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Extraction and characterization of chitin and chitosan from local sources
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Bioresource Technology 99 (2008) 1359–1367
Extraction and characterization of chitin and chitosan from local sources Entsar S. Abdou a, Khaled S.A. Nagy a, Maher Z. Elsabee a
b,*
Food Technology Research Institute, Agriculture Research Center, Cairo 12631, Egypt Department of Chemistry, Faculty of Science, Cairo University, Cairo 12631, Egypt
b
Received 20 November 2006; received in revised form 27 January 2007; accepted 30 January 2007 Available online 26 March 2007
Abstract Chitin has been extracted from six different local sources in Egypt. The obtained chitin was converted into the more useful soluble chitosan by steeping into solutions of NaOH of various concentrations and for extended periods of time, then the alkali chitin was heated in an auto clave which dramatically reduced the time of deacetylation. Chitin from squid pens did not require steeping in sodium hydroxide solution and showed much higher reactivity towards deacetylation in the autoclave that even after 15 min of heating a degree of deacetylation of 90% was achieved. The obtained chitin and chitosan were characterized by spectral analysis, X-ray diffraction and thermo gravimetric analysis. 2007 Elsevier Ltd. All rights reserved. Keywords: Chitin extraction; Local sources; Deacetylation conditions; Characterization
1. Introduction Chitin, a naturally abundant polymer consists of 2-acetamido 2-deoxy-b-D-glucose through a b(1 ! 4) linkage. In spite of the presence of nitrogen, it may be regarded as cellulose with hydroxyl at position C-2 replaced by an acetamido group. Like cellulose, it functions as structural polysaccharides. Its natural production is inexhaustible; arthropods, by themselves, count more than 106 species from the 1.2 · 106 of total species compiled for animal kingdom, constitute permanent and large biomass source. Chitin is a white, hard, inelastic, nitrogenous polysaccharide and the major source of surface pollution in coastal areas. Chitin is usually isolated from the exoskeletons of crustaceans and more particularly from shrimps and crabs where a-chitin is produced (Minke and Blackwell, 1978; Austin et al., 1989). Squid is another important source of chitin in which it exists in the b form which was found to be more amenable for deacetylation. It also shows higher *
Corresponding author. Tel.: +20 26352316. E-mail address: [email protected] (M.Z. Elsabee).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.01.051
solubility, higher reactivity and higher affinity towards solvents and swelling than a-chitin due to much weaker intermolecular hydrogen bonding ascribable to the parallel arrangement of the main chains (Pawadee et al., 2003; Gardner and Blakwell, 1975; Hunt and Elsherief, 1990; Chandumpaia et al., 2004). Many authors (Tolaimate et al., 2000, 2003; Pawadee et al., 2003; Gardner and Blakwell, 1975; Hunt and Elsherief, 1990; Chandumpaia et al., 2004; Acosta et al., 1993; Rege and Block, 1999; Galed et al., 2005; Paulino et al., 2006) have tackled the problem of extracting chitin from its natural sources followed by its deacetylation to obtain the much more useful material chitosan. Potential and usual applications of chitin, chitosan and their derivatives are estimated to be more than 200 (Sandford, 1989; Ravi Kumar, 2000). This wide range of applications include cosmetics, agriculture, food, biomedical, and textile, as chelating agents and refinement industrial effluents (Rathke and Hodson, 1994; Chassarya et al., 2005). The production of chitosan from crustacean shells obtained as a food waste is economically feasible, especially if it includes the recovery of carotenoids. The shells contain
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considerable amount of astaxanthin, a carotenoids that has so far not been synthesized, and which is marked as a fish food additive in aquaculture. Chitosan itself was directly extracted from fungi by alkaline and acid treatment (Rane and Hoover, 1993; Cai et al., 2006; Suntornsuk et al., 2002; Chatterjee et al., 2005). Some authors (Wang et al., 2006; Gagne and Simpson, 1993; Oh et al., 2000; Yang et al., 2000) have developed methods to use microorganisms or proteolytic enzymes for the deproteinization of the crustacean chitin wastes in this way a more economic production of chitin and chitosan can be achieved. The major procedure for obtaining chitosan is based on the alkaline deacetylation of chitin with strong alkaline solution. Isolation of chitin itself from different sources is affected by the source. Generally the raw material is crushed, washed with water or detergent and cut into small pieces. The mineral content of the exoskeleton of the different crustaceous is not the same and consequently different treatments may be used. The present work is the first systematic trial to investigate the extraction of chitin and chitosan from different indigenous sources in Egypt. 2. Experimental part 2.1. Isolation of chitin Chitin was isolated from six sources, two kinds of shrimp (brown and pink) shells, two kinds of squid pens, crabs shells and shells of fresh water lobster (crayfish), Procambarus clarkii which is a species of freshwater crayfish, native to the south-eastern United States, but found also on other continents, where it is often an invasive pest. It is known variously as the red swamp crawfish, red swamp crayfish, Louisiana crawfish or Louisiana crayfish. This crayfish is an intriguing species besides being a source for chitin it is also an environmental problem in Egypt, the crayfish has become an abundant resident in the River Nile because of its high reproduction rate and its strong adaptability. This crayfish is a voracious carnivore, preying upon various crustaceans, mollusks and small fish, as well as their eggs and fries causing thus a serious obstacle for aquaculture industry. Other evidence shows that they feed well upon some benthic vector snails, and so, it might be used as a potential biological Schistosome control agent if it is intentionally introduced into the irrigation and drainage canals which are largely infested with these snails. The raw materials were obtained in solid form from the different sources, washed with water, desiccated at room temperature and cut into small pieces. Demineralization was carried out at room temperature using 1 M hydrochloric acid bathes. The number of bathes and their duration were dependent upon the source; it was observed that the emission of CO2 gas was more or less an important indicator according to the studied species. It is for example strong in case of crabs and shrimp and crayfish and weak in case of one kind of squid. De-proteinization was performed
using alkaline treatments with 1 M sodium hydroxide solutions at 105–110 C. This treatment was repeated several times. The number of bathes depends on clarity of the solution; the absence of protein was indicated by the absence of color of the medium at the end of the last treatment. Washing with distilled water was then carried out up to neutrality after which the samples were dried. At this stage, chitin isolated from squid pens is perfectly white unlike those isolated from other sources which were highly pink. Pigment traces responsible for color are removed using a mild oxidizing treatment (KMnO4 + oxalic acid + H2SO4). Refluxing in ethanol for 6 h was also used to eliminate traces of protein and coloring materials. Chitin content was determined from weight difference of the raw material and that of the chitin obtained after acid and alkaline treatments.
