Talanta 45 (1998) 713 – 719

The degree of deacetylation of chitosan: advocating the first

derivative UV-spectrophotometry method of determination
Su Ching Tan b, Eugene Khor a,*, Teck Koon Tan b, Sek Man Wong b
a
Department of Chemistry, The National Uni6ersity of Singapore, 10 Kent Ridge, Singapore 119260, Singapore
b
School of Biological Sciences, The National Uni6ersity of Singapore, 10 Kent Ridge, Singapore 119260, Singapore

Received 5 February 1997; received in revised form 24 July 1997; accepted 28 July 1997

Abstract

The degree of deacetylation (DD) is increasingly becoming an important property for chitosan, as it determines
how the biopolymer can be applied. Therefore, a simple, rapid and reliable method of determining the DD for
chitosan is essential. In this report, the DD of chitosan was determined by nuclear magnetic resonance (NMR), linear
potentiometric titration (LPT), ninhydrin test and first derivative UV-spectrophotometry (1DUVS). The DD was
calculated on a per mol basis instead of on a per mass basis. This is important as the molecular weights of
N-acetyl-D-glucosamine and D-glucosamine are different. By converting the mass of N-acetyl-D-glucosamine and
D-glucosamine into mols and calculating for the percentage of D-glucosamine present in the chitosan sample, a more
accurate estimation of the DD can be obtained. Of the four methods, there is good correlation between 1DUVS and
NMR. The concentration of chitosan solution for 1DUVS analysis was standardised as 0.1000 mg chitosan per ml
of 0.0100 M acetic acid solution. The presence of D-glucosamine was corrected for by a reference curve for
N-acetyl-D-glucosamine. 1DUVS is easy to perform, sensitive and the interference of other contaminants to the
results is minimal compared with the other three methods. Therefore, we advocate 1DUVS to be used as the standard
methods for routine determination of DD of chitosan. © 1998 Elsevier Science B.V.

Keywords: Chitosan; Degree of deacetylation; First derivative UV-spectrophotometry

1. Introduction include wastewater treatment, food, agriculture,

Over the past 30 years, chitosan, the (b-1,4)-
linked D-glucosamine derivative of the polysac-
charide chitin, has been extensively advanced as a
promising renewable polymeric material. Wide
ranging applications in many areas for chitosan
* Corresponding author. Tel.: +65 7722836; fax: +65
7791691; e-mail: chmkhor@nus.edu.sg
cosmetic/personal care and biotechnological and
pharmaceutical industries [1]. While chitosan has
lived up to some of these expectations, significant
barriers to its broader usage exist. These include
supply cost, variability in quality and poor meth-
ods of characterisation of its properties [2]. One of
the more important property is the degree of
deacetylation (DD), which determines whether the
biopolymer is chitin or chitosan. The arbitrary

0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 2 8 8 - 9
714 S.C. Tan et al. / Talanta 45 (1998) 713–719
DD of ] 40 defining chitosan [3], plays an impor-
tant role in defining the use of chitosan as DD
influences the physical, chemical and biological
properties of chitosan such as the tensile strength
of films [4], ability to chelate metal ions [5] and
immunoadjuvant activity [6,7].
Many methods have been used to determine the
DD of chitosan. These include infrared spec-
troscopy (IR) [8 – 10], near infrared spectroscopy
(NIR) [11], UV-spectrophotometry [12], first
derivative UV-spectrophotometry (1DUVS) [13],
colloidal titration [14], linear potentiometric titra-
tion (LPT) [15], enzymatic determination [16], nu-
clear magnetic resonance (NMR) [17], ninhydrin
test [18] and circular dichroism measurements
[19]. The choice of method appears to be arbitrary
and often does not correlate well with others [12].
A standard method used to determine the DD of
chitosan that satisfies the producers and end users
is essential, if the wider exploitation of chitosan is
to be realised. A standard method has to be
simple, rapid, cost effective and reliable yet toler-
ate the presence of impurities, especially protein, a
common contaminant. From our survey, the use
of the 1DUVS as proposed by Muzzarelli and
Rocchetti is the simplest and most convenient
among all the presently available methods. The
1DUVS method requires only very small amounts
of sample and relies on simple reagents and in-
strumentation. In addition, the results obtained
from 1DUVS are reasonably independent of
protein contamination. Therefore, we wish to ad-
vocate the use of 1DUVS as the standard method
for determining the DD of chitosan.
In this paper, we have evaluated and refined the
1DUVS method. The DD of commercial and
purified commercial chitosan were determined and
the results compared with those of NMR, LPT
and ninhydrin test.
2. Materials and methods
Chitosan was obtained from Fluka (Switzer-
land) and Tokyo Kasei (Japan). D-Glucosamine
(GlcN) and N-acetyl-D-glucosamine (GlcNAc)
were obtained from Sigma (USA). All other
chemicals were of reagent grade.
2.1. Purification of chitosan
Chitosan (4.0 g) and 160 ml of 40% (w/v)
NaOH were placed in a 500 ml round bottom
flask. NaBH4 (0.05 g) was added and the suspen-
sion was refluxed, with stirring, at 95°C for 4 h.
The suspension was cooled, the residue retained
by filtration and washed with distilled water until
the pH of the filtrate was same as the pH of
distilled water. The residue was submitted to an-
other round of reflux. The residue retained from
the second round of reflux was dissolved in 250
ml of a 2% acetic acid solution. The chitosan
solution was centrifuged at 16 000× g for 5 min
and the supernatant retained. Chitosan was then
precipitated from the supernatant by increasing
the pH (to pH 8–9) with 1 N NaOH. The suspen-
sion was centrifuged at 16 000× g for 5 min and
the chitosan pellet retained. The chitosan was
washed with distilled water until the pH of the
supernatant was the same as the pH of distilled
water. The chitosan was finally washed once with
95% ethanol and freeze dried.
2.2. Calculation of the degree of deacetylation of
chitosan
Besides NMR, the DD in all methods were
calculated on a per mol basis instead of on a per
mass basis. This is important as the molecular
weights of chitin (acetylated form of chitosan)
and chitosan are different. By converting the mass
of chitin and chitosan into mols and calculating
for the percentage of chitosan present in the sam-
ple, a more accurate estimation of the DD is
realised.
2.3. Nuclear magnetic resonance [17]
A chitosan sample was dissolved in
CD3COOD/D2O solution and the NMR spectrum
(300 MHz) was obtained at 90°C (Bruker; Model
ACF 300). The DD of the chitosan was deter-
mined by the formula below.
DD= 1−
  1/3ICH3 n × 100,
1/6IH2 – H6
 S.C. Tan et al. / Talanta 45 (1998) 713–719
Table 1
Degree of deacetylation of commercial chitosan measured by first derivative spectrophotometry, nuclear magnetic resonance, linear
potentiometric titration and ninhydrin test
Chitosan sample
Degree of Deacetylation (%) First derivative uv-spectrophotometry
Nuclear magnetic resonance
Linear potentiometric titration
Ninhydrin test
* No replicate was done.
where ICH3 is the integral intensity of CH3 and
IH2 – H6 is the summation integral intensities of H2,
H3, H4, H5, H6 and H6%.
2.4. Linear potentiometric titration [15]
The determination of DD by LPT was a mod-
ification of the method of Ke and Chen. Chitosan
(0.20–0.25 g) was dissolved in 20 ml of standard-
ised 0.10 N HCl (to give a non-viscous solution)
and diluted with 10 ml of distilled water. The pH
of the solution was adjusted to : 2 with standard
0.01 M NaOH and taken as the start point.
Under continuous stirring, 1 ml of standard
NaOH (0.5 ml for purified chitosan) 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) of the corresponding
volume of NaOH added was calculated using the
following formula:
f(x)=
 V0 + V×([H + ]− [OH − ]),
NB
where V0 is the volume of chitosan solution (ml);
V is the volume of NaOH added (ml); NB is the
concentration of NaOH (N); [H + ] is the concen-
tration of H + (M); [OH − ] is the concentration of
OH − (M).The linear titration curve was obtained
by plotting f(x) vs. the corresponding volume of
NaOH. The volume of NaOH at the end point of
the titration, Ve, was estimated by extrapolating
the linear titration curve to the x-axis. The DD of
the chitosan sample was calculated using the fol-
lowing formula. Five replicates were performed
for each sample.
DD(%)=¥ / [(W −161¥) / 204 +¥] ×100,
715
Fluka Kasei Purified Kasei
