Gas chromatograph

SCFA concentrations were measured by gas chromatography as described previously (Richardson et al., 1989). Following derivatization of the samples using N‐tert‐butyldimethylsilyl‐N‐methyltrifluoroacetamide, the samples were analysed using a Hewlett Packard gas chromatograph (GC) fitted with a silica capillary column using helium as the carrier gas. Ethanol concentrations were also measured by gas chromatography using the same GC but fitted with a ZB WAX column.

Short chain fatty acids (SCFAs) and lactate were extracted from samples collected from gut compartments using diethyl ether and derivatised using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide, then quantified by analysing these extracts by gas–liquid chromatography using a Hewlett Packard gas chromatograph (GC) fitted with a silica capillary column using helium as the carrier gas as described previously (Richardson et al., 1989 ). For each experiment, 2-ethyl butyrate as used as an internal standard and an SCFA mixture as an external standard. SCFA concentrations were determined relative to standards of known SCFA concentration, and relative to the internal standard.

Short chain fatty acids (SCFAs) present in each infant faecal sample were identified by gas chromatography. Weighed dried samples (150 mg) were resuspended in sterile distilled water (1.1mL) and the concentration of SCFAs measured in duplicate in 1:16 dilution of 250 μl supernatant. Samples were prepared as described previously [38 (link)]. Briefly, 25 μl of internal standard, 0.25 ml HCl and 1 ml of ether was added to 0.5 ml of diluted sample. Standards were included in each analysis set and contained 25 μl of internal standard, 0.5 ml of external standard, 0.25 ml HCl and 1 ml of ether. All tubes were vortexed for 1 min and centrifuged for 10 min at 3000 rpm. The upper ether layer was then transferred to a new tube and the sample was re-extracted with a further 0.5 ml of ether. Samples were vortexed and centrifuged as before and the ether layers combined. 400 μl of the combined ether layer was transferred to a Wheaton screw cap vial containing 50μl of MTBSFA (Sigma Aldrich). Samples were heated at 80°C for 20 min, then left at room temperature for 48 hr to derivatize before being run on a Hewlett Packard (Palo Alto, CA, USA) gas chromatograph (GC) fitted with a fused silica capillary column, with helium as the carrier gas.