2.2. Deacetylation of chitin Preliminary experiments were carried out by refluxing chitin in strong NaOH solution at normal atmosphere. The experiments took more than 20 h producing low deacetylation content and the reaction was accompanied by drastic degradation of the final chitosan. To avoid long heating times, the refluxing in alkaline solution was tried in an autoclave under two atmospheres pressure. The heating lasted for several hours (10–15 h) and still the resulting chitosan was partially soluble in acetic acid indicating low deacetylation extent. Kurita (2001) has indicated that deacetylation of chitin can be highly facilitated by steeping in strong sodium hydroxide solution at room temperature before heating. This approach was then adapted and the effect of steeping time on the feasibility of deacetylation was investigated.
3. Determination of the deacetylation percent 3.1. Potentiometric titration Chitosan (0.5 g) was dissolved in 25 ml of 0.1 M standard HCl aqueous solution. The solution was then toped up to 100 ml with distilled water and calculated amount of KCl was added to adjust the ionic strength to 0.1. The titrant was a solution of 0.05 M NaOH. pH meter was used for pH measurements under continuous stirring. The titrant was added until the pH value reached 2.00, the standard NaOH was then added stepwise and the pH values of solution were recorded and a curve with two inflection points was obtained. The difference of NaOH solution volumes between these points corresponds to the acid consumed for salification of the amine groups of chitosan and allows the determination of DDA% of the chitosan. The DA was calculated from the relation (Broussignac, 1968):
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DDA% ¼
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1 161Q 1 þ 42Q
where Q = NDV/m, DV is the volume of NaOH solution between the two inflection points (in l), N is the concentration of NaOH (in mol/l in this paper 0.05 mol/l) and m is the dry weight of chitosan (in g). 3.2. Elemental analysis The DDA% value of chitosan samples was calculated from the following formula (Kasaai et al., 2000; Jiang et al., 2003): DDA% ¼
6:857 C=N 1:7143
where C/N is the carbon/nitrogen ratio measured from the elemental composition of the chitosan samples. 3.3. Fourier transform infrared FTIR spectroscopy The FTIR spectra were measured in KBr pellets in the transmission mode in the range 400–4000 cm1 using Perkin–Elmer 2000 spectrophotometer. The DDA of the samples were calculated from the IR spectra following the method in Ref. (Brugnerottoa et al., 2001). The bands at 1650 and 1320 cm1 were chosen to measure the DA. As internal reference, the intensities at 3450 and 1420 cm1 were evaluated. The absorption band ratios involving the reference at 3450 cm1 had poorer fit. The absorption ratio A1320/A1420 shows superior agreement between the absolute and estimated DA-values according to Brugnerottoa et al. (Brugnerottoa et al., 2001) and therefore was adapted in this work: ½DA% ¼ 31 : 92 ðA1320 =A1420 Þ 12 : 20;
r ¼ 0 : 990
3.4. NMR method Hirari et al. (Hirari et al., 1991) reported the chemical shift of methyl protons in acetamide group to appear at 2.07 ppm in acidic deuterium oxide solutions while the methyl group of glacial acetic acid appears at 2.12 ppm (The Aldrich Library of NMR Spectra). Therefore, the peak around 2.1 ppm in the spectra (Fig. 1) can be safely assigned to the methyl protons in the acetamide group, and that around 2.2 ppm is due to the methyl group in acetic acid moiety. The chemical shift is affected by solvent, concentration and temperature, and taking these into account, one can explain the difference between the chemical shift value reported by Lavertu (lavertu et al., 2003) and those reported here. In the obtained spectra, the difference in chemical shifts between peaks at about 2.1 and 2.2 ppm is constant (about 0.15 ppm) within experimental errors. The setting of TMS peak to be 0 ppm is sometimes slightly affected by operational and spectral variations, and for peaks with many
Fig. 1. 1HNMR spectrum of chitosan derived from pink shrimp (0.5 h heating in autoclave and 1 day steeping in 40% NaOH).
‘‘spikes’’ as in our case, the reproducibility might be lowered. Therefore, it can be safely stated that the chemical shift for amide methyl protons can be set to 2.06 ppm and that for acetic acid moiety to 2.21 ppm. It is described (Hirari and Lavertu) that the peak at 4.9 ppm is assigned to C1 proton of glucosamine unit in chitosan and the peaks in 3–4 ppm to C2–C6 protons of glucosamine and N-acetylglucosamine units. They also showed that C1 proton of N-acetylglucosamine unit appears around 4.6 ppm, and this peak is overlapped with the strong solvent peak in our spectra and can be seen as a shoulder in the representative example shown in Fig. 1 for pink shrimp chitosan obtained after 0.5 ppm heating in the autoclave the chitin was steeped for 1 day in 40% NaOH solution. For the determination of the degree of deacetylation (DDA) the method of Lavertu was adapted in which H4:9 DDA ð%Þ ¼ 100 H4:9 þ H2:06=3 Based on these, one can calculate DDA unequivocally from the obtained NMR spectra. NMR and potentiometric titration were found to be the most reliable methods and the values reported here are usually the average of these methods. 3.5. Viscosity measurements The viscosities of all prepared chitosan samples (except the insoluble samples) were measured using an Ostwald capillary viscometer and the intrinsic viscosities were determined, the solvent was 5% acetic acid and 0.1 M KCl the obtained intrinsic viscosity was used to calculate molecular
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weight for the prepared samples from the Mark–Houwink– Sakurada relation: ½g ¼ KM a where K and a are constants which were determined in the literature by many authors. K = 1.38 · 104 and a = 0.85 (Gamzazade et al., 1985), and K = 2.14 · 103 and a = 0.657 (Pankaj et al., 1999) another values were K = 1.4 · 104 and a = 0.83 (Wan et al., 2003). Another formula for squid chitosan has been adapted (Chandumpaia et al., 2004) with K = 8.93 · 104 a = 0.71. Due to the variation of the K and a constants it seemed reasonable to use the intrinsic viscosity data [g] directly since converting it to molecular weight values will yield the same behavior pattern in addition to the variation in the values of the constants. 3.6. X-ray diffraction X-ray diffraction analysis (XRD) was applied to detect the crystallinity of the extracted samples of chitin and their corresponding chitosan. A Scintag powder diffractometer was used for this purpose between 2h angles of 5 and 40. Ni-filtered Cu Ka-radiation was used as the X-ray source. The relative crystallinity of the polymers was calculated by dividing the area of the crystalline peaks by the total area under the curve. 3.7. NMR Nuclear magnetic resonance NMR spectra were measured using JNMAl 300, Jeol spectrometer at 300 MHz in D2O in which drops of DCl was added to enhance solubility. 3.8. Thermal analysis Thermo-gravimetric analysis TGA was carried out using Shimadzu TGA-50H instrument at a heating rate of 10 C/ min under nitrogen atmosphere.
