Sulfuric acid

Short-chain fatty acids (SCFAs) in cecal contents were measured in cecum samples collected using gas chromatography (7890 B, 5977 B; Agilent Technologies, Inc., Santa Clara, CA, USA). SCFAs were extracted from 50 mg of cecal contents by acidifying the cecal contents with sulfuric acid (Fujifilm Wako Pure Chemical Co., Osaka, Japan) and shaking them in 50 μL of 2 N sulfuric acid, 400 μL of diethyl ether (Fujifilm Wako Pure Chemical Co., Osaka, Japan), and 200 μL of ethanol (Fujifilm Wako Pure Chemical Co., Osaka, Japan). The mixture was centrifuged at 14,000 rpm for 30 s at room temperature. Next, 300 μL of the supernatant was mixed with 100 μL of TMSI-H (GL Science Inc., Tokyo, Japan), and the mixture was heated at 60? for 30 min. After heating, the mixture was placed on ice for 10 min and centrifuged at 14,000 rpm for 30 s at room temperature. A total of 2 μL of the organic phase was injected into a capillary column [InertCap Pure WAX (30 m × 0.25 mm, df = 0.5 μm), GL Science, Tokyo, Japan], followed by analysis by gas chromatography with a flame ionization detector. The incipient and final temperatures were 80? and 200?, respectively. Helium was used as the carrier gas. We have previously established a quantitative analysis of SCFAs from mouse cecal contents (25 (link)).

The GO dispersion was synthesized as follows. Graphite (15 g, FUJIFILM Wako Pure Chemicals Co., Ltd., Osaka, Japan) and 640 g of sulfuric acid (FUJIFILM Wako Pure Chemicals Co., Ltd.) were mixed, and then 45 g of potassium permanganate (FUJIFILM Wako Pure Chemicals Co., Ltd.) was added at room temperature. The mixture was allowed to react for 2 h, and then 1070 mL of water and 42 mL of 30% aqueous hydrogen peroxide (FUJIFILM Wako Pure Chemicals Co., Ltd.) were added to stop the reaction. The product was purified by static sedimentation to remove the supernatant and repeated redispersion in ion-exchanged water. After purification, the reaction solution was homogenized using a homogenizer (10,000 rpm, HG-200, AS ONE Corporation, Osaka, Japan) to obtain the GO dispersion.

Pieces of Japanese cypress (Chamaecyparis obtuse) (Yamani, Co., Ltd., Nagano, Japan) with dimensions of 5.0 mm (longitudinal direction, L) × 20 mm (radial direction, R) × 20 mm (tangential direction, T) were used as wood specimens. The specimens were cut from a larger piece with a transverse section of 20 mm (R) × 20 mm (T). The initial mass of these specimens was ~0.7 g. Methanol, propionic anhydride (PA), trifluoroacetic anhydride (TFAA), propionic acid, 4-dimethylaminopyridine (DMAP), N-methyl pyroridone, sodium hydrogen sulfate, and sulfuric acid (SA) were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan) and utilized as obtained.