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Indian Journal of Chemical Technology Yoi.IO, July 2003, pp. 350-354

Articles

Preparation and characterization of lobster shell chitosan: Modification of traditional method

* S Chatterjee, A K Guha & B P Chatterjee

Department of Biological Chemistry, Indian Assoc iation for the Cultivation of Science, Jadavpur, Kolkata 700 032, India

Received 7 January 2003; revised received 13 May 2003; accepted 27 May 2003

Preparation of chitosan from lobster shells by Hackman method lll)d modified Hackman method was compared with re­spect to, their physico-chemical properties, molecular we\ght, degree of deacetylation, p<ilydispersity and crystf}llinity. In modified method maximum yield (78%) of chitosan with almost 91% degree of deacetylation determined by ftrst derivative J!;:! spectra w~s achieved. ~olecular size distribution as well as average size c.f chitosan. produced by Hackman method was

higher (PI 1.205, particle. size 1448.6 nm) and of M w1 10 x 105 wheri. compared to chitosan produced by modified Hackman

method (PI 0.8832, particle size 95.3 nm) of M w1 3.98 x 105• X-ray powdered diffraction showed that chitosan prepared by

;~ackman method was found to be more crystalline, Bragg refractions 13.3°28 (d = 6.66A) and 19.7°28 (d = 4.51A), re­spectively than chitosan produced by modified method, Bragg refraction 13.6°28 (d = 6.51A). This method provides quality product in less time and is economical.

Chitosan is a natural polycationic biopolymer that occurs in the cell wall of fungi (Basidomycetes sp.) or is obtained by N-deacetylation of chitin by alkali treatment. Chitin is the most abundant biopolymer on earth comprising of ~- 1,4 linked N-acetyl-0-glucos­amine, [GlcNAc-~-1,4-GlcNAc]n residue and is pres­ent as a major structural component of exoskeleton or cuticles of many invertebrates, insects and cell wall of fungi .

Chitosan having three-dimensional cx:-helical con­figuration is stabilized by intramolecular hydrogen bonding'. It is biodegradable and non-toxic in nature. Due to such unique properties, it is widely used in various industrial applications, such as water purify­ing agent, viscosity-controlling agent, paper strength­ening agent, flocculating agent, antimicrobial agentsn, biological adhesive8

, antitumour agents9 and also in cosmetic industry.

Chitosan can be obtained from different sources. Two well known sources are exoskeletons of different crustacea viz, shrimp, prawn, lobster, crab and cell wall of zygomycetes group 10

-14

• Although chitosan obtained from fungi is more reliable as per quality of chitosan is concerned, the yield is inadequate for the industrial use and therefore crustacea are considered

*For correspondence (E-mail: bcbpc @mahendra.i acs.res.in; Fax: 91 33 2473 2805)

the best source for large-scale preparation of chitosan. Thi s investigation shows not only the modification

of commonly used Hackman method since it is cost benefited but also delineates comparison of the ctual­ity of chitosan prepared before and after modification of the above method.

Experimental Procedure Lobster shells were collected from New Market,

Kolkata. All other chemicals were procured from E. Merck.

Preparation of chitosan In Hackman method, lobster shells after deaning

and drying were treated with 2 N HCI (sheli:HCl; 1:10, wlv) for 5 h at 25°C, washed again and dried. The dried mass was pulverized and treated with 2 N HCl (mass:HCl; 1 :6) at 0°C for 48 h. The resulting mass was freed from acid and refluxed with 1 N NaOH at 95°C for 12 h. Last step was repeated four more times, washed and solid mass containing chitin was collected. Chitin was deacetylated to chitosan by refluxing with 50% NaOH (chitin :NaOH; 1:20, w/v)

at 120°C for 7 h, thoroughly washed and dried . The mixture was dissolved in 7% AcOH and filtered through celite bed to remove any unreacted chitin. The pH of the filtrate was maintained at 8.5 with 1 N KOH, whereby chitosan was preci pitated as a white

Chatterjee et al.: Preparation and characterization of lobster shell chitosan: Modification of traditional method Articles

mass. It was washed several times with water, fol­lowed by ether and dried at 80°C. The final product was stored in vacuum in the presence of PzOs.

The method described above was very lengthy. Therefore, modification of this method was essential to prepare chitosan directly from lobster shell omit­ting chitin preparation step. In this method dried and pulverized lobster shell was treated with 2 N HCl (shell:HCl; 1:10, wlv) at 0°C for 48 h under vigorous stirring condition, washed vigorously with water and treated with varying concentrations viz. 20, 30, 40, 50 and 60% of NaOH (solid:NaOH; 1:20, wlv) for 6 h. to optimize the NaOH concentration and then for vary­ing times (3, 4, 5, 7, 8 h) at optimized NaOH concen­tration for optimization of the reaction time. Both the experiments were performed at l20°C under N2 at­mosphere. Solid mass thus obtained were washed with water, triturated with warm (- 40°C) acetone 3-4 times. Chitosan was dried and preserved for further use as described above.