As seen from Table 1 two types of squid pens were used, one which was opaque white in color (CT) and was found to consist almost of CaCO3, while the other pens were transparent and much smaller in size with very small amount of carbonates and more than double the chitin content of any other source (SQ). The chitin content in Crayfish (CF) ranging about 20–21% warrants its use as an economic way of producing chitin on an industrial scale due to the availability and the low price of the source. The chitin extracted from squid pen was found to be much more amenable to deacetylation than that from shrimp, crab and crayfish. That is why the effect of steeping on deacetylation was studied only for the four a-chitin i.e. that from shrimp, crabs and crayfish. 4.1. Effect of steeping time on DDA% of chitosan The extracted chitin was heated into an autoclave after being steeped in sodium hydroxide solution of various concentrations at a solid: liquor ration of 1:20. The effect of steeping time in 40% NaOH solution and heating in autoclave (at 2 atmosphere pressure) is illustrated in Fig. 2. From Fig. 2, it can be seen that after 1 day steeping in the NaOH solution, the deacetylation takes only 1 h to yield high DDA reaching almost 92% for the crayfish chitin while 4 days steeping led to further increase in the DDA up to 92–97%. Therefore, the use of the autoclave leads to dramatic reduction in the time of deacetylation and conservation of energy. 4.2. Effect of NaOH concentration on the DDA% of chitosan To study the effect of alkaline concentration of the steeping solution on the DDA%, different concentrations of sodium hydroxide solutions (10%, 20%, 30% and 40% (w/w) were used for steeping of chitin for 24 h) the reaction time in the autoclave was kept at 0.5 h. It was found that the DDA% increases as the concentration of NaOH increases and Fig. 3 shows the DDA% as a function of 98
4. Results and discussion
96
Table 1 contains data of the percent of chitin present in the exoskeleton of different local sources.
Source Brown shrimp (penaeus aztecus) shells (BS) Pink shrimp (penaeus durarum) shells (PS) Cuttlefish pens (CT) Squid pens (SQ) Crabs shells (CR) Crayfish shells (CF)
% Of CaCO3
% Of protein
% Of chitin
48.97
29.50
21.53
42.26
34.02
23.72
88.48 4.74 66.58 63.94
6.12 46.23 16.68 15.46
5.40 49.00 16.73 20.60
94
DDA%
Table 1 Percent of chitin in different local sources
BS RS CF CR
92 90 88
The values depicted above are the average value of 3–5 experiments.
86 1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Steeping time, days Fig. 2. Effect of steeping time in 40% NaOH upon the DDA% after heating in an autoclave for 1 h.
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1363
96
100 BS PS CF CR
80
94 92
DDA%
DDA%
90
60
40
88 86 84 BS PS CF CR
82
20
80
0 10%
78
20%
30%
0.0
40%
0.5
1.0
1.5
2.0 Time, h
NaOH wt %
the concentration of the steeping bath before heating in the autoclave. It seems that below 40% sodium hydroxide concentration the steeping has little effect. While Fig. 4 shows how reactive the b-chitin towards deacetylation since even 30% NaOH can achieve up to 90% DDA even without pretreatment in NaOH. 4.3. Effect of the heating time in autoclave on DDA% of chitosan
3.0
100 98 96 94 92 90 88
The effect of the reaction time on the DDA% of chitin from different sources in the autoclave using 40% NaOH and a steeping time of 1 day is shown in Fig. 5. It seems that the crayfish chitin is relatively more reactive than the other forms of chitin. The chitin extracted from squid pen required no steeping and it readily undergoes deacetylation in the autoclave to high values of DDA as shown in Fig. 6. Even after 15 min, the degree of deacetylation reaches 87–89%. It has been established that chitin and chitosan have many biological and biomedical applications which are 120 100
DDA%
80 60 40 20
CT SQ
0 10%
20%
30%
40%
NaOH wt% Fig. 4. Effect of NaOH concentration on the extent of hydrolysis of the b-chitin from the two types of squid pens.
3.5
Fig. 5. Effect of the heating time in autoclave on DDA% of the different sources using 40% NaOH and steeping time 1 day.
DDA%
Fig. 3. Effect of NaOH concentration of the steeping bath on the DDA% after heating for 1 h in the autoclave.
2.5
CT SQ
86 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Time, h Fig. 6. Effect of reaction time in the autoclave on the DDA% for the b-chitin form squid pens, using 40% NaOH.
highly dependent on both the DDA% and the molecular weight of the polymer. Therefore, after investigating the effect of the different parameters on the DDA%, it is essential to see how the different procedures affect the molecular weight of the final chitosan polymer. The molecular weight was determined by measuring the intrinsic viscosity in 5% acetic acid solution where the chitosan was freely soluble. Figs. 7 and 8 illustrate how the intrinsic viscosity of the obtained chitosan is affected by the reaction time in the autoclave and the time of steeping in 40% NaOH solution prior to heating. As a representative example the equation of (Wan et al., 2003) K = 1.4 · 104 and a = 0.83 has been used to calculate the molecular weight since it is the most recent one; the chitosan of CF after 0.5 h in the autoclave has a molecular weight of 8.13 · 105 was reduced to 4.60 · 105 after heating for 2.5 h. Heating time has more pronounced effect on the viscosity than the steeping time which has almost an erratic effect as shown in Fig. 9 while the reaction time is always followed by gradual decrease in viscosity. The same trend was found for the case of b-chitin from squid pen, a gradual decrease of viscosity with
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8
heating time in the autoclave has been observed in 40% NaOH solution the molecular weight (K = 8.93 · 104 a = 0.71 (Chandumpaia et al., 2004)) was reduced from 6.4 · 105 to 3.9 · 105 after heating for 0.25 h and 1.25 h, respectively, for SQ sample.
6
4.4. FTIR
4
A representative example of chitin and its chitosan is given in Fig. 10. The DDA was calculated as was explained in the experimental part using the ratio of the bands at 1320 and 1420 cm1.
12 BS PS CF CR
[η] dl/g
10
2
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.5. X-ray diffraction
Reaction Time, h Fig. 7. Effect of the reaction time in the autoclave on the intrinsic viscosity [g] values of the chitosan obtained from the a-chitin, using 40% NaOH and steeping for 1 day.
X-ray diffraction (XRD) analysis was applied to detect the crystallinity of the isolated chitin and the obtained chitosan. Relative mass crystallinity of the different chitin and chitosan are listed in Table 2. The structure of a-chitin
8 BS PS CR CF
7
[η] dl/g
6 5 4 3 2 1 day
2 days
3 days
4 days
Steeping Time, days Fig. 8. Effect of the steeping time on the intrinsic viscosity of chitosan after heating for 1 h in the auto clave in 40% NaOH solution.
14 CT SQ
12
[η] dl/g
10 8
Fig. 10. FTIR spectra of CF upper curve chitosan lower curve chitin.
6 4
Table 2 Crystallinity of chitin and chitosan (X-ray analysis)
2 0 0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time, h Fig. 9. Effect of the reaction time in the autoclave on the intrinsic viscosity [g] values of the chitosan obtained from b-chitin using 40% NaOH and no steeping.
BS chitin chitosan
PS chitin CT chitin chitosan chitosan
SQ chitin chitosan
CR chitin chitosan
CF chitin chitosan
Crystallinity (%) 64 66 66.6 48.9 70.82 43.15 59.22 41.78 59.86 40.99 56.94 36.43 Crystallinity percent was calculated by dividing the area of the crystalline peaks by the total area under the curve.
E.S. Abdou et al. / Bioresource Technology 99 (2008) 1359–1367
has been determined by Minke and Blackwell (Minke and Blackwell, 1978) using X-ray diffraction analysis, based on the intensity data from deproteinized lobster tendon. Least-squares refinement shows that adjacent chains have alternating sense (i.e. are antiparallel). In addition, there
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is a statistical distribution of side-chain orientations, such that all the hydroxyl groups form hydrogen bonds. The unit cell is orthorhombic. The chains form hydrogenbonded sheets linked by C@OÆÆÆH–N bonds are approximately parallel to the a-axis, and each chain has an
Fig. 11. X-ray diffraction patterns of (a) a-chitin from (1) BS (2) PS (3) CR (4) Cray Fish (b) the corresponding chitosan.
Fig. 12. X-ray diffraction patterns of (a) b-chitin from (1) CT (2) SQ (b) the corresponding chitosan.
E.S. Abdou et al. / Bioresource Technology 99 (2008) 1359–1367
O-3 0 –HÆÆÆO.5 intra-molecular hydrogen bond, similar to that in cellulose. The results indicate also that a statistical mixture of CH2OH orientations is present, equivalent to half oxygen on each residue, each forming inter- and intra-molecular hydrogen bonds. As a result, the structure contains two types of amide groups, which differ in their hydrogen bonding, and account for the splitting of the amide I band in the infrared spectrum. The Inability of this chitin polymorph to swell on soaking in water is explained by the extensive intermolecular hydrogen bonding. In Fig. 11, the X-ray diffraction patterns of the obtained a-chitin (A) and the corresponding hydrolyzed chitosan (B) are given. All chitin samples show strong reflections at 2h around 9–10 and 2h of 20–21 and minor reflections at higher 2h values e.g. at 26.4 and higher. The band at ˚ and is due to 9.9 corresponds to a d spacing of 8.92 A the incorporation of bound water molecules into the crystal lattice (Ogawa et al., 1992). The reflection at 2h 19.4–20 ˚ (Harish Prashcorresponds to a d spacing of about 4.41 A anth et al., 2002). Generally, the sharpness of the bands are higher in the chitin samples than in their chitosan analogue with slight decrease in the crystallinity percent. Fig. 12 illustrates the chitin and chitosan obtained from squid pens (b-chitin) the band at 2h 9.9 decreases after deacetylation followed by dramatic decrease in crystallinity percent as seen in Table 2. 4.6. Thermal analysis TGA curves of chitin and chitosan are shown in Fig. 13 as representative examples. Two endothermic peaks are observed. The first peak appears around 90–99 C corresponds to loss of water. The second one emerges at about 303 C in chitosan and at about 372 C in the corresponding chitin as seen in the DTA curves in Fig. 14. All the extracted samples showed similar trend; the chitin has higher thermal stability than the corresponding chitosan.
120 chitosan chitin CF chitin PS Chitin Sq
Weight loss%
100 80 60 40 20 0 0
100
200
300
400
500
600
Temperature, ºC Fig. 13. TGA curves for chitosan and chitin.
700
0.000 -0.002
DTGA
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-0.004 -0.006 -0.008
CF, chitosan CF Chitin
-0.010 0
100
200
300 400 Temperature, ºC
500
600
Fig. 14. DTA curves for chitosan and chitin from CF.
