1. International Conference On Chemical Sciences (ICCS-2007)
Innovation In Chemical Sciences For Better Life
Yogyakarta-Indonesia, 24-26 May, 2007
MAT/19-6P
SYNTHESIS OF CATIONIC WATER SOLUBLE CHITOSAN HAVING
QUARTERNARY AMMONIUM GROUP SIDE CHAIN
Agus Haryono* and Dewi Sondari
Polymer Chemistry Group, Research Center for Chemistry
Indonesian Institute of Sciences
Kawasan Puspiptek Serpong, Tangerang 15314
ABSTRACT
Cationic water-soluble chitosan was synthesized by conjugating 3-chloro-hydroxy-propyltrimethylammonium
chloride onto chitosan chain. Varying the molar ratio of 3-chloro-hydroxy-propyltrimethylammonium chloride to
chitosan from 3:1 to 6:1 produced chitosan with variation of substitution degree. The molecular weight of chitosan
was determined by intrinsic viscosity measurements in 0.25 M acetic acid / 0.25 M sodium acetate at 25oC. FTIR
measurements were performed to analyze the chemical composition of the cationic water-soluble chitosan. 1H NMR
spectra were obtained to confirm the successful conjugation onto chitosan chain. Water solubility of obtained
cationic polymers was measured using UV-vis spectrophotometer at 600 nm.
Keywords: Water soluble, chitosan, cationic polymer, quarternary ammonium
INTRODUCTION limited water-solubility would be too substantive and
would be adsorbed almost completely onto skin.
Chitosan is a (1,4)-linked 2-amino-2deoxy-β-D- Recently, the use of polymeric surfactant as
glucan and can be obtained from chitin, a kind of waste emulsifiers has been suggested as non-irritant
material from the ocean food industry, by alkaline or alternative to low molecular weight surfactants.5 Due to
enzymatic deacetylation. This natural polysaccharide their large molecules, polymeric surfactants do not
possesses useful characteristics such as non-toxicity, penetrate into the skin, nor do they enhance an
high contability, and non-antigeneticy that offers unwanted penetration of other formulation compounds.6
advantages for the possibility of medical use.1 Despite In addition, polymeric emulsifiers have the advantage
all these properties, chitosan is still a polymer that lacks of low usage level, excellent emulsion stability,
good solubility at physiological pH values. Chitosan has simplified emulsion formation procedure and possibility
an apparent pKa of 5.5, as measured by potentiometric to formulate water proof systems.7
titration.2 The solubility of chitosan is poor when the pH Hydrophobically modified polymers have been
value is above 6, and it will lose charge when extensively investigated as emulsion stabilizers.8 An
precipitating from solution. emulsion is a dispersion of one immiscible liquid in
Thanou et al. prepared the chitosan nanoparticles another in the form of droplets.9 It is usually the case
that consisted of quarternized chitosan derivatives using that the droplet size is in the region of about 10 μm for
N-trimethyl chitosan chloride.3 Chitosan nano- and emulsions prepared using polymeric surfactants.10
micro-systems can be employed in a wide range of Some of these polymers have been reported to form
biomedical applications, such as cosmetics and drug stable emulsions with oil droplets of 50-100 m in
delivery systems.4 diameter.11 Some researcher stated, that an o/w
As is generally the case with high polymers, better emulsion based on polymeric surfactants can provide
efficiency is realized with higher molecular weight the release of active ingredients.12 These new
chitosan. The conditioning effect is obtained because the opportunities make the study of polymeric surfactants,
positively charged ammonium groups of the cationic their properties and interactions with other raw
chitosan derivatives are able to bind to anionic sites that materials an interesting and indeed necessary step
are present on natural keratins, such as skin. The towards their extensive use in both pharmaceutical and
cationic chitosan derivatives impart smooth feel, cosmetic formulations.
emolliency and skin protection. The proposed mechanism of emulsion
On the other hand, monomeric cationic surfactant stabilization by polymeric surfactants is named
conditioning active compounds have been said to have electrosteric stabilization, and represents a combined
numerous deficiencies. Products of good water-solubility effect of electric repulsion and steric stabilization.7 In
and adequate compatibility with anionic detergent bases the present of oil phase, hydrophobic alkyl chains
are thus said to have too weak an action, which would anchor to the surface of oil droplets by hydrophobic
make a high dosage necessary. Other products of interactions. At the same time, the long, hydrated and
* Corresponding author.
