S Camy

S Montanari

A Rattaz

M Vignon

J.-S Condoret

Oxidation of cellulose in pressurized carbon dioxide

This work presents first results upon oxidation of type II cellulose by nitrogen dioxide dissolved in carbon dioxide at high pressure. This reaction leads to oxidized cellulose, a natural-based bioresorbable fabric used for biomedical applications. The oxidation reaction takes place in a heterogeneous fluid-solid system. Kinetics of oxidation is presented here and effects of operating conditions such as pressure, temperature and initial moisture content of cellulose are investigated. Results are presented in terms of degree of oxidation of cellulose and quality of the final oxidized cellulose, which has been characterized using liquid-state and solid-state 13 C NMR. The experimental results show the existence of possible secondary reactions which may lead to oxidized cellulose with insufficient mechanical strength. An attempt is made to evidence and understand the role of CO 2 as a solvent in this system. Indeed, although supercritical CO 2 appears to be a suitable candidate as a solvent for oxidation reactions, some inhibiting effect on nitrogen dioxide activity are observed in this case.

Introduction

Oxidized cellulose is a very interesting material for biomedical applications, because it is degradable in human body [START_REF] Stilwell | Oxidized cellulose: chemistry, processing and medical applications, Drug Target[END_REF] and also exhibits haemostatic and antibacterial properties. Oxidized cellulose is thus used as a raw material for medical devices such as adhesion barriers, sutures, absorbable haemostats or scaffolds for tissue engineering. To obtain this product, nitrogen dioxide (NO 2 ) is the most suitable oxidant because, in this case, oxidation does not affect secondary hydroxyl groups of the cellulose, and thus secondary reactions are avoided. In this case oxidized cellulose maintains acceptable mechanical properties, allowing its use as a biomedical device. The global reaction is illustrated by the following scheme:

The first industrial process for oxidation of cellulose was based on the use of gaseous nitrogen dioxide, where the gas was circu-lated across layers of fabrics [START_REF] Yackel | The oxidation of cellulose by nitrogen dioxide[END_REF]. But this process was abandoned because of the difficulty of handling a gas (especially when its atmospheric condensation point is 21 • C, as it is the case for NO 2 ), as well as because mastering the exothermicity of the reaction in a gaseous system at industrial conditions is uneasy. Consequently, solvent based processes were proposed. In this case, nitrogen dioxide was dissolved in an organic solvent, and thus the oxidation took place in the liquid phase [START_REF] Ashton | Oxidized cellulose product and method for preparing the same[END_REF]. Nevertheless, the oxidant could cause some damage to the solvent, and solvent degradation products were often present. Therefore, the biomedical company Johnson and Johnson proposed the use of inert halogenated solvents, and patented the use of perfluorocarbons as solvents for this reaction [START_REF] Boardman | Cellulose oxidation by a perfluorinated hydrocarbon solution of nitrogen dioxide[END_REF].

Recently the use of supercritical carbon dioxide as a suitable inert solvent to perform oxidation of cellulose was proposed and patented [START_REF] Vignon | Method for the controlled oxidation of polysaccharides[END_REF]. Because of its peculiar physical properties and its specific "green" character, supercritical CO 2 is now growing interest as a reaction medium for many chemical syntheses, such as hydrogenation, hydroformylation, oxidation or polymerization [START_REF] Beckman | Supercritical and near-critical CO2 in green chemical synthesis and processing[END_REF] Nomenclature AC carboxylic acid content of the cloth (%w/w) DO degree of oxidation of cellulose V a

HCl solution volume used to neutralise basic cellulose solution (L) V b

HCl solution volume used to neutralise basic solution without cellulose (L) C

HCl solution concentration (mol L -1 ) M molar weight of COOH group = 45 g mol -1 m weight of the cellulose sample (g) for instance. Especially, as it is the result of complete oxidation of carbon, CO 2 is a solvent of choice for oxidation reactions considering the absence of by-products possibly issued from degradation of the solvent by the oxidant. In the view of biomedical applications for oxidized cellulose, using CO 2 has the major advantage to yield processed fabrics as "solvent residue free". More widely, carbon dioxide is considered as a green non-expensive solvent, and, in this case, compares very favourably with perfluorocarbons. This work focuses on the main aspects of oxidation of cellulose in high-pressure CO 2 and using nitrogen dioxide as the oxidant.

Generalities on cellulose oxidation by nitrogen dioxide

Nitrogen dioxide does not exist as pure molecular specie, but is in equilibrium with its dimer, nitrogen tetroxide, as follows:

N 2 O 4 ⇆ 2NO 2
When temperature is less than 262.15 K, nitrogen dioxide is completely dimerized giving nitrogen tetroxide (N 2 O 4 ) and this dimer dissociates into 2 mol of NO 2 as temperature increases, proportion of each compound depending on conditions of temperature and pressure. Under atmospheric pressure, the mixture boils at 294.25 K, the liquid being yellowish brown and the vapour reddish brown. If moisture is present, N 2 O 4 reacts with water forming nitric and nitrous acids which are thus involved in complex equilibria mechanisms [START_REF] Pascal | Traité de Chmie Minérale[END_REF]. This decomposition phenomenon makes it very aggressive to numerous metals. Acute toxicity of this reactant makes it quite delicate to handle, so individual protective equipments must be used.

