The presence of phytoconstituents, β-sitosterol, lupeol and quercetin were investigated by HPLC using Waters HPLC instrument (Kyoto, Japan) fitted with a dual pump LC-20AD binary system with photodiode array (PDA) detector SPD-M20A, Merck RPC18 column (I.D. 4.6 x 250 mm, 5 mm). MCU was dissolved in methanol and injected. Gradient elution was carried out with methanol: phosphate buffer (50 M) at pH 3, (70:30) and the flow rate was adjusted to 1.0 mL/min with 20 µL injection volume, detection by UV at 250 nm.
Hplc instrument
Manufactured by Waters Corporation
Sourced in United States
The HPLC (High-Performance Liquid Chromatography) instrument is a laboratory equipment used for the separation, identification, and quantification of chemical compounds in a liquid sample. It is a highly versatile analytical tool that employs a high-pressure liquid system to separate and analyze complex mixtures.
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Lab products found in correlation
Lc 20ad binary system, by Merck Group (1 mentions)
Spd m20a, by Merck Group (1 mentions)
Rp c18 column, by Merck Group (1 mentions)
Membrane filter, by Merck Group (1 mentions)
1525 binary hplc pump, by Waters Corporation (1 mentions)
1525 hplc system, by Waters Corporation (1 mentions)
Calf intestinal phosphatase, by New England Biolabs (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Nuclease p1, by Merck Group (1 mentions)
8 protocols using hplc instrument
1
HPLC Analysis of Phytoconstituents
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The presence of phytoconstituents, β-sitosterol, lupeol and quercetin were investigated by HPLC using Waters HPLC instrument (Kyoto, Japan) fitted with a dual pump LC-20AD binary system with photodiode array (PDA) detector SPD-M20A, Merck RPC18 column (I.D. 4.6 x 250 mm, 5 mm). MCU was dissolved in methanol and injected. Gradient elution was carried out with methanol: phosphate buffer (50 M) at pH 3, (70:30) and the flow rate was adjusted to 1.0 mL/min with 20 µL injection volume, detection by UV at 250 nm.
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The presence of phytoconstituents, β-sitosterol, lupeol and quercetin were investigated by HPLC using Waters HPLC instrument (Kyoto, Japan) fitted with a dual pump LC-20AD binary system with photodiode array (PDA) detector SPD-M20A, Merck RPC18 column (I.D. 4.6 x 250 mm, 5 mm). MCU was dissolved in methanol and injected. Gradient elution was carried out with methanol: phosphate buffer (50 M) at pH 3, (70:30) and the flow rate was adjusted to 1.0 mL/min with 20 µL injection volume, detection by UV at 250 nm.
2
Purification and Characterization of CAG RNA
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The CAG motif containing RNA 5´ r(UUGGGCCA A A
3
HPLC Analysis of Metabolite Quantification
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Chromatographic analyses were performed with an HPLC instrument (Waters Corporation, Milford, DE, USA) equipped with a 1525 binary HPLC pump and a 2998 photodiode array detector. A Symmetry® RP C18 column, 4.6 × 75 mm, 3.5 μm column (Waters, Milford, USA) was used. As previously reported [69 (link)], a linear methanolic gradient was applied from 20 to 80% v/v in 12 min (A: water with 0.1% v/v acetic acid, and B: methanol with 0.1% v/v acetic acid), with a flow rate of 1 mL/min. Peaks were detected in the range of 210–400 nm. All samples were filtered with membrane filters (0.22 μm pore size, Millipore, Merck KGaA, Darmstadt, Germany) and then injected (10 μL). Standard stock solutions were prepared in methanol. The amount of each compound was calculated using a standard calibration curve. The protein content of the lysate was detected using the Lowry protein assay [70 (link)], to express the results as nmol/mg protein.
