The determination was carried out as described.[24] Briefly, adapting the conditions of the FAAH assay the test compound was incubated in PBS buffer (pH 7.4) containing Triton X‐100 (0.2 %) and EDTA (1 mM) at 37 °C for 60 min. After centrifugation, the concentration of the compound was determined by reversed‐phase HPLC with UV‐detection at 220 nm. The relative amount of the test compound found in the aqueous sample after 60 min incubation at 37 °C was determined with the aid of a freshly prepared reference solution prepared analogously. Separation was achieved using a Synergi Polar‐RP 80 Å column (4.6 mm (I.D.)×150 mm, particle size 4 μm) (Phenomenex, Aschaffenburg, Germany) protected with a Phenomenex phenyl guard column (3 mm (I.D.)×4 mm) or an Aqua C18 125 Å column (4.6 mm (I.D.)×150 mm, particle size 3 μm) (Phenomenex, Aschaffenburg, Germany) protected with a Phenomenex C18 guard column (3 mm (I.D.)×4 mm).
Aqua c18 125 column
Manufactured by Phenomenex
Sourced in Germany
The Aqua C18 125 Å column is a chromatographic column designed for liquid chromatography. The column features a stationary phase with a pore size of 125 Ångström and a C18 functional group. The core function of this column is to provide separation and analysis of a variety of compounds in liquid samples.
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3 protocols using aqua c18 125 column
1
Determination of compound solubility
2
HPLC-MS Analysis of P. dioica Seed Compounds
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The EAE of P. dioica seeds was analysed using a Hewlett-Packard 1200 chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a binary pump and a diode array detector (DAD) coupled to an HP Chem Station (rev. A.05.04) data-processing station. The HPLC system was connected via the DAD cell outlet to an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany) mass spectrometer (MS) consisting of an ESI source and a triple quadrupole-ion trap mass analyser, which was controlled by the Analyst 5.1 software. An Aqua C18 125 Å column (250 × 4.6 mm, 5 μm; Phenomenex) thermostated at 35 °C was used. The solvents were: (A) 0.1% formic acid and (B) acetonitrile. The elution gradient was the same as previously described27 . Compound identification was made based on their absorption and mass spectral characteristics (both positive and negative modes) and comparison with previously published data.
3
FAAH Enzyme Assay Protocol
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Adapting the
conditions of the FAAH assay,36 (link) a solution
of the test compound in DMSO (1 mM) (4 μL) was diluted with
a solution of Triton X-100 (0.2%, v/v) in PBS containing EDTA (1 mM)
(196 μL) and incubated at 37 °C for 60 min. Immediately
thereafter, the sample was centrifuged at 12 000g and 20 °C for 5 min and analyzed by HPLC. Separation was achieved
using a Synergi Polar-RP 80 Å column (4.6 mm inner diameter ×
150 mm, particle size 4 μm) (Phenomenex, Aschaffenburg, Germany)
protected with a Phenomenex phenyl guard column (3 mm inside diameter
× 4 mm) or an Aqua C18 125 Å column (4.6 mm inner
diameter × 150 mm, particle size 3 μm) (Phenomenex, Aschaffenburg,
Germany) protected with a Phenomenex C18 guard column (3
mm inside diameter × 4 mm). An aliquot of each sample (30 μL)
was injected into the HPLC/UV system. Autosampler and column oven
temperatures were set to 20 °C. The mobile phase consisted of
acetonitrile/water/H3PO4 conc. (58:42:0.1, v/v/v).
The flow rate was 1.0 mL/min, and the absorption wavelength was set
to 238 nm. The relative amount of the test compound found in the aqueous
sample after 60 min incubation at 37 °C was determined with the
aid of a freshly prepared reference solution obtained by dilution
of a DMSO solution (1 mM) of the compound (4 μL) with a solution
of Triton X-100 (0.2%, v/v) in PBS containing EDTA (1 mM) (196 μL).
conditions of the FAAH assay,36 (link) a solution
of the test compound in DMSO (1 mM) (4 μL) was diluted with
a solution of Triton X-100 (0.2%, v/v) in PBS containing EDTA (1 mM)
(196 μL) and incubated at 37 °C for 60 min. Immediately
thereafter, the sample was centrifuged at 12 000g and 20 °C for 5 min and analyzed by HPLC. Separation was achieved
using a Synergi Polar-RP 80 Å column (4.6 mm inner diameter ×
150 mm, particle size 4 μm) (Phenomenex, Aschaffenburg, Germany)
protected with a Phenomenex phenyl guard column (3 mm inside diameter
× 4 mm) or an Aqua C18 125 Å column (4.6 mm inner
diameter × 150 mm, particle size 3 μm) (Phenomenex, Aschaffenburg,
Germany) protected with a Phenomenex C18 guard column (3
mm inside diameter × 4 mm). An aliquot of each sample (30 μL)
was injected into the HPLC/UV system. Autosampler and column oven
temperatures were set to 20 °C. The mobile phase consisted of
acetonitrile/water/H3PO4 conc. (58:42:0.1, v/v/v).
The flow rate was 1.0 mL/min, and the absorption wavelength was set
to 238 nm. The relative amount of the test compound found in the aqueous
sample after 60 min incubation at 37 °C was determined with the
aid of a freshly prepared reference solution obtained by dilution
of a DMSO solution (1 mM) of the compound (4 μL) with a solution
of Triton X-100 (0.2%, v/v) in PBS containing EDTA (1 mM) (196 μL).
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