85.82 9 0.51 82.66 90.74 98.08 90.03
82.20* N/A N/A
75.00 9 0.41 73.29 90.62 89.25 90.38
51.01 91.88 44.92 91.75 55.41 9 2.91
where ¥ =(NAVA − NBVe)/1000; NA is the con-
centration of HCl (N); VA is the volume of HCl
(ml); NB is the concentration of NaOH (N); Ve is
the volume of NaOH at the end point (ml); W is
the sample mass (g).
2.5. Ninhydrin test [18]
Ninhydrin test is another test which estimates
the amount of chitosan by direct detection of the
–NH2 group on the glycoside repeat unit of chi-
tosan. Standard solutions were prepared by pipet-
ting 0.1, 0.2, 0.3, 0.4, and 0.5 ml of GlcN solution
(0.1 mg ml − 1 2% acetic acid) into separate test
tubes. Acetic/acetate buffer (0.5 ml; pH 5.5, 4 M)
was added to each tube and the volume adjusted
to 1 ml with distilled water. Finally, 2 ml of
ninhydrin reagent (250 ml acetic/acetate buffer,
pH 5.5, 4 M, 20 g ninhydrin, 3 g hydrindantin,
750 ml methyl cellosolve) were added to each tube
and the solution heated in a boiling water bath
for 10 min. The solution was allowed to cool for
15 min before the UV absorbance at 570 nm of
each solution was recorded. The calibration curve
was obtained by plotting the absorbance against
the concentration of standard solutions. Sample
solutions consisted of 0.5 ml of chitosan solution
(0.1 mg ml − 1 2% acetic acid) to which was added
the buffer, topped up with distilled water and the
ninhydrin reagents. The solution was heated as
above and the absorbance value recorded. The
amount of chitosan in the sample was estimated
and the DD of the chitosan samples were deter-
mined by the formula below. Five replicates were
performed for each sample.
DD= ¥ / [ (W−161¥) / 204+ ¥ ] ×100,
716 S.C. Tan et al. / Talanta 45 (1998) 713–719
where ¥ is the amount of GlcN determined/161
and W is the sample mass.
2.6. First deri6ati6e UV-spectrophotometry [13]
UV-vis absorption spectra were obtained using
a Shimadzu 1601 UV-vis spectrophotometer. The
zero crossing point (ZCP) was determined by
superimposing the first derivative spectra of
0.0100, 0.0200 and 0.0300 M of acetic acid solu-
tions at 203 nm.
2.6.1. Calibration cur6e
Solutions of 0.0050 – 0.0500 mg of GlcNAc per
ml of 0.0100 M acetic acid solution were prepared
and their first derivative spectra obtained. Each
concentration had five replicates. The spectra of
0.0100, 0.0200 and 0.0300 M of acetic acid solu-
tions were superimposed and the vertical distance
from ZCP to each GlcNAc solution spectrum, H,
was measured (mm). A linear calibration curve
was obtained by plotting the H values against the
corresponding GlcNAc concentration.
2.6.2. Correction of the effect of D -glucosamine
on H 6alues
As proposed by Muzzarelli and Rocchetti, the
presence of GlcN may give rise to a larger H
value for GlcNAc than expected. Therefore, a
reference curve for correcting this discrepancy was
necessary. A 0.1000 mg GlcNAc per ml 0.0100 M
acetic acid solution was prepared and varying
amounts of GlcN was dissolved in the GlcNAc
solution to give a series of different percentages of
GlcNAc solutions (w/w). The H values of the
pure GlcNAc solution, H1, and the H values of
the solutions of different percentages of GlcNAc,
H2, were measured. The reference curve was ob-
tained by plotting H1/H2 vs. the corresponding
GlcNAc percentage.
2.6.3. Determination of the DD of chitosan
Freeze-dried chitosan samples (0.0100 g) were
dissolved in 10 ml of 0.1000 M acetic acid solu-
tion and topped up to 100 ml with distilled water.
Five replicates were performed for each sample.
The H values of the chitosan samples were mea-
sured and the contribution due to GlcNAc was
obtained from the calibration curve. The DD of
the samples were determined by the formula:
DD= 100− [A / (W− 204A) / 161+ A] × 100),
where A is the amount of GlcNAc determined/204
and W is the mass of chitosan sample used.
3. Results and discussion
In the estimation of the DD, methods which
assess the amine or acetyl amine groups on the
glycoside unit of chitosan directly would be pre-
ferred. Methods such as circular dichroism,
NMR, GPC and thermogravimetry are not suit-
able for routine purposes because of the cost,
specialist considerations and sophistication which
renders them more appropriate for research pur-
poses. Elemental analysis has long been the com-
mon method to ascertain the DD of chitosan. The
method requires very pure samples for accurate
estimation. This is not often attainable in the
extraction of chitin and chitosan from shellfish.
Furthermore, the hygroscopic nature of chitosan
will always include water in the derivation of the
empirical formula. IR and NIR spectroscopy are
methods that are also commonly used. However,
they are primarily a solid state method and may
not be appealing because variations can be found
in the results obtained using different baselines
[8–11]. Different baselines have been suggested
for samples with different ranges of DD [6] but
the choice of the baseline is debatable, especially
when its range for individual samples are un-
known. Therefore, we feel that IR and NIR are
not suitable as standard methods to determine the
DD of chitosan. Thus the choice of the standard
method to determine the DD on a routine basis
appears to rest on a solutions based approach. In
dilute solutions, it is expected that the amine
group would be at its most accessible for opti-
mum quantification.
We have reviewed four solution based methods
and are advocating the use of 1DUVS. Table 1
presents the results of the DD obtained from
NMR, LPT, ninhydrin test and 1DUVS. The DD
of 1DUVS, LPT and ninhydrin methods do not
correlate well with each other. Of the four meth-
 S.C. Tan et al. / Talanta 45 (1998) 713–719 717

