A volume of 0.05 g of obtained paramyosin powder was mixed with 10 ml PBS buffer (0.1 M KCl, pH 7.5) at 4°C under stirring for 3 h and then centrifuged at 5,000 × g for 10 min at 4°C. The supernatant was collected and diluted down to 1 mg/ml with 0.05 M phosphate buffer and was measured with the Bradford assay. This solution was passed through a 0.45 μm filter (Millex-HA, 25 mm, MCA) to remove dust and was then sonicated in an ultrasound cleaner (KQ-50DE, Shumei Co., Kunshan, Jiangsu, China) at 25°C with an ultrasonic frequency of 40 kHz and an input power of 100 Wm, in order to remove air bubbles. A Shodex SEC/GPC protein KW-804 column (300 × 8.0 mm i.d.), an Agilent 1,260 Quaternary pump with an Agilent 1260 VWD detector (λ = 280 nm) was used to separate and detect the components of the protein solutions. A PBS solution with 0.5 M KCl was used as the mobile phase with a flow rate of 1 ml/min. The elution time was 20 min, and the column temperature was 25°C.
Agilent 1260 vwd detector
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1260 VWD (Variable Wavelength Detector) is a high-performance liquid chromatography (HPLC) detector that measures the absorbance of analytes at a specified wavelength. It provides precise detection of compounds in liquid chromatography applications.
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Lab products found in correlation
Refractive index detector, by Agilent Technologies (3 mentions)
Hpx 87h column, by Bio-Rad (3 mentions)
Agilent 1260 infinity lc system, by Agilent Technologies (2 mentions)
Agilent 1 260 quaternary pump, by Agilent Technologies (1 mentions)
Capcell pak c18 column, by Shiseido (1 mentions)
Agilent 1260 hplc, by Agilent Technologies (1 mentions)
Lck 304, by HACH (1 mentions)
Dr 3900 spectrophotometer, by HACH (1 mentions)
Lck 341, by HACH (1 mentions)
Orion 4 star, by Thermo Fisher Scientific (1 mentions)
1260 hplc, by Bio-Rad (1 mentions)
Libra s11, by Harvard Bioscience (1 mentions)
Sod kit, by Nanjing Jiancheng (1 mentions)
S 4800, by Hitachi (1 mentions)
8 protocols using agilent 1260 vwd detector
1
Paramyosin Purification and Characterization
2
Organic Acid Content Determination
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For the determination of the organic acid content, the method of Tian Ya [26 ] was used with slight modifications. HPLC was performed on an Agilent Z0RBAX SB-AQ (4.6 mm × 250 mm, 5 μm, American Agilent Corporation (Santa Clara, CA, USA) column with an Agilent1260 VWD detector. In the mobile phase, according to the following parameters: KH2PO4 (0.02 mol/LKH2PO4 pH 2) the ratio of methanol was 95:5, the flow rate was 0.8 mL/min, the column temperature was 35 °C, the detection wavelength was 210 nm, and the sample volume was 10 uL. The experiment was conducted three times for each sample.
3
Screening of Solid Matrix Formers for Drug Solubility
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For the selection of a suitable solid matrix former, various hydrophilic polymers and surfactants were screened using solubility testing published guidelines with slight modifications [34 ]. An excess of drug was added to 1 mL of distilled water containing 1% (w/v) polymer or surfactant and vortexed for a few seconds. The microtubes were shaken at 25 °C and 100 rpm for five days in an isothermal water bath shaker. The resulting suspensions were centrifuged at 10,000 g for 10 min and the supernatant was filtered through a 0.45 µm membrane filter. The filtrate was diluted with methanol as needed and then assayed using an Agilent 1260 Infinity LC system (Agilent Technologies; Santa Clara, CA, USA) equipped with Agilent ChemStation software (version B.04.02), an Agilent 1260 Quat pump, and an Capcell Pak C18 column (Shiseido, 250 mm × 4.6 mm I.D., 5 μm). The mobile phase consisted of methanol, acetonitrile, and distilled water (v/v/v: 40/40/20), at a flow rate of 1 mL/min. The mobile phase was monitored using an Agilent 1260 VWD detector (Agilent Technologies; Santa Clara, CA, USA) at a wavelength of 240 nm, and the injection volume was 10 μL. All the experiments were carried out in triplicate (n = 3), and the solubility data are represented as mean ± standard deviation.
4
Metabolite and Nitrogen Quantification
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Metabolite concentrations in culture supernatants were analysed by high-performance liquid chromatography (HPLC) on an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA) fitted with a Bio-Rad HPX 87 H column (Bio-Rad). The flow rate was set at 0.6 mL/min, 0.5 g/L H2SO4 was used as eluent and the column temperature was set at 65°C. An Agilent refractive-index detector and an Agilent 1260 VWD detector were used for metabolite quantification (Verhoeven et al. 2017 ). Nitrate, nitrite and ammonium concentrations in culture supernatants were measured with a Hach DR3900 spectrophotometer and Hach kits LCK 339, LCK 341 and LCK 304 (Hach Lange, Düsseldorf, Germany), according to the manufacturer's instructions.
5
Rapid Quenching and HPLC Analysis
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During the runs, rapidly temperature‐quenched samples were taken from the fermenters to instantly stop metabolism and obtain representative measurements of extracellular metabolites such as formic acid. Immediately after sampling the samples were cooled in syringes filled with precooled steel beads for fast cooling of the sample (Mashego et al., 2003 (link)). The cells were immediately removed by filtration and samples were stored frozen.
