Natural lignin, as contained in plants, is fundamentally altered with regard to its chemical structure and properties upon pulping of wood or other biomass processing (Berlin and Balakshin, 2014). The products of these conversions, technical lignins, have only rather limited resemblance to native lignins – this is why the distinction between technical and native lignins is made. Technical lignins need to be retrieved from complex products mixtures, so-called “black liquors”, which also contain other components, such as process chemicals, salts and other inorganics, degradation products from lignin and carbohydrates (Niemelä and Alén, 1999).

Lignin isolation from their production matrices reduces or eliminates interference from non-lignin constituents and thus plays a crucial role in lignin characterization. ALICE offers several approaches for the isolation and purification of lignin from spent pulping liquors generated in the kraft and sulfite pulping processes as well as from other biorefinery streams: precipitation, ultrafiltration and accelerated solvent extraction.


1) Berlin, A. & Balakshin, M. (2014) Industrial Lignins: Analysis, Properties, and Applications. In: Bioenergy Research: Advances and Applications, Chapter 18, pp. 315-336. doi: 10.1016/B978-0-444-59561-4.00018-8

2) Niemelä, K. & Alén, R. (1999) Characterization of Pulping Liquors. In: Analytical Methods in Wood Chemistry, Pulping, and Papermaking. Sjöström, E. & Alen, R. (eds.). Berlin, Heidelberg: Springer Berlin Heidelberg, pp. 193-231. doi: 10.1007/978-3-662-03898-7_7

1) Lignin isolation

A) Lignin isolation by precipitation

Kraft lignin is usually recovered from black liquor in the laboratory and on an industrial scale by acid precipitation and heat coagulation (Zhu and Theliander, 2015, Sewring et al., 2019). The isolation procedure consists of three steps: acidification, filtration, and washing. Acidification of black liquor is done with a mineral acid, such as sulfuric or hydrochloric acid, under intensive mixing. The filter cake generated upon filtration is exhaustively washed with deionized water until the residual ash content of the isolated kraft lignin is reduced to less than 2%.


  1. Sewring, T., Durruty, J., Schneider, L., Schneider, H., Mattsson, T. & Theliander, H. (2019) Acid Precipitation of Kraft Lignin from Aqueous Solutions: The Influence of pH, Temperature, and Xylan. J. Wood Chem. Tech., 39, 1-13. doi: 10.1080/02773813.2018.1488870
  2. Zhu, W. & Theliander, H. (2015) Precipitation of Lignin from Softwood Black Liquor: An Investigation of the Equilibrium and Molecular Properties of Lignin. BioResources, 10. doi: 10.15376/biores.10.1.1696-171

B) Lignin isolation by ultrafiltration or adsorption methods

The development of semipermeable membranes has led to a wide use of ultrafiltration (UF) in many fields, including, the isolation and fractionation of technical lignins (Zinovyev et al., 2017, Sulaeva et al., 2019, Sevastyanova et al., 2014). UF also allows for the quantitative isolation of lignosulfonates with high purity (Sumerskii et al., 2015).

The application of membranes with different molecular weight cut-offs provides selected lignin fractions with low dispersity, which are of interest both for analytical purposes and in development of lignin applications.

ALICE core facility offers analytical and preparative scale lignin purification by means of UF with molecular-weight cut-off of 1, 3, 5, 10, 30 and 100 kDa.

Additionally, ALICE offers a novel approach for isolation of lignosulfonates from spent sulphite liquor by an adsorption method (Sumerskii et al., 2015). We offer this method in three variants: small analytical scale, preparative scale, and large preparative scale, allowing to isolate up to 1 gram, 10 grams, and 100 grams of purified lignosulfonate, respectively.


