Acid hydrolysis of chitin and chitosan

Article abstract of DOI:10.1023/B:RJAC.0000031297.24742.b9

Acid hydrolysis of chitin and chitosan recovered from shells of sea Crustacea was studied by exclusion high-performance liquid chromatography.

Full text of DOI:10.1023/B:RJAC.0000031297.24742.b9

Russian Journal of Applied Chemistry, Vol. 77, No. 3, 2004, pp. 484 487. Translated from Zhurnal Prikladnoi Khimii, Vol. 77, No. 3,
2004, pp. 490 493.
Original Russian Text Copyright
2004 by Novikov.
MACROMOLECULAR CHEMISTRY
AND POLYMERIC MATERIALS
Acid Hydrolysis of Chitin and Chitosan
V. Yu. Novikov
Knipovich Arctic Research Institute of Fishery and Oceanography, Murmansk, Russia
Received December 17, 2002; in final form, January 2004
Abstract Acid hydrolysis of chitin and chitosan recovered from shells of sea Crustacea was studied by
exclusion high-performance liquid chromatography.
Antiarthrosis preparations based on chitin oligo-
mers and glucosamine salts attract growing attention
[1, 2]. The yield and quality of glucosamine hydro-
filtered through a glass filter with pore diameter of no
more than 10 m (POR 10).
The degree of CSN deacetylation was determined
by potentiometric titration of a 2% CSN solution with
0.1 M HCl by the procedure in [10]. The absolute
error of the determination was 0.5%.
1
chloride produced from chitin depend in a complex
manner on the chitin properties [3]. To understand this
dependence, the mechanism of acid hydrolysis of
chitin was studied in more detail.
The hydrolysis products of CTN and CSN were
Acid hydrolysis of chitin (CTN) and chitosan
(CSN) was examined in [4 8]. The aim of this work
was to determine the limiting degree of chitosan
deacetylation suitable for preparing glucosamine. For
this purpose, the influence of the degree of chitosan
deacetylation on its acid hydrolysis was studied by
exclusion high-performance liquid chromatography
(HPLC).
separated by HPLC on an LC10A chromatograph
vp
(Shimadzu, Japan) with TSK-gel Alpha-2500 (30
0.78 cm) column and TSK-guardcolumn Alpha (6
0.4 cm) precolumn (TOSOH, Japan). The hydrolyzate
samples were applied in the form of 0.1% solutions in
the eluent (0.3 M NaCl acidified with HCl to pH 3).
The hydrolysis products were detected by the absorp-
tion at 210 nm.
The UV detection of the hydrolysis products al-
lowed us to monitor changes in the concentration of
acetylated compounds and acetic acid. The consider-
ably weaker absorption of deacetylated CSN oligo-
mers and D-glucosamine (GA) was obscured by the
strong band of the acetylated products. Monomeric
N-acetylated glucosamine (AcGA) was identified by
comparing its retention time (9.78 min) with that of
pure GA (9.86 min). For this purpose, a 5% GA solu-
tion in the eluent was used.
EXPERIMENTAL
We used CTN prepared by deproteinization and
demineralization of shell of North shrimp (Pandalus
borealis) by the procedure in [9]. The chitosan content
in the sample was 97 wt %; the ash content was lower
than 0.1 %; the particle size was no larger than 2 mm.
The chitosan samples were prepared by deacetyla-
tion of CTN and CSN. Chitosan with the 68.0%
degree of deacetylation (DD) (CSN-1) was obtained
by treatment of CTN with 50% aqueous NaOH at
95 5 C for 30 min. The samples with DD of 88.0
(CSN-2) and 92.0% (CTN-3) were prepared under
similar conditions from CSN-1 and CSN-2, respec-
tively.
Based on the published chromatographic data on
CTN oligomers [5 7], we assigned the other chro-
matographic peaks to the acetylated oligomers (Fig. 1).
If peak 2 is due to AcGA, then peaks 3 and 4 are
assigned to N,N -diacetylchitobiose and N,N ,N"-tri-
acetylchitotriose, respectively. The semilog plot of the
molecular weight (MW) of these compounds on the
elution time is linear. The nonlinear deviation for
GA can be due to either characteristics of the linearity
the column or structural difference of the deacetylated
monomer from AcGA. Hence, GA cannot be used to
construct the calibration curve.
Chitin and chitosan were hydrolyzed with 36.5%
HCl at 50 and 70 C. The resulting hydrolyzates were
diluted with distilled water, neutralized to pH 3, and
1
Ekobiotek-Murmansk Research and Technical Center, Limited
Liability Company.
1070-4272/04/7703-0484 2004 MAIK Nauka/Interperiodica