5. Conclusion Chitin has been extracted from different sources indigenous for Egypt. It was found that Crayfish which constitute a pollution hazard in Egypt could be an economic source of chitin. The obtained chitin was hydrolyzed using an autoclave after being steeped in strong sodium hydroxide solution for different period of time which reduced the deacetylation time dramatically to produce chitosan with reasonably high molecular weight, and high deacetylation percent. It has also been shown that b-chitin is much more amenable to deacetylation than the a form. The chitin and chitosan have been characterized as to their degree of deacetylation and their relative degree of crystallinity was measured by X-ray diffraction method. The thermal behavior of the obtained polymers revealed that they both decompose by two step patterns and that chitin is more thermally stable than chitosan. References Acosta, A., Junenez, C., Borau, V., Heras, A., 1993. Extraction and characterization of chitin from crustaceans. Biomass and Bioenergy 5 (2), 145–153. Paulino, Alexandre T., Simionato, Julliana I., Garcia, Juliana C., Nozak, Jorge, 2006. Characterization of chitosan and chitin produced from silkworm crysalides. Carbohydrate Polymers 64, 98–103. Austin, P.E., Castle, J.E., Albisetti, C.J., 1989. In: Skjak-Braek, G., Anthonsen, T., Sandford, P. (Eds.), Chitin and Chitosan. Elsevier, Essex, p. 749. Broussignac, P., 1968. Chemi-Genrie Chim 99, 1241–1247. ` elles-Monalc, W., Brugnerottoa, J., Lizardib, J., Goycooleab, F.M., ArguE ´ resa, J., Rinaudo, M., 2001. An infrared investigation in DesbrieA relation with chitin and chitosan characterization. Polymer 42, 3569– 3580. Cai, J., Yang, J., Du, Y., Fan, L., Qui, Y., Li, J., Kennedy, J.F., 2006. Enzymatic preparation of chitosan from the waste Aspergillu niger mycelium of citric acid production plant. Carbohydrate Polymers 64, 151–157. Chandumpaia, A., Singhpibulpornb, N., Faroongsarngc, D., Sornprasit, P., 2004. Preparation and physico-chemical characterization of chitin and chitosan from the pens of the squid species, Loligo lessoniana and Loligo formosana. Carbohydrate Polymers 58, 467–474.
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Bioresource Technology 99 (2008) 1359–1367
Extraction and characterization of chitin and chitosan from local sources Entsar S. Abdou a, Khaled S.A. Nagy a, Maher Z. Elsabee a
b,*
Food Technology Research Institute, Agriculture Research Center, Cairo 12631, Egypt Department of Chemistry, Faculty of Science, Cairo University, Cairo 12631, Egypt
b
Received 20 November 2006; received in revised form 27 January 2007; accepted 30 January 2007 Available online 26 March 2007
Abstract Chitin has been extracted from six different local sources in Egypt. The obtained chitin was converted into the more useful soluble chitosan by steeping into solutions of NaOH of various concentrations and for extended periods of time, then the alkali chitin was heated in an auto clave which dramatically reduced the time of deacetylation. Chitin from squid pens did not require steeping in sodium hydroxide solution and showed much higher reactivity towards deacetylation in the autoclave that even after 15 min of heating a degree of deacetylation of 90% was achieved. The obtained chitin and chitosan were characterized by spectral analysis, X-ray diffraction and thermo gravimetric analysis. 2007 Elsevier Ltd. All rights reserved. Keywords: Chitin extraction; Local sources; Deacetylation conditions; Characterization
1. Introduction Chitin, a naturally abundant polymer consists of 2-acetamido 2-deoxy-b-D-glucose through a b(1 ! 4) linkage. In spite of the presence of nitrogen, it may be regarded as cellulose with hydroxyl at position C-2 replaced by an acetamido group. Like cellulose, it functions as structural polysaccharides. Its natural production is inexhaustible; arthropods, by themselves, count more than 106 species from the 1.2 · 106 of total species compiled for animal kingdom, constitute permanent and large biomass source. Chitin is a white, hard, inelastic, nitrogenous polysaccharide and the major source of surface pollution in coastal areas. Chitin is usually isolated from the exoskeletons of crustaceans and more particularly from shrimps and crabs where a-chitin is produced (Minke and Blackwell, 1978; Austin et al., 1989). Squid is another important source of chitin in which it exists in the b form which was found to be more amenable for deacetylation. It also shows higher *
Corresponding author. Tel.: +20 26352316. E-mail address: [email protected] (M.Z. Elsabee).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.01.051
solubility, higher reactivity and higher affinity towards solvents and swelling than a-chitin due to much weaker intermolecular hydrogen bonding ascribable to the parallel arrangement of the main chains (Pawadee et al., 2003; Gardner and Blakwell, 1975; Hunt and Elsherief, 1990; Chandumpaia et al., 2004). Many authors (Tolaimate et al., 2000, 2003; Pawadee et al., 2003; Gardner and Blakwell, 1975; Hunt and Elsherief, 1990; Chandumpaia et al., 2004; Acosta et al., 1993; Rege and Block, 1999; Galed et al., 2005; Paulino et al., 2006) have tackled the problem of extracting chitin from its natural sources followed by its deacetylation to obtain the much more useful material chitosan. Potential and usual applications of chitin, chitosan and their derivatives are estimated to be more than 200 (Sandford, 1989; Ravi Kumar, 2000). This wide range of applications include cosmetics, agriculture, food, biomedical, and textile, as chelating agents and refinement industrial effluents (Rathke and Hodson, 1994; Chassarya et al., 2005). The production of chitosan from crustacean shells obtained as a food waste is economically feasible, especially if it includes the recovery of carotenoids. The shells contain
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considerable amount of astaxanthin, a carotenoids that has so far not been synthesized, and which is marked as a fish food additive in aquaculture. Chitosan itself was directly extracted from fungi by alkaline and acid treatment (Rane and Hoover, 1993; Cai et al., 2006; Suntornsuk et al., 2002; Chatterjee et al., 2005). Some authors (Wang et al., 2006; Gagne and Simpson, 1993; Oh et al., 2000; Yang et al., 2000) have developed methods to use microorganisms or proteolytic enzymes for the deproteinization of the crustacean chitin wastes in this way a more economic production of chitin and chitosan can be achieved. The major procedure for obtaining chitosan is based on the alkaline deacetylation of chitin with strong alkaline solution. Isolation of chitin itself from different sources is affected by the source. Generally the raw material is crushed, washed with water or detergent and cut into small pieces. The mineral content of the exoskeleton of the different crustaceous is not the same and consequently different treatments may be used. The present work is the first systematic trial to investigate the extraction of chitin and chitosan from different indigenous sources in Egypt. 2. Experimental part 2.1. Isolation of chitin Chitin was isolated from six sources, two kinds of shrimp (brown and pink) shells, two kinds of squid pens, crabs shells and shells of fresh water lobster (crayfish), Procambarus clarkii which is a species of freshwater crayfish, native to the south-eastern United States, but found also on other continents, where it is often an invasive pest. It is known variously as the red swamp crawfish, red swamp crayfish, Louisiana crawfish or Louisiana crayfish. This crayfish is an intriguing species besides being a source for chitin it is also an environmental problem in Egypt, the crayfish has become an abundant resident in the River Nile because of its high reproduction rate and its strong adaptability. This crayfish is a voracious carnivore, preying upon various crustaceans, mollusks and small fish, as well as their eggs and fries causing thus a serious obstacle for aquaculture industry. Other evidence shows that they feed well upon some benthic vector snails, and so, it might be used as a potential biological Schistosome control agent if it is intentionally introduced into the irrigation and drainage canals which are largely infested with these snails. The raw materials were obtained in solid form from the different sources, washed with water, desiccated at room temperature and cut into small pieces. Demineralization was carried out at room temperature using 1 M hydrochloric acid bathes. The number of bathes and their duration were dependent upon the source; it was observed that the emission of CO2 gas was more or less an important indicator according to the studied species. It is for example strong in case of crabs and shrimp and crayfish and weak in case of one kind of squid. De-proteinization was performed
using alkaline treatments with 1 M sodium hydroxide solutions at 105–110 C. This treatment was repeated several times. The number of bathes depends on clarity of the solution; the absence of protein was indicated by the absence of color of the medium at the end of the last treatment. Washing with distilled water was then carried out up to neutrality after which the samples were dried. At this stage, chitin isolated from squid pens is perfectly white unlike those isolated from other sources which were highly pink. Pigment traces responsible for color are removed using a mild oxidizing treatment (KMnO4 + oxalic acid + H2SO4). Refluxing in ethanol for 6 h was also used to eliminate traces of protein and coloring materials. Chitin content was determined from weight difference of the raw material and that of the chitin obtained after acid and alkaline treatments.
