A Novel Method for the Determination of Carbonyl Groups in

Biomacromolecules 2002, 3, 959-968

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A Novel Method for the Determination of Carbonyl Groups in Cellulosics by Fluorescence Labeling. 1. Method Development ¨ rgen Ro ¨ hrling,† Antje Potthast,*,† Thomas Rosenau,† Thomas Lange,† Gerald Ebner, Ju Herbert Sixta,‡ and Paul Kosma*,† Christian-Doppler-Laboratory, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria, and Lenzing AG, R & D, A-4860 Lenzing, Austria Received March 18, 2002; Revised Manuscript Received May 27, 2002

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. Introduction Cellulose is the most abundant polymer from renewable resources. While the chemical structure of the cellulose backbone does not seem to offer major challenges anymore, the supramolecular architecture of cellulose and its biosynthesis remain the prominent topics in today’s cellulose research. However, cellulose is not only the “ideal” homopolymer, which is built up of anhydroglucose units linked by β-1,4-glycosidic bonds, but it contains small amounts of various other, “irregular” structures, which are mainly oxidized groups. This applies especially to the material that has undergone a number of process steps in the pulp and paper industries andsalbeit to a lesser extentsalso to genuine, untreated cellulose. The reliable and accurate determination of those oxidized functionalities in cellulose represented a largely unsolved problem in cellulose chemistry,1 and is the topic of the present paper. Oxidized groups in cellulosics, such as keto and aldehyde groups,2 are introduced by pulping and bleaching processes, according to the respective conditions chosen.3 Especially bleachingsboth chlorine based and oxygen basedsis known to affect the integrity of the cellulose backbone by generation of oxidized positions and subsequent chain cleavage, which can be a result of both homolytic reactions, e.g., attack of oxygen or oxygen-derived radicals, and heterolytic processes, such as β-elimination. The creation of oxidized groups along the cellulose chain is thus a highly undesired process, as these positions constitute “hot spots” along the carbohydrate chain, where a pronounced chemical instability is introduced and where subsequent cleavage will primarily occur. Oxidized † ‡

University of Agricultural Sciences Vienna. Lenzing AG.

positions in cellulose are a main reason for strength loss and decreased performance parameters in textiles, paper, and other cellulosic materials, and they are chiefly responsible for general aging processes of cellulosics4 and are furthermore assumed to be the cause and promoter of thermal and light-induced yellowing processes.5 The determination of carbonyls in cellulose has to contend with several inherent difficulties. First, the extremely low average contents of carbonyls, which range in the order of µmol/g, require very sensitive means of detection since conventional, direct instrumental techniques, such as IR, Raman, UV, or NMR spectroscopy, fail to report such minor amounts. Hence, a chemical method must be applied in which the carbonyl structures are either “titrated” by a reagent or converted in a suitable reaction into structures, which can then be monitored by traditional spectroscopic techniques. The second main problem follows directly from the necessity to use such chemical methods: the derivatization of the carbonyl group will be a heterogeneous reaction with inherent problems of accessibility and reagent adsorption. But even when the reaction is carried out in solution, the solvents will be “exotic”, such as metal amine complexes, N,N-dimethylacetamide/LiCl or N-methylmorpholine-N-oxide, so that conventional carbonyl reactions cannot easily be transferred into those media. And third, the situation is additionally complicated by the lack of means to report the completeness of derivatization reactions since kinetic data are not available; this applies to both the heterogeneous and homogeneous reaction of carbonyls in cellulosics. All these facts contribute to the inaccuracy of traditional measurements. Three conventional approaches to estimate the amount of cellulosic carbonyls are rather common: the copper number, applying copper(II) salts, which are reduced in turn and

10.1021/bm020029q CCC: $22.00 © 2002 American Chemical Society Published on Web 07/17/2002

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determined titrimetrically.6 According to the oxime method7 carbonyls react with hydroxylamine, and the carbonyl content is reflected by the nitrogen content as determined by elemental analysis or the Kjeldahl procedure. The third approach aims at converting the carbonyls into cyanohydrins by reaction with cyanide.8 All these approaches suffer from clear and distinct drawbacks, either they are based on conversions whose underlying mechanisms are far from being understood (copper number), they provide data which are only an indirect measure of carbonyls (copper number), or they suffer from limited reproducibility (oxime and cyanohydrin method). In our approach, we started from two prerequisites: first, fluorescence spectroscopy was to be used as the reporting technique, as it is highly sensitive and allows the detection even of smallest amounts of oxidized structures. Second, the method to be developed should be implemented into gel permeation chromatography (GPC) measurements of cellulose (with fluorescence detection) and thus provide not only the carbonyl content as a sum parameter but also a carbonyl group profile in relation to the molecular weight of the cellulosic material. This approach has to accomplish the following individual tasks, which will be described in detail in this first part of our studies: selection of an appropriate fluorophore and label; optimization of the derivatization reaction by means of oxidized carbohydrate model compounds, which includes comprehensive kinetic studies in terms of reaction temperature, reagent concentrations, reagent stability, product stability, catalysts, solvent composition, and side reactions; transfer of the elaborated methodology from model compounds to genuine cellulosic material, optimization for both homogeneous and heterogeneous reaction conditions and kinetic studies; incorporation of the method into a GPC system; calibration and optimization for GPC conditions. The use of the novel method to quantify the content of carbonyl groups in pulps, the application to several topics, such as monitoring bleaching sequences of pulps, and the thorough validation of the method will be discussed in the forthcoming second part. Materials and Methods Chemicals. Chemicals were obtained from commercial sources and were of the highest purity available. DMAc was obtained from Promochem chemicals, Germany. The water content in DMAc/LiCl was determined according to the previously developed method based on the solvatochromic shift of a fluorescent dye.9 Pulps from different origins and sources were used. General Analytics. UV spectra were obtained on a HITACHI U-3010 photometer in quartz cuvettes (d ) 1 cm) at 20 °C, fluorescence spectra on a HITACHI F-4500 device under otherwise identical conditions. GPC and highperformance liquid chromatography (HPLC) measurements used the following components: online degasser, Dionex DG-2410 and Gynkotek DG-1310; pumps, Dionex P580 (HPLC), Kontron 420 (GPC); pulse damper; autosampler, HP series 1100; column oven, Gynkotek STH 585; fluores-

