Metabolite Analysis by HPLC

Samples were collected from 2 mL culture samples after centrifugation of the samples (14,000 rpm for 5  min at 4°C) and filtering of the supernatant using Nylon syringe filters of 0.22 µm. Samples were measured by HPLC through an Alliance Waters HPLC equipped with an Aminex HPX-87H column (7.8 mm × 300 mm). The mobile phase was H₂SO₄ (25 mM), the column temperature was 75°C, and the eluent flow rate was 0.7 mL min⁻¹. Instrument linearity was evaluated with each of the compounds (obtained from Sigma-Aldrich; ReagentPlus, 99%) in the concentration range of 0.8–90 mM.

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Bordel S., Martín-González D., Börner T., Muñoz R, & Santos-Beneit F. (2024). Genome-scale metabolic model of the versatile bacterium Paracoccus denitrificans Pd1222. mSystems, 9(2), e01077-23.

Publication 2024

HPLC HPX-87H

Corresponding Organization : Universidad de Valladolid

Other organizations : HES-SO Valais-Wallis

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Variable analysis

independent variables

Centrifugation speed (14,000 rpm)
Centrifugation duration (5 minutes)
Centrifugation temperature (4°C)
Filtration method (Nylon syringe filters of 0.22 µm)
Mobile phase (H2SO4 25 mM)
Column temperature (75°C)
Eluent flow rate (0.7 mL min-1)
Concentration range (0.8–90 mM)

dependent variables

Compound concentrations measured by HPLC

control variables

Culture sample volume (2 mL)
HPLC instrument (Alliance Waters HPLC)
HPLC column (Aminex HPX-87H column, 7.8 mm × 300 mm)

controls

Positive control: Compounds obtained from Sigma-Aldrich (ReagentPlus, 99%)

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