Craveiro, A. A., Craveiro, A.C., Queiroz, D.C., 1998, “Quitosana: A fibra do futuro”. Editora Universitária.261-266. Li, J. K.; Wang, N. & Wu, X. S. - J. Control Release, 56, p. 117 (1998). Jaworska, M., Sakurai, K., Gaudon, P., Guibal, E., 2003, “Influence of chitosan characteristics on polymer properties. I: Crystallographic properties, Polymer International”, pp. 198–205. Rinaudo, M., 2006, “Prog. Polym. Sci. 31, 603-632”. Roberts, G. A. F., 1992, “Chitin Chemistry”, Macmillan: London. Rocha, I.P., Rodrigues, J., Amorim, L.A., 2004, “Carcinicultura brasileira. Revista da Associação Brasileira de Criadores de Camarão”, pp. 30. SOARES, N. M.; MOURA, C. M.; RIZZI, J.; VASCONCELOS, S. R.; SANTOS, V. O. B.; PINTO, L. A. A., 2001, “Estudo da produção de quitosana a partir de resíduos de camarão em escala piloto”. Trabalho apresentado no XVI CRICTE – Congresso Regional de Iniciação Científica e Tecnológica em Engenharia, Ijuí, RS, Brasil., Tonhi, E., Plepis, A. M. G., 2002, “Quim. Nova”, pp. 25, 943. Zhang, M., Haga, A., Sekiguchi, H., Hirano, S., 2000, “Int. J. Biol. Macromol”. 27, 1. Wu, F, C., Tseng, R, L., Juang, R, S., 2001, “Enhanced abilities of highly swollen chitosan beads for colour removal and tyrosinase immobilization”, journal of hazardous materials” 81, pp. 167-177. Corresponding Author: Rasiah Ladchumananandasivam.

Figure 11b, shows the characteristic bands of chitosan from grey shrimp (Litopenaeus Vannameis). It can be observed in the region of 3357 cm-1, the corresponding to the axial OH stretch and at 1646 cm-1 is the band of C = O of amide I. The region 1375.72 corresponds to axial deformation of the C-N of amide. At 2875.97, it corresponds to the stretching of C-H and the region near 1150 cm-1 corresponds to COC (polysaccharide).

Figure 11 b. FTIR Spectrogram of the chemical structure of chitosan from grey shrimp - Litopenaeus Vannameis.

All the characteristic bands are very similar and show that all of them have basically the same functional groups.

4. ACKNOWLEDGEMENTS

The authors would like to thank CNPq (Brazilian National Research Council) for the financial support as well as Dr. Rajesh Anandjiwala of CSIR-Port Elizabeth for letting the author Rasiah Ladchumananandasivam to use the equipments (FTIR, TG/, DTG) as well as the help from Dr. Rakesh Kumar, Dr. Maya John and Mrs. Valencia Jacobs of the same Institution. 5. REFERENCES Abram, P., 2004, Chitin e Chitosan: Obtención caracterización y aplicaciones, Pontificia Universidad Católica- Perú. Canella, K. M. N. C., Garcia, R. B., 2001, “Chitina caracterization por cromatografia de permeação em gel – influência” do método de preparação e do solvente”, Química Nova, Vol. 24, No. 1, pp. 13-17.

c) Chitin from crab d) Chitosan from crab e) Industrial chitosan from

shrimp Figure 10. a). and b). Curves of TG/DTG for shrimp chitin and Chitosan; c) and d) Curves of TG/DTG for crab chitin and chitosan; e). curve for industrial Chitosan from shrimp.

3.4 Fourier transform infrared (FTIR) spectroscopy The spectroscopy in the infrared region was one of the techniques used for

characterization of chitin and chitosan. The spectrographs for chitin and chitosan from crab - Ucides cordatus are shown in Figure 11a. The infrared spectrum of chitin shows the band with peak at 3264.47 cm-1 due to axial stretching vibrations of OH groups present in chitin, which is superimposed to the stretching band of N-H. The peak at 1627.44 corresponds to the characteristic absorption band of amide I, attributed to axial deformation of the carbonyl C = O present in chitin. The band at 1400 cm-1 is attributed to the symmetrical angular deformation of CH3 group.