Figure 1. Setup of the GPC system used.

cence detector, TSP FL2000; multiple-angle laser light scattering (MALLS) detector, Wyatt Dawn DSP with argon ion laser (λ0 ) 488 nm), refractive index (RI) detector, Shodex RI-71; UV detector, Dionex UVD 340. Data evaluation was performed with standard Chromeleon and Astra software. GPC System. For GPC measurements, the system as described by Schelosky et al.10 was modified; see Figure 1 for the experimental setup. DMAc/LiCl (0.9%, w/v), filtered through a 0.02 µm filter, was used as the eluant. The sample was injected automatically, chromatographed on four serial GPC columns, and monitored by fluorescence, MALLS, and RI detection. For measurements with UV detection (290 nm), the detector was inserted between fluorescence and MALLS detector. Molecular weight distribution and related polymerrelevant parameters were calculated by software programs, based on a refractive index increment of 0.140 mL/g for cellulose in DMAc/LiCl (0.9% w/v) at 25 °C and 488 nm. The following parameters were used in the GPC measurements: flow, 1.00 mL/min; columns, four, PL gel mixedA ALS, 20 µm, 7.5 × 300 mm; detectors, fluorescenceMALLS-RI; fluorescence detection, 290 nm excitation, 340 nm emission; injection volume,: 100 µL; run time, 45 min. HPLC Methods. The following parameters were used in HPLC measurements: temperature, 25 °C; fluorescence detection with excitation at 286 nm and emission at 330 nm; injection volume, 10 µL. (Method 1) Check of purity of CCOA: eluant, 57% 0.05 M NH4OAc, 43% acetonitrile; flow, 1.00 mL/min; columns, Nucleosil 120-7 C18, 250 × 4 mm; peaks, tR ) 5.29 min ((0.3%, k ) 1.42, N ) 1274). (Method 2) Labeled cyclohexanone: eluant, 35% 0.05 M NH4OAc, 65% acetonitrile; flow, 1.00 mL/min, columns, Nucleosil 120-7 C18, 250 × 4 mm, peaks, tR ) 5.38 min ((0.4%, k ) 1.83, N ) 2436). (Method 3) Separation of carbazole-9-carbonyloxyamine (CCOA) and N-acetylated CCOA: eluant, 50% 0.05 M NH4OAc, 50% acetonitrile; flow, 0.40 mL/min; columns, Aquasil C18, 150 × 3 mm, 3 µm (Keystone Scientific Inc.); peaks, tR ) 4.29 min ((0.8%, k ) 1.84, N ) 1617, R ) 1.21) for N-acetylated CCOA, tR ) 4.87 min ((1.3%, k ) 2.26, N ) 1733, R ) 1.29) for CCOA. Activation of Pulp Samples. To achieve a “good”, i.e., molecular disperse dissolution in DMAc/LiCl (9%, w/v), the pulp samples had to be activated, no matter if genuine pulp, heterogeneously labeled pulp, or precipitated pulp from the homogeneous derivatization procedure had to be dissolved. The pulp samples were activated by solvent exchange (H2O

Determination of Carbonyl Groups in Cellulosics

to DMAc) followed by agitating in DMAc and filtration, which produced efficiently activated, i.e., readily soluble samples. Optimization of Reaction Conditions. The optimization of the conditions for the labeling of carbohydrate model compounds and the optimization of the homogeneous and heterogeneous labeling procedure for pulp required multiple variations of the reaction conditions, aimed at providing generally applicable procedures.11 General Procedure for the Determination of Carbonyls in Pulp by Homogeneous Fluorescence Labeling. Activated pulp (0.50 g, corresponding to 0.20 g o.d. pulp) was dissolved in 20 mL of DMAc/LiCl (9%, w/v) overnight at room temperature. DMAc (50 mL) containing CCOA (50.0 mg) and triethylamine (19 µL) was added. The reaction mixture was shaken in a temperature-controlled water bath at 40 °C. After reaction times of 48, 96, 168, 214, 262, 382, 430, 502, and 568 h, aliquots (7 mL) were taken and injected into water (100 mL) under efficient stirring. After 15 min, the precipitated pulp was removed by filtration, activated, and redissolved in 2 mL of DMAc/LiCl (9%, w/v) at room temperature. Aliquots of the solution were diluted with DMAc, filtered through a 0.45 µm filter, and analyzed by GPC. For evaluationsand to shorten reaction timessthe fluorescence data obtained were extrapolated toward complete conversion based on first-order kinetics (c/c∞ ) 1 e-kt). For the determination of the optimum base concentrations to be used, the procedure was varied by adding different amounts of triethylamine. Reproducibility was determined by measurements in duplicate of four independent pulp samples, labeled with four independently prepared CCOA solutions. General Procedure for the Determination of Carbonyls in Pulp by Heterogeneous Fluorescence Labeling. Pulp, corresponding to 20-25 mg of dry pulp, was suspended in 2 mL of 20 mM zinc acetate buffer, pH 4.0, and shaken for 2 h. A CCOA stock solution was prepared by dissolving the label (125 mg) in 50 mL of zinc acetate buffer, and 2 mL was added to the pulp. The suspension was agitated in a water bath with temperature control for 168 h at 40 °C. The pulp was removed by filtration, activated (see above), and dissolved in 2 mL of DMAc/LiCl (9%, w/v) at room temperature. Samples of the solution were diluted with DMAc, filtered through 0.45 µm, and analyzed by GPC yielding the carbonyl content directly without extrapolation. For the determination of the optimum pH value of the reaction medium, the procedure was modified by using buffers of different pH (acetate buffer for pH 4, 5, and 6 and phosphate buffer for pH 7 and 9).11 Results and Discussion 1. Selection of the Fluorophore, Structure and Synthesis of the Carbonyl Label. The selection of the fluorophore in the fluorescence label depends strongly on the conditions of the GPC measurement. As MALLS (multiangle laser light scattering) detection is indispensable, the fluorescent light must not interfere with this detection mode, which means