Physical properties of the mixture and equilibrium constant value depend on temperature and have been the subject of several studies. For example, Reamer and Sage [START_REF] Reamer | Volumetric behaviour of nitrogen dioxide in the liquid phase[END_REF] have done density measurements in the liquid-vapour coexistence region. Values of equilibrium constants in the gas phase as a function of temperature can be found in works of Verhoek and Daniels [START_REF] Verhoek | The dissociation constants of nitrogen tetroxide and of nitrogen trioxide[END_REF] or Chao et al. [START_REF] Chao | Gas phase equilibrium in dinitrogen trioxide and dinitrogen tetroxide[END_REF]. James and Marshall [START_REF] James | An electron spin resonance study of the dinitrogen tetroxide-nitrogen dioxide system[END_REF] have determined equilibrium data for the liquid mixture and Redmond and Wayland [START_REF] Redmond | Dimerization of nitrogen dioxide in solution: a comparison of solution thermodynamics with the gas phase[END_REF] have been interested in equilibrium constant data for the nitrogen dioxide dissolved in organic solvents.

In order to run the reaction in a homogeneous mixture of oxidant and solvent, fluid phase equilibria involved in the reactional mixture have been previously experimentally investigated [START_REF] Belkadi | Modelling the vapour-liquid equilibrium and association of nitrogen dioxide/nitrogen tetroxide and its mixtures with carbon dioxide[END_REF]. These measurements give us information upon solubility of NO 2 in highpressure CO 2 , and about interactions which exist between NO 2 and CO 2 . Such interactions may have a dramatic effect on the reaction of oxidation of cellulose, as mentioned by some authors who studied this reaction in organic solvents [START_REF] Pavlyuchenko | Effect of organic solvent nature on the interaction of cellulose with nitrogen tetroxide[END_REF]. Indeed, the NO 2 specie is suspected to be the only active oxidant specie [START_REF] Nevell | Oxidation of cotton cellulose by nitrogen dioxide[END_REF][START_REF] Nevell | Qualitative X-ray study of the oxidation of cotton cellulose by nitrogen dioxide[END_REF] and its concentration depends upon the dimerization equilibrium. In their investigations upon the oxidation of cellulose by nitrogen dioxide dissolved in various solvents, it was shown that the degree of dissociation of N 2 O 4 increases in non-polar solvents, leading to an increase in the degree of oxidation of cellulose. Considering that supercritical CO 2 is considered as a non-polar solvent, this observation seems a favourable clue for its use for the reaction of oxidation.

Mechanisms of oxidation of cellulose with NO 2 /N 2 O 4 in gas phase or in solvent phase have been the subject of different studies that have been summarized by Stilwell et al. [START_REF] Stilwell | Oxidized cellulose: chemistry, processing and medical applications, Drug Target[END_REF] where it appears that formation of cellulose nitrites as intermediates is necessary to achieve oxidation, which occurs then through physical and chemical adsorption of the NO 2 onto the cellulose. Cellulose must contain initially between 5.5 and 11% (w/w) of moisture to ensure adsorption of the reactant. The proposed stoichiometry of the oxidation reaction is then:

RCH 2 OH + NO 2 → RCOOH + H 2 O + (1/2)N 2
Considering nitric acid formation by the reaction of NO 2 with H 2 O, Stilwell et al. [START_REF] Stilwell | Oxidized cellulose: chemistry, processing and medical applications, Drug Target[END_REF] proposed the overall stoichiometry of the reaction to be:

RCH 2 OH + 2N 2 O 4 ⇆ RCOOH + 2HNO 3 + NO + (1/2)N 2
Because of the by-products of this reaction, an important step of the production process lies in the cleaning of the oxidized cellulose at the end of the reaction, because the remaining acidic by-products, as well as the remaining adsorbed NO 2 are responsible of degradation of the product during storage.

Materials and methods

Cellulose

Regenerated cellulose, i.e., man-made cellulose fibres produced by the cuprammonium process [START_REF] Krässig | Cellulose, Ullmann's Encyclopedia of Industrial Chemistry[END_REF] from Bemberg company, was used as the starting raw material for all this study. This regenerated cellulose, usually termed rayon, consists in fibres that are spun in such a way to form a continuous filament. Such continuous filaments can be knitted together to form simple braid or meshes.

Chemicals

NO 2 /N 2 O 4 was supplied by Air Liquide (water content max 0.5%wt) and CO 2 TP by Linde Gas.

Oxidation experimental set-up

Experiments were performed using the set-up presented in Fig. 1. The reactor (250 mL), from Parr Instrument Company is thermo-regulated and comprises two sapphire windows allowing visual control of the reactional mixture. Homogeneity of the mixture is ensured due to a magnetic stirrer. A Teflon ® made supporting grid is used to maintain the piece of cellulose to be oxidized in the upper part of the reactor volume. Pressure and temperature sensors are used to control operating conditions and are connected to a digital recorder (Memo-graph RSG10, Endress + Hauser). Liquid CO 2 is pumped due to an air operated reciprocating piston pump (Highpressure pump ref. 0902 10000, Top Industrie). A Coriolis type mass flow meter (Micro-Motion) allows the control of the amount of CO 2 introduced in the reactor.

