The phenolic and aliphatic hydroxyl groups in lignin are fundamental functionalities affecting the physical and chemical properties of lignins, such as reactivity in condensation reactions or antioxidative properties. Quantitative determination of hydroxyl moieties provides crucial information on the structure, the degree of lignin degradation vs. condensation, antioxidant activity, thermal and oxidative behavior, compatibility with other solvents or polymer matrices, as well as the reactivity of lignin in various reactions involved in the development of lignin-based applications.
Only a rather limited number of methods is able to precisely monitor the hydroxyl group content in lignin, especially in lignins from various biorefinery streams. We are offering several methods for hydroxy group quantification.
Hydroxyl groups by 31P NMR
Phosphitylation followed by quantitative 31P NMR spectroscopy of lignin enables the quantification of different types of hydroxyl groups, such as aliphatic, different phenolic and carboxyl groups (Balakshin & Capanema, 2015). Due to the high sensitivity of the 31P NMR the method is able to cope with very small sample amounts. Usually phosphorous derivatives possess limited stability which, in practice, represented a bottleneck when it came to high-throughput measurements. However, with appropriately selected derivatization reagents and internal standards as well as properly optimized derivatization and NMR conditions, the approach provides hitherto unparalleled precision, accuracy, and robustness (Kortner et al., 2015). The analysis is performed on a Bruker Avance II 400 NMR spectrometer, operating at 162 MHz (31P frequency).
Sample amount: 30 mg
Balakshin, M., & Capanema, E. (2015).
On the Quantification of Lignin Hydroxyl Groups with P-31 and C-13 Nmr Spectroscopy.
Journal of Wood Chemistry and Technology, 35(3), 220-237. doi: 10.1080/02773813.2014.928328
Korntner, P., Sumerskii, I., Bacher, M., Rosenau, T., & Potthast, A. (2015).
Characterization of technical lignins by NMR spectroscopy: optimization of functional group analysis by P-31 NMR spectroscopy.
olzforschung, 69(6), 807-814. doi: 10.1515/hf-2014-0281
Hydroxyl groups by 1H NMR after peracetylation
If only information on the total amount of aliphatic and aromatic hydroxy groups is required, the 1H NMR of peracetylated lignins provides reproducible results combined with good long-term sample stability (Lundquist, 1992). In this case, a relatively long sample preparation (exhaustive acetylation) which may take up to 10 days, has to be taken into account. Due to the overall simplicity and fast NMR acquisition, the method offers the potential for analysis of large sample numbers. The analysis is performed on a Bruker Avance II 400 NMR spectrometer, operating at 400.13 MHz (1H frequency).
Sample amount required: 30 mg.
Lundquist, K. (1992).
Proton (1H) NMR Spectroscopy.
In S. Y. Lin & C. W. Dence (Eds.), Methods in Lignin Chemistry (pp. 242-249). Berlin, Heidelberg: Springer Berlin Heidelberg.
Hydroxyl groups by methylation headspace isotope-dilution GC-MS
A brand-new method for the fast and precise determination of total hydroxyl groups in lignins involves a quantitative methylation, followed by accurate and reproducible robotic lignin isolation and purification with subsequent lignin demethylation and headspace-isotope dilution GC-MS analysis. The methoxy groups present in the original sample serve as the internal standard, which renders the method predestined to be applied in tandem with the lignin methoxy group determination. The high robustness of the method was achieved by the implementation of the isotope dilution approach, which allows compensating for any error or inaccuracy. The method provides the overall hydroxyl group content, i.e. it cannot differentiate between different hydroxyl groups present in the lignin structure. It requires just a few milligrams of the sample and possesses high robustness and sufficient high-throughput features which positively distinguishes it from alternative methods.
The analysis is performed on an Agilent 6890N gas chromatograph equipped with an Agilent 7697A headspace autosampler and coupled with an Agilent 5975B inert XL mass selective detector.
Sample amount required: 20 mg.