A Novel Method for the Determination of Carbonyl Groups in

according to Funk et al.3 with the help of statistics software.4. Naturally, also the present labeling method cannot provide the “true” values for...
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Biomacromolecules 2002, 3, 969-975

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A Novel Method for the Determination of Carbonyl Groups in Cellulosics by Fluorescence Labeling. 2. Validation and Applications ¨ rgen Ro ¨ hrling,† Antje Potthast,*,† Thomas Rosenau,† Thomas Lange,† Andrea Borgards,‡ 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

Fluorescence labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide was shown to be a promising approach toward the accurate determination of carbonyls in cellulosic materials. Combined with gel permeation chromatography in DMAc/LiCl with fluorescence/multiple-angle laser light scattering/refractive index detection, the method yields carbonyl profiles relative to the molecular weight of the cellulosic material. The derivatization procedure can be carried out either homogeneously in DMAc/ LiCl or advantageously as heterogeneous derivatization in aqueous buffer. The heterogeneous carbonyl group determination, offering shorter reaction times and increased simplicity as compared to the homogeneous approach, was comprehensively validated. The carbonyl content in numerous dissolving pulps of different provenience has been determined, including pulps with carbonyl contents additionally increased by oxidative treatment. The method was also applied to follow bleaching sequences and oxidative treatments of pulps. Introduction Responding to a lack of reliable and accurate methods for the determination of carbonyl groups, such as keto, 1,2diketo, and aldehyde structures, in pulps and other cellulosics, a novel approach was developed, based on fluorescence labeling of carbonyls with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide (“CCOA”). It was considered imperative that the procedure can be incorporated into gel permeation chromatography (GPC) systems, so that the labeling procedure would provide carbonyl profiles of the respective cellulosics relative to their molecular weight, when combined with multiangle laser light scattering (MALLS), refractive index (RI), and fluorescence detection. Furthermore, it was demonstrated that the labeling does not lead to any cellulose degradation under the prevailing conditions.1 Two versions of the labeling method were elaborated, a homogeneous procedure in DMAc/LiCl (2.5%, w/v) and a heterogeneous derivatization in aqueous buffer solution. Both variants have been thoroughly optimized with regard to reaction conditions (temperature, reagent ratios, catalysts, stability of reagent, and labeled products), completeness of conversion, and reproducibility. The homogeneous procedure requires prolonged reaction times, which can, however, be reduced by recording a reaction kinetics and subsequently extrapolating toward complete conversion. In addition, for each sample the laborious and time-consuming cycle of precipitation, washing, and redissolution of the pulp is required to remove excess marker. The heterogeneous † ‡

University of Agricultural Sciences Vienna. Lenzing AG.

labeling is advantageous for various reasons: it is completed at much shorter derivatization times, recording kinetics is unnecessary, the precipitation-redissolution procedure becomes obsolete, and the results are as consistent as those obtained according to the homogeneous working procedure. The heterogeneous method (and only this one) thus clearly has the potential to become a routine method in pulp and cellulose chemistry. To place the labeling approach at the cellulose chemists’ disposal as a general analytical method, several tasks, must be performed beforehand, which will be delineated in this second part of our studies: validation of the heterogeneous labeling procedure; selection of reference pulps; testing of the general applicability of the method by determining the carbonyl content of multiple pulps; application of the method in examples to demonstrate its use in cellulose and pulping chemistry. Materials and Methods Chemicals, general analytics, GPC, and high-performance liquid chromatography (HPLC) systems components were used as described previously.1 The GPC system, a modification of the setup described by Schelosky et al.,2 consisted of fluorescence, MALLS, and refractive index (RI) detectors, with automatic injection and four serial columns. Molecular weight distribution (MWD) and related polymer-relevant 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). The following general GPC parameters were used: eluant, DMAc/LiCl (0.9%, w/v); flow, 1.00 mL/min; columns, four, PL gel mixedA ALS, 20

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µm, 7.5 × 300 mm; fluorescence detection, 290 nm excitation, 340 nm emission; injection volume, 100 µL; run time, 45 min. Activation of Pulp Samples. To achieve a “good” solubility in DMAc/LiCl (9%, w/v), the pulp samples had to be activated, no matter if genuine pulp or labeled pulp had to be dissolved. The pulp samples were activated by solvent exchange (H2O to DMAc) followed by agitating in DMAc and filtration, which produces efficiently activated, i.e., readily soluble samples. General Procedure for the Determination of Carbonyls in Pulp by Heterogeneous Fluorescence Labeling. A CCOA stock solution was prepared by dissolving the label (62.50 mg) in 50 mL of 20 mM zinc acetate buffer, pH 4.00. Pulp, corresponding to 20-25 mg of dry pulp, was suspended in the acetate buffer containing the label (4 mL). 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 filters, and analyzed by GPC directly yielding the carbonyl content in the respective pulp. Analysis of Pulps. For the analysis of the carbonyl profiles in genuine pulps and chemically treated pulps, the general analytical procedure was used. Calibration of the system was done by means of reference pulps. For the determination of the overall carbonyl content, the carbonyl peak area was normalized with regard to the injected mass. After five samples, a standard was injected. All data result from at least duplicate runs. Results and Discussion 1. General. If the determination of carbonyls in pulp by heterogeneous labeling is to be introduced as a routine method, the main analytical process parameters must be known, including precision of the method, detection limit, limit of determination, robustness toward changes of process parameters (temperature, pH, reaction time, marker concentration), as well as constancy of the procedure over a certain period of time. Determination of these data was performed according to Funk et al.3 with the help of statistics software.4 Naturally, also the present labeling method cannot provide the “true” values for the carbonyl contents in cellulosics, but the comprehensive method development performed and the thorough validation make it reasonable to assume that the method reports “better” values than the traditional approaches. The procedure to be validated encompasses the heterogeneous derivatization of pulp and the subsequent analysis by GPC. The derivatization was performed according to the general working procedure described in the Materials and Methods section. The GPC analysis provides the molecular weight distribution and absolute mass from the MALLS and RI detector signals, and a fluorescence detector output. The integrated fluorescence signal and the amount of pulp injected (from the RI signal) yield the overall carbonyl content. The validation assumes that a constant overall carbonyl content

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Figure 1. Reaction kinetics of the six reference standard pulps: A, BS Z4P4; B, BS; C, SS; D, ES; E, eucalyptus PHK; F, cotton linters. Abbreviations: BS ) beech sulfite; SS ) spruce sulfite; ES ) eucalyptus sulfite; Z ) ozone stage; P ) peroxide stage; PHK ) prehydrolysis kraft.

means an unchanged peak form of the fluorescence signal and thus a carbonyl distribution being unchanged by reaction parameters. 2. Selection and Analysis of Reference Pulps. The calibration of the GPC system with CCOA solutionseven though readily applicableshas two distinct drawbacks: the injection volume must be quartered to obtain a peak width comparable to that of pulps, and the run time must be extended since the low-molecular marker elutes very slowly only.5 The calibration with pulps of known carbonyl contents would offer an advantageous alternative. With such reference pulps, a calibration curve can be obtained which can then be used for the determination of the CdO content of unknown pulps. However, reference cellulosic material with exactly known carbonyl contents is not commercially available, so that the reference pulps themselves must be derivatized and thoroughly analyzed beforehand. For that purpose, such pulps were selected as calibration standards, whose carbonyl content would cover the whole range expectable in relevant cellulosic material. To make sure that the labeling of carbonyls was quantitative within the normal reaction time of 168 h, kinetics of the respective reactions were recorded for all selected reference pulps (Figure 1). It was demonstrated that in all cases the conversion was complete. Moreover, a complete conversion was achieved much earlier, so that small changes in the reaction time would not affect the conversion. To determine the carbonyl content of the reference pulps, the material was labeled according to the standard procedure in sextuplicate (three different runs in duplicate), with calibration against CCOA solutions. The mean value of the outlier-free measurements was taken as the overall carbonyl content in these pulps, which were used as standards for the determination of CdO amounts in unknown samples; see Table 1 for the data. The use of reference pulps for the calibration has another advantage besides shorter analysis times. The reference pulps have principally the same reaction behavior as the analytes. This means that the procedure becomes less sensitive toward changes in the reaction conditions, since changes in the

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Determination of Carbonyl Groups in Cellulosics Table 1. Determination of the Carbonyl Content in Reference Standard Pulps pulpsa

mean valueb

SDc

CIMd

rel. CIMe

A B C D E F

40.58 23.85 16.57 10.02 4.63 3.49

3.12 1.62 1.36 1.19 0.42 0.29

3.28 1.70 1.43 1.25 0.44 0.30

8.08 7.13 8.61 12.45 9.53 8.61

a For abbreviations see caption to Figure 1. b In µmol/g. c Standard deviation in µmol/g. d Confidence interval of the mean (95%) in µmol/g. e Relative confidence interval of the mean (95%) in %.

Figure 2. Method calibration with reference pulps, fluorescence detection at 340 nm.

conversion due to changed reaction parameters should have the same effect on all pulps, i.e., reference pulps and unknown samples. 3. Calibration. The calibration was carried out by means of the six reference pulps, giving a calibrated range of 3.4940.58 µmol/g for the carbonyl content. The carbonyl content of almost all pulpsswith the exception of some “exotic” examples with little practical relevancesranges within these dimensions. The calibration curves are shown in Figure 2, the corresponding statistical parameters (confidence interval 95%) for the calibration line Y ) AX + B are as follows: sensitivity A ) 58.49 ( 3.37; intercept B ) 59.83 ( 70.38; N ) 6 data points; mean of working range X, 16.52; mean of working range Y, 1026.36; correlation coefficient R, 0.99914; residual standard deviation sy, 38.06; process standard deviation sx0, 0.6507; process variation coefficient ()relative process standard deviation) Vx0, 3.94%. The calibration curve is linear. By means of the Mandel test (P ) 99%) it was demonstrated that a quadratic calibration curve produced no significantly better results. 4. Validation of the Method. The detection limit, based on a signal/noise ratio of 3, was determined to be 0.0142 µmol/g; the limit of quantification, based on a signal/noise ratio of 10, was 0.0476 µmol/g.6 By means of recovery functions, the robustness of the method was evaluated, i.e., the influence of changes in the analysis parameters on the resulting analysis value. For that purpose, the reaction parameters were varied, and the result was compared to that of the standard procedure. Thus, the reaction temperature was increased to 42 °C and decreased

Table 2. Determination of the Carbonyl Content (µmol/g) in Control Pulpsa

mean standard deviation sw standard deviation sb standard deviation st

BS Z1

PHK Z1

SS Z1

26.49 0.9395 0.9282 0.9371

26.47 0.7403 0.3819 0.6787

30.91 1.1088 1.3345 1.1616

a Abbreviations: Z ) ozone stage; BS Z1, beech sulfite Z1; PHK Z1, eucalyptus prehydrolysis kraft Z1; SS Z1, spruce sulfite Z1.

to 38 °C, pH value was changed to 3.8 and 4.2, the reaction time was prolonged to 192 h and shortened to 144 h, and the amount of marker used, generally 5 mg/4 mL, was set to 4 mg/4 mL and 6 mg/4 mL. The process standard deviation of the calibration function of the fundamental procedure and the residual standard deviation of the recovery functions showed no significant differences (P ) 99%). It was shown that in all cases a proportional systematic error was absent as the confidence interval of the slope included the value 1 (P ) 99%). Similarly, a constant systematic error was proven to be absent by showing that the confidence interval of the intercept included the value 0 (P ) 99%). An optimum routine analytical method should continuously produce values as accurate and precise as possible. Over longer periods of time, the quality can be affected by environmental or human influences. To determine the sensitivity of the method toward such effects, the test for time dependency was performed to detect time-dependent systematic trends. Thus, six independent series, each with four determinations were measured for three control pulps, beech sulfite Z1 (BS Z1), eucalyptus prehydrolysis kraft Z1 (PHK Z1), and spruce sulfite Z1 (SS Z1). The standard deviation within a series sw, the standard deviation between the series sb, and the total standard deviation st were determined (for the data see Table 2). In no case a statistically significant increase in sb relative to sw was observed. 5. Simultaneous Determination of the Molecular Weight Distribution and the Carbonyl Content of Selected PulpssApplications. 5.1. Analysis of the Molecular Weight Distribution. As already shown, the derivatization reaction does not change the molecular weight distribution (MWD) of the cellulose sample, which is an indispensable precondition.7 This was additionally confirmed by a comprehensive analytical series on 41 different pulps, whose MWD was determined before and after heterogeneous CCOA labeling in duplicate runs.8 Figure 3 gives a graphic representation of the results. If the Mw of labeled pulps is plotted versus the Mw of the respective unlabeled sample, a regression line with slope 1 and intercept 0 should result in the idealized case for exactly unchanged Mw values. The pulps investigated produced a regression line with the slope 1.03, and 0 fell within the 95% confidence interval of the intercept. These results demonstrate that CCOA derivatization of pulps does not systematically affect the Mw of the pulp samples. Provided that an unchanged Mw reflects an unchanged MWD, it can be concluded that also the MWD remains unaffected by the labeling procedure. 5.2. Comparison to Traditional Methods. The new approach was compared with the copper number (Cu#) determination as a traditional method for carbonyl group

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Figure 3. Comparison of Mw before and after derivatization for 41 selected pulps.

Figure 4. Comparison of carbonyl contents according to the CCOA method with the copper number.

determination in pulps. The copper number relates the reduction of two-valent copper to the amount of carbonyl groups in cellulosics, which are oxidized in turn, but the method suffers from deficient knowledge of underlying mechanisms and detectable functionalities. Regression analysis showed a linear correlation between the CCOA values and the copper numbers, based on 40 pulps investigated (Figure 4). The relationship allows calculation of the CCOA values if only the copper numbers are available. However, the mathematical conversion according to CCOA ) (Cu# - 0.07)/0.06 remains a rather rough approximation. Also the agreement between the CCOA method and the oxime method was tested.9 According to the latter, the carbonyl content is related to the nitrogen content as determined by elemental analysis or by the Kjeldahl procedure. No statistical data, such as standard deviation, limit of detection, or reproducibility, were available. For pulps with a low carbonyl content (Figure 5, pulps B-D), the CCOA method correctly relates increasing overall carbonyl contents with increasing bleaching intensity, while the oxime method fails to report such differences. Only for higher carbonyl contents do the values become comparable (Figure 5). 5.3. Analysis of the Overall Carbonyl Content. The ability of the CCOA method to determine the overall

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Figure 5. Comparison of CCOA and oxime method: A, Eucalyptus PHK; B, BS Z1P1; C, BS Z3P3; D, BS Z4P5; E, BS F, BS H25; G, BS H50; H, BS H75. Abbreviations: BS ) beech sulfite; Z ) ozone stage; P ) peroxide stage; H ) hypochlorite stage; PHK ) prehydrolysis kraft; increasing numbers reflect increasing intensity of the respective treatment.

Figure 6. Overall carbonyl content (bars) and Mw (bullets) of a beech sulfite (BS) pulp, measured along a bleaching sequence with ozone and ozone/peroxide, respectively (kappa numbers are given in parentheses): A, BS EO (1.6); B, BS Z1 (1.09); C, BS Z1P1 (0.73); D, BS Z2 (0.42); E, BS Z2P2 (0.18); F, BS Z3 (0.23); G, BS Z3P3 (0.11); H, BS Z4 (0.20); I, BS Z4P4 (0.10); J, BS Z4P5 (0.10). Abbreviations: Z ) ozone stage; P ) peroxide stage; increasing numbers reflect increasing intensity of the respective treatment.

carbonyl content was used to monitor oxidative treatments of pulps, two examples of which, ozone/peroxide bleaching sequences and hypochlorite treatment, are reported here. For all pulps, the carbonyl contents were determined according to the given standard procedure. It was experimentally shown, that oxidative treatment of the pulps causes an increase in the carbonyl content, as expected. In ozone bleaching (“Z stage”), this increase was clearly dependent on the ozone charge. A subsequent peroxide treatment (“P stage”), comprising peroxide treatment and alkaline extraction, decreases the carbonyl content in turn. This is due to oxidative transformations of carbonyl groups and extraction of carbonyl containing lower molecular weight material, as well-known from bleaching chemistry. Figure 6 presents the data of bleached beech sulfite pulps, which had undergone different treatments with either ozone (Z) or ozone/peroxide (ZP). Likewise, Figure 7 describes the bleaching of a spruce sulfite and a eucalyptus prehydrolysis kraft pulp. Both figures clearly demonstrate that cellulose degradation, i.e., a DP loss, increases with increas-

Determination of Carbonyl Groups in Cellulosics

Figure 7. Overall carbonyl content (bars) and Mw (bullets) of a spruce sulfite (SS) pulp and a eucalyptus prehydrolysis kraft (PHK) pulp, measured along a bleaching sequence with ozone and ozone/ peroxide, respectively (kappa numbers are given in parentheses): A, PHK (0.37); B, PHK Z1 (0.21); C, PHK Z1P1 (0.14); D, PHK Z1 Z2 (0.23); E, PHK Z2P2 (0.22); F, SS (0.55); G, SS Z1 (0.15); H, SS Z1P1 (0.11); I, SS Z2 (0.16); J, SS Z2P2 (0.09). Abbreviations: Z ) ozone stage; P ) peroxide stage; increasing numbers reflect increasing intensity of the respective treatment.

ing bleaching intensity, with ozone/peroxide combinations being especially critical. Similar observations were made for a hypochlorite bleaching sequence (“H stage”), as shown in Figure 8 for a beech sulfite pulp. Also here, the carbonyl content increased with increasing hypochlorite charge: from a carbonyl content of 24 µmol/g in the original pulp to a content of 149 µmol/g in the hypochlorite-treated beech pulp sample H100. At the same time, a severe DP loss was observed. Reductive treatment of pulp by sodium borohydride effectively decreases the number of detectable carbonyls: the CdO content of the original pulp was lowered to approximately one-third after a reaction time of 1 h; cf. Figure 8. The borohydride reduction seemed to be complete within this time span, as an extended reduction over 72 h did not further diminish the carbonyl content. Independent of the reaction time applied, the Mw remained unchanged by the reduction. 5.4. Carbonyl Group Profiles. If only the overall carbonyl content of a cellulose sample is consideredsas important as this value might besinformation is lost, since only a sum parameter is provided and possible differences between fractions of different molecular weight are leveled. Only carbonyl group profiles would convey the full amount of information. With the CCOA labeling having been elaborated into a general method, it became possible for the first time to determine the results of chemical treatments of cellulosics not only in terms of the overall carbonyl content but also with regard to the carbonyl content relative to the molecular weight distribution. As the CCOA labeling is actually a precolumn derivatization procedure, the subsequent GPC analysis yields the carbonyl profiles for the respective samples and reports possible changes in these profiles caused by chemical treatments.10 In some cases, the carbonyl content determined by the fluorescence method ranges below the number, which is derived from the theoretical content of reducing end groups, i.e., one end group per chain. This might be due to two

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Figure 8. Overall carbonyl content (bars) and Mw (bullets) of a beech sulfite (BS) pulp, measured along a hypochlorite bleaching sequence and a reductive treatment with NaBH4: A, BS (starting material); B, BS B1; C, BS B2; D, BS H25; E, BS H50; F, BS H75; G, BS H100. Abbreviations: H ) hypochlorite stage; B ) borohydride reduction; increasing numbers reflect increasing intensity of the respective treatment.

reasons: first, it could be possible that the analytical method does not cover all carbonyl groups so that the derivatization remains incomplete, and second, some of the end groups could not be present in detectable form, for instance as carboxyl groups or as acetals. The comprehensive model compound experiments, reaction optimization, and kinetics provide evidence for the fact that the derivatization of carbonyl groups is indeed complete. The observed differences would thus likely be due to end groups having been chemically modified into structures which do not react with the fluorescence label. This is supported by the fact that the sum of carbonyl groups, determined according to the CCOA method, and carboxyl groups, determined by the methylene blue method, is for all pulps larger than the theoretical “oneper-chain” content of reducing end groups. In cellulose chemistry, the average degree of substitution (DS) denotes the number of substituted OH groups per anhydroglucose unit; it is most frequently used for cellulose ethers or ethers. The DS thus reflects the completeness of a chemical modification at the hydroxyl groups of the polysaccharide. In the following, the term “carbonyl DS” shall be used to describe the average content of CO groups per anhydroglucose unit.11 The main prerequisite to the analysis of the carbonyl DS relative to the chain length is the simultaneous recording of a mass-proportional signal and a substituent-proportional signal, which are provided by the RI detector and the fluorescence detector, respectively, in the present method. Both outputs were used to calculate the carbonyl DS profiles. In general, DS or ∆DS plots are very suitable graphic representations to report even slight differences in carbonyl contents. The typical decaying curve form of the DS graphs results from the higher number of reducing end carbonyl groups per anhydroglucose units in shorter chains. Especially ∆DS plots, which simply give the difference between two DS curves, facilitate the comparison of two samples with regard to their carbonyl contents relative to the molecular weight. They allow, for instance, straightforward analysis of how a chemical treatment increases or decreases the carbonyl content in certain molecular weight ranges.