  1. Sevastyanova, O., Helander, M., Chowdhury, S., Lange, H., Wedin, H., Zhang, L., Ek, M., Kadla, J. F., Crestini, C. & Lindström, M. E. (2014) Tailoring the molecular and thermo–mechanical properties of kraft lignin by ultrafiltration. Journal of Applied Polymer Science, 131, 40799. doi: 10.1002/app.40799
  2. Sulaeva, I., Vejdovszky, P., Henniges, U., Mahler, A. K., Rosenau, T., & Potthast, A. (2019). Molar Mass Characterization of Crude Lignosulfonates by Asymmetric Flow Field-Flow Fractionation. ACS Sustainable Chemistry & Engineering, 7(1), 216-223. doi: 10.1021/acssuschemeng.8b02856
  3. Sumerskii, I., Korntner, P., Zinovyev, G., Rosenau, T., & Potthast, A. (2015). Fast track for quantitative isolation of lignosulfonates from spent sulfite liquors. Rsc Advances, 5(112), 92732-92742. doi:10.1039/c5ra14080c
  4. Zinovyev, G., Sumerskii, I., Korntner, P., Sulaeva, I., Rosenau, T., & Potthast, A. (2017). Molar mass-dependent profiles of functional groups and carbohydrates in kraft lignin. Journal of Wood Chemistry and Technology, 37(3), 171-183. doi: 10.1080/02773813.2016.1253103

C) Lignin isolation by accelerated solvent extraction (ASE)

Lignin isolation by means of precipitation, ultrafiltration or adsorption methods may not guarantee absolute purity of the final sample. Quite often lignins are still contaminated with non-lignin impurities, such as extractives, carbohydrates, tannins, or lignin degradation products (Brunow et al., 1999), which may interfere with further analysis. Therefore, crude isolated lignin can additionally be purified by means of solvent extractions prior to lignin characterization for accurate quantitative data.

Conventional lignin purification is based on solvent extraction in a Soxhlet apparatus with different solvent systems. Beside this technique, ALICE provides a more efficient and flexible purification approach – in terms of process conditions and high throughput – by means of a Dionex™ ASE™ 350 Accelerated Solvent Extractor (Thurbide et al., 2000).

Required sample amount: 1-2 g


  1. Brunow, G., Lundquist, K., & Gellerstedt, G. (1999). Lignin. In E. Sjöström & R. Alén (Eds.), Analytical Methods in Wood Chemistry, Pulping, and Papermaking (pp. 77-124). Berlin, Heidelberg: Springer Berlin Heidelberg.
  2. Thurbide, K. B., & Hughes, D. M. (2000). A rapid method for determining the extractives content of wood pulp. Industrial & Engineering Chemistry Research, 39(8), 3112-3115. doi: 10.1021/ie0003178

2) Total dry solids in biorefinery process liquors

As simple as the solid content determination in various biorefinery streams by means of conventional drying might appear at a first glance, there are major pitfalls and a significant risk or errors. Usually the solid content is determined by drying a sufficiently large amount of sample to constant weight in an oven at 105 ± 3°C. However, most lignocellulosic materials and products from biorefinery streams are comprised of quite reactive compounds and/or process chemicals, such as bases or acids, which inevitably bring about reactions, in particular when exposed to air, and inaccurate results.

Within the ALICE core facility, we offer an optimized and very accurate approach for the determination of solids content and for dry sample preparation for further analysis in general. The drying process is conducted by means of freeze-drying and/or vacuum oven drying.

3) Lignin content in biorefinery process liquors

The reliable determination of the lignin content in lignocellulosic materials or biorefinery streams is far from being a trivial task. This analysis is performed for characterizing lignocellulosic materials, for assessing the effects of chemical, physical, and biological treatments of wood and pulp, for monitoring effluents in wood-processing biorefineries, etc. There are several methods for lignin determination. We offer Klason lignin determination and acetyl bromide lignin determination (Dence, 1992, Iiyama & Wallis, 1988). Klason total lignin content of biomass or pulps is estimated from the sum of the acid-insoluble and acid-soluble lignin components obtained after digesting the samples with sulfuric acid. The acid-insoluble and acid-soluble constituents are determined by gravimetric and UV- techniques, respectively. 

Alternatively, lignin can be quantified by measurement of UV absorbance of wood and wood pulp samples after complete digestion and dissolution in acetyl bromide/acetic acid mixture. In contrast to the Klason approach, the acetyl bromide method requires much smaller amounts.

Sample amount needed: 100 mg.


