The HPLC system was used to monitor the reaction products, equipped with a quaternary pump (Waters e2695), a controller (Waters e2695), an autosampler (Waters e2695), and a photodiode array detector (2998 PDA detector) coupled to a data processing computer (EmpowerTM 2 chromatography data software). The column was Innoval C18 (5 μm, 4.6 × 250 mm) and the temperature was set at 30 °C. The flow rate of the mobile phase was fixed at 0.7 mL/min. Two elution solvents, A (water:formic acid; 98:2, v/v) and B (water:acetonitrile:formic acid; 68:30:2, v/v), were used with the gradient elution program as follows: 0 min, 18% B; 42–48 min, 47% B; 78–110 min, 100% B; and re-equilibration of the column for 10 min. The detection wavelength was 525 nm for the detection of anthocyanins and their derivatives and 280 nm for all polyphenols.
E2695
Manufactured by Waters Corporation
Sourced in United States, United Kingdom, Germany, China
The E2695 is a high-performance liquid chromatography (HPLC) system manufactured by Waters Corporation. It is designed to separate, identify, and quantify components in a liquid sample. The E2695 system includes a solvent delivery module, an autosampler, and a column compartment to maintain temperature control. It is capable of performing a wide range of analytical techniques with a focus on precise and accurate sample handling and separation.
Automatically generated - may contain errors
Lab products found in correlation
Empower 3, by Waters Corporation (8 mentions)
Sunfire c18 column, by Waters Corporation (4 mentions)
Acquity qda, by Waters Corporation (4 mentions)
Symmetry c18 column, by Waters Corporation (4 mentions)
E2489, by Waters Corporation (4 mentions)
C18 column, by Shiseido (4 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (4 mentions)
Phosphoric acid, by Sangon (3 mentions)
Analytical grade methanol, by Sangon (3 mentions)
C18 column, by Agilent Technologies (3 mentions)
Nexion 350, by PerkinElmer (3 mentions)
C18 column, by Waters Corporation (3 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (3 mentions)
Hpx 87h column, by Bio-Rad (3 mentions)
Sb c18 column, by Agilent Technologies (3 mentions)
Q exactive, by Thermo Fisher Scientific (3 mentions)
Hplc system, by Waters Corporation (3 mentions)
Rezex roa organic acid h column, by Phenomenex (2 mentions)
Dawn heleos 2, by Wyatt Technology (2 mentions)
Autoflex 3, by Bruker (2 mentions)
264 protocols using e2695
1
HPLC Analysis of Anthocyanins and Polyphenols
2
Analytical Techniques for Chemical Characterization
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HPLC was performed using a Waters e2695 (Waters, USA). Preparative HPLC was manufactured by Agela technologies (Tianjin, China). NMR spectra were measured using Bruker-600 with tetramethylsilane (TMS) as the internal standard (Bruker, USA). High-resolution electrospray ionization mass spectrometry (HR-ESIMS) data were obtained using an Agilent 1290 infinity 6540 UHD accurate mass Q-TOF MS (Agilent, USA). A constant temperature incubator shaker (ZHWY-2012C) was obtained from Shanghai Zhicheng Analytical Instrument Co. Ltd (China).
3
HPLC Quantification of Organic Acids in Loquat Jam
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HPLC (E2695, Waters, Milford, CT, USA) was used to determine the content of organic acids [25 (link)]. An amount of 25 g of loquat jam was added to ultrapure water and diluted into a 50 mL volumetric flask, heated and extracted in a 75 °C water bath for 1 h, cooled to room temperature and then filtered. The filtrate was centrifuged at 16,000× g r/min for 20 min, and 1 mL of the supernatant was filtered through a 0.45 μm microporous membrane (Anavo Technology Co., Ltd., Beijing, China) into a sample bottle for testing.
The HPLC conditions were as follows: The instrument was equipped with a 717+ automatic injector with 20 μL once and a 2996 Photodiode Array detector (PDA) UV at 210 nm. Separation was achieved using an Agilent Zorbax SB-C18 column (5 μm, 4.6 mm × 250 mm) at 25 °C. The mobile phase was 20 mmol/L NaH2PO4 (0.01 mol/L potassium dihydrogen phosphate solution was used with a pH of 2.60), with a flow rate of 1.00 mL/min. Each organic acid was quantified by the standard curve of the authentic product. The standard curves for the six measured organic acids are shown inTable 1 below.
The HPLC conditions were as follows: The instrument was equipped with a 717+ automatic injector with 20 μL once and a 2996 Photodiode Array detector (PDA) UV at 210 nm. Separation was achieved using an Agilent Zorbax SB-C18 column (5 μm, 4.6 mm × 250 mm) at 25 °C. The mobile phase was 20 mmol/L NaH2PO4 (0.01 mol/L potassium dihydrogen phosphate solution was used with a pH of 2.60), with a flow rate of 1.00 mL/min. Each organic acid was quantified by the standard curve of the authentic product. The standard curves for the six measured organic acids are shown in
4
HPLC Analysis of Chemical Compounds
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A Waters e2695 high performance liquid chromatography (HPLC) system equipped with a Waters 2489 UV detector was used for the products analysis (Waters Technology Co. Ltd., USA). Chromatographic separation was carried out on a silica gel column (4.6 × 250 mm × 5 μm) (Waters Technology Co. Ltd., USA). The mobile phases were n-hexane and 2-propanol (20 : 1, v/v) with a flow rate of 0.8 mL min−1. The injection volume was 10 μL, and the column temperature and detection wavelength were set at 30 °C and 280 nm, respectively.
5
Quantification of Cell Wall Sugars
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All experimental strains were treated with different concentrations of acetic acid (0 and 150 mM) for 2 h after reaching exponential phase in SC media (pH 3.0), harvested and washed thrice with distilled water by resuspension and centrifugation at 12,000×g for 5 min at 4 °C. Cells were collected and broken by ultrasonic cell disruptor completely. After twice centrifugation and resuspension, the underlayer deposition containing cell wall was gathered and freeze-dried. About 20 mg specimen was treated with 150 μl 72% (W/V) concentrated sulfuric acid overnight and boiled 4 h. Saturated Ba(OH)2 added to terminated reaction until reaching neutral pH. Supernatant was injected into HPLC (Waters E2695, USA) for detection after centrifugation at 12,000×g for 5 min at 4 °C and filtration of 0.22 μ membrane as previously described.
6
Characterizing Deproteinized Calf Blood Extractive Injection
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As described in previous studies [47 (link)–50 (link)], we determined the molecular weight distribution and the nucleotide and ribose contents of Deproteinized Calf Blood Extractive Injection (DCBEI)(CAS: 20160803; obtained from Jilin Connell Pharmaceutical Co. LTD., Jilin, China) samples using high-performance liquid chromatography (HPLC) (E2695, Waters Co., Ltd., USA) systems and gel permeation chromatography (1515, Waters Co., Ltd., USA).
DCBEI was hydrolyzed by the addition of 6 mol/L of hydrochloric acid at 110° C for 24 h, and then dried under vacuum. Deionized water was added to prepare DCBEI test samples and amino acid standards. Seventeen distinct free amino acids were detected using the HPLC system as described in a previous study [51 (link)].
DCBEI was hydrolyzed by the addition of 6 mol/L of hydrochloric acid at 110° C for 24 h, and then dried under vacuum. Deionized water was added to prepare DCBEI test samples and amino acid standards. Seventeen distinct free amino acids were detected using the HPLC system as described in a previous study [51 (link)].
7
Quantification of 2'-FL and related metabolites in microbial fermentation
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During cultivation, the culture broth was sampled at intervals and growth conditions were determined by OD600 analysis using a spectrophotometer (P7 Double Beam UV‐VIS Spectrophotometer, Mapada, China). Dry cell weight (g l−1) was calculated from OD600 using conversion factor of 0.36 (Chin et al., 2013 (link)).