Comparing the spectra of chitosan with chitin, it appears that there were significant changes in the region between 1400 cm-1, a reduction in the peak. The band at 1655 cm -

1 associated with carbonyl (C = O) decrease, as the degree of deacetylization of the Chitosan increases.

The peak at 1378.21 cm-1 refers to the vibration of C-H of the CH3 group, referring to the waste, acetamine of chitin. The spectrum still shows absorption peak at 3264.5 cm-1, associated with the N-H, the hydrogen bond and the stretching of O-H.

─ crab chitosan ─ crab chitin Figure 11a. FTIR Spectrogram of the chemical structure of chitin and chitosan from

crab - Ucides cordatus.

Figure 9. TG/DTG curves for exoskeleton of crab - Ucides cordatus demineralized in HCl, ratios: a) 1:3; b). 1:6 and c). 1:10.

It was observed that the profiles of the thermogravimetric curves (TG/DTG) were

similar. According to the results obtained, it is preferable to use a lower concentration in the demineralization process, thus avoiding degradation.

From Figures (10a, 10b, 10c, 10d and 10e) the profile of the thermal decomposition of a TG / DTG samples of chitin and Chitosan can be seen. From the thermograms of chitins, Figures 10a and 10c, two stages of decomposition can be observed. The first occurs in the range of 50-100 oC and is attributed to evaporation of water molecules and the second stage of decomposition occurs in the range of 400-500 oC and could be attributed to the degradation of saccharide structure of the molecule, including dehydration of saccharide rings and polymerization and decomposition of the acetylated and deacetylated units of chitin.

In the thermogram of chitosan, Figure 10b, there are two stages of decomposition, similar to that found in chitin, occurring respectively in the ranges of 40-100 oC and 400-500 oC. In Figure 10d there are four stages of decomposition, occurring respectively in the ranges of 40-100 oC, 300-400 oC, 400-500 oC and 600-700 oC. In Figure 10e, it occurs in two stages in the range 40-100 oC and in the range of 300-400 oC.

The second stage of decomposition of chitosan from crab, Figure 10d, and industrial chitosan, Figure 10e, occurred at a temperature lower than that observed for the decomposition of chitin. This suggests that chitosan has a lower thermal stability. The peak that appears around 390 oC and 329 oC respectively may be due to the degradation of the part of the molecule that has been deacetylated. Thus, it could be seen that the sample showed a dehydration process, followed by decomposition of the biopolymer, generating carbonized material.

a). Chitin from Shrimp b). Chitosan from shrimp

Ucides cordatus Chitosan from crab

Ucides cordatus 72 566 87,0 71,8

The results of the crystallinity index (ICR) showed very close values to the

chemical to chitin under treatment with sodium hydroxide. Chitin has led to low crystallinity of chitosan, as well as and the conditions of the treatments used for chitin and chitosan. It should also be noted that the crystallinity of the samples depends on several factors, such as the nature of the organism from which the chitin was extracted and the conditions employed in the extraction of the polymer. Thus, samples of chitin obtained in this study have a diffraction pattern with well defined peaks and higher crystallinity. The chitosan extracted from crab carapace showed crystallinity index of 87% while that of shrimps varied between 66 and 85.8%.

3.3. Thermogravimetric analysis (TG/DTG) The curves were obtained with a heating rate of 10 oC min-1 under a dynamic

atmosphere of air in the temperature range of 25-700 oC. TG curves of the samples were obtained to verify the profile of thermal decomposition and to determine the temperature intervals corresponding to the percentage of hydration, decomposition of organic material and for the residue formed DTG curves corresponding to the first derivative curves of TG are used. Initially, a study on the effect of HCl concentration in samples from exoskeleton of shrimp and crab were carried out in order to determine the carbonate content.

The demineralization process, Figures 8 and 9 was carried out in three ratios: 1:3, 1:6 and 1:10, as can be seen in the curves of weight loss as a function of temperature in exoskeletons of shrimps and crabs.

a). Demineralisation - ratio 1:3 b). Demineralisation - ratio 1:6 c). Demineralisation - ratio 1:10

Figure 8. TG/DTG curves for exoskeleton of shrimp - Litopenaeus Vannamei

demineralised in HCl, ratios: a). 1:3; b). 1:6 and c). 1:10.

a. Demineralisation - ratio 1:3 b. Demineralisation - ratio 1:6 c. Demineralisation - ratio 1:10

a b

Figure 7. X-ray diffractogram showing the peaks 10 to 80º (2θ). a. Chitin from crab- Ucides cordatus b. Choitosan from crab –Ucides cordatus

Chitosan itself has no absolute standard of crystallinity; therefore, from the two

peaks of highest intensity of X-ray diffraction of this material, the crystallinity index was determined. The crystallinity index (ICR) could be determined with the use of equation (1) (LI et al, 1998).

(1) Where: IC and IA are the intensities of the signals from the crystalline regions (2θ ≈

20) and amorphous (2θ ≈ 10-13 °), respectively. The relationship between the degree of deacetylation (DD) and the relative crystallinity index is inversely proportional. The higher is the crystallinity index the lower is the degree of deacetylation, which is characteristic to chitins that possess high degree of crystallinity.

Data correlating the samples with their crystallinity index and degree of deacetylation is shown in Table 3.

Tabele 3. Results for chitin and chitosan samples of shrimp and crab.

Amostras

IA IC % I CR %GD

Chitin from ash coloured shrimp Litopenaeus Vannamei

116 514 77,4 -

Chitin from red coloured shrimp Aristeus antennatus

151 954 84,2 -

Chitin from red coloured shrimp Aristeus antennatus

274 2396 88,5 -

Chitosan from red coloured shrimp Aristeus antennatus

290 2050 85,8 73,0

Chitosan from ash coloured shrimp Litopenaeus Vannamei

431 1566 72,4 70.2

Chitosan from ash coloured shrimp Litopenaeus Vannamei

474 1404 66,2 69,4

Chitin from crab 37 1262 97,0 -

corresponding to 2θ = 19 280 o and 2θ = 31 900 o, respectively, while in Figure 6b the crystalline peaks, with the intensities of 954 and 334 cps, correspond to 2θ = 19 256 o and 2θ = 32 040 o. In Figure 6c, the crystalline peaks for the intensities 2396 and 1524 cps correspond to 2θ = 19 040 o and 2θ = 26 500 o. In Figure 6d the crystalline peak with the intensity of 2050 cps, corresponds to 2θ = 19 100 o. In Figure 6e crystalline peaks with the intensities of 1566 cps correspond to 2θ = 18 729 o. By analyzing the Figure 6f the spectrum of chitosan crystalline peak with the intensity of 140) cps corresponding to 2θ = 19 840 o could be observed. By analyzing the diffractograms of chitin and Chitosan samples, it could be verified that Chitosan has a semi-crystalline structure.

a b c

d e f

Figure 6. X-ray difratogramas from 10 to 80 º (2θ). a). chitin from ash coloured shrimp -

Litopenaeus Vannamei; b) and c) chitin from red shrimp - Aristeus antennatus; d). chitosan from red shrimp - Aristeus antennatus; e) and f). chitosan from ash coloured

shrimp - Litopenaeus Vannamei .

