Peptide stock solutions (1 μL of 3 mM) were injected into an Agilent 1260 Infinity HPLC system equipped with a Poroshell 120 EC-C18 2.7 μm (Agilent #699975–902) analytical column, and run with a linear gradient of a mobile phase composed of eluent A (99.9 % v/v H2O, 0.1% v/v TFA) and eluent B (99.9% v/v acetonitrile, 0.1% v/v TFA) from 5% to 95% over 6 minutes at a flow rate of 0.6 ml/min. The absorbance at a wavelength of 220 nm was used to generate plots of peptide purity.
Poroshell 120 ec c18 2.7 μm
Manufactured by Agilent Technologies
Sourced in United States
The Poroshell 120 EC-C18 2.7 μm is a type of high-performance liquid chromatography (HPLC) column. It features a 2.7 μm particle size and a C18 stationary phase, which is commonly used for the separation and analysis of a wide range of organic compounds.
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Lab products found in correlation
5 protocols using poroshell 120 ec c18 2.7 μm
1
Peptide Purity Analysis by HPLC
2
UPLC-DAD-MS Analysis of Marfey's Derivatized Analyte
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An aliquot of Marfey’s derivatized analyte (3 μL) (see method #1) was subjected to UPLC-DAD-MS analysis using the same binary solvent system and detection as described above (method #2), but with an isocratic 0.8 mL/min elution at 23% Phase B in A using an Agilent Poroshell 120 EC-C18 2.7 μm, 3.0 × 150 mm column at 50 °, with comparison to authentic standards of Marfey’s derivatized amino acids (Figure 3 and Figure S38 ).
3
HPLC-MS Identification of Bioactive Compounds
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The High Performance Liquid Chromatography Mass Spectrometry (HPLC/MS) was used for identification of RA, TMF, SIN and EUP in different extracts of OS. An Accela Thermo Finnigan LCQ DUO (Mundelein, IL, USA) system equipped with an ESI source was used in this study. The standard compounds of RA, TMF, SIN and EUP were prepared separately in MS grade methanol and 20 μL of each compound was injected through HPLC into ESI probe. The operating conditions were as follows: Colum; Poroshell 120 EC-C18, 2.7 μm (Agilent, Santa Clara, CA, USA), column dimension 2.1 × 100 mm, part number: USCGC 01268; mobile phase, methanol and 0.1% formic acid (90:10) at a flow rate 0.3 mL/min; the capillary temperature, 220 °C; capillary voltage, 4 kV; drying gas (N2) flow, 12 L/min; nebulizer (N2) pressure, 35 pis. Full scan data acquisition was performed from m/z 105 to 500 units in MS scan mode.
4
Analytical Characterization of Organic Compounds
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1H and 13C NMR spectra
were recorded on a Varian Mercury-300 NMR spectrometer or a Bruker
AVANCE III HD NanoBay 400 MHz spectrometer, and chemical shifts were
measured in ppm relative to the specific solvent signal. Routine mass
and purity analyses (LRMS) were performed on an HP Agilent LC/MS series
1100 system equipped with a reverse phase column (Agilent Poroshell
120 EC-C18, 2.7 μm, 50 × 2.1 mm) and photodiode array detector
coupled to an Agilent 1946 DSL quadrupole mass selective detector
using electrospray ionization (ESI). The gradient mobile phase consisting
of acetonitrile/water with 0.1% formic acid and UV detection at 254
and 210 nm were used to confirm all final products to be ≥95%.
The melting point was measured on an Electrothermal 9100 apparatus.
Most reagents used in the synthetic procedure were purchased from
Sigma-Aldrich, Alfa Aesar, and TCI. The progress of the reaction was
monitored using thin-layer chromatography (TLC) (silica gel 60 F254
0.25 mm), and the products were visualized by UV light (254 and 365
nm). SiliaFlash P60 (40–60 μm) used in flash column chromatography
was purchased from Silicycle Inc. Other solvents were purchased from
commercial vendors and used without further purification unless otherwise
mentioned.
were recorded on a Varian Mercury-300 NMR spectrometer or a Bruker
AVANCE III HD NanoBay 400 MHz spectrometer, and chemical shifts were
measured in ppm relative to the specific solvent signal. Routine mass
and purity analyses (LRMS) were performed on an HP Agilent LC/MS series
1100 system equipped with a reverse phase column (Agilent Poroshell
120 EC-C18, 2.7 μm, 50 × 2.1 mm) and photodiode array detector
coupled to an Agilent 1946 DSL quadrupole mass selective detector
using electrospray ionization (ESI). The gradient mobile phase consisting
of acetonitrile/water with 0.1% formic acid and UV detection at 254
and 210 nm were used to confirm all final products to be ≥95%.
The melting point was measured on an Electrothermal 9100 apparatus.
Most reagents used in the synthetic procedure were purchased from
Sigma-Aldrich, Alfa Aesar, and TCI. The progress of the reaction was
monitored using thin-layer chromatography (TLC) (silica gel 60 F254
0.25 mm), and the products were visualized by UV light (254 and 365
nm). SiliaFlash P60 (40–60 μm) used in flash column chromatography
was purchased from Silicycle Inc. Other solvents were purchased from
commercial vendors and used without further purification unless otherwise
mentioned.
5
Peptide Purity Analysis by HPLC
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