We used Waters Delta600 HPLC system, which equipped with an XTerra C18 RP column and an in-line diode array UV detector to purify the product. LC-MS machine we used was a Waters Acquity Ultra Performance LC with Waters MICROMASS detector. Hydrogen nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity Inova 400 with DMSO as solvent. We used Morgagni 268 transmission electron microscope to take Transmission electron microscope (TEM) images. MTT assay for cell cytotoxicity was test on DTX880 Multimode Detector.
Rp c18 column
Manufactured by Waters Corporation
Sourced in United States
The RP-C18 column is a reversed-phase chromatography column used for the separation and analysis of a wide range of organic compounds. It features a stationary phase of chemically bonded C18 alkyl groups on a silica substrate, which provides highly selective retention of non-polar and moderately polar analytes.
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Lab products found in correlation
Acquity ultra performance lc, by Waters Corporation (1 mentions)
Unity inova 400, by Agilent Technologies (1 mentions)
Delta 600 hplc system, by Waters Corporation (1 mentions)
Micromass detector, by Waters Corporation (1 mentions)
Accq fluor reagent, by Waters Corporation (1 mentions)
Wat088122, by Waters Corporation (1 mentions)
Analytical hplc system, by Waters Corporation (1 mentions)
Prism v5, by GraphPad (1 mentions)
2487 dual λ absorbance uv detector, by Waters Corporation (1 mentions)
No 2 filter paper, by Cytiva (1 mentions)
Perkin elmer series 200, by PerkinElmer (1 mentions)
Microtof focus, by Bruker (1 mentions)
Avance 3 hd 600, by Bruker (1 mentions)
E2695 separation module, by Waters Corporation (1 mentions)
2998 photodiode array detector, by Waters Corporation (1 mentions)
Whatman filter paper number 1, by Cytiva (1 mentions)
11 protocols using rp c18 column
1
Purification and Characterization of Product
2
Amino Acid Composition Analysis of Meat
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The crude protein content was determined by Kjeldahl method [6 ]. Amino acid composition was determined following Ishida et al. [7 (link)] and has been described earlier [8 (link)]. Briefly, muscle protein was hydrolyzed with 6N hydrochloric acid at 110°C under anaerobic condition for 24 h. The hydrolyzed samples were neutralized with 6N NaOH and were derivatized using a kit (AccQ-Fluor Reagent, WAT052880, Waters). The derivatized samples were injected in high performance liquid chromatography (HPLC) (1525, Waters) equipped with a C18 RP column and a fluorescence detector (2475, Waters). The amino acids were identified and quantified by comparing with the retention times and peak areas of standards (WAT088122, Waters). For the tryptophan analysis, minced meat was digested with 5% (w/v) NaOH for 24 h and neutralized to pH 7.0 with 6N HCl. Tryptophan content was measured spectrophotometrically at 530 nm [9 ]. All data have been presented as mean ± standard deviation.
3
Quantification and Identification of Acidic Rhamnogalacturonan Polysaccharides
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LC analysis was applied for quantification and identification of ARPS. The monosaccharide derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP) was implemented. ARPS (3 mg) was hydrolyzed with 0.02 mmol/L of trifluoroacetic acid at 100 °C for 12 h and then cooled. After centrifugation at 8000 rpm was conducted for 10 min, the pH of supernatants was mediated by adding 0.1 mol/L of NaOH as hydrolyzed sample solutions. Then, 0.5 mol/L 100 µL PMP and 0.1 mol/L of 30 µL NaOH were added into the hydrolyzed samples of ARPS and various standard monosaccharides (mannose, ribose, rhamnose, glucose, arabinose, xylose, and galacturonic acid). The mixture was mixed and reacted in a bath at 70 °C. Through the above procedure of hydrolysis and derivatization, the reaction solution was extracted with chloroform to remove PMP, and then the aqueous layer was filtered through a 0.45 µm membrane for HPLC analysis. This analysis was performed using an HPLC instrument equipped with RP-C18 column (4.6 × 250 mm, 5 μm, Waters) at a wavelength of 254 nm. The eluotropic procedure was as follows: the concentrations of acetonitrile were 7%, 19%, 25%, and 45%; the other solvent system was distilled water; the time changes were 0, 4, 13, and 28 min; and the flow rate was 0.8 mL/min.
4
Quantification of Entomopathogenic Beauveria Compounds
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The 2 × 106E. rugulosa spores were grown in 50 ml Arg medium for 10 d. Only Arg medium was used for comparative ECB production and transcriptional studied of ecdB knockout background. ECB production was initially tested by confrontation assay (susceptibility) against C. albicans followed its detection by HPLC. Briefly, confrontation assay was done by placing filter disc containing extract on pre-inoculated C. albicans cells in YEPD agar plates. Zone of inhibition formed by E. rugulosa extract against C. albicans was measured. Samples for HPLC analysis were performed as method opted by Hu et al., with minor modifications [26 (link)]. The harvested culture was lyophilized followed by methanol extraction at 30 °C for overnight. The filtered sample was run on analytical HPLC system (Waters) using an RP-C18 column (4.5 × 250 mm, I.D.WAT054375) using 20 μl sample injection. The standard of ECB (Santa Cruz Biotechnology: SC-362020) was run and monitored at 222 nm. The ECB concentration was calculated by peak(s) area and statistical analysis was applied using GraphPad Prism v5.01 (GraphPad Software, Inc.).
