The experiments were performed with Waters HPLC system equipped with a pump, auto sampler, column oven, photodiode array UV/VIS detector (Waters HPLC system, USA) and Empower software program was used to data acquisition and process. The chromatographic columns used in this experiment are commercially available; obtained from Phenomenex Luna C18 (0 4.6 × 250 mm, 5μm, Daejeon, Korea). The column oven temperature was kept at 40°C. The injection volume was 10 μL and the flow rate of the mobile phase was 1.0mL/min. The wavelength of the UV detector was set at 254nm. The mobile phase composed of acetonitrile 100% (A) water containing 0.1% trifluoroacetic acid (B). The run time was 60 min and the mobile phase program was the step gradient elution as follows: Time 0-10 min A-B (10-90), 10-25 min (20-80), 25-33 min (30-70), 33-43 min (33-67), 43-50 min (40-60) and 50-60 min (44-56). The chromatographic conditions were presented in Table 3 .
Hplc system
Manufactured by Waters Corporation
Sourced in United States, Germany, Japan, Ireland, United Kingdom, India, France
The HPLC system is a high-performance liquid chromatography instrument used for the separation, identification, and quantification of various chemical compounds in complex mixtures. It employs a liquid mobile phase to carry the sample through a stationary phase within a column, enabling the separation and analysis of the components based on their unique interactions with the stationary phase.
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Lab products found in correlation
717 plus autosampler, by Waters Corporation (24 mentions)
1525 binary hplc pump, by Waters Corporation (14 mentions)
Empower 3, by Waters Corporation (10 mentions)
Empower 2, by Waters Corporation (10 mentions)
1525 binary pump, by Waters Corporation (9 mentions)
Sunfire c18 column, by Waters Corporation (9 mentions)
Breeze 2, by Waters Corporation (8 mentions)
Symmetry c18 column, by Waters Corporation (7 mentions)
996 photodiode array detector, by Waters Corporation (7 mentions)
Wat038040, by Waters Corporation (6 mentions)
C18 column, by Waters Corporation (6 mentions)
2707 autosampler, by Waters Corporation (6 mentions)
2998 photodiode array detector, by Waters Corporation (5 mentions)
717 plus, by Waters Corporation (5 mentions)
717 autosampler, by Waters Corporation (5 mentions)
Luna c18 column, by Phenomenex (5 mentions)
C18 reverse phase column, by Waters Corporation (5 mentions)
Sequencing grade trypsin, by Promega (4 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (4 mentions)
Hplc grade acetonitrile, by Merck Group (4 mentions)
523 protocols using hplc system
1
Analytical HPLC Method for Compound Separation
2
Copolymer Molecular Weight Analysis by GPC
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The number-average molecular weight and polydispersity of copolymers were determined by gel permeation chromatography (GPC) equipped with a Waters HPLC system. The Waters HPLC system consists of a 510 pump, a 717 autosampler, and a 410 refractive index detector. Separations were achieved using a Waters Stryragel HT3, HT4, and HT5 columns in series. Tetrahydrofuran (THF) was the solvent, and the flow rate was 0.8 ml min−1. The system was calibrated with polystyrene molecular weight standards from 2830 K to 2640 K (from Polymer Laboratory). The sample concentration was 3–4 mg ml−1, the injection volume was 10 μL, and then the data were analysed by Trisec GPC software version 3.
3
Carotenoid Profiling by HPLC-PDA
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Carotenoids profiles were determined by liquid chromatography according to Pacheco et al. (2014) , using a Waters TM HPLC system, controlled by the Empower software program with photodiode array detector (PDA). Carotenoid separation was obtained in a C30 column (S-3 Carotenoid, 4.6 mm × 250 mm, YCMTM) at 33 °C, by a gradient elution of methanol and methyl tert-butyl ether. The elution started with a mix of 80% methanol and 20% methyl tert-butyl ether. At 0.5 min the concentration of ether was increased to 25%, at 15.00 min to 85% and at 15.05 to 90% ether. The concentration of ether was maintained at 90% until 16.50 min and then, at 16.55 min, it returned to the initial condition (20%), remaining constant up to 28 min point. Flow rate was 0.8 mL/min and running time was 28 min. Sample injection volume was 15 µL. Carotenoids were identified based on their retention times and UV/Vis absorption spectra, compared to the standard retention times and UV/Vis absorption spectra of each carotenoid.
4
Characterization of Antimicrobial Hemolymph Compounds
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The MGW extracted hemolymph was examined via reverse-phase high-pressure liquid chromatography (RP-HPLC; Waters TM HPLC system) with a C-18 Prep column. Two solvent sets (Solvent A & Solvent B) were used, solvent A: 0.1% TFA in water (v/v) and solvent B: 0.1% TFA in 100% acetonitrile (v/v). A linear gradient of 0–100% acetonitrile was used to collect different fractions over 30 min, flow rate of 8 mL min−1, monitored at 254 nm. All the fractions were collected and freeze-dried to perform antibacterial assay. The active fraction showing antimicrobial activity was further purified through C-18 Prep column as described above, collected and stored at -80 °C for further characterization.
