Sil 10 autoinjector

Ion-exclusion high-performance liquid chromatography was carried out to measure the concentrations of fecal organic acid, as per Tsukahara, et al. [30 (link)]. In brief, 0.3 g of feces was mixed with 0.6 mL of distilled water and the diluents was further mixed with 90 µL of 12% perchloric acid (v/v). After centrifugation (13,000× g, 4 °C, 10 min), the supernatants were filtered using a 0.45 μm cellulose acetate membrane filter (Cosmonice Filter W, Nakalai Tesque, Kyoto, Japan) and transferred to a vial. Then, it was injected into the high-performance liquid chromatography apparatus with an SIL-10 autoinjector (Shimadzu, Kyoto, Japan). Organic acids were separated by two serial organic acid columns (Shim-pack SCR-102H, Shimadzu) with a guard column (SCR-102HG; Shimadzu) at 45 °C with isocratic elution (0.8 mL/min) of 5 mmol/L ρ-toluene sulfonic acid aqueous solution using a solvent delivery pump (LC-10ADvp; Shimadzu) with an online degasser (DGU-12A; Shimadzu). Organic acids were detected with an electronic conductivity detector (Waters 431; Waters, Tokyo, Japan) after post-column dissociation (0.8 mL/min) with 5 mmol/L ρ-toluene sulfonic acid, 20 mmol/L bis-Tris and 100 μmol/L ethylenediaminetetraacetic acid using a solvent delivery pump (LC-10ADvp; Shimadzu). Organic acids were quantified with a system controller (CBM-20A; Shimadzu).

Preprocessing of cecal digesta and analysis of organic acid concentrations were based on the methods of Tsukahara et al. [24 (link)]. Samples (0.3 g) were mixed with 0.6 mL distilled water. Diluents were mixed with 90 μL 120 g/L perchloric acid After centrifugation (15,000× g, 10 min, 4 °C), the supernatant fractions were filtered through a 0.45-μm cellulose acetate membrane filter (Cosmonice Filter W, Nakalai Tesque, Kyoto, Japan) and degassed under vacuum. The supernatant fractions (5 μL) were injected into an SIL-10 autoinjector (Shimadzu, Kyoto, Japan). Organic acids were separated by two serial organic acid columns (Shim-pack SCR-102H, Shimadzu, Kyoto, Japan) with a guard column (SCR-102HG; Shimadzu, Kyoto, Japan) at 45 °C with isocratic elution (0.8 mL/min) of 5 mM p-toluenesulfonic acid aqueous solution using a solvent delivery pump (LC-10ADvp; Shimadzu, Kyoto, Japan) with an online degasser (DGU-12A; Shimadzu, Kyoto, Japan). Organic acids were detected with an electronic conductivity detector (Waters 431; Waters Corporation, Milford, MA, USA) after post-column dissociation (0.8 mL/min) with 5 mM p-toluenesulfonic acid, 20 mM bis-Tris, and 100 μM ethylenediaminetetraacetic acid by using the solvent delivery pump. Organic acids were quantified with a system controller (CBM-20A; Shimadzu, Kyoto, Japan).