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Determination of degree of deacetylation of chitosan - Comparision of

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 DETERMINATION OF DEGREE OF DEACETYLATION
OF CHITOSAN - COMPARISION OF METHODS

Renata Czechowska-Biskup*1,2, Diana Jarosińska1, Bożena Rokita1,2,

Piotr Ulański1,2, Janusz M. Rosiak1,2.

1) Institute of Applied Radiation Chemistry, Faculty of Chemistry, Technical University

of Lodz, Wroblewskiego 15, 93-590 Lodz, Poland

2) European Centre of Bio- and Nanotechnology, Technical University of Lodz,

Zeromskiego 116, 90-924 Lodz, Poland

Abstract
Degree of deacetylation (DD) is one of the main parameters characterizing chitosan. The most
precise measurements of DD require sophisticated equipment (NMR spectrometer), not
available at many laboratories worldwide working on chitosan. There is a need for low-cost,
simple, yet study and reliable methods and procedures for DD determination. The aim of this
work was to test and compare – on the same set of chitosan samples - a few of the existing
analytical techniques and provide recommendation for selecting DD determination methods.
Tests were performed on four chitosans of nominal DD in the range 70 - 95 %. Three different
methods of titration, two different methods of spectroscopy UV / Vis and infrared spectroscopy
using various calculation approaches were used. The results are summarized and compared with
values obtained by 1H NMR, considered as the reference method. Moreover, evaluation of the
ease of performance and availability of reagents in the given methods was performed. On that
basis, recommendations for selection of DD determination methods were formulated.
Keyword: chitosan, degree deacetylation, methods

1. Introduction
Chitosan is typically obtained by partial deacetylation of chitin. The product is a
copolymer of N-acetylglucosamine units and D-glucosamine units. Molar fraction of N-
acetylglucosamine units in the chain, defined as:

(1)

is called the degree of deacetylation (DD), where, n GlcN – average number of D-

glucosamine units, n GlcNAc - average number of N-acetylglucosamine units. In some
works, degree of acetylation is used, DA = 100 – DD. Several methods for determining
the degree of deacetylation have been elaborated, from simple, such as pH-metric
titration, UV-Vis spectroscopy [1], infrared spectroscopy [1-2], elemental analysis [3],

*
Corresponding author: czechow@mitr.p.lodz.pl

1
to complex ones, which require complicated and expensive equipment, such as 1H NMR
spectroscopy [4-6] and 13C NMR spectroscopy [7,8]. Within the same method, there are
usually many analytical procedures for performing measurements and calculations and
many ways of interpretation of results. It is obvious that not every research laboratory
has an NMR spectrometer. Moreover, this method, although probably the most precise,
is not suitable for routine measurements of DD due to high costs (deuterated solvents,
depreciation of equipment, specialized staff time) and time-consuming sample
preparation procedure. Most laboratories must work on less advanced, simpler and
cheaper techniques. Therefore it is important to determine which of these methods lead
to reliable, reproducible results in line with 'standard' by NMR. Indication of such
methods is also important in the case of parallel research conducted by many
laboratories (e.g., international projects, collaboration with industrial research
laboratories, etc.) to provide the ability to compare the results. The selected technique
must be precise, accurate, reproducible and inexpensive.
Authors’ interest in this topic has been stimulated by participation in a large
international project led by the International Atomic Energy Agency (IAEA) -
Co-ordinated Research Programme “Development of Radiation-Processed Products of
Natural Polymers for Application in Agriculture, Healthcare, Industry and
Environment”, where interlaboratory comparison of results emerged as an important
problem.
The aim of this work was to test and compare – on the same set of chitosan samples - a
few of the existing analytical techniques and procedures, and to provide
recommendation for selecting DD determination methods. Tests were performed on
four chitosans of nominal DD in the range 70 - 95 %.

2. Materials and Methods:

2.1 Materials
In this study four chitosan samples were used, of similar nominal viscosity (ca. 1000
mPas at 1 % in 1 % acetic acid, 20 C) but different degrees of deacetylation. Samples
have been manufactured and donated by Heppe Medical Chitosan GmbH. Chitosans
were of high purity (Chitoscience® product line) and had individual certificates of

2
analysis. These samples are denoted here as S1, S2, S3 and S4. DD values of these
samples determined by 1H NMR technique were taken as the reference values for all
other determination methods. Chitosan samples were dried at 40 °C in vacuum until
constant mass. All reagents were of analytical purity.
2.2 Methods
Some of the most commonly applied methods of DD determination have been used in
our tests. They are described below.
1
H NMR Spectroscopy [6]

Dilute solutions (5–7 mg cm-3) of chitosan samples in a deuterated aqueous acid

DCl/D2O, at about pH 4 were prepared. 0.05 g - 0.07 g chitosan was weighted in plastic
tube. Next, 6 cm3 D2O and 1 cm3 deuterium chloride was added to every sample to
obtain a solution. D2O and DCl were further added to the final volume of 10 cm3 and
pH of ca. 4. The samples were twice freeze-dried using D2O (99.9%) to exchange labile
protons by deuterium atoms. Measurements were made for three samples of every kind
of chitosan. Spectra between 0 - 6 ppm were recorded using a NMR spectrometer
Bruker Avance II (700 MHz) at the temperature 27 °C and 60 °C. Integrals under
characteristic signal are used to calculation degree of deacetylation.

Titration methods
Titration I [9-11]

Dried chitosan (0.2 g) was dissolved in 20 cm3 0.1 M hydrochloric acid and
25 cm3 deionized water. After 30 minutes continuous stirring, next portion of deionized
water (25 cm3) was added and stirring continued for 30 minutes. When chitosan was
completely dissolved, solution was titrated with a 0.1 mol·dm-3 sodium hydroxide
solution using automatic burette (0.01 cm3 accuracy). Degree of deacetylation (DA) of
chitosan was calculated using formula:

[ ] (2)

where: m – weight of sample

V1, V2 – volumes of 0.1 mol·dm-3 sodium hydroxide solution corresponding to the
deflection points

3
2.03 – coefficient resulting from the molecular weight of chitin monomer unit
0.0042 – coefficient resulting from the difference between molecular weights of chitin
and chitosan monomer units.
Titration II [12]
Titration II was based on modified procedure described by Tan et al. [12]. Chitosan
(0.20–0.25 g) was dissolved in 20 cm3 of 0.1 mol·dm-3 HCl and diluted with 10 cm3 of
deionized water. Under continuous stirring, 0.5 cm3 of 0.1 mol·dm-3 sodium hydroxide
was added, allowed to equilibrate and the pH recorded. This sequence was repeated
until the pH reached a value of 3.
A value of f(x) corresponding to the volume of NaOH added, was calculated using the
following formula:

( ) [ ]- -
(3)

where:
V0 is the volume of chitosan solution (cm3) (30 cm3)
V is the volume of NaOH added (cm3)
NB is the concentration of NaOH (0.1 mol·dm-3)
[H+] is the concentration of [H+] (mol·dm-3)
[OH-] is the concentration of [OH-] (mol·dm-3).
The dependence of f(x) on the hydroxide sodium volume is linear. By extrapolating the
linear titration curve to the x-axis, the volume of NaOH at the end point can be
determined.
The DD of the chitosan sample was calculated using the following formula [12]:

-
(4)

where:
-
(5)

NA is the concentration of HCl (mol·dm-3)

VA is the volume of HCl (cm3)
NB is the concentration of NaOH (mol·dm-3)
VB is the volume of NaOH at the end point (cm3)
W is the sample mass [g].

4
Titration III [13]
Chitosan of 0.125 g were dissolved in 25 cm3 aqueous solution of 0.1 mol·dm-3
hydrochloric acid and stirring by 30 minutes until totally dissolved. The solution was
titrated with 0.1 mol·dm-3 NaOH. The degree of deacetylation was calculated as follow:

-
(
(6)
- )

where:
C1 – HCl concentration [mol·dm-3]
C2 – NaOH concentration [mol·dm-3]
V1 – volume of HCl solution [cm3]
V2 – volume of NaOH solution [cm3]
0.016 – molecular weight of NH2 in 1 cm3 0.1 mol·dm-3 HCl [g].
G – the sample weight [g]
W – the water percentage of sample [%].

⁄ (7)

where 9.94 % is the theoretical NH2 percentage.

UV-VIS Spectroscopy methods

Method I [14]
Establishing the calibration curve:
The calibration curve was made by plotting the first derivative UV values at 203 nm as
a function of N-acetyl-D-glucosamine (GlcNAc) and D-(+)-glucosamine hydrochloride.
Standard solutions of GlcNAc and GlcN were prepared in 0.85 % phosphoric acid at
concentrations of 0, 12.8; 25.6; 38.4; 51.2 and 64.0 μg·cm-3. 0.85 % H3PO4 was used as
the reference liquid.