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Figure 9. Carbonyl DS and differential MWD of a beech sulfite (BS) pulp after ozone-bleaching stages (Z). Increasing numbers reflect increasing intensity of the ozone treatment: A, starting pulp (BS EO); B, BS Z2; C, BS Z3; D, BS Z4.

Figure 10. Carbonyl DS and differential MWD of a beech sulfite (BS) pulp after a ozone (Z) and a peroxide (P) treatment: A, starting pulp (BS EO); B, BS Z3; C, BS Z3P3.

A first example of the value of DS profiles is displayed in Figure 9, which delineates the curves for ozone-bleached beech sulfite pulps. With increasing bleaching intensity, the carbonyl DS was increased for medium and high molecular weight regions but dropped below the value of the starting material for low molecular weights. The boundary between DS increase and DS decrease ranged around an Mw of 10000-50000 g/mol. Thus, the ozone treatment is an interplay between carbonyl-generating and carbonyl-consuming processes. Such detailed descriptions of the carbonyl profiles would be impossible to make simply by the data of the overall carbonyl content. Reducing end groups represent the major part of the total amount of carbonyl groups in cellulose chains with a low DP. Upon ozone treatment, these reducing end groups are oxidized to lactones and carboxylic acids, so that an ozone bleaching stage lowered the carbonyl DS in the lower molecular weight regions. In cellulose chains with a high molecular weight, however, the contribution of reducing end groups to the total amount of carbonyls becomes much smaller, so that the oxidation of reducing end groups is overcompensated by the introduction of new carbonyl functions. The generation of carbonyls predominantly into higher molecular weight material might explain the well-

known observation that ozone-bleached pulps suffer a severe DP loss in a subsequent P stage: when the carbonyls introduced upon ozone treatment are reduced by NaBH4, the pulp is rendered largely insensitive toward the subsequent peroxide treatment.12 With the pulp used, a very low ozone charge did not significantly increase the carbonyl content, since preferably the residual lignin present in the material is attacked by the oxidant.13 The progressing degradation of the polysaccharide material upon further oxidation, as reflected by the decreasing Mw, is clearly visible in Figure 9. The effect of a peroxide bleaching stage is demonstrated in Figure 10: a standard beech sulfite pulp was subjected to an ozone treatment followed by a peroxide bleaching stage. Again, it is obvious that the initial ozone treatment increased the carbonyl content at molecular weights above 20000 g/mol and decreased it below this value. Also the DP loss is evident. The subsequent peroxide stage has a quite beneficial effect with regard to the CdO content: in all molecular weight ranges the carbonyl content is decreased as compared to the ozone-bleached material. Moreover, the DS profile falls even below the level of the unbleached starting material at molecular weights below 105 and ranged only slightly above the curve for the initial pulp above 105 g/mol.

Determination of Carbonyl Groups in Cellulosics

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Figure 11. Carbonyl DS and differential MWD of a beech sulfite (BS) pulp after hypochlorite (H) treatment. Increasing numbers reflect increasing intensity of the hypochlorite treatment: A, starting pulp (BS); B, BS H25; C, BS H50; D, BS H100.

