The activity assay samples were analyzed using an Agilent 1200 series HPLC with a Phenomenex Luna 5 μm C18(2), 100 Å, 150 × 4.6 mm column. The following method was used: 20 min at 52% solvent B, gradient of 52% to 95% solvent B over 1 min, 95% solvent B for 3 min, gradient of 95% solvent B to 52% solvent B over 1 min, and 2 min at 52% solvent B. Solvent A is HPLC grade water + 0.1% trifluoroacetic acid (TFA), and solvent B is HPLC grade acetonitrile + 0.1% TFA. Absorbance was monitored at 220 nm, and the tribromopyrrole product peak was integrated and normalized to the amount produced by WT Bmp8.
1200 series
Manufactured by Phenomenex
Sourced in United States
The 1200 series is a modular HPLC instrument designed for analytical separation and detection of chemical compounds. It features a variable wavelength UV/Vis detector, a quaternary pump, and an autosampler. The system is capable of performing high-performance liquid chromatography analysis with good accuracy and precision.
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Lab products found in correlation
7 protocols using 1200 series
1
Quantification of Tribromopyrrole Synthesis
2
LC-MS Decolorization and Compound Separation
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For LC-MS, MG decolorization was performed at 25°C in water instead of buffer. Aliquots (2 μL) were injected into an HPLC system (Agilent 1200 Series, equipped with a Phenomenex Luna C-18 analytical column of 2.0 mm x 150 mm length and 3 μm particle size) coupled with Agilent 6224 Accurate-Mass Time of Flight (TOF) MS. The compounds were resolved by using solvent A: 5 mM ammonium acetate supplemented with 0.5% formic acid and solvent B: acetonitrile. The flow rate was kept at 0.2 mL min-1. A linear gradient was set as follows: t = 0–2, A = 95; t = 4–5, A = 40; t = 7–11, A = 10; t = 12–15, A = 95. The column effluent was introduced into the electrospray ionization source of the mass spectrometer in positive ion mode. The MS parameters were as follows: capillary voltage 3.5 kV; nebulizer pressure 50 psi; drying gas flow 11 L min-1; drying gas temperature 360°C; fragmentor voltage 130 V. LC-TOF MS accurate mass spectra were recorded across the range 70–400 m/z. Data processing was carried out with Applied Biosystems/MDS-SCIEX Analyst QS software (Frankfurt, Germany) with accurate mass application-specific additions from Agilent MSD TOF software. Accurate-mass internal mass calibration was performed automatically using a dual-nebulizer ion source. The reference masses were 121.0509 m/z and 922.0098 m/z.
3
Quantifying Rutin in Buckwheat Samples
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HPLC method was used to determine the content of rutin [44 ]. The samples (0.5 g) were extracted with 70% methanol (9 mL) in a shaking water bath at 70 °C for 120 min. After being centrifuged at 3000 rpm for 5 min, the supernatant was taken out and the residue was cleansed with 80% methanol (1 mL). The buckwheat samples were recentrifuged, and the volume of bulked supernatants was made to 10 mL with 70% ethanol. The extracts from buckwheat samples were filtered through a 0.45 µm Millipore Teflon filter, and then separated using Agilent Technologies 1200 Series liquid chromatography with UV-VIS (DAD) detector and column Phenomenex Synergi RP C18 (250 × 4.6 mm). As a mobile phase, a mixture of 2.5% acetic acid, methanol, and acetonitrile was applied in a ratio of 35:5:10 at a constant flow rate (1 mL min−1) with isocratic elution. The detection was carried out at 360 nm, and the results were interpreted by the comparison of the peaks of the analyzed samples against the standard separation.
4
HPLC Quantification of KMTB
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The amount of KMTB in the reaction mixture was determined with HPLC (Agilent 1200 series, Santa Clara, CA, USA) using a Phenomenex 5 µm Kromasil C8 column (150 mm and 4.6 mm; Phenomenex, Torrance, CA, USA) at a flow rate of 1 mL min−1 and a fixed wavelength of 210 nm in a mobile phase consisting of 15 mM KH2PO4, 30% methanol, and 10 mM tetrabutylammonium hydrogen sulfate, pH 6.5. The column temperature was maintained at 35°C, and the injection volume was 10 µL. KMTB was detected with a diode detector at a wavelength of 210 nm.
