The crude peptide (0.02 mmol) dissolved in 25 mL of MilliQ water were loaded on Vydac C18 (5 μm, 300 Å, 10 × 250 mm, Grace) and separated in the following conditions: eluent A, 0.05% TFA in MilliQ water; eluent B, 0.05% TFA in CH3CN; gradient, from 0%B to 5%B in 2 min, and from 5%B to 15%B in 40 min; flow rate, 4 mL/min; detection at 214 nm. The chromatogram of purified peptide was carried out in the following conditions: column, Vydac C18 monomeric (5 μm, 300 Å, 4.6 × 250 mm, Grace); injection volume, 20 μL of 1 mg/mL peptide solution; flow rate, 1 mL/min; eluent A, 0.05% TFA in water; eluent B, 0.05% TFA in CH3CN; gradient, from 8%B to 18%B in 20 min, detection at 214 nm. The retention time results 12.4 min and the purity grade, 97.9%. Experimental mass: 2448.32 Da, Theoretical mass: 2447.82 Da (ESI-TOF, Mariner System 5220, Applied Biosystem, Perkin-Elmer, California, USA).
C18 vydac
Manufactured by Grace Bio-Labs
Sourced in United States
The C18 Vydac is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of organic compounds, including peptides, proteins, and small molecules. The column features a C18 stationary phase, which provides high selectivity and resolution for the separation of analytes.
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4 protocols using c18 vydac
1
Purification and Characterization of Peptide
2
Characterization of C. vicina AMP Complex
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Natural compound containing C. vicina AMP complex was characterized by a combination of reversed phase HPLC, MS and bacterial growth inhibition assays. 1 mg of the lyophilized compound was dissolved in deionized water and applied to Shimadzu LC20 Prominence HPLC system equipped with analytical column C18 Vydac (4.6 х 250 mm, 5 μm, Grace), equilibrated with 0.05% TFA. The column was eluted with a linear gradient of acetonitrile (ACN) from 0 to 50% in acidified water (0.05% TFA) for 50 min [17 ]. Chromatographic fractions were automatically collected with 1 min intervals. The fractions’ optical densities were registered by means of a UV detector at two fixed wavelengths 214 and 280 nm. The fractions were lyophilized, dissolved in deionized water and tested against M. luteus A270 and E. coli D31 using the plate growth inhibition assay described below. Active antibacterial fractions were analyzed by MS (MicroTOF ESI, Bruker Daltonics) and experimentally determined masses were compared with the previously published characteristics of C. vicina individual AMPs [8 (link), 10 ]. The peptides were sequenced by Edman degradation method as described [10 ].
3
Purification and Characterization of Complex Antimicrobial Peptides
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The complex AMPs were previously isolated and characterized by a combination of reversed phase HPLC, MS and Edman degradation methods [25 , 35 ]. In that work 1 mg of the lyophilized compound was dissolved in deionized water and applied to Shimadzu LC20 Prominence HPLC system equipped with analytical column C18 Vydac (4.6 × 250 mm, 5 μm, Grace), equilibrated with 0.05% TFA. The column was eluted with a linear gradient of acetonitrile (ACN) from 0 to 50% in acidified water (0.05% TFA) for 50 min. Chromatographic fractions were automatically collected with 1 min intervals. The fractions’ optical densities were registered by means of a UV detector at two fixed wavelengths 214 and 280 nm. The fractions were lyophilized, dissolved in deionized water and tested against planktonic M. luteus A270 and E. coli D31strains using the plate growth inhibition assay. Anti-biofilm activity of the fractions against one-day biofilms formed by E. coli ATCC 25922 и S. aureus 203 was analyzed by TTC and crystal violet assays as described below.
4
Reversed-Phase HPLC for Insulin Quantification
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Insulin was assayed by a reversed-phase HPLC–UV method. The HPLC system (1260 Series, Agilent Technologies, USA) was composed of a quaternary pump, a degasser, an autosampler, a column heater and a tunable ultraviolet detector. The separation was achieved on the Vydac C18 analytical column (150 mm×4.6 mm i.d., 5 μm; Grace, USA) maintained at 35 °C. The mobile phase was composed of acetonitrile and 0.1% trifluoroacetic acid solution using gradient elution and the flow rate was set to 1.0 mL/min. The detection wavelength of the detector was set at 214 nm. The HPLC method was of good specificity and accuracy, and the linear range was 5–400 μg/mL.
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