Single-Standard Quantification Strategy for Lignin Dimers by Supercritical Fluid Chromatography with Charged Aerosol Detection

Abstract

The increased interest in utilizing lignin as a feedstock to produce various aromatic compounds requires advanced chemical analysis methods to provide qualitative and quantitative characterization of lignin samples along different technology streamlines. However, due to the lack of commercially available chemical standards, routine quantification of industrially relevant lignin oligomers in complex lignin samples remains a challenge. This study presents a novel method for universal quantification of lignin dimers based on supercritical fluid chromatography with charged aerosol detection (CAD). A series of lignin-derived dimeric compounds that have been reported from reductive catalytic fractionation (RCF) were synthesized and used as standards. The applicability of using linear regression instead of quadratic calibration curves was evaluated over a concentration range of 15-125 mg/L, demonstrating that the former calibration method is as appropriate as the latter. The response factors of lignin dimeric compounds were compared to assess the uniformity of the CAD signal, revealing that the CAD response for the tested lignin dimers did not differ substantially. It was also found that the response factors were not dependent on the number of methoxy groups or linkage motifs, ultimately enabling the use of only one calibrant for these compounds. The importance of chromatographic peak resolution in CAD was stressed, and the use of a digital peak sharpening technique was adopted and applied to address this challenge. The developed method was verified and used for the quantification of lignin dimers in an oil obtained by a RCF of birch sawdust.

Figure 1

Structures of lignin reference materials synthesized and used in this study. D1: 3,3′-dimethoxy-5,5′-dipropylbiphenyl-2,2′-diol (dehydrodidihydroeugenol); D2: 4-[2-(4-hydroxy-3,5-dimethoxyphenyl)ethyl]-2,6-dimethoxyphenol; D3: 4,4′-(ethane-1,2-diyl)bis(2-methoxyphenol); D4: 4-(4-hydroxy-3-methoxyphenethyl)-2,6-dimethoxyphenol; D5: 5-[(3S,3aR,6S,6aR)-3-(1,3-benzodioxol-5-yl)-1,3,3a,4,6,6a-hexahydrofuro[3,4-c]furan-6-yl]-1,3-benzodioxole (sesamin); D6: 4-[(3S,3aR,6S,6aR)-6-(4-hydroxy-3-methoxyphenyl)-1,3,3a,4,6,6a-hexahydrofuro[3,4-c]furan-3-yl]-2-methoxyphenol (pinoresinol); D7: 4-[6-(4-hydroxy-3,5-dimethoxyphenyl)-1,3,3a,4,6,6a-hexahydrofuro[3,4-c]furan-3-yl]-2,6-dimethoxyphenol (syringaresinol); D8: (E)-4-(3-(hydroxymethyl)-5-(3-hydroxyprop-1-en-1-yl)-7-methoxy-2,3-dihydrobenzofuran-2-yl)-2-methoxyphenol; M1: 2-methoxy-4-[(E)-prop-1-enyl]phenol (isoeugenol); M2: 2-methoxy-4-propylphenol; M3: 4-(3-hydroxypropyl)-2-methoxyphenol (dihydroconiferyl alcohol); M4: (E)-2,6-dimethoxy-4-(prop-1-en-1-yl)phenol; M5: 2,6-dimethoxy-4-propylphenol; and M6: 4-(3-hydroxypropyl)-2,6-dimethoxyphenol.

Figure 2

Scheme of the SFC-DAD/CAD system. MeOH: methanol; DAD: diode array detector; BPR: backpressure regulator; and CAD: charged aerosol detector.

Figure 3

Gel permeation chromatogram of the birch sawdust oil. Column: Agilent PLGel 500 Å (300 × 7.5 mm) at 50 °C; eluent: THF (1 mL/min). Mass calibration was performed with linear polystyrene standards.

Figure 4

Comparison of peak resolution obtained using different column chemistries, as determined by the DAD (A) and CAD (B) signals. R_s = 0 was assigned when no shoulder was observed between two peaks. Resolutions were calculated from chromatograms with the best performing gradients for each column (see Supporting Information Figure S3 for the gradients).

Figure 5

Scaled effects of variables (F: mobile phase flow rate; T: mobile phase temperature; and p: backpressure) on the resolution of selected peak pairs in the CAD chromatogram. Insignificant effects were removed.

Figure 6

DAD at 210 nm, blank-corrected (A) and CAD (B) chromatogram of lignin phenolic standard mixture. In (B), the red chromatogram is the sharpened version of the original black one. Monomer concentrations: M2 (800 mg/L); M1 (200 mg/L); M5 (200 mg/L); M4 (800 mg/L); M3 (400 mg/L); and M6 (160 mg/L). All dimers are present at 100 mg/L concentration. Method: Torus 1-AA column (100 × 3 mm, 1.7 μm), BPR: 130 bar, column temperature: 30 °C. Mobile phase: liquid CO₂ (solvent A) in MeOH (solvent B), 1.25 mL/min. Gradient: 0–1 min: 2% B, 1–3.5 min: 2–12% B, 3.5–7 min: 12–20% B, 7–9.5 min: 20–35% B, and 9.5–10 min: 2% B. Makeup solvent: 5 mM NH₃ in MeOH, 0.85 mL/min.

Figure 7

Response factors for linear calibration curves of the tested lignin dimers. Error bars represent 95% confidence intervals (n = 3).

Figure 8

SFC/CAD chromatogram of the complete lignin oil (left), dimer fraction (right, black chromatogram), and monomer fraction (right, red chromatogram) after peak sharpening. M = monomer, D = dimer, and N = nonclassified compound.