Biomacromolecules, Vol. 3, No. 5, 2002 961 Scheme 1. Reaction of Carbonyl Compounds with Hydroxylamine

that the difference between emission wavelength of the label and working wavelength of the laser used (λMALLS ) 488 nm) should be as large as possible. This is all but trivial, since most of the common, commercially available markers do not meet these requirements. The first experiments pointed out a second problem, which arose from the necessity to perform the labeling reaction in a medium that is the cellulose solvent as well. In fact, N,Ndimethylacetamide/LiCl, which has found broadest use for GPC of cellulosic material,12 appeared to be the only practicable choice available. The conventionally used carbonyl labels carry hydrazine or hydrazide structures as reactive anchor groups,13 which react with ketones or aldehydes under formation of a hydrazone structure in aqueous, mostly slightly acidic media. Unfortunately, derivatizations of simple carbonyl model compounds with dansyl hydrazine, which proceed quantitatively in aqueous media, gave only very low conversions in N,N-dimethylacetamide/LiCl. Another problem originated in the influence of the spatial environment of the fluorophore on the fluorescence emission wavelength. If the fluorophore is directly attached to the carbonyl compound via the anchor group, the fluorescence emission depends (although weakly) on the spatial surroundings of the chromophore: it will vary according to the type of carbonyl labeled (aldehyde or ketone) and the structure of neighboring groups (polar or less polar moieties). The resulting shift of the fluorescence maximum might finally result in incorrect values of the carbonyl content determined. This problem can be overcome by using a spacer that links the anchoring group to the actual fluorophore, so that the fluorophore is always kept in defined distance from the carbonyl group. The resulting emission spectrum consequently will be independent of type and direct environment of the carbonyl. In the recent literature, O-substituted hydroxylamines have been described as structures for carbonyl markers having increased reactivity as compared to hydrazines.14 The derivatization reaction is a two-step process, consisting of the nucleophilic addition of the nitrogen at the CO group, followed by the acid-catalyzed release of water. The mechanism (Scheme 1) is thus similar to the reaction of CO with hydrazines. Both the hydroxylamine anchor group and the polar ethylene glycol spacers of the derivatives reported in the literature seemed to be well suitable for the present case. However, the fluorophores used, dansyl and rhodamine B derivatives, were inappropriate, due to their emission wavelengths interfering with MALLS detection.

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Scheme 2. Structure of the Fluorescence Label Carbazole-9-carboxylic Acid [2-(2-Aminooxy-ethoxy)ethoxy]amide, “CCOA”

Finally, a novel carbonyl-selective fluorescence label, which meets all the requirements addressed above, was synthesized in an expeditious three-step approach as described previously.15 The marker, termed “CCOA” according to its trivial name carbazole-9-carbonyloxyamine (Scheme 2), is based on the fluorophore carbazole, with its fluorescence emission of 340 nm in DMAc/LiCl (0.9% w/v) at an excitation of 290 nm being sufficiently remote from the working wavelength of the MALLS laser. As additional advantages, CCOA offers the reactive O-substituted hydroxylamine structure as the anchor group, which binds to carbonyls as an oxime, and carries an ethylene glycol spacer that ensures the fluorescence characteristic to be independent of type and environment of the group labeled. By means of model compounds, such as cyclohexanone as well as selectively oxidized carbohydrates as keto sugar models16 and oligosaccharides with reducing end groups, it was demonstrated that CCOA reacts quantitatively with keto and with aldehyde groups.15 All labeled model compounds exhibited identical fluorescence emission maxima. With the demonstration of the suitability of CCOA as a fluorescence label for keto and aldehyde groups in low-molecular, oxidized sugar units under the conditions needed for the chromatography of cellulosics, the fundament was laid for the labeling of carbonyl structures also in macromolecular cellulosic materials. 2. GPC of Labeled Pulps. 2.1. Marker-GPC System Interactions. For homogeneous derivatization, it is essential to use excess marker to achieve a conversion as fast and complete as possible (see below). However, the use of excess marker posed some serious problems: First, the separation efficacy of the columns used was insufficient to achieve a neat baseline separation between polymer and salt peak (Figure 2). The very strong fluorescence signal of the salt peak containing the excess CCOA sometimes caused a shutdown of the fluorescence detector by photomultiplier overload. Second, a part of the excess marker eluted very slowly only, so that purging with eluant for 90 min or longer was necessary to return the fluorescence background level to the initial low value, prior to the next GPC run. The most serious problem, however, was that high loads of marker affected the long-term stability of the GPC columns. The lifetime of the columns was significantly shortened by extreme marker overloads, probably by reaction or adsorption of CCOA at the column filling. Thus, removal of excess marker prior to GPC measurements not only was advisable