Fig. 1. First derivative UV-spectra for various concentrations of chitosan solutions. A1, 0.01 M acetic acid solution; A2, 0.02 M
acetic acid solution; A3, 0.03 M acetic acid solution; a, 0.05 mg chitosan/0.01 M acetic acid solution; b, 0.10 mg chitosan/0.01 M
acetic acid solution; c, 0.25 mg chitosan/0.01 M acetic acid solution; d, 0.50 mg chitosan/0.01 M acetic acid solution; e, 1.00 mg
chitosan/0.01 M acetic acid solution.

ods, we believe that the results obtained by
1DUVS are the closest to the actual DD of the
chitosan samples. This is because the results from
NMR, LPT and ninhydrin test may be affected by
the presence of protein contaminants, which is
commonly present in crude chitosan samples. The
maximum UV absorbance of proteins is typically
at the wavelength 280 nm, far enough from 203
nm, the wavelength at which the H value was
measured. Therefore, the interference from the
proteinaceous contaminant in the sample to the
results obtained by 1DUVS is kept at a minimal
level.
Although the result from NMR correlates well
with 1DUVS, 1DUVS has several advantages
over the NMR method. The most important is
1DUVS can tolerate a higher level of proteina-
ceous contaminants. Second, chitosan samples
used for NMR has to be dissolved in deuterated
solvent (CD3COOH/D2O) which is usually more
expensive than the solvent (CH3COOH/H2O)
used in 1DUVS. Last, the cost of NMR instru-
mentation is typically prohibitive compared with
UV-spectrophotometry for the small producer.
The advantage of using ninhydrin test is that a
very small amount of chitosan is required. How-
ever, the values of DD obtained were the lowest
among the three methods compared. There was a
large difference between the results obtained by
ninhydrin test and other methods. This may be
attributed to the fading of colour intensity of the
sample solution with time, after the boiling pro-
cess. This decolourisation was observed visually
about 30 min after the boiling process and there-
718 S.C. Tan et al. / Talanta 45 (1998) 713–719

