Effects and kinetics of a novel temperature cycling treatment on the N-deacetylation of chitin

Effects and kinetics of a novel temperature cycling treatment on the N -deacetylation of chitin in alkaline solution

T.G.Liu a,c ,B.Li a,b,*,W.Huang a,*,B.Lv a ,J.Chen a ,J.X.Zhang a ,L.P.Zhu a

a

College of Food Science and Technology,HuaZhong Agricultural University,WuHan 430070,China b

Department of Food Science and Engineering,Ningbo University,Ningbo 315211,China c

Department of Chemisty and Food Science,Chizhou College,Chizhou 247000,China

a r t i c l e i n f o Article history:

Received 14March 2008

Received in revised form 2December 2008Accepted 9December 2008

Available online 24December 2008Keywords:Chitin Chitosan

Temperature cycling treatment N -deacetylation

Pseudo-?rst-order kinetics

a b s t r a c t

The in?uences of alkaline concentration,cold-treatment temperature,cold-and heat-treatment method,reaction time and the ration of chitin powder to NaOH solution on the N -deacetylation of shrimp chitin were investigated.The effects of the alkaline concentration,cold-and heat-treatment method and reac-tion time were signi?cant;nevertheless,the effects of temperature and the ration of chitin to solution were insigni?cant.The N -deacetylation followed the pseudo-?rst-order kinetics.The apparent rate con-stants of the reaction ranged from 3.3?10à3h à1to 22.8?10à3h à1,and the apparent activation energy was preliminary estimated about 9.76kJ/mol at aqueous 35%NaOH solution,in the temperature range of à5to à35°C,which was much lower than that of previous literatures.The effect of novel temperature cycling treatment on the reaction process and structure of chitin,compared with the single alkali-freez-ing treatment,was greater.So the novel temperature cycling treatment could be regarded as a more ef?-cient novel pretreatment method for further modi?cation of chitin.

ó2009Published by Elsevier Ltd.

1.Introduction

Chitin is an ideally (1–4)-linked linear polysaccharide com-posed of 2-acetamino-2-deoxy-b -D -glucopyranosyl residues.Chitosan composed of 2-amino-2-deoxy-b -D -glucopyranosyl resi-dues is the N-deacetylated derivative of chitin (Shigemasa,Matsu-ura,Sashiwa,&Saimoto,1996)and usually prepared from chitin.Chitin is the second most abundant natural biopolymer on earth after cellulose,and found mainly in invertebrates,insects,marine diatoms,algae,fungi,and yeasts.Approximately 10billion tons of chitin is produced by these organisms each year (Rege &Block,1999;Tolaimate et al.,2000).Chitin and chitosan are bio-renew-able,biocompatible,environmentally friendly,biodegradable,and bio-functional such as antibacterial activity,hypocholesterolemic acitivity,antitumor activity,immuno-stimulating effect,antioxi-dant activity and antihypertensive activity (Chae,Jang,&Nah,2005;Je,Cho,&Kim,2006),and these unique properties offer much potential applications in many ?elds.Recently,it has been widely applied in the ?elds of agriculture,medicine,pharmaceuti-cals,dentistry,functional food,food processing,textile production,environmental protection,biotechnology industry,cosmetic/

personal care,and so on (Baxter,Zivanovic,&Weiss,2005;Tan,Khor,Tan,&Wong,1998;Sandford,1988).

