Molar Mass Characterization of Crude Lignosulfonates by Asymmetric

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Molar Mass Characterization of Crude Lignosulfonates by Asymmetric Flow Field-Flow Fractionation Irina Sulaeva, Philipp Vejdovszky, Ute Henniges, Arnulf Kai Mahler, Thomas Rosenau, and Antje Potthast ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02856 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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ACS Sustainable Chemistry & Engineering

Molar Mass Characterization of Crude Lignosulfonates by Asymmetric Flow Field-Flow Fractionation Irina Sulaeva†, Philipp Vejdovszky†, Ute Henniges†, Arnulf Kai Mahler‡, Thomas Rosenau†, Antje Potthast†* †

Department of Chemistry, Division of Chemistry of Renewable Resources, University of

Natural Resources and Life Sciences, Konrad-Lorenz-Str. 24, 3430 Tulln, Austria ‡

SAPPI Europe, Gratkorn Mill, SAPPI Paper Holding, Brucker Str. 21, A-8101 Gratkorn,

Austria * [email protected]

ABSTRACT Lignosulfonates (LS) are the main component of the by-product of sulfite pulping processes accumulated in large quantities of hundreds of kilotons per year. Furthermore, they are currently the most widely utilized form of lignin due to their chemical and physical properties. LS molar mass characteristics determine their functional properties; however, there is a lack of fast and precise methods for LS molar mass analysis. The complex LS structure after pulping as well as the presence of considerable amounts of impurities in the raw sulfite spent liquor require tedious

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and time-consuming sample purification steps if conventional size exclusion chromatography (SEC) is used for analysis. Asymmetric flow field-flow fractionation (AsFlFFF) represents a good alternative to the conventional SEC technique, as 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. The method´s applicability was tested using various industrial lignin samples. The results obtained demonstrated that the AsFlFFF approach in combination with multi-angle laser light scattering (MALLS) detection is an efficient tool for rapid screening of the molar mass of technical LS. The essential parameters of the AsFlFFF/MALLS approach are presented in this paper. For chromatographic setups that do not have a MALLS detection available, a calibration approach is provided.

KEYWORDS: industrial lignin, black liquor, molar mass, asymmetric flow field-flow fractionation, multi-angle laser light scattering

INTRODUCTION Worldwide, the pulp and paper industry utilize various chemical methods to separate the polysaccharide components of wood from lignin to convert different lignocellulosic materials into cellulosic fibers. As a consequence, large quantities of spent liquor with lignin being the major by-product are generated.1 The lion´s share of these industrial spent liquors is converted into steam and energy. However, the current interest in a wider utilization of renewable biomass broke new ground in the development of novel lignin utilization pathways.2-4 Depending on the pulp processing conditions, the structure of the lignin obtained differs, affecting its chemical and physical properties. The most commonly used industrial delignification procedures are based on the kraft process operating under highly alkaline conditions and, to a much smaller extent, the sulfite process, which utilizes sulfite ions at different pH levels.5 As a result, kraft or lignosulfonate (LS) lignins are generated, respectively. When organic solvents are used in pulping, organosolv lignins are produced.5 Although the kraft process dominates the overall production of pulp worldwide, LS are the most widely utilized form of lignin concerning the production of value-added products.6 The broad spectrum of its commercial applications


