Hp innowax fused silica column

The supernatants (500 µl) of the samples collected at 0, 24 and 48 hours were spiked with an internal standard (4-methyl valeric acid). This was further diluted in a 70% (v/v) ethanol and 0.1% (v/v) trifluoroacetic acid (TFA) solution to obtain a final concentration of the internal standard in the mixture at 100 ppm. The solution was then vortexed and filtered through a 0.2 µm membrane (Millipore, Australia) prior to analysis using a gas chromatograph with a flame ionisation detector (GC-FID, Shimadzu GC-17A). Samples were separated on a 30 m × 0.25 × 0.5 µm i.d. HP-INNOWax fused silica column (Hewlett-Packard) as per the manufacturer’s instructions. GC-FID analysis for each of the 270 samples was performed with further instrument specific technical triplicates (n = 810). SCFA concentrations were normalised for the weight of the fecal inoculum in each biological sample.

The concentration of SCFAs (acetate, propionate, and butyrate) was quantified using fecal samples collected at weeks 17, 23, and 32. Approximately 20–50 mg of feces was extracted with 500 µL of 70% (v/v) ethanol and 0.1% (v/v) trifluoroacetic acid (TFA) solution spiked with an internal standard (4-methyl valeric acid) at a final concentration of 100 ppm. The solution was mixed thoroughly, then centrifuged at 14,000 x g at 4°C for 30 minutes to pellet the fecal material. The top 200 µL was removed and analyzed using a Shimadzu GC-17A gas chromatograph with a flame ionization detector (GC-FID, Shimadzu GC-17A). Samples were separated on a 30 m x 0.25 × 0.5 µm i.d. HP-INNOWax fused silica column (Hewlett-Packard, Australia) as per the manufacturer’s instructions. GC-FID analysis for each sample was performed in three technical replicates (n = 450). All measurements were normalized for the weight of fecal samples used for SCFA quantification.

The supernatants of the liquid fraction samples (500 μL) collected at 0, 24, and 48 h were spiked with an internal standard (4-methyl valeric acid). This was further diluted in a 70% (v/v) ethanol and 0.1% (v/v) trifluroacetic acid (TFA) solution to obtain a final concentration of the internal standard at 100 ppm. The solution was vortexed then filtered through a 0.2 μm membrane filter (Millipore, Australia). Analysis was performed using a GC-FID (Shimadzu GC-17A). Samples were separated on a 30 m × 0.25 × 0.5 μm i.d. HP-INNOWax fused silica column (Hewlett-Packard, Australia) as per the manufacturer's instructions. GC-FID analysis for each sample was performed in three technical replicates (n = 636). The concentrations of SCFAs are reported in mmolL⁻¹ per gram of feces.