However,chitin is insoluble in water,acid,alkaline solution,and common organic solvents because of its strong intra-and in-ter-molecular hydrogen bonds.Its derivative,chitosan,prepared by deacetylation of native chitin,is only soluble in some speci?c di-lute acids because of free amino groups in its chemical structure,such as organic acids including formic,acetic,propionic,lactic,cit-ric,and succinic acid,and a very few inorganic solvents,such as hydrochloric,phosphoric,and notric acid (Wang,Turhan,&Gun-asekaran,2004),but it is still insoluble in the neutral or basic range (Koide,1998).The water-insoluble property of chitin/chitosan is disadvantageous to its widespread application.Therefore,improv-ing the solubility of chitosan is crucial if this plentiful resource is to be utilized across a wide pH range (Chung,Kuo,&Chen,2005).To fully improve the solubility of chitin,many methods can be used,such as homogeneous phase reaction (Kurita,Kamiya,&Nishim-ura,1991;Sannan,Kurita,&Iwakura,1976),reducing the molecu-lar weight of chitosan (Chang,1996),introducing a hydrophilic functional group to the chitosan (Holme &Perlin,1997).Alkali-freezing treatment of chitin is one of the methods,which has al-ready been chosen as a convenient precursor for ef?cient modi?ca-tions (Sannan,Kurita,&Iwakura,1975;Sannan et al.,1976;Dong,Wu,Wang,&Wang,2001;Feng,Liu,&Hu,2004;Tokura &Tamura,2001).

The kinetics of homogeneous alkaline deacetylation of a -chitin was reported to be a pseudo-?rst-order reaction (Sannan,Kurita,&

0144-8617/$-see front matter ó2009Published by Elsevier Ltd.doi:10.1016/j.carbpol.2008.12.006

*Corresponding authors.Address:Department of Food Science and Technology,Huazhong Agricultural University,Wuhan 430070,China.Tel.:+862763730040;fax:+862787282966.

E-mail addresses:liutg@5b15517b7375a417866f8f7f (T.G.Liu),libinfood@5b15517b7375a417866f8f7f (B.Li).

Carbohydrate Polymers 77(2009)

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Carbohydrate Polymers

j o u r n a l ho m e p a g e :w w w.e l s e v i er.c om/loc

ate/carbpol

Iwakura,1977).Similar results were obtained from heterogeneous deacetylation at150°C(Castelli,Bergamasco,Beltrame,&Focher, 1996).However,Chang,Tsai,Lee,and Fu(1997)has reported that under different concentrations of alkali heterogeneous deacetyla-tion of shrimp chitin appeared to be more complicated than a pseudo-?rst-order reaction.It might be controlled by a higher-or-der reaction and a diffusion controlled reaction(Chang et al., 1997).Methacanona,Prasitsilpa,Pothsreea,and Pattaraarchachaib (2003)reported that heterogeneous N-deacetylation of squid chitin also followed pseudo-?rst-order kinetics at the initial period and leveled off after1h(Methacanona et al.,2003).

However,deacetylation kinetics of chitin in alkaline solution at low-temperature was still in blank.In this study,we used a novel temperature cycling treatment and the N-deacetylation reaction conditions were controlled.The temperature we chose combined the industrial possibility of producing water-soluble chitosan and the properties of alkali solution.Because the normal low-tempera-ture cold storage was usually kept atà35°C,and most of the domestic refrigerator was atà18°C.Here,we chose the two tem-peratures.In fact,we measured the approximate frozen tempera-ture of aqueous35%NaOH,and it began to turn to ice crystal belowà13.5°C(it can not be regard as physical concept‘‘freezing point”),so we selected theà15°C,since ice stress might be stron-ger when the temperature was close to the ice crystal forming tem-perature.We also selectedà5°C only for detecting the case when the chitin was in low-temperature but not low enough to freeze. The aim of this study was to investigate the effects of alkali-tem-perature-cycling treatment on the solid state structure of chitin, and the kinetics of the process was also investigated.

2.Materials and methods

2.1.Materials

Chitin derived from shrimp shells with a degree of deacetyla-tion(DDA,%)of21.11±0.59%,as determined by alkalimetric esti-mation,was purchased from Zhejiang Yuhuan Biochemical Co. (PRC).All other commercially available solvents and reagents(ana-lytical grade)were used without further puri?cation.Double dis-tilled water was used throughout unless otherwise speci?ed.

