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A method for the determination of the sulfonic acid content in lignosulfonates via sulfur dioxide quantification upon treatment with H3PO4 by headspace gas chromatography is presented and compared to two already established alternatives, conductometric titration and elemental analysis. Several lignosulfonates, purified from various industrial sources, were examined by all three methods. Limitations and possible interference by other functional groups, such as carboxylic acids, are discussed, and suitable solutions are presented. Results of the novel approach are comparable to those of the established techniques in terms of accuracy and precision. The limit of detection (LOD) is 0.88 μmol of SO3H and the corresponding limit of quantification (LOQ) is 3.78 μmol of SO3H. At the same time, this method outperforms conductometric titration in terms of a higher sample throughput and a much smaller sample amount needed for analysis. Treatment of the sample with highly concentrated phosphoric acid and simple heati...

A novel method for the precise and accurate quantification of wood monosaccharides by gas-chromatography mass-spectrometry in complex lignocellulosic biomass matrices is presented. Instead of using the syn- or the anti-peak obtained by blocking the anomeric center using e.g. oximation, the sum of each syn- and anti-peak pair is used for quantification rendering the approach of the apparently constant syn- and anti-peak area ratio. Each wood monosaccharide syn- and anti-peak could essentially be distinguished upon O-ethoximation followed by trifluoroacetylation and separation on a 14% cyanopropyl/phenyl- 86% dimethylpoly-siloxane column. Additionally, internal standardization was carried out applying isotopically labeled compounds. Hence, the analytical figures of merit, such as precision and accuracy, could be substantially improved compared to standard gas-chromatography mass spectrometry as well as high-performance anion exchange chromatography (HPAEC) coupled to pulsed-amperometric detection (PAD). The applicability of the novel gas-chromatography mass-spectrometry approach is demonstrated by analysis of five selected lignocellulosic biomass sample matrices typically occurring in a biorefinery scenario. Even in exceedingly complex sample matrices precise and accurate data are obtained according to the methodology presented.

The detection of individual chromophores that contribute to the overall discoloration of paper or pulp ("yellowing") is a challenge because these substances are only present in extremely small amounts (ppm to ppb range). In this work, paper spray (PS) coupled with mass spectrometry was used to detect a low-concentrated cellulosic key-chromophore, 2,5-dihydroxyacetophenone (DHAP). Sensitivity was enhanced by derivatization with Girard's reagent T (GT). DHAP was successfully detected in historic paper samples and also was applied to model papers in order to investigate different factors that influence its generation: temperature, time, relative humidity, and the presence of iron ions, by means of a full factorial design. The main factors, temperature and relative humidity, have the most impact on the generation of DHAP, but the interactions between the factors are also significant and are therefore important for the degradation process. The historical papers containing DHAP were then compared to the artificially aged samples. The results were confirmed by independent, accurate mass measurements.

Solvents constitute a major portion of a chemical laboratory’s waste and cause immediate costs for proper disposal and reordering. Next to reducing solvent usage, the recycling of used solvents by distillation is an obvious measure. In practice, commercially available equipment for distillation often offers insufficient separation and is inappropriately sized, overly costly, or simply not solvent-resistant. We constructed and put into service two distillation units for 6 L batches to recover analytical grade solvents–ethyl acetate and toluene for preparative chromatography and N,N-dimethylacetamide for analytical size exclusion chromatography. The systems separate fractions automatically at freely programmable vapor temperatures with high accuracy (0.1 K). The recovered solvents are therefore highly pure and are reused for analytical applications without any problems. Common laboratory glassware and standard electric components were used for construction. All wetted parts of the distillation systems are s...

A method based on headspace-isotope dilution (HS-ID) GC-MS for the quantitative analysis of methoxyl and ethoxyl groups in any kind of lignin has been developed. The method involves the application of an isotopically labeled internal standard (4-(methoxy-d3)-benzoic acid and 4-(ethoxy-d5)-benzoic acid), which, together with lignin, undergoes standard hydroiodic acid cleavage of methoxyl and ethoxyl groups, followed by headspace GC-MS analysis of the respective deuterated and non-deuterated iodomethanes and iodoethanes. As also the internal standard is generated in situ during ether cleavage, any variations in the procedure are levelled out. The application of the isotopically labeled internal standard essentially increased the robustness of the overall approach. The accuracy and precision of methoxyl and ethoxyl group quantification in lignins have been assessed, and the data were compared to the classical Zeisel–Viebock–Schwappach method for methoxyl groups in lignins. The novel approach for methoxyl and ethoxyl group determination showed a more satisfactory precision, accuracy, and also a much higher sample throughput, with approximately 40 samples per day.