5
HPLC Analysis of Compounds
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The HPLC system (Waters, Milford, MA, USA) consisted of a waters 1525 binary HPLC pump, a Waters 2998 Photodiode Array Detector, and a Waters 2707 Autosampler. The data were acquired and processed using Windows XP-based Waters Breeze 2 software. Ultrapure water (18 MΩ/cm) was produced by a Milli-Q® Advantage A10® Water Purification System (Billerica, MA, USA).
The chromatographic separations were carried out on a reverse phase Waters Symmetry®C18 column (150 × 4.5 mm i.d., particle size 3.5 μm). The mobile phase was a mixture of acetonitrile and 10 mM potassium dihydrogen orthophosphate (65:35, v/v; pH adjusted to 7 with sodium hydroxide) delivered at a flow rate of 1 ml/min. The mobile phase was filtered through 0.45-µm Whatman®filterpaper and sonicated for 20 min. Analysis was performed at ambient temperature, and the elution of the compounds was monitored by diode array detection (DAD) from 190 to 400 nm. The chromatograms were recorded at 254 nm, and the injection volume was 20 µl.
The chromatographic separations were carried out on a reverse phase Waters Symmetry®C18 column (150 × 4.5 mm i.d., particle size 3.5 μm). The mobile phase was a mixture of acetonitrile and 10 mM potassium dihydrogen orthophosphate (65:35, v/v; pH adjusted to 7 with sodium hydroxide) delivered at a flow rate of 1 ml/min. The mobile phase was filtered through 0.45-µm Whatman®filterpaper and sonicated for 20 min. Analysis was performed at ambient temperature, and the elution of the compounds was monitored by diode array detection (DAD) from 190 to 400 nm. The chromatograms were recorded at 254 nm, and the injection volume was 20 µl.
6
SEC-MALS Analysis of Macromolecular Complexes
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SEC-MALS analysis was performed with an HPLC system (Waters, Milford, MA, USA), coupled with UV- (Waters), Dawn8+ MALS- (Wyatt, Dernbach, Germany), and RI- (Shodex, Tokyo, Japan) detectors. Samples were filtered using 0.1 μm centrifugal filters (Millipore, Burlington, MA, USA) and injected onto Superdex 200 Increase 10/300 column (Cytiva, Marlborough, MA, USA), previously equilibrated with 20 mM Tris pH 7.5, 150 mM NaCl. Data were analyzed using Astra 7.0.
7
HPLC Analysis of Asperuloside from Senna obtusifolia
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AO content was determined via HPLC. The extraction method and processing referred to the method described by the Chinese Pharmacopoeia (2015 Edition) [22 ]. A Waters HPLC system equipped with a 1525 binary pump and a Waters 2487 Dual λ detector was used to analyze the extracts from S. obtusifolia seeds. A Waters Sunfire C18 column (4.6 mm × 250 mm, 5 μm) was employed for all separations. AO standard compound (111,900–201,504) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
8
HPLC Analysis of Glycerol and Ethanol
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The samples (1.2 mL culture supernatant) were filtered through 0.2-μm Minisart RC membrane filters (Sartorius, Göttingen, Germany) and stored at − 20 °C until analysis. Detection and quantification of glycerol and ethanol were performed using a Waters HPLC system (Eschborn, Germany) consisting of a binary pump (Waters 1525), an injector system (Waters 2707) and a Waters column heater module WAT038040 and a refractive index detector (Waters 2414). An Aminex HPX-87H cation-exchange column (Biorad, München, Germany) coupled to a Micro-guard® cation-exchange column (Biorad) was used for chromatography. As a solvent, 5 mM H2SO4 at a flow rate of 0.6 mL min−1 was used. The column was kept at 45 °C. A sample volume of 20 μL was injected. Under these conditions, the retention times were about 13.5 min for glycerol and 22.7 min for ethanol. Data were analyzed using the Breeze 2 software (Waters).
9
SEC analysis of HA, Fab, and complexes
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TSK Gel PW5000xl 7.8×300-mm columns (TOSOH Corporation) were equilibrated in PBS using the Waters HPLC system. Samples of HA, Fab and the immune complexes were dialyzed against PBS and diluted to 1 mg/mL before loading. The flow rate was maintained at 0.5 mL/min and the absorbance at 280 nm was recorded.
10
HPLC Analysis of Maltooligosaccharides
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A Waters HPLC system comprising an ELS detector (Waters, Wexford, Ireland) was used to detect MOS. MOS was separated on an NH2P-50 4E amine column (250 × 4.6 mm ID) using 64% acetonitrile as the mobile phase at a flow rate of 1.0 mL/min [32 (link)]. DP1 (glucose) and DP2 (maltose), which were used as standards, were purchased from Sigma-Aldrich and DP3 (maltotriose), DP4 (maltotetraose), and DP5 (maltopentaose) were purchased from Elicityl-oligotech (Crolles, France).
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