2.2.Alkali-temperature-cycling treatment of chitin

Chitin powder was soaked in different concentration aq NaOH (35,40,45,and50%,wt.%)at room temperature for8h,chitin pow-der to NaOH solution ratio was1:4,1:6,and1:10(w/V).The mix-ture in the sealed containers were kept freezing at different temperature(à5,à15,à18,andà35°C)for about22h,the sam-ples processed at low-temperatures were then taken out and heated by various methods:(I)processed at room temperature; (II)320W microwave(WP800TL23-K1,Galanz,China)assisted process for5min;(III)heated for15min in a boiling water bath; (IV)heated at40°C for2h.The total time of low-temperature treatment and heat-treatment was designed as24h(1day),after the?rst cold-treatment and heat-treatment cycle,the sample was coded as1st day.Repeated the temperature cycling treatment for2,3,4,5or6times,the samples obtained were coded as2nd, 3rd,4th,5th,and6th day correspondingly,and the total treatment time was48h,72h,96h,120h,and144h,respectively.The rate of heating will depend not only on temperature but also the quan-tity of material being treated.All the samples treated with the same type of container,and the total mass of samples was the same except for1:6and1:10(the ratio of chitin and alkali solu-tion)samples.And all the data used for kinetic analysis and regres-sion analysis was obtained under the same conditions.

After the temperature cycling treatment,the heat-treated sam-ples were then dispersed in70%(V/V)aqueous ethanol(EtOH)and neutralized using diluted HCl in ice-water bath carefully.The pre-cipitated samples were?ltered and washed with70%EtOH until no Clàremained(determined by AgNO3method),?nally the samples were dried at60°C under reduced pressure.Each sample was car-ried out in triplicate.

The reference sample prepared as the following method and was coded as V:chitin was soaked in aq45%(wt%)NaOH at room temperature,the chitin to solution ratio was1:10,and then kept freezing atà18andà35°C for various time,ranging from1to5 days,the sample was thawed at room temperature without further treatment(Feng et al.,2004).

2.3.Determination of degree of deacetylation(DDA)

The acid-base titration method(Gu,Wang,Liu,&Xia,2003;Wu, Zeng,Zeng,&Zhang,2004)with a little modi?cation was used to determine the degree of deacetylation of the samples.All the sam-ples were desiccated for24h at80°C under reduced pressure,and then0.1500g of the various samples were dissolved in20ml 0.1mol/L HCl standard solutions,and stirred for3h at 25±0.1°C.The mixture was diluted with20ml double distilled water,and then titrated with0.1mol/L NaOH of the standard solu-tion using two drops of0.1%methyl orange–aniline blue(1:2,V/V) water solution as an indicator(Chen&Guo,1990).All experiments were carried out in triplicate.DDA was calculated using the follow-ing equation:

DDAe%T?

eC1V1àC2V2T?0:016

Ge1àWT?0:0994

?100%

where C1and C2are the concentration of the standard solution of HCl and NaOH(mol/L),respectively.V1was the volume of HCl (ml),and V2was the volume of NaOH at the end point of titrated (ml).0.016was the–NH2%content equal to1ml1mol/L HCl stan-dard solution(g),0.0994was the ideal–NH2%content of chitin(16/ 161),G was the sample mass(g),W was the water content(%).

2.4.FT-IR spectra analysis

The deacetylated samples were ground and blend well with KBr,the sample to KBr ratio was1:60,the mixture were dried overnight at60°C under reduced pressure,and then KBr discs were prepared under27MPa pressure for2min,each sample was prepared6discs.The KBr pellets of the mixed powder were desiccated for24h at110°C under reduced pressure,and then their IR spectra were recorded on a Nicolet NEXUS470FT-IR spectrophotometer.At32accumulate scans with a resolution of 4cmà1were averaged and referenced against air.The intensity of selected IR absorption bands were determined by the baseline method using the OMNIC software package of the instrument (Brugnerotto et al.,2001;Duarte,Ferreira,Marvao,&Rocha, 2002;Shigemasa et al.,1996).Crystalline Index(CrI)was calcu-lated from the ratio of absorbance at A1382/A2920cmà1(Focher, Beltranme,Naggi,&Torri,1990).

