Determination of deacetylation degree of chitosan: a comparison between conductometric titration and CHN elemental analysis

Carbohydrate Research 344 (2009) 2591–2595

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Note

Determination of deacetylation degree of chitosan: a comparison between conductometric titration and CHN elemental analysis Z. M. dos Santos, A. L. P. F. Caroni, M. R. Pereira, D. R. da Silva, J. L. C. Fonseca * Departamento de Química, Universidade Federal do Rio Grande do Norte, Campus Universitário, Lagoa Nova, Natal, RN 59078-970, Brazil

a r t i c l e

i n f o

Article history: Received 31 March 2009 Received in revised form 17 August 2009 Accepted 25 August 2009 Available online 28 August 2009 Keywords: Chitosan Degree of deacetylation Conductometry CHN elemental analysis

a b s t r a c t Chitosan is a polysaccharide used in a broad range of applications. Many of its unique properties come from the presence of amino groups in its structure. A proper quantification of these amino groups is very important, in order to specify if a given chitosan sample can be used in a particular application. In this work, a comparison between the determination of chitosan degree of deacetylation by conductometry and CHN elemental analysis was carried out, using a rigorous error analysis. Accurate expressions relating CHN composition, conductometric titration, and degree of deacetylation, in conjunction with their associated errors, were developed and reported in this note. Error analysis showed conductometric analysis as an inexpensive and secure method for the determination of the degree of deacetylation of chitosan. Ó 2009 Elsevier Ltd. All rights reserved.

Chitosan is a linear polyaminosaccharide obtained from the deacetylation of chitin, the latter being the second most abundant polymer in nature, occurring as the main constituent of the exoskeleton of crustaceans and insects.1 Because chitosan is non-toxic, biodegradable, and biocompatible,2 it has been used in several applications such as drug releasing systems,3 in the treatment of skin injuries,4 heavy metal sorption,5 and textile industry effluent treatment.6 In dilute acid solutions, chitosan behaves as a cationic polyelectrolyte, due to the protonation of amine groups present in this biomacromolecule.7 The amount of these protonated groups is a parameter of extreme importance for the understanding of its molecular association/dissociation mechanisms as well as in the control of biomaterials processing.8,9 Furthermore, Rinaudo et al. have found that the protonation degree of chitosan also depends on the pKa of the acid used in its solubilization.10 The degree of deacetylation, XD, is the parameter that indicates the molar percentage of monomeric units that have amino groups and vary from 0 (chitin) to 100 (fully deacetylated chitin).11 Figure 1 shows the structure of chitosan and the relationship between the degree of deacetylation and the forming units of chitosan: b(1?4)-2-amino-2-deoxy-D-glucopyranose (deacetylated unit) and b-(1?4)-2-acetamido-2-deoxy-D-glucopyranose (acetylated unit). Berth and Dautzenberg have shown that XD is a parameter that can be related to physicochemical properties of chitosan, such as

* Corresponding author. Tel.: +55 84 3215 3828x215; fax: +55 84 3211 9224. E-mail address: [email protected] (J.L.C. Fonseca). 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.08.030

solubility and chain conformation,12 making it an important parameter for the characterization of chitosan. Several techniques have been used for the determination of XD, such as conductometric titration,13 potentiometric titration,14 IR spectroscopy,15 1H NMR spectrometry,16 UV–vis spectroscopy,17 and CHN elemental analysis.14 Despite conductometric titration being a very inexpensive method that is dependent upon a simple instrument, the conductivimeter,18 there is a lack of work consistently describing the relationship between data obtained from this method and XD, the same being true for CHN elemental analysis. In this work a rigorous analysis of the use of conductometric titration and CHN elemental analysis will be carried out. A comparison between the more established (but more expensive and equipmentdependent) technique of CHN elemental analysis and the simpler and inexpensive conductometric titration is carried out. Additionally, mathematical expressions for calculating XD experimental errors will be derived for both methods, as described below. CHN was used to determine carbon, hydrogen, and nitrogen mass percentages of three samples of chitosan of different molecular weights: Sample A was purified chitosan, Sample B was the result of acid hydrolysis under mild conditions (12 h at room temperature in HCl 0.6 mol L1), and Sample C was the result of acid hydrolysis at more severe conditions (2 h in reflux with HCl 2 mol L1). Average viscometric molar mass and chitosan mass fraction in the sample [WCHI, as defined by Eq. 23] for these samples are given in Table 1. In the next paragraphs we develop an adequate description of the exact relationship between XD and CHN-derived data, unambiguously taking into account the experimental details/constraints.