Email: haryonolipi@yahoo.com

2. 2 Proceeding of ICCS 2007, Yogyakarta-Indonesia, 24-25 May 2007
negatively charged hydrophilic chains form microgels
around dispersed oil droplets and thus prevent their
coalescence.12 It has been reported in the literature, that
the combination of polymeric emulsifier and surfactant
has been shown to improve physical stability of model
emulsions, especially when polar oil phase were
emulsified in water.13 However, it is also known that
polymer/surfactant interactions sometimes occur and
they may in turn lead to decreased physical stability of
an emulsion.14
In this work we consider water-soluble Scheme 1
polysaccharides that enable preparation of stable
emulsions. The aim of this work was first to synthesize ammonium chloride from the mixture. Furthermore
water-soluble chitosan as a polymeric surfactant, then to acetone was used to purify the cationic chitosan. The
formulate emulsions using many oil types like vegetable purification process was repeated three times, and the
oils, mineral oil, and synthetical oils. The aim of the obtained cationic chitosan was dryed under vacuum at
present study is to synthesize N-trimethyl chitosan 25oC for 5 days.
chloride with an intermediate degree of substitution and The same preparation method was carried out for
to compare it with high degree of substitution derivative the molar ratio 1:4 and 1:3 between chitosan and 3-
concerning its effect on the stabilizing of emulsion chloro-2-hydroxypropyltrimethyl ammonium chloride.
system. The calculation of the degree of substitution was
The compounds are prepared by reacting chitosan performed by using 1H NMR spectra (JEOL 500MHz)
with 3-chloro-hydroxy-propyltrimethylammonium chloride assigning the quarternized peak at 3.4 ppm and the
in a suitable ratio in the presence of a solvent. The tertiary peak at 2.6 ppm.15 1H NMR spectra were
cationic polysaccharides based on chitosan have a obtained on a JEOL 500 spectrometer (500 MHz for
1
possibility exhibit better solution properties in water and H) in D2O at 25oC.
which have better technological properties, in particular
when used ion cosmetic formulations for the skin. FTIR spectrum Analysis
The chemical composition, molecular weight, ionic Infrared absorpsion spectra of chitosan and the
strength, and degree of substitution of the materials obtained water-soluble chitosan were studied by FTIR
were determined using FTIR, 1H NMR, pH and using IR Prestige-21 Shimadzu (Shimadzu Co., Japan).
conductivity measurements. The water solubility of the The polymers were mixed with KBr and pressed to a
obtained water-soluble chitosan was measured at 25oC. plate for measurement. The degree of deacetylation of
chitosan as starting material was determined by FTIR
MATERIALS AND METHODS analysis.16 All of the spectra were measured 16 scans
at a resolution 4 cm-1. A background spectrum
Materials containing no sample was substracted from all spectra.
Chitosan was purchased from Brataco Chem Co.
(Jakarta, Indonesia). 3-Chloro-2-hydroxypropyltrimethyl Characterization of polymers
ammonium chloride was purchased from Aldrich The chitosan films were prepared by casting 1.0%
(Singapore). Silver nitrate, acetone, acetic acid, sodium w/v chitosan in 1% acetic acid solution, followed by
acetate, and sodium chloride were reagent grade. drying in a vacuum air for 12 hr. The chitosan films
Ethanol and methanol were distilled before using. were deprotonated by washing 2-3 times with
methanol. The chitosan films were kept in desiccator
Methods for 12 hr and then placed in plates. The degree of
Synthesis of water soluble chitosan deacetylation of chitosan was established using a FTIR
Chitosan was mixed with 3-chloro-2- instrument. The degree of deacetylation was calculated
hydroxypropyltrimethyl ammonium chloride (molar ratio using the baseline of absorbance at 1655 cm-1 and
1:6) in a 0.5% solution of acetic acid at 60oC for 18 hr 3450 cm-1. The equation for degree of deacetylation
(Scheme 1). The flake of chitosan 2.5 gram was was described as follow:
dissolved in 100 mL acetic acid 0.5% aqueous solution.