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originates from its structural characteristics and, in particular, from the presence of sulfonic acid groups, which ensure solubility in water and thus provide unique colloidal properties.7 Great progress in understanding the basic lignin structure has been made in the last decades. The development of novel analytical protocols for lignin characterization, which include the implementation of GC-MS,8, 9 FTIR,10 one dimensional (1H, 13C, 31P),11-13 and multidimensional (1H-13C HSQC) NMR14-16 methods and allow for a routine analysis of lignin structural peculiarities, including the determination of substructures in lignin, typical linkages, and end groups. Less progress, however, was achieved in the field of lignin molar mass characterization, where conventional approaches, such as size exclusion chromatography (SEC), are still the predominantly used ones.17 Even the basic question related to the molar mass of natural lignin remains unanswered so far. Various studies suggest that the molar mass values differ by several orders of magnitude.18, 19 When it comes to technical lignin samples, the knowledge of molar mass parameters is important, as the structure and molar mass are altered during pulping depending on the conditions applied.18 However, the development of novel lignin utilization pathways requires the simultaneous development of fast and precise techniques for the characterization of molar masses, as some material applications favor the utilization of lignins with specific and narrow molar mass distribution ranges, and others may require a broader distribution.20 SEC remains as the most prevalent technique due to the ease of implementation and the large choice of commercially available columns responding to most demands. When it comes to the characterization of technical lignins, SEC based on calibration with commercially available standards is the method of choice.17 There are different SEC systems described in the literature, which are suitable for analysis either of water-soluble lignin samples, e.g., LS,21, 22 or for lignins soluble in organic solvents, like organosolv or kraft lignin.23,

24

Recently, we proposed a

universal SEC methodology that can cope with all different lignin types in a single system.25 Apart from that, it was shown that significantly improved system performance could be achieved when using porous ethylene-bridged hybrid inorganic/organic columns. Implementation of this advanced polymer chromatography (APC) approach allows for a tenfold gain in analysis speed compared to the conventional SEC method utilizing gel-packed columns.25


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However, the actual time limiting step in lignin SEC characterization is the preliminary sample preparation, which is particularly true for lignosulfonates. Industrial spent LS liquors contain (apart from lignin) large amounts of impurities, which usually account for around 50% of its total mass. These impurities include inorganic salts utilized during processing, as well as degraded organic wood components, sugar monomers, and oligomers and phenolic extractives.26,

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Analysis by SEC requires lignin isolation from this mixture due to the menace of column damage and system contamination when unpurified samples are injected. Lignin isolation is usually performed by ultrafiltration (UF) or adsorption, and both require chemicals, equipment, and time.28 The lack of approaches for lignin molar mass characterization, which are fast to implement, provide an incentive to develop an alternative system that would not require sample pre-cleaning steps and would be easy to establish at industrial sites and scientific laboratories. To find such an analytical method, we tested the asymmetric flow field-flow fractionation (AsFlFFF) as an alternative to the conventional SEC approach. In case of AsFlFFF, the separation occurs in an empty narrow channel without a stationary phase, which reduces the possible risk of nonspecific interactions to a minimum.29 This can be particularly beneficial when it comes to the direct analysis of industrial liquors, as it may help to avoid their preliminary purification. Moreover, the low molar mass impurities, in this case, can be isolated directly in the channel during sample analysis. To achieve this, the system parameters should be adjusted allowing the impurities to penetrate through the permeable membrane; meanwhile, the analyte passes the separation channel being purified from the other substances. Should this approach be feasible, it will lead to a significantly simplified sample preparation procedure as sample purification and molar mass analysis happen almost simultaneously. A black liquor sample from, e.g., the sulfite process could directly be injected without any lignin isolation step. To realize this approach, the optimization of the AsFlFFF separation conditions is the most challenging part of establishing an efficient measuring protocol. The variety of critical parameters prone to alteration includes the adjustment of separation flows (detector, focusing, and cross-flows), as well as the selection of a suitable membrane and eluent.30 These challenges relate to finding a proper measuring procedure applicable to the different lignin types that have been addressed in this account.


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Another question is linked to the selection of a suitable sample detection technique, which is certainly required for lignin molar mass characterization after its separation. The autofluorescence problem is a well-known issue in lignin analysis, which hampers its characterization by different techniques, including multi-angle laser light scattering (MALLS).31 While MALLS analysis is the most common approach for the absolute molar-mass determination of polymers, it often fails when it comes to lignin characterization, as the fluorescence adds to the detected scattered light and results in incorrectly calculated molar masses.19 Compared to other lignins, LS are the least troublesome samples due to the reduced amount of fluorescence inducing functionalities.32 Many of them are suitable for direct characterization using MALLS enhanced with band-pass filters. However, in the case of other technical lignins, the filters are incapable of eliminating the occurring fluorescence, and therefore the method is not suitable for the characterization of the better part of technical lignin samples.19 In case of SEC, the problem is normally solved by implementing a calibration with commercially available narrow standards. However, in the case of AsFlFFF, the calibration represents a significantly more complicated procedure. In contrast to SEC, where the sample retention time is determined by a single system flow, the sample elution behavior in the case of AsFlFFF is affected by a combination of flows, and minor deviations in the flow rates sum up in higher deviations in the elution patterns of the samples. The applicability of calibration in AsFlFFF analysis and the reproducibility of the obtained results were thoroughly investigated in this study as well.

