 mentions)
Empower pro software, by Waters Corporation (1 mentions)
Esquire 6000, by Bruker (1 mentions)
Cary eclipse fluorescence spectrophotometer, by Agilent Technologies (1 mentions)
Spectrometer, by Bruker (1 mentions)
8453 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
2695 separations module, by Waters Corporation (1 mentions)
Esi source, by Bruker (1 mentions)
Microtof q type qq tof ms instrument, by Bruker (1 mentions)
Hypersil gold reversed phase c18 column, by Thermo Fisher Scientific (1 mentions)
Alliance 1525, by Waters Corporation (1 mentions)
Sil 20a autosampler, by Shimadzu (1 mentions)
Sunfire c18, by Waters Corporation (1 mentions)
Lc solution chromatography workstation, by Shimadzu (1 mentions)
Lc 20a, by Shimadzu (1 mentions)
Spd m20a diode array detector, by Shimadzu (1 mentions)
14 protocols using 2996 diode array detector
1
Analytical Techniques for Chemical Analysis
2
Flavonoid Extraction and Quantification
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The extraction of total flavonoids was conducted according to previous study [5 (link)] with minor modification. A Waters HPLC system equipped with a 2695 quaternary pump and a 2996 diode array detector (Waters Corp., Milford, MA, USA) was used in the chromatographic experiments. A Sunfire C18 ODS column (4.6 × 250 mm, 5 μm, Waters Corp.) was used to separate the compounds at room temperature with a flow rate of 1 mL min−1. The mobile phases were water (A) and acetonitrile (B) with a linear gradient program as follows, 0/80, 5/80, 10/73, 15/73, 25/60, 35/40, 40/20, 42/0, 45/80, and 50/80 (min/A%) [40 (link)]. The compounds were monitored under 280 nm and 330 nm, and quantitated according to the standard curves of the authentic standards [40 (link)]. The enzymatic reaction products were detected using an Agilent 1290 Infinity HPLC system (quaternary pump, a DAD detector) and the compounds were identified combining ion fragments, retention time, and characteristic spectrum.
3
Amino Acid Extraction and Analysis
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Cells were disrupted in 80% (vol/vol) ethanol using a bead beater followed by a 30-min extraction at 70 °C using a Büchi Rotavapor (26 (link)). The extract was brought to dryness and redissolved in 5 mL H2O. This extract was then separated into neutral, cationic, and anionic fractions using tandem ion exchange chromatography as previously described (48 (link)). One-mL aliquots of the basic fractions were evaporated to dryness before resuspension in 100 µL 20% (vol/vol) acetonitrile in 20 mM HCl. A total of 20 µL of this solution was analyzed by high-performance liquid chromatography (HPLC) after derivatization with the AccQ reagent (Waters). The Waters HPLC system was controlled by the Empower Pro software and equipped with a 600 controller. Detection was done using the Waters 2996 Diode Array Detector. Amino acids were separated on a Waters AccQ•Tag Amino Acid Analysis column (3.9 × 150 mm) equipped with a Nova-Pak C18 precolumn cartridge (3.9 × 20 mm). Quantification was performed using calibration curves generated from commercial standards (Thermo Scientific and Sigma-Aldrich). Results were corrected for recovery using l -norvaline as an external standard.
4
HPLC Determination of DMAT Compound
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The HPLC determination of DMAT was performed using the Waters 2695 Separations module equipped with a thermostable autosampler (5 °C), a column module (35 °C), and a Waters 2996 diode array detector (DAD). The separation of the compounds was achieved using a VDSphere PUR C18-M-SE (4.6 × 150 mm, 5 µm) (Agilent technologies, Palo Alto, CA, USA) column. For HPLC-MS measurements, the HPLC instrument was coupled with a MicroTOF-Q-type Qq-TOF MS instrument equipped with an ESI source from Bruker (Bruker Daltoniks, Bremen, Germany). The flow rate and run time were 1.0 mL/min and 12 min, respectively. The active component DMAT was detected with DAD at 260 nm and MS. For the separation of the compounds, isocratic eluent composed of 25% methanol, 40% AcN/water (9/1) containing 0.1% trifluoroacetic acid (TFA), and 35% water was used.
5
HPLC-DAD Analysis of Hepatodamianol
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High-performance liquid chromatography with diode array detection was performed as previously described [12 (link)] with minor modifications. A Waters Alliance 1525 LC system equipped with an online degasser, binary pump, autosampler, and a 2996 diode array detector (Waters, Milford, MA, USA) was used. The separation was carried on a Hypersil Gold C18 reversed-phase column (4.6 × 150 mm, 5 μm; Thermo Fisher Scientific, Waltham, MA, USA) in gradient mode with methanol and aqueous 0.1% formic acid solution as mobile phases at a flow rate of 0.4 mL/min. The elution program commenced with 30% methanol, changing to 60% methanol in 20 min with a linear gradient, then increasing to 70% methanol in 5 min, held for 5 min, and subsequently returned to the initial conditions in 10 min with an equilibration time of 5 min. The injection volume was 10 μL. Samples were always analyzed in duplicate. Data acquisition in the diode array detector was recorded from 210 to 400 nm with a resolution of 1.2 nm per diode. Hepatodamianol quantitation was carried out at 254 nm.
