The high resolution mass spectroscopy (HRMS), infrared spectroscopy (IR), and high-performance liquid chromatography (HPLC) analyses were performed to examine the chemical profiling of HIET. The HRMS analysis was performed on an ESI-Q-TOF micro mass spectrometer (Waters, Germany) in the electrospray ionization mode (positive mode). The IR spectrum analysis was done by placing a small amount of sample on the ATR accessory using an ALPHA FTIR spectrometer (Bruker, Germany). HPLC analysis was performed on a WatersTM HPLC System (Waters, Germany) equipped with a C30 column (4.6 × 250 mm with 5 µm pore size) followed by Waters 2489 UV/Visible Detector (λ 248 nm). The mobile phase was composed of methanol:ethanol (95:5 v/v), with an elution volume of 0.5 ml/min. The temperature was maintained at 23 °C.
2489 uv visible detector
Manufactured by Waters Corporation
Sourced in United States, Ireland
The 2489 UV/Visible Detector is a laboratory instrument designed to measure the absorption of ultraviolet and visible light by samples. It is capable of detecting and quantifying various compounds based on their characteristic light absorption properties.
Automatically generated - may contain errors
Lab products found in correlation
Hplc system, by Waters Corporation (4 mentions)
1525 binary hplc pump, by Waters Corporation (4 mentions)
Breeze 2 hplc system, by Waters Corporation (3 mentions)
1525 binary pump, by Waters Corporation (3 mentions)
E2795 separations module, by Waters Corporation (2 mentions)
2545 binary gradient module instrument, by Waters Corporation (2 mentions)
2767 sample manager, by Waters Corporation (2 mentions)
Sunfire c18 column, by Waters Corporation (2 mentions)
Waters alliance e2695 separation module, by Waters Corporation (2 mentions)
Esi q tof micro mass spectrometer, by Waters Corporation (1 mentions)
Alpha ftir spectrometer, by Bruker (1 mentions)
Sunfire prep c18 column, by Waters Corporation (1 mentions)
Autopurification system, by Waters Corporation (1 mentions)
Avance 3 500 spectrometer, by Bruker (1 mentions)
2545 binary gradient module, by Waters Corporation (1 mentions)
Masslynx 4.1 workstation, by Waters Corporation (1 mentions)
Gemini c18 column, by Phenomenex (1 mentions)
High performance liquid chromatography system, by Waters Corporation (1 mentions)
600 controller, by Waters Corporation (1 mentions)
Sunfire c18 obd prep column, by Waters Corporation (1 mentions)
40 protocols using 2489 uv visible detector
1
Chemical Profiling of HIET via HRMS, IR, and HPLC
2
Extraction and Isolation of Compounds from Sargassum horneri
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The frozen specimen of S. horneri (3.0 kg) was lyophilized, and the dried algal sample (344 g) was extracted with 7 L of a mixture of dichloromethane and methanol (DCM:MeOH, 1:1) 7 times successively, and the filtrate was concentrated in a vacuum to yield 20.63 g of crude extract that was suspended in MeOH and extracted with n-hexane. The methanol portion was separated and concentrated, then suspended in water followed by sequential partitioning between EtOAc and butanol to obtain 4.03, 8.25, and 2.8 g, respectively. The EtOAc fraction was subjected to size exclusion chromatography on a Sephadex LH-20 column and eluted with DCM:MeOH (1:1), yielding 5 fractions as follows: 1 (2193.8 mg), 2 (1973.8 mg), 3 (648.7 mg), 4 (266.6 mg), and 5 (327.8 mg). EtOAc fraction number 4 was further fractionated by reverse phase chromatography using a C18 preparative column (Hector C18-M510, 16H1001, 250 × 21.2 mm), using a gradient elution system of 10% ACN for 5 min, and 10–100% ACN (5–60 min) with acetonitrile and 0.1% TFA in HPLC grade water as the mobile phase at a flow rate of 10 mL/min. The ultraviolent absorbance was observed at 220 and 250 nm by a Waters 2489 UV/Visible detector (Milford, MA, USA) coupled with a binary HPLC pump (Waters 1525, Milford, MA, USA). The isolated compound (2.7 mg) was eluted at 25.55 min with 95% purity.