ACID HYDROLYSIS OF CHITIN AND CHITOSAN
It is known [4, 8] that the rate of formation of
AcGA and GA in the course of acid hydrolysis of
CTN and CSN decreases with an increase in their DD.
485
MW
The influence of DD on the rate of acid hydrolysis
of CTN and CSN is seen from the chromatograms of
their hydrolyzates. At 50 C, CTN is hydrolyzed in-
completely to form a mixture of macromolecular and
low-molecular-weight products (Fig. 2). As the hy-
drolysis time increases, the peaks of the macromolecu-
lar products shift toward lower molecular weights and
decrease in the integral intensity; the intensity of the
peaks of the oligomers and monomers increases. It
should be noted, however, that the integral intensity of
peaks cannot be a measure of chitin depolymerization,
since depolymerization is accompanied by formation
of deacetylation products whose molar extinction co-
efficient is lower than that of the acetylated products.
, min
Fig. 1. Calibration curve for chromatographic determination
of mono- and polysaccharides. (MW) Molecular weight and
( ) retention time. (1) GA, (2) AcGA, (3) chitobiose, and
(4) chitotriose.
The content of the macromolecular compounds in
the products of hydrolysis of CTN and CSN-1 (DD =
68%) at 70 C sharply decreases (Fig. 3). The products
of hydrolysis performed for more than 10 min contain
mainly oligomers, monomers, and acetic acid; the
polymer concentration is very low.
In the products of hydrolysis of CSN-2 and -3
(DD 88 and 92%), the peak of macromolecular frac-
tions is preserved even after hydrolysis for 300 min. It
should be noted that a chitosan precipitate in the form
of a fine dispersion and with the outward appearance
of the initial sample was observed after 5-h hydrolysis
of CSN-2 and CSN-3, respectively.
, min
Fig. 2. Chromatograms of CTN hydrolyzates obtained at
50 C, normalized to the equivalent amount of acetyl groups
corresponding to DD = 0%; the same for Fig. 3. (D ) Op-
210
tical density at 210 nm, and ( ) retention time. Hydrolysis
time, min: (1) 20 and (2) 40.
The degree of hydrolysis was estimated from the
AcGA yield. The AcGA amount was recalculated to
the number of acetyl groups in CTN with DD = 0%,
taking the initial DD into account. The AcGA yield
increases in the first hydrolysis steps owing to cleav-
age of glycoside bonds of the macromolecules and
then decreases owing to deacetylation (Fig. 4). The
AcGA yield in hydrolysis of CTN and CSN, per-
formed for the same time, sharply decreases with an
increase in DD of the polysaccharide (Fig. 5).
, min
Fig. 3. Chromatograms of hydrolyzates of (1) CTN,
This result agrees with the fact that the glycoside
bond in acetylated monomeric chains is more readily
hydrolyzed than the bond in deacetylated chains [8].
Based on relative deceleration of depolymerization
with respect to the deactylation rate at high acid con-
centrations, Varum et al. [8] suggested different hy-
drolysis mechanisms of N-acetyl and glycoside bonds.
(2) CSN-2, and (3) CSN-3 at 70 C (120 min). (D ) Opti-
210
cal density at 210 nm and ( ) retention time.
molecules decreases and the reaction decelerates. The
glycoside bond is hydrolyzed by the S 1 mechanism
N
in which the rate-determining step is the proton attack
to form carbocation. In this case, the reaction rate is
independent of the water concentration.
Probably the N-acetyl bond is hydrolyzed by the S N2
4
mechanism in which the rate-determining step is addi-
tion of water molecule to the carbocation. As the acid
concentration increases, the concentration of water
However, the influence of the acetyl substituent at
the nitrogen atom of a CTN molecule on the depoly-
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 77 No. 3 2004