Sample preparation and determination of DA:

Aliquots of 100 ± 10 mg chitin or chitosan were heated in 20 cm3 85 % phosphoric acid

for 40 min at 60 C with constant stirring. After 40 min, when chitin/chitosan was

5
completely dissolved, 1 cm3 clear solution was taken and diluted to 100 cm3 with
deionized water. The dilution was necessary to get the chitin/chitosan concentration to
the range detectable by a spectrophotometer. The diluted solutions were incubated at
60 C for 2 h prior the UV measurement. Next the UV/Vis spectra were carried out in
range 190 – 400 nm. 0.85% H3PO4 was used as the reference liquid.
The degree of acetylation of chitin and chitosan samples was calculated as:

(8)
where:
m1 is the mass of acetyl-glucosamine in 1 cm3 chitin/chitosan solution, calculated from
the calibration curve by the corresponding  = 203 nm
m2 is the mass of glucosamine in 1 cm3 chitin/chitosan solution,
m2 = M - m1.
M - the mass of chitin/chitosan in the 1 cm3 solution
M = (M1  M3)/(M1 + M2),
where M1 is mass of solid chitin/chitosan sample taken for analysis (100 ± 10 mg)
M2 is mass of 20 cm3 85 % phosphoric acid (34 g)
M3 is mass of 1 cm3 chitosan solution in concentrated phosphoric acid (1.7 g).
Method II [15]
Establishing the calibration curve:
Aqueous solutions containing various proportions of of glucosamine (GlcN) and acetyl-
glucosamine (GlcNAc) in 0.1 mol·dm-3 HCl are prepared to simulate chitosan samples
of various DD. UV spectra of these solutions are recorded. Calibration curve is
generated by relating the ratio absorbance / total concentration (CS, see below) to the
degree of acetylation (DA = 1 - DD). Absorbance is measured at λ = 201 nm. The DA
of the solution is defined as the concentration of acetylglucosamine divided by the total
concentration of acetylglucosamine and glucosamine hydrochloride.

Preparation of chitosan solution and determination degree of deacetylation

7-9 mg chitosan is dissolved in 25 cm3 of 0.1 mol dm-3 HCl. Next UV spectrum
is collected in the range w of wavelength 190 – 230 nm. On the basis of the absorbance
of chitosan solution at λ = 201 nm, the DD is calculated by equation:

6
 -
- (9)
-

where:
161.1 – the molecular weight of GlcN residue [g·mol-1]
42.1 – difference between the molecular weight of N-acetylglucosamine and the
molecular weight of GlcN residue [g·mol-1]
A – absorbance at the λ = 201 nm
V – the volume of solution [dm3]
m – mass of chitosan sample [mg]
C – intercept with x-axis in the standard curve
k – slope in the standard curve

Infrared spectroscopy
Measurements have been performed in the transmission mode, with chitosan contained
in potassium bromide (KBr) tablets. Potassium bromide was mixed with chitosan in
mass ratio 100:1 (200 mg KBr and 2 mg chitosan) [16]. KBr was placed in an oven at
300 °C for 24 h before mixing. Substances were mixed in agate mortar and pressed to
tablet form. Tablets were dried for 24 hours at 50 °C in order to remove moisture. For
every kind of chitosan 3 tablets were produced. The spectra of chitosan samples were
obtained within a frequency range of  = 400 – 4000 cm-1, each spectrum is an average
of 64 scans with a resolution of 2 cm-1.

3. Results and Discussion:

1
H NMR Spectroscopy
NMR spectroscopy is one of the most accurate methods for determining the
degree of deacetylation, 1H NMR spectroscopy has been chosen as a standard method
by the American Standard Test Method [17]. Interpretation of results is clear, and the
values obtained are reproducible. Figure 1 shows 1H NMR spectra of chitosan with
different degrees of deacetylation. It can be seen that with increasing DD the signal
from the methyl group and hydrogen H-1GlcNAc decreases because the molar content
of N-acetylglucosamine in chitosan molecule goes down.

7
 S4

S3

S2

H1-GlnNAc H2-H6
H2-GlnN CH-
H1-GlnN S1

Figure 1. 1H NMR spectra of chitosan with different degrees of deacetylation in DCl/D2O at 60○C
(700MHz).