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in terms of user friendliness of the method but also became an absolute must as a matter of system integrity. 2.2. Calibration of the System. Determining the absolute content of carbonyls in pulp requires calibration of the method relative to a reference standard, which should exhibit the same excitation and emission spectra as labeled pulp and also the same fluorescence yield. It had been shown for the model compounds that the fluorescence characteristic of the label is independent of the location of the attachment due to the spacer link (see above); the same independence was also assumed for labeled pulp. Pure CCOA was chosen as the reference standard. When CCOA solutions of different concentrations were injected, the fluorescence was found in the salt peak, with the peak width being rather narrow (about 3 min, in contrast to approximately 15 min elution time for labeled pulp) and the peak height ranging sometimes beyond the dynamic range of the detector. To circumvent this problem, the reference standard was not injected at once, but the injection volume was quartered and injected in 1-min intervals. The peaks obtained by this injection mode were comparable to peaks from labeled pulp with regard to both peak width and height. For calibration, the run time was doubled because of the slow elution of the low molecular weight compounds. The injected mass of cellulose was calculated from the integral of the pulp peak in the RI signal, with the RI constants having been thoroughly determined beforehand. 3. Optimization of the Labeling TechniquesHomogeneous Reaction in DMAc/LiCl. 3.1. Spectroscopic Properties of the Fluorophore. The fluorescence behavior of a marker generally depends not only on the chemical structure of the fluorophore but also on the solvent used. To obtain reproducible results, the dependence of the fluorescence absorption and emission maxima (i.e., solvatochromic effects) and the fluorescence intensity (i.e., fluorescence quenching) on the composition of the solvent must be known. The solvent DMAc/LiCl, as routinely used for GPC of cellulosics, is subject to differing contents of LiCl, water, acetic acid, and N,N-dimethylamine, with the latter two originating in the LiCl-catalyzed hydrolysis of DMAc.9,17 With the pure label CCOA and CCOA-labeled cyclohexanone as model compounds, the influence of composition changes in the solvent DMAc/LiCl on the fluorescence properties was determined. Generally, a solvatochromic shift was never observed. For the CCOA derivatives used, it can thus be stated that changes in the concentration of LiCl, water, and byproducts (acetic acid, HNMe2) exert an influence only on the fluorescence intensity but not on the absorption/emission maxima. To avoid incorrect fluorescence data, the eluant should consequently not be changed during a series of GPC runs, unless it is made sure that the LiCl and water content indeed remain unchanged. In addition, calibration standards must be prepared with the same solvent that is used as the eluant, and the calibration must be repeated in the case of the eluant being changed. 3.2. Stability of the Marker. The stability of both marker and labeled carbonyl compounds in the respective reaction medium is essential for a quantitative analysis. To test the


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Figure 2. Chromatogram of a labeled beech sulfite pulp with and without removal of excess marker. Scheme 3. N-Acetylation of CCOA by Thermal Treatment in DMAc

Figure 3. Thermal stability of CCOA in DMAc/LiCl.

stability of CCOA, the label was heated in DMAc containing 2.5% (w/v) LiCl, which was used as the reaction medium for the derivatization, at different temperatures. The degradation was followed by means of HPLC, monitoring the amount of unchanged CCOA. The experiments showed the marker to be completely stable at temperatures below 40 °C (Figure 3). At 60 °C a slow degradation sets in, which became faster at higher temperatures so that at 100 °C CCOA was completely decomposed within 8 h. At 80 °C in the absence of LiCl, CCOA was completely decomposed within 25 h, producing the N-acetylated derivative in 93% yield. In the presence of LiCl under otherwise identical conditions, the degradation process was decelerated, so that LiCl seems to increase the thermal stability of the marker in DMAc. After 25 h, 27% of the starting material was still present. The main degradation product was formed by acetylation of the terminal NH2 group by the solvent DMAc, so that an O-substituted hydroxamic acid was formed (see Scheme 3). The structure of the byproduct was proven by independent synthesis from CCOA and acetic anhydride, by NMR, and by co-injection of the synthesized product with the degradation product from the heating experiments. In contrast to water as the reaction medium, CCOA-labeled carbonyls showed no tendency to react back to CCOA and carbonyl compounds in the solvent DMAc at ambient

temperature in the absence of marker. The thermal behavior of the labeled compounds was in principle identical to CCOA itself, as demonstrated by experiments with CCOA-labeled cyclohexanone, which were carried out under the same conditions as used for pure CCOA. Like CCOA, also the CCOA-labeled compounds underwent a slow degradation at temperatures above 40 °C but were completely stable at 40 °C and below as demonstrated in tests run over 525 h. 3.3. Optimization of Reaction Conditions by Means of Model Compounds. Optimization of the reaction conditions for the derivatization in terms of a fast and complete conversion was carried out by means of cyclohexanone as the model compound. The influence of reaction temperature, water content in the solvent system DMAc/LiCl (2.5%, w/v), and presence of acids or bases on the reaction rate was systematically investigated. In all experiments, the concentration of the carbonyl model corresponded to the expected carbonyl concentration in the GPC injection solution, the fluorescence label was used in an at least 20-fold excess, and the formation of the labeled compound was followed by HPLC. In general, primary and secondary amines being present in the reaction mixture decrease the amount of carbonyls available for labeling by formation of the corresponding Schiff’s bases or enamines, respectively. The reaction of CCOA with carbonyl compounds in DMAc/LiCl is catalyzed by bases. Triethylamine, N,N,Nethyldiisopropylamine (Hu¨nig’s base), 1-methylimidazol, and 1,4-diazabicyclo[2.2.2]octane were used as catalysts. The optimum catalyst amount was determined for each base by varying the concentration from 0 to 10 equiv relative to the marker employed. Triethylamine proved to be the most suitable catalyst, with 1 equiv of base giving the fastest reaction. Under optimized conditions, 95% (or more) of the carbonyl compound could be labeled after 22 h, and the

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Figure 4. Homogeneous labeling of a beech sulfite pulp with CCOA: the influence of the catalyst amount (triethylamine) and reaction kinetics.

