A simple preparation of N-acetylated chitosan highly soluble(2)

naP

Untreated

0.010.010.010.050.51.02.0sat.sat.sat.sat.

r.t.3050505050505050505011111111248

1.4×1039.3×1029.0×1025.9×1024.0×1023.1×1022.0×1021.7×1028.2×105.1×103.0×102.3×10

n;Mn=SHi/S(Hi/Mi),whereHiisthesampleconcentrationandNumber-averagedegreeofpolymerizationcalculatedfromM

MiisthemolecularweightofeachfractionofSEC.

270N.Kubotaetal./CarbohydrateResearch324(2000)268–274

Fig.2.1HNMRspectraofpartiallyN-acetylatedchitosaninD2O(top)andinDsignalsindicatedbyarrows2O-containingCFareassigned3COOD(bottom).ThetotheH-2,H-3,andH-1oftheGlcNresiduefromtheright.

Fig.3.RelationshipbetweenthedegreeofN-acetylationandthemolarratioofAc2OtoGlcNunits.TheerrorbarrepresentsthemaximumandminimumofN-acetylationde-grees,regardlessofmolecularweight.

Degradationofchitosan.—Puri edchitosan(0.5g)wasdissolvedin5%aqAcOH(30mL),andaqNaBOcentrationwas3(100mL)ofaprescribedcon-added.Afterstirringatapre-determinedtemperatureforapredeterminedtime,thereactionmixturewascooled,andaqKOHwasaddeduntilthepHreached10–11.Theprecipitateobtainedwascentrifugedand

washedwithdeionizedwateruntilitwasneu-tral,rinsedwithacetone,driedinvacuoatroomtemperature(rt),anddriedfurtherun-dervacuumat50°C.

Determinationofmolecularweight.—Weight-averagemolecularweightsofthede-gradedchitosansamplesweredeterminedusingaTosohSECsystem(column:TSKgelGMPWXL,7.8mmi.d.×30cm)equippedwithalow-anglelaserlight-scatteringphoto-meter(LS-8000)andadifferentialrefractome-ter(RI-8011).Eachsamplewasdissolvedinacetatebuffer(1M,pH4.8),whichwastheeluent,and lteredthroughamicro lter(0.45

Fig.4.Molecular-weightdependenceofwatersolubilityofhalfN-acetylatedchitosan.Watersolubilitywascharacterizedastransmittanceofthedispersionsat600nm.

Fig.5.1HNMRspectraintheregionforresonancesofanomericprotonsofpartiallyN-acetylatedchitosaninDcontainingDClat80°C:(a)GlcN–GlcN,(b)GlcN–Glc-2ONAc,(c)GlcNAc–GlcN,and(d)GlcNAc–GlcNAc.

N.Kubotaetal./CarbohydrateResearch324(2000)268–274271

Table2

Fractionsoffourpossibledisaccharidesforwater-solublehalfN-acetylatedchitosanSamplecode600k-50380k-54340k-52230k-52150k-50110k-5178k-5163k-4833k-4719k-5112k-488.8k-53

DegreeofN-acetylation(%)505452525051514847514853

FGlcN–GlcN0.240.200.230.220.240.220.230.280.260.220.240.21

FGlcN–GlcNAc(FGlcNAc–GlcN)0.260.260.250.260.260.270.260.240.270.270.280.26

FGlcNAc–GlcNAc0.240.280.260.260.240.230.250.240.200.240.200.27

mm).The nalconcentrationofthesampleswas1.0g/L.Thecolumntemperaturewas40°C,the owratewas1.0mL/min,andtheinjectedsamplesizewas100mL.

N-Acetylationofchitosan.—Chitosan(0.3g)wasdissolvedin10%aqAcOH(50mL),andAc2Owasadded.Afterstirringatambienttemperaturefor5h,aqNaOHwasaddeduntilthepHreached8–9inordertostopthereaction.Thereactionmixturewasdialyzedagainstdeionizedwaterfor2daystoremoveanymicroionsandlyophilized.TheacetylatedchitosanwasthentreatedwithmethanolicKOHfor5hatrtandrepeatedlywashedwithMeOHusingacentrifuge.Finally,itwasdis-solvedindeionizedwaterandlyophilized.NMRspectroscopy.—1HNMRspectrawererecordedonaBrukerARX300spec-trometerinD2Oat25°C.ThepartiallyN-acetylatedchitosanwaslyophilizedtwicefromD2OsolutionbeforepreparingthesamplesforNMRanalysis.Allsamplesweredissolvedin5mmdiametertubes,andthesampleconcen-trationswere20mg/mL.1HNMRspectrawerealsorecordedinthepresenceof6%CF3COOD.ThechemicalshiftsweregivenonthelscalerelativetoMe4Si.Themeasure-mentconditionswereasfollows:aspectralwindowof8064Hz,32kdatapoints,apulseangleof30°,anacquisitiontimeof2.03s,and32scanswithadelayof1sbetweenscans.Inordertodecreasethelinewidthoftheanomericprotonsignals,eachsampleofthehalfN-acetylatedchitosanwasdissolvedinD2Ocontaining1%DClandthe1HNMRspectrawererecordedat80°C.

Estimationofsolubility.—WatersolubilityofthehalfN-acetylatedchitosanwasevalu-atedfromtheturbidity.Afterbeingpulverizedgently,thehalfN-acetylatedchitosan(50mg)wasdissolvedindeionizedwater(5mL),andthetransmittanceofthesolutionwasrecordedonaHitachiU-1000spectrophotometerusingaquartzcellwithanopticalpathlengthof1cmat600nm.ThepHdependenceofthewatersolubilityofthehalfN-acetylatedchi-tosanwasalsoestimatedfromthetransmit-tanceofthesolution,whichwaspreviouslypreparedwithaqAcOH(10%),bythestep-wiseadditionofconcdNaOH.Thesampleconcentrationswere0.5%.ThesolubilityofthehalfN-acetylatedchitosaninaqorganicsolventswassimilarlyestimatedfromthe

Fig.6.pHdependenceofwatersolubilityofhalfN-acetylatedchitosan:( )8.8k-53,( )19k-51,( )33k-47,( )78k-51,( )150k-50,( )340k-52,and( )600k-50.SamplecodescorrespondtothoseinTable2.

272N.Kubotaetal./CarbohydrateResearch324(2000)268–274

Fig.7.DependenceofsolubilityofhalfN-acetylatedchitosanonDMAcontent:( )8.8k-53,( )12k-48,( )19k-51,( )33k-47,and( )63k-48.SamplecodescorrespondtothoseinTable2.

Fig.8.DependenceofsolubilityofhalfN-acetylatedchitosanonMe2SOcontent:( )8.8k-53,( )12k-48,( )19k-51,( )33k-47,and( )63k-48.SamplecodescorrespondtothoseinTable2.

transmittanceofthesolution,whichwaspre-paredinadvancewithdeionizedwater,byaddingorganicsolventsstepwise.Thesampleconcentrationswere1.0%.3.Resultsanddiscussion

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