To the solution was added 35.9 gram of 3-chloro-2- DD = {(A1655 / A3450) x 100 / 1.33}
hydroxypropyltrimethyl ammonium chloride, and stirred
for 30 minutes at room temperature. The mixture was where A1655 and A3450 were the absorbance of amide
heated until 60oC with vigorous stirring (300 rpm) for 18 band as a measure of the N-acetyl group content and
hours. The product was washed with methanol in order 3450 cm-1 of the hydroxyl band as an internal standard
to remove the excess 3-chloro-2-hydroxypropyltrimethyl to correct for film thickness. The factor 1.33 denoted
Agus Haryono and Dewi Sondari

3. Proceeding of ICCS 2007, Yogyakarta-Indonesia, 24-25 May 2007 3
the value of the ratio of A1655/A3450 for fully N-acetylated
chitosan.
For the determination of viscosity-average
molecular weight (Dalton), the chitosan was dissolved in
a mixture of 0.1 M acetic acid with 0.2 M NaCl. Eight
different dilute solutions were used to do the
experiments on measurement of intrinsic viscosity (η).
The Mark-Houwink equation relating to intrinsic viscosity
with empirical viscometric constant K = 1.49 x 10-4 cm3/g
and α = 0.79 for chitosan was used to calculate the
molecular weight using this equation.17 Figure 1: FTIR spectrum of chitosan
Measurement of Water Solubility of Quarternized
Chitosan
Series of concentration of the obtained chitosan in
aqueous solution were prepared by the addition of
quarternized chitosan to water such the concentration
ranged from 0.2 to 20 g/dL. The transmittance of each
solution was measured at 600 nm using a UV-visible
spectrophotometer. The materials are considered
insoluble when the transmittance of the polymer solution
is less than 50% of the transmittance for deionized
water. Figure 2: FTIR spectrum of the ammonium quarternized
chitosan after reaction
RESULT AND DISCUSSION 1
H NMR analysis of ammonium quarternized
To overcome the problem of solubility, a chitosan chitosan were performed in D2O solution (Figure 3).
derivative N,N,N-trimethyl chitosan chloride has been From the obtained NMR spectra, peaks at δ = 1.9 ppm
synthesized and characterized.18 Chitosan can be (-COCH3 from chitin) and δ = 3.5 – 3.8 ppm (H from
quarternized in different degrees, dependent upon the chitosan backbone) were observed. The peaks
conditions, steps and duration of synthesis reaction.18 N- represent the -+N(CH3)3 at δ = 3.2 ppm and –N-CH2-
(2-hydroxyl) propyl-3-trimethylammonium chitosan groups at 3.3 ppm were observed in the obtained
chloride can be prepared by a relatively easy chemical products. The peak represents the –CH(OH)- group
reaction of chitosan and glycidyl-trimethyl-ammonium was observed at δ = 4.5 ppm. In addition the peak at
chloride. Quarternized chitosan has excellent water 3.1 ppm, which is attributed to the –CH(NH2)- in
solubility over wide pH range as mentioned at previously chitosan, shifted to 2.9 ppm following the chemical
published report.19 reaction.
