A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 1. Method development

A novel method to accurately determine the carbonyl content in cellulosic materials by fluorescence labeling with carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide has been developed. The procedure can readily be incorporated into a gel permeation chromatography (GPC) system with refractive index and multiple-angle laser light scattering detection. Both a homogeneous procedure, working in DMAc/LiCl (2.5%, w/v), and a heterogeneous derivatization approach, using aqueous buffer pH 4.0, for determination of carbonyls in pulps have been optimized with regard to reaction conditions, presence of catalysts, reproducibility, and completeness of conversion. The homogeneous labeling requires prolonged reaction times and removal of excess marker prior to GPC analysis by a time-consuming precipitation-washing-redissolution sequence, which is not needed in the heterogeneous approach. The heterogeneous procedure offers the additional advantages of higher efficiency, shortened analysis times, increased simplicity, and widest applicability.     

A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 3. Monitoring oxidative processes

The fluorescence-based CCOA method for determination of carbonyl group profiles in cellulosic substrates was employed to study the mechanisms of various oxidative and degradation processes involving celluloses in greater detail. The approach comprises labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide (CCOA), followed by gel permeation chromatography in DMAc/LiCl with fluorescence, multiangle laser light scattering, and refractive index detection. At first, the CCOA method was applied to study solutions of pulp in N-methylmorpholine-N-oxide monohydrate (NMMO), as occurring in the production of Lyocell-type fibers. NMMO is a rather strong oxidant that on one hand converts reducing end groups to carboxyl structures, thus lowering the overall carbonyl content, but generates new keto structures along the chain by nonselective oxidation on the other hand. The CCOA method allowed for the first time to distinguish the carbonyl course in different molecular weight ranges. Second, alkalization and aging of pulp, which are used in the industrial preparation of cellulose derivatives, e.g., as an element of the preripening process in viscose rayon production, were investigated. The CCOA method shows a clear reduction of the molecular weight, accompanied by a fast loss of carbonyls in the first phase, which is due to removal of low-molecular weight material by dissolution, and a slow decrease in the second phase, which is caused by further oxidation of carbonyl groups. Also here, differences in the carbonyl course in different molecular weight regions were monitored. Third, electron beaming, proposed as a means of pulp activation, was shown to decrease and narrow the molecular weight distribution, under generation of comparatively low amounts of carbonyls, which, however, are also introduced into high molecular weight, crystalline domains, as shown by a comparison of homogeneous and heterogeneous CCOA labeling approach. Finally, as the fourth application, thermal treatment of cellulose at temperatures between 105 and 165 degrees C was shown to bring about a small reduction of the molecular weight, which only at higher drying temperatures is accompanied by an introduction of carbonyls over the whole molecular weight range.     

Dissolution behavior of different celluloses

Celluloses from different origins were dissolved stepwise in N,N-dimethylacetamide/lithium chloride (9% v/w; DMAc/LiCl) with the aim to study the time course of the dissolution process, completeness of dissolution in the dissolved fractions, possible discrimination effects, and differences between the celluloses. Cellulosic pulps from both annual plants and different wood species were analyzed. The obtained fractions were subject to gel permeation chromatography (GPC) with multiple detection to monitor the development of molecular mass distribution (MMD), molecular mass, and recovered mass. The dissolution behavior of accompanying xylans was followed by quantitative analysis of the uronic acids by fluorescence labeling--GPC. The morphological changes at the remaining fibers in the stepwise dissolution were addressed by SEM. The time needed to dissolve completely the cellulosic pulp differed from species to species, mainly between pulps from annual plants and pulps from wood. Annual plants generally needed much longer to dissolve completely. In the beginning of the dissolution, the dissolved fractions of annual plants showed a distinct discrimination effect because they were enriched in hemicellulose. By contrast, wood pulps dissolve fast and without distinct changes in the MMD of the dissolved fractions over time. Bagasse pulp is an exception to the observation for annual plants and rather resembled the behavior of wood celluloses. Prolonged dissolution times, as often practiced in cellulose GPC, do not lead to any improvements regarding the determination of molecular mass, MMD, and recovered mass of injected sample, so that the dissolution times required for reliable GPC analysis can be significantly shortened, which will be important for biorefinery analytics with high numbers of samples.     

Cross-sectional analysis of the polysaccharide composition in cellulosic fiber materials by enzymatic peeling/high-performance capillary zone electrophoresis

A combined enzymatic, chemical, and analytical approach was used to determine the cross-sectional carbohydrate composition in cellulosic fibers. The outer surface of cellulosic fibers was enzymatically removed layer-by-layer with precise quantitative control, and the monosaccharides in the peelings were subsequently analyzed by high-performance capillary electrophoresis (HPCE) after precolumn derivatization with a UV label. This method was applied to dissolving pulps and regenerated cellulose fibers, with special emphasis on the cross-sectional distribution of hemicelluloses. Commercially available enzyme solutions were used, resulting in a reproducible peeling. Significant differences were found in the hemicellulose distribution across the fiber of different dissolving pulps, dependent on both natural source (beech or spruce) and preparation process (acidic sulfite cook or prehydrolysis kraft cook). Among the dissolving pulps, beech prehydrolysis kraft pulp showed the highest enrichment of surface xylan. Similar, albeit smaller, differences were noticed between various regenerated fibers (viscose, viscose Modal, and Lyocell): a thin hemicellulose-rich outermost layer was found in all the regenerated fibers studied.     

Structural comparison and enhanced enzymatic hydrolysis of the cellulosic preparation from Populus tomentosa Carr., by different cellulose-soluble solvent systems

This study aims to establish an efficient pretreatment process using cellulose-dissolution solvents to enhance the enzymatic saccharification. LiOH/urea, LiCl/DMAc, concentrated phosphoric acid, ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and N-methyl-morpholine-N-oxide (NMMO) were selected as the cellulose dissolution agents. Except the cellulosic sample regenerated from LiCl/DMAc system, all the other treated samples exhibited lower cellulose crystallinity and degree of polymerization (DP), and consequently, exhibited a significant enhancement on enzymatic hydrolysis kinetic. Ionic liquid pretreatment offered unique advantages in the hydrolysis rate in the first 10 h, probably due to the extensively structural transformation of cellulose from the crystalline to the amorphous region. Meanwhile, the regenerated cellulose from concentrated phosphoric acid almost completely consisted of cellulose II, and achieved the highest saccharification yield.     

Theory and applications of DMAC/LICL in the analysis of polysaccharides

DMAc/LiCl has become a favored solvent in the analysis of polysaccharides. Although much is understood about its interaction with carbohydrate molecules, a great deal remains to be known in order for a comprehensive mechanism of dissolution to be discerned. These limitations, however, have not precluded the extended use of DMAc/LiCl in the study of chitin, cellulose, etc. This article reviews the theory of DMAc/LiCl as a solvent and new developments in this area, as well as the variety of applications which have been found for it.     

Chitin fractionation and characterization in N,N-dimethylacetamide/lithium chloride solvent system

A detailed study of the interaction of chitin molecular species with the solvent system N,N-dimethylacetamide (DMAc)/lithium chloride (LiCl) allowed the development of a new method for chitin fractionation by coacervate extraction. The controlled increase of the extracting power of the solvent was carried out using slight modification of the solvent composition. Partial extractions of molecular species were done between coacervation and complete dissolution limits using different mixtures of DMAc/LiCl of increasing extracting power. Fractions were characterized in DMAc/LiCl 5% (w/w) by viscometry and size exclusion chromatography with refractive index and multi-angle laser light scattering detectors. Fractions obtained by coacervate extraction range from 80,000 to 710,000 g mol⁻¹ with polydispersity index between 1.28 and 1.44. The Mark–Houwink–Sakurada equation constants a and K for chitin in DMAc/LiCl 5% (w/w) were found to be 0.95 and 7.6×10⁻⁵ dl g⁻¹, respectively.     

New approach for upgrading pulp & paper quality: Mild potassium permanganate treatment of already bleached pulps

The present work introduces mild – room temperature – potassium permanganate treatment of cellulosic materials, namely already bleached pulps. Such treatment represents a new approach for upgrading pulp and paper quality, which is lacking in the literature. Potassium permanganate was investigated as a purifying and mild oxidizing agent for commercial already bleached softwood and bagasse pulps. It was found that treatment of the bleached beaten pulps, with 0.25–2% KMnO₄ (based on pulp weight), led to significant improvement in paper properties. The strength (breaking length) increased greatly and the brightness increased significantly due to treatment. The improvements were related to the degree of polymerization, and to the alphacellulose content of pulps.Moreover, potassium permanganate serves as a disinfectant and deodorizer. Thus treatment of bleached pulps with KMnO₄ is a promising remedy for the side effects which pulps suffer, during transportation and storage, before papermaking.     

微波条件下LiCl/DMAc溶解稻秆纤维素制备还原糖

利用氯化锂/N,N-二甲基乙酰胺(LiCl/DMAc)溶剂体系在微波控制条件下对稻秆纤维素进行溶解预处理以提高纤维素酶解糖化效率。考察了微波时间和微波强度对产糖量及还原糖转化率的影响。通过扫描电镜(SEM)和热重分析仪(TG)对预处理前后稻秆纤维素的微观形貌及热稳定性进行表征,并利用高效液相色谱仪(HPLC)对酶解糖液进行糖成分鉴定和含量分析。结果表明,微波加热能够有效促进LiCl/DMAc对稻秆纤维素的溶解。与原生稻秆相比,经微波-LiCl/DMAc法溶解后再生纤维素出现明显解聚,热分解温度由290℃降至220℃。在微波功率为385 W、加热溶解时间为7 min时,所得稻秆纤维素还原糖转化率由30.90%上升至98.67%;HPLC谱图表明,糖液中主要成分为葡萄糖和木糖,分别占所得还原糖总量的43.74%和48.55%。     

Cellulose acetates from linters and sisal: correlation between synthesis conditions in DMAc/LiCl and product properties

We report the acetylation of celluloses from sisal (untreated and alkali treated) and cotton linters (alkali treated), under homogeneous solution conditions, using DMAc/LiCl as solvent system. Our target was to evaluate the effects of cellulose dissolution and reactions conditions on the product properties. The products were characterized in terms of degree of substitution (DS) by 1H NMR, and molar weight distribution (MWD) by size exclusion chromatography. Changes in the DS of the products were correlated with reaction conditions and solution properties. It was found that the dissolution of celluloses and degree of substitution of cellulose derivatives depends on a fine adjustment of the dissolution/derivatization conditions, as well as on the origin (sisal or linters) of celluloses.     

The assessment of chromophores in bleached cellulosic pulps employing UV-Raman spectroscopy

UV-Resonance Raman (UV-RR) coupled with UV-visible Diffuse Reflectance (UV-vis DR) spectroscopy was applied to a solid-state study of chromophores in Eucalyptus globulus kraft cellulosic pulps bleached by chlorine dioxide and hydrogen peroxide. The UV-RR spectra were acquired at 325 nm laser beam excitation, which was shown to be appropriate for selective analysis of chromophore structures in polysaccharides. The proposed approach allowed the monitoring of chromophores in pulps and to track the extent of polysaccharide oxidation. However, precaution was suggested while performing a quantitative analysis of chromophores at the characteristic band of ∼1600 cm⁻¹ because of charge transfer complexes (CTCs) that exist in the pulp. These CTCs can affect the intensity of the aforementioned band by diminishing the conjugate state in the chromophore moieties. The amount of carbonyl and carboxyl groups in polysaccharides correlated with the intensity of the band at 1093 cm⁻¹. The analysis of UV-RR spectra revealed xylan as an important source of chromophores in eucalypt kraft pulp.     

Effect of solvent exchange on the solid structure and dissolution behavior of cellulose

Effects of solvent exchange and milling on the solid structure of cellulose were investigated, using small- and wide-angle X-ray scattering and solid-state NMR. The solvent exchange facilitated the dissolution of cellulose in LiCl/DMAc with no change of the crystalline structure of cellulose. In contrast, the milling never facilitated the dissolution of cellulose, though the crystalline structure was almost destroyed. These facts show that the crystalline structure of cellulose hardly affects the dissolution in LiCl/DMAc. The fractal dimensions determined by the small-angle X-ray scattering measurements were increased by the solvent exchange, suggesting that the aggregation state of the cellulose microfibril is affected. It was also suggested by the NMR (1)H spin relaxation time measurements that the solvent exchange enhances the molecular mobility of cellulose and shortens the characteristic length along the microfibril, which allows easier access of the solvent molecule to cellulose.     

Thermogravimetry study of xylanase- and laccase/mediator-treated eucalyptus pulp fibres

Thermogravimetric analyses (TGA) was applied to study the effects of enzymatic bleaching of eucalyptus pulp with xylanase and a laccase-mediator system. The thermal degradation profile of the pulps was sensitive to the enzymatic treatments. Xylanase treatment produced an ordered and clean microfibril, whereas laccase oxidized surface cellulose chains and increased the amorphous (paracrystalline) cellulose content. In this case, pulp viscosity decreased from 972 to 859 mL/g and apparent pulp crystallinity calculated from TGA data decreased almost 50%. Alkaline extraction was necessary to recover pulp crystallinity and to remove oxidized lignin in the laccase-treated samples. TGA data allowed differentiating and quantifying crystalline and amorphous cellulose. This thermogravimetric approach is a simple method in order to monitor superficial changes in cellulosic microfibrils.     

SEC-MALS-QELS study on the molecular conformation of cellulose in LiCl/amide solutions

The SEC-MALS-QELS (size-exclusion chromatography equipped with multiangle light scattering and quasi-elastic light scattering detectors) method using lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) and LiCl/1,3-dimethyl-2-imidazolidinone (LiCl/DMI) as mobile phases was applied to cellulose and cellulose tricarbanilate (CTC) samples with various average degree of polymerization (DP) values. Molecular conformations of cellulose and CTC in the solvents were then discussed and compared on the basis of the relationships between the radii of gyration (R(g,z) or S(2)(z)(1/2)), the hydrodynamic radii (R(h,z)), and weight-average DP (DP(w)) or the contour lengths (L(w)). The Benoit-Doty theory for wormlike polymer chains was applied to the R(g) vs L(w) data obtained, and the theoretical curves with Kuhn segment lengths l(K) of around 18 nm were found to fit the data of both cellulose and CTC molecules in the solvents. It was concluded from the obtained results that both cellulose and CTC molecules have conformations essentially identical to each other in the solvents; they behave as typical semiflexible chains in good solvents.     

Novel cellulose derivatives. Part VI. Preparation and thermal analysis of two novel cellulose esters with fluorine-containing substituents

Two novel cellulose esters were prepared with fluorine (F)-containing substituents using homogeneous phase reaction chemistry in DMAc/LiCl. The partially substituted derivatives and their corresponding perpropionates proved to be thermoplastic polymers. The 2,2-difluoroethoxy and 2,2,3,3,4,4,5,5-octafluoropentoxy substituents were easily identified by ¹H- and ¹⁹F-NMR spectroscopy without disclosing their precise location on the anhydroglucose unit. Thermal analysis revealed modest or no crystallinity; glass transition temperatures between 53 and 113°C; and improved thermal stability as compared to their F-free counterparts.     

Preparation of C-6 substituted chitin derivatives under homogeneous conditions

Tosylation of chitin under homogeneous conditions was achieved by the reaction of tosyl chloride with chitin in a DMAc/LiCl solvent system. The resultant tosyl-chitin was fully N-acetylated with acetic anhydride in methanol. The fully acetylated tosyl-chitin was subsequently reacted with the sodium salts of ethyl p-hydroxybenzoate, diethyl malonate, and diethyl phosphite in DMAc to give the corresponding chitin derivatives of 6-O-ethyl benzoate-chitin, 6-deoxy-diethyl malonate-chitin, and 6-(deoxydiethyl) phosphite-chitin, respectively. Subsequent hydrolysis of the chitin-ester derivatives with tert-butoxide in dimethyl sulfoxide (DMSO) generated 6-O-carboxyphenyl-chitin and 6-(deoxydicarboxy)methyl-chitin. The structures of the chitin derivatives were assessed by FT-IR, (13)C NMR, and (31)P NMR, while the degree of substitution of the S(N)2 reaction was estimated by elemental analysis. All the chitin derivatives were found to be soluble or swellable in water, DMAc, or DMSO.     

Fluorescent hydrazides for the high-performance liquid chromatographic determination of biological carbonyls

Methods for the determination of carbonyl compounds of biological origin by high-performance liquid chromatography were improved by the use of new fluorescent derivatizing agents. Eight fluorescent hydrazides were either synthesized or obtained commercially and compared to dansyl hydrazine (1-dimethylaminonaphthalene-5-sulfonylohydrazide). Four of the compounds yielded carbonyl hydrazones with a higher relative fluorescence quantum yield than dansyl hydrazine in acetonitrile:water mixtures. Darpsyl hydrazide [(3-phenylpyrazoline-1-yl)-4-phenylsulfonylohydrazide] and apmayl hydrazide [N-(2-aminophenyl-6-methylbenzthiazole)-acetylohydrazide] both yielded an increase of greater than 20-fold in sensitivity over dansyl hydrazine in determinations of abscisic acid and jasmonic acid from plant tissues. Different hydrazides and derivatizing conditions were found to be optimum for the determination of different carbonyl compounds. Also, a simple method for precolumn purification of the hydrazones of acidic carbonyls was developed to remove contaminants arising during derivatization and from the tissue source.     

Fluorimetric screening assay for protein carbonyl evaluation in biological samples

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.     

Determination of plasma, urine, and bovine serum albumin low-molecular-weight carbonyl levels by capillary gas chromatography with electron-capture and mass-selective detection

Peroxidation of lipids produces low-molecular-weight carbonyl compounds, which are reactive with biological nucleophiles. The analysis of these compounds is often difficult. A multicomponent method for the determination of 11 of them in biological samples is reported. The samples are subjected to a pretreatment-derivatization procedure followed by gas chromatographic analysis with either electron-capture detection (ECD) or mass-selective detection (MSD) in the selected-ion monitoring mode. The procedure involves derivatization of the analyte with 2,4,6-trichlorophenylhydrazine, extraction with n-hexane, and separation of the derivatization products on a nonpolar gas chromatographic column. The concentration of the derivatization reagent, pH, reaction time, temperature, and presence of extraneous ions were investigated to determine the optimal derivatization conditions. Under these conditions, the method allows for the selective detection of low-molecular-weight carbonyl compounds at femtomole levels in several biological materials such as plasma, urine, and bovine serum albumin without interferences. The limits of detection were in the ranges 0.01-0.2 microM for ECD and 0.15-1.5 microM for MSD. The mean procedural recoveries obtained during the method validation were within the range 85-95% and the intra- and interassay standard deviations do not exceed 4.6 and 6.1%, respectively.     

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization.