International Journal of Mechanical Engineering and Technology (IJMET)
Volume 10, Issue 01, January 2019, pp. 2011-2020, Article ID: IJMET_10_01_196
Available online at http://www.iaeme.com/ijmet/issues.asp?JType=IJMET&VType=10&IType=1
ISSN Print: 0976-6340 and ISSN Online: 0976-6359
© IAEME Publication
Scopus Indexed
EXTRACTION, CHARACTERIZATION AND
KINETICS OF DEMINERALIZATION OF
CHITIN PRODUCED FROM SNAIL SHELLS OF
DIFFERENT PARTICLE SIZES USING 1.2 M
HCL
1
*Daniel T. Oyekunle and James A. Omoleye
Department of Chemical Engineering, College of Engineering,
Covenant University, Ota, Nigeria
*Corresponding Author
ABSTRACT
In this study, kinetics of demineralization of chitin extraction from snail shells was
investigated. Chitin was extracted from snail shells by demineralizing the
deproteinized shells in 1.2 M HCl solution. Prior to demineralization, the raw snail
shells were deproteinized using 1 M NaOH solution to remove proteins and organic
matter present in the shells. The product was dried before the demineralization
process was carried out. The results showed that based on the R2 values obtained for
each of the shrinking core models considered which include; fluid film diffusion
(FFD), ash layer diffusion (ALD), and chemical reaction control (CRC), it was noted
that the CRC model was prevalent for all the various range of particle sizes analyzed
(6.3 – 4.75 mm, 4.75 – 2 mm, 2 – 1 mm, and 600 – 300 μm). The surface morphologies
and the Fourier Transform Infra-Red (FTIR) bands of the extracted chitin were
similar to previous studies..
Keywords: Kinetics, chitin, demineralization, particle sizes, HCl.
Cite this Article: Daniel T. Oyekunle and James A. Omoleye, Extraction,
Characterization and Kinetics of Demineralization of Chitin Produced from Snail
Shells of Different Particle Sizes using 1.2 M Hcl, International Journal of
Mechanical Engineering and Technology, 10(1), 2019, pp. 2011-2020.
http://www.iaeme.com/IJMET/issues.asp?JType=IJMET&VType=10&IType=1
1. INTRODUCTION
Chitin is white, dense, nitrogenous compound that is abundant in nature. It is an Nacetylglucosamine biopolymer that compose of some glucosamine, which is a major
component of fungi cell walls, arthropods exoskeletons such as mollusks radulas, insects,
crustaceans and cephalopods beaks [1]. It is a waste product in fish industries and it’s
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Extraction, Characterization and Kinetics of Demineralization of Chitin Produced from Snail
Shells of Different Particle Sizes using 1.2 M Hcl
regarded as a renewable raw material that’s second in abundance to cellulose. Its abundance
in nature accounts for about 1000 tons per annum, and 70 % of it is produced from marine
species. Chitin is a major constituent in crusetaceans shells which include shrimps, crabs and
lobsters. It is found in insects as well as mollusks exoskeletons. It’s a major component of
fungi cell wall mass of about 1 – 15 %, with yeast having an average component of 1 – 2 %
chitin and fungi filaments being 15 % of chitin. Often times it is commercially produced by
the removal of the actetyl groups from the chitin polymer known as deacetylation process by
alkali [2]. Chitin has been applied in several applications such as agriculture, biotechnology,
wastewater treatment, cosmetics, photography, food, pharmaceutical, environmental and
biomedical industries due to its biological and physiochemical properties [3, 4]. Its most
essential use is as a nutrient supplement. USA food and beverage industry used about 2288
metric tons in 2018. It is a biomaterial that is valuable due to its non-toxicity,
biocompatibility, antimicrobial, antioxidant and biodegradable properties [3, 5].
The occurrence of chitin in three different polymorphic forms which are α-, β-, and γforms, because of the presence of sturdy hydrogen bond among the carbonyl and amide
groups of the chains nearby. However, the process of producing chitin and its derivatives
from available raw materials are required to be ecofriendly, simple and economical, this
determines the final properties such as purity, DA (Degree of acetylation), and structural
morphologies that largely affect its applications. Chemical methods are usually applied on a
large scale [6, 7]. Two steps are usually considered in this technique which includes,
deproteinization process carried out by treatment with alkali, and demineralization process by
treatment with acids, then decolorization of the product was carried out as this ensures the
removal of lipids and pigments. Further studies have compared the conventional method
alongside stirring chitin repeatedly in NaClO solution for few minutes prior to
deproteinization and demineralization step. Although, this has brought comparable results as
it saves energy and time, it’s efficient in rapid extraction of chitin from marine sources as
demonstrated by Kaya et al. [8] in extracting chitin from shrimp, crayfish and crab shells.
Demineralization process has reportedly led to detrimental effects on the DA and molecular
mass which invariably have adverse effects on the purified chitin intrinsic properties [4, 9]. In
order to prevent depolymerization of chitin, the use of stirred bioreactors at ambient/room
temperatures have been encouraged, this has resulted to shorter processing time at greater
quality [10, 11].
DA of chitin is important because, it affects its physical, biological and chemical
properties such as solubility, biodegradability and production of its derivatives such as
chitosan. The ratio of 2-acetamido-2-deoxy-D-glucopyranose to 2-amino-2-deoxy-Dglycopyranose units is known as the degree of N-acetylation (DA). The DA can be
determined by XRF (X-ray Floreesence) elemental analysis of chitin as recorded in various
literatures [12 – 14]. FTIR (Fourier Transform Infra-Red) analysis as reported by Kumari et
al. [15] and Yen et al. [16], or by NMR analysis [12, 17 – 18]. Because of chitin’s limited
solubility, IR, 13C CP / MAS NMR, and DSC spectroscopy has successfully been used for
chitin analyses in the solid state [19].
The aim of this study is to synthesize chitin from snail shells using chemical methods
which involves deproteinization and demineralization. Kinetics of the demineralization
process was studied using shrinking core models of heterogeneous reaction system. The
reaction was assumed to occur first on the external surface of the particle. As the reaction
proceeds the zone of reaction moves into the internal layer of the solid particle leaving behind
a completely converted product and an inert solid referred to as “ash” [20]. Three different
diffusion models were considered in this study which are the fluid film diffusion (FFD), ash
layer diffusion (ALD), and chemical reaction control (CRC). X-ray florescence (XRF) was
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Daniel T. Oyekunle and James A. Omoleye
used to determine the amount of calcium content converted as the demineralization process
proceeds. Hence, this was used to determine the kinetic model of fluid particle reaction for
each range of snail shell particle sizes considered. The structural morphology and FTIR bands
of the chitin produced was reported and compared with previous studies.
2. MATERIAL AND METHOD
2.1. Raw material
Snail shells used in this study was purchased from, Ilorin, Kwara state, Nigeria. The shells
were washed carefully to remove tissues and sundried for about 2 weeks. The dried shells
were then pulverized using an industrial grinder, it was carefully collected and sieved using
the following mesh sizes: 6.3 mm, 4.75 mm, 2 mm, 1 mm, 600 μm, and 300 μm. The particle
sizes were separated into the following ranges 6.3 – 4.75 mm, 4.75 – 2 mm, 2 – 1 mm, and
600 – 300 μm (Fig. 1), and kept at room temperature prior to further processing.
Raw
shells
6.3 - 4.75 mm
(4.75 - 2 mm)
2 - 1 mm
600 -300 μm
Figure. 1 Raw snail shells of different particle sizes
2.2. Kinetics of demineralization process using 1.2 M HCl.
Deproteinization process was carried out at 80 0C on a hot plate magnetic stirrer for 2 hours at
2 ml of acetic to 1 g the shells to reduce the amount of proteins. The product was dried at 70
0
C in an oven, until constant weight was achieved. XRF analysis was carried out on the
deproteinized product (Fig. 2). 40 g of deproteinized shells were demineralized using 3 ml of
HCl to 1 g of shell. Reaction times were for 2, 4, 6, 8, 10, 12 and 14 minutes at room
temperature on a magnetic stirrer with constant revolution per minute. At the end of each runs
XRF analysis was used to determine the calcium oxide content.
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Extraction, Characterization and Kinetics of Demineralization of Chitin Produced from Snail
Shells of Different Particle Sizes using 1.2 M Hcl
120.00
96.73
Concentration
100.00
80.00
60.00
40.00
20.00
0.04 0.54 1.76 0.09 0.14 0.10 0.11
0.11
0.39
0.00
Figure 2 XRF analysis of the deproteinized snail shells
3. KINETIC MODELLING
The conversion-time experimental data obtained were used to test for the rate controlling step
using unreacted shrinking core model. Equations (1), (2) and (3) represent the modelling
expressions for the film (FLD), Ash (ALD), and chemical reaction controlling (CRC)
mechanisms respectively [20].
𝑡
𝜏
= 𝑋𝐵
(1)
2⁄
3
𝑡
𝜏
= 1 − 3(1 − 𝑋𝐵 )
𝑡
𝜏
= 1 − (1 − 𝑋𝐵 )
+ 2(1 − 𝑋𝐵 )
(2)
1⁄
3
(3)
Where t is the reaction time, τ is the time required for complete reaction and conversion of
Ca2+ ion is given by XB.
3.1. Characterization tests
Scanning electron microscope (SCM) was used to observe the surface morphologies and
microstructure of the samples. Chitin produced from snail shells were characterized by FTIR8400S spectrophotometer. The FTIR wavelength spectrum was between 700 cm-1 to 4000 cm1
with a spectral resolution of 2.0 cm-1. About 10 scans were conducted during analysis. DA
of the isolated chitin samples was calculated by the ratio of measured peak absorbance to
reference peak absorbance. The DA was determined from the absorbance ratio given by
Baxter et al. [21] as stated in equation (4).
𝐴
𝐷𝐴(%) = 𝐴1655 × 115
(4)
3450
4. RESULTS
Fig. 3 shows the rate of calcium conversion against time. A rapid increase in calcium
conversion was observed between 2 to 6 minutes reaction time for the largest particle sizes
(6.3 – 4.75 mm), while other particle sizes demonstrate a steady increase. This can be due to
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the fact that a large amount of calcium had been converted in the first 2 minutes as a result of
the exposure of a larger surface area of the shells to the acid. It also demonstrates that the rate
of reaction rapidly occurs for all the particle sizes which may be due to the high reactivity of
HCl acid.
0.6
Calcium conversion
0.595
0.59
0.585
6.3-4.75 mm sizes
4.75 – 2 mm sizes
0.58
2 – 1 mm sizes
600 -300 μm sizes
0.575
0.57
0.565
2
4
6
8
Time
10
12
14
Figure 3 Calcium conversion against time
The kinetic model of fluid film diffusion (FFD), ash layer diffusion (ALD), and chemical
reaction control (CRC) were determined from conversion of each particle sizes (6.3-4.75 mm,
4.75-2 mm, 2 – 1 mm, 600 -300 μm) illustrated in Fig. 4 – 7. From the figures, it can be
deduced that for each range of particle sizes considered the R2 values of each shrinking core
model was determined. R2 values of FFD, CRC and ALD for 6.3-4.75 mm range of particle
sizes were reported to be 0.8098, 0.9504, and 0.9118, this clearly shows that based on this
particle sizes, CRC controls the reaction process due to the closeness of its R2 value to 1. Also
for 4.75 – 2 mm, the R2 values of FFD, CRC and ALD were reported to be 0.8204, 0.9763,
and 0.9307, this also shows that for this particle sizes CRC controls the reaction process due
to the closeness of its R2 value to 1. In addition for 2 – 1 mm range of particle size, R2 values
of FFD, CRC and ALD were reported to be 0.8266, 0.9430, and 0.9010, this also implies that
for this range of particle size CRC controls the reaction process due to the closeness of its R2
value to 1. Furthermore, the R2 value for 600 – 300 μm particle sizes were considered for
FFD, CRC and ALD and they were reported to be 0.8993, 0.9661, and 0.9509, this clearly
shows that based on the particle size CRC controls the reaction process due to the closeness of
its R2 value to 1.
It can be concluded that the CRC mechanism of the shrinking core model with the highest
2
R values best describes the kinetics of demineralization of snail shell using 1.2M
hydrochloric acid for all the particle size used in this work.
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Extraction, Characterization and Kinetics of Demineralization of Chitin Produced from Snail
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y = 0.0038x + 0.9522
R² = 0.8098
1.05
1
y = 0.0211x + 0.6817
R² = 0.9118
FFD, CRC, ALD
0.95
0.9
FFD
0.85
y = 0.0193x + 0.6103
R² = 0.9504
0.8
ALD
CRC
Linear (FFD)
0.75
Linear (ALD)
Linear (CRC)
0.7
0.65
0.6
0
2
4
6
8
10
Reaction Time (mins)
12
14
16
Figure 4 Graph of FFD, ALD and CRC against time using 6.3 – 4.75 mm particle sizes
y = 0.0017x + 0.9783
R² = 0.8204
1.05
1
y = 0.0137x + 0.7973
R² = 0.9307
FFD, CRC, ALD
0.95
0.9
FFD
0.85
ALD
y = 0.0153x + 0.6972
CRC
R² = 0.9763
0.8
Linear (FFD)
0.75
Linear (ALD)
0.7
Linear (CRC)
0.65
0.6
0
2
4
6
8
10
Reaction Time (mins)
12
14
16
Figure 5 Graph of FFD, ALD and CRC against time using 4.75 – 2 mm particle sizes
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y = 0.0017x + 0.9788
R² = 0.8266
1.05
1
FFD, CRC, ALD
0.95
y = 0.0146x + 0.7993
R² = 0.901
0.9
FFD
0.85
ALD
y = 0.0168x + 0.6981
R² = 0.943
0.8
CRC
Linear (FFD)
0.75
Linear (ALD)
Linear (CRC)
0.7
0.65
0.6
0
2
4
6
8
10
Reaction Time (mins)
12
14
16
Figure 6 Graph of FFD, ALD and CRC against time using 2 – 1 mm particle sizes
1.05
y = 0.0017x + 0.9731
R² = 0.8993
1
FFD, CRC, ALD
0.95
y = 0.0112x + 0.7761
R² = 0.9509
0.9
0.85
FFD
ALD
0.8
y = 0.0105x + 0.6882
R² = 0.9661
CRC
Linear (FFD)
0.75
Linear (ALD)
0.7
Linear (CRC)
0.65
0.6
0
2
4
6
8
10
Reaction Time (mins)
12
14
16
Figure 7 Graph of FFD, ALD and CRC against time using 600 – 300 μm particle sizes
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Extraction, Characterization and Kinetics of Demineralization of Chitin Produced from Snail
Shells of Different Particle Sizes using 1.2 M Hcl
4.1. Analysis on chitin produced
4.1.1. FTIR analysis
Fig. 8 displays FