Preparation of Good Quality Chitosan
Deacetylation of Chitin to Chitosan (Autoclave Method)
Deacetylation of chitin under autoclaving conditions (15 psi/121 °C) was evaluated for the preparation of chitosan under different NaOH concentrations and reaction times. Deacetylation was effectively achieved by treatment of chitin under elevated temperature and pressure with 45% NaOH for 30 min and a solids/solvent ratio of 1:15. Treated chitosan showed similar nitrogen content (7.42%), degree of deacetylation (90.4%), and molecular mass (1560 kDa) but significantly higher viscosity values (2025 cP) compared with those (7.40%, 87.6%, 1304 kDa, and 143 cP, respectively) of a commercial chitosan. Reduction of the solids/solvent ratio from 1:15 to 1:10 did not affect degree of deacetylation, viscosity, and molecular mass of chitosan.
Chitin (3 g) was mixed with various concentrations of NaOH (40%, 50%, and 60%,
w/v) at a ratio of 1:10 (w/v) and was left to steep in NaOH solution for two days. After
that, the samples were treated under a pressure of 101.3 kPa at 121°C for 30 minutes in
an autoclave (Hirayama, HVE-50) (Hong et al. 2000). After autoclaving, the solid
products were washed to neutrality under running tap water, rinsed with distilled water,
and filtered to remove excess NaOH. The obtained product was dried for 24 h at 60 o C,
after which it was further treated with 2% acetic acid solution (1:15 ratio, w/v) under
stirring for 10 minutes which dissolved the chitosan, leaving the remaining chitin as an
insoluble solid. The mixture was filtered using a sieve and chitosan collected in a beaker.
by extraction with acetone and dried for 2 hr at ambient temperature, followed by
bleaching with 0.315 % (v/v) sodium hypochloride solution (containing 5.25% available
chlorine) for 5 min with a solid to solvent ratio of 1:10 (w/v), based on dry shell. But,
the color of chitin products varied from cream white to intermediate pink color (No et
al., 1989). Without prior acetone extraction, bleaching for more than 1 hr was needed to
obtain a commercially acceptable white product.
Deacetylation is the process to convert chitin to
chitosan by removal of acetyl group. It is generally achieved by treatment with
concentrated sodium or potassium hydroxide solution (40-50%) usually at 100oC or
higher for 30 min or longer to remove some or all of the acetyl groups from the polymer
(No and Meyers, 1989). The N-acetyl groups cannot be removed by acidic reagents
without hydrolysis of the polysaccharide, thus, alkaline methods must be employed for
N-deacetylation (Muzzarelli, 1977).
Depolymerization of chitosan
Chitosan was selectively depolymerized following the
method developed by Huang et al. 26 Briefly, 100 mL of a 2%
(w/v) chitosan (400,000 g/mol) solution in 6% (v/v) acetic
acid solution was prepared. Then the dissolved chitosan
was depolymerized at room temperature under stirring with
10 mL of NaNO 2 solutions in water at different concen-
trations (7.0, 2.7, and 1.6 g/L), to obtain the desired final
molecular weight of 20,000, 50,000, and 100,000 g/mol,
respectively. After 1 hour of reaction, precipitated chitosan
was obtained by raising the pH to 9.0 by addition of 4N NaOH. The white-yellowish solid was filtrated, washed thor-
oughly with acetone, and re-dissolved in a minimum volume
of acetic acid 0.1 N (40 mL).
Purification was carried out by subsequent dialyses
against purified water (Sigma dialysis tubes, molecular
weight cutoff, 12 kDa). The dialyzed product was lyophilized
using a LyoTrap plus Freeze dryer (LTE Scientific, Oldham,
UK), and the yellowish lyophilized product was then stored
at 4°C until use.
Thiolated chitosan was prepared with covalent attachment of
reduced glutathione to different molecular weight of chito-
san according to the slightly modified method described by
Atyabi et al and Kafedjiiski et al as follows: 5,27
One gram of chitosan was dispersed in 50 mL demineral-
ized water by stirring, then 10 mL HCl (1 mol/L) was added
and finally dissolved by the addition of demineralized water
to obtain a 1% (w/v) polymer solution. The pH was adjusted
to 6.0 by the addition of NaOH (5 mol/L). Afterwards,
5 g of reduced glutathione in 10 mL demineralized water
was added to the above solution under continuous stirring.
EDC and NHS were then added in a final concentration of
200 mmol/L each. The pH was readjusted to 6.0 by adding
NaOH (5 mol/L). The reaction mixture was incubated for
15 hours at room temperature under constant stirring. To
eliminate unbound reagents, the resulting polymer conjugate
was dialyzed using dialysis tubing (molecular weight cutoff
12 kDa), first against 5 mmol/L HCl, twice against 5 mmol/L
HCl containing 1% NaCl, and finally twice against 1 mmol/L
HCl. Controls were prepared in the same way but omitting
EDC and NHS during the coupling reaction. Finally, the
frozen aqueous polymer solutions were lyophilized at −50°C
and 0.01 mbar (LyoTrap plus; LTE Scientific, UK) and stored
at 4°C until further use
Preparation of thiolated
chitosan-pMMA NPs
The NPs were prepared by using a modif ied radical
polymerization method. Conjugated chitosan (37.5 mg) with different theoretical molecular weight (20, 50, and 100 kDa)
was dissolved in 4 mL nitric acid (0.2 mol/L) in a two-necked
flask at 40°C, under gentle stirring and nitrogen bubbling.
After 10 minutes, under vigorous magnetic stirring, a solu-
tion of 0.08 mol/L cerium IV ammonium nitrate (CAN) in
0.2 mol/L nitric acid and MMA was added to obtain a 5-mL
solution. Nitrogen bubbling was continued for an additional
10 minutes. The reaction was allowed to continue at 40°C
under gentle stirring for 40 minutes. After cooling to room
temperature, the pH of the obtained suspension was adjusted
to 4.5 by addition of sodium hydroxide (1 N) drop-wise. The
NP suspension was then purified by dialysis (Sigma dialysis
tubes, molecular weight cutoff, 12 kDa) against 1 L of acetic
acid solution (16 μmol/L) in demineralized water, twice for
90 minutes and once overnight. The frozen NP suspensions
were lyophilized at −50°C and 0.01 mbar and stored at 4°C
until further use.