3
HPLC Quantification of Doxorubicin in Liposomes
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DOX was quantified using a Waters HPLC system, equipped with a Waters e2795 separations module, a Waters 2489 UV/Visible detector and a C-18 column: XSELECT CSH column XP, 2.5 μm 3.0×75 mm (Waters, Dublin, Ireland). Detection wavelength was 254 nm, and the injection volume was 10 μL. The flow rate was set to 0.5 mL/min and the temperature was set to 25 °C. Two mobile phases were applied for gradient flow condition. Mobile phase A was Milli-Q water with 0.1% (v/v) formic acid and Mobile phase B was acetonitrile with 0.1% (v/v) formic acid. The mixing ratio of Mobile phase A and Mobile phase B was changed linearly from 95:5 to 5:95 (v/v) during 10 min, and with succeeding equilibration sequence of 5 min. Liposome samples and standard samples were prepared in triplicate in Mobile phase A and Triton X-100 95:5 (v/v). Each DOX standard solution (concentration range 5-100 μg/mL) was injected three times into the HPLC (linear standard curves R2 value was 0.9997). DOX retention time (RT) was 4.8 min, whereas the Triton X-100 top had a RT of approximately 10.0 min.
4
HPLC Analysis of Pancreas Tissue Adenosine and AMP
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Filtered supernatants were analyzed by high‐performance liquid chromatography using the Waters Breeze 2 HPLC System (Waters 2489 UV/Visible Detector and Waters 1525 Binary HPLC Pump). Flow rate was 1 ml/min and 100 μl per sample was injected in the column (XSElect HSS C18 SB 5 μm 4.6 × 250 mm) with a mobile phase 100% A (0.02M NH4H2PO4) for 0–4 min, which then was switched to 100% B (0.02M NH4H2PO4 containing 20% methanol) from 4 to 8 min, then stayed in 100% B from 8 to 18 min, and finally switched back to 100% A from 18 to 20 min. Absorbance was measured at a wavelength of 260 and 280 nm, and adenosine and AMP peaks were determined using a standard HPLC curve. Pancreas tissue adenosine and AMP levels were normalized to lysate protein levels.
5
Preparative HPLC Purification of TCM Extracts
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The TCM materials were powdered using an electric blender and then extracted with a 10-fold volume of MeOH using ultrasonication (three times, 1 h each). After filtration, the percolates were combined, and the organic solvent was removed by vacuum evaporation at 45 °C. The residue was suspended in water, and separated by ODS column chromatography, eluting with water, MeOH, and acetone, successively. The methanolic fraction (200 mg) was further fractionized using preparative HPLC, running at a flow rate of 30 mL/min with the mobile phases of water/0.1% formic acid (A) and ACN (B). The LC gradient was 0–3 min, B at 10%; 3–50 min, B at 10–80%; 51–60 min, B at 95%. Preparative HPLC was performed on a Waters 2545 Binary Gradient Module instrument with a Waters 2489 UV/Visible Detector using a Waters Sunfire Prep C18 column (5 µm, 30 × 150 mm). The subfractions were collected according to the UV and ELSD responses. Finally, they were powdered with freeze-drying and stored at − 40 °C for use.
6
HPLC Quantification of Pancreas Tissue Adenosine and AMP
7
Preparative HPLC Purification and Structural Elucidation
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The preparative high-performance liquid chromatography (HPLC) was performed on a Waters Auto-Purification system, which consisted of a Waters 2545 binary gradient module, a Waters 2767 sample manager and a Waters 2489 UV/visible detector. Data were collected using a Masslynx 4.1 workstation (Waters, Milford, MA, USA)
The isolated compounds were identified by HRMS (Agilent, Palo Alto, CA, USA) and NMR (Bruker, Karlsruhe, Germany). The HRMS analysis was performed on Agilent 1290 Infinity LC/6530 Q-TOF MS (Agilent, Palo Alto, CA, USA). 1H-NMR spectra and 13 C-NMR spectra were measured on a BRUKER AVANCE III-500 spectrometer (1H-NMR at 500 MHz; 13 C-NMR at 125 MHz) with DMSO-d6 as the solvent.