4
Biofilm Matrix Extraction and Quantification
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The WT and mutant strains were each grown in the wells of two 48-well plates for 48 h to grow biofilms, as described above. The biofilm biomass from the two 48-well plates was then scraped and pooled together, and biofilm lysates were generated by SDS treatment, as described above for the isolation of cell protein. The biofilm matrix was isolated as previously described24 (link), except that the biofilm lysate was generated by repeated passages of the biofilm biomass through an 18-G needle and included several steps of centrifugation and washes to isolate the insoluble biofilm matrix fraction. The sample was lyophilized and its EPS component was acid hydrolysed, as previously described1 (link). EPS content was calculated as glucose equivalents using a published protocol5 (link), except that the glucose content in the acid-hydrolysed sample was estimated by HPLC, using a Waters HPLC instrument (Milford, MA) equipped with the refractive index and ultraviolet detectors, and operated as described elsewhere58 .
5
HPLC Analysis of Dendrimer Drug Conjugates
6
HPLC Purification of Peptides
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The HPLC instrument was from Waters Corporation (Waters, Milford, MA, USA). Purification was performed on a Delta Pak C18 column using a Waters 1525 HPLC system and absorbance was monitored at 220 nm and 280 nm using a PDA detector. The column was equilibrated with eluent A (95% H2O, 5% ACN, and 0.1% TFA) at a flow rate of 3 mL/min.
Each lyophilized peptide sample was dissolved in a suitable solvent (tIK-9mer: 95% H2O, 5% ACN, and 0.1% TFA; tIK-14mer and 18mer: 100% H2O and 0.1% TFA). When the dissolved solution was injected together with a polar mobile phase, the peptide could interact with the column. The ratio of the nonpolar mobile phase was then gradually increased to elute the peptide. The gradient method of increasing eluent B composition by 1% per minute from 100% eluent A was used. Before injecting the protein (20 mg) dissolved in the appropriate solvent (10 mL) into the HPLC injector, the supernatant was recovered after centrifugation at 14,500× g rpm for 30 min at 4 °C. The sample was injected through a 0.45 μm syringe filter.
Each lyophilized peptide sample was dissolved in a suitable solvent (tIK-9mer: 95% H2O, 5% ACN, and 0.1% TFA; tIK-14mer and 18mer: 100% H2O and 0.1% TFA). When the dissolved solution was injected together with a polar mobile phase, the peptide could interact with the column. The ratio of the nonpolar mobile phase was then gradually increased to elute the peptide. The gradient method of increasing eluent B composition by 1% per minute from 100% eluent A was used. Before injecting the protein (20 mg) dissolved in the appropriate solvent (10 mL) into the HPLC injector, the supernatant was recovered after centrifugation at 14,500× g rpm for 30 min at 4 °C. The sample was injected through a 0.45 μm syringe filter.
7
HPLC Analysis of 2-MPPA and D-2MPPA Conjugate
8
Quantifying Global DNA Methylation
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Global 5-mC levels were estimated using reversed-phase high-performance liquid chromatography (RP-HPLC) as described earlier. [27] [28] [29] Briefly, 2 µg of genomic DNA was digested with DNase1 (Ambion, USA), denatured at 100°C for 5 min, chilled on ice, and incubated with 2 U each of Nuclease P1 (Sigma, USA) and calf intestinal phosphatase (New England BioLabs, USA) according to manufacturer's instruction. The sample was passed through Quia quick spin column (Qiagen, USA), lyophilized, and subjected to chromatographic separation (10 µL) under ambient column temperature, maintaining the samples at 4°C using isocratic conditions with a flow rate of 1 mL/min in HPLC instrument (Waters, USA), consisting of dipotassium hydrogen phosphate buffer (K 2 HPO 4 , pH 4.1) supplemented with 10% methanol and C-18 Grace Vydac smart column at 260 and 280 nm. Standards such as dC, 5-dmC, dT, dA, and dG were used to identify the retention time of these nucleosides. Peaks corresponding to 5-mC and other nucleosides were monitored over 30 min interval. The relative degree of methylation in the DNA samples was measured using the following formula: % of 5-mdC = A5mdC / (A5mdC + AdC) × 100.
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