There are three characteristic peaks of chitosan at Chitosan is not soluble in media at physiological
3434 cm-1 of ν (OH), 1094 cm-1 of δ (C-O-C) and 1603 pH (i.e., pH = 7.4) but is soluble in acidic environment
cm-1 of ν (NH2) (Figure 1).20 Compared with chitosan, the (below pH of 6.5) due to the protonation of its amine
ammonium quarternized chitosan shows the group (pKa = 6.5).23 Some acid solvent such as acetic
disappearance of a new band at 1482 cm-1, which is acid, lactic acid, or hydrochloric acid solution is often
attributed to the methyl groups of ammonium (Figure 2). used to dissolve chitosan. Chitosan in acidic media
The IR spectra were consistent with the reported becomes a polyelectrolyte because of the protonation
spectra.21 Characteristic peaks of alcohol and second of the –NH2 groups. The following equilibrium reaction
alcohol between 1160 and 1030 cm-1 are not changed in described the state of inonization:
quarternized chitosan confirming the lack of the CS-NH2 + H3O+ CS-NH3+ + H2O
introducing the alkyl group at chitosan backbone.22 Acetic acid is commonly used to solubilize
Hydroxyl group absorption of chitosan at 1243 cm-1 chitosan at a concentration of 0.1 M or 1%. The
almost disappears in quarternized chitosan, which average viscometric molecular weight Mv = 262,000
indicates that free hydroxyl groups from hydrogen was estimated from the intrinsic viscosity determined in
bonding. Degree of deacetylation of chitosan from FTIR the solvent 0.3 M CH3COOH/0.2 M CH3COONa using
spectra was obtained 50-70%. the Mark-Houwink parameters α = 0.79, Kη = 1.49 x 10-
4
at 25oC when the intrinsic viscosity is expressed in
ml/g.24
Agus Haryono and Dewi Sondari

4. 4 Proceeding of ICCS 2007, Yogyakarta-Indonesia, 24-25 May 2007
between chitosan and 3-chloro-2-
hydroxypropyltrimethyl ammonium chloride. The
obtained ammonium quarternized chitosan was
evaluated their water solubility with measurement with
UV-visible spectrophotometry at 600 nm (Figure 5).
As shown in Figure 5, the transmittance was
greatly dependent on the polymer concentration. When
the polymer concentration was less than 2 g/N, the
transmittance was constant. In the condition of polymer
concentration between 2-7 g/N, the transmittance
decreased slightly. Increasing the polymer
concentration above 7 g/N abruptly decreased the
transmittance.
The experimental data were fitted using a
polynomial equation, and tha curve was extraplotted to
50% transmittance in order to estimate the solubility of
the ammonium quarternized chitosan in water. In this
way, the water solubility of the quarternized chitosan
obtained by the reaction between chitosan and 3-
chloro-hydroxy-propyltrimethylammonium chloride in
Figure 3: 1H NMR spectra of the obtained ammonium molar ratio of 1:6 was 40 g/N. The water solubility of
quarternized chitosan (from upper to lower are the 1H NMR the obtained chitosan derivatives was showed as a
spectra for the products of reaction between 3-chloro-hydroxy- high solubility in water.
propyltrimethylammonium chloride with molar ratio of 3:1, 4:1
and 6:1, respectively).
CONCLUSION
1.4
A water-soluble chitosan was prepared by the
1.2 y = 0.1732x + 0.4611 substitution reaction of 3-chloro-hydroxy-
η sp/concentration
1
R2 = 0.9597
propyltrimethylammonium chloride onto the chitosan
0.8 chains. The obtained ammonium quarternized chitosan
0.6 was succesfully characterized using FT-IR and 1H
0.4 NMR. The water solubility of the obtained was 40 g/N,
0.2 which was high solubility compared with the reported
0 water-soluble chitosan.
0 1 2 3 4 5 6
polymer concentration (M) ACKNOWLEDGMENTS
Figure 4: Viscosity and polymer concentration of quarternized The authors are thankful for the financial support
chitosan from Indonesia Toray Science Foundation (ITSF)
100 Science and Technology Research Grant 2006. This
work also supported by DIPA tematik from Indonesian
Transmittance (%)
90
80 Institute of Sciences (LIPI).
70
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