The isolated compounds were identified by HRMS (Agilent, Palo Alto, CA, USA) and NMR (Bruker, Karlsruhe, Germany). The HRMS analysis was performed on Agilent 1290 Infinity LC/6530 Q-TOF MS (Agilent, Palo Alto, CA, USA). 1H-NMR spectra and 13 C-NMR spectra were measured on a BRUKER AVANCE III-500 spectrometer (1H-NMR at 500 MHz; 13 C-NMR at 125 MHz) with DMSO-d6 as the solvent.
8
Quantitative Analysis of Bioactive Compounds in Tea
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The catechins, theanine, and caffeine were analyzed on a Waters High Performance Liquid Chromatography (HPLC) system supported with a Waters 600 controller and Waters 2489 UV/Visible Detector (280 nm). Chromatographic separation was performed on a Phenomenex Gemini C18 column. The column temperature was set at 25 °C. The injection volume of sample was 5 μL, the elution rate was 1 mL/min, and the detection wavelength was set at 278 nm. The mobile phase consisted of mobile phase A (deionized water with 0.17% acetic acid) and mobile phase B (100% acetonitrile). The linear gradient at a flow rate of 1.0 mL/min was set as follows: mobile phase B from 8–28.4% (v/v) in 30 min was initiated, from 28.4–100% (v/v) for 8 min, and from 100–8% (v/v) for another 10 min.
9
Simultaneous HPLC Detection of 6-APA, PenV, and POA
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In this study, the HPLC method for simultaneous detection of 6-APA, PenV, and POA molecules was developed using C18 analytical column X-Bridge, 4.6 × 250 mm in size with 5 µm particle size, 1525 binary pump, 2489 UV–Visible detector, and 2707 autosampler system (Waters, USA). The mobile phase consisting of MeOH: water, ACN: water, and MeOH: ACN: water with varying concentrations and pH were tested for better separation of these molecules. The chromatography analysis was processed by injecting 20 µL of the test sample with a flow rate of 1 mL/min and detected at 210 nm. The separation and quantification of the test sample were compared with the standard graph prepared with standard molecules, 6-APA, PenV, and POA. The limit of detection (LOD), the limit of quantification (LOQ), and the precession of the method were measured by preparing calibration curves of commercial standards.
10
Enantioselective Resolution of Nitropropranolol
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Resolution of the racemic mixtures containing (±)-4-nitropropranolol 2a or (±)-7-nitropropranolol 2b was performed using a Waters 2535 Quaternary Gradient Mobile equipped with Waters 2489 UV/Visible Detector set to a dual-wavelength UV detection at 254 and 280 nm. The chiral resolutions were achieved on the Kromasil 5-Amycoat column Phenomenex (150 mm × 21.2 mm, 5 μm particle size). Two mobile phases, in isocratic condition, were used. Mobile phase A: n-Hexane (Chromasolv Sigma-Aldrich) and mobile phase B: Isopropanol (Chromasolv Sigma-Aldrich) + 0.1% Diethylamine. Ratio of the mobile phases was 86(A):14(B). The racemic mixtures were dissolved in ethanol at concentration of 75 mg/mL, and the injection volume was 200 μL (repeated 5 times); the sample was eluted from the column at a flow rate of 15.0 mL/min at room temperature (pressure: ≈500 psi). At the end of racemic resolutions, 30 mg of each enantiomer was collected. Purity of (+) and (−) enantiomers was assessed by chiral HPLC using the Kromasil 5-Amycoat column Phenomenex (150 mm × 4.6 mm, 5 μm particle size) with the same solvents and conditions as for preparative purification, but with a flow rate of 1 mL/min.
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