Biogas production often competes with food and feed production for the raw materials and cropland required for cultivation. Common reed offers an alternative source of biomass for biogas production, alleviating this conflict. Effective microbiological conversion of this type of lignocellulosic biomass requires a pretreatment process. This study aims to determine the specific methane yields of steam-exploded reed as well as to identify how pretreatment conditions influence its physico-chemical characteristics. For this purpose, reed was pretreated with steam explosion at severity factors ranging from 2.47 to 4.83. The effects on methane yields were analyzed in batch experiments. Scanning electron microscopy (SEM) images were captured and detailed chemical analyses of the substrates carried out. Results show that the digestibility of reed biomass improved remarkably after pretreatment. Compared to the untreated sample, steam explosion increased the specific methane yield up to 89% after pretreatment at 200 °C for 15 min. However, methane yield decreased under harsher conditions, which may be due to the formation of degradation compounds during the pretreatment.

The detection of individual chromophores that contribute to the overall discoloration of paper ("yellowing") is a challenge because those substances are only present in very small amounts. In this research, two analytical approaches based on ambient ionization techniques, namely, desorption electrospray ionization and paper spray, both coupled to mass spectrometry, are compared to each other with regard to their suitability for detecting acetylated cellulosic key chromophores. The paper spray approach proved to be the more sensitive and versatile method. Subsequently, paper spray (PS)-mass spectrometry was applied to model papers and historical papers in which the acetylated chromophores were detected successfully. Independent accurate mass measurements confirmed the results obtained from reference compounds, model samples, and real-world specimens.

When a fire strikes libraries or archives, physical deterioration of the paper is so severe that its chemical properties are often regarded as less important. However, knowledge of the chemical changes of the papers upon nearly burning is necessary to design a subsequent conservation treatment. In the present study, we have examined a rag paper object, which partially survived a fire, and analysed its chemical properties by various methods. The polymeric state of cellulose, as well as its low-molar mass degradation products, were assessed. Comparison to an identical, unharmed duplicate provided a more profound understanding of the changes caused by the fire. Light scattering analysis revealed conformational changes of the cellulose molecule after high-temperature impact, and a chemical cross-linking was observed. In our study, we found the integrity of cellulose to depend on the temperature profile induced by the fire. The low thermal conductivity of cellulose protects the material even in close proximity to the burned edges.

The binding mechanisms of two reactive sizing agents, alkenyl succinic anhydride (ASA) and maleated sunflower oil high-oleic (MSOHO), with cellulose were studied. While ASA is produced from olefins out of fossil resources, MSOHO is a green sizing agent based on renewable plant materials. In contrast to common assumptions, that ASA is mostly covalently bound to cellulose, this study showed the largest part of ASA to be bound only by physisorption and only a rather small fraction, typically about 0.5 %, to be covalently attached by ester bonds. In the case of MSOHO, the covalent binding was only slightly higher, with about 3 % of the total amount being covalently linked. In both cases, covalently bound sizing agents were found to be almost uniformly distributed over the whole DP range of the cellulose as seen by carboxyl-selective fluorescence labeling in combination with size exclusion chromatography (“FDAM method”).

The acetylating action of ionic liquids frequently used in biomass and polysaccharide processing, namely 1,3-dialkylimidazolium acetates, on cellulose has been studied. Analysis was based on an accurate method to quantify even minute amounts of organic acetates in polysaccharides. The acetylation is caused by impurities in the ionic liquids, specially purified ionic liquids do not show an acetylating effect. 1-Acetylimidazole has been proven to be the actual acetylating agent, and its presence in aged ionic liquids has been confirmed. Formation of the reagent, mechanism of its action—being an acetyl transfer rather than a typical esterification—kinetics of the acetylation and the effect of water in the system are studied in detail. The implications on processing of biomaterials in ionic liquids are discussed as well.

A novel approach to measure the degree of acetylation in biopolymers applying a combination of Zemplen-deacetylation by sodium methanolate and GC-MS methodology is introduced. The development focuses on very low limits of detection to cover also samples with extremely low degrees of acetylation which hitherto eluded accurate determination. Free acetic acid or inorganic acetates, often present in biopolymer samples, do not disturb the quantification. Two techniques to measure the Zemplen-released methyl acetate were comparatively assessed, direct injection of the liquid phase and a SPME-based approach, the former being more straightforward, but being inferior to the latter in sensitivity. By applying isotopically labeled methyl acetate released from 4-O-(13C2-acetyl)-vanillin as the internal standard, influences, such as varying moisture contents, are corrected, improving the overall method reliability to a large extent. The combination of Zemplen-release of acetyl groups in biopolymers as methyl acetate, in connection with its accurate quantification by SPME-GC-MS, was found to be the method of choice for routine, yet very accurate analysis of a wide range of acetylation degrees of biopolymers, showing satisfying analytical parameters along with easy handling and widest applicability. Limit of detection for acetylated cellulose samples is 0.09nmol/mg, for hemicellulose samples 0.48nmol/mg.

