The cell‐free medium supernatant was applied to 40 g of pre‐equilibrated Amberlite XAD‐16 resin packed in a column. The XAD‐16 column was subsequently washed with 1 L of H2O and then eluted with 1 L of 100% methanol. The methanolic elution was evaporated to dryness by rotary evaporation, and the brown residue was redissolved in 5 ml of purified water (MilliQ system; Millipore). The concentrated solution was then applied onto a 12 gram Biotage® SNAP Ultra C18 column for fractionation using reverse‐phase flash chromatography on a Biotage Isorela One instrument (Stockholm, Sweden). The separation was performed using a linear gradient of acetonitrile from 5% to 100% over 50 min at 15 ml/min. To identify column fractions containing active compounds, each fraction was evaporated to dryness by rotary evaporation and redissolved in 2 ml of MilliQ water. The bioactivity of these fractions was further determined by performing agar disk diffusion assays.
Snap ultra c18 column
Manufactured by Biotage
Sourced in Sweden
The SNAP Ultra C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and purification of a wide range of organic compounds. The column features a silica-based stationary phase with chemically bonded C18 functional groups, which provide effective retention and separation of analytes based on their hydrophobicity. The column is suitable for use in various HPLC applications, including pharmaceutical, environmental, and biochemical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Milli q system, by Merck Group (1 mentions)
Acquity uplc h class system, by Waters Corporation (1 mentions)
Pu 2080, by Jasco (1 mentions)
Jnm ecz400 spectrometer, by JEOL (1 mentions)
Ods 3, by GL Sciences (1 mentions)
Isolera one purification system, by Biotage (1 mentions)
Xevo tqd, by Waters Corporation (1 mentions)
Acquity uplc beh c18 1.7 μm, by Waters Corporation (1 mentions)
Isolera one flash chromatography, by Biotage (1 mentions)
4 protocols using snap ultra c18 column
1
Fractionation of Bioactive Compounds
2
Comprehensive Analytical Techniques for Chemical Compounds
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NMR spectra were recorded using a JEOL JNM-ECZ400 spectrometer at 400 MHz for 1H NMR and 100 MHz for 13C NMR. Mass spectra (MS) were obtained using JEOL JMS-T100LP AccuTOF LC-plus 4 G (ESI).
Preparative HPLC was performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2015). Preparative MPLC was performed on an Isolera One purification system (Biotage) equipped with a Biotage SNAP Ultra C18 column (for reverse phase separation) or on an MPLC system comprising a pump and detector (EPCLC AI-580S, Yamazen) and equipped with a silica gel column (silica gel 40 μm or Amino 40 μm, Yamazen) (for normal phase separation). LC-MS analysis was performed on an Acquity UPLC H-Class system (Waters) equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column (Waters) and an MS detector (QDa or Xevo TQD, Waters).
Preparative HPLC was performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2015). Preparative MPLC was performed on an Isolera One purification system (Biotage) equipped with a Biotage SNAP Ultra C18 column (for reverse phase separation) or on an MPLC system comprising a pump and detector (EPCLC AI-580S, Yamazen) and equipped with a silica gel column (silica gel 40 μm or Amino 40 μm, Yamazen) (for normal phase separation). LC-MS analysis was performed on an Acquity UPLC H-Class system (Waters) equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column (Waters) and an MS detector (QDa or Xevo TQD, Waters).
3
Column Chromatography for Compound Separation
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Automated column chromatography was performed on a Büchi Reveleris X2 with a binary pump and ELSD Detector using a Biotage Snap Ultra C18 column with a gradient (4 min at 5% B, up to 25% B in 14 min, in 1 min up to 100% B for 2 min and flushing with 80% B for 5 min, total run time 27 min) at a flow rate of 30 ml /min. Solvent A consisted of 99.9% water and 0.1% TFA, solvent B of 99.9% acetonitrile and 0.1% TFA.
4
Purification of Crude Peptides and TSC-Conjugates
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Crude peptides and TSC-conjugates were purified by Reverse-Phase Flash Liquid Chromatography (RP-HPLC) on Isolera One Flash Chromatography (Biotage, Uppsala, Sweden) using a SNAP Ultra C18 column (40 g) at 20 ml/min flow. Eluent systems: 0.1% TFA in H2O (A), 0.1% TFA in ACN (B). Due to the poor solubility in water of final compounds, the samples for purification were dissolved in the mixture of solvents A and B at the initial percentage of the purification gradient and treated with ultrasounds. The linear gradient applied for each purification was: 35–65% B in A in 30 min.
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