2.5.Statistical analysis

All collected data were expressed as mean±SD.The statistical analysis method such as ANOVA was adopted to analyse the data obtained from the experiments,and the least signi?cant difference (LSD)at5%was applied to de?ne signi?cant difference between mean values.Polymultiple non-linear regression analysis was also used to search the optimal condition for N-deacetylation reaction, the backward method was used.G3D model was employed to gen-

T.G.Liu et al./Carbohydrate Polymers77(2009)110–117111

erate three-dimensional graphs.The data were statistically ana-lysed by using the SAS System for Windows V8(SAS Institute Inc.,Cary,NC27513,USA,2000).

3.Results and discussions

3.1.Alkaline N-deacetylation of chitin

In traditional methods,abundant high concentration NaOH solution was usually employed to make chitin deacetylating,which caused environmental pollution and made the following puri?ca-tion of chitosan more dif?cult.Therefore,it was meaningfulness to develop a method to reduce the amount of NaOH.Alkali-freez-ing method was usually adopted,à18°C was a common used tem-perature(Dong et al.,2001;Feng et al.,2004).

Effects of chitin to solution ratio,NaOH concentration,cold-treatment temperature,heated methods and reaction time on the N-deacetylation reaction were investigated.By using acid-base titration method for determination of DDA,the process of N-deacetylation could be monitored.Table1showed the change of DDA in several conditions.The effect of chitin to solution ratio was signi?cant(chitin:solution<1:6,p<0.05),but it was insignif-icant when there was an excess of NaOH solution(chitin:solu-tion>1:6,p>0.05).At high concentration of NaOH with long reaction time,there were no signi?cant changes on the alkali N-deacetylation of chitin,while it was signi?cant with a short reac-tion time.The effects of cold-treatment temperature and heated methods were signi?cant not only at the initial treatment,but also in the later reaction period.The greatest impact on the DDA was microwave-assisted heat method and the second greatest was boil-ing water treatment with short times reaction such as the1st day treatment.Nevertheless,long time of treatment such as heat meth-od of5days at the40°C became the greatest.An interesting result showed that underà15°C cold-treatment temperature,DDA in-creased signi?cantly,and it might be related to the freezing con-centration effects,which might made the NaOH in?ltrate more easily.In a word,the DDA of most samples was still not increased too much even with long time of alkali-temperature-cycling treat-ment,which provided the possibility for preparing partially deacetylated water-soluble chitin using alkali-temperature-cycling treatment as a pretreatment method.3.2.Optimal conditions for N-deacetylation

In order to investigate the optimum conditions of N-deacetyla-tion,DDA of samples(Nos.4–7and10–12,the No.is the same as Table1)obtained from various conditions was analysed with the RSREG procedure of SAS and the Backward Elimination Stepwise method was employed,and the results were shown in Table2. Reaction time,NaOH concentration,interaction of temperature and time,interaction of time and NaOH concentration played dom-inant role on the degree of deacetylation(p<0.05).The quadratic term of temperature,NaOH concentration and time((Temp.)2, (Con.)2and(Time)2)also showed signi?cant effects on the DDA (p<0.05).However,the effect of other factors including the cold-treatment temperature,the interaction of temperature and NaOH concentration were insigni?cant(p>0.05).Response surface for the change in DDA as a function of temperature and time,time and NaOH concentration,and temperature and NaOH concentra-tion were shown in Fig.1a–c,respectively.The best-?t regression equation for the optimum deacetylation within the experiment range was obtained from the statistical analysis.

DDAe%T?à260:2205t12:7571?Con:t0:6936?Time

t0:0019?Temp:?Timeà0:0074?Con:?Time

à0:0053?eTemp:T2à0:1337?eCon:T2à0:0010

?eTimeT2

where DDA was the degree of deacetylation(%);Temp.,cold-treat-ment temperature(°C);Con.,NaOH concentration(%);Time,tem-perature cycling treatment time(h).