The concentrations of glycerol, lactose and 2′‐FL in the fermentation broth were quantified with an HPLC system (Waters e2695) equipped with a refractive index (RI) detector and a Rezex ROA‐Organic Acid H+ column (Phenomenex, Torrance, CA, USA). The column was eluted with 0.01 M H2SO4 at a flow rate of 0.6 ml min−1 at 50°C (Chin et al., 2016 (link)). To assay 2′‐FL, culture broth samples (1 ml) were boiled for 10 min to break the cells completely and centrifuged at 12 000 g for 10 min. The supernatant was used to estimate the total oligosaccharide content. To identify the product of 2′‐FL, 2′‐FL standards and samples were analysed by electro‐spray ionization‐mass spectroscopy (ESI‐MS) in negative ion mode under standard conditions on a Waters MALDI SYNAPT Q‐TOF MS (Milford, MA, USA). The content of GDP‐l ‐fucose in fermentation broth was determined according to Huang et al. (2017 (link)).
The concentrations of glycerol, lactose and 2′‐FL in the fermentation broth were quantified with an HPLC system (Waters e2695) equipped with a refractive index (RI) detector and a Rezex ROA‐Organic Acid H+ column (Phenomenex, Torrance, CA, USA). The column was eluted with 0.01 M H2SO4 at a flow rate of 0.6 ml min−1 at 50°C (Chin et al., 2016 (link)). To assay 2′‐FL, culture broth samples (1 ml) were boiled for 10 min to break the cells completely and centrifuged at 12 000 g for 10 min. The supernatant was used to estimate the total oligosaccharide content. To identify the product of 2′‐FL, 2′‐FL standards and samples were analysed by electro‐spray ionization‐mass spectroscopy (ESI‐MS) in negative ion mode under standard conditions on a Waters MALDI SYNAPT Q‐TOF MS (Milford, MA, USA). The content of GDP‐
8
Optimizing Epitope Peptide Loading Capacity
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To determine the optimum epitope peptide loading capacity, six different concentrations (0, 250, 500, 1000, 1500, and 2000 μg/mL) were examined. NEs for each of the six concentrations were prepared using our previously described method (Smix = 4:1). The average size and zeta potential were then measured as described above. The encapsulation efficacy of these six concentrations was determined by HPLC (E2695, Waters, MA, USA) with a ZORBAX SB-C18 Chromolith column (5 μm, 4.6 mm × 250 mm) at a wavelength of 220 nm according to our previously described method [21 (link)].
9
Molecular Characterization of Polysaccharide Fractions
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The Mn, Mw and PDI for the two polysaccharide fractions in 0.9% NaCl aqueous solution were measured using HPSEC-MALLS-RID. HPSEC-MALLS-RID measurements were carried out on a multi-angle laser light scattering detector (DAWN HELEOS-II, Wyatt Technology Co., Santa Barbara, CA, USA) with a separation module (Waters e2695) equipped with a TSK-Gel G4000SWXL column (300 mm × 7.8 mm, i.d., Tosoh Bioscience, Tokyo, Japan) at 35 °C. A differential refractive index detector (RID, Optilab T-rEX, Wyatt Technology Co., Santa Barbara, CA, USA) was connected simultaneously. The mobile phase was 0.9% NaCl aqueous solution and was passed through the HPSEC column at a flow rate of 0.5 mL/min. Astra software (Version 7.1.3) was utilized for data acquisition and analysis. The molecular weight was calculated using the Zimm model.
10
Quantification of MK by HPLC
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Determination of MK by HPLC was in accordance to the method in QB/T 2847-2007 and reference (Huang C. F. et al., 2019 (link)) with minor modification. Before chromatographic analysis, the supernatant was filtered through a 0.22 μm filter. The supernatant was measured by Waters e2695 with a chromatographic column (Zorbax SB-C18 column, 5 μm × 250 mm × 4.6 mm) and a UV detector at 238 nm. The mobile phase was composed of methanol:water:phosphoric acid in a ratio of 385:115:0.14 (v/v), with an accompanying flow rate of 1.0 ml/min.
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