Widening the methodology of chromophore analysis in pulp and paper science, a sensitive gas-chromatographic approach with electron-capture detection is presented and applied to model samples and real-world historic paper material. Trifluoroacetic anhydride was used for derivatization of the chromophore target compounds. The derivative formation was confirmed by NMR and accurate mass analysis. The method successfully detects and quantifies hydroxyquinones which are key chromophores in cellulosic matrices. The analytical figures of merit appeared to be in an acceptable range with an LOD down to approx. 60 ng/g for each key chromophore, which allows for their successful detection in historic sample material.

This study presents a liquid chromatography– ultraviolet (LC –UV) method for the accurate analysis of formose reaction products with regard to sum parameters. Limitations encountered in gas chromatography (e.g., thermal stability of analytes) were overcome. First, alditols are O-benzoylated and subjected to reversed-phase chromatography. They are then detected by UV and by electrospray ionization-mass spectrometry (ESI-MS) for validation purposes. Compared with standard 2,4-dinitrophenylhydrazine labeling, the accuracy of results from LC– UV is dramatically improved due to enhanced chromatographic resolution and validation by ESI-MS. For the first time ever, LC– UV may be used to accurately quantify 2-hydroxymethylglycerol, a branched chain alditol, D,L-glyceraldehyde, D,L-galactose, which represents the group of D,L-iditol/galactose/glucose/ mannose and D,L-erythrose in formose reaction products.

Off-the-shelf computer cooling hardware was used to construct a closed-loop air cooling system (CLACS) that is distinguished by scalability, low energy, and no tap water consumption. Constructed to be generally used with laboratory condensers, the system was tested with several common low and high boiling solvents and showed a condensation performance equivalent to conventional tap water cooling. Reaction yields were therefore unaffected. Also, long-lasting Soxhlet extractions showed no performance loss relative to conventional cooling. Optionally, the device can be assembled from low-voltage components and be powered from a battery, rendering it independent of the main power. Both investment and running costs are minimal, allowing a lab-wide adoption and elimination of the two major drawbacks of commonly employed tap water cooling: waste of drinking water and the risk of flooding.

Degraded celluloses are becoming increasingly important as part of product streams coming from various biorefinery scenarios. Analysis of the molar mass distribution of such fractions is a challenge, since neither established methods for mono- or disaccharides nor common methods for polysaccharide characterization cover the intermediate oligomer range appropriately. Size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS), the standard approach for celluloses, suffers from decreased scattering intensities in the lower-molar mass range. The limitation in the low-molecular range can, in principle, be overcome by calibration, but calibration standards for such “short” celluloses are either not readily available or structurally remote and thus questionable. In this paper, we present the calibration of a SEC system— for the first time – with monodisperse cellooligomer standards up to about 3400 g mol −1 . These cellooligomers are “short-chain celluloses” and can be seen as the “true” standard compounds, by contrast to commonly used standards that are chemically different from cellulose, such as pullulan, dextran, polystyrene, or poly(methyl methacrylate). The calibration is compared against those commercial standards and correction factors are calculated. Calibrations with non-cellulose standards can now be adjusted to yield better fitting results, and data already available can be corrected retrospectively.

The separation of C3-carbohydrates (DL-glyceraldehyde and dihydroxyacetone) present after the aldol-condensation of formaldehyde and glycolaldehyde was examined using the membrane processes nanofiltration (NF) and reverse osmosis (RO), which exemplifies the crucial problem area of the retention of small organic compounds. Two RO- and one NF-membrane were tested in order to obtain two fractions, namely a C3-enriched and a fraction containing mainly formaldehyde, methanol, and formic acid for a subsequently repeated aldol-condensation. In the case of a RO-process, operated until a volumetric concentration factor of 4 and two sequenced diafiltration steps, approximately 80% of formaldehyde, methanol, and formic acid were removed. Meanwhile, only about 40% of C3-carbohydrates were found in the permeate.

