The culture supernatants were thawed on ice, diluted, and passed through 0.45 μm membrane syringe filters (Millipore; Merck-Millipore, Burlington, MA, USA). A high-performance liquid chromatograph, Agilent Technologies 1200 series (Agilent Technologies, Santa Clara, CA, USA), was used to determine the concentration of glycerol (GLY; [g L−1]) and metabolites (erythritol, ERY; mannitol, MAN; citric acid, CA; α-ketoglutaric acid, α-KG [g L−1]) contained in the culture liquid. The apparatus was equipped with a refractive-index detector (G1362A) and a Rezex ROA-Organic Acid H+ column (Phenomenex, Torrance, CA, USA). Operating conditions were as follows: 0.005 N H2SO4 as eluent at a flow rate of 0.6 [mL min−1]; the column temperature was set at 40 °C. External standards (purchased from Sigma-Aldrich) were used for identification and quantification of the peaks areas in chromatograms, which were analyzed using ChemStation for LC 3D software (Agilent).
Chemstation for lc 3d software
Manufactured by Agilent Technologies
ChemStation for LC 3D software is a data analysis and instrument control platform for Agilent's liquid chromatography systems. It provides tools for data acquisition, processing, reporting, and system management.
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3 protocols using chemstation for lc 3d software
1
HPLC Analysis of Microbial Metabolites
2
RP-HPLC Analysis of TPA, MHET, and BHET
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The RP-HPLC was a Hewlett Packard (ChemStation 1100 series) with a diode array detector, equipped with an ODS-L optimal column (250 x 4.6 mm) packed with C18 particles, 5 µm in diameter size. Injection volume was 20 µL and samples were eluted with 24 % acetonitrile over 25 minutes at a flow rate of 0.5 mL/min and 40 °C. UV detection at 240 nm and peak analysis was performed using the Agilent ChemStation for LC 3D software. Known concentrations of TPA, MHET and BHET were used to determine retention times and as standards for quantification.
3
Simultaneous Quantification of Neurotransmitters
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The concentrations of NA, MHPG, DA, DOPAC, HVA, 5-HT and HIAA were determined Mobile phases were adjusted to pH 4 using H 3 PO 4 . The eluent flow rate was set to 0.8 ml/min. The method is based on a previously published one using isocratic elution [39] . A binary HPLC-pump was used for gradient elution. The starting composition of the mobile phase was 88 % A and 12 % B and was kept for 10 min. During the following 20 min gradual adjustement to 30 % A and 70 % B was performed and this percentage kept for 10 min. For column reequilibration, percentages of A and B were returned to initial conditions within 4 min and kept for 6 min before the next sample injection. Total acquisition time was 50 min per sample. Analytes were detected applying an oxidation potential of 0.72 V versus the reference electrode. Chromatograms were acquired with Agilent ChemStation for LC 3D software.
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