Besides different functional groups, the molar-mass distribution of lignins and technical lignins is an analytical key parameter which significantly influences physicochemical properties and reactivity.

The lignin´s  molar mass is highly dependent on the source of lignin and the process of its extraction from lignocellulosic biomass, such as pulping conditions, further isolation and purification. The knowledge on lignin molar-mass distribution and dispersity are imperative for establishing sustainable biorefineries, elaboration of lignin depolymerization or fractionation approaches, monitoring of lignin quality and finally for its effective utilization in various applications.

A) Size exclusion chromatography

Size exclusion chromatography (SEC) represents the most important and advantageous technique for determination of the molar mass distribution of lignins, due to the ease of implementation and the large choice of commercially available chromatographic columns responding to most demands. There are different SEC systems, which are suitable for analysis of either water-soluble lignins, such as lignosulfonates, or lignins soluble in organic solvents, such as organosolv or kraft lignin (Ringena et al. 2006, Baumberger et al. 2007). The ALICE core facility offers a universal SEC methodology that can cope with all different lignin types in a single system, requiring no special lignin sample preparation or time-consuming derivatization pretreatment. Still, the method requires the lignin to be liberated from its liquor matrix by means of precipitation, ultrafiltration or other approaches. For the most accurate characterization an additional lignin purification by means of solvent extraction can be recommended.

In order to achieve a reliable and reproducible determination of the molar mass distributions of all types of lignins an appropriate set of detectors has to be applied. In addition to the conventional detection techniques, such as ultraviolet-visible (UV-Vis) and refractive index (RI), we apply the most advanced and accurate Multi-Angle Light Scattering (MALS), requiring no instrument calibration and providing absolute molar mass characterization (Zinovyev et al., 2018).

SEC-MALS analysis is performed using an Ultimate 3000 autosampler, column oven, UV detector (all Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a Dionex HPLC Pump Series P580 (Dionex Softron GmbH, Germering, Germany), Dawn HELEOS II MALS detectors with lasers operating at either 658 or 785 nm; and an Optilab T-rEX differential refractive index detector, λ = 633 nm (all Wyatt Technology, Santa Barbara, CA, USA). Both MALS detectors are equipped with 18 photodiodes at different measuring angles, with narrow band pass filters (±10 nm for the respective wavelength used, installed on every second photodiode).

Sample amount required: 20 mg.


Baumberger, S., Abaecherli, A., Fasching, M., Gellerstedt, G., Gosselink, R., Hortling, B., Li, J., Saake, B. & De Jong, E. (2007).
Molar mass determination of lignins by size-exclusion chromatography: towards standardisation of the method.
Holzforschung, 61(4), 459-468. doi: 10.1515/Hf.2007.074

Ringena, O., Lebioda, A., Lehnen, R., & Saake, B. (2006).
Size-exclusion chromatography of technical lignins in dimethyl sulfoxide/water and dimethylacetamide.
Journal of Chromatography A, 1102(1-2), 154-163. doi: 10.1016/j.chroma.2005.10.037

Zinovyev, G., Sulaeva, I., Podzimek, S., Rossner, D., Kilpelainen, I., Sumerskii, I., Rosenau, T., Potthast, A. (2018).
Getting Closer to Absolute Molar Masses of Technical Lignins.
Chemsuschem, 11(18), 3259-3268. doi: 10.1002/cssc.201801177

B) Ultra-performance liquid chromatography (Advanced polymer chromatography, APC)

When molar mass characterization of larger number of lignin samples is required, for example when monitoring processes, an ultra-performance liquid chromatography (UPLC) approach is recommended which uses on porous ethylene-bridged hybrid inorganic/organic columns. Under optimized instrument parameters and settings, the implementation of this advanced approach allows for a tenfold gain in analysis speed compared to the size exclusion chromatography method utilizing gel-packed columns (Sulaeva et al., 2017).

The UPLC system is comprised of an Acquity UPLC H-Class instrument (Waters Corp., Milford, MA, USA), consisting of UPLC pump, sample manager, column compartment, photodiode array (PDA) detector (280 nm), and RI detector.

Sample amount required: 10 mg.


Sulaeva, I., Zinovyev, G., Plankeele, J. M., Sumerskii, I., Rosenau, T., & Potthast, A. (2017).
Fast Track to Molar-Mass Distributions of Technical Lignins.
ChemSusChem, 10(3), 629-635. doi: 10.1002/cssc.201601517

C) Asymmetric Flow Field-Flow Fractionation

Asymmetric flow field-flow fractionation (AsFlFFF) represents a good alternative to the conventional size exclusion chromatography (SEC) technique. The absence of a stationary phase allows purification and molar mass analysis of non-purified industrial black liquors in one step, resulting in a drastically simplified procedure (Wahlund & Nilsson, 2012). AsFlFFF separation conditions have been carefully optimized and a Multi-Angle Light Scattering (MALS) detector equipped with 785 nm laser is used, so that the method is able to provide precise and absolute molar mass values of all lignin types (Sulaeva et al., 2019).

The following setup is applied for AsFlFFF analysis: Dionex DG-1210 online degasser; Agilent Technologies 1260 Infinity pump; Agilent Technologies G1367C autosampler; Wyatt Technology Eclipse AF4 separation system. The detectors: Knauer 101 UV detector at 280 nm, Wyatt Technology DAWN HELEOS II, (λ = 658 nm or λ = 785 nm laser) multi-angle light scattering detector with band-pass filters installed on all even numbered detectors, and a Wyatt TReX RI detector.

Sample amount required: 50 mg.


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

Wahlund, K.-G., & Nilsson, L. (2012).
Flow FFF – Basics and Key Applications.
In S. K. R. Williams & K. D. Caldwell (Eds.), Field-Flow Fractionation in Biopolymer Analysis (pp. 1-21). Vienna: Springer Vienna.