3.3.Kinetics of N-deacetylation

3.3.1.Effects of alkaline concentration on N-deacetylation

The effect of alkaline concentration on N-deacetylation was shown in Fig.2a,b.It seemed that at a low concentration condition, such as35%(wt%),the N-deacetylation followed the pseudo-?rst-order kinetics at the?rst5days treatment,and the apparent reac-tion rate constant was4.3?10à3hà1(R2value=0.9879).Never-theless,with the increasing of NaOH concentration,the deacetylation process did not followed the?rst-order kinetics at the total treatment.There was a sudden jump point on the?rst

Table1

Degree of N-deacetylation of chitin obtained from various conditions.

No.RCS.(w/V)He.Con.(wt%)Temp.(°C)DDA(%)

24h48h72h96h120h

11:4IV35à1835.41±0.6442.17±0.6548.12±0.4053.69±1.2462.59±0.43a 21:6–56.10±0.74b

31:10–55.36±0.15b

41:4I35à1825.73±0.25b33.46±1.15c42.45±0.18c47.6±0.25c51.31±0.46b 54045.58±6.57a49.37±0.66b52.07±0.71b61.77±0.74a64.75±0.42a 64544.10±0.11a50.41±0.21a55.83±0.23a60.33±0.44b63.94±1.14a 75049.33±0.53a52.83±0.52a56.64±0.68a59.87±0.77b64.81±0.85a

41:4I35à1825.73±0.25d33.46±1.15d42.45±0.18d47.60±0.25d51.31±0.46d 8II47.24±0.39b53.79±0.59a60.12±0.78a64.55±0.70a60.49±1.87b 9III54.32±0.30a49.77±0.41b45.74±0.98c50.32±0.57c56.85±0.89c 1IV35.41±0.64c42.17±0.65c48.12±0.40b53.69±1.24b62.59±0.43a

101:4I35à3523.44±0.68d32.69±1.86c39.27±1.05d41.34±1.81d46.38±0.15d 4à1825.73±0.25c33.46±1.15c42.45±0.18c47.60±0.25c51.31±0.46c 11à1535.05±0.64a44.30±0.57a49.38±0.98a53.30±0.51b59.67±0.84a 12à527.54±0.58b41.51±0.61b47.69±0.84b55.21±0.22a57.81±0.41b

131:10V45à3523.03±0.34b23.11±0.69b24.12±0.64a22.43±0.74b23.65±0.67a 14à1824.29±0.30a25.18±0.52a23.91±0.32a24.23±0.77a23.91±1.41a

The values were presented as mean±SD,n=3;values with different superscripts within a column indicate signi?cant differences(p<0.05).RCS.,ratio of chitin and NaOH aq. solution;He.,heat methods;Con.,NaOH concentration(wt%);Temp.,cold-treatment temperature(°C),respectively.

112T.G.Liu et al./Carbohydrate Polymers77(2009)110–117

day’s treatment,but the reaction reduced gradually and followed the?rst-order kinetics again at the subsequently treatment,and the reaction rate constant decreased as a function of concentration rapidly(R2=0.9985).It could be seen,at the1st day’s treatment, the higher NaOH concentration led to the faster the loss of acetyl group.For example,at50%concentration,the reaction rate was four times more than that of at35%concentration,but at the sub-sequently treatment it was lower than the latter.The heteroge-neous N-deacetylation of chitin was a typical solid–liquid phase reaction,and deacetylation reaction?rst occurred in the chitin par-ticles surface and shallow surface.At the beginning,the DDA was low,many acetyl groups on the chitin particles surface.With increasing the NaOH concentration,more NaOH contacted with the acetyl groups,the reaction rate was bigger.Thereafter,the sur-face acetyl groups decreased and the in?ltration of NaOH into the chitin particles was farther more dif?cult,thus the reaction rate decreased.In a sense,it was not an effective method to improve the deacetylation rate by only increasing the concentration of NaOH.

3.3.2.Effects of cold-treatment temperature on N-deacetylation

Fig.3showed the effects of cold-treatment temperature on N-deacetylation.At the same alkaline concentration,the N-deacetyla-tion process also followed the pseudo-?rst-order kinetics,and the reaction rate constants declined as a function of temperature de-creased.At35%NaOH conditions,the apparent activation energy was preliminary estimate as about9.76kJ/mol(R2=0.9247)with Arrhenius equation and plot method.This is lower than the 35.56–57.74kJ/mol reported for heterogeneous N-deacetylation between80and120°C(Castelli et al.,1996),35.63and46.02kJ/

Table2

Regression analysis between the degree of N-deacetylation and the alkaline N-deacetylation variables.

Variable Parameter estimate Standard error Type II SS F value Pr>F

Interceptà260.220530.7185647.195771.7600<.0001 Con.12.7571 1.4678681.242675.5400<.0001 Time0.69360.0801675.475374.9000<.0001 Temp.?Time0.00190.000943.0119 4.77000.0316 Con.?Timeà0.00740.0017175.439219.4500<.0001 (Temp.)2à0.00530.001785.95169.53000.0027 (Con.)2à0.13370.0173541.162360.0000<.0001 (Time)2à0.00100.0003105.271711.67000.0010 R20.9289

Temp.,cold-treatment temperature(°C);Con.,NaOH concentration(wt%);Time,temperature cycling treatment time(h).

T.G.Liu et al./Carbohydrate Polymers77(2009)110–117113

mol reported for heterogeneous N-deacetylation between51.0and 82.5°C,and80–100°C,respectively(Chen,1992;Focher et al., 1990).Furthermore,this is also lower than the92.05kJ/mol for homogeneous N-deacetylation in the temperature range25–40°C reported by Sannan et al.(1977).So it means that repeated cold-and heat-treatment may break the crystal structure of chitin and improve the process of deacetylation.It was similar with the reac-tion rate constants atà5andà15°C,but was higher than the con-stants atà18°C andà35°C,respectively.At approximately à15°C,which was1–2°C below the freezing temperature of chi-tin–alkali mixture,there was a so called freezing concentration ef-fect.The mechanical force induced by the repeatedly formed and recrystallized of ice crystals during the slow cold-treatment and low-temperature heat-treatment cycle process,might destruct the condensed matter especially the crystalline state structure,so that the mass-transfer process was aggrandizement.NaOH was forced to penetrate into the chitin particles,and the rate constants were larger.When the temperature deviated from the freezing temperature,most ice microcrystals formed rapidly,the crystalline

114T.G.Liu et al./Carbohydrate Polymers77(2009)110–117

destructed effect decreased,and then the concentration effect transferred as cryogenic effect,the resistance of mass-transfer de-creased only to an extent far from enough,the rate of mass-trans-fer were lower than that cold-treatment atà15°C,so the reaction rate constants were lower than that cold-treatment atà15°C. 3.3.3.Effects of heating methods on N-deacetylation

The effects of heating methods on the N-deacetylation were shown in Fig.4a–c.All the deacetylation process followed pseu-do-?rst-order kinetics.The results showed the effects of heating methods on N-deacetylation were signi?cant(Table1,p<0.05), and the more intense heating methods were,the higher DDA were. Fig.4a showed the effects of boiling water treatment,it could be seen clearly that on the1st day of treatment(1st day),the DDA in-creased fast,the rate constant was16.8?10à3hà1,which was much higher than the treatment of the following days,because N-deacetylation was a typical nucleophilic substitute reaction, the contents of substrates and products could affect the reaction rate.At the initial day,the DDA was lower,and increased with the boiling water treatment rapidly.On the following days,the free acetyl-content of products in the reaction solution was increased with the increasing of DDA,while the acetyl residues on the chitin molecular chain was decreased,and it was dif?cult for nucleophilic substitutions reaction.With the increasing of resistance of mass-transfer,the inhibitory effect of deacetylation became the main resistance to improve the DDA,so the reaction rate declined and slower than the initial day’s.Fig.4c showed the effects of micro-wave-assisted heating method,it was similar to the effects of boil-ing water treatment.Nevertheless,the initial day’s rate constant (22.8?10à3hà1)was higher than that of the boiling water treat-ment(16.8?10à3hà1),for the microwave heating model is a di-rect and internal heating model,and it could produce thermal-effect as well as non-thermal-effect.Microwave heating is the use of constantly rotating of molecular dipole moment,heating the materials internal and external simultaneously.It is more effective than traditional heating methods,which rely on thermal conduction and radiation model heating the materials from the surface to internal.Subsequently,the rate constants were lower than the boiling water method.On1st day,the initial DDA was low,ef?cient microwave-assisted method can signi?cantly im-prove the deacetylation process.DDA was already high in the fol-lowing days,which could not improve too much on the basis of the original even use of microwave-assisted method.The rate con-stants decreased with longer time reaction,however,it was still higher than that of using room temperature treatment and40°C heated method,respectively.Of particular interests,the DDA of referenced samples had a very little change throughout the whole treatment process,it indicate that the heating method and the heating process was very important for improving the reaction rate of N-deacetylation at a low-temperature.It was also interesting that using this novel temperature cycling treatment could obtain a homogeneous chitin alkali solution with4%chitin and10%NaOH, which could be employed to prepare partially deacetylated water-soluble chitin with homogeneous 5b15517b7375a417866f8f7fpared with traditional homogeneous method,the chitin concentration in-creased3times while the use of NaOH reduced to1=4(Sannan et al.,1975).

3.4.FT-IR spectra analysis

Infrared spectroscopy is a useful method to study hydrogen bonding and other interactions.Fig.5showed an FT-IR spectrum of chitin and baselines(Duarte et al.,2002)for determining CrI with FT-IR spectroscopy.FT-IR spectra of samples treated with al-kali-temperature-cycling were shown in Fig.6.It could be seen,the band at3448cmà1became broader and moved to a lower number-waves due to the O–H stretching vibrations and the absorption of intra-hydrogen bonds.The concealed of the N–H stretching vibra-tion(Amide III)absorption appeared at about3264and3104cmà1 with increasing DDA,which could also indicate the reduction of in-ter-molecular C(21)NHáááO@C(73)and C(61)OHáááHOC(62)hydro-gen bonds,respectively(Cho,Jang,Park,&Ko,2000).It meant this novel temperature cycling treatment could break the chitin strong intra-and/or inter-molecular hydrogen bonds,which could also explain the increasing reaction rate displayed in the kinetics study.The absorption bands of chitin at about1662(1628),

T.G.Liu et al./Carbohydrate Polymers77(2009)110–117115

1560,and1312cmà1were,respectively,referenced as amide I, amide II,and amide III.The absorption band at about1597cmà1 in chitosan was ascribed to N A H bending mode in the primary amine.The decrease of relative absorption intensity of amide II and amide III suggested the increase of DDA.The absorption bands of chitin at about2932,2891,1415,and1378cmà1were assigned to the C A H stretching and rocking bands,the relative absorption intensity varied with the CrI,and the1415cmà1peak became acu-ity with increasing DDA.The absorption band at about1158cmà1 was assigned to the bridge oxygen stretching,the absorption bands at about1073and1026cmà1was considered as the contribution of the C A O stretching,and the bands became broader with the fur-ther treatment.

Fig.7Showed the CrI of the treated chitin samples.During the temperature cycling treatment period,the crystallinity decreased rapidly in the initial3days and then underwent a few changes. It showed the initial days were useful for small molecules to per-meate and make further reaction to take place more thoroughly; this was coincided with the kinetics study.4.Conclusion

Under most conditions,semi-logarithmic plots between the amount of N-acetyl-D-glucosamine residues and the deacetylation time had well correlation coef?cients(R2>0.9).The N-deacetyla-tion process followed the pseudo-?rst-order kinetics,and the apparent activation energy was preliminary estimated to be 9.76kJ/mol which was lower than the activation energy reported for heterogeneous N-deacetylation at high temperature and for homogeneous N-deacetylation at low-temperature reported by Sa-nan et al.,respectively.It means it has much higher reactively with this novel temperature cycling treatment.

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