Reactant NO 2 (note that we refer here to the NO 2 /N 2 O 4 mixture) is introduced into the reactor by using a 50 mL high-pressure air operated syringe injector (Injector 1644 1000, Top Industrie). This part of the set-up is equipped for nitrogen circulation in order to dry and inert the system, and this is necessary to avoid nitric acid formation during NO 2 introduction. At the outlet of the reactor, the closing valve is followed by a heated back-pressure regulator (Tescom). This system is used to maintain a constant reactor pressure, especially during the CO 2 washing procedure at the end of the reaction (washing by dilution, see next paragraph).

All parts of the experimental set-up in contact with NO 2 are made of 316L stainless steel, and gaskets are made of Teflon. Because of the toxicity of NO 2 , in case of accidental leakage, operators are protected using individual protecting equipment.

Operating procedure

The reactor is firstly heated to the desired temperature (around 313 K) and the cellulose fabric sample is placed on the Teflon grid. After closing the reactor, sweeping with gaseous CO 2 is done before filling with CO 2 until the desired pressure is reached. From measurement of the course of the injector piston, a given amount of reactant NO 2 is then introduced into the reactor and oxidation starts.

Post-oxidation washing of oxidized samples is fastidious step of the process. Indeed, if a simple depressurization of the reactor is carried out, residual NO 2 may condense onto cellulose and could cause irreversible damage to the product. As a consequence, a washing by dilution with CO 2 under pressure is carried out in order to maintain the reacting mixture as a homogeneous phase and avoid condensation of NO 2 onto cellulose. This washing is done by pumping CO 2 into the reactor, while the diluted reacting mixture is released through the back-pressure regulator.

Then, a second washing step is necessary to remove acidic byproduct of the oxidation and residual adsorbed NO 2 . In accordance with industrial oxidation processes ( [START_REF] Yackel | The oxidation of cellulose by nitrogen dioxide[END_REF][START_REF] Ashton | Oxidized cellulose product and method for preparing the same[END_REF]), this washing is done at atmospheric pressure outside the reactor with an isopropyl alcohol-ethanol mixture (50/50 vol.) until pH is neutral. Residual water is then removed by washing three times with anhydrous alcohol (isopropyl alcohol or ethanol). Finally, the sample is dried in an oven at 333 K during 24 h.

Titration by colorimetric method

The degree of oxidation is directly related to the carboxylic acid content of the oxidized cellulose samples. This latter is determined by a back-titration method: a small amount of dried oxidized sample (about 200 mg) is dissolved in 2 mL of 0.5N solution of sodium hydroxide diluted with 8 mL of distilled water. The solution is then titrated with standard 0.05N HCl to a phenolphthalein end point. A blank is also run using only the NaOH solution without the cloth. The titration is repeated at least three times. This titration allows determination of the carboxylic acid content of the cloth (AC), 100% of oxidation corresponding to 25.5% carboxylic acid content, and a degree of oxidation equal to unity. The degree of oxidation (DO) represents the number of oxidized hydroxyl groups per anhydroglucose unit of the cellulose sample, following the relations:

AC = (V b -V a ) × C × M m × 100 (1) DO = M anhydroglucose × (V b -V a ) × C m -14 × (V b -V a ) × C (2)
In relation ( 2), M anhydroglucose is the molar mass of one anhydroglucose unit of the cellulose (162.15 g mol -1 ), and 14 is the difference between the molecular weight of a glucuronic acid unit and an anhydroglucose unit.

Characterization of the cellulose by solid-state NMR

A Brucker MSL spectrometer was used to obtain solid-state 13 C NMR spectra, operating at 50 MHz 13 C frequency.

The solid-state NMR spectrum of unprocessed rayon is presented in Fig. 2. Peaks corresponding to each carbon group of the sample can be observed, as referred on the anhydroglucose group above it. Characteristic peak of "-CH 2 OH" group is observable at 62.3 ppm, peaks of "-CH" groups at positions 2, 3 and 5 of the cycle are between 68 and 80 ppm, and carbons at position 4 and 1 are characterized by peaks between 80 and 90 ppm and 105 ppm, respectively. Shoulders which appear on C1 and C4 peaks evidence existence of crystalline parts.

Characterization of the cellulose by liquid-state NMR

13 C NMR spectra of oxidized samples are obtained on a Brucker AC 300 spectrometer (operating frequency of 75.47 MHz). Samples are studied after dissolution in NaOD solution (60 mg in 1 mL of NaOD 2 M, followed by neutralisation (pD ∼ 7) with DCl) at 298 K in 5 mm o.d. tubes. The methyl signal of acetone was used as internal standard ( 13 C (CH 3 ) at 31.5 ppm relative to Me 4 Si). 13 C spectra were recorded using 30 • pulses, 7500 Hz spectral width, 0.5 s acquisition time, 0.5 s relaxation delay and 50,000 scans were accumulated.