MATERIALS AND METHODS Chemicals and lignin samples The following chemicals were obtained from Sigma-Aldrich Handels-GmbH, Schnelldorf, Germany: sodium chloride (99.5%), lignosulfonic acid sodium salt (average Mw ~ 54000, Mn ~ 6000 g/mol), and lignosulfonic acid acetate sodium salt. Polystyrene sulfonate (PSS) sodium salt standards were purchased from Polymer Standard Service (PSS, Mainz, Germany). Technical lignin samples were either received from industrial partners in the form of unpurified liquor from commercial pulping processes or present as isolated LS. Kraft lignin was supplied by MeadWestvaco Corp. (Glen Allen, VA, USA); LS from different sulfite processes - Ammonia


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LS (Borregaard, Norway), Magnesium LS (Lenzing AG, Austria), Calcium LS (Lignotech, South Africa). Instrumental set up for AsFlFFF analysis The following setup was 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. Separation was accomplished in a long (275 mm) separation channel with a 350 µm spacer. The membranes used for separation were obtained either from Wyatt Technology: regenerated cellulose (RC) 2 kDa, RC 5 kDa, RC 10 kDa; polyether sulfone (PES) 1 kDa, PES 5 kDa, PES 10 kDa, and PES 30 kDa; or from Merck KGaA, Darmstadt, Germany: RC Ultracel 1Da and RC Ultracel 3Da. The membranes from Merck were obtained in the form of square sheets (20x20 cm) and were manually precut to fit the AsFlFFF separation channel. The mobile phase was 0.1M NaCl (+250 ppm NaN3). All technical lignin samples were injected without additional purification or filtration unless otherwise stated. Sample recovery was determined by the analysis of the amount of the sample eluted from the channel after the standard separation procedure. A 100 µl probe of lignin sample (20 mg/ml) was injected into a channel and further collected in a 50 ml volumetric flask. The actual sample concentration was analyzed by UV–vis absorption spectra using a Perkin Elmer Lambda 35 UV–vis spectrophotometer and an external calibration curve. Separation conditions used for AsFlFFF/MALLS analysis An analysis was performed using 10 kDa PES membranes purchased from Wyatt Technology Corporation. To ensure a decent separation, we applied a gradual decrease of the cross-flow for 20 min from 4 to 0.1 ml/min followed by a 10 min channel flushing with a constant cross-flow of 0.1 ml/min. A subsequent washing step without any cross-flow was performed for 5 min to ensure the complete sample elution from the channel. Other system parameters: 100 µl injection volume; 1 mL/min detector flow; 0.2 ml/min injection flow; 5 min total focusing time (including


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2 min injection); 40 min total run time. Data evaluation was performed with Astra 6.1 software (Wyatt Technology, US). Determination of lignin dn/dc values The analysis of (dn/dc)µ values of lignin samples in 0.1M NaCl was performed using the 100% sample mass recovery approach. The samples were chromatographed on three GPC columns (300 mm × 7.5 mm Agilent-OH Mixed-H columns; pore size 5µm), and the (dn/dc)µ was measured by integrating the DRI peak area after sample elution from the columns using Astra 6.1 software (Wyatt Technology, US). To assure the accuracy of the measurements, the injection volume of the autosampler, as well as the actual sample recovery from the columns, were preliminary determined. The injection volume was controlled by multiple injections of Rhodamine B followed by the analysis of the eluted sample mass using offline UV measurements (Perkin Elmer Lambda 35) and an external calibration curve. Similarly, the sample recovery from the columns was controlled via offline UV measurements of the lignin samples collected after the separation. Separation conditions used for channel calibration Ultracel 3 kDa membranes from Merck KGaA were used for channel calibration. The cross-flow was kept constant at 2 ml/min for the first 5 min of elution, and afterward linearly decreased for 10 min until 0.1 ml/min. The channel was then flushed for 5 min at 0.1 ml/min cross-flow and an additional 5 min without cross flow. Other system parameters: 100 µl injection volume; 0.5 ml/min detector flow; 0.2 ml/min injection flow; 5 min total focusing time (including 2 min injection); 30 min total run time. In order to improve the data reproducibility, the calibration was repeated with every new sequence. Data evaluation was performed with Astra 6.1 software (Wyatt Technology, US).