In the analysis of Figure 7, it could be observed that the spectrum of crab chitin showed several crystalline peaks, the most intense ones attains an intensity of 1232, 1262, 474, 380 and 228 cps, corresponding to 2θ = 34 140 o, 2θ = 18 040 o, 2θ = 47 160 o, 2θ = 50 820 o and 28.780 o respectively, while in Figure 7b the crystalline peak relating to the intensity of 566 cps, corresponds to 2θ = 29 520 o.

a. shrimp chitin x150 b. crab chitin x1000 c. shrimp chitin x1000

d. shrimp chitosan x150 e. shrimp chitosan x200 f. shrimp chitosan x1000

Figure 5. g. crab chitin x 250 h. crab chitin x500 i. crab chitosan x1000. Figure 5. Micrograms (a) shrimp chitin x150, (b) crab Chitin x1000, (c) shrimp chitin x1000, (d) shrimp chitosan x150, (e) shrimp chitosan x200 and f) shrimp chitosan x1000.

3.2 X-ray diffraction

X-ray diffraction studies were carried out using a Universal X-ray diffractometer, model Shimadzu XRD-6000 with Cu radiation, with power of 30 kV and 30 mA. The samples were scanned from 10 to 80 º.

The use of X-ray diffraction also allows clearly distinguishing between chitin and its derivative deacetylated chitosan. In fact, the diffractograms of chitin showed best resolved signs and in greater numbers, Figures 6a, 6b and 6c, than observed in the diffractograms of chitosan, Figures 6d, 6e and 6f, which could be attributed to the presence of higher crystalline areas in chitin than Chitosan (ROBERTS, 1992 and Zhang et al., 2000).

By observing Figure 6a, the spectrum of chitin from shrimp shows several crystalline peaks, the most intense ones are related to the intensity of 514 and 318 cps,

Figure 4. Samples dilution 1:3, 1:6 e 1:10. a. shrimp b. Crab. c. demineralised washed and dried crab chitin

d. demineralised washed and dried shrimp chitin.

A solution of sodium hydroxide 5% w/v was used for the deproteinization to reduce the nitrogen content of protein, under stirring and finally the samples were repeatedly washed to remove any traces of sodium hydroxide. In the deodorization process the deproteinized material was mixed with a solution of sodium hypochlorite 0.36% v/v under stirring to reduce the odour of the material and to remove any pigments. The samples were filtered, washed many times to remove any traces of the chemicals used as well as any soluble impurities left in the sample. The filtered samples were then dried in an oven at 80oC for 4 hours.

The dried deproteinized and deodorized samples of chitin were treated with NaOH 45 °Bé (42.3%) and the solution was heated for 2 hours while stirring. At the end of the reaction time the samples were filtered and then washed until pH was neutral. Acetic acid (1%) was added to the Chitosan to dissolve it and the pH was recorded as 6.0. The solution was precipitated in an alkaline solution of NaOH to obtain a pH of 12.5, and the samples were neutralized using acid until the pH of 7.0 was attained. The purified Chitosan was obtained after drying it in an oven at a constant temperature.

3. RESULTS AND DISCUSSION

3.1. Scanning electron microscope The morphological characteristics of the samples of chitin and chitosan were

evaluated by electronic microscopy (SEM) and the results showed that they had similar structures presenting various aspects of geometrically irregular fine particles and loosely attached to it, as seen in Figures (5a, 5b (chitin) and 5f and 5i (chitosan) and they still present a heterogeneous surface. In Figures 5b, 5g and 5h it could be seen that that the crab chitin analyzed is porous in structure.

The micrographs of the samples analyzed still showed enough rough surface morphologies and fibre, as observed in Figures (5c, 5d and 5e).

d

2.2. Methods 2.3. Extraction of Chitin and Chitosan

The procedure for obtaining chitin from shells of crustaceans was similar to the process used by Smith, which follows the steps of pretreatment, demineralization, deproteinization, deodorizing and drying. Such steps were performed in the textile laboratory, LABTEX/UFRN, Brazil and some analysis at CSIR, Port Elizabeth - South Africa.