6
HPLC-DAD Analysis of Compound Separation
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HPLC-DAD was performed using a Waters Alliance 1525 liquid chromatography system equipped with an online degasser, binary pump, autosampler, and a 2996 diode array detector (Waters, Milford, Massachusetts, USA). The separation was carried out in an inverse phase Luna Phenomenex column (C-18, 150 × 4.6 mm, 5 μm) with a Phenomenex precolumn (C-18 4 × 3.0 mm). The mobile phase consisted of a gradient of (A) MeOH and (B) formic acid 0.1%, elution starting with 30% A; 30–60% A, 0–20 min; 60–70% A, 20–25 min; and 70% A, 25–60 min. The flow rate was 0.4 mL/min; 0–20 min, 0.2 mL/min; 20–45 min; 0.4 mL/min, and 45–60 min. Injection volumes were 20 μL. All samples were injected in duplicate.
7
HPLC Analysis of Compounds
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Analyses were performed using HPLC system, which were Shimadzu LC-20A including LC-20AT solvent delivery unit, SIL-20A autosampler, SPD-M20A diode array detector, and LC solution chromatography workstation (Shimadzu Corporation, Japan); Waters Alliance HPLC, including 2695 solvent management systems, 2996 diode array detector, Empower 3 chromatography workstation (Waters Corporation, USA). Columns used in the analysis were Inertsil ODS-3 C18 (250 mm × 4.6 mm, 5 μm, Shimadzu), Venusil XBP C18 (250 mm × 4.6 mm, 5 μm, Agela), and SunFire C18 (250 mm × 4. 6 mm, 5 μm, Waters). Mobile phase consisted of (a) acetonitrile and (b) water and separation was achieved using the following gradient: 0–28 min, 19% A; 28–58 min, 19%–29% A; 58–85 min, 29% A; 85–125 min, 29%–40% A. The flow rate was set at 1.0 mL/min and the column temperature was maintained at 30°C. The detection wavelength was at 203 nm, and the injection volume was 20 μL. Chromatograms are shown in Figure 2 .
8
Carotenoid Separation and Analysis
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Column chromatography separation was performed using an ODS silica gel open tubular column in the stepwise elution mode. HPLC analysis was conducted on a Waters Alliance 2695 HPLC system, equipped with a quaternary solvent delivery system, an autosampler, a 2996 diode array detector (Waters, Milford, MA, USA), and a 250 mm × 4.6 mm i.d., 3 µm, YMC C-30 carotenoid column (Waters Co., Ireland). Freeze drying was carried out using a laboratory freeze dryer (Scientz®Scientz-10N, freeze dryer, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China)
9
Ropivacaine Quantification by HPLC
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The total concentration of ropivacaine was determined with high-performance liquid chromatography (Chen et al. 2020 (link); Gromov et al. 2021 (link)) (HPLC, 2995 pump, 2996 diode array detector, Waters, USA). The HPLC column was a shim-pack C18 column (250 mm × 6 mm, 5 microns) and the pre-column was Alltech C18 (10 mm × 4.6 mm, 5 microns). The mobile phase was potassium dihydrogen phosphate/acetonitrile (75/25) buffer (pH 3.0). The column temperature was maintained at 25 ℃, flow rate at 1.0 mL/min, detection wavelength at 210 nm, and sample injection amount at 20 μL. Lidocaine (Zhaohui Pharmaceutical Co., Ltd., Shanghai, China) was used as the control substance in this work.
10
HPLC Analysis of Organic Acids
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HPLC analyses of organic acid content was carried out using 2695 Alliance Separation Module (Waters, Milford, MA, USA) equipped with a 2996 diode array detector (Waters, Milford, MA, USA). A 10 µL aliquot of filtered sample was separated using Synergi Hydro-RP80A column (250 × 4.6 mm, 4 µm particle size) (Phenomenex, Torrance, CA, USA) with temperature controlled at 30 °C. The mobile phase consisted of mobile phase A (20 mM KH2PO4 with adjusted pH 2.9) and mobile phase B (water) with a flow rate of 0.6 mL/min. Gradient elution was performed as follows: 0–30 min, maintained at 100% A; 30–31 min, from 100% to 0% A; 31–45 min, maintained at 0% A; 45–46 min, from 0 to 100% A; 46–55 min, maintained at 100%. Peak identification was made by comparing retention times and UV spectra at 190, 210 and 254 nm with authentic organic acids compounds. Quantification was performed using calibration curves obtained by injecting known amounts of pure organic acids (tartaric acid, lactic acid, acetic acid, citric acid, succinic acid, oxalic acid, L-mallic acid, kojic acid and ascorbic acid) as external standards.
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