RESULTS AND DISCUSSION For a few decades, AsFIFFF has been and still is considered a universal separation system which is easily applicable for routine analysis of various synthetic and biopolymers with several


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advantages over the conventional SEC approach.30 However, there is not a single study reported on AsFlFFF implementation for lignin characterization. The reason for this is the limited system flexibility when it comes to mobile phase selection. The AsFlFFF systems are easily adjustable to aqueous and mild organic solvents; however, they are not stable when more aggressive (e.g. salt containing) organic solvents are utilized, as the hardware itself or separation membranes may not be stable under these conditions. At the same time, most of the technical lignin samples require specific solvent systems to reach the desired solubilization. Currently, the most widely used solvents in lignin analysis are THF,17 DMSO/LiBr,25 and NaOH (0.1 M).33 The utilization of these solvents in SEC demands specific corrosion-stable hardware, which is not commercially available for AsFlFFF setups. In addition, the commercially available permeable membranes for the AsFlFFF channels are not stable for utilization with all solvents, as it would cause their swelling, partial hydrolysis, or even complete dissolution. Nevertheless, the AsFlFFF approach is suitable for the characterization of water-soluble LS. As most of the attainable LS are also accessible for MALLS characterization, the first trials in the AsFlFFF/MALLS method optimization were performed using these easy-to-handle samples. AsFlFFF/MALLS analysis of lignosulfonates: method optimization To achieve a satisfying sample recovery and a good separation, a careful optimization of the AsFlFFF separation parameters is essential. In case of lignin analysis, the membrane selection is particularly crucial, as the desired elimination of the low molar mass impurities should not be accompanied by the partial loss of lignin sample material. The most common commercially available membranes are made of RC and PES. PES membranes have the advantage of providing a good thermal and hydrolytic stability as well as a high mechanical resistance. However, numerous cases have been reported demonstrating the presence of physical effects, which are affecting the sample elution behavior. In this context, the most frequently reported cases are related to the fouling of the PES membrane caused by its hydrophobicity.34,

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especially pronounced in case of nonpolar solutes and hydrophobic particles, which tend to adhere to a hydrophobic surface.36-38 Nevertheless, the opposite effect of sample early elution has repeatedly been observed in case of charged molecules or nanoparticles occurring due to the electrostatic repulsion of the analyte with the negatively charged membrane surface.38,

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membranes are more hydrophilic and have a lower negative surface charge density compared to


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the PES membranes, but the mentioned sample/membrane interaction effects have also been reported in this case.40,

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Therefore, the choice of the membrane for AsFlFFF analysis is

normally determined by the particular properties of the analyte and the eluent. To optimize lignin recovery, we tested both types of membranes in this study. The analysis was carried out using 0.1 M NaCl to provide a sufficient ionic strength to the eluent and to reduce possible electrostatic interaction effects. To have a better overview of the recovery of low molar mass fractions, we used the narrow polystyrene sulfonate (PSS) standards for the membrane selection experiments. At first, the PES and RC membranes specified by the vendor as the specific membranes for AsFlFFF analysis have been tested in all available molecular weight cut-offs (MWCO). The elution profiles of the standards obtained for different membranes are shown in Figure 1.

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As can be seen, the separation efficiency in case of PES membranes is not ideal. A comparatively early elution was observed for the low molar mass standards even at the maximum possible cross-flow of 4 ml/min (Figure 1c). An early elution is often an indicator of the occurrence of electrostatic repulsion between the analyte and the membrane. Considering the presence of the negatively charged sulfonic acid groups in the PSS structure (which resembles the structure of LS), the assumption can be made that the electrostatic repulsion of the sample and the membrane indeed takes place. This conjecture was additionally confirmed when a 30 kDa PES membrane was used for analysis. The recovery of the low molar mass standards was rather high considering the large pore size of the membrane so that only the smallest standard with a molar mass below 1 kDa was penetrating the membrane (Figure 1d). Without the counteracting repulsion effects, a much higher loss of the low molar mass standards due to penetration through the membrane with the cross-flow would have been observed. Similar elution behavior was observed in case of lignin samples.In lignin ultrafiltration, RC membranes are known to induce weaker repulsion effects compared to PES membranes.42, 43 The same effect was observed when RC membranes were used for the AsFlFFF lignin analysis. To reduce the sample loss caused by its leakage from the channel, we reduced the cross-flow to 2 ml/min during the first trials. However, these precautions did not help to prevent poor recovery of the low molar mass fractions. Although surprisingly, the 5 kDa RC membrane demonstrated better performance compared to the 2 kDa membrane (Figure 1 a and b), none of the RC membranes purchased from the AsFlFFF instrumentation vendor can be used for lignin characterization. Therefore, despite the fact that PES membranes cause early elution of standards and lignin samples, the membranes of this type were utilized in the first trials of LS AsFlFFF/MALLS analysis. In this case, the repulsion effect can be considered as a positive feature, as it minimizes the lignin loss caused by sample penetration through the membrane. At the same time, the major impurities in the industrial spent liquors are salts and degraded sugars, which are due to their structural peculiarities easily passing through the membrane. Therefore, the separation on PES membranes assures an effective lignin purification and its good recovery. The utilization of MALLS in this case excludes the possible risk of molar mass miscalculation, as the detector provides the absolute molar mass values for every elution slice. To estimate the performance of both types of membranes regarding lignin recovery, we determined the actual concentration of the sample after passing the separation channel using


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offline UV measurements. The results of the analysis are shown in Table 1. As expected, the repulsion effects taking place when PES membranes are used for the analysis result in a minimal sample loss and close to 100% recovery. The RC membranes are not always suitable for lignin characterization, and the lignin recovery should be carefully determined in additional experiments prior to actual analysis.

Table 1. Analysis of lignin mass recovery from the AsFlFFF channel depending on the membrane type RC 5 kDa membrane PES 5 kDa membrane Lignin sample Injected Eluted Sample Injected Eluted Sample mass, mg mass, mg recovery, % mass, mg mass, mg recovery, % 1 1.00476 0.13761 13.7 1.00476 1.08541 108.0* 2 1.00476 0.12850 12.8 1.00476 1.08541 108.0* 3 1.00476 0.13761 13.7 1.00476 1.07623 107.1* 4 1.00476 0.12850 12.8 1.00476 1.08541 108.0* 5 1.00476 0.11939 11.9 1.00476 1.08541 108.0* * The recovery values of above 100% are related to the sum of errors originating from:  the fluctuation in the actual injection volumes of the sample performed by an autosampler, which affects the injected mass value;  possible miscalculations of the eluted mass due to the imperfection of the UV calibration curve.

AsFlFFF/MALLS analysis of lignosulfonates: results The first trials to analyze industrial LS samples were performed using a 10 kDa PES membrane. Although LS are known to exhibit a reduced fluorescence compared to other technical lignins, their characterization by means of MALLS is not straightforward. The MALLS data has to be critically evaluated for each particular sample in order to assure that the optical effects, such as absorption and fluorescence, are eliminated and do not affect the final results. The study of Zinovyev et al., provides a detailed description of the MALLS data evaluation procedure which has to be taken into account when performing this type of analysis.19 The adjusted AsFlFFF parameters which make use of the highest possible cross-flow (detailed description is provided in the materials and methods) resulted in a symmetrical peak with a good resolution (Figure 2a). To assure the absence of fluorescence effects impairing the analysis, we installed band-pass filters


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on half of the light acquisition detectors of the MALLS device. A comparison of data obtained when detectors with filters and without filters, respectively, were used showed a significant difference in the molar mass values. As expected, the signals were constantly stronger when filters were not applied compared to the signals generated when filters were in use, which is in accordance to the literature data.19 Therefore, the detectors equipped with filters were used for the analysis of unknown samples. Additionally, the data correction for the possible absorption effect was performed evaluating the scattered light intensity based on the intensity of the signal from forward monitor.19 The dn/dc values have been preliminarily determined for each LS sample using the 100% mass recovery approach. The example of the molecular weight distribution of the industrial calcium LS is presented in Figure 2b. Additionally, a kraft lignin sample was analyzed using the same approach. The solubility of the sample in 0.1 M NaCl was achieved after sample isolation by ultrafiltration. The results of the AsFlFFF analysis for all technical lignin samples is given in Table 2.

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Table 2. Results of the (dn/dc)µ analysis by SEC and AsFlFFF/MALLS molar mass measurements


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(dn/dc)µ* (ml/g) from MALLS-658 nm 100% mass recovery Mn, Da Mw, Da Sigma Sugared LS 0.2034 20000 43500 Sigma LS-NaAc 0.1929 5600 15200 Ammonia LS 0.2209 16200 56900 Magnesium LS 0.2148 8400 30400 Calcium LS 0.2217 5800 14800 Kraft lignin after UF 0.2286 61100 68400 * (dn/dc)µ was analyzed using a DRI operating at 658 nm Lignin sample

MALLS-785 nm Mn, Da Mw, Da 18100 48800 4400 16300 13500 49800 10200 41000 5800 15100 8100 13200

The established AsFlFFF/MALLS measuring protocol provides reliable results for all tested LS samples, which proves that the method represents a promising alternative to the standard SEC analysis as a fast, reliable, and precise tool for LS routine analysis without prior tedious sample preparation. The results obtained for kraft lignin are obviously overestimated, which is a common issue due to the high concentration of the fluorescence emitting units. Recent studies have demonstrated that the utilization of the MALLS detector equipped with a laser operating at 785 nm in lignin analysis helps to reduce the fluorescence input to a minimum and to provide accurate results. Additionally, these same precise results even show for samples with high fluorescence activity, such as kraft lignin, which cannot be analyzed using a standard MALLS detector with a 658 nm laser.19 The MALLS-785 nm was tested in the present study to compare the results derived from two MALLS devices and to estimate the possible error of utilization of MALLS-658 nm. The results of the measurements are presented in Table 2. The results obtained for LS samples are similar for both MALLS detectors. These samples can be analyzed using an ordinary MALLS-658 system due to their reduced fluorescence and do not necessarily require the use of the MALLS-785 nm. The utilization of MALLS-785 nm can help to expand the areas of AsFlFFF implementation making the system suitable not only for LS characterization but also for the analysis of other lignin types. Possibility to use calibration in AsFlFFF analysis Although most of the LS can be measured using the AsFlFFF/MALLS approach, channel calibration can still be useful for the analysis of some specific samples. An accurate channel calibration cannot be performed using PES membranes due to the sample repulsion effects affecting the elution behavior of both the lignin samples and standards used for calibration. At


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the same time, the MWCO range of the commercially available RC membranes specifically fabricated for the AsFlFFF channel is above the requirements for the small lignin molecules. To establish a decent calibration, we purchased the UF RC membranes (Ultracel 1 kDa and 3 kDa) in the form of 20x20 cm square pieces from Merck KGaA, manually pre-cut to fit the AsFlFFF channel, and tested them regarding their applicability for lignin analysis. The 1 kDa membrane was observed to create a high back pressure in the channel, which required the reduction of the flow rates to 0.5 ml/min for detector flow and 1.0 ml/min for the cross-flow. Such an operating regime was not capable of ensuring a satisfying sample separation. The issue was resolved by the implementation of a 3 kDa membrane. It allowed for an increase of the cross-flow to 2 ml/min at a constant detector flow of 0.5 ml/min. The elution behavior of the standards was not affected by the electrostatic repulsion effects, which resulted in a much more stable and reproducible calibration plot (Figure 3a). The established calibration was utilized to estimate the relative molar masses of the samples, which cannot be measured using the MALLS approach. As an example, the analysis of a kraft lignin sample is shown in Figure 3b.

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Figure 3. The elution profiles of the PSS standards obtained using Ultracel 3kDa membrane (a) and the implementation of the obtained calibration curve to the lignin molar mass calculation (b)

The established calibration using Ultracel RC membranes could be considered as a suitable approach, as the obtained molar mass value for the kraft lignin sample of 21000 g/mol is within a reasonable range. The additionally determined lignin recovery in the case of Ultracel RC


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membranes is 93%, which, considering the smaller size of kraft lignins compared to LS, is an appropriate value for routine analysis. At the same time, the use of calibration results in relative molar masses which are slightly overestimated compared to the absolute values obtained using MALLS-785 nm detection. Apparently, the reason for this overestimation is the loss of the low molar mass fraction due to its leakage through the utilized RC membrane, whereas such loss was minimized in case of PES membranes used during MALLS measurements. On the other hand, the derived values are significantly more reasonable than the results obtained from MALLS-658 nm, which makes the calibration approach a preferable option for samples exhibiting strong fluorescence and when specific MALLS instrumentation operating at a higher wavelength is not available.

CONCLUSIONS Based on the results obtained, a careful selection of appropriate and suitable AsFlFFF separation conditions for lignin analysis is strictly determined by the specific characteristics of the sample. First, one should ascertain whether or not the molar mass of the respective technical lignin sample can be measured by MALLS. If MALLS turns out to be applicable, i.e., in case of samples showing reduced fluorescence that can be eliminated by the band-pass filters, PES membranes in an AsFlFFF/MALLS setup is the best option. MALLS will provide absolute molar mass values, and PES 10 kDa will lead to a lower lignin loss through the membrane, whereas the low molar mass impurities will be directed to the waste flow during the sample run. In a situation, when MALLS cannot be applied, a channel calibration with narrow standards has to be performed. However, PES membranes cannot be used for calibration, as the elution times of the PSS standards and the lignin samples are determined not only by the basic AsFlFFF elution theory based on the size of the molecules but can vary depending on the amount of the sulfonic acid groups in the structure of the analyte. Considering that the sulfonic acid group content in LS varies from sample to sample, a unique elution profile will be observed for each lignin, making the channel calibration approach unreliable. Therefore, it could be shown that the RC membranes with a low MWCO are better suited for the channel calibration, as their


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utilization helps to prevent repulsion effects and thus uncontrollable elution of the standards and lignin samples. In addition, a variety of other critical AsFlFFF parameters, including the laminar and the crossflow rate, were thoroughly adjusted toward the ultimate goal of the best possible sample separation conditions in combination with a satisfying sample recovery. By using such a fine adjustment and considering the flow conditions in combination with the careful selection of a suitable membrane type, the method could be shown to have a good performance in the analysis of various industrial LS samples. It could be demonstrated that AsFlFFF is a powerful alternative to conventional SEC-approaches, as it allows for a direct injection of the samples and avoids the time-consuming sample isolation procedures while still providing equally reliable results.

AUTHOR INFORMATION Corresponding Author * [email protected] Note The authors declare no competing financial interest.

ACKNOWLEDGMENTS We would like to gratefully acknowledge the financial support of the “FLIPPR2” project supported by the Austrian Research Promotion Agency, FFG, and the associated companies SAPPI, MONDI, and Heinzel Pulp. Dr. Roessner and Wyatt Technology Europe, Dernbach, Germany are acknowledged for providing a MALLS-785 detector.

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For Table of Contents Use Only

SYNOPSIS AsFlFFF is an efficient approach that allows for the direct molar mass analysis of non-purified technical lignins directly from black liquors


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Molar Mass Characterization of Crude Lignosulfonates by Asymmetric

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