fore, contribute to errors in the absorbance values trations of chitosan solution for analyses are to be
[16]. The procedure of this method is also rela- avoided as it would require more sample which
tively more complicated than LPT and 1DUVS as may add more experimental errors whenever dilu-
the ninhydrin reagent needs to be freshly prepared tions are made. Chitosan solutions of too low
for each test. Furthermore, the ninhydrin reagent concentrations are also not advisable as this may
is carcinogenic and extra precautions in handling also incur larger experimental errors.
are necessary. Finally, since the chemical basis for The reference curve for the correction of the
the ninhydrin test is reaction with the amine effect of GlcN on the H value is shown in Fig. 2.
group, the presence of protein (which also con- Muzzarelli and Rocchetti showed that the pres-
tains amines) in the sample may adversely inter- ence of GlcN contributes to the H value, when the
fere with the results. GlcNAc to GlcN ratio is below 0.11 (GlcNAc is
LPT is easier to perform than the ninhydrin test less than 10%). However, our results showed that
and the results are often more consistent. Unlike even when the GlcNAc is 20%, contributions of
the traditional potentiometric titration, the ana- the GlcN to the H value is also present. The
lyte is not titrated all the way to its end point. discrepancy between our data and that of Muz-
Therefore, interference from the precipitation of
zarelli and Rocchetti may lie in the sensitivity of
chitosan is avoided. The equipment and reagent
the UV-spectrophotometer used. Therefore, our
used in LPT are also simple and readily available
current data suggests that corrections up to 20%
in a chemistry laboratory. The major source of
are necessary.
error for LPT is the reliability of the pH measure-
Based on the results we obtained, the DD of
ment. The results from LPT are very dependent
chitosan determined, varied with the methods
on the [H + ] and [OH − ] concentrations in the
analyte as the pH measured directly affects the used. Each of these methods has its own advan-
DD determined. The amine and thiol groups in tages and disadvantages. Therefore, it is difficult
amino acids will affect the [H + ] concentration of to tell which method has given us the most accu-
the analyte, and therefore, the presence of protein rate results. However, all of them did show the
contaminant can also be a major interference. relative DD between different chitosan samples.
1DUVS is very sensitive for GlcNAc detection, Up to date there is still no standard method for
the detectable concentration of GlcNAc in 0.01 M
acetic acid solution was found to be as low as
0.0005 mg ml − 1. The concentration of the chi-
tosan sample solution to be used for analysis was
standardised as 0.1000 mg chitosan per ml of
acetic acid solution. This permitted a wider range
of DD (0–50%) to be ascertained based on the
calibration plot ranging from 0.0050 to 0.0500 mg
GlcNAc per ml acetic acid solution. In this range,
the plot was linear and obeyed Beer’s law. This is
contrary to the concentration of 1 mg chitosan
per ml of acetic acid solution used by Muzzarelli
and Rocchetti [13]. Using this concentration, only
DD in the range 0 – 5% can be determined, which
is quite narrow. The dip of the 1DUVS spectrum
of chitosan solution is shifted to the right with
increasing chitosan concentration (Fig. 1). If the
concentration of chitosan solution is too high, the
dip of its first derivative spectrum may be shifted
beyond the valid range. Therefore, high concen- Fig. 2. Correction curve for GlcNac determination.
 S.C. Tan et al. / Talanta 45 (1998) 713–719
the determination of DD of chitosan yet. We
advocate 1DUVS to be used as the standard
method for routine determination of the DD of
chitosan due to its high sensitivity, minimal inter-
ference of results from contaminants and easy to
be performed.
Acknowledgements
The authors acknowledge the National Univer-
sity of Singapore for financial sponsorship of this
work (RP 920633) and the award of a research
scholarship for S.C. Tan.
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