reaction time was shortened to 6 h by working at 40 °C. Moreover, experiments with NEt3 as the base at room temperature showed that water in the concentration range between 0.1 and 2.5% had no influence on both conversion and reaction rate. 3.4. Optimization of Reaction Conditions for the Homogeneous Labeling of Pulp. A complete derivatization is a prerequisite for the quantification of carbonyl groups in pulp. To find the best parameters, the conditions found to be optimal for model compounds were used as starting conditions for the reaction with pulps. For that purpose, the activated pulp was dissolved in DMAc/LiCl (9%, w/v), diluted to a LiCl content of 2.5% (w/v), and finally subjected to labeling with CCOA. Aliquots of the mixture were taken at different reactions times to determine the progress of the reaction, which required precipitation of the pulp with water, thorough washing to remove excess label, activation, and redissolution for GPC analysis. With the reaction temperature being set to 40 °C, the reaction conditions had to be optimized only with respect to catalyst and reaction time. For the catalyst amounts to be used, the result was the same as for the model compounds: 1 equiv relative to the marker worked best by giving the fastest conversion (see Figure 4). However, pulp reacted rather slowly with the marker, in contrast to the carbohydrate model compounds and cyclohexanone. For example, a beech sulfite pulp showed a degree of conversion of 88% after 502 h of reaction time. The long periods until completion of the conversion are a minor drawback only, as the labeling of carbonyls in pulp strictly follows first-order kinetics with regard to the labeled material (Figure 4), so that extrapolation toward maximum conversion is possible. The integrated firstorder rate law yields the conversion c/c∞ in dependence on the reaction time: c/c∞ ) 1 - e-kt. Thus, for determination of the carbonyl content of an unknown pulp, the kinetics of the labeling must be recorded over about 500 h by repeated GPC runs with aliquots and extrapolated toward complete conversion, requiring the rather tedious precipitationredissolution procedure for each sample taken. The overall carbonyl content of an unknown pulp [A]∞ can be determined also after shorter reaction times (with incomplete conversion) relative to a reference pulp. This

approach is based on the assumption that the reaction rate for different pulps is similar. Kinetic data points for the unknown pulp and the reference pulp, i.e., an [A] and a [R] value, and the overall carbonyl content of the reference pulp ([R]∞) must be known. For this purpose, the overall carbonyl of a standard beech sulfite reference pulp was accurately measured, so that this pulp could function as reference standard. To determine the overall content of an unknown pulp at shortened reaction times, both this pulp and a reference pulp were subjected to the homogeneous labeling procedure. With the actual amount of carbonyls determined for the unknown pulp ([A]) and the reference pulp ([R]), and with the overall amount of carbonyls in the reference pulp as taken from the calibration data ([R]∞), the overall carbonyl amount in the unknown analyte can be calculated according to the equation [A]∞ ) [A][R]∞/[R]. According to this procedure the carbonyl contents of numerous pulps were determined at reaction times of 96, 268, and 524 h (Figure 5). It became obvious that the extrapolation approach was valid and that a shortening of the reaction time by about 50% and even by about 80% had no strongly negative effect of the accuracy of the determination: the results obtained at the three different times are largely similar for the respective pulp samples. The error of the measurement was larger for pulps with high carbonyl content. In summary, the data extrapolation with the help of a reference pulp is an alternative to the homogeneous derivatization up to complete conversion. The analysis times can be shortened by more than 50%, albeit at the expense of accuracy. However, the error introduced by the extrapolation is rather small. Extrapolation could be completely avoided by using reaction times of approximately 700 h. It was experimentally verified that no degradation of the pulp under the prevailing conditions occurred, even within such long reaction times. 3.5. Side Reactions of the Marker (Reaction with Lactones and Hexenuronic Acids). To be able to attribute the fluorescence measured after labeling exclusively to carbonyl groups, the selectivity of the labeling reaction must be verified. The two structures in pulp, which might react


Figure 5. Carbonyl contents in different pulps: Results of homogeneous labeling over different reaction times followed by extrapolation toward complete conversion: A, BS EO; B, BS Z1; C, BS Z2; D, BS Z3; E, BS Z4; F, BS Z1P1; G, BS Z2P2; H, BS Z3P3; I, BS Z4P4; J, BS Z4P5; K, cotton linters; L, eucalyptus PHK. Abbreviations: BS ) beech sulfite, E ) E stage (extraction), O ) oxygen stage, Z ) ozone stage, P ) peroxide stage, PHK ) prehydrolysis kraft; numbers reflect the intensity of the respective treatment. Scheme 4. Examples of the Reaction of CCOA with Lactones and R,β-Unsaturated Acids

with the hydroxylamine anchor group of CCOA in side reactions, are lactones and hexenuronic acids. Reactions of hydroxylamines with lactones produce hydroxamic acids under suitable conditions.18 Also pulps contain lactone structures, which might therefore interfere with the carbonyl determination by consuming marker and increasing carbonyl contents. In the oxime method to determine carbonyls in pulp, the reaction of lactones in pulp with hydroxylamine is prevented by opening the lactones by means of zinc salts prior to derivatization.19 As models for lactone structures, δ-valerolactone (tetrahydropyran-2one) and γ-valerolactone (5-methyl-dihydrofuran-2-one) were used (see Scheme 4). Both compounds were allowed to react with equimolar amounts of CCOA in DMAc/LiCl (2.5%, w/v) in the presence of 1 equiv of triethylamine over 500 h; the progress of the reaction was followed by HPLC. While the CCOA concentration was constant in the absence of a model compound, γ-valerolactone decreased the amount of marker by 10%, δ-valerolactone by 20% after 500 h. However, the concentration of lactones in pulp is about 2 orders of magnitude (factor 100) lower than the one used for the model reactions, and the reaction rate of pulp is generally lower than that of low-molecular models (see