Results and discussion

Influence of the pressure

When considering phase equilibria of the CO 2 -NO 2 /N 2 O 4 mixture at 313 K issued from [START_REF] Belkadi | Modelling the vapour-liquid equilibrium and association of nitrogen dioxide/nitrogen tetroxide and its mixtures with carbon dioxide[END_REF] and presented in Fig. 3 we can deduce that, if reaction is run above 8.5 MPa, it takes place in a homogeneous reacting mixture, whatever its composition. Indeed, the biphasic zone where liquid and vapour are in equilibrium, has to be avoided in order to insure that the reaction is run in a homogeneous phase to obtain homogeneously oxidized samples. Under 8.5 MPa, depending on the composition, the reaction may take place either in a "dense gaseous phase" (CO 2 rich mixtures) or in an "expanded liquid" (NO 2 /N 2 O 4 rich mixtures).

Oxidation of a sample of rayon (3.3 g) has been studied under conditions where the reacting mixture is a monophasic at 313 K for two values of the oxidant concentration in the reactor, i.e., 132 g L -1

Fig. 3. P-x, y data of CO2-NO2 mixture around 313 K. Experimental ( ) and calculated with Peng-Robinson equation of state with k ij = 0 (-) (from [START_REF] Belkadi | Modelling the vapour-liquid equilibrium and association of nitrogen dioxide/nitrogen tetroxide and its mixtures with carbon dioxide[END_REF]). Because the oxidation reaction is run under constant volume condition, at a given NO 2 concentration, an increase in the CO 2 amount in the mixture leads to an increase of the total pressure of the reaction. At low pressure, i.e., low CO 2 and high NO 2 mass fractions, oxidation is actually performed in a dense medium that can be regarded as an "expanded" liquid NO 2 -phase. Conversely, reactions performed at high pressure, i.e., low NO 2 mass fraction, correspond to dense gaseous NO 2 /CO 2 mixtures. Evolution of the degree of oxidation of rayon samples with the pressure is presented in Fig. 4.

At constant NO 2 concentration, we clearly observe that when the pressure, i.e., the amount of CO 2 in the system, is increasing, the degree of oxidation of rayon globally decreases, whatever the initial NO 2 concentration. This behaviour is quasi-linear for pressure below 9 MPa, but seems to level off above this value. Same results, expressed in terms of mass fraction of CO 2 inside the reactor, as done in Fig. 5, are more explicit because they clearly reflect effect of the amount of CO 2 in the system. Indeed, above the critical pressure of CO 2 , an increase of the amount of CO 2 has a drastic effect on the pressure because of the high density of CO 2 as com- pared to its gaseous density. Anyway, the more CO 2 is introduced in the system, the less effective is the oxidation.

In Figs. 4 and5, we observe that a large increase in the NO 2 mass concentration does not lead to an important improvement of oxidation. Oxidation is mostly dependant on the fraction of NO 2 in the NO 2 /CO 2 mixture, i.e., of the amount of CO 2 . However, this behaviour is not reproduced if CO 2 is replaced by N 2 for example, as it can be seen in Fig. 6. In the case of N 2 , it is observed that the degree of oxidation is not significantly influenced by the total pressure. At 313 K and [NO 2 ] = 12 g L -1 , 4 h oxidation leads to DO values around 0.6, whatever the amount of N 2 in the system. We can conclude that in the case of N 2 , oxidation of rayon does not depend on the quantity of N 2 introduced into the system. Fig. 7 shows solid-state 13 C NMR spectra of non-oxidized and oxidized samples both in N 2 and CO 2 ([NO 2 ] = 12 g L -1 in both cases). Spectra of oxidized samples show a decrease of the peak corresponding to -CH 2 OH group and present the subsequent appearance of the peak corresponding to the carboxylic acid function, around 170 ppm. Moreover, we can deduce from these spectra that cellulose seems to maintain its partial crystallinity during oxidation in CO 2 /NO 2 mixtures, while it looses it during oxidation in N 2 /NO 2 mixtures. Indeed, with CO 2 , no important modification in size or shapes of C1 and C4 peaks is observed, and, especially, shoulders are still present on oxidized samples. This is not the case for samples that have been oxidized in N 2 media. Another observation concerns the increase of a peak comprised between C1 and C4 peaks (around 90 ppm). A hypothesis could be a possible breakage of cellulose chains during oxidation, leading to degradation of cellulose, and/or to secondary reactions of hydroxyl groups (C2 and C3) leading to formation of hemiacetal groups. This peak is particularly important for samples that have been oxidized in N 2 /NO 2 mixtures. Thus, these samples are both more oxidized and more degraded. Finally, these spectra are consistent with values of DO obtained by colorimetric titration, since they show that samples are oxidized but degraded also.

As a conclusion, from these results of oxidation in CO 2 and N 2 , it appears that CO 2 seems to have an "inhibiting" effect on oxidation mechanism. Several hypotheses may be proposed concerning the role of CO 2 in this system.