Various expressions have been worked out to calculate degree of deacetylation. In our
calculations we have chosen five of them, the most often used. Formulas (10) [5, 12)]
and (14) [18] are used in all range of DD, (11) is recommended for chitosans in DD
range 79-98% [19], the (12) for chitosan with DD > 48 % [20]; and the (13) for
DD < 90 % [18].

(10)

(11)

(12)

(13)

(14)

were:
IH1-GlnN – integrals for H1 (GlcN)
IH1-GlnNAc – integrals for H1 (GlcNAc);
8
ICH3 – integral of – CH3 signal
I(H2-H6) – the summation of integrals of H2, H3, H4, H5, and H6.

DD values calculated on the basis these formulas are shown in collective Table 1.
Results from formula (10) were taken as reference point to compare with data from
other methods.

Titration methods

To determine the degree of deacetylation by titration methods, three different methods

were selected. The first one (Titration I) seems to be the simplest in terms of
performance and calculation. By using this method, on the basis of titration of chitosan
solution, a curve with two inflexion points is obtained [Fig.2]. The difference of the
volumes of these two points (V1 i V2) corresponds to the acid consumed by the amine
groups and allows to calculate degree deacetylation. Determination of the first
derivative helps in precise reading of V1 and V2. Measurements were carried out for
minimum five samples. Average values were shown in Table 1.

12 pH
derivative
10

0 5 10 15 20 25
3
Volume of NaOH solution [cm ]
Figure. 2. Determination of the degree of deacetylation DA by pH-metric titration of chitosan
solution (10-1mol dm-3 hydrochloric acid by 10-1 mol dm-3 sodium hydroxide).

In method Titration II, the linear relation between function f(x) and corresponding
volume of NaOH was shown in Fig. 3. Calculation procedure is described in the

9
Experimental section above. Measurements were performed for five samples of every
kind of chitosan. Average values are shown in Table 1.

6
y = 7,35 - 0,89x
f(x)

0
2 4 6 8
3
V NaOH/cm

Figure 3. Determination of the final point of titration chitosan solution (sample S3) (7,22·mg
cm-3 0,1 mol dm-3HCl).

Third method of titration, in terms of the measurement procedure, is similar to

the first method (Titration I). However, the calculations are made based on the position
of the first point on the titration curve, and not based on the difference between the
position of the first and second inflection point as in method "Titration I". Figure 4
shows a titration curve for chitosan solution using method of Titration III. The first
maximum corresponds to neutralization of HCl, the second to the amount of NaOH
needed for reaction with H+ on amine groups. Because chitosan was earlier dried to the
constant mass, expression (100-W) was omitted. Degrees of deacetylation were
collected in Table 1.

10
 titration curve
14
the first derivative

12

10

pH
8

0
0 5 10 15 20 25 30 35
3
V NaOH /cm
Figure 4. Determination of degree of deacetylation DA by pH-metric titration of chitosan
solution (4,99·mg cm-3 in 10-1 mol dm-3 hydrochloric acid by 10-1 mol dm-3 sodium hydroxide).

Methods of determination of degree deacetylation by titration are cheap and based on

readily accessible reagents and apparatus, while a disadvantage is the relatively long
time used for sample preparation and for the titration itself. The difficulty of mixing for
higher values of pH, due to precipitation of chitosan, can be the cause of error in
Titration I. The advantages of Titration II is lack of potential sources of error due to
precipitation of chitosan (titration is limited to low pH range) and the relatively short
measurement time, compared to the Titration I where the full range of pH is used.
Additionally, the values are closer to the reference values obtained by 1H NMR,
standard deviations are small. This does not mean that the agreement of results with the
results of the reference is very good throughout in all the range. It seems that this
method gives more relevant results for samples with a low degree of deacetylation.
However, disadvantage of this method is, as before, quite long time for sample
preparation and complex procedure of calculation. The advantages of method Titration
III is, as for other methods of this group: low cost, readily available reagents and
apparatus.
Potentiometric titration seems to be the simplest and most robust method for
determination of DD, when proper protocol is chosen and care is taken to use acid and
base solutions of precisely known concentrations (ready to use volumetric standards;
base solutions either used at once or stored in a way which prevents reaction with

11
atmospheric carbon dioxide). If we consider Titration I, II and III, an interesting
observation is that the obtained results were (in all cases except one) lower than those
obtained by 1H NMR (on average, by 3.53 %), which seems to indicate some
systematic difference, independent of the titration protocol. Our preference is protocol I
and protocol III, since laborious calculations and extrapolations based on a relatively
narrow range of data such as in method II are not necessary. Protocol I yields results
that are closest to those obtained by 1H NMR; on the other hand measurements
according to protocol III are not disturbed by precipitation at pH > 7.