above). Hence, the contribution of labeled lactones to the overall fluorescence will be negligibly small, and the reaction of CCOA and lactones in pulp can be disregarded. Unbleached pulps and pulps with higher kappa numbers may contain R,β-unsaturated acids in the form of hexenuronic acids (4-deoxy-β-L-threo-hex-4-enopyranosyluronic acids), which are formed in alkaline media, for instance from 4-Omethyl-R-D-glucuronic acids in xylans by elimination of methanol. Reactions of hydroxylamines with R,β-unsaturated acids are known in organic chemistry,20 so that a reaction of CCOA with these structures appeared possible (see Scheme 4). The behavior of CCOA toward R,β-unsaturated acids was tested by means of acrylic acid as the model compound under conditions identical to those used for the testing of lactones. Also in the case of acrylic acid, 20% of the label was consumed after 500 h of reaction time. However, the same restrictions apply as for lactones: the concentration of hexenuronic acids in pulp is 2 (or more) orders of magnitude lower than the concentration of model compound used, and the reaction rate in pulp will be much lower than in the case of acrylic acid. Therefore, also hexenuronic acids can be neglected as an influencing factor for the determination of carbonyls by CCOA. Moreover, the dissolving pulps used within this work did not contain measurable amounts of hexenuronic acids, as determined according to the Gellerstedt method.21 Hexenuronic acids can easily be destroyed by treatment with Hg(II) salts, which can be used in the case of pulps with a supposedly high content of hexenuronic acids to exclude even the slightest influence on the measured carbonyl content (CAUTION! Mercury salts are known to be highly toxic!) For a prehydrolysis kraft pulp, which did not contain detectable amounts of hexenuronic acids, it was verified that the carbonyl content was unchanged by the Hg(II) treatment, as expected. Furthermore, it was demonstrated that the mercury salt treatment did not change the molecular weight. 3.6. Reproducibility of the Pulp Labeling Reaction. A main criterion for the applicability of a new analytical method is its reproducibility. To check the consistency of the results, a beech sulfite pulp was analyzed in four independent experiments in duplicate under the above standard conditions (DMAc/LiCl (2.5% w/v), 40 °C, 1 equiv of triethylamine, 504 h) and subsequently analyzed by GPC. The standard deviation was determined to be 2.88%, which attests to a very good reproducibility. 4. Optimization of the Labeling TechniquesHeterogeneous Reaction in Water. As an alternative to the labeling in homogeneous phase, also the applicability of heterogeneous labeling in aqueous media was tested. Heterogeneous derivatization would circumvent the time-consuming and laborious removal of excess marker by precipitation and redissolution of the labeled pulp and thus allow much shorter times for analysis. 4.1. Stability of the Marker. Also for aqueous media, the stability of the marker was verified. For that purpose, solutions of CCOA in buffer pH 4.0 were heated to different temperatures between 25 and 80 °C. The content of unchanged CCOA was determined by HPLC. The thermal behavior of CCOA in water is largely similar to that in

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Figure 6. Heterogeneous labeling of a beech sulfite pulp with CCOA in aqueous medium: the influence of the pH value.

DMAc/LiCl. It is completely stable at 40 °C, and at 60 °C a relatively slow decomposition sets in, which becomes quite fast at 80 °C. At this temperature, 95% of the label had decomposed within 48 h. From these results, the heterogeneous derivatization must be performed at a temperature of 40 °C or below. 4.2. Optimization of Reaction Conditions for the Labeling of Pulp. The pH value is an important influencing factor in the reaction of hydroxylamines and carbonyls in aqueous media. Experiments with low-molecular-weight model compounds have shown that the reaction rate is largely independent of the structure of the carbonyl compound in the pH range 2.5-5.0.22 Therefore, the optimization of the reaction conditions was started directly with pulp. For that purpose, beech sulfite pulp samples were suspended in water, transferred into aqueous buffer solution, and labeled with CCOA in a heterogeneous reaction at 40 °C. The CCOA concentration was 1.25 mg/mL corresponding to a 34-fold excess, calculated for an average carbonyl content of 20 µmol/g. The reaction was strongly dependent on the pH value of the reaction medium, with lower pH values increasing the reaction rate (Figure 6). At a pH of 4.0 the reaction was complete within 170 h, giving a carbonyl content of 24.6 µmol/g for the standard pulp, which agrees very well with the number of 26.1 µmol/g as determined by the homogeneous labeling procedure via extrapolation. It should be noticed that the final value for the carbonyl content was obtained directly after 168 h, whereas the homogeneous determination required prolonged reaction times or extrapolation toward complete conversion. For determination of the carbonyl content of an unknown pulp by heterogeneous derivatization in aqueous medium, the pulp was treated with excess marker at pH 4.0 at 40 °C for 168 h, then thoroughly washed to remove adhesive marker, and finally activated and dissolved for GPC analysis. Recording of reaction kinetics and extrapolation toward complete conversion, as required for the homogeneous labeling procedure, is not necessary, and the repeated precipitation-redissolution procedure becomes equally obsolete. Lowering the pH value below 4.0 was considered inappropriate due to possible cellulose chain degradation with resulting DP loss. Increasing the reaction temperature to

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Figure 7. The reaction of CCOA with lactones and acrylic acids in heterogeneous labeling of pulp.