Extracellular concentrations of glucose and formic acid in culture filtrates were analysed by high‐performance liquid chromatography (HPLC) on an Agilent 1260 HPLC, equipped with a Bio‐Rad HPX 87H column, operated at 60°C with 5 mM H2SO4 as mobile phase at a flow rate of 0.600 ml/min. Detection was performed by means of an Agilent refractive index detector and an Agilent 1260 VWD detector at 210 nm. The NH3 concentration was measured in supernatant samples using an Orion 4 Star with the Orion 95‐12 Ammonia Electrode (Thermo Fisher Scientific).
Extracellular concentrations of glucose and formic acid in culture filtrates were analysed by high‐performance liquid chromatography (HPLC) on an Agilent 1260 HPLC, equipped with a Bio‐Rad HPX 87H column, operated at 60°C with 5 mM H2SO4 as mobile phase at a flow rate of 0.600 ml/min. Detection was performed by means of an Agilent refractive index detector and an Agilent 1260 VWD detector at 210 nm. The NH3 concentration was measured in supernatant samples using an Orion 4 Star with the Orion 95‐12 Ammonia Electrode (Thermo Fisher Scientific).
6
Yeast Culture Optical Density and Composition
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Optical densities of yeast cultures were measured with a Libra S11 spectrophotometer (Biochrom, Cambridge, UK) at a wavelength of 660 nm. Biomass dry weight was measured by filtering 10-mL culture samples over pre-weighed nitrocellulose filters with a pore size of 0.45 μm. Filters were washed with 10 mL water, dried in a microwave oven (20 min at 350 W) and reweighed. Each measurement was performed in duplicate. For glucose, maltose, maltotriose, and ethanol analysis, culture samples were centrifuged 5 min at 10,000 g and supernatants were analyzed by high-performance liquid chromatography (HPLC) analysis on an Agilent 1260 HPLC equipped with a Bio-Rad HPX 87 H column (Bio-Rad, Hercules, CA). Elution was performed at 65°C with 5 mM H2SO4 at a flow rate of 0.8 mL min−1. Detection was by means of an Agilent refractive-index detector and an Agilent 1260 VWD detector.
7
Phytochemical Analysis of Rubus Fructicosus Var.
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Total phenolic content was measured by Folin–Ciocalteu reagent using gallic acid as a standard [7 (link)]. Total flavonoid content was measured by NaNO2-Al(NO3)3-NaOH reagent using rutin as a standard [7 (link)]. The measurement of total acid was performed by acid-base titration method [12 (link)]. The content of VC was measured by colorimetric method [12 (link)]. Measurement of SOD content was performed via SOD kit (SOD kit purchased from Nanjing Jiancheng Institute of Biological Engineering, Jiangsu, China) according to the kit instructions.
HPLC was used to analyze organic acids in RFV based on the literature [25 (link)] with slight modifications. Separation was conducted using an Agilent Z0RBAX SB-AQ column (250 mm × 4.6 mm, 5 µm, American Agilent Corporation (Santa Clara, CA, USA) with an Agilent1260 VWD detector. The relevant parameters of the mobile phase are as follows: monopotassium phosphate buffer solution (0.02 mol/L, pH 2) the ratio of methanol was 95:5, the injection volume was 10 µL and the flow rate was 0.8 mL/min, the spectra were recorded at 210 nm, the column temperature was 35 °C. The experiment was conducted three times for each sample.
HPLC was used to analyze organic acids in RFV based on the literature [25 (link)] with slight modifications. Separation was conducted using an Agilent Z0RBAX SB-AQ column (250 mm × 4.6 mm, 5 µm, American Agilent Corporation (Santa Clara, CA, USA) with an Agilent1260 VWD detector. The relevant parameters of the mobile phase are as follows: monopotassium phosphate buffer solution (0.02 mol/L, pH 2) the ratio of methanol was 95:5, the injection volume was 10 µL and the flow rate was 0.8 mL/min, the spectra were recorded at 210 nm, the column temperature was 35 °C. The experiment was conducted three times for each sample.
8
Characterization of CNM-Loaded Solid Dispersions
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An excess amount of CNM-loaded solid dispersions (equivalent of about 100 mg of CNM powder) was added to distilled water, agitated at 25°C for 7 d and centrifuged at 13000×g for 5 min (5415C; Eppendorf, Hamburg, NY, U.S.A.). The supernatant layer was diluted and passed through a 0.45 µm membrane filter. The concentration of CNM in the filtrate was assayed by an Agilent 1260 Infinity LC system (Agilent Technologies; Santa Clara, CA, U.S.A.) equipped with Agilent ChemStation computer software, an Agilent 1260 Quat pump, an Agilent 1260 VWD detector and an Inertsil ODS-4 reverse-phase C18 column (GL Science, 250 mm×4.6 mm i.d., 5 µm). The mobile phase consisted of methanol and distilled water (90 : 10, v/v). The mobile phase was monitored at 220 nm with a flow rate of 1 mL/min. In this HPLC method, the inter-and intra-day variance were within the acceptable range (R 2 =0.999). 3) (link) Morphological Characterization of Surface-Modified Solid Dispersion The surface morphology of the drug and solid dispersion was examined by SEM (S-4800; Hitachi; Tokyo, Japan). Samples were affixed onto a brass specimen holder using double-sided adhesive tape, and the powders were made electrically conductive by coating with platinum (6 nm/min) in a vacuum (0.8 Pa) using an EmiTeck Sputter Coater (K575 K) for 4 min at 15 mA.
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