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O HOH2C *

NHCCH3 O

HO

O

*

O HO

O NH2

HOH2C

nD

nA

Figure 1. Chemical structure of chitosan: for 1 mol of chitosan, nD is the number of moles of b-(1?4)-2-amino-2-deoxy-D-glucopyranose units and nA is the number of moles of b-(1?4)-2-acetamido-2-deoxy-D-glucopyranose units. For this structure, the degree of deacetylation is XD = 100nD/(nD + nA) [Eq. 2].

Table 1  V (g mol1), and solid mass fraction, WCHIT, of the Average viscometric molar mass, M samples used in this study Sample

 V (g mol M

A B C

1.9  105 1.1  105 9.7  105

1

)

WCHIT 0.875 ± 0.007 0.889 ± 0.009 0.879 ± 0.008

When purifying/hydrolyzing chitosan, there is a substance that is practically impossible to eliminate: water. Full dehydration of chitosan would involve procedures such as lyophilization, which was not available to our work. Even after complete cryodesiccation, one could not guarantee that in the process of sample manipulation chitosan would not gain back some of its released water molecules. As a consequence, it does not seem sensible to use the elements mass percentage to determine XD. In other words, one should use the ratio between the mass percentages of two elements (apart from H and O, which are also present in water) to determine XD. It rests to us the carbon and nitrogen mass percentage. The mass ratio between carbon and nitrogen present in a given chitosan sample, wC/N, can be given by

total mass of carbon in sample total mass of nitrogen in sample M C nC;D  nD þ nC;A  nA  ; ¼ MN nN;D  nD þ nN;A  nA

wC=N ¼

ð1Þ

where MC is the molar mass of carbon, nD is the number of moles of deacetylated units in the sample, nC,D is the number of carbon moles per mole of deacetylated unit, nA is the number of moles of acetylated units in the sample, nC,A is the number of carbon moles per mole of acetylated unit, MN is the molar mass of nitrogen, nN,D is the number of nitrogen moles per mole of nonacetylated unit, and nN,A = nN,D is the number of nitrogen moles per mole of acetylated (or deacetylated) unit. Since the degree of deacetylation is given by

X D ¼ 100 

nD ; nD þ nA

ð2Þ

nD is proportional to XD as nA is proportional to 100  XD, so that Eq. 1 could be rewritten as

wC=N ¼

M C nC;D  X D þ nC;A  ð100  X D Þ  : MN nN;D  X D þ nN;A  ð100  X D Þ

ð3Þ

ð4Þ

where Dn = nC,A  nC,D If one uses data listed in Table 3 in Eq. 4, it follows that

ð5Þ

To our knowledge, the simplicity resultant in Eq. 5 has not been reported in the literature. Following a rigorous error analysis, for a quantity y calculated from quantities x1, x2, . . ., xn, it can be established that19

y ¼ yðx1 ; x2 ; . . . ; xn Þ;  n  X   @y   dxi ; dy 6 @x  i¼1

ð6Þ ð7Þ

i

where dy is the propagated error of y due to the uncertainties dxi from the measured quantities xi. Application of Eq. 7 to 5 yields the following expression for the associated error of XD

dX D 6 58:3093  dwC=N ;

