TEMPO-Mediated Oxidation of Cellulose III

TEMPO-Mediated Oxidation of Cellulose III

Denilson da Silva Perez,† Suzelei Montanari, and Michel R. Vignon*

Centre de Recherches sur les Macromolecules Vegetales, (CERMAV-CNRS), Universite Joseph Fourier,BP 53, 38041, Grenoble Cedex 9, France

Received May 12, 2003; Revised Manuscript Received July 10, 2003

Various cellulose samples converted into cellulose III by two different ammonia treatments, either liquid orgaseous, were reacted with catalytic amounts of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), sodiumhypochlorite, and sodium bromide in water. A substantial increase in the reactivity of cellulose III sampleswas observed in comparison to those in cellulose I, and a relationship between oxidation conditions andcellulose primary hydroxyl groups accessibility was directly established. For the characterization, we haveused several methods, mainly13C NMR, methylene blue adsorption, FTIR, and conductometric titration. Inall samples, the primary alcohol groups were selectively oxidized into carboxyl groups, provided the sodiumhypochlorite is added dropwise and the reaction is performed at constant pH 10.

Introduction

The selective oxidation of the primary alcohol group ofpolysaccharides, yielding polyuronic acids, has been studiedfor more than a half-century.1,2 Such derivatives are of greatinterest because they yield not only polyelectrolytes but alsovaluable intermediates. A number of applications have beendescribed for these oxidized carbohydrates. They rely onsome of their specific properties, ranging from gelation, tocomplexation, antiflocculation, adhesion, as well as a numberof biological activities.

The oxidation of primary alcohol groups in naturalpolysaccharides, catalyzed by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), has been recently proposed as amore selective, faster, and better-controlled method,3-5 asopposed to the traditional procedure using nitrite/nitrate inconcentrated phosphoric acid.1,2 NaOBr, generated in situby NaOCl and NaBr, is used to regenerate the catalyst. Thismethod was first proposed for water-soluble polysac-charides,3-7 namely, starch, inulin, amylodextrin, pullulan,alternan, amylopectin, chitosan, and galactomannan, and laterextended to water-insoluble products,6-11 such as cellulose,amylose, and chitin.

The success of using the TEMPO-NaCl-NaBr oxidationmethod for cellulose to produce water soluble polyglucuronicacid seems to depend on the accessibility and on thecrystalline state of the starting material. In the case of nativecellulose, Chang and Robyt6 have observed a significantincrease in the water-solubility of the oxidized productobtained from native cellulose (cellulose I). In fact, crystallinenative cellulose seems to become oxidized only at the crystalsurfaces, a phenomenon that can be interesting for subsequentgrafting or derivatization purposes.11 It has been shown thatthe cellulose decrystallization leads to a substantial increase

in the polyglucuronic acid yield, but still minor quantitiesof water-insoluble material remained in all preparations.9 Itseems that cellulose can be fully oxidized to yield purepolyglucuronic acid only if regenerated or mercerizedcellulose samples (cellulose II) are used.8

The swelling of cellulose in liquid ammonia (L-NH3) orin molecules such as amines, diamines, or polyamines is asimple and classical way to increase the accessibility ofcrystalline cellulose.12,13 This procedure that leads to thecellulose III allomorphs,14,15 III I from cellulose I and IIIIIfrom cellulose II, has been used frequently to improve thereactivity of crystalline cellulose for the preparation ofderivatives.16,17 The conversion of cellulose to cellulose IIIis essentially a solid-state process that keeps the integrity ofthe cellulose microfibrils while achieving a substantialdecrystallization and a reorganization of the intracrystallinehydrogen bond pattern of cellulose.18,19 It has been shownthat ammonia or the amines enter the cellulose crystals asguests, which not only distort the crystals but also modifythe conformation of the hydroxymethyl group within thecellulose lattice itself.20-22 Upon release of the guestmolecules these hydroxymethyl groups do not return to theirinitial stable conformation but remain distorted, leavingtherefore the cellulose in an “activated state”. In addition,the once swollen crystals adopt a crumpled morphology whenthe guest departure is effective.18

To our knowledge, cellulose samples activated in the IIIallomorph have not been probed by the TEMPO techniquefor the production of polyglucuronic acid. In this work, wehave subjected different cellulose III samples resulting fromtwo different ammonia treatments to the TEMPO oxidation.These derivatized samples were analyzed in terms of watersolubility, carboxyl content, and yield in polyglucuronic acid.

Experimental Section

Materials. Cellulose samples from different sources wereused in this work. They included cotton linters from Tubize

* To whom correspondence should be addressed. Telephone: 33-47603 76 14. Fax: 33-476 54 72 03. E-mail: [email protected].

† Present address: AFOCEL, Laboratoire Bois-Process, Domaine del’Etancon, 77370, Nangis, France.

1417Biomacromolecules 2003,4, 1417-1425

10.1021/bm034144s CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 08/16/2003

Plastics, Rhodia (Belgium), Avicel microcrystalline cellulosefrom FMC Europe, viscose fibers from Spontex (Beauvais,France), alphacellulose rich-softwood sulfite pulp fromBorregaard Pulp Division (Sarpsborg, Norway), and driedsugar beet pulp (SBP) from Saint Louis Sucre (Nassandres,France). This last sample was purified according to theprocedure of Dinand et al.23 The set of samples wascompleted with bacterial cellulose produced in the laboratoryfollowing the method of Hestrin.24 All samples were usedas received, with the exception of bacterial cellulose andSBP, which were microfibrillated before further processing.

Preparation of Microfibril Suspensions. Purified SBPas well as bacterial cellulose samples were dispersed in waterand disrupted in a Waring Blender operated at full speedfor 5 min at a concentration between 1% and 2%. The slurrywhich had reached a temperature of 60°C was immediatelytreated with a laboratory scale Manton-Gaulin homogenizer15MR-8TBA, from APV Gaulin Inc., Wilmington, Mass.Fifteen passes were applied at a pressure of 500 bar, andthe temperature was kept below 95°C to avoid cavitation.The resulting creamy suspensions were freeze-dried forfurther use.

Ammonia Treatments.Two different protocols were usedto prepare cellulose III. The first process involved the useof L-NH3 at atmospheric pressure. Gaseous ammonia wasslowly liquefied (boiling point-33 °C) by means of eitherdry ice or a liquid-nitrogen-acetone slush bath kept at-50 °C. The resulting L-NH3 dripped into a vesselcontaining the cellulose and placed in a bath filled with thesame cooling agent. After total immersion of the cellulosesample, the liquefaction was stopped and the specimen waskept in L-NH3 for 4 h. The cooling bath was then removed

and the temperature allowed to slowly increase. Because theresidual ammonia reacts with the TEMPO reagents, itscomplete elimination was needed. The samples were there-fore evaporated overnight, and the residual traces of ammoniawere removed either by a vacuum pumping during a fewhours or washing with methanol followed by water. Thesecond protocol involved the use of exploded gaseousammonia (EG-NH3) and corresponded to samples processedby Rhodia Acetow, following their patented process,25,26

which consists of treating cellulose samples with gaseousammonia under high pressure followed by a rapid decom-pression.

Oxidation. Oxidation experiments were carried out aspreviously published with minor modifications.9 In a typicalrun, cellulose samples (0.648 g, 4 mmol glycosyl units) weredispersed in distilled water (60 mL) for 3 min with a highspeed T25 basic Ultra-Turax homogenizer (Ika-Labortechnik,Staufen, Germany). A total of 30 mL of water was used towash the homogenizer. TEMPO (10 mg, 0.065 mmol), NaBr(0.21 g, 2 mmol), and NaOCl (1.76 M, 0.5 mL, 0.88 mmol)were stirred in 10 mL of water until complete dissolution.This solution was then added to the cellulose suspension,which was mechanically stirred and maintained at 20°C,and the remaining NaOCl (Table 1) was added dropwise inorder to maintain the pH at 10 during the addition. The pHwas maintained constant by adding 0.5 M NaOH solutionuntil no more variation was observed, indicating that thereaction was finished. A total of 5 mL of methanol was thenadded to destroy the residual NaOCl and the pH adjusted to7 with 0.5 M HCl. After centrifugation, we could separatethe supernatant, containing the water-soluble oxidized cel-lulose, from the water-insoluble fraction. The supernatant,

Table 1. TEMPO-Mediated Oxidation of Cellulose Samples

cellulose samplesNaOCl

(molar ratio)a

soluble fraction(%)b

gel fraction(%)b

insoluble fraction(%)c

global yieldd

(%) DO

avicel (I) 1.3 17 17 64 98 0.20avicel (III) L-NH3 1.3 50 18 23 91 0.22avicel (III) EG-NH3 1.3 54 22 15 91 0.23linters (I) 0.73 8 2 84 94 0.22linters (I) 1.4 10 4 86 100 0.22linters (I) 2.1 10 2 87 99 0.23linters (I) 2.5 9 3 85 97 0.21linters (IIl) L-NH3 0.64 8 2 91 101 0.20linters (III) L-NH3 1.4 29 13 58 100 0.28linters (IIl) L-NH3 2.1 37 19 39 95 0.39linters (IIl) L-NH3 2.5 58 16 21 95 0.39linters (III) EG-NH3 0.7 10 3 87 100 0.26linters (III) EG-NH3 1.4 43 21 36 100 0.32linters (III) EG-NH3 2.5 92 6 2 100bacterial (I) 1.4 40 23 33 96 0.25bacterial (III) L-NH3 1.4 72 13 6 91SBP primary wall (I) 1.2 45 45 10 100 0.24SBP primary wall (III) L-NH3 1.4 64 23 9 96 0.30borregaard (I) 1.3 24 76 0 100borregaard (III) L-NH3 1.2 47 52 0 99borregaard (III) EG-NH3 1.3 59 39 0 98rayon (II) 1.3 36 62 2 100 0.37(gel)rayon (IIIII) L-NH3 1.2 54 38 0 92 0.39(gel)

a Mol NaOCl/mol glycosyl unit. b Molar yields were calculated with the molecular weight of the oxidized products as the sodium salt of the glucuronicacid units considering the soluble fraction as 100% oxidized and the gel fraction as 40% oxidized. c Molar yield of insoluble fraction was calculated usingthe carboxyl content (DO) values measured by conductimetry. d Global yield corresponded to the sum of soluble, gel and insoluble fractions.

1418 Biomacromolecules, Vol. 4, No. 5, 2003 da Silva Perez et al.

referred to as thesoluble fraction, was precipitated by addingan excess of ethanol, centrifuged, redissolved in water,dialyzed against water, and finally freeze-dried. The water-insoluble fraction was dispersed into a large volume of water(250 mL), stirred for 1 h, and recentrifuged. The oxidizedmaterial recovered in this second supernatant was water-soluble in dilute solutions but yielded a gel when concen-trated. This product after dialysis and freeze-drying wasreferred as thegel fraction. Finally, the solid recovered fromthe second centrifugation step was suspended in water andcentrifuged to obtain theinsoluble fraction.

NMR Spectroscopy.Carbon-13 spectra were recorded ona Bruker AC 300 spectrometer (operating frequency of75.468 MHz) using the Bruker INVGATE pulse sequence.Samples were studied as their sodium salts in D2O solution(30 mg in 0.5 mL of D2O, pD∼ 7) at 60°C in 5 mm o.d.tubes. The methyl signal of acetone was used as internalstandard (13C (CH3) δ ) 31.5 ppm relative to Me4Si). 13Cspectra were recorded using 90° pulses, 15 000 Hz spectralwidth, 8000 data points, 0.54 s acquisition time, 3 s relaxationdelay. 20 000 up to 150 000 scans were accumulateddepending on the sample solubility.

X-ray Diffraction. X-ray measurements were made onpellets of cotton linters. The X-ray diagrams were recordedon a Warhus flat film vacuum X-ray camera mounted on aPhilips PW 1720 X-ray generator operated with Cu KRradiation at 20 mA and 30 kV.

Infrared Spectroscopy. Infrared spectra were recordedon a FT-IR Perkin-Elmer 1720X spectrometer. Samples werestudied as KBr pellets (1% in anhydrous KBr). Spectra wererecorded using 3600 cm-1 spectral width (between 400 and4000 cm-1), 2 cm-1 resolution, and 20 scans were ac-cumulated.

Conductimetry. The carboxyl content of oxidized cel-lulose samples was determined by conductometric titrations.The cellulose samples (30-40 mg) were suspended into 15mL of 0.01 M hydrochloric acid solution. After 10 min ofstirring, the suspensions were titrated with 0.01 M NaOH.The titration curves showed the presence of strong acid,

corresponding with the excess of HCl and weak acidcorresponding to the carboxyl content, as shown in Figure1.

The carboxyl groups content or degree of oxidation (DO)is given by the following equation:

where V1 and V2 are the amount of NaOH (in L) as shownin Figure 1,c is the NaOH concentration (mol/L), andw isthe weight of oven-dried sample (g).

Methylene Blue Adsorption.A sample of water-insolubleoxidized cellulose (10 to 15 mg) is dispersed in 25 mL of aborate buffer solution (pH) 8.5). Methylene blue (25 mLof a 300 mg/L solution) is added, and the dispersion is keptunder stirring for 1 h. After filtration or centrifugation, 1mL of the nonadsorbed methylene blue is added to 1 mL of0.1 M HCl, and water is added to a final volume of 10 mL.Determination of methylene blue concentration is then carriedout by photometry using a Dynatech MR5000 multi-samplerapparatus. The carboxyl groups content (mmol/g) is givenby the following equation:14

where MBna is the nonadsorbed methylene blue (in mg) andw is the weight of oven-dried sample (g).

Determination of the Degree of Polymerization.Thewater insoluble oxidized cellulose samples were dissolvedin 1 M cupriethylenediamine diluted from the 3 M stocksolution purchased from Prolabo (France). The viscosimetricaverage degree of polymerization (DPv) of the oxidizedcellulose was calculated from the intrinsic viscosities of thecorresponding solutions at 25°C, using the relation

according to Rinaudo.27

The oxidized water soluble samples were dissolved in 0.1M NaCl solution. The viscosimetric average degree ofpolymerization (DPv) of the oxidized cellulose was calcu-lated from the intrinsic viscosities of the corresponding

Figure 1. Conductometric titration curve of oxidized cellulose insoluble sample.

DO ) 162(V2- V1)c[w - 36(V2 - V1)c]-1 (1)

COOH(mmol/g)) (7.5- MBna)0.00313w-1 (2)

[η] ) 0.891DPv0.936 (3)

TEMPO-Mediated Oxidation of Cellulose III Biomacromolecules, Vol. 4, No. 5, 2003 1419

solutions at 25°C, using the relation

according to Dantas et al.28

Results and Discussion

Conversion of Cellulose I into Cellulose III. TEMPO-oxidation of native cellulose (cellulose I) is difficult and oftenincomplete. Even after 24 h under oxidation, at pH 10, partof native cellulose samples remain insoluble.9 On the otherhand, regenerated cellulose (cellulose II) gives larger amountsof totally oxidized water-soluble polyglucuronans. Suchbehavior is usually attributed to the poor accessibility of theprimary hydroxyl groups, engaged in both intra- andintermolecular hydrogen bonds, in the native cellulose Istructure.

The treatment of both native and regenerated celluloseswith L-NH3 leads to a swelling of the crystalline cellulosefollowed by a rearrangement in a different crystallineconfiguration, the cellulose III, when the ammonia isevaporated. This is a classical process for activating cellulosefor further derivatization reactions. In this context, we studiedhere the TEMPO-mediated oxidation of cellulose III, whichwere activated in two different ways: (i) immersion inL-NH3 (-33 °C) at atmospheric pressure; (ii) gaseousammonia treatment under high pressure followed by a rapiddecompression (EG-NH3).

The conversion of the samples into cellulose III wasfollowed by X-ray diffraction, as shown in Figure 2 in thecase of cotton linters. The diffraction patterns of cellulose Ishown in Figure 2a, characterized by the 4 sharp rings withd spacing at 6.0, 5.4, 4.38, and 3.9 Å, disappeared completelywhen the materials are treated by EG-NH3 as shown inFigure 2b, characterized by the 3 sharp rings withd spacingat 7.6, 5.15, and 4.2 Å of cellulose III. Moreover, the aspectof the new patterns (identical to cellulose III obtained bysupercritical ammonia treatment in the laboratory19) is very

well defined. The experiments showed that the transformationof crystalline cellulose into allomorph IIII was achievedwithout generating too much amorphous cellulose. On theother hand, samples treated at atmospheric pressure presentedfeatures from both cellulose I and cellulose III. Someamorphous cellulose must also be present because thedefinition was poorer than in the other X-ray patterns. Anestimation of the ratio of cellulose I and III could be deducedfrom the measurement of the radial profile of the X-raypatterns and by comparing with cellulose I and III standard.For the cellulose shown in Figure 2c, the distribution isroughly 30% of cellulose I and 70% of cellulose III. Theconversion of the samples into cellulose III was furtherconfirmed by a CP/MAS solid state13C NMR study thatwill be published separately.29

TEMPO-Mediated Oxidation. To evaluate the influenceof parameters such as crystallinity, morphology, and degreeof polymerization on the conversion into cellulose III,samples from different origins, with DP covering a largerange, from 170 (Avicel microcrystalline) to 1450 (bacterial),were used during this work. Nevertheless, the major aim ofthis study was the comparison of the reactivity betweencellulose III and native or regenerated cellulose. The oxida-tion runs were carried out according to the scheme presentedin Figure 3. Sodium hypochlorite, used to regenerate thecatalyst, is added dropwise during the reaction.

Depending on the conditions, the insoluble materialprogressively disappeared and the solution became hazy,evidencing that the oxidation had occurred. The evolutionof the reaction was followed by the sodium hydroxideconsumed to neutralize the carboxylic acids generated in C-6,because the pH must be rigorously kept at 10 in order toprevent a severe degradation of the water-soluble polymer.At the end of the reaction (when no more variation of pHwas observed), methanol was added to react with theremaining TEMPO, and the reaction mixture was thenneutralized and treated with NaBH4 in order to reducecarbonyl carbons eventually formed during the oxidationreaction.

The isolation and purification of products include cen-trifugation, dialysis, precipitation, and freeze-drying steps.In most of the cases, three fractions, one water-soluble, agel, and the insoluble residue, were recovered after theoxidation was completed (see the Experimental Section). Themolar fractions distribution for each experiment is presentedin Table 1. Because of the small size of milled Borregaardpulp particles, the gel and the water-insoluble fractions cannotbe separated during centrifugation and are given together.

Figure 2. X-rays diffraction patterns of cotton linters: (a) CelluloseI; (b) Cellulose III EG-NH3; (c) Cellulose III L-NH3.

[η] ) 0.05Mw0.8 (4)

Figure 3. Simplified oxidation scheme.


The analysis of the data shown in Table 1 clearly indicatesthat cellulose III is more reactive than native cellulose. Inall of the cases, the yield of soluble fraction is higher forthe ammonia-activated samples than for the original samples.The same tendency is observed for the gel fraction.

The oxidation of untreated cotton linter samples onlyyielded 8-10% of water-soluble (in molar basis) product.When the same samples were converted to cellulose IIIthrough atmospheric pressure L-NH3 or EG-NH3 treat-ments, the molar percentages of water-soluble productsreached 58% and 92%, respectively. Moreover, an increasein the NaOCl/primary hydroxyl groups molar ratio does notshow any influence on the reactivity of native cellulose. Inopposition, ammonia-treated cotton linter samples yieldedhigher water soluble and gel fractions amounts when thesodium hypochlorite concentration is increased. For thesamples treated at atmospheric conditions, the water-solublefractions yields increased from 8 to 58% when the NaOCl/primary OH ratio was changed from 0.64 to 2.5, whereasfor samples obtained by EG-NH3, the variations were from10 to 92% if the mentioned ratio was increased from 0.7 to2.5. A similar behavior was observed for the gel fraction,and as a consequence, for the EG-NH3 cotton linter samples,practically no insoluble residual was obtained at the end ofthe reaction. So far, such result had only been obtained formercerized or regenerated cellulose (cellulose II).

For the microcrystalline cellulose, the water-soluble frac-tion yield obtained from native cellulose was slightly higherthan that obtained with cotton linters because of a betteraccessibility of the sample. Indeed the acidic treatment ledto crystals with smaller size and, hence, with a greater surfaceratio of cellulose chains. Because of their direct expositionto the chemical reagents, they are the first to be oxidized.However, at very similar NaOCl/OH ratios, the samplestreated with ammonia gave an incomparably higher amountof water-soluble fractions, confirming the cellulose activation.

Borregaard is an alpha cellulose-rich pulp mainly used inthe cellulose derivatives market. For this study, the pulp wasmilled and sieved and only a fine powder (40-60 mesh)was used for oxidation tests. The presence of some hemi-celluloses (amorphous polysaccharides present in wood) andthe increase in the accessibility due to grinding of samplesexplain the large amount of soluble and gel fractions whenuntreated Borregaard cellulose was submitted to the oxidationwith TEMPO. We observed once again that the ammoniatreatments greatly increase the yield of soluble polyglucu-ronan fraction. For Borregaard pulp runs, gel and insolublefractions could not be separated because the finely dividedmaterial presents similar behavior during the centrifugationstep and forms a very compact gel.

An increase of reactivity is also observed for samplesconverted from cellulose II to cellulose IIIII, as confirmedby the higher soluble/gel fractions ratio obtained for thesamples treated with L-NH3 when compared to untreatedrayon (54/38 to 36/62, respectively).

The different results observed here confirm that thecrystallinity is not the only factor governing the efficiencyof the oxidation, but other parameters influencing theaccessibility of TEMPO oxoammonium ion to the hydroxyl

groups such as molecular weight and fiber morphology, playan important role.

As it will be discussed later in this article, water-solublefractions are composed of fully C-6 oxidized water-solublepolymer chains (polyglucuronan), whereas gel and insolublefractions are only partially oxidized. Amorphous celluloseis rapidly and fully oxidized yielding polyglucuronan.However, the oxidation of the strongly crystalline arrangedcellulose I is very difficult, and it only occurs on the surfaceand in a moderate extension. The conversion into celluloseIII increases the accessibility because the distance betweenchains in this crystalline form is increased as confirmed bythe density variation from 1.61 (cellulose I) to 1.54 (celluloseIII) g/cm3 and also because hydroxyl groups are less involvedin hydrogen bonds network. Indeed, because of the swellingduring the ammonia treatment and the reorganization intocellulose III crystals, the hydroxylmethyl moieties adopt agt conformation, instead of a tg conformation in cellulose I,with a better conformational freedom, and therefore animproved accessibility.19 This explains the higher solubleproduct contents for ammonia treated samples. Moreover, ahigher reactivity is observed for the samples treated by EG-NH3, when compared to the L-NH3 treated samples. As wehave shown earlier, these samples have been fully convertedto cellulose III, which explains their higher reactivity vis-a-vis the cellulose treated by atmospheric L-NH3. It is likelythat the insoluble fractions are composed of crystallinecellulose I partially oxidized at the surface, whereas the gelfractions are formed either by self-organization of partiallyoxidized cellulose chains or by partially oxidized cellulosechain still attached to very small crystallites.

Kinetics of Oxidation. We also have observed that theoxidation of EG-NH3 samples occurred slightly faster thanL-NH3 treated samples, both of them being considerablyfaster than cellulose I ones. In Figure 4, the oxidation kineticsof seven cotton linters samples (native, EG-NH3, andL-NH3) are shown. The reaction was followed through theconsumption of sodium hydroxide added to keep the pH at10.0 ( 0.2, the total sodium hypochlorite being added atthe beginning of the reaction. During the first 15 min ofreaction no difference of soda consumption was observed.Native cellulose was probably being oxidized at the surface,which explains the similar reactivity of ammonia-treatedsamples. However, from this point until the end of thereaction, a considerable increase in the reactivity of celluloseIII compared to cellulose I was clearly observed.

The concentration of sodium hypochlorite does not seemto affect the reaction kinetic for neither cellulose I norcellulose III, confirming the catalyst role of TEMPO andNaBr. Nevertheless, as expected the final reaction kineticdepends on the NaOCl concentration. An increase from 0.73to 1.4 and 2.5 equiv of sodium hypochlorite yielded verysimilar curves for the first 120 min of reaction. However, aclear plateau was not observed, suggesting either a slowcellulose oxidation by the remaining NaOCl or the degrada-tion of cellulose by side reactions (i.e.,â elimination). Forcellulose III samples, on the other hand, the shape of thecurves suggests a leveling-off oxidation value is reached afterall the added NaOCl has been consumed.


The oxidation of EG-NH3 samples is slightly faster thanof those treated with L-NH3, which confirms the higherpolyglucuronans yields mentioned earlier. This difference issurely connected with the total conversion of cellulose I intocellulose III, and it confirms the increase of the reactivitywhen this transformation takes place.

Characterization of Products. The three fractions ob-tained after the TEMPO-oxidation of cellulose are apparentlyoxidized to different levels. The water-soluble fractionsobtained here seem to be fully oxidized, whereas insolubleand gel fractions are only partially derivatized, whichindicates that a critical carboxyl content must be reached toallow the product to become soluble. However, the separationof fractions also depends on the molecular weight of startingmaterial. Thus, the water-soluble fractions obtained fromcotton linters or bacterial cellulose seem to be fully oxidized,according to1H and13C NMR characterization.9 However,when low DP cellulose was used, the product became water-soluble even if it was partially oxidized.30 We have in factobtained some partially oxidized water-soluble oligosaccha-rides at the laboratory, but their molecular weights were verysmall (20-35 glycosyl units). The same behavior has beenobserved for the TEMPO-oxidation of cyclodextrins,31,32

where a great increase in the water solubility was noticedafter the formation of one or two carboxyl groups.

We have tried several methods traditionally used for thedetermination of carboxyl groups in carbohydrates to controlthe degree of oxidation such as carbazole chemical method,methylene blue adsorption, quantitative infrared and13CNMR, and conductometric titration.

The traditional carbazole method involves the hydrolysisof the polysaccharide before the sugar analysis through acolorimetric determination. However, in a previous work,9

we have shown that this method gave degrees of oxidationranging from 55 to 90% for totally oxidized cellulosesamples, as measured by13C NMR. To explain this under-estimation, we have studied both acidic and enzymatic

hydrolysis of oxidized cellulose and we obtained a complexmixture of mono, di, tri, tetra, and other oligosaccharides.Therefore, it is evident that the hydrolysis of glycosidiclinkages in polyglucuronic is much more difficult than inthe original cellulose. Further optimization for the hydrolysisof such units is then needed before validating the monosac-charide analysis methods for the carboxyl content determi-nation.

The adsorption of methylene blue by the carboxyl groupshas been widely used in the industry to determine carboxylgroups in pulps before being replaced by the ion exchangemethod.9 The methylene blue adsorption method tends togive lower carboxyl content values than13C NMR or FTIR,especially for low DP fractions. In fact, we have observedthat when the water-insoluble fraction is dispersed in a largevolume of water part of the product becomes “soluble-like”and can be recovered in the supernatant after centrifugation.In the conditions used for the methylene blue method (10 to50 mg of sample dispersed in 50 mL of water), the sameprocess could occur, and in this case, the measured carboxylgroup content would be lower than expected. The advantagesof the methylene blue method are the simplicity and therapidity for the determinations. The main disadvantages arethe impossibility of using water-soluble samples, the lowvalues obtained for samples partially soluble during themeasurement, the difficulties in weighting small amountsrequired for the analysis, and the problems associated to thelatter (moisture content, nonrepresentative sampling, etc.).

Quantitative13C NMR can be used for the determinationof functional groups in complex naturally occurring macro-molecules.16,33NMR spectra of one partially and one totallyoxidized sample are presented in Figure 5, parts a and b,respectively. We can observe that the signals of carbons C1at 103.1 ppm, C2 at 73.5 ppm, C3 at 75.0 ppm, and C4 at81.7 ppm of oxidized units are weakly affected by theoxidation of primary hydroxyl, as compared to chemicalshifts of C1 at 102.9 ppm, C2 at 73.6 ppm, C3 at 75.6 ppm,

Figure 4. Effect of the amount of NaOCl (eq NaOCl/eq glycosyl units) on the oxidation kinetics of cellulose samples: (A) Cellulose I (0.73); (B)Cellulose I (1.4); (C) Cellulose I (2.5); (D) Cellulose III L-NH3 (0.64); (E) Cellulose III L-NH3 (1.4); (F) Cellulose III L-NH3 (2.5); (G) CelluloseIII EG-NH3 (1.4).


and C4 at 81.1 ppm of non-oxidized units. On the other hand,the signals of C5 at 79.2 ppm and particularly C6 at 60.8ppm from glucose moiety are strongly shifted by theoxidation. We observe the C5 and C6 signals at 76.1 and175.3 ppm, respectively, for the glucuronic unit. When thespectra are obtained under quantitative conditions by usingthe Bruker INVGATE pulse sequence, the integration ofsignals gives the degree of oxidation. However, not all ofthe signals are useful for such a purpose because of the longrelaxation time (C6 in carboxyl groups) or overlapping withother signals (C3 and C5). Therefore, the degree of oxidationis obtained from integration of the C4 signals in oxidizedand nonoxidized units relative to the C1 signal and can bechecked by the C6 signal of glucose units. However, theacquisition time to get useful spectra, especially for gelsamples, is too long for routine analyses, because sometimesup to 150 000 scans are needed for quantification purposes.Moreover, the determination of carboxyl groups by quantita-tive 13C NMR gives higher values than infrared spectroscopyand the methylene blue method, especially for water-insoluble fractions of low DP cellulose. The existence ofdifferent microheterogeneous environments in the gel leadsthe fractions with higher mobility to be preferentiallyobserved in NMR, and this obviously corresponds to chainshaving a higher degree of oxidation. For the water-solubleproducts, a very good correlation is obtained between FTIRand NMR measurements. The nondestructive character ofthe technique and the direct measurement of carbon-13population are the major advantages of this technique. Themain inconveniences are the too long acquisition time, theimpossibility of determining the degree of oxidation forwater-insoluble samples, and the over estimation of carboxylgroups content in the gel.

Infrared spectroscopy has been used for a long time as atool for characterizing cellulose. Complete attribution ofbands can be found in a cellulose textbook.16 When celluloseis selectively oxidized in position 6, some changes in bandsare observed, as shown in Figure 6. The most importantchanges are the appearance of a CdO stretching band at 1608cm-1 (carboxylate) or 1730 cm-1 (acid), that will be usedfor quantification purposes, and a C-O stretching band at1400 cm-1. On the other hand, only a slight reduction ofthe bands related to hydrogen bonds stretching vibration ofOH groups at 3338 cm-1 and to stretching vibrations of CHat 2900 cm-1 is observed.

Both water-soluble and insoluble samples were obtainedas a sodium salt, and in this case, the band to be used forquantification overlapped with the band arising from tracesof adsorbed water at 1640 cm-1. Because we have used thesame amount of product for the acquisition of all of the

Figure 5. Carbon-13 NMR spectra in D2O: (a) Partially oxidized fraction; (b) totally oxidized fraction.

Figure 6. FTIR spectra of different fractions obtained after oxidationand ion exchange through an acidic treatment.


spectra and normalized them, we considered the area of theband of cellulose spectrum as 0% and of a fully oxidizedsample as 100%. Therefore, the carboxyl content of partiallyoxidized samples could be determined by interpolation.However, variation in moisture content of the sample canlead to uncertainty in the measurement. Another possibilityis using the displacement of the band to a higher wavenumberwhen the oxidized products are in the acid form, as presentedin Figure 6. In this case, no interference with the absorbedwater is observed and the carboxyl content can be obtainedfrom the area of the band at 1730 cm-1. The infraredtechnique is a fast, weakly time and sample consumingmethod for the determination of carboxyl groups in bothwater-soluble and insoluble fractions of partially oxidizedcellulose. The disadvantages of this technique are thoseassociated to the quantitative infrared like low repeatability,heterogeneity of pellets, need for baseline correction andnormalization, among others.

The carboxyl content of oxidized cellulose samples canalso be determined by conductometric titrations. In mostalkalimetric methods, the metallic cations bound to thecarboxyl groups are exchanged by hydrogen ions throughtreatment with acid in excess. We carried out a direct titrationwith alkali and the titration curves show the presence of astrong acid, corresponding to the excess of HCl and a weakacid, corresponding to the carboxyl content. We haveobtained very reproducible results, both on water-soluble andwater-insoluble cellulose oxidized samples.

The average degree of polymerization (DPv) of water-insoluble samples was calculated from the viscosity incupriethylenediamine (cuene) solutions. Although TEMPO-oxidation of polysaccharides have been described in theliterature as a more selective and less aggressive method thanthe traditional nitrite/nitrate method, a severe depolymeri-zation took place either during the viscosity measurementsin cuene or during the oxidation reaction, as shown in Table2. For the water-soluble fractions, measurements were carriedout in 0.1 M NaCl solutions, because unstable substitutegroups such as carboxyls seem to cause DP loss in thealkaline cuene solvent used for the DP measurement, asshown by Jewell et al.34 Their results indicated that the cuenemeasurement method appeared to be producing lower valuesthan the actual DP of the sample before dissolution in cuene.Our data in Table 2 confirmed that degradation occurred inthe alkaline cuene solvent, as the DP of the gel and insolublefractions are lower than the fully oxidized soluble samples,whereas they should be higher or at least in the same range,as they are less oxidized.

Conclusions

From the results presented here, we conclude that apretreatment of cellulose sample with ammonia aiming atconversion of cellulose I into III improves its reactivity withrespect to the TEMPO-mediated oxidation system. Samplestreated by the EG-NH3 process are more reactive than thoseobtained by the traditional atmospheric L-NH3 process. Thiscan be easily explained, first because, in the atmosphericprocess, the samples are not completely converted intocellulose III as shown in Figure 2c, and second because theexplosion treatment is known as an efficient destructuratingprocess. This was clearly established in all the steamexplosion applications.35,36The TEMPO-mediated oxidationof cellulose III samples is highly selective for primaryhydroxyl groups, whereas secondary hydroxyl groups werein most cases insensitive toward oxidation.

We noticed a depolymerization of the cellulose samplesduring the oxidation, but at a lower level compared to ourresults obtained on TEMPO oxidation of amorphous samples.9

Furthermore, we observed an important decrease of DPvalues of the gel and insoluble fractions, obtained byviscosity measurements in cuene that can be more likelyexplained byâ-elimination degradation in the alkaline cueneconditions.

We have presented here a comparative study of thedetermination of carboxyl groups by four methods, theadsorption of methylene blue, quantitative13C NMR andinfrared spectroscopy, and conductometric titration. Themethylene blue adsorption method seems to underestimateand13C NMR to overestimate the carboxyl groups contentwhen intrinsic solubility problems occur during the handling.Moreover, the methylene blue method cannot be used withwater-soluble fractions and quantitative13C NMR only workswith liquid samples. FTIR seems to be a general method forboth water-soluble and insoluble samples despite of theproblems associated to the quantification by this technique.Among all of these procedures, the conductometric titrationappears to be the most attractive and reproducible method,either on soluble or insoluble oxidized cellulose samples.

Acknowledgment. The authors acknowledge Mrs. M.-F. Marais for preparing the bacterial cellulose sample. Theythank also Mr. T. Karstens, Rhodia Acetow Aktiengesell-schaft, Freiburg, Germany for the ammonia high pressurerapid decompression cellulose samples.

References and Notes

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Table 2. Degree of Polymerization of TEMPO-Mediated OxidizedCellulose Samples

cellulosesamples

startingsamplesa

solublefractionb

gelfractiona

insolublefractiona

avicel 170 75 50 85cotton linters 850 120 105 80bacterial 1450 130 55 100primary wall 980 110 130borregaard 450 105 80rayon 360 100 65

a Measured in cuene. b Measured in 0.1 M NaCl.


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TEMPO-Mediated Oxidation of Cellulose III