First of all, although chemically inert towards oxidant, CO 2 may interact with NO 2 in such a way that reactivity may be reduced, i.e., through the modification of the NO 2 /N 2 O 4 equilibrium towards N 2 O 4 . CO 2 is considered as non-polar, especially as compared to polar solvents which were shown to have a dramatic negative effect on oxidation [START_REF] Pavlyuchenko | Effect of organic solvent nature on the interaction of cellulose with nitrogen tetroxide[END_REF]. Nevertheless, the CO 2 molecule has a significant quadratic dipolar moment, and recent studies suggest that supercritical CO 2 can participate in conventional or non-conventional hydrogen-bonding interactions [START_REF] Raveedran | Polar attributes of supercritical carbon dioxide[END_REF]. As a consequence, specific interactions may exist between CO 2 and NO 2 /N 2 O 4 or between CO 2 and cellulose. In this latter case, CO 2 may compete with adsorption of NO 2 onto cellulose.

A conventional approach would consider that carbon dioxide, as well as nitrogen, acts as a diluting agent in the system. The mass action law, applied to the dimerization equilibrium, indicates that, in the case of addition of a diluting agent at constant temperature and volume (thus inducing a pressure increase), the equilibrium reaction is not affected, if the mixture can be considered as a mixture of perfect gases. This is not the case for real gases for which the equilibrium constant must be expressed in terms of fugacities of the components in the mixture, instead than in terms of partial pressures. Modification of fugacities of NO 2 and N 2 O 4 may occur with the addition of CO 2 , under high pressure, depending upon interactions between the components. A possible explanation to our results, which have shown a decrease of oxidation when CO 2 is added to the system, could be a displacement of the NO 2 /N 2 O 4 chemical equilibrium in favour of the presumed inactive oxidant compound, N 2 O 4 . This is consistent with experimental results obtained in the case of N 2 , where no effect of N 2 pressure has been detected. Indeed, it is well known that the thermodynamic behaviour of N 2 at high pressure, which is far from its critical temperature, is not very different from the behaviour of a perfect gas.

Another possible explanation of the negative effect of CO 2 could be its role as a drying agent for cellulose. Stilwell et al. [START_REF] Stilwell | Oxidized cellulose: chemistry, processing and medical applications, Drug Target[END_REF] reported an optimum of moisture content for cellulose oxidation, in the range 5.5-11% (w/w), this optimum being related to an effective adsorption of nitrogen dioxide onto cellulose. Several hypotheses are reported upon physisorption and/or chemisorption of nitrogen dioxide onto cellulose, but moisture content is clearly evidenced to be a key parameter for oxidation. To interpret this result, the supermolecular structure of cellulose must be considered. Indeed, most of the reactions on cellulose fibres can be considered as nonhomogeneous because the solid is indeed a kind of two-phase solid system, composed of less-ordered (amorphous) zones (mainly located as the surface of the fibrils or in the interlinking regions between crystallites) and well-ordered elementary crystallites. As a consequence, any reaction starts in the less-ordered regions (firstly at the surface and then in the regions interlinking elementary crystallites), and then proceeds into the crystallites [START_REF] Krässig | Cellulose, Ullmann's Encyclopedia of Industrial Chemistry[END_REF]. However, a quasi-homogeneous oxidation can only be achieved when cellu- lose looses its crystalline structure, due for instance to a swelling pre-treatment. Water can be regarded as a swelling agent because it inserts between cellulose chains in amorphous zones, breaking hydrogen bonds between hydroxyl groups of cellulose, forming its own hydrogen bonds. This insertion induces an inter-crystalline swelling of the textile in amorphous zones, and can facilitate penetration of reactants in these zones, but not in crystalline zones of the cellulose [START_REF] Krässig | Cellulose: structure, accessibility and reactivity[END_REF]. Water alone is not sufficient to induce a complete swelling, including an intra-crystalline swelling of the cellulose. However, the primordial role of water had been already underlined for the treatment of cellulose with supercritical CO 2 in the domain of textile dyeing [START_REF] Van Der Kraan | Equilibrium distribution of water in the two-phase system supercritical carbon dioxide-textile[END_REF]. In this case, the drying effect of CO 2 is taken into account through controlled pre-addition of water in the system. In the context of cellulose oxidation, we may expect an analogous role for water, which allows the oxidant penetrating amorphous zones of cellulose and adsorbing onto the textile to react. Indeed, because solubility of water in scCO 2 is significant (around 1 g/L of CO 2 at 10 MPa and 313 K), a part of the water initially present onto cellulose dissolves in the supercritical phase. Influence of initial water content will be discussed later.

Kinetics of oxidation

Kinetics of oxidation in high-pressure CO 2 /NO 2 mixtures has been studied, and, as previously, oxidations have been run at two reactant concentrations. Temperature of oxidation was set at 313 K and pressure was chosen to be 10 MPa.

