Vitamin C was determined by high-performance liquid chromatography (Waters, USA) according to the method of “GB5009.86-2016 Determination of Ascorbic Acid in Foods” (National Health and Family Planning Commission, China). The determination of vitamin B1 was performed by high-performance liquid chromatography (Waters Corporation, USA), according to the method of “GB 5009.84-2016 Determination of vitamin B1 in foods” (National Health and Family Planning Commission, China).
High performance liquid chromatography (hplc)
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Sourced in United States, France, United Kingdom, China, Japan
HPLC (High-Performance Liquid Chromatography) is a analytical technique used for the separation, identification, and quantification of various chemical compounds. It utilizes a liquid mobile phase to carry the sample through a stationary phase within a column, facilitating the separation of the components based on their interactions with the stationary and mobile phases.
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Lab products found in correlation
Empower software, by Waters Corporation (12 mentions)
1525 binary pump, by Waters Corporation (7 mentions)
Api 4000 qtrap, by Thermo Fisher Scientific (5 mentions)
717 plus autosampler, by Waters Corporation (5 mentions)
In line degasser af, by Waters Corporation (5 mentions)
Xbridge c18 column, by Waters Corporation (5 mentions)
C18 column, by Waters Corporation (4 mentions)
2998 photodiode array detector, by Waters Corporation (4 mentions)
Empower 3, by Waters Corporation (4 mentions)
Refractive index detection, by Waters Corporation (3 mentions)
H835 50, by Hitachi (3 mentions)
Refractive index detector, by Shimadzu (3 mentions)
Sunfire c18 column, by Waters Corporation (3 mentions)
Acquity qda detector, by Waters Corporation (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Micro guard carbo c guard column, by Bio-Rad (2 mentions)
Hplc system, by Waters Corporation (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Rezex roa organic acid h column, by Phenomenex (2 mentions)
Ellagic acid, by Merck Group (2 mentions)
445 protocols using high performance liquid chromatography (hplc)
1
Quantifying Vitamins C and B1 in Foods
2
HPLC Analysis of Compound Purity
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The purities of compounds were analyzed using HPLC (Waters Corporation, Milford, MA). The HPLC is equipped with a 1525 binary pump, an In-Line degasser AF, a 717 plus autosampler, a 2998 photodiode array detector, and a 2475 multi-λ fluorescence detector interfaced with Waters Empower software. A Symmetry C18 reversed-phase column (Waters) was used, having a particle size of 5 μm, a length of 25 cm, and an internal diameter of 4.6 mm. The HPLC chromatograms were monitored at 210 nm using a photodiode array (PDA) detector, and the fluorescently labeled conjugate was monitored at both 650 and 210 nm using both PDI and fluorescence detectors. The injection was run using a gradient flow starting with 90:10 (H2O/ACN), gradually increasing to 10:90 (H2O/ACN) in 20 min and returning to 90:10 (H2O/ACN) in 25 min maintaining a flow rate of 1 ml/min.
3
HPLC Characterization of Dendrimer Conjugates
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The purities of the intermediates and the final conjugates were analysed using HPLC (Waters Corporation, Milford, MA) equipped with a 2998 photodiode array detector, a 2475 multi λ fluorescence detector, a 1525 binary pump, and an in-line degasser AF. The HPLC was interfaced with Waters Empower software. A C18 symmetry 300, 5µm, 4.6x250mm column from Waters was used. The HPLC chromatograms were recorded at 210nm (dendrimer and 2-PMPA absorption), for D-2PMPA and at 650nm wavelength (Cy5 absorption) for Cy5-D-2PMPA. A gradient flow was used using a mobile phase consisting of buffer A: 0.1% TFA and 5% ACN in water and buffer B: 0.1% TFA in ACN). The gradient started from 100:0 (A:B) gradually increasing to 50:50 (A:B) at 20 min, finally returning to 100:0 (A:B) at 40 minutes maintaining a flow rate of 1 mL/min. For the purification of drug linker, semi-preparative HPLC from Shimadzu was used using same method with a flowrate of 5 mL/min.
4
Extraction and Characterization of A. bidentata Root ABPPs
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A. bidentata Blume root was purchased from a Chinese medicine grocery, and identified by Dr. Haoru Zhao, and its powder was soaked in 80°C ultrapure water. ABPPs were subsequently prepared by the Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education as described by Shen et al. (2008). The ammonium sulfate precipitate was desalted using a 1000 MW cutoff tubing with ultrapure water. The dialysate was freeze-dried to obtain a powder of ABPPs, which is soluble in water. The ABPPs were characterized by high performance liquid chromatography (Waters, Milford, MA, USA).
5
Xylose Fermentation with Engineered Yeast
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Example 24
Fermentation Procedure:
Cells aerobically grown overnight on YNB medium supplemented with 20 g/L glucose were used to inoculate a shake flask containing 50 mL of YNB medium supplemented with xylose (70 g/L) as carbon source. When the cells were growing exponentially they were used to inoculate a serum flask containing 30 mL of YNB medium supplemented with xylose (55 g/L) and a solution of ergosterol and Tween80 with final concentration of 0.03 and 1.2 g/l, respectively. 7 ml mineral oil was added on top of each serum flask to keep culture anaerobic. Concentrations of xylose, ethanol, glycerol, and xylitol were determined by high performance liquid chromatography (Waters, Milford, Mass., USA).
The results are shown in Table 8 and
6
Xylose Utilization and Ethanol Production
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Example 14
Cells aerobically grown overnight on YNB medium supplemented with 20 g/L glucose were used to inoculate a serum flask containing 30 mL of YNB medium supplemented with glucose (20 g/L) and xylose (50 g/L) as carbon source and acetic acid (8 g/L) at initial concentration of 1 g cell dry weight/L.
Concentrations of glucose, xylose, ethanol, glycerol, xylitol and acetic acid were determined by high performance liquid chromatography (Waters, Milford, Mass., USA). The compounds were separated with a Shodex SUGAR SP0810 Pb2+ copolymer-based column (Showa Denko America, NY, USA), or a Rezex H+ column, preceded by a Micro-Guard Carbo-C guard column (Bio-Rad, Hercules, Calif., USA). Separation was performed at 80° C., with H2O at a flow rate of 0.6 ml min-1 as mobile phase. Compounds were quantified by refractive index detection (Waters). A seven-point calibration curve was made for each compound to calculate concentrations.
Results are presented graphically in
7
Evaluating Xylose Consumption and Ethanol Production
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Example 27
Cells aerobically grown overnight on YNB medium supplemented with 20 g/L glucose were used to inoculate a serum flask containing 30 mL of YNB medium supplemented with glucose (20 g/L) and xylose (50 g/L) as carbon source and formic acid (4.5 g/L) at initial concentration of 1 g CDW/L.
Concentrations of glucose, xylose, ethanol, glycerol, and xylitol were determined by high performance liquid chromatography (Waters, Milford, Mass., USA). The compounds were separated with a Shodex SUGAR SP0810 Pb2+ copolymer-based column (Showa Denko America, NY, USA) preceded by a Micro-Guard Carbo-C guard column (Bio-Rad, Hercules, Calif., USA). Separation was performed at 80° C., with H2O at a flow rate of 0.6 ml min-1 as mobile phase. Compounds were quantified by refractive index detection (Waters). A seven-point calibration curve was made for each compound to calculate concentrations. Results are presented graphically in
In conclusion, these data show that strains carrying PGM1 or PGM3 consumed more xylose and produced more ethanol than the control strain.
8
Extraction and Purification of DPHC from I. okamurae
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The phenolic compound, DPHC, was obtained from I. okamurae. The separation and identification methods were described in the previous study [42 (link)]. Briefly, I. okamurae was extracted with aqueous ethanol (50%, v/v), and the ethanol extract of I. okamurae (IOEE) was obtained. IOEE was fractionated by a centrifugal partition chromatography (Tokyo, Japan) with a two-phase solvent system, which was composed of n-hexane: ethyl acetate: methanol: water (1:9:4.5:6.5, v/v). DPHC was further purified by a high-performance liquid chromatography (Waters, Mailford, USA) and identified by a mass spectrometer (Bruker Daltonics, Breman, Germany). DPHC stock solution (100 mM, DMSO) was diluted to different concentrations (0.5 mM, 1 mM, and 2 mM) with 1X PBS for experiments.
9
In vitro Assay of LDC Activity
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LDC activity was assayed in vitro using purified proteins and muropeptide substrates. Muropeptide substrates (M4 and M5) were extracted from sacculi isolated from Caulobacter crescentus , which are known to have high M4 and M5 content (76 (link)), and solubilized as described above. Soluble muropeptides were separated using a high-performance liquid chromatography (Waters) system, and each individual muropeptide was collected manually. Collected fractions were washed from solvents by evaporation using a SpeedVac vacuum concentrator followed by resuspension with MilliQ water. All reactions were performed in 100 mM Tris-HCl, mixing a fraction of M4 or M5 and 10 ng of the corresponding protein (ElsLHis, LdcAHis, or none in the negative control). Reaction mixtures were incubated for 2 h at 37°C and stopped by incubation at 100°C for 5 min. After inactivation, the samples were reduced, and their pH was adjusted for injection on the UPLC. Muropeptides were identified according to their retention time, and their identities were validated through MS analysis.
10
Determination of Mg-Proto IX via HPLC
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Mg-Proto IX was analyzed by high performance liquid chromatography method according to Strand et al. [37 (link)]. Leaf material (0.2 g) was homogenized in 1 mL acetone (0.1 M NH4OH, 9:1 [v/v]) and homogenized and centrifuged with ice-cooling. The residue was resuspended again, and the same procedures described above were repeated. The collected supernatants were mixed and centrifuged prior to high performance liquid chromatography (Waters, Milford, USA). The eluate (0–10 min) was passed through an ACQUITY Ultra Performance Liquid Chromatography (UPLC) Fluorescence (FLR) Detector (Waters, Milford, USA), with initial excitation wavelength of 417 nm and an emission wavelength 595 nm, and secondary excitation and emission at 402 nm and 633 nm, respectively [68 (link)]. Mg-Proto IX was identified and quantified using authentic standards.
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