ð8Þ

where dwC=N is the wC/N associated uncertainty, which, according to Eq. 7, is given as

dwC=N 6

1 %C d% þ d% : %N C %2N N

ð9Þ

The parameter wC/N was determined from the average values of %C and %H from 4 CHN analysis runs (d%C and d%N being their standard deviations), as shown in Table 2. Following the rest of the calculations, XD was determined for the three samples and is displayed in Table 4. One can see that at mild hydrolysis conditions, although molar mass decreased (due to hydrolysis of glycoside links), XD remained the same, while at severe hydrolysis conditions, it increased to around 100%, suggesting that in these conditions chitosan is deacetylated by acid hydrolysis. Figure 2 shows a typical curve of conductometric titration of an acidic chitosan solution by NaOH as obtained in this work. This curve can be divided into three regions.13,18 The first region of the curve is related to the neutralization of free HCl present in the solution; because of that, conductivity decreases as NaOH is added (Na+ is a bigger ion than H+). The second region is connected to the neutralization of the protonated amino groups present in chitosan (conductivity increases, since Na+ ions have a higher mobility than protonated chitosan). Finally the third region is associated to the addition of Na+ and OH ions to the solution (conductivity increases because there is an increase in ionic species, as NaOH is added). The three regions are described as curves with constant slope: HCl neutralization:

jHþ ¼ AHþ þ BHþ V NaOH

XD can be expressed, then, as

  100 MN XD ¼  wC=N ;  nC;A  nN;A  Dn MC

X D ¼ 100  ð4  0:583093  wC=N Þ:

ð10Þ

NH3 þ neutralization:

jNH3 þ ¼ ANH3 þ þ BNH3 þ V NaOH +



Addition of Na and OH ions:

ð11Þ

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Z. M. dos Santos et al. / Carbohydrate Research 344 (2009) 2591–2595 Table 2 CHN results for the samples analyzed in this work Sample

Run

%C

%H

%N

A

1 2 3 4 Average

40.05 39.85 39.87 40.2 40.0 ± 0.2

7.59 7.62 7.89 7.68 7.7 ± 0.1

7.41 7.37 7.39 7.46 7.41 ± 0.04

B

1 2 3 4 Average

39.27 39.33 39.34 39.39 39.33 ± 0.05

7.67 7.71 7.73 7.7 7.70 ± 0.03

7.25 7.31 7.25 7.24 7.26 ± 0.03

C

1 2 3 4 Average

39.66 39.62 39.7 39.69 39.67 ± 0.04

7.46 7.44 7.53 7.5 7.48 ± 0.04

7.73 7.72 7.74 7.73 7.73 ± 0.01

wC/N = 5.40 ± 0.05

wC/N = 5.42 ± 0.03

wC/N = 5.14 ± 0.03

Table 3 Parameters related to the chemical structure of chitosan

12.0107 g mol1 1.00794 g mol1 15.9994 g mol1 14.0067 g mol1 161.1558 g mol1 203.1925 g mol1 42.0367 g mol1 6 8

4

-1

Value

Carbon molar mass Hydrogen molar mass Oxygen molar mass Nitrogen molar mass Deacetylated unit (C6H11O4N) molar mass, MD Acetylated unit (C8H13O5N) molar mass, MA DM Number of carbons per deacetylated unit, nC,D Number of carbons per acetylated unit, nC,A

κ (mS cm )

Parameter

Table 4 Values of XD and their associated errors obtained with CHN analysis Sample

XD (%)

A B C

85 ± 3 84 ± 2 100 ± 2

3

VNaOH,i

2

jOH ¼ AOH þ BOH V NaOH

ð12Þ

The intercept between Eqs. 10 and 11 gives the volume in which protonated amino groups begin to be neutralized, VNaOH,i (when jHþ ¼ jNH3 þ ), and the intercept between Eqs. 11 and 12 gives the volume in which the amino groups are totally neutralized, VNaOH,f (when jNH3 þ ¼ jOH ):

V NaOH;i

AHþ  ANH3 þ ¼ BNH3 þ  BHþ

1 0

ð13Þ

10

20

V (mL) Figure 2. Typical conductometrical titration curve for the chitosan used in this work. VNaOH,i and VNaOH,f [Eqs. 13 and 14] are indicated by arrows.

and

V NaOH;f ¼

VNaOH,f

ANH3 þ  AOH : BOH  BNH3 þ

ð14Þ

The number of moles of aminated groups (or deacetylated units, nD) is given by

nD ¼ DV  C NaOH ;