2.2. Deacetylation of chitin Preliminary experiments were carried out by refluxing chitin in strong NaOH solution at normal atmosphere. The experiments took more than 20 h producing low deacetylation content and the reaction was accompanied by drastic degradation of the final chitosan. To avoid long heating times, the refluxing in alkaline solution was tried in an autoclave under two atmospheres pressure. The heating lasted for several hours (10–15 h) and still the resulting chitosan was partially soluble in acetic acid indicating low deacetylation extent. Kurita (2001) has indicated that deacetylation of chitin can be highly facilitated by steeping in strong sodium hydroxide solution at room temperature before heating. This approach was then adapted and the effect of steeping time on the feasibility of deacetylation was investigated.
3. Determination of the deacetylation percent 3.1. Potentiometric titration Chitosan (0.5 g) was dissolved in 25 ml of 0.1 M standard HCl aqueous solution. The solution was then toped up to 100 ml with distilled water and calculated amount of KCl was added to adjust the ionic strength to 0.1. The titrant was a solution of 0.05 M NaOH. pH meter was used for pH measurements under continuous stirring. The titrant was added until the pH value reached 2.00, the standard NaOH was then added stepwise and the pH values of solution were recorded and a curve with two inflection points was obtained. The difference of NaOH solution volumes between these points corresponds to the acid consumed for salification of the amine groups of chitosan and allows the determination of DDA% of the chitosan. The DA was calculated from the relation (Broussignac, 1968):
E.S. Abdou et al. / Bioresource Technology 99 (2008) 1359–1367
DDA% ¼
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1 161Q 1 þ 42Q
where Q = NDV/m, DV is the volume of NaOH solution between the two inflection points (in l), N is the concentration of NaOH (in mol/l in this paper 0.05 mol/l) and m is the dry weight of chitosan (in g). 3.2. Elemental analysis The DDA% value of chitosan samples was calculated from the following formula (Kasaai et al., 2000; Jiang et al., 2003): DDA% ¼
6:857 C=N 1:7143
where C/N is the carbon/nitrogen ratio measured from the elemental composition of the chitosan samples. 3.3. Fourier transform infrared FTIR spectroscopy The FTIR spectra were measured in KBr pellets in the transmission mode in the range 400–4000 cm1 using Perkin–Elmer 2000 spectrophotometer. The DDA of the samples were calculated from the IR spectra following the method in Ref. (Brugnerottoa et al., 2001). The bands at 1650 and 1320 cm1 were chosen to measure the DA. As internal reference, the intensities at 3450 and 1420 cm1 were evaluated. The absorption band ratios involving the reference at 3450 cm1 had poorer fit. The absorption ratio A1320/A1420 shows superior agreement between the absolute and estimated DA-values according to Brugnerottoa et al. (Brugnerottoa et al., 2001) and therefore was adapted in this work: ½DA% ¼ 31 : 92 ðA1320 =A1420 Þ 12 : 20;
r ¼ 0 : 990
3.4. NMR method Hirari et al. (Hirari et al., 1991) reported the chemical shift of methyl protons in acetamide group to appear at 2.07 ppm in acidic deuterium oxide solutions while the methyl group of glacial acetic acid appears at 2.12 ppm (The Aldrich Library of NMR Spectra). Therefore, the peak around 2.1 ppm in the spectra (Fig. 1) can be safely assigned to the methyl protons in the acetamide group, and that around 2.2 ppm is due to the methyl group in acetic acid moiety. The chemical shift is affected by solvent, concentration and temperature, and taking these into account, one can explain the difference between the chemical shift value reported by Lavertu (lavertu et al., 2003) and those reported here. In the obtained spectra, the difference in chemical shifts between peaks at about 2.1 and 2.2 ppm is constant (about 0.15 ppm) within experimental errors. The setting of TMS peak to be 0 ppm is sometimes slightly affected by operational and spectral variations, and for peaks with many
Fig. 1. 1HNMR spectrum of chitosan derived from pink shrimp (0.5 h heating in autoclave and 1 day steeping in 40% NaOH).
