Application of a New pH-metric Method to the Kinetic Study of Copolymerization of Polysaccharides Fucan N1 with PMMA in Presence of Cerium Ce

The copolymerization of a Poly-Acrylic PMMA with polysaccharide, Fucan N1 and Dextran T70,in presence of a ceric salt used as initiator in acidic medium,was carried out under the following conditions: Fucan N1=0,5 g; [ ] M . , CAN 3 10 6 3 − = , [ ] M 2 , 0 HNO 3 = ; temperature =40°C with a relative ratio between the initiator and the monomer, initiator/monomer =1/10. Besides a pH study of the reaction medium, an infrared spectroscopy (IR) proved the appropriatness of these best conditions of synthesis, and this by showing the presence of a characteristic peak of the carbonyl grouping situated at 1733cm for the copolymer Fucan PMMA in respect to the individual PMMA spectra and Fucan N1. In addition, a C NMR study has been conducted on the copolymer. A viscosimetry study of two Fucan fractions was performed in aqueous medium (H20), and also of the obtained copolymer in DMSO. An outcome of this work allows for us the suggestion of a plausible reaction mechanism, of which the copolymerization rate -Rpis first order dependant to the monomer concentration, and to the square –concentration of both the initiator and the polysaccharide.


Introduction
The essential aim of this work is to apply the pH-metric measuring method to the study of the PMMA copolymerization with polysaccharides Fucan N1 and Dextran T70 (Fucan N1 is a sulfated polysaccharide extracted from a brown seaweed of the Ascopohyllum nodosum kind), in presence of ceric ions ce 4+ in nitric acidic medium pH=1, at a temperature 4OC° during 40mn, under argon atmosphere (Chowdhury and Pal, 1999;Dalton et al., 2002;Masci et al., 2004;Fuoss and Cathers, 1949;Costa and Vasconcelos, 2002), with a relative ratio between the initiator and the monomer "Acros" leading first to the formation of a white-milky complex that disappears immediately, giving a radical (F) of Fucan to a proton (H + ) and an ion Ce 3+ ,as it is shown in the following reaction equation : The kinetic study of this copolymerization, of which the polymerization rate measure is performed by pH metric measuring method -that we had put in evidence -and with comparison to the method of extraction by soxhelt with acetone.

Products
Dextran of different molecular weights (MW= 10,40,70,80.7,and 264 kg/mol) were purchased from Sigma for molecular weight assessment.Dextran (70 kg/mol) was also used for synthesis.It was dried in a vacuum oven at 60°C for 24h.Methyl methacrylate monomers were obtained from Acros France and were purified by washing with 5% NaOH and 20% NaCl, followed by distilled water.Ammonium cerium (IV) nitrate (Acros France) was dried at 80°C under vacuum for 24h.Solvents were of the highest commercially available purity.
Fucan N1 and Fucan BBP-2, having a mass of 95500g/mol and 46600g /mol respectively, are two fractions of Fucan obtained by HPLC chromatography on a S 5300 HR gel after an acidic extraction followed by an acidic hydrolysis (Hoppe HA, 1979), ( see Figure 1).
The Dextran is, like the Fucan, soluble in water, whereas its copolymer with PMMA is soluble in the water /THF (20/ 80 v/v) mixture and in the dimethylsulfoxide (DMSO).
The polymerization of the methyl methacrylate « MMA »with Fucan N1 and Dextran T-70 depends closely on the concentration of the initiator (Colliec, 1993;Mulloy, B, 2002) of the monomer (MMA) and the concentration of the used polysaccharide.This polymerization is performed by cycle -opening forming active radicals F or D that are likely to be bound by covalent links to the methyl poly-methacrylate (PMMA) forming Fucan copolymer(PMMA) and Dextran (PMMA).This copolymerization is conducted in a highly acidic medium to avoid a possible formation of the ceric hydroxide, which can take place at a medium in which pH=4 to 5. In such a medium, the IR spectrum shows no anomaly.
The kinetic polymerization is performed by applying the PH-Metric measuring method, as an experimental technique, we know that the variation of the PH in the medium is linked to the initiator concentration at instant "t", with the equation: where : -variation of the hydrogen concentration during the synthesis .
0 pH pH and ∞ pH are respectively the variation of the pH at the instant "t=0", at time t (we take t=30mn) and the instant when the whole monomer will be converted into copolymer ( The equation (2) permits to determine directly the polymerization rate -Vp-; monitoring the variation of the monomer concentration in function of pH, and time.We have also determined the polymerization rate by applying the extraction by soxhlet with the acetone method.We determined the percentage (P g %) of the PMMA grafting, the grafting rate (Vg) of the copolymerization, polymerization, the homo -polymerization (V h ), and the total rate (V P ) of the polymerization that are calculated from the weight of the grafted poly-acrylic of the obtained products (Lepoutre P, 1975).(5) The total rate of the polymerization is: We have determined the polymerization rate-V P -by applying two methods independent the one from the other (equations 2 and 6) (table III), for a comparison purpose.

Synthesis and Reaction of the copolymerization
In a (250 ml) reactor equipped with three flasks, we take a polysaccharide mass of 0, 5 to 1g (Fucan N1 or Dextran )dissolved in HNO 3 at 0, 2M. after 10minutes of agitation under an atmosphere of Argon (or N 2 ) at a temperature of 40 ± 1°C, we add at the same time 3,6.10 -3 M/L of cerium and 0,5M of MMA (of which the relative ratio 10 1 = Monomer initiator ), The time variation of the pH is measured at a sampling interval of 5mn.The reaction is considered finished when the pH does not change any more, and this is the case after 30 minutes of reaction ( The obtained product is poured into 500ml of methanol with the PH adjusted to a volume of 8, and NaOH at 10M.After that we concentrate the solution to a volume of 20/30ml approximately.We decant the solution into a spectra-membrane of a diameter of mm ,4 20 = φ to be cleaned /washed in EDTA solution at 0, 05 M during 72hours, under permanent agitation.Then, the solution is put under lyophilization, and later on in a vacuum dryer till a constant weight. First of all, we have monitored pH evolution in time of the reaction medium of the copolymerization .This pH variation is generally not higher than the unity,as it is shown on Figure (2) bellow .

Extraction of Homo-polymer of PMMA
We weight exactly 2,00gr of each raw product-mentioned above -that was extracted with the acetone by soxhlet during 24 h, to eliminate the homo-polymer-poly (Methyl Methacrylate).The pure copolymer is dried at 50°C till a constant weight.

Preparation OF Three-Dimensional Structures
We prepared discs or films by using dried homopolymer PMMA (15 mg) solubilized in THF.Copolymer DM was dissolved in 1 ml of THF/H 2 O (80/20) that was treated in an ultrasonic bath for 1 h .Dextran gave any 3D structures.To obtain discs, solutions were poured in Petri dishes for 24 h at room temperature in a satured atmosphere of THF and in the presence of CaCl 2 to absorb water.Thin films were stripped from the mold and were dried in an oven at 30 °C.A morphological analysis of the cross section of films was carried out by the of a Leica S-440scanning electron microscope (SEM) .All 3D structures were extensively washed with before any biological assays.

13
C NMR spectra of dextran, PMMA and copolymer DM were recorded by the use of a Varian Gemini 200 Mhz spectrometer in deutered dimethyl sulfoxide-d 6 at ambient temperature.Tetramethylsilane (TMS) was used as an internal standard.Before use, all products were lyophilized twice with deuterium oxide (D 2 O).

Copolymers Analysis by Infrared Spectroscopy
This technique allows us to identify some chemical groupings that may be present in the polymers and the copolymers.Tablets of 150mg of potassium bromide (KBr), with infrared quality (Fluka).
The sample is mixed in the KBr, ground and then vacuum dried at 45°C during 6hours, pressed under 10 tons for 2minutes, and then stored at a 45°C in a humidity-free environment .These tablets are analyzed by means of a Fourier transform based infrared spectrophotometer (Perklin Elmer, 1600) .
Every spectrum is taken to be an average of the cumulus of 16 increments, in order to decrease the effect of background noise.Figure 3 shows the spectra of MMA, and Dextran taken individually, in addition to that of the synthesized Dextran-PMMA copolymer.
Curve (A) presents many strips characteristic of the PMMA : one at about 2953cm -1 which is assigned to the (CH 2 ) group, another strip is situated at 1732cm -1 which represents the carbonyl ( 0 c = 〉 ) group and a third band is located at 1380cm -1 assigned to the(CH 3 ) Methyl group .
Curve ( B) presents strips that are specific for Dextran T70: the presence of the radical ( OH)is attested by the absorption band that appears at about 3400 cm -1 , the strip at 2930cm -1 is assigned to the methylene group (CH 2 ), the strip arround1650corresponds to the grouping (OH) of the Dextran T70.
Curve (C) illustrates the spectrum of the product obtained by copolymerization of the Dextran and the MMA monomer by Cerium (Ce 4+ ).This spectrum presents strips that are common to both the Dextran and PMMA .We can notice an important intensity at 1733Cm -1 that corresponds to the carbonyl grouping ( 0 c = 〉 ) .,by keeping the same concentration of monomer and polysaccharide,we notice the formation of a copolymer Fucan PMMA characterized by a peak located at 1725cm -1 for PMMA with respect to the spectrum of Fucan N1alone, as shown on curve (C).

Viscosity Measurements
In order to determine the molar mass of the different obtained products, we have measured the viscosity of Fucan N1, BBP-2 solutions as well as the viscosity of copolymer solutions for different cerium (Ce 4+ ) concentrations.In this technique, we use a well adapted viscometer (the Ubbelohde Viscometer ) with a capillary diameter of 0,7mm, was used for two different polysaccharides; Fucan N1 and Fucan BBP-2 having a mass 95500 g/mol and 46600g/mol respectively We determine η and the specific viscosity for a set of an increasing concentration in the percentage of Fucan N1 and BBP-2 dissolved in water, as follows (0% , 0, 45 %, 0,65%, 0, 75%, 1,0%, 1,25%, 1,45%, 1,65%, and 2 %).This is why we have synthesized tow Fucan PMMA copolymers; one with no monomer ; the other with the presence of 0,4% of monomer MMA, keeping the same concentration values in the initiator and the polysaccharide (0,5 of Fucan ), soluble in the mixture water/ THF (20/80 v/v) at T° = 40°C.

On the same graph (
We found the intrinsic viscosity equal to 0,16 dl/g for the first copolymer, but equal to 0,244 dl/g, for the second copolymer .The viscometric mass Mv corresponding to the viscosity could be formulated through the following equation : [ ] where [ ] 0, 244; 98900 / M g mol We notice that the increase of the viscosimetric mass is due to the increase of PMMA formed in shapes of grafted connections on the polysaccharide, on the one hand, and this prove the effective formation of a copolymer between the Fucan and PMMA for Dextran T70, on the other hand.We can use the following formula: [ ] By the same manner, knowing [ ] η we can deduce the molar mass M .We can interpret these results on Figure 5.

Results and Discussion
This research consists of applying the pH-metric measuring method in the kinetic study the MMA polymerization of the methacrylate methyl, with the polysaccharides Fucan N1 and Dextran T70, in presence of ceric ions ( ) Ce in the best conditions .We must take the quantity of the initiator and the monomer in a reactor we have found equal to 1/10.The infrared analysis (IR )of the copolymers obtained after radical synthesis, shows that the polymerization depends on the nature of each polysaccharide, and on the concentration of the cerium ( ) The radical II is chained to the monomer and engendering copolymers.The first stage consists of forming a complex between the polysaccharide and the cerium ( ) Ce .This complication is performed between the cerium ( ) Ce and hydroxide groups in position 2and 3 of the Polysaccharide.After a laps of time, it is decomposed giving a Radical I, ion Ce 3+ and a proton H + .The second stage, however, consists of forming an oxydium from a radical I with the cerium, making the appearance of aldhydic groups in position 2 and 3 of the polysaccharide, an ion Ce 4+ and a proton H + .As concerns the third stage, it consists of forming a radical II, which is Dextran radical (D or F) responsible for the propagation with the monomer ( )

D
is generated by the reaction between Oxidium and Cerium (Ce 4+ ).
The termination is achieved by classical termination.
Under the light of the obtained results and the discussion we have led, we may establish that our polymerization follows a reactional mechanism a little complex; the stages of which are governed by the following reaction equation: 3 4 2 (13) To put in evidence this polymerization mechanism,we should suppose that the states between the following different initiating species,are quasi-stationary .That is to say: By summing together the equations (20to23) and ignoring the equation ( 22) which is less likely to take place; because we did not notice any considerable attack by the cerium (Ce 4+ ) on the monomer .By assuming that the termination constants are equal to the global ( ) We deduce: The polymerization rate Rp of the Fucan N1 or Dextran in presence of a ceric initiator, is expressed in this formula: By substituting the expression of eq. ( 25) into eq.( 26), we obtain: The expression of the polymerization rate Rp of the Fucan N1 and Dextran T70 with the MMA in presence of a ceric salt used as initiator, in acidic medium a simple ratio between Rp and the order equal to the concentration unity of the monomer MMA, and to the square -root of both the polysaccharide and initiator ( Figures 7 to 10).
And also, the speed of polymerization is: ] max (28) The grafting speed is expressed as: Consequently, the rate of grafting is given by : PG% = ( A R 2 P + B R P + C) .100 (30) where A, B,C and K are constants which can be determined ultimately.
We also know that the speed of polymerization 'R p ' is proportional to the square-root of initiator concentration R P = K ' [ Ce + 4] 1/2 , and the same relationship is found for the quantities R h , R g and PG. ( The method applied in measuring the polymerization rate (Rp) is the PH-metric measuring method, compared to the extraction by soxhlet with the acetone (method of " Le Poutre and Hui (1975) ( see Table III).

Reliability Control of the PH-Metric Method
In order to control the reliability of the PH-metric method, we compared it to a more exact and precise method; which is the "extraction by soxhlet with the acetone method.By comparing these two methods, we can see that same values of PG, V g , V h and the universal constants A, B, C and K are exactly found by both methods.The variation of PG reaches the same maximum for a maximal speed of R p =3,39. 10 -5 mol.l -1 .s - and a initiator concentration [CAN] =1,197.10 -3 M for both methods .The obtained results are given in the following table (see Table III).We notice that the pH-metric method is in good concordance with the extraction by soxhlet with the acetone method, at strong concentration values of the initiator.
( ) (a)   h , R (a)  g and PG (a) polymerization rate determined by the pH-metric method, that we have worked out .
As  show, the quantity kp (kk 0 /k t ) 1/2 is equal to =5,32; 10,22 and to 13,71 for soluble starch (Mansor and Haron, 2000) ; as concerns Dextran T70 and Fucan N1 respectively .This quality varies increasingly with a rate of nearly 2.5 when going from the soluble starch to the Fucan and this in proportion with the mass of each polysaccharide used, namely : M n = (164,4) n g / mol For the soluble starch ; 73000g/ mol for the Dextran T70,and finally 95500g/ mol for the Fucan N1 .

Conclusion
We can summarize the obtained results in the following points: Polymerization between polysaccharides and poly-acrylics is generally achieved through opening the polysaccharide cycle.This kinetic polymerization is considered to be a complex one.The pH-metric method used to determine Rp is found to be very precise and with great concordance with the extraction by the acetone method .The infrared spectroscopy shows that the copolymer can't be formed if the initiator concentration is .The viscometry shows the effective formation of a copolymer between the polysaccharide and the PMMA, due to the intrinsic viscosity increase of 0,16 dl/g to 0,244dl/g in absence and presence of 0,4 % of monomer .The polymerization rate Rp of polysaccharide / poly-acrylic depends on a kinetic complex, first order,equal to the concentration of the monomer and, and to the square-root of the initiator and polysaccharide.
The grafting rate of PMMA (PG%) on the polysaccharide increases with the increase of (CAN) concentration, it reaches a maximum concentration equal to : [ CAN] =1,197 .10 -3 Mol/l, and realizes 196,.60%(this maximum value is found to be equal to 260% at 0.5.10 -3 M/l ceric ion).
Regarding difficulties that may face researchers for determining the speed of polymerization R P , we propose to use the pH-metric method because it is simple and precise, and also does not require sophisticated means.In addition, it opens doors for great perspectives in this domain of research.Raw acidic extract ( ) ⎯→ ⎯ 3 Fucans.
conducted in suspension,and initiated by the ions ce 4+ (Hexanetratace rate of ammonium )

Δ
So we can write the equation (1) as follows :

Figure 4
Figure 4 shows that the individual Fucan N1 spectrum in curve (A) is identical to curve (B) with a concentration o f cerium [ ] L / M 10 .8 , 1 Ce 5 4 − + = we have simultaneously plotted the specific viscosity ( C sp η ) in function of polysaccharide concentration.The intrinsic viscosity [ ] η is obtained by extrapolation and intersections of these two curves at a zero-point concentration .On the other hand, this intrinsic viscosity is related to the molar mass through the formula of Mark Houwkin: [ ] a M .K = η , where a and K are constants.As for the two fractions of Fucan N1 and BBP-2 with known mass, the values of the constants were deduced from measures of the intrinsic viscosity [ ] η at a zero-point concentration.For example, as concerns the first fraction [ Dextran T70 (with a mass=73 000g/mol), in water and in DMSO, we have found the following values: values of 'a' for the Dextran T70 and Fucan, is present only in aqueous solutions, the Dextran is manifested therefore, more flexible than the Fucan.3.4.1 Viscosity and mass measurements of the obtained copolymersThe measurement of the molar mass by applying the viscometric method is performed through measuring the intrinsic viscosity by extrapolation of the two curves at [ ]

Table 1 .
Measurement of the relative viscosity

Table 3 .
Comparative table of the two (Vp) measuring methods: the pH-metric method and the extraction by soxhlet with the acetone method .