Observation of Fig. 8 shows a two-step kinetics of oxidation, which appears to be rapid until around 4 h, and then undergoes a strong slow-down, whatever the initial oxidant concentration. Indeed, with a large excess of NO 2 in the reactor, oxidation degree is greater, but we always observe a levelling off after about 4 h of reaction. The second step is a slow reaction, and we observe that the maximum DO value of 1 was never reached in our experiments, even for high [NO 2 ] or long reaction time.

Once again, in the case of oxidation in CO 2 /NO 2 mixtures, we can observe from solid-state 13 C NMR analysis, that cellulose maintains its crystalline structure during oxidation. Fig. 9 shows spectra of oxidized celluloses obtained from experiments in CO 2 /NO 2 mixtures, with [NO 2 ] = 132 g L -1 . Indeed, no modification in size or shapes of C1 and C4 peaks is observed. Main modifications concern the decrease of the size of CH 2 OH peak and the subsequent appearing and increasing, as far as the reaction proceeds, of the COOH peak, so as the increase of the peak of secondary reactions (around 90 ppm).

Additional observations can be extracted from 13 C NMR spectra obtained by dissolving oxidized sample in NaOD solution. Spectra of dissolved oxidized samples (experiments with [NO 2 ] = 132 g L -1 ), shown in Fig. 10, present some slight signal displacement of C2, C3, C4 and C5 between carbons from anhydroglucose units and carbon from oxidized units. As long as the reaction proceeds, we observe an increase in the number of signals around 92 ppm, corresponding to a possible secondary reaction on cellulose chains. Peaks of residual iso-propanol from the washing step are also present in the spectra.

As mentioned above, in CO 2 /NO 2 mixtures at 10 MPa and 313 K (see Fig. 8) the reaction proceeds as a two-step phenomenon. The first step corresponds presumably to oxidation of easily reachable zones of cellulose fibrils, and the second step to zones where primarily hydroxyl groups are less easily reachable. However, as observed on solid-state NMR spectra, these two types of zones do not correspond to amorphous and crystalline zones since these latter do not seems to be oxidized. As a conclusion, under these operating conditions, NO 2 dissolved in CO 2 seems to be unable to sufficiently swell the cellulose in order to destroy its crystalline structure to yield effective oxidation (cf. Fig. 9).

The influence of CO 2 as the solvent has been investigated once again by operating the reaction without any solvent. In this case only liquid NO 2 is injected in the air filled reactor at 313 K, and the quantity of injected NO 2 is adjusted in order to ensure that NO 2 vaporizes completely. This phenomenon can be observed experimentally because the recorded pressure inside the reactor increases all along the injection. After a given value of the injected quantity, the pressure inside the reactor is no longer increasing and stabilises, indicating the presence of a liquid-vapour equilibrium for NO 2 . In this case, the total pressure remains constant because, whatever the quantity of NO 2 , its partial pressure is equal to its vapour pressure for the experimental temperature. This limiting value could be predicted by simple calculations using the perfect gas law, but the computed theoretical value is indeed smaller (around 7 g L -1 ) than the experimental observed one. This may be because a significant part of gaseous NO 2 adsorbs onto the cellulose sample present in the reactor and do not participate to the partial pressure of NO 2 . Consequently the oxidation was done in low pressure gaseous N 2 /NO 2 mixture at a NO 2 concentration of 12 g L -1 , corresponding to the observed maximum gaseous concentration which can be obtained at 313 K. We also performed oxidation in pressurized N 2 /NO 2 mixtures at 7 MPa, 313 K and 12 g L -1 of NO 2 . For these two kinds of experiments, the same amount of cellulose is placed inside the reactor. Kinetic curves are presented in Fig. 11. These curves represent kinetics of oxidation ([NO 2 ] = 12 g L -1 at 313 K) in different reacting media. An obvious conclusion from these curves is that the presence and nature of the 'solvent' has a drastic influence on reaction of oxidation. Indeed, when oxidation is performed in low pressure gaseous N 2 /NO 2 mixture, a 0.85 value of the degree of oxidation is reached after 4 h of oxidation. This is a higher value than in CO 2 /NO 2 mixture or pressurized N 2 /NO 2 mixture. However, operation in pressurized N 2 /NO 2 mixture seems to have a lower impact on oxidation as compared to operation in pressurized CO 2 /NO 2 mixture. Nevertheless, in all cases, we observe that reaction is more rapid during the four first hours of reaction. On 13 C NMR spectra of oxidized samples obtained in low pressure gaseous N 2 /NO 2 mixture (Fig. 12), we notice the disappearance of shoulders on C1 and C4 peaks, (that we previously attributed to crystalline zones of cellulose). This tends to show that structure of cellulose is rapidly loosened in that case. Increase of "COOH" peak and decrease of "CH 2 OH" one, evidence oxidation of primary hydroxyl groups of cellulose. Collateral reaction can be detected from the increase of the peak around 90 ppm. This peak is quite large for highly oxidized sample, such as the one obtained after 250 min, and thus such samples could not be used for application of biomedical device, because cellulose would not have satisfactory mechanical properties. Indeed, oxidation in low pressure gaseous N 2 /NO 2 mixture allowed obtaining oxidized cellulose with higher DO (even completely oxidized at DO = 1) as compared to oxidation in N 2 /NO 2 mixture. In low pressure gaseous N 2 /NO 2 mixture, oxidation seems to be a quasi-homogeneous phenomenon, probably because of a rapid swelling and loosening of cellulose crystallinity, allowing oxidation of all zones of cellulose.