‘‘spikes’’ as in our case, the reproducibility might be lowered. Therefore, it can be safely stated that the chemical shift for amide methyl protons can be set to 2.06 ppm and that for acetic acid moiety to 2.21 ppm. It is described (Hirari and Lavertu) that the peak at 4.9 ppm is assigned to C1 proton of glucosamine unit in chitosan and the peaks in 3–4 ppm to C2–C6 protons of glucosamine and N-acetylglucosamine units. They also showed that C1 proton of N-acetylglucosamine unit appears around 4.6 ppm, and this peak is overlapped with the strong solvent peak in our spectra and can be seen as a shoulder in the representative example shown in Fig. 1 for pink shrimp chitosan obtained after 0.5 ppm heating in the autoclave the chitin was steeped for 1 day in 40% NaOH solution. For the determination of the degree of deacetylation (DDA) the method of Lavertu was adapted in which H4:9 DDA ð%Þ ¼ 100 H4:9 þ H2:06=3 Based on these, one can calculate DDA unequivocally from the obtained NMR spectra. NMR and potentiometric titration were found to be the most reliable methods and the values reported here are usually the average of these methods. 3.5. Viscosity measurements The viscosities of all prepared chitosan samples (except the insoluble samples) were measured using an Ostwald capillary viscometer and the intrinsic viscosities were determined, the solvent was 5% acetic acid and 0.1 M KCl the obtained intrinsic viscosity was used to calculate molecular
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weight for the prepared samples from the Mark–Houwink– Sakurada relation: ½g ¼ KM a where K and a are constants which were determined in the literature by many authors. K = 1.38 · 104 and a = 0.85 (Gamzazade et al., 1985), and K = 2.14 · 103 and a = 0.657 (Pankaj et al., 1999) another values were K = 1.4 · 104 and a = 0.83 (Wan et al., 2003). Another formula for squid chitosan has been adapted (Chandumpaia et al., 2004) with K = 8.93 · 104 a = 0.71. Due to the variation of the K and a constants it seemed reasonable to use the intrinsic viscosity data [g] directly since converting it to molecular weight values will yield the same behavior pattern in addition to the variation in the values of the constants. 3.6. X-ray diffraction X-ray diffraction analysis (XRD) was applied to detect the crystallinity of the extracted samples of chitin and their corresponding chitosan. A Scintag powder diffractometer was used for this purpose between 2h angles of 5 and 40. Ni-filtered Cu Ka-radiation was used as the X-ray source. The relative crystallinity of the polymers was calculated by dividing the area of the crystalline peaks by the total area under the curve. 3.7. NMR Nuclear magnetic resonance NMR spectra were measured using JNMAl 300, Jeol spectrometer at 300 MHz in D2O in which drops of DCl was added to enhance solubility. 3.8. Thermal analysis Thermo-gravimetric analysis TGA was carried out using Shimadzu TGA-50H instrument at a heating rate of 10 C/ min under nitrogen atmosphere.
As seen from Table 1 two types of squid pens were used, one which was opaque white in color (CT) and was found to consist almost of CaCO3, while the other pens were transparent and much smaller in size with very small amount of carbonates and more than double the chitin content of any other source (SQ). The chitin content in Crayfish (CF) ranging about 20–21% warrants its use as an economic way of producing chitin on an industrial scale due to the availability and the low price of the source. The chitin extracted from squid pen was found to be much more amenable to deacetylation than that from shrimp, crab and crayfish. That is why the effect of steeping on deacetylation was studied only for the four a-chitin i.e. that from shrimp, crabs and crayfish. 4.1. Effect of steeping time on DDA% of chitosan The extracted chitin was heated into an autoclave after being steeped in sodium hydroxide solution of various concentrations at a solid: liquor ration of 1:20. The effect of steeping time in 40% NaOH solution and heating in autoclave (at 2 atmosphere pressure) is illustrated in Fig. 2. From Fig. 2, it can be seen that after 1 day steeping in the NaOH solution, the deacetylation takes only 1 h to yield high DDA reaching almost 92% for the crayfish chitin while 4 days steeping led to further increase in the DDA up to 92–97%. Therefore, the use of the autoclave leads to dramatic reduction in the time of deacetylation and conservation of energy. 4.2. Effect of NaOH concentration on the DDA% of chitosan To study the effect of alkaline concentration of the steeping solution on the DDA%, different concentrations of sodium hydroxide solutions (10%, 20%, 30% and 40% (w/w) were used for steeping of chitin for 24 h) the reaction time in the autoclave was kept at 0.5 h. It was found that the DDA% increases as the concentration of NaOH increases and Fig. 3 shows the DDA% as a function of 98
4. Results and discussion
96
Table 1 contains data of the percent of chitin present in the exoskeleton of different local sources.
Source Brown shrimp (penaeus aztecus) shells (BS) Pink shrimp (penaeus durarum) shells (PS) Cuttlefish pens (CT) Squid pens (SQ) Crabs shells (CR) Crayfish shells (CF)
% Of CaCO3
% Of protein
% Of chitin
48.97
29.50
21.53
42.26
34.02
23.72
88.48 4.74 66.58 63.94
6.12 46.23 16.68 15.46
5.40 49.00 16.73 20.60
94
DDA%
Table 1 Percent of chitin in different local sources
BS RS CF CR
92 90 88
The values depicted above are the average value of 3–5 experiments.
86 1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Steeping time, days Fig. 2. Effect of steeping time in 40% NaOH upon the DDA% after heating in an autoclave for 1 h.
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1363
96
100 BS PS CF CR
80
94 92
DDA%
DDA%
90
60
40
88 86 84 BS PS CF CR
82
20
80
0 10%
78
20%
30%
0.0
40%
0.5
1.0
1.5
2.0 Time, h
NaOH wt %
the concentration of the steeping bath before heating in the autoclave. It seems that below 40% sodium hydroxide concentration the steeping has little effect. While Fig. 4 shows how reactive the b-chitin towards deacetylation since even 30% NaOH can achieve up to 90% DDA even without pretreatment in NaOH. 4.3. Effect of the heating time in autoclave on DDA% of chitosan
3.0
100 98 96 94 92 90 88
The effect of the reaction time on the DDA% of chitin from different sources in the autoclave using 40% NaOH and a steeping time of 1 day is shown in Fig. 5. It seems that the crayfish chitin is relatively more reactive than the other forms of chitin. The chitin extracted from squid pen required no steeping and it readily undergoes deacetylation in the autoclave to high values of DDA as shown in Fig. 6. Even after 15 min, the degree of deacetylation reaches 87–89%. It has been established that chitin and chitosan have many biological and biomedical applications which are 120 100
DDA%
80 60 40 20
CT SQ
0 10%
20%
30%
40%
NaOH wt% Fig. 4. Effect of NaOH concentration on the extent of hydrolysis of the b-chitin from the two types of squid pens.
3.5
Fig. 5. Effect of the heating time in autoclave on DDA% of the different sources using 40% NaOH and steeping time 1 day.
DDA%
Fig. 3. Effect of NaOH concentration of the steeping bath on the DDA% after heating for 1 h in the autoclave.