UV-VIS Spectroscopy methods

Figure 5 shows the first derivative UV spectra of standard solutions containing
acetyl-glucosamine and glucosamine at different concentrations, in the wavelength
range 190–220 nm, obtained as proposed by Wu et al. (UV method) [14]. The linear
regression was made at 203 nm.
The first derivative of absorbance

0,00

3
GlnNAc 12,8 m/cm
3
GlnNAc 25,6 m/cm
-0,05 GlnNAc 38,4 m/cm
3

3
GlnNAc 51,2 m/cm
3
GlnNAc 64,0 m/cm
3
GlnN 12,8 m/cm
3
GlnN 25,6 m/cm
3
GlnN 38,4 m/cm
3
-0,10 GlnN 51,2 m/cm
3
GlnN 64,0 m/cm

190 200 210 220 230

/nm
Figure 5. First derivative UV spectra of acetyl-glucosamine (GlcNAc) and glucosamine (GlcN)
standards at concentrations ranged from 12.8 to 64 μg·cm-3.

The presence of glucosamine at the tested concentrations did not impose noticeable
interference to the calibration curve, whereas acetylglucosamine resulted in a good
linear regression in the study range of concentration [Fig.6].

12
 -5
y=-1,69x10 x

Absorbance of the first derivative

0,00

for  = 203nm
GlnNAc
GlnN
-0,05
y = -0,0016x

-0,10
0 10 20 30 40 50
-3
C /g cm
Figure 6. The first derivative value at 203 nm against concentrations of glucosamine (GlcN) and
acetyl-glucosamine (GlcNAc).

Figure 7 illustrates the absorbance and the first derivative of absorbance for tested
chitosans in the range of 190-220 nm. Derivative values were read at 203 nm. Using
standard curve concentrations of acetyl-glucosamine, degree of deacetylation of
chitosan samples were determined and calculated. Results obtained by this method were
shown in Table 1.

0.4
S1
The first derivative of absorbance

S1
0,01 S2
S2
S3
S3 203 nm
0.3 S4
S4
0,00
Absorbance

0.2
-0,01

0.1
-0,02

0.0
-0,03
190 195 200 205 210 215 220 190 195 200 205 210 215 220
 /nm  /nm

a) b)

Figure 7. UV spectra of the examined chitosan samples a) absorbance b) the first derivative of
absorbance. Chitosans were dissolved in 0.85 % aqueous solution phosphoric acid.

13
Method II of UV/ViS spectroscopy is similar to the method I. Both of them require
preparation of calibrations curves using glucosamine and acetyl-glucosamine at various
molar ratios. Figure 8 shows the UV spectra of the standards solution. It shows that the
N-acetyl-glucosamine and glucosamine hydrochloride in common solution have a
maximum at 201 nm in a 0.1 mol dm-3 hydrochloric acid solution, and this maximum is
mainly due to N-acetyl-glucosamine.

DA=0,763
DA=0,616
DA=0,376
0,4 DA=0,285
DA=0,167
Absorbance

DA=0,131
DA=0,091
DA=0,048
DA=0,010

0,2

0,0
195 200 205 210 215 220
 [nm]
Figure 8. UV spectra N-acetylglucosamine and glucosamine hydrochloride admixture in 0,1 mol
dm-3 hydrochloric acid solution, DA of chitosan is defined as the mole fraction of acetylated
units in the mixture.

Standard curve [Fig.9] indicates a linear dependence of the ratio of absorbance to the
total molar concentration of repeating units (A/CS) on degree of acetylation at 201 nm.

1,4

1,2

1,0

0,8
A/Ct

0,6

0,4 y = 1,633x + 0,032

2
R = 0,9949
0,2

0,0
0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8

DA
Figure 9. A/Ct versus DA of standard solutions.

14
Degrees of deacetylation determined by this method for all chitosan samples are shown
in Table 1. Corresponding UV/Vis spectra for these chitosan are presented in Fig. 10.