5
Quantitative Phytochemical Analysis of D. longicolla
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High-performance liquid chromatography analysis was performed for the treated crude extract and the non-treated crude extract of D. longicolla, using RP-C18 column (Waters, 4.6 mm × 100 mm, 3.5 μm, made in Netherlands). Pre-run sample stabilization was done for about 20 min with mobile phase consisting of 0.1% TFA:acetonitrile (60:40 v/v). The flow rate was maintained at 1.0 ml/min and the injected volume was 20 μl. The peak detection was done at 346.5 nm for berberine, 259.4 nm for caffeine, and 264.1 nm for theobromine, respectively, and the retention time (RT) was recorded duly maintaining the ambient experimental conditions. The presence of caffeine, theobromine, and berberine chromatogram in the BRD4770-treated sample was matched with the chromatogram of standards solution. The identification of each compound was based on a combination of RT, absorption maxima at a given wavelength, and chromatogram matching.
6
HPLC Quantification of Markers in MEF
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For quantification of markers by HPLC (Waters, 2487, Dual λ Absorbance UV Detector), MEF (500 μg/ml) was dissolved in methanol and water (80 : 20) and filtered using 0.22 µm syringe filter. The RP-C18 column (5 μm, 250 × 4.6 mm, Waters, USA) was used. Sample (200 μl) was injected and run in an isocratic solvent system with 1.0 ml/minute flow rate. Peaks were monitored using a UV detector at 254 nm. All peaks area and retention time (RT, minutes) were calculated using Empower 2 software. Similarly, HPLC fingerprints of all MEFs including water extract were compared. The external calibration method was used to quantify the markers in MEF using pure compounds (5–20 μg) by HPLC [10 ].
7
Organic Synthesis with Anhydrous Solvents
8
Surfactin Extraction and Quantification in Cheonggukjang
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The extraction and analysis of surfactin were performed according to Lee et al. [4 (link)] methods. Ten grams of ground cheonggukjang were extracted using 30 mL of methanol and adjusted to pH 2.0 with concentrated HCl by shaking (160 rpm) at 30 °C for 12 h. The extract was filtered through Whatman No. 2 filter paper and dried under a vacuum. The dried material was redissolved in 10 mL of 80% methanol and filtered through a 0.45-μm Millipore PVDF filter (Schleicher & Schuell, GmbH, Dassel, Germany). The filtrate was used for HPLC analysis to determine the surfactin concentration during the fermentation of cheonggukjang. The injection volume of the sample was 20 μL. The surfactin was analyzed through HPLC (Perkin–Elmer 200 series, Perkin–Elmer Corp., Norwalk, CT, USA) using an RP C18 column (4.6 × 250 mm, 5 μm, Waters Corp., Milford, MA, USA). The acetonitrile:water (1:1, v/v) was eluted at a flow rate of 1 mL/min at 40 °C. Surfactin was measured at 214 nm using a UV detector (Perkin–Elmer UV 200 series, Perkin–Elmer Corp., Norwalk, CT, USA). The concentration of surfactin was determined using a standard curve with standard solutions at 25, 50, 75, and 100 μg/mL.
9
Quantitative Analysis of Alkaloids
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For the mobile phase, 60 parts of 0.1% TFA and 40 parts of acetonitrile were mixed to get 0.1% TFA:acetonitrile (60:40 v/v) as a final solution. The mixed mobile phase was then filtered through nylon membrane vacuum filtration (0.22 μm) and degassed by sonication. Samples were analyzed on a detector at wavelength of 346.5 nm for berberine, 259.4 nm for caffeine, and 264.1 nm for theobromine and an injection volume of 10 μl using RP-C18 column (Waters, 4.6 mm × 100 mm, 3.5 μm) with a run time of 20 min.
10
Spectroscopic Characterization of Compound 1
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1 H, 13 C, COSY, HSQC, and HMBC NMR spectra were recorded on a Bruker Avance III HD 600 (Bruker Daltonics, Bremen, Germany) instrument. Accurate electrospray ionization mass spectra (ESI) were obtained by a micrOTOF focus (Bruker Daltonics, Bremen, Germany. TLC was performed on TLC plates precoated with silica gel F254 (Merck, Darmstadt, Germany). HPLC separation and purification were performed on a Agilent Technologies Series 1100 (ALS, UV Detect00719, USA) on a semi-preparative RP-C18 column (5 µm, 10 × 250 mm, Waters XBridge, city, Germany). MR 700 Microplate Reader (optical density measurement) (Dynatech Engineering Ltd., Willenhall, UK). The NMR spectral data of compound 1 were acquired using a 600 MHz instrument: 1 H, 13 C, 13 C-DEPT135, 1 H-1 H COSY, HSQC, and HMBC (optimized to J = 8.3 Hz) in DMSO-d 6 (Table 1).
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