Hp 5890 series 2

All samples were centrifuged and the supernatant was acidified by addition of ortho-phosphoric acid to obtain a final pH value of 2.0. Total VFA, i.e. C₁–C₆ short chain fatty acids, alcohols, lactic acid, and phenyl acetic acid, were determined gaschromatographically using an Agilent HP 5890 Series II gas chromatography system equipped with a flame ionization detector (FID) and a BP 21 bonded FFAP fused silica column (length 250 mm, diameter 0.53 mm, thickness of the immobilized phase 0.5 µm) [21 (link)]. The FID was used in the automatic and splitless mode. An automatic sampler injected 1 µl sample under the temperature program of 240/70–235/260 °C for injector, column and detector, respectively. H₂ was applied as carrier gas with a H₂-flow of 30 ml min⁻¹. The air-flow of the FID was 300 ml min⁻¹ with a makeup-flow of 25 ml nitrogen per min. The total run time was adjusted to 30 min. A weekly calibration was performed with a commercial external standard (Supelco 46975-U, Sigma-Aldrich, Germany). DM and VS content were determined according to the standard guidelines, i.e. VDI 4630 protocols [22 ].

Concentrations of SCFA and BCFA were measured by gas chromatography at the German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Nuthetal, Germany. The stool sample aliquot was weighed, diluted with 350 μL of water, and centrifuged (14,000× g, 5 min). The supernatant (100 μL) was mixed with 20 μL of 8.6 mM ethyl butyrate (internal standard), 280 μL of 0.36 M perchloric acid, and 270 μL of 1 M sodium hydroxide. After freeze-drying overnight, the residue was dissolved in 100 μL of 5 M formic acid and 400 μL of acetone and centrifuged (14,000× g, 5 min). An aliquot (1 µL) of the organic phase was used for the quantification of SCFA and BCFA by a gas chromatograph (HP 5890 series II; Agilent, Waldbronn, Germany) equipped with a HP-20 M column and a flame ionization detector (FID), as described previously [26 (link)].
Ammonia levels were determined at DIfE in fecal supernatants following ultrafiltration (Vivacon 500 centrifugal units, Sartorius, Göttingen, Germany) by using a colorimetric assay (Ammonia Colorimetric Assay Kit II, BioVision, Milpitas, CA, USA). Amounts of all compounds refer to fecal wet weight.

The following parameters were measured daily on influent and effluent of both full-scale reactors: TOC was analyzed using a TOC analyzer (TOC-L CPN Basic System, Shimadzu, Japan). TSS was measured following the procedure of APHA (1998) [19 ]. For U1: Methanol and methyl acetate were detected using gas chromatography with FID on an HP5890 with a RTX-1 nonpolar column. Acetate is detected on a hp 5890 Series II with a DB-Wax polar column; and TA, BZ, IA, OA, PT, and TMA were detected using an Agilent 1200 HPLC System with multiple wavelength detector, or equivalent; the HPLC column was an Agilent SB-C18, 4.6 mm i.d. x 50 mm, 1.8 μm particle diameter (p/n 822975–902). For reactors E and F: TA, BZ, IA, OA, PT, and TMA were aromatic compounds, fatty acids and methyl compounds were detected using a high-performance liquid chromatography (Agilent ZORBAX Eclipse XDB-C18, Rapid Resolution HT 4.6 mm i.d. x50 mm, 1.8 μm particle diameter, operated at 600 Bar).

The organic phases containing FAMEs were injected (0.5 μL) into a GC apparatus (HP5890 Series II, Agilent, Waldbronn, Germany) equipped with the column (100 m × 0.25 mm, 0.25 μm) CP-Select FAME (Varian, Palo Alto, CA, USA). The injector was kept at 270°C. To determine the whole profile of FAs, the oven temperature ramped from 135°C to 250°C (2.5°C/min) and was maintained at 250°C for 23 min. The same program was followed, starting from 160°C, when only LA and CLA had to be determined. Elution was performed with high-purity helium, with constant column head pressure of 200 kPa. Qualitative analyses of FAMEs were performed with a MS quadrupole detector (HP5972, Agilent). Analytes were identified by comparison with standards (O5632, Sigma Aldrich) and by analysis of their fragmentation patterns (EI, 70 eV) with the mass spectrum library NIST 2005 (Gatesburg, USA). Quantitative analyses were performed with a flame ionization detector held at 300°C.

FFAs and FAMEs were extracted by addition of 6 mL of a 2:1 chloroform/methanol mixture (spiked with 0.15 mg/L of either methyl tridecanoate or methyl heptadecanoate as an internal control) to 5 ml of culture. For consistency in data analysis, 1 mL of dodecane layer was similarly treated with 6 mL of a 2:1 chloroform/methanol mixture before gas chromatography (GC) analysis. Quantification of FAs/FAMEs was conducted by GC-FID using an HP 5890 Series II gas chromatograph equipped with an HP-Innowax Column (0.32 mm x 30 m x 0.25 μm, Agilent). All samples were analyzed using the following parameters: inject: 1 μl; inlet temperature 250 °C with split ratio 1:1; carrier gas: helium; flow: 5 ml/min; oven temperature: initial temperature of 160 °C, hold 3 min; gradient to 255 °C at 5 °C/min; hold 3 min; inlet temp: 270 °C, detector temp: 330 °C. The amount of FAs/FAMEs was determined by comparison to a standard curve of various FAs and FAMEs and methyl tridecanoate or methyl heptadecanoate concentrations. To identify all FA/FAME products, GC/mass spectrometry analysis was additionally performed using an Agilent 6890-5975 equipped with HP-Innowax Column (0.32 mm x 30 m x 0.25 μm, Agilent). Peak identification was performed through comparison with GC retention time, known standards and mass spectra with the National Institute of Standards and Technology (NIST) database.