1,0 S1
S2
S3
Absorbance S4

0,5

0,0
190 200 210 220 230
 [nm]

Figure 10. UV spectra of chitosans in 0.1 mol dm-3 hydrochloric acid solution.

Method II UV/Vis is similar to methods I UV/Vis. Both of them require preparation of

calibrations curves using glucosamine and acetyl-glucosamine in a different molar
relationships. The advantages of these methods are: generally available equipment (UV-
Vis spectrometer) and easy measurements. Disadvantages include: using expensive
chemicals, laborious and time-consuming procedure (calibration curve, etc.), complex
procedures of calculation and the fact that the presence of contaminants (absorbing
below 210 nm) can influence spectra and hence the final results.
Unfortunately, Method II UV/Vis, being somewhat easier to perform than Method I (no
need for re-calculating the spectra into first derivatives), failed to produce results
comparable with the standard data obtained by 1H NMR. The obtained DD data were
systematically on the low side. Therefore, based on our experience, we cannot
recommend Method II as reliable.

Infrared spectroscopy
Like in the NMR spectroscopy, also in the FTIR spectroscopy, several
procedures and equations are described in literature for calculation of degree of
deacetylation. These equations were derived on the basis of calibration curves, where

15
the calibration values of DD were determined by absolute methods like NMR.
Calculation procedures are based on absorbance ratios of various spectral bands
[21, 22]. In this project four procedures were chosen. The equations used in these
procedures are listed below.
⁄ [17,23] (15)

⁄ [23] (16)

[17] (17)

- [22] (18)
where:
A3450, A2870, A1655, A1420, A1320, – values of absorbance from baseline 1, 2, 3, 4, 5 to
maximum, respectively. In Figure 11, on the basis IR spectrum of chitosan S2, baseline
settings and individual bands ascribed for characteristic groups in chitosan are
presented.
Our choice of expressions used for calculation of DD was based on literature
recommendations. Expressions 15 and 17 were chosen because some authors maintain
that calculations based on equations proposed by Baxter et al. [24] are - in a sense -
absolute methods. These equations were set up without comparing the DA values to a
range of samples with known DA and were with good agreement with values from
NMR. Next two formulas (16, 18) were taken for DD calculations because: in (16), the
stretching band at 2870 cm-1, as a reference band, is convenient – its position and
intensity reportedly do not change with water content; in (18): DD obtained from the
ratio A1320/A1420 was in agreement with DD determined from 1H NMR and 13
C NMR
-1 -1
[22] and positions at 1320 cm and 1420 cm do not change with humidity.
Exemplary FTIR spectra for all chitosan samples are presented in Figure 12. It can be
seen that with an increase of degree of deacetylation the band 1655 cm-1 (>C=O)
undergoes changes and finally decays, the shape of bands between 1500 - 1750 cm-1 is
changed too. Significant differences can also observed in shape of 3000 - 3500 cm-1
band. Using the four above formulas degrees of deacetylation were calculated, the
results are shown in Table 1.

16
 OH
0,30
3416.95 CH
2879.35
0,25
C=O CH 2

1420.41
0,20 1658.97 amide III band
1322.13
A

0,15 2

3 5
0,10
4
0,05
1

0,00
4000 3000 2000 1000 0
-1
/cm

Figure 11. Rereferences bands and corresponding baselines, based on Duarte et al. [21] (1-3)
and Brugaretto et al. [22] (4-5) for FTIR spectrum of chitosan sample S2.

S1
S2
S3
S4
A

4000 3500 3000 2500 2000 1500 1000 500

-1
/cm

Figure 12. Exemplary FTIR spectrum of the chitosans: S1, S2, S3 and S4.

Our general impression is that the FT-IR method, although undoubtedly having some
advantages, can be recommended only for rough estimation of the DD of chitosan, and
even when used for this purpose it must be performed with care. This is mainly due to
problems with reproducibility. Absorbance of some of the peaks may be influenced by
humidity. Baseline setting is troublesome and operator-dependent (in our opinion this is

17
one of the major sources of error). Signals resulting from impurities may overlap with
the signals from chitosan. Probably also instrument settings (like spectral resolution)
may influence the results. Out of the four tested calculation procedures, we found No.
(16) as producing totally unreliable results (which may be a result of some systematic
error either in derivation of this procedure itself or in our understanding). The best
results (i.e., being systematically closest to the 1H NMR data) were obtained by
applying procedure (15).