486
m, g
NOVIKOV
bonds in a chitin molecule, more comprehensive
studies are required.
It is of practical importance that partially deace-
tylated CTN or CSN are completely hydrolyzed to
AcGA and GA in a time longer than that required for
hydrolysis of CTN with minimal DD.
The GA concentration in the CTN hydrolyzate is
low, and this product cannot be detected by UV ab-
sorption since its molar extinction coefficient is small.
Therefore, deacetylation of the acetylated products
was monitored by the rate of acetic acid formation
(Fig. 6).
, min
Fig. 4. Kinetics of AcGA formation in hydrolysis of
(1) CTN, (2) CSN-1, (3) CSN-2, and (4) CSN-3. Tempera-
ture 70 C; the same for Figs 5 and 6. (m) AcGa weight and
( ) hydrolysis time.
The AcOH amount was estimated in relative units
from chromatographic peak area (retention time
14.14 min). The amount of AcOH that could be
released upon complete acid hydrolysis of CTN with
DD = 0% was taken as the maximal. A sample con-
taining the calculated amount of AcOH was intro-
duced in the column, and the peak area was measured.
m, g
As seen from Fig. 6, the deacetylation rate is
almost independent of the initial DD. This is also the
case for the deacetylation of CTN and CSN in alkaline
solution [11].
Thus, Figs. 4 and 6 show that depolymerization of
CTN and CSN depends on the initial degree of their
deacetylation. The higher DD, the lower is AcGA
yield. The deacetylation course depends only on the
experimental hydrolysis conditions and is independent
of DD of CTN and CSN. Hence, the steps of depro-
teinization and demineralization of chitin production
should be performed with care to avoid uncontrolled
deacetylation affecting the GA yield.
DD, %
Fig. 5. Yield of AcGA as a functions of DD of CTN and
CSN hydrolyzed for (1) 30 and (2) 60 min. (m) AcGa
weight and (DD) degree of deacetylation.
AcOH/AcOHmax, %
The limiting degree of deacetylation providing
optimal hydrolysis of CTN can be estimated from the
dependence of the AcGA yield on DD, assuming that
this dependence is linear in the DD range from 15 to
68%. We suggest that a decrease in the monomer
yield in the course of hydrolysis should not exceed
10% of its yield in hydrolysis of commonly used CTN
with DD 15%. Our calculations show that the DD of
CTN should not exceed 26%. The yield estimated by
extrapolation to zero DD is higher by 14% than that
of AcGA formed in hydrolysis of commonly used
CTN. Hence, the production process of glucosamine
can be further improved.
, min
Fig. 6. Rate of AcOH formation in hydrolysis of
(1) CTN, (2) CSN-1, (3) CSN-2, and (4) CSN-3.
([AcOH]/[AcOH]
( ) hydrolysis time.
) Relative AcOH concentration and
max
merization rate cannot be explained within the frame-
work of the proposed hydrolysis mechanism of the
glycoside bond. Computer simulation of the electron
density distribution in the molecule showed no ap-
preciable increase in the excess negative charge on the
atoms of the glycoside bond after acetylation of the
amino group. This charge promotes the proton attack.
To understand the hydrolysis mechanism of glycoside
CONCLUSIONS
(1) The study of acid hydrolysis of chitin and chi-
tosan by HPLC shows that accumulation of oligomers
and monomers of chitin and chitosan decelerates with
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 77 No. 3 2004

ACID HYDROLYSIS OF CHITIN AND CHITOSAN
487
2002, no. 18, pp. 76 78.
increase in the degree of deacetylation and the de-
acetylation rate is independent of the initial degree of
deacetylation of chitin and chitosan.
4. Horowitz, S.T., Roseman, S., and Blumental, H.J.,
J. Am. Chem. Soc., 1957, vol. 79, no. 18, pp. 5046
5049.
(2) The minimal degree of deacetylation of chitin
suitable for glucosamine production is 26%.
5. Capon, B. and Foster, R.L., J. Chem. Soc., Sect. C,
1970, no. 12, pp. 1654 1655.
6. Chitin Handbook, Muzzarelli, R.A.A. and Peter, M.G.,
ACKNOWLEDGMENTS
Eds., Grottamare (Italy): Atec, 1997, pp. 195 198.
7. Chang, K.L.B., Lee, J., and Fu, W.-R., J. Food Drug
Anal., 2000, vol. 8, no. 2, pp. 75 83.
We are grateful to Yu.B. Ripak (Ekobiotek-Mur-
mansk Scientific and Technical Center) for putting
HPLC columns (TOSHO, Japan) at our disposal.
8. Varum, K.M., Ottoy, M.H., and Smidsrod, O., Carbo-
hydr. Polym., 2001, vol. 46, no. 1, pp. 89 98.
9. Chitin Handbook, Muzzarelli, R.A.A. and Peter, M.G.,
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