Sh rxi 5sil ms column

Pyrolysis coupled with gas chromatography mass spectrometry was used to determine the S/G ratio, as previously described [39 (link), 40 (link)]. Sub-samples of ca. 0.1 mg were pyrolyzed at 650 °C for 30 s using the pyroprobe 6200 (CDS Analytical, Oxford, PA) connected to a gas chromatography system (GC-2010 Plus, Shimadzu Scientific Instruments, Columbia, MD) using a Shimadzu SH-Rxi-5Sil MS column (30 m × 0.25 mm ID × 0.25 DF) attached to a mass spectrometer (GCMS-Q P2010, Shimadzu Scientific Instruments, Columbia, MD) system operated using He as carrier (1 mL min⁻¹). The chromatograph was operated at a split ratio of 10 and the program was set at 50 °C for 1 min followed by ramping to 300 °C at 20 °C min⁻¹ and finally maintained at 300 °C for 10 min. Released products of S and G origin were identified on the basis of their mass spectra using the NIST08 mass spectrum library and quantified from the chromatogram using the peak area.

The hydrogenation reaction was conducted in a 20 mL stainless steel reactor. Typically, the HMF aqueous solution (2.00 g, HMF: 126.0 mg), catalyst (20.0 mg), and magneton were put into a glass lining. The lining was set in the reactor and sealed and purged with H₂ for 4 times to displace the air. Then the reactor was filled with H₂ at a specified pressure and put in an oil bath set at a certain temperature. After the reaction, 0.5 mL ethanol solution of the internal standard (n-decane) was added and the mixture was diluted to 10 mL by ethanol. After centrifugation, the liquid was used for analysis. The qualitative analysis was conducted by GC on a Shimadzu GC-2014 equipped with a SH-Rtx-1701 column (30 m × 0.32 mm × 0.25 µm). The oven temperature was started from 353 K for 2 min and raised to 523 K with 20 K/min heating rate. The oven was kept at 523 K for 1.5 min. The GC-MS was performed on Shimadzu GC/MS-TQ8040 equipped with an SH-Rxi-5Sil MS column (30 m × 0.25 mm × 0.25 µm). The oven temperature was started from 323 K for 1 min and raised to 473 K with 40 K/min heating rate and then raised to 553 K and kept at the temperature for 5 min.

Each 10 mg reaction mixtures with corresponding amounts of standard compound were dissolved into 800 μL ethyl acetate in a GC vial, then pyridine (100 μL) and N,O-bis trimethylsilyl trifluoroacetamide (BSTFA, 98%, 100 μL) were added, the mixtures were trimethylsilyl derivatized at 50 °C and kept for 40 min. At last, the mixtures were analyzed by a GC-MS-TQ-instrument (Shimadzu GCMS-TQ8040 triple quadrupole GC/MS/MS, Kyoto, Japan) equipped with a SH-Rxi-5Sil MS column (Shimadzu, 30 m × 0.25 mm × 0.25 μm).

Pyrolysis coupled with gas chromatography mass spectrometry was used to determine lignin S/G ratio on cell wall residues, as previously described (Tian et al., 2021 (link)). Specifically, sub-samples of ≈ 0.1 mg were pyrolyzed at 650°C for 30 s using the pyroprobe 6200 (CDS Analytical, Oxford, PA) connected to a gas chromatography system (GC-2010 Plus, Shimadzu Scientific Instruments, Columbia, MD) using a Shimadzu SH-Rxi-5Sil MS column (30 m × 0.25 mm ID × 0.25 DF) attached to a mass spectrometer (GCMS-Q P2010) system operated using helium as carrier (1 ml min⁻¹). The chromatograph was operated at a split ratio of 10 and the program was set at 50°C for 1 min followed by ramping to 300°C at 20°C min⁻¹ and finally maintained at 300°C for 10 min. Released products of S and G origin were identified on the basis of their mass spectra using the NIST08 mass spectrum library and quantified from the chromatogram using the peak area.

Free formaldehyde in GCS reaction mixtures was derivatized with 2,4-dinitrophenylhydrazine (2,4-DNPH) to form 2,4-dinitrophenylhydrazone, before quantified by HPLC (Fig. 2). To this end, 0.2 mL of a reaction mixture was mixed with 0.2 mL of 10% (v/v) trifluoroacetic acid (TFA), 0.1 mL of 2,4-DNPH (1 g/L) and 0.5 mL of acetonitrile. Derivatization occurred at 40 °C for 30 min. The 2,4-dinitrophenylhydrazone derivative was analyzed using a Wondasil C₁₈ column (4.6 mm × 150 mm, 5 μm, Shimadzu, Japan) with acetonitrile/water (50,50) containing 0.095% TFA as the mobile phase at a flow rate of 1.0 mL/min, and detected at the wavelength of 352 nm.
Fig. 2

HPLC analysis of formaldehyde from different samples. a 1 mM formaldehyde standard. b GCS reaction mixture after 2 h. c GCS reaction mixture without adding GCS enzymes after 2 h

1,3-PDO was analyzed by GC-MS as described in Wang et al. using a QP2020 system (Shimadzu, Japan) equipped with a SH-Rxi-5Sil-MS column (Shimadzu, Japan), with helium as the carrier gas. The oven temperature was programmed to be held at 100 °C for 2 min, raised at a gradient of 15 °C min^− 1 to 270 °C and held for 12 min at 270 °C.

The bio-catalytic system contained 10 µM P450_BsβHI enzyme (or its variants), 500 µM fatty acid substrate (from a 100 mM stock solution in ethanol) and 1 mM H₂O₂ in a final volume of 1 mL of 100 mM potassium phosphate buffer (pH = 8.0). The reaction was carried out at 30 ℃ for 2 h. After reaction, 200 µM of the tridecene was added as internal standard compound (particularly, undecene was used as internal standard when using myristic acid as the substrate), then additional 50 µL of 6 M HCl was used for reaction quenching. The mixture was extracted by 800 μL hexane. Following extraction, the alkene products were analyzed by gas chromatography–mass spectrometry (GC–MS-QP2020, Shimadzu, Japan) equipped with a Sh-Rxi-5Sil-Ms column (Shimadzu, Japan) using helium as carrier gas. In particular, for detection of hydroxyl fatty acid products, the extracted samples were additionally derivatized with 100 μL N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) at 50 ℃ for 2 h before GC–MS analysis. The oven temperature was controlled initially at 50 ℃ for 2 min, then increased at the rate of 10 ℃ min⁻¹ to 280 ℃, and held for 10 min. The injecting temperature was 280 ℃. The concentration of alkene and hydroxyl fatty acid products was calculated by calibration curves with internal standards (Additional file 1: Figure S5).