5
Isoflavones Quantification in Plant Extracts
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The isoflavones content was determined according to Ma et al. (Ma et al., 2018 (link)). The lyophilized sample (0.2 g) was extracted with 6 mL 80% methanol solution at 50 °C for 1 h, centrifuged at 12,000 g for 20 min. The supernatant was filtered with a 0.45 μm micropore filter prior to the high performance liquid chromatography (HPLC) analysis. The HPLC system (Agilent Technologies 1200 series, USA) was equipped with a LC Column (Luna® 5 µm C18(2) 100 A, 250×4.6 mm, Phenomenex, USA). The test parameters were as follows: solvent A, 0.1% acetic acid in water; solvent B, 0.1% acetic acid in acetonitrile; elution gradients, the ratio of solvent A was decreased (87–65%, 50 min), and then increased (65–87%, 1 min); flow rate, 1 mL/min; oven temperature, 35 °C.
6
Quantifying Insulin Absorption via HPLC
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The absorption spectra generated by the fluorescein sodium solutions were obtained employing 10mm path length cuvettes in a Perkin Elmer Lambda 365 at a nominal resolution of 1nm. The spectra were recorded at room temperature (20 o C), using deionised water as reference. The spectra were employed to quantify the concentration of the dilute.
J o u r n a l P r e -p r o o f 2.12 High-performance liquid chromatography (HPLC)
The amount of insulin collected from the receptor fluid of the Franz cells was determined by HPLC (Agilent Technologies, 1200 series, Cheshire, UK) equipped with a Phenomenex Jupiter 5u c18 300 Å, LC Column (250×4.60 mm, particle size 5 μm, Macclesfield, UK). The mobile phase consisted of water with 0.1% Trifluoroacetic Acid (TFA) and acetonitrile with 0.1% TFA (66:34 v/v), with a 1 mL min -1 flow rate. The column was equilibrated at 35 °C, the injection volume was 20 μL and the eluent was analysed with a UV detector at 214 nm. The results were integrated using Chemstation® software and the samples were analysed in triplicates.
J o u r n a l P r e -p r o o f 2.12 High-performance liquid chromatography (HPLC)
The amount of insulin collected from the receptor fluid of the Franz cells was determined by HPLC (Agilent Technologies, 1200 series, Cheshire, UK) equipped with a Phenomenex Jupiter 5u c18 300 Å, LC Column (250×4.60 mm, particle size 5 μm, Macclesfield, UK). The mobile phase consisted of water with 0.1% Trifluoroacetic Acid (TFA) and acetonitrile with 0.1% TFA (66:34 v/v), with a 1 mL min -1 flow rate. The column was equilibrated at 35 °C, the injection volume was 20 μL and the eluent was analysed with a UV detector at 214 nm. The results were integrated using Chemstation® software and the samples were analysed in triplicates.
7
HPLC Analysis of Pharmaceutical Compounds
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The concentrations of tested compounds (ibuprofen, mefenamic acid and MCPA sodium monohydrate) were monitored using high-performance liquid chromatography (HPLC) (Agilent Technologies 1200 series, USA) equipped with an Eclipse XDB-C 18 (3 μm particle size, 4.6 mm × 150 mm) column (Phenomenex, USA), using a diode array detector at a wavelength of 230 nm. The mobile phase was 50% of 0.1% phosphoric acid solution/50% acetonitrile. The flow rate was 1.0 mL min -1 . Several concentrations (from 0.05 to 1.0 mg L -1 ) of mix solution containing all tested compounds were injected into the HPLC and analysed; peak areas vs. concentrations of each compound were plotted and the calibration curves were obtained with determination coefficients (R 2 ) of 0.998, 0.999 and 0.998 for ibuprofen, mefenamic acid and MCPA sodium monohydrate, respectively. The limit of detection (LOD) of studied compounds for this method was ranging 0.012-0.017 mg L -1 , and the limit of quantitation (LOQ) was 0.03-0.05 mg L -1 .
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