Comparison of results

The mean values of degree of deacetylation obtained by all tested methods are presented
in Table 1.

18
 Table 1. Comparison degree of deacetylation obtained by different methods

Chitosan 1
H NMR2 Titration3 UV/Vis FTIR4
sample

Formula Formula

10 115 12 136 14 I II III I7 II8 15 16 17 18

79,72 ± 77,92 ± 78,99 ± 81,93 ± 81,92 ± 76, 65 75,27 ± 74,67 ± 80,14 ± 67,83 ± 76,61 ± 64,22 ± -8,50 ± 64,49 ±
S1 0,05 0,38 0,20 1,15 0,26 ±0,48 0,87 1,80 0,27 1,19 1,92 2,94 13,03 7,14

81,23 ± 80,23 ± 80,84 ± 82,42 ± 83,49 ± 81, 47 76,29 ± 77,28 ± 82,55 ± 72,47 ± 77,84 ± 66,11 ± -12,50 ± 74,60 ±
S2 0,19 0,47 0,29 0,79 0,33 ±0,91 1,15 1,51 1,58 0,28 2,36 2,36 23,30 3,60

89,44 ± 90,42 ± 91,27 ± 84,87 ± 91,26 ± 87, 67 85,29 ± 85,59 ± 90,55 ± 81,09 ± 85,61 ± 78,00 ± 43,86 ± 89,50 ±
S3 0,32 0,69 0,25 1,27 0,57 ±1,41 0,63 0,35 0,92 0,19 2,04 2,04 9,19 5,85

97,47 ± 98,00 ± 98,20 ± 100,00 ± 98,04 ± 94, 69 91,47 ± 94,65 ± 100,29 ± 96,79 ± 96,20 ± 94,19 ± 83,16 ± 76,48 ±
S4 0,31 0,38 0,19 0,00 0,36 ±1,25 0,67 1,11 0,73 0,34 0,89 1,35 0,80 10,77

2
n = 3, t = 60C
3
n≥5
4
n=3
5
for DD 79 - 98%
6
for DD < 90%
7
n=6
8
n =3
n – number of measurements

19
Conclusions:
The aim of this study was to test and select the method of determining the degree of
deacetylation of chitosan, which could be easily performed in most laboratories dealing
with this polysaccharide. There seem to be a general agreement in the literature that 1H
NMR is the best and most accurate method currently available for determination of DD
of chitosan. While this technique itself cannot be treated as ideal or absolute, it
definitely deserves to be the reference method, to which other techniques, better suited
for general lab use and not requiring access to very expensive equipment, can be
compared. In our study we have tested, on the same set of chitosan samples,
determination of DD by potentiometric titration, FT-IR spectroscopy and UV-Vis
spectroscopy. For each of these techniques, numerous detailed procedures exist in
literature. By direct comparison of these procedures on the same set of samples which
have been also analyzed by the benchmark method of 1H NMR we were able to indicate
which of those procedures yield reliable results, and also to assess their advantages and
disadvantages (see final parts of the corresponding chapters). Similarly, we tried to
compare the techniques themselves, in terms of their reliability, weak and strong sides.
As expected, there are no ideal solutions, but some general recommendations can be
formulated. In our opinion, potentiometric titration, when based on selected procedures
and standard analytical solutions of well-known concentrations, is the most reliable and
robust of the non-NMR methods. Here we indicate procedures I and III to yield best
results in our hands. The titration technique is followed by UV-Vis (procedure I) and
FT-IR spectroscopy (procedure (15)). UV-Vis when carefully performed seems to
produce good results, but the procedure is quite time-consuming (the need of
calibration) and potentially sensitive to impurities. FT-IR offers faster measurements,
but quantitative reproducibility of the spectra and the somewhat operator-dependent
procedures for setting the baselines for reading absorbances are the weak sides of this
technique.
Acknowledgements:
The authors would like to thank the Heppe Medical Chitosan GmbH for donating chitosan
samples used in this study, as well as Dr. Torsten Richter (HMC GmbH, Halle, Germany), Prof.
Murat Sen (Hacettepe University, Ankara, Turkey) and Prof. Saphwan Al-Assaf (Glyndwr
University, Wrexham, U.K.) for valuable discussions.
This work has been financed in part by International Atomic Energy Agency ((UN) IAEA CRP
F22046) and Ministry of Science and Higher Education, Poland (2379/IAEA/2012/0).

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