Pivalic acid

Polysulfone (PSU, Mw 35,000 Da in transparent pellet form, Sigma Aldrich, St. Louis, MO, USA), N,N-Dimethylformamide (DMF, 99% Fisher, Loughborough, UK), and 1-Methyl-2-pyrrolidone (NMP, 99% Acros Organics, New Jersey, NY, USA) were used for membrane preparation.
For the synthesis of 1-butyl-3-methylimidazolium chloride, 1-chlorobutane (99% Acros Organics, Geel, Belgium), 1-methylimidazole (99% Sigma-Aldrich, St. Louis, MO, USA), acetonitrile (CAN, 99.8% Fisher, Loughborough, UK), and ethyl acetate (99.9% VWR, Fontenay-Sous-Bois, France) were used. The task-specific ILs were prepared by the ion exchange process using Amberlite IRA-402(Cl) ion exchange resin (Alfa-Aesar, Kandel, Germany), Sodium hydroxide (NaOH, 97% Fisher, Geel, Belgium), and acidic compounds: benzoic acid (99% Acros Organics, New Jersey, USA), pivalic acid (99% Sigma-Aldrich, Steinheim, Germany), DL-proline (99% Sigma-Aldrich, St. Louis, USA), formic acid (99% Merck, Darmstadt, Germany), and malonic acid (99% Alfa-Aesar, Kandel, Germany)
The membrane gas solubility was evaluated with CO₂ (99.995%) and N₂ (99.999%) gases purchased from Linde (Barcelona, Spain).

Short chain fatty acid profiles of the ileal digesta were determined by Alimetrics Diagnostics (Espoo, Finland) using gas chromatography (GC; Agilent Technologies, Santa Clara, CA, USA) with pivalic acid (Sigma-Aldrich, St. Louis, MO, USA) used as an internal standard. The chromatography procedure used a glass column packed with 80/120 Carbopack B-DA/4% Carbowax stationary phase, helium as a carrier gas, and a flame ionization detector (FID) and has been described previously by Apajalahti et al.^{86 (link)}. Lactic acid and the VFA (acetic acid, propionic acid, isobutyric acid, butyric acid, 2-methylbutyric acid, isovaleric acid and valeric acid) were derivatised to the respective phenyl esters using phenyl chloroformate reagent. The resulting esters were analysed by Agilent GC-FID (Agilent Technologies). Matrix-matched internal standard calibration with butyric-d7- and acetic-d3 acids was used in quantitation.

3-Hexylthiophene (3HT) was purchased from TCI (Tokyo, Japan). triphenylamine, benzo [c] [1,2,5] thiadiazole, tetrahydrofuran (99.9%) and N-bromosuccinimide were purchased from Acros Organics. Palladium(II) acetate (Pd(OAc)₂) (98%), tricyclohexylphosphine tetrafluoroborate (97%, PCy₃·HBF₄), 3,3′dibromo-2,2′bithiophene, benzamide, N,N′-dimethylethylenediamine (85%, DMEDA) and pivalic acid (PivOH) were purchased from Sigma-Aldrich. Potasium acetate (KOAc), sodium carbonate (99%), magnesium sulfate (98%), and copper iodine (CuI) were purchased from Acros and used as received. Chloroform (CHCl₃, 99.5%), toluene (99.5%), and dimethylacetamide (DMAc, 99%) were purchased from Fisher/Acros and dried using molecular sieves under N₂. Dichloromethane (99.8%), n-heptane (99%), methanol (99.8%), ethyl acetate (99%) and diethyl ether (99%) were purchased from Fisher/Acros and used as received.

Volatile fatty acids and lactic acid, referred to in combination as short-chain fatty acids (SCFA), were analyzed in six replicate fermentation vessels per treatment at the 4-, 10-, and 24-h time-points. The SCFA were analyzed as free acids, using pivalic acid (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. For this, 400 μL of fermentation fluid and 2.4 mL of 1.0 mM pivalic acid solution were mixed, vigorously shaken for 5 min, and then centrifuged at 3,000 × g for 10 min. Then 800 μL of the supernatant and 400 μL of saturated oxalic acid solution were mixed, incubated at 4°C for 60 min, and centrifuged at 18,000 × g for 10 min. The supernatant was analyzed by gas chromatography (Agilent Technologies, Santa Clara, CA, USA) using a glass column packed with 80/120 Carbopack B-DA/4% Carbowax stationary phase, helium as a carrier gas, and a flame ionization detector. The acids quantified were acetic, propionic, butyric, valeric, isobutyric, 2-methylbutyric, isovaleric, and lactic acid.

Zirconium propoxide (70 w% in 1-propanol) and hafnium butoxide (99%) was provided by Sigma-Aldrich and stored in a Straus flask upon arrival. Acetic acid (>99%) was purchased from Sigma-Aldrich and vacuum distilled after which it was stored in a Schlenk flask. Zirconium isopropoxide isopropanol complex (99.9%), propionic acid (>99.5%), butyric acid (99%), hexanoic acid (99%), octanoic acid (99%), decanoic acid (>99.5%), dodecanoic acid (98%), oleic acid (90%), methyl-butyric acid (98%), methyl-heptanoic acid (>98.5%), pivalic acid (99%) and benzyl alcohol (anhydrous, 99.8%) were bought from Sigma-Aldrich and used without any further purification. Acetone, diethylether (BHT stabilized) and dichloromethane (DCM) were bought from Biosolve and used without any further purification. HPLC grade acetonitrile (ACN) was bought from VWR and used without any further purification. All yields reported here are calculated without any co-precipitated molecules unless otherwise specified. Centrifugation was always performed at 5000 rcf for 3 minutes, unless otherwise specified. After purification all clusters are stored in a desiccator.

Volatile fatty acids (VFA) and lactic acid were analysed as free acids in both the ileum and caecum simulation vessels after 9-h incubation using pivalic acid (Sigma-Aldrich, St. Louis, MO, United States) as an internal standard (Apajalahti et al., 2019 (link)). For this, 400 μL of sample and 2.4 mL of 1.0 mM pivalic acid solution were mixed, shaken vigorously for 5 min, and then centrifuged at 3000 × g for 10 min. Then, 800 μL of the supernatant was mixed with 400 μL of saturated oxalic acid solution, incubated at 4°C for 60 min, and then centrifuged at 18,000 × g for 10 min. The supernatant was analysed by gas chromatography (Agilent Technologies, Santa Clara, CA, USA) using a glass column packed with 80/120 Carbopack B-DA/4% Carbowax stationary phase, helium as the carrier gas, and a flame ionization detector. The quantified acids were acetic, propionic, butyric, valeric, isobutyric, 2-methylbutyric, isovaleric, and lactic acid.

A BioFreeze™ vial with intestinal content [distal small intestine (last 25%), caecum and mid-colon] was shaken vigorously and centrifuged as described in section 2.2.6. The formed supernatant was used as the SCFA sample. The SCFA profiles were analyzed by gas chromatography (Agilent Technologies, Santa Clara, CA, USA) with pivalic acid (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. The chromatography procedure used a glass column packed with 80/120 Carbopack B-DA/4% Carbowax stationary phase, helium as a carrier gas, and a flame ionization detector, and it has been described previously by Apajalahti et al. (22 (link)).