ð15Þ

where DV = VNaOH,f  VNaOH,i, and CNaOH is the sodium hydroxide concentration. The number of acetylated units may be expressed by

mA mchit  nD  M D mchit  DV  C NaOH  MD ¼ ¼ MA MA MA W CHIT  mS  DV  C NaOH  M D ¼ ; MA

nA ¼

ð16Þ

where mA is the mass of acetylated units, mchit is the mass of chitosan used in the analysis (in a dried basis), mS is the sample mass (absorbed water included), MA is the molar mass of the acetylated units, and MD is the molar mass of the deacetylated units. Here there is a disadvantage of using conductometric titration for the determination of water content: since one has to work with the total mass of pure chitosan (without absorbed water), another parameter, with its associated error, must enter in the calculus: WCHI. Substitution of 15 and 16 in the expression that defines XD [Eq. 2] results in

X D ¼ 100MA 

DV  C NaOH ; DV  C NaOH  DM þ W CHIT  mS

ð17Þ

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Z. M. dos Santos et al. / Carbohydrate Research 344 (2009) 2591–2595

where DM = MA  MD. Substitution of MA and DM by the values listed in Table 3 results in

X D ¼ 20319:25 

C NaOH  DV : 42:0367  C NaOH  DV þ W CHIT  mS

ð18Þ

In the case of Eq. 18, application of Eq. 7 to calculate error propagation yields

        @X D @X D dC DV þ  dW m ; dX D 6  NaOH   @ðC NaOH DVÞ @ðW CHIT mS Þ CHIT S

ð19Þ

where

dC NaOH DV ¼ C NaOH dDV þ DVdC NaOH ;

ð20Þ

and

dW CHIT mS ¼ W CHIT dmS þ mS dW CHIT :

ð21Þ

Applying 19–21 on 18 yields   1 dX D 6 20319:25 42:0367  C NaOH DV þ W CHIT  mS   42:0367  C NaOH DV   d 2  C NaOH DV ð42:0367  C NaOH  DV þ W CHIT  mS Þ     C NaOH  DV   þ20319:25 d 2  ð42:0367  C NaOH  DV þ W CHIT  mS Þ  W CHIT mS  XD  ¼ j1  0:00206881  X D jdC NaOH DV þ 4:921381  105  X D  dW CHIT mS : C NaOH DV

ð22Þ The use of Eq. 18 and 22 with data, listed in Table 5, resultant from conductometric analysis results in the values of XD, shown in Table 6. It can be clearly seen that conductometric titration gives values that are in agreement with those obtained via CHN elemental analysis, so that this method can safely be used for the determination of chitosan deacetylation degree. The results also confirmed that the procedures involved in the purification of the chitosan samples used in this study were able to eliminate improbable protein residues present in these samples (it has been shown that protein may occur as an impurity in untreated chitin samples20).

Table 5 Results related to conductometric analysis Sample

Run

VNaOH,i (mL)

A

1 2 3 4

5.896 13.136 5.825 13.124 5.753 13.141 5.779 13.095 DV = (7.31 ± 0.06) mL

7.24 7.299 7.388 7.316

1 2 3 4

6.185 6.188 6.268 6.149

13.247 13.239 13.198 13.337 DV = (7.1 ± 0.1) mL

7.062 7.050 6.930 7.188

1 2 3 4

5.168 5.054 5.096 5.138

12.923 13.119 13.160 13.080 DV = (8.0 ± 0.2) mL

7.755 8.065 8.064 7.942

mS = (0.2286 ± 0.0001) g

B mS = (0.2250 ± 0.0001) g

C mS = (0.2275 ± 0.0001) g

VNaOH,f (mL)

DV (mL)