Nevertheless, the overall carbonyl content was slightly increased from 25 to 30 µmol/g, which shows the limitations of this sum parameters with regard to a detailed interpretation of the results. The increase in the overall carbonyl content is exclusively due to a DP loss, with concomitant increase in reducing end groups. A comparison of the graphs for the starting pulp and the Z/P-treated pulp shows that this DP loss occurs predominantly in the higher molecular weight region: the differential MWD curves below a Mw of 10000 g/mol are nearly identical, whereas the curves above 10000 g/mol are shifted to lower values; cf. Figure 10. Thus, the degradation produces mainly fragments of a molecular weight larger than 104. This interpretation is justified since the yield loss upon bleaching is only 0.5% so that almost no material is lost. Thus, a chain cleavage occurs chiefly in the same molecular weight ranges, in which carbonyl groups are introduced during the ozone treatment. In the low molecular weight range, which is only little affected by the Z stage, also the chain degradation is small. As a last example for the applicability of the CCOA method, the effect of an intensive hypochlorite treatment of pulp (“H stage”) at pH 7.0, aimed at producing highly oxidized material, is presented; see Figure 11. The progressing DP loss with increasing bleaching intensity was apparent. More intriguing is the effect of the treatment on the carbonyl profile, however. The carbonyl DS generally ranged above the values for the genuine pulp but increased only slightly with enhanced hypochlorite charge in higher molecular weight regions. In contrast, carbonyl functions were mainly introduced into shorter chains below a molecular weight of about 30000. This is a major difference to the abovediscussed ozone and peroxide-treated pulps. Conclusions The analytical method for determination of carbonyl groups in cellulosiscs by heterogeneous labeling with CCOA followed by GPC with fluorescence, MALLS, and RI detection was validated. The system was calibrated by means of six reference pulps covering a wide range of carbonyl contents. Already the overall carbonyl content is a valuable parameter to monitor the effect of oxidative chemical treatments. However, the method even provides carbonyl

profiles relative to the molecular weight, which allow a much more precise evaluation of oxidative changes. Evaluation of carbonyl profiles can conveniently be done by means of “carbonyl DS” or “carbonyl ∆DS” plots. Outlook. As carbonyl groups play a key role in many cellulose reactions, the CCOA method can realistically be expected to find ample applicability in the various fields of cellulose chemistry. In future studies, we will employ the labeling procedure to help clarify the role of carbonyls in cellulose-related processes, e.g., chromophore generation in cellulose/NMMO solution as used in the Lyocell process and other phenomena. Acknowledgment. The financial support by the Austrian Christian-Doppler-Forschungsgesellschaft and Lenzing AG, Lenzing, Austria, is gratefully acknowledged. References and Notes (1) See part 1 of this study. (2) Schelosky, N.; Ro¨der, T.; Baldinger, T. Das Papier 1999, 53, 728738. (3) Funk, W.; Dammann, V.; Donnevert, G. Quality Assurance in Analytical Chemistry; Wiley-VCH: Weinheim, 1995. (4) Lernhardt, U.; Kleiner, J. SQS98, Build 4.2.4, Perkin-Elmer Germany, October 1998. (5) Cf. part 1: method development. (6) Riley, C. M.; Rosanske, T. W. DeVelopment and Validation of Analytical Methods; Elsevier Science Ltd.: Oxford, 1996. (7) Cf. part 1: section 5 and Figure 8. (8) Detailed MWD data and carbonyl contents of these pulps will be published elsewhere. (9) Samples were analyzed in a different laboratory. (10) The same principle was used, for instance, by Fischer et al. to determine the xanthogenate groups in viscose samples or methyl groups in methyl cellulose: Fischer, K.; Koch, R.; Fischer, M.; Schmidt, I. Das Papier 1999, 53, 722-727. (11) Strictly speaking, introduction of carbonyl groups by oxidative modifications of cellulose is no “substitution”, so that usage of the expression “degree of substitution” would be inappropriate. Nevertheless, the term shall be maintained for convenience. (12) Chirat, C.; Lachenal, D. Holzforschung 1994, 48 Suppl., 133-139. (13) (a) Olkkonen, C.; Tylli, H.; Forsskahl, I.; Fuhrmann, A.; Hausalo, T.; Tamminen, T.; Hortling, B.; Janson, J. Holzforschung 2000, 54, 397-406. (b) Reference 12.

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