One of the preliminary operations in obtaining chitin was aimed at the separation of unwanted material in the collected samples. In the case of residues of crustaceans, such pre-processing includes milling, in order to obtain smaller particles. Particle size used in this study was 0.297 mm. The demineralization step was aimed at reducing the ash content found in them. In the demineralization stage of the exoskeletons of shrimp and crab, ratios of 1:3, 1:6 and 1:10, were analyzed with the aim to verify which ratio would best eliminate the carbonates and phosphates. The steps are described in Table (2a and 2b).

Table 2a. Demineralisation stages of shrimp exoskeleton - Litopenaeus Vannamei –

dilution ratio of HCl 1:3, 1:6 e 1:10. (for 1000 cm3 of water 30, 60 and 100 cm3 of HCl)

Process Weight

(g) HCl (%)

Temperature (ºC)

Time (h) Ratio sol/liq

Demineralization 10 2,5 23 1 1:3 Demineralization 10 2,5 23 1 1:6 Demineralization 10 2,5 23 1 1:10

Table 2b. Demineralisation stages of crab exoskeleton - Ucides cordatus in the ratios of

1:3, 1:6 e 1:10. (for 1000 cm3 of water 30, 60 and 100 cm3 of HCl)

Process Weight

(g) HCl (%)

Temperature (ºC)

Time (min)

Ratio sol/liq

Demineralization 10 7,0 23 1 1:3 Demineralization 10 7,0 23 1 1:6 Demineralization 10 7,0 23 1 1:10

2.5% v/v hydrochloric acid (HCl) for shrimp and 7.0% v/v for crabs were used to

wash the samples, Figures 4a and 4b. Both samples were washed repeatedly until the neutral pH stage was attained. After filtering, the samples were placed in an oven at 80 oC for 1 hour and 25 minutes and then transferred to a desiccator.

found in nature thus its characteristics and possible uses are not well defined (Wu et. al, 2001).

Japan, the U.S. and China are the world's largest producers of chitin, but the polymer is also produced, albeit to a lesser extent in India, Norway, Canada, Italy, Poland, Chile and Brazil, among others (Abram, 2004) and from the waste from canning industries in various parts of the world including the USA (Oregan, Washington, Virginia), Japan and several fishing fleets in Antarctica (Rinaudo. M. 2006). Several investigations have been conducted over the past 20 years in medical research aiming at producing biomaterials that could improve both the quality as well as the life expectation of patients.

In the present work, chitin was extracted from the wastes of crustaceans (shrimp: Litopenaeus vannamei, Aristeus antennatus and crabs: Ucides cordatus. In the process of obtaining chitin, collected shells were de-mineralized, de-proteinized and deodorized. Once these processes were completed, the chitin was subjected to de- acetylisation to obtain Chitosan. SEM, XRD, thermal analysis (TG and DTG), FTIR were used to analyse the morphology of chitin and Chitosan. The Chitosan was used to spin fibres in a Electrospinning apparatus.

It was observed from thermogravimetric analysis that maximum degradation temperatures for chitin is higher than that of Chitosan, which showed that the structure of Chitosan is semi-crystalline, compared to that of chitin. All bands observed in the FTIR spectrum of Chitosan showed very similar in characteristics and all have basically the same functional groups.

2. MATERIALS AND METHODS 2.1. Materials

The raw materials used in this work to obtain chitin and chitosan were exoskeletons of crustaceans (shrimp: Litopenaeus vannamei, Aristeus antennatus and crab: Ucides cordatus) Figures (3a and 3b). They were collected from local restaurants in the city of Natal / RN-Brazil and all reagents used in this work were of analytical grade, Table 1.

a. b. c.

Figure 3 a). Shrimp - Litopenaeus Vannamei b). Shrimp - Aristeus antennatus c). Crab - Ucides cordatus.

Table 1. Reagents used.

REAGENTES Formula Supplier

Sodium Hydroxide (analytical grade) NaOH Vetec Hydrochloric acid (analytical grade) HCl Quimex

Sodium Hypochlorite (analytical grade) NaClO QM Acetic acid (analytical grade) Hac Vetec

Acetona propana (analytical grade) (CH3)2CO Vetec

1. INTRODUCTION Recently, researchers in various fields of knowledge have shown great interest in

new materials, especially natural biopolymer, in particular chitin and chitosan. Chitin and chitosan, Figure (1), are used as functional polymeric materials, which are nontoxic, biodegradable, biocompatible and produced from renewable natural source, for applications in several industrial areas: agriculture, food industry, textiles, pharmaceuticals, cosmetics and development of biomaterials such as gels, films, membranes and polymer nanofibers (Tonhi; Plepis, 2002).

Figure 1. Chemical structure of chitin and chitosan.

Chitin is a natural linear polymer with monomer unit β-(1-4)-N-acetyl-D-glucosamine. Source of Chitosan, it is the second most abundant biopolymer found in nature after cellulose (Canella and Garcia, 2001). It is present in cell walls of fungi, crustaceans (shrimp, crab (siri), crabs, and lobsters) and is insoluble in water, organic solvents, dilute acids and alkalis. Depending on the source from where it is extracted, chitin can be found in three polymorphic conformations (α, β and γ), according to the orientation of the polymer chains, Fig (2), depending on their crystal structures (Jaworska et. al, 2003).

Figure 2. Orientation of polymeric chains in different forms of chitin.

The α conformation is found in shells of crustaceans, insects and the cell walls of fungi, with features such as a structure where there is an alternate arrangement of parallel and anti-parallel chains. In the β form, predominant in microscopic squids and seaweeds, the arrangement is of parallel chains. α and β chitins are mostly known, α being the most common and consequently the most studied one. The γ chitin is rarely

ELECTROSPINNING OF BIOPOLYMER CHITOSAN NANOFIBRES AND THEIR

APPLICATION TO MEDICAL TEXTILES.

Sânia Maria Belísio de Andrade; Rasiah Ladchumananandasivam.

UFRN - Federal University of the State of Rio Grande do Norte – Post Graduate Programme in Mechanical Engineering - PPGEM, Campus Universitário, Av. Senador

Salgado Filho, 3000, Lagoa Nova, Natal, RN 59078-970 - Brazil. ABSTRACT:

Chitin is the second most abundant biopolymer found in nature next to cellulose, and is mainly found in the exoskeletons (belong to crustacea family) of shrimps, crabs and lobsters. Chitin is also found in insects, mollusks and fungal cell wall. About 5 to 7% and 15 to 20% of chitin are found in shrimps and crabs respectively. The exoskeletens of shrimps (Litopenaeus Vannamei & Aristeus antennatus) and crabs (Ucides cordatus) have been used as raw materials for the extraction of chitin and their utilization in the production of nanofibres constitutes the main objective of this work. Chitin is used as a functional material in medical applications, food, pharmaceutical and chemical industries. The choice of chitin is justified due to its excellent biomedical properties: biodegradability, biocompatibility, nontoxicity, antibacterial as well as emulsificants and quelants. In the present work chitin was extracted from shells of crustaceans using demineralisation, deproteinization and desordorisation processes. Chitosan was obtained from chitin through deacetylation process. In the demineralisation processes samples of exoskeletons of crustaceans were used at different proportins of 1:3, 1:6 and 1:10 to verify the degree of carbonate and phosphate elimination. Surface topography analysis was carried out using Scanning Electron Microscope (SEM), Differential X-ray Diffraction (DRX), and thermal analysis was carried out using Thermogravimetric analysis (TGA) and Differential Thermal Analysis (DTA). DRX results show that the chitosan has semicrystalline structure. The lowest cristalinity of chitosan could be explained from the results of thermogravimetric analysis, as the chitosan shows very low degradation temperatures. Regarding morphology, surface characteristics of the samples of chitin and chitosan were observed in the micrograms from SEM. The nanofibres produced by electrospinning were also analysed for further studies.

.