Influence of initial moisture content of the cellulose

As mentioned above, initial moisture content of cellulose is suspected to influence the oxidation mechanism. So an attempt has been made to evaluate its importance on oxidation results. Thermogravimetric analysis of raw cellulose has been done due to differential scanning calorimetry and to thermogravimetric analysis apparatus (DSC-TGA, TA Instruments Q600 SDT) in order to evaluate the initial moisture content of cellulose under normal atmospheric conditions. This reveals that rayon at ambient naturally contains around 11% (w/w) of water.

To evaluate the importance of initial moisture of cellulose on oxidation with NO 2 , we have modified the initial moisture content of the samples. Firstly, this was done by simply drying the cellulose during one night in an oven at 333 K before oxidation. Two other samples have been equilibrated with saturated salt solutions. In that case, non-oxidized cellulose samples have been maintained inside a closed vessel during several days in the atmosphere equilibrated with of a solution saturated with a given salt. A first solution has been saturated with NaOH, providing a water activity equal to 0.0698 at 293.15 K, and the second one has been saturated with NaCl, providing a water activity of equal 0.7542 at 293.15 K. In all cases, moisture content of cellulose before oxidation is evaluated by gravimetric method.

Oxidation of these equilibrated cellulose samples has been done in CO 2 /NO 2 mixture at 10 MPa, 313.15 K, at [NO 2 ] = 132 g L -1 during 4 h, and results of oxidation are presented in Fig. 13. On this figure, we reported also degree of oxidation of cellulose obtained without further modification of initial humidity (initial moisture content equal to 11%).

Results presented in Fig. 13 clearly show that the degree of oxidation of cellulose is, in a certain range, directly related to its initial moisture content. This result is consistent with previously reported results indicating an optimum moisture content range for cellulose [14] between 5.5 and 11% (w/w) for oxidation, an excess of moisture in the cellulose leading to a predominant nitration phenomenon. As a matter of fact, water seems to have an important role in the mechanism of oxidation. This has been confirmed by solid-state 13 C NMR analysis of oxidized samples obtained from non-equilibrated cellulose, and from previously dried sample (in the oven), showing that the pre-dried sample is less oxidized and less-damaged than the non-equilibrated one, and as a consequence of the low degrees of oxidation, crystalline structure seems to be still present in both samples.

Moreover, oxidation having been realized in CO 2 at high pressure, it has to be considered that water could be stripped out because of the significant solubility of water in the CO 2 /NO 2 mixture (solubility of water is around 0.74 g L -1 in pure CO 2 at 313 K and 10 MPa). The lowering of the water content of cellulose, due to the presence of CO 2 , is then likely to influence the reaction of oxidation. Surprisingly, no effect was observed when preliminary addition of water in the system is done, in a similar way which we mentioned for dyeing experiments.

Finally, from these results, we observe that optimum degree of oxidation was actually obtained from cellulose samples where no modification regarding water content had been done. Consequently, these results confirm the importance of water in the system, but they do not evidence the suspected negative effect of CO 2 on the distribution of water.

Influence of the temperature

Temperature can influence several parameters of oxidation like reaction rate and selectivity, chemical equilibrium between NO 2 and N 2 O 4 , thermodynamic behaviour of the reacting mixture and adsorption of NO 2 onto cellulose.

Although literature upon oxidation of cellulose by nitrogen dioxide is very scarce and not recent, Ermolenko et al. [START_REF] Ermolenko | The mechanism of oxidation of cellulose by oxides of nitrogen[END_REF] early observed that oxidation rate was surprisingly slower when the temperature was increased, in contradiction with a simple approach based on the conventional Arrhenius law. These authors attributed this phenomenon to the detrimental effect of the temperature on the necessary adsorption of N 2 O 4 onto cellulose. Note that, according to these authors, the active oxidant specie was N 2 O 4 . Moreover, they argued that, considering NO 2 /N 2 O 4 equilibrium, an increase of temperature favoured dissociation into NO 2 , which was in their sense detrimental for oxidation. However, the same research team later called this hypothesis into question by showing that the reactive specie was actually NO 2 . This last result arose from the observation that an increase in the degree of oxidation of cellulose was obtained when non-polar solvents were used for oxidation with NO 2 [START_REF] Stilwell | Oxidized cellulose: chemistry, processing and medical applications, Drug Target[END_REF], assuming that these non-polar sol- vent favoured an increase in the degree of dissociation of N 2 O 4 into NO 2 .

In our case, we performed oxidation with temperature varying between 307 and 333 K at 10 MPa in CO 2 /NO 2 mixture during 4 h and [NO 2 ] = 132 g L -1 . This range of temperature has been chosen in such way that the reaction is run in a single-phase mixture. Results obtained in these conditions are presented in Fig. 14.

Our results clearly show an increase in the degree of oxidation of cellulose when temperature is increased, and, in this case, these results are consistent considering the Arrhenius law. Moreover, these results tend to show that NO 2 is the active oxidant since an increase of temperature is favourable to NO 2 .

A more complete characterization of the oxidized samples, due to solid and liquid 13 C NMR spectroscopy, has revealed that increasing the temperature had lead to strongly damaged cellulose. Liquid-state 13 C NMR spectra of cellulose samples are shown in Fig. 15. At 333 K, we observe that the peak corresponding to -COOH group (around 178 ppm) is not only important but "multimodal" too, and this could be due to occurrence of a carboxylation reaction on carbon atoms other than C6. In addition, peaks attributed to breakage of cellulose chains (between 90 and 100 ppm) are important. These analyses are confirmed by tactile analysis where oxidized samples were found fragile and brittle. As a conclusion, temperature of oxidation has to be kept as low as possible in order to limit degradation of the product mechanical properties.

Conclusion

Results upon oxidation of cellulose with nitrogen dioxide in high-pressure CO 2 medium are reported here for the first time. Kinetics of oxidation, influence of pressure, temperature and initial moisture content of cellulose were investigated. Although highpressure CO 2 matches all the requirements to be a green solvent for the reaction of oxidation with nitrogen dioxide, it indeed appears that its role is not as neutral as expected and the degree of oxidation of cellulose depends on the amount of CO 2 introduced in the system. Our first attempts to understand the role of CO 2 have not allowed clarifying completely its action on cellulose or/and the oxidant but it can be suspected that CO 2 interacts with NO 2 , inhibiting the reactivity of this latter towards cellulose. The nature and the intensity of the interactions between NO 2 and CO 2 have still to be investigated, for example through spectroscopy measurements in parallel with ab initio calculations.

Nevertheless, in the specific process of production of oxidized cellulose for biomedical application, CO 2 , although not the most efficient solvent for this system, can be regarded as an efficient "moderator" agent, allowing soft and homogeneous oxidation, which could be easily controlled by varying the pressure of the system. Furthermore, if we remind that the post-oxidative washing procedure is a very important step of the process, use of highpressure CO 2 is a very good way to improve and simplify the whole process. At last, one of the major advantages of such a system is also related to the green aspect of using CO 2 as a solvent, especially for biomedical applications where biocompatibility of the product must be strictly ensured.

Fig. 1 .

 1 Fig. 1. Scheme of the high-pressure oxidation experimental set-up.

Fig. 2 .

 2 Fig.2. Solid-state13 C NMR spectrum of original rayon.

Fig. 4 .

 4 Fig. 4. Influence of the pressure on oxidation of rayon with NO2 in CO2 at 313 K during 4 h.

Fig. 5 .

 5 Fig. 5. Influence of mass fraction of CO2 (zCO 2 ) on the degree of oxidation of rayon in CO2/NO2 mixtures at 313 K during 4 h with [NO2] = 12 g L -1 ( ) and [NO2] = 132 g L -1 ( ).

Fig. 6 .

 6 Fig. 6. Influence of pressure of N2 on the degree of oxidation of rayon in NO2/N2 mixtures at 313 K, 4 h and [NO2] = 12 g L -1 .

Fig. 7 .

 7 Fig. 7. 13 C NMR spectra of non-oxidized and oxidized samples of rayon obtained in N2 and CO2 at 313 K, 4 h and [NO2] = 12 g L -1 .

Fig. 8 .

 8 Fig. 8. Kinetics of oxidation of rayon in CO2/NO2 mixtures at 10 MPa and 313 K.

Fig. 9 .

 9 Fig. 9. 13 C NMR spectra of non-oxidized and oxidized samples of rayon obtained in CO2/NO2 mixtures.

Fig. 10 .

 10 Fig. 10. 13 C NMR spectra of oxidized samples of rayon dissolved in NaOD/D2O solution.

Fig. 11 .

 11 Fig. 11. Kinetics of oxidation at 313 K in low pressure gaseous N2/NO2 mixture ( ), N2/NO2 mixture at 10 MPa ( ), in CO2/NO2 mixture at 10 MPa ( ).

Fig. 12 .

 12 Fig.12.13 C NMR spectra of non-oxidized and oxidized samples of rayon obtained in low pressure gaseous N2/NO2 mixture.

Fig. 13 .

 13 Fig. 13. Influence of initial moisture content of cellulose upon the degree of oxidation in CO2/NO2 mixture at 313 K, 10 MPa, 4 h and [NO2] = 132 g L -1 .

Fig. 14 .

 14 Fig. 14. Influence of the temperature on the degree of oxidation in CO2/NO2 mixture at 10 MPa, 4 h and [NO2] = 132 g L -1 .

Fig. 15 .

 15 Fig. 15. 13 C NMR Spectra of oxidized samples of cellulose dissolved in NaOD/D2O solution. Oxidation in CO2/NO2 mixture at various temperatures at 10 MPa, during 4 h and [NO2] = 132 g L -1 .