Determination of degree of deacetylation The degree of deacetylation of chitosan was meas­

ured by first derivative UV spectroscopic method as recorded by Muzzarelli 15.

Determination of weight average molecular weight Chitosan solutions of varying concentrations rang­

ing from 0.0125-0.2 g/100 mL were prepared using 2% acetic acid. Intrinsic viscosity (TJ;n) of the solu­tions was obtained by Oswald viscometer at 25°C. Using the value of fl ;n in double logarithmic plot of the intrinsic viscosity ([TJ;n]/,dVg) and weight average

molecular weight, M wt of chitosan at 25°C was de­termined16.

Estimation of protein Chitosan (50 mg) was incubated with I mL 5 M

urea at 95°C for 30 min with periodic mixing. To the mixture, cooled at 25°C, 1 mL water was added fol­lowed by centrifugation at 10,000 rpm for 15 min . The supernatant (300 J.!L) was diluted with an equal volume of water. The protein content of the solution was determined by Bradford method 17

•

Co-infrared spectroscopy Approximately 2-3 mg of chitosan was mixed with

100 mg of KBr. About 40 mg of the mixture was used to prepare KBr pellet. The pellets were subjected to Co IR in Shimadzu FT IR 8300 spectrophotometer.

Dynamic light scattering Chitosan (1 g) was dissolved in 2% acetic acid and

filtered through millipore (0.22 Jlm). Light scattering experiment was performed in phenol using He-Ne laser at 632.8 nm at an angle 90° (DLS-700 Otusuka Electronic, Japan).

X-ray diffraction studies Powder X-ray diffraction patterns of chitosans pro­

duced from two different methods were obtained us­ing Seifert C3000 instrument with the following oper­ating condition - 40 KV and 30 rnA, with Cu/Ni ra­diation at A=1.5406. The relative intensity was re­corded in a range of (28) of 10-100°.

Ash, moisture content and specific rotation were measured by the standard method 18.

Results and Discussion The yield of chitosan with respect to NaOH con­

centration was standardized by modification of Hackman method, by fixing reaction time at 6 h. It showed that 50% NaOH was optimal for the prepara­tion of chitosan, since beyond that concentration on both sides there was no increase of chitosan yield. In the next step, yield of chitosan was standardized with respect to reaction time by fixing NaOH concentration at 50% (wlv). Optimum reaction time was 7 h, since before or after that period, yield did not increase. Thus, 50% NaOH (wlv) and 7 h reaction time are op­timum for chitosan production.

The modified method produced chitosan which was completely soluble in acetic acid and the yield was higher. Following original Hackman method, only 32% of lobster shell was converted to chitosan, whereas by modified Hackman .method, 78% lobster shell was converted to chitosan. The increase of chi to­san yield at remarkably high percentage conversion, presumably due to reaction being conducted in nitro­gen atmosphere is notable feature of modified method.

Purity of chitosan prepared by original and modi­fied method along with standard procured from Sigma was compared by Co FT IR spectra as shown in Fig. 1. It was found that all chitosans showed band at 2900 cm-1 (NH bond stretching), at 1650 cm-1 (C=O bond stretching) and at 1550 cm-1 (NH vibrational mode) , indicating that all samples are of the same grade of purity and degree of deacetylation of these polymers could be the same. This was further con­firmed by the first derivative UV spectra15 (Table 1).

35 1

Articles lndian J. Chern. Technol. , July 2003

100.0 -- -----------------·- - -·--....-

I . / i

0 .0

'·,,- ·

I .

a I ' I I

/, ( i

( \_.J /

b [ I

I I c

-----,---,-,~ ---. -. -·---r-- - ~ --- -

4000.0 3500.0 3000.0 2500.0 2000.0

r' \J,- j ._i

1750.0 1500.0 1250.0 1000.0 750.0

Wave number in cm-1

Fig. 1- CoFT IR spectra of chitosan from (a) H;:ckman's modified method (b) Hackman's method and (c) standard (Sigma)

The degree of deacetylation of chitosan by Hackman method was slightly higher than that in case of chito­san prepared by Hackman's modified method and other parameters such as ash and protein contents, specific rotation, which reflect the quality of chitosan were found to be almost same (Table 1). Dynamic light scattering (DLS) experiments (Figs 2a and 2b) showed molecular distribution patterns of chitosan produced by two different methods. From Table 2 it can be observed that molecular distribution of chito­san produced by Hackman method was very high (polydispersity index 1.205), whereas that in chitosan produced by modified Hackman method is very low (polydispersity index 0.833). This indicated that qual­ity of the chitosan produced by Hackman's modified method was better compared to that produced by Hackman 's method. DLS experiment, also indicates that averaoe molecular size distribution in chitosan by b