  1. Dence, C. W. (1992). The Determination of Lignin. In S. Y. Lin & C. W. Dence (Eds.), Methods in Lignin Chemistry (pp. 33-61). Berlin, Heidelberg: Springer Berlin Heidelberg.
  2. Iiyama, K., & Wallis, A. F. A. (1988). An Improved Acetyl Bromide Procedure for Determining Lignin in Woods and Wood Pulps. Wood Science and Technology, 22(3), 271-280. doi: Doi 10.1007/Bf00386022

4) Carbohydrate components in lignins

A) Methanolysis

Hemicellulose is the second most abundant polysaccharide constituent of lignocellulosic biomass, which is present in almost all terrestrial plant cell walls and also found as the major chemical “impurity” in technical lignins (Vuorinen & Alén, 1999). Hemicellulose is an amorphous polymer with a much lower degree of polymerization compared to cellulose which is therefore more susceptible to hydrothermal extraction and/or hydrolysis. This is also the reason why hemicellulose and its degradation products accumulate in different biorefinery streams, effluents, or black liquors. Hemicelluloses usually accompany isolated lignins, no matter whether from a laboratory preparation or an industrial process.

Depending on the source, hemicelluloses are comprised of D–xylose, D–glucose, D–galactose, D–mannose, L–arabinose, and D–glucuronic acid to name but a few major monosaccharidic constituents. The composition and structure of hemicelluloses varies significantly depending on the origin of the lignocellulosic biomass and the conditions of its processing. Knowledge of the hemicellulose content and composition provides information about raw materials, processing conditions, product composition or side stream composition. Moreover, monitoring and optimization of biomass processing based on chemical changes of the hemicellulose fraction is possible. 

ALICE core facility offers an accurate and robust determination of the hemicellulose content and the hemicellulose composition (monosaccharide analysis) by means of methanolysis (Sundberg et al. 1996). The protocol requires just milligram quantities of dry sample, which are digested under carefully optimized conditions in an anhydrous methanolic solution of hydrochloric acid, fowwoed by GC-MS-FID quantification of the liberated methyl glycosides. The method has a high selectivity towards hemicelluloses, but covers also the amorphous parts of celluloses. The advantage of methanolysis ocer acidic hydrolysis is the preservation and quantification of uronic acids, which are otherwise degraded under total hydrolysis conditions.

Sample amount required: 100 mg.


  1. Sundberg, A., Sundberg, K., Lillandt, C., & Holmhom, B. (1996). Determination of hemicelluloses and pectins in wood and pulp fibres by acid methanolysis and gas chromatography. 11(4), 216-2019. doi: 10.3183/npprj-1996-11-04-p216-219
  2. Vuorinen, T., & Alén, R. (1999). Carbohydrates. In E. Sjöström & R. Alén (Eds.), Analytical Methods in Wood Chemistry, Pulping, and Papermaking (pp. 37-75). Berlin, Heidelberg: Springer Berlin Heidelberg.

B) Fully automated high-performance thin layer chromatography (HPTLC)

Biorefinery streams quite often contain some amounts of carbohydrates and their degradation products with low degree of polymerization. When those compounds need to be characterized, high-performance liquid chromatography (HPLC) is the traditional method of choice which however, soon comes to its limits when encountering the overly complex product mixtures typical of biorefineries. ALICE core facility offers a much better solution for the characterization of low molecular weight carbohydrates and carbohydrate degradation products, namely high-performance thin layer chromatography (HPTLC), in a fully automated setup. The method is much faster than HPLC or HPLC-derived approaches, it offers the same accuracy and precision, and – most importantly - it is largely insensitive towards (insoluble and insoluble) impurutues and thus does not require sophisticated sample purification (Oberlerchner et al., 2018). Matrix compounds, which are usually a major part of biomass, do not interfere with the analysis. The method is highly selective, has good repeatability and has limits of detection and quantification in the nanogram range. Therefore, also common minor compounds of biorefinery streams – such as glucuronic acid, galacturonic acid, rhamnose, cellobiose and hydroxymethylfurfural – can easily be determined. The visual fingerprint offers information on the samples’ constituents in the case of unknown samples. Also, hydrolysis of biomass can be followed by the oligosaccharide patterns.

Sample amount required: 200 mg.


  1. Oberlerchner, J. T., Böhmdorfer, S., Rosenau, T., & Potthast, A. (2018). A matrix-resistant HPTLC method to quantify monosaccharides in wood-based lignocellulose biorefinery streams. Holzforschung, 72(8), 645-652. doi: 10.1515/hf-2017-0170