2.5
CT SQ
86 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Time, h Fig. 6. Effect of reaction time in the autoclave on the DDA% for the b-chitin form squid pens, using 40% NaOH.
highly dependent on both the DDA% and the molecular weight of the polymer. Therefore, after investigating the effect of the different parameters on the DDA%, it is essential to see how the different procedures affect the molecular weight of the final chitosan polymer. The molecular weight was determined by measuring the intrinsic viscosity in 5% acetic acid solution where the chitosan was freely soluble. Figs. 7 and 8 illustrate how the intrinsic viscosity of the obtained chitosan is affected by the reaction time in the autoclave and the time of steeping in 40% NaOH solution prior to heating. As a representative example the equation of (Wan et al., 2003) K = 1.4 · 104 and a = 0.83 has been used to calculate the molecular weight since it is the most recent one; the chitosan of CF after 0.5 h in the autoclave has a molecular weight of 8.13 · 105 was reduced to 4.60 · 105 after heating for 2.5 h. Heating time has more pronounced effect on the viscosity than the steeping time which has almost an erratic effect as shown in Fig. 9 while the reaction time is always followed by gradual decrease in viscosity. The same trend was found for the case of b-chitin from squid pen, a gradual decrease of viscosity with
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8
heating time in the autoclave has been observed in 40% NaOH solution the molecular weight (K = 8.93 · 104 a = 0.71 (Chandumpaia et al., 2004)) was reduced from 6.4 · 105 to 3.9 · 105 after heating for 0.25 h and 1.25 h, respectively, for SQ sample.
6
4.4. FTIR
4
A representative example of chitin and its chitosan is given in Fig. 10. The DDA was calculated as was explained in the experimental part using the ratio of the bands at 1320 and 1420 cm1.
12 BS PS CF CR
[η] dl/g
10
2
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.5. X-ray diffraction
Reaction Time, h Fig. 7. Effect of the reaction time in the autoclave on the intrinsic viscosity [g] values of the chitosan obtained from the a-chitin, using 40% NaOH and steeping for 1 day.
X-ray diffraction (XRD) analysis was applied to detect the crystallinity of the isolated chitin and the obtained chitosan. Relative mass crystallinity of the different chitin and chitosan are listed in Table 2. The structure of a-chitin
8 BS PS CR CF
7
[η] dl/g
6 5 4 3 2 1 day
2 days
3 days
4 days
Steeping Time, days Fig. 8. Effect of the steeping time on the intrinsic viscosity of chitosan after heating for 1 h in the auto clave in 40% NaOH solution.
14 CT SQ
12
[η] dl/g
10 8
Fig. 10. FTIR spectra of CF upper curve chitosan lower curve chitin.
6 4
Table 2 Crystallinity of chitin and chitosan (X-ray analysis)
2 0 0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time, h Fig. 9. Effect of the reaction time in the autoclave on the intrinsic viscosity [g] values of the chitosan obtained from b-chitin using 40% NaOH and no steeping.
BS chitin chitosan
PS chitin CT chitin chitosan chitosan
SQ chitin chitosan
CR chitin chitosan
CF chitin chitosan
Crystallinity (%) 64 66 66.6 48.9 70.82 43.15 59.22 41.78 59.86 40.99 56.94 36.43 Crystallinity percent was calculated by dividing the area of the crystalline peaks by the total area under the curve.
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has been determined by Minke and Blackwell (Minke and Blackwell, 1978) using X-ray diffraction analysis, based on the intensity data from deproteinized lobster tendon. Least-squares refinement shows that adjacent chains have alternating sense (i.e. are antiparallel). In addition, there
1365
is a statistical distribution of side-chain orientations, such that all the hydroxyl groups form hydrogen bonds. The unit cell is orthorhombic. The chains form hydrogenbonded sheets linked by C@OÆÆÆH–N bonds are approximately parallel to the a-axis, and each chain has an
Fig. 11. X-ray diffraction patterns of (a) a-chitin from (1) BS (2) PS (3) CR (4) Cray Fish (b) the corresponding chitosan.
Fig. 12. X-ray diffraction patterns of (a) b-chitin from (1) CT (2) SQ (b) the corresponding chitosan.
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O-3 0 –HÆÆÆO.5 intra-molecular hydrogen bond, similar to that in cellulose. The results indicate also that a statistical mixture of CH2OH orientations is present, equivalent to half oxygen on each residue, each forming inter- and intra-molecular hydrogen bonds. As a result, the structure contains two types of amide groups, which differ in their hydrogen bonding, and account for the splitting of the amide I band in the infrared spectrum. The Inability of this chitin polymorph to swell on soaking in water is explained by the extensive intermolecular hydrogen bonding. In Fig. 11, the X-ray diffraction patterns of the obtained a-chitin (A) and the corresponding hydrolyzed chitosan (B) are given. All chitin samples show strong reflections at 2h around 9–10 and 2h of 20–21 and minor reflections at higher 2h values e.g. at 26.4 and higher. The band at ˚ and is due to 9.9 corresponds to a d spacing of 8.92 A the incorporation of bound water molecules into the crystal lattice (Ogawa et al., 1992). The reflection at 2h 19.4–20 ˚ (Harish Prashcorresponds to a d spacing of about 4.41 A anth et al., 2002). Generally, the sharpness of the bands are higher in the chitin samples than in their chitosan analogue with slight decrease in the crystallinity percent. Fig. 12 illustrates the chitin and chitosan obtained from squid pens (b-chitin) the band at 2h 9.9 decreases after deacetylation followed by dramatic decrease in crystallinity percent as seen in Table 2. 4.6. Thermal analysis TGA curves of chitin and chitosan are shown in Fig. 13 as representative examples. Two endothermic peaks are observed. The first peak appears around 90–99 C corresponds to loss of water. The second one emerges at about 303 C in chitosan and at about 372 C in the corresponding chitin as seen in the DTA curves in Fig. 14. All the extracted samples showed similar trend; the chitin has higher thermal stability than the corresponding chitosan.
120 chitosan chitin CF chitin PS Chitin Sq
Weight loss%
100 80 60 40 20 0 0
100
200
300
400
500
600
Temperature, ºC Fig. 13. TGA curves for chitosan and chitin.
700
0.000 -0.002
DTGA
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-0.004 -0.006 -0.008
CF, chitosan CF Chitin
-0.010 0
100
200
300 400 Temperature, ºC
500
600
Fig. 14. DTA curves for chitosan and chitin from CF.
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