Hackman 's modified method was 95.3 nm, which was small with respect to chitosan produced by Hack­man's method (1448 .6 nm) (Table 2). Variation of molecular size of chitosan obtained by two different methods gave an insight to an average molecular

weight, M wt which was found to be in good agree-

352

ment with experimental results obtained. From the above it may be concluded that higher the average molecular size, higher will be the average molecular

weight, M wt· Molecular weight of chitosan (Table 2) produced by Hackman's method is very high i.e, 10xl05

, while chitosan produced by modified method is very low i.e, 3.98x105

. X-ray diffraction is com­monly used to determine polymorphic forms of a compound having different crystaliine structures for which distinct powdered X-ray diffraction patterns are obtained. These patterns are indicative of different spacings of the crystal planes, which provide a strong evidence for polymorphic difference. In addition, it provides accurate measurements of crystallinic con­tents, which greatly affect physical and biological properties of the polymer. Figs 3a and 3b showed powder di lffraction pattern of chitosan prepared by Hackman and Hackman's modified method respec­tively. Strong Bragg refractions were observed at an angle 13.3"28 (d = 6.66 A) and 19.7°28 (d = 4.51 A), the second one showing stronger intensity in case of chitosan obtained from Hackman method. The same for chitosan isolated by modified method showed strong Bragg refraction at 13.6°28 (d = 6.51 A).

Chatterjee et al.: Preparation and characterization of lobster shell chitosan : Modification of traditional method Articles

Table !-Physico-chemical properties of chitosans

Method Degree of deacety-adopted lation

(%)

Hackman method 89.7

Modified Hackman 90.8 method

6

5 ....... .. .. .. ....... .. .

4

2 - - - ....

168 614 2245

Diameter (run)

Ash (%)

0.60

0.49

30000

,Fig. 2a-Dynamic light scattering of chitosan produced by Hackman 's method

Table 2-Polydispersity, average molecular size and weight aver­age molecular weight of chitosans

Method

Hackman method

Modified Hackman method

Conclusions

Polydis­persity index

1.205

0.833

Av. mo­lecular size

(nm)

1448.6

95.3

Weight av. Molecular

weight

( M w) Da x 105

10.00

3.98

From the above results, it may be concluded that chitosan from Hackman 's method is more crystalline than that from the modified method, though with re­gard to polydi spersity, particle size and degree cf deacetylation, chitosan from modified method is bet­ter. It has molecular weight 2.5 times less than the

8

Protein (%)

0.05

0.06

Moisture (%)

5.12

4.73

Specific rotation [a]2s

... ........ . ; .....

.... ..... .. __ _ __ _, , _ ___ , __ _____ , _

6 ..... ..... ... .. .... .... .... .. . .. r ........... .. ........ ........ .. .. . ....... .. ...... ... ........... ...

----·--·· ---- ·- -·· --·· ···· ---··· .. ··- UitHit-1-.. ......... .. . .... ..... .... .. .

5 ... .. ... ... ........... .. ............ 1·1-1-li~H·Hit - ........... .. ..... .... .. .. .................. ... .......

-... ..... ... .. .......... .. .... ... tlltHIII'H -11·1 .... -.. ... --- ..... .. ....... ·

3 · ...... .. ...... · • ...... .. .... · --HI•H ·H-Itl-l-1·1-11·1-1- ............ ..

t- .. .. ........... -.. .... .. .. . .. ~0~1 ...... 1-I·Htl·l-1- · . ~ · · ~ -. -. -. --~ · ... · _ ...... ~ --· ·_-

2 ........ " ...... -.... .. .. .. ffi·I1HtlffltHI'HI1tiH·• · .... .... .. · · .. ......

. ... ... _ :--·~ -:-<·~-~--~~·.:.·· -li-11ti11·H·IH-HI·I-1·1111tH·Hili.1•1 ... ~-~ :.~~-:-: . -:·:·:·:---: 0~-+--~~~U£~~~~~~~~+-~~

4 7 15 31 66 137 286 5fi7 1245 2600

Diameter (run) Fig. 2b--Dynamic light scattering of chitosan produced by Hackman' s modified method

2(0)

Fig. 3a-X-ray diffraction pattern of chitosan by Hackman's method

Fig. 3b

2(8)

Fig. 3b--X-ray diffraction pattern of chitosan by Hackman ' s modified method

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Articles

chitosan prepared by Hackman's method and the time required was less.

Acknowledgement Authors gratefully acknowledge Department of

Biotechnology, New Delhi for financial support.

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Indian J. Chem. Techno!. , July 2003

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