achieve faster conversions was equally unsuitable since already a temperature of 60 °C produced a lower overall carbonyl content and caused a slow decrease in the carbonyl content which indicates the occurrence of degradation reactions, in addition to the thermal lability of the marker itself. 4.3. Side Reactions of the Marker with Lactones and Hexenuronic Acids. The influence of lactones and hexenuronic acids on the selectivity of the carbonyl labeling was also investigated for the heterogeneous procedure in aqueous media. CCOA was allowed to react with equimolar amounts of γ-valerolactone, δ-valerolactone, and acrylic acid, respectively, in aqueous buffer pH 4.0 at 40 °C. In all cases, the buffer solution additionally contained zinc ions, as Zn(II) was known to hydrolyze lactones in pulps.19 No influence of lactones on the result of the carbonyl labeling was found in these experiments (Figure 7). However, CCOA was consumed in the presence of acrylic acid to a significant extent. This is a clear indication that also hexenuronic acids in pulp would react with CCOA, so that the measured carbonyl contents of pulps containing hexenuronic acids could be too high. Since hexenuronic acids can be easily removed by treatment with mercury salts (CAUTION! Mercury salts are known to be highly toxic!)sand commercial dissolving pulps, as those used within this work, do not contain hexenuronic acids anywaysthe reaction with CCOA means no restriction to the general applicability of the method. 4.4. Reproducibility of the Pulp Labeling Reaction. As the heterogeneous procedure appeared most convenient, promising widest applicability in all fields of pulp and cellulose chemistry, a simple reproducibility test seemed to be insufficient, so that a comprehensive validation was performed, which will be reported in due course. 5. Comparison of the Homogeneous and the Heterogeneous Labeling Procedure. The homogeneous and heterogeneous derivatization was comprehensively compared by means of a set of 15 pulp samples. Each derivatization type for each pulp was analyzed in duplicate, evaluating Mw, the peak form of the fluorescence signal, and the overall carbonyl content. According to the optimized procedures, homogeneous derivatization was carried out in DMAc/LiCl


Figure 8. Comparison of the molecular weight data (Mw) before and after derivatization with CCOA for the homogeneous and the heterogeneous variant.

(2.5%, w/v) with 1 equiv of triethylamine relative to CCOA for 524 h at 40 °C, heterogeneous labeling in an acetic acid/ zinc acetate buffer pH 4.0 for 168 h at 40 °C. Any labeling method can only be useful if the molecular weight of the pulp is not negatively affected by the procedure, i.e., if no degradation of the pulp occurs. For testing, the data of unlabeled pulps were compared with those of labeled pulp. The Mw data were used, assuming that changes in the molecular weight distribution are reflected in the value of Mw. The results are reported in Figure 8, where a regression line with the slope of 1 means an Mw being unaffected by the reaction and thus represents the case of a degradationfree process. For both the heterogeneous and the homogeneous procedures, no statistically significant change of Mw was observed, which means that in both labeling procedures the reaction proceeded without noticeable cellulose degradation. It might be assumed that heterogeneous derivatization would not report all carbonyl groups equally well and that especially carbonyls in higher-molecular weight cellulose chains might be “disfavored” by accessibility and steric factors. However, a comparison of the fluorescence signals of homogeneously and heterogeneously labeled pulps revealed merely marginal differences for most samples as shown in Figure 9 for a beech sulfite pulp, indicating that the heterogeneous labeling gives only slightly decreased values for higher-molecular weight parts (and insignificantly increased values for lower-molecular weight parts) as compared to the homogeneous procedure. The derivatization procedures were also compared with regard to the total amount of carbonyls determined. In theory, both the heterogeneous and the homogeneous approach should produce the same values, but also in practice the results came very close to each other. This was demonstrated for a variety of different pulps (Figure 10). In the vast majority of cases, both methods yield nearly identical values. Homogeneous reaction with CCOA tends to give the larger values, the opposite behaviorswith the heterogeneous method reporting a higher carbonyl contentswas only observed for very few pulps. In some rare cases, the carbonyl content determined in water ranged up to 10% below the values determined by homogeneous labeling, which can only be


Figure 9. Homogeneous vs heterogeneous labeling, comparison of elution profiles.

Figure 10. Comparison of the homogeneous and the heterogeneous labeling procedure (overall carbonyl contents determined): A, BS EO; B, BS Z1; C, BS Z2; D, BS Z3; E, BS Z4; F, BS Z1P1; G, BS Z2P2; H, BS Z3P3; I, BS Z4P4; J, BS Z4P5; K, BS; L, cotton linters; M, eucalyptus PHK. Abbreviations: BS ) beech sulfite, E ) E stage (extraction), O ) oxygen stage, Z ) ozone stage, P ) peroxide stage, PHK ) prehydrolysis kraft; numbers reflect the intensity of the respective treatment.

attributed to accessibility or inaccuracy problems in the respective sample. Conclusions Two approaches aimed at accurate determination of carbonyl groups in cellulosics have been developed, both employing fluorescence labeling to convert the carbonyl structures into groups detectable by instrumental analysis. One method is working in homogeneous phase, the other in heterogeneous suspension. Both labeling procedures can be readily incorporated into a GPC system with RI and MALLS detection, as the fluorescence label CCOA does not interfere with the MALLS detection. Labeling of the carbonyls under the conditions used neither changes the solution state of the pulp nor causes cellulose degradation. The labeling procedures have been systematically elaborated, based on experiments with cellulose model compounds and optimization toward the most favorable reaction parameters.