The aim of this study is to emphasize the potential of membrane technologies and the specific performance-limiting borders of pressure-driven (microfiltration, ultrafiltration, nanofiltration, reverse ssmosis) as well as electro-membrane (electrodialysis, electrodialysis using bipolar membranes) techniques for the separation of valuable substances from silage press-juice obtained in green biorefineries. Depending on the product, nanofiltration can be considered a partially fractionating technique with great future potential. Electrodialysis turns out to be a suitable separation technique for removing huge amounts of salt and isolating individual valuable substances. However, residual impurities must be taken into account for subsequent separation steps. In any case, further separation processes (e.g. chromatography) must be integrated in future green biorefinery production plants. © 2008 Society of Chemical Industry and John Wiley & Sons, Ltd

In the present work, a viable protocol was developed to prepare monodisperse cellooligomers up to a degree of polymerisation (DP) of 20. Peracetylated cellooligosaccharides were obtained from cellulose by acetolysis and subsequently purified by Normal Phase-High Performance Liquid Chromatography using toluene, ethyl acetate and acetone as eluents. In addition, we demonstrated how to efficiently monitor the purity and dispersity of the obtained compounds by High Performance Thin Layer Chromatography, Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry and Nuclear Magnetic Resonance. With this approach, it is possible to isolate cellooligomer standards up to DP 20 on a preparative scale (dozens of mg). The column chromatographic separation proved to be robust over several months and to be scalable from a analytical to a preparative column. The isolated oligomer standards allow a more precise description of cellooligomer distributions typically emerging from biorefinery process streams after hydrolysis of lignocelluloses. They can be used to calibrate the oligomeric region in size-exclusion chromatography where light scattering detection fails due to limited scattering intensities.

This review provides a general overview of preparation, separation, and analytical methods for cello- and xylooligosaccharides. Arising as side-stream products of different biorefinery processes, these compounds have increasingly gained the interest of researchers and engineers in the last few decades. Beside their application as additives in the food, feed, and pharmaceutical industries, these oligomeric carbohydrates are of key importance as model compounds for studying the dependence of physicochemical properties on the degree of polymerization (DP). First, different preparation methods for mixtures of oligosaccharides with DPs between 1 and 30 are discussed. These methods include acetolysis, acid and enzymatic hydrolysis, and glycoside synthesis. Then, separation techniques, including size exclusion chromatography, normal phase and hydrophilic interaction chromatography, and chromatography on cation exchange resins, are presented. Analysis of oligosaccharides by different techniques is described.

Highlights • Sucrose is transported symplastically towards developing and growing nodosities. • Starch is accumulated and metabolized during nodosities growth and development. • Nodosity formation has systemic effects on non-infected root tips of phylloxerated plants. • Gall formation reprograms processes of the secondary metabolism as demonstrated transciptionally., Gall forming phylloxera may compete for nutrients with meristematic tissues and develop heterotrophic structures that act as carbon sinks. In this work, we studied the underlying starch metabolism, sink-source translocation of soluble sugars towards and within root galls. We demonstrated that nodosities store carbohydrates by starch accumulation and monitored the expression of genes involved in the starch metabolic. Thereby we proved that the nodosity is symplastically connected to the source tissues through its development and that the starch metabolism is significantly affected to synthesize and degrade starch within the gall. Genes required for starch biosynthesis and degradation are up-regulated. Among the carbohydrate transporters the expression of a glucose-6-phosphate translocater, one sucrose transporter and two SWEET proteins were increases, whereas hexose transporters, tonoplast monosaccharide transporter and Erd6-like sugar transporters were decreased. We found general evidence for plant response to osmotic stress in the nodosity as previously suggested for gall induction processes. We conclude that nodosities are heterogenous plant organs that accumulate starch to serve as temporary storage structure that is gradually withdrawn by phylloxera. Phylloxera transcriptionally reprograms gall tissues beyond primary metabolism and included downstream secondary processes, including response to osmotic stress.

Grasslands are often abandoned due to lack of profitability. Extensively cultivating grassland for utilization in a biogas-based biorefinery concept could mend this problem. Efficient bioconversion of this lignocellulosic biomass requires a pretreatment step. In this study the effect of different steam explosion conditions on hay digestibility have been investigated. Increasing severity in the pretreatment induced degradation of the hemicellulose, which at the same time led to the production of inhibitors and formation of pseudo-lignin. Enzymatic hydrolysis showed that the maximum glucose yields were obtained under pretreatment at 220 °C for 15 min, while higher xylose yields were obtained at 175 °C for 10 min. Pretreatment of hay by steam explosion enhanced 15.9% the methane yield in comparison to the untreated hay. Results indicate that hay can be effectively converted to methane after steam explosion pretreatment.