1. Experimental Acetic acid (99.5%, Cromato Produtos Químicos Ltd, Brazil), sodium hydroxide (P.A., Cromoline Química Fina Ltd, Brazil), and hydrochloric acid (PA, 37%, Cromato Produtos Químicos Ltd, Brazil) were used as received. Chitosan (Polymar Ltd, Brazil) was purified following the procedure described below  A given mass of chitosan was dissolved in a 2 (v/v) % acetic acid solution under stirring, for 24 h, at room temperature, in order to obtain a solution with a polymer concentration of 20 g L1. Afterwards, the solution was filtered using a Millex MilliporeTM filter with an average pore diameter of 41 lm. Following, a 5 (w/v) % NaOH solution was slowly added under stirring, until all chitosan was precipitated. The precipitate was separated from the supernatant and washed with water until neutral pH. Finally, the wet precipitate was dried in air at 40 °C and grounded to its final form. This sample was named Sample A. Another two samples of chitosan were produced by hydrolysis of this sample:  Sample B: a given mass of chitosan was solubilized in HCl 0.6 mol L1, in order to obtain a solution of chitosan with a concentration of 20 g L1 and was left under mechanical stirring for 12 h, at room temperature. Finally, the previous procedure used to purify Sample A was carried out in the solution to obtain the purified hydrolyzed Sample B.  Sample C: this sample was obtained by a more severe hydrolysis, as reported by Qandil et al.:21 10 g of chitosan was solubilized in 830 mL of HCl 0.1 mol L1, 170 mL of HCl 37% was added to the mixture (resulting in a final HCl concentration of 2 mol L1), which was left under reflux for 2 h, cooled down to room temperature, and hydrolyzed chitosan was precipitated by the addition of ethanol. The precipitate was separated from the supernatant, re-dissolved in 2 (v/v) % acetic acid solution, and the same process of purification applied to Sample A and Sample B was applied to this solution in order to obtain solid Sample C.  V ), was determined Chitosan viscometric average molar mass, (M using Mark–Houwink–Sakurada equation as described elsewhere,22 and chitosan mass fraction for a given sample, WCHIT, was determined by leaving a sample mass mS in an oven, at 105 °C, until a constant mass mchit was reached, with the chitosan mass fraction given by

W CHIT ¼

mchit : mS

ð23Þ

1.1. CHN elemental analysis CHN elemental composition of chitosan was determined using a Perkin Elmer analyzer, model 2400. All the determinations were done in quadruplicate. 1.2. Conductometric titration

Table 6 Values of XD and their associated errors obtained with conductometry Sample

XD (%)

A B C

88 ± 2 86 ± 2 95 ± 3

A given chitosan solution was prepared by the dissolution of a given mass of chitosan (in order to have 0.2 g of it, in a dried basis) in 40 mL of a 0.05 mol L1 HCl solution, which remained under stirring for 18 h, at room temperature. After the addition of 100 mL of water, conductometric titration was carried out with NaOH [CNaOH = (0.1456 ± 0.0007) mol L1] using a conductivimeter Digimed, model DM 31 (K = 0.1 cm1). All the titrations were done in quadruplicate and milli-Q water was used in these experiments.

Z. M. dos Santos et al. / Carbohydrate Research 344 (2009) 2591–2595

Acknowledgments The authors thank Brazil’s Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundo Setorial do Petróleo e Gás Natural (CT-PETRO), and Pró-Reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte (PROPESQ-UFRN) for financial support during the course of this work. The authors also thank Ms. Camila R.M. de Lima for her help in carrying out the experiments as her part in an undergraduate student scientific initiation program. References 1. Kumar, M. N. V. R. React. Funct. Polym. 2000, 46, 1–27. 2. Dodane, V.; Vilivalam, V. D. Pharm. Sci. Technol. Today 1998, 1, 246–253. 3. Liu, G.-Y.; Zhai, Y.-L.; Wang, X.-L.; Wang, W.-T.; Pan, Y.-B.; Dong, X.-T.; Wang, Y.-Z. Carbohydr. Polym. 2008, 74, 862–867. 4. Muzzarelli, R. A. A.; Morganti, P.; Morganti, G.; Palombo, P.; Palombo, M.; Biagini, G.; Mattioli Belmonte, M.; Giantomassi, F.; Orlandi, F.; Muzzarelli, C. Carbohydr. Polym. 2007, 70, 274–284. 5. Babel, S.; Kurniawan, T. A. J. Hazard. Mater. 2003, 97, 219–243.

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