SCFA Quantification by Gas Chromatography

The SCFA were analysed by gas chromatography as previously described^{55 (link)}. Briefly, 1 g of ceca was mixed 1 ml of H₂O and 1 ml of 20 mmol/l pivalic acid solution as an internal standard. The solution was mixed and 1 ml of HClO₄ (perchloric acid) was added in order to extract SCFA by shaking by vortexing for 5 min. The HClO₄ acid was precipitated by adding 50 ml of 4 mol KOH into 500 ml of supernatant. The addition of saturated oxalic acid, at 40C for 60 min, and centrifugation at 18,000g for 10 min. Samples were analysed by gas chromatography using SCION-456-GC with a flame ionization detector.