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Critical Comparative Evaluation of the Methods: Advantages and Drawbacks. The homogeneous labeling procedure requires reaction times of 500 h and more to achieve a complete conversion of carbonyls. The determination of the carbonyl content at shorter reaction times requires, first, monitoring of the labeling kinetics, which is a rather laborious process involving numerous sample preparations (precipitation, washing, activation, and redissolution of pulps) and GPC runs, and, second, extrapolation toward complete conversion. Thus, an “indirect” mathematical step is involved in this case, even though the extrapolation is very simple due to the clear first-order kinetics, which produces highly consistent values. Nevertheless, the homogeneous procedure demands either long reaction times or the laborious derivatization procedure for numerous samples, which renders the homogeneous labeling approach largely impracticable for routine use with a high throughput. Heterogeneous labeling of pulp with CCOA is a direct process which is much faster and less laborious than the homogeneous alternative: it demands no extrapolation or calculation steps, the conversion is completed within 168 h, and monitoring of multiple samples with the concomitant sample preparation requirements is not needed. It must be stressed that the heterogeneous method represents no oversimplification that achieves speed at the expense of accuracy but is an individual method that provides similar consistent results as the homogeneous procedure. The heterogeneous approach thus appeared to be well suited to be used as a general method for the determination of carbonyls in cellulosics. Outlook. As will be described in a forthcoming report, the heterogeneous labeling method was validated, demonstrating that the approach is more reliable than any other method hitherto available. The results were compared to those from conventional methods, and carbonyl contents were determined for a variety of different pulps. The value of the method was demonstrated by means of several applications in the field of cellulose chemistry. Acknowledgment. The financial support by the Austrian Christian-Doppler-Forschungsgesellschaft and Lenzing AG, Lenzing, Austria, is gratefully acknowledged. References and Notes (1) Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U.; Wagenknecht, W. ComprehensiVe Cellulose Chemistry; Wiley-VCH: Weinheim, Ger-

many, 1998; Vol. 2, pp 302-314. (2) Included are also 1,2-dicarbonyls (-CO-CO-) and dialdehyde groups. Carboxyl groups (COOH), which have a much lower carbonyl reactivity, are explicitly not covered in the following. (3) (a) See any text book of pulping and bleaching chemistry. (b) Schleicher, H.; Lang, H. Das Papier 1994, 12, 765-768. (c) Sixta, H. Habilitation thesis, Technical University of Graz, 1995. (4) Lewin, M. Macromol. Symp. 1997, 118, 715-724. (5) See for instance: Beyer, M.; Ba¨urich, D.; Fischer, K. Das Papier 1995, 10A, 8-14. (6) Tappi Test Methods 1989-1999, Tappi Press: Atlanta, 1998, T430. (7) Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U.; Wagenknecht, W. ComprehensiVe Cellulose Chemistry; Wiley-VCH: Weinheim, Germany, 1998; Vol. 1, p 236. (8) Lewin, M.; Epstein, J. A. J. Polym. Sci. 1962, 58, 1023. (9) Potthast, A.; Rosenau, T. Buchner, R.; Ro¨der, T.; Ebner, G.; Sixta, H.; Kosma, P. Cellulose 2002, 9, 41-53. (10) Schelosky, N.; Ro¨der, T.; Baldinger, T. Das Papier 1999, 53, 728738. (11) Ro¨hrling, J. PhD thesis, University of Agricultural Sciences/University of Technology, Vienna, 2002. (12) Dawsey, T. R.; McCormick, C. L. J. Macromol. Sci.-ReV. Macromol. Chem. Phys. 1990, C30 (3&4), 405-440. (13) Anderson, J. M. Anal. Biochem. 1986, 152, 146-153. (14) (a) Boturyn, D.; Boudali, A.; Constant, J. F.; Defrancq, E.; Lhomme, J. Tetrahedron 1997, 53, 5485-5492. (b) Houdier, S.; Legrand, M.; Boturyn, D.; Croze, S.; Defrancq, E.; Lhomme, J. Anal. Chim. Acta 1999, 382, 253-263. (c) Houdier, S.; Perrier, S.; Defrancq, E.; Legrand, M. Anal. Chim. Acta 2000, 412, 221-233. (15) Ro¨hrling, J.; Potthast, A.; Rosenau, T.; Lange, T.; Borgards, A.; Sixta, H.; Kosma, P. Synlett 2001, 5, 682-684. (16) Ro¨hrling, J.; Potthast, A.; Rosenau, T.; Adorjan, I.; Hofinger, A.; Kosma, P. Carbohydr. Res. 2002, 337, 691-700. (17) Rosenau, T.; Potthast, A.; Hofinger, A.; Kosma, P. Holzforschung 2000, 55 (6), 661-666. (18) Connell, R. D. N-Heterosubstituted Amines. In ComprehensiVe Organic Functional Group Transformations; Katritzky, A. R., MethCohn, O., Rees, C. W., Eds.; Pergamon Press: Cambridge, U.K., 1995; Vol. 5, pp 313-392. (19) Rehder, W. PhD thesis, University of Rostock, German Democratic Republic, 1965. (20) (a) Zeeh, B.; Metzger H. Methoden zur Herstellung und Umwandlung von Hydroxylaminen. In Methoden der organischen Chemie (HoubenWeyl); Mu¨ller, E., Ed., Georg Thieme: Stuttgart, 1971; Vol. X/1, pp 1091-1279. (b) Bowman, W. R.; Mormon, R. J. Alkylnitrogen Compounds: Compounds with N-Halogen, N-O, N-S, N-Se, and N-Te Functional Groups. In ComprehensiVe Organic Functional Group Transformations; Katritzky, A. R., Meth-Cohn, O., Rees, C. W., Eds.; Pergamon Press: Cambridge, U.K., 1995; Vol. 2, pp 333370. (21) (a) Gellerstedt, G.; Li, J. Carbohydr. Res. 1996, 294, 41-51. (b) Tenkanen, M.; Gellerstedt, G.; Vuorinen, T.; Teleman, A.; Perttula, M.; Li, J.; Buchert, J. J. Pulp Pap. Sci. 1999, 25, 306-311. (22) (a) Jencks, W. P. J. Am. Chem. Soc. 1959, 81, 475. (b) Houdier, S.; Legrand, M.; Boturyn, D.; Croze, S.; Defrancq, E.; Lhomme, J. Anal. Chim. Acta 1999, 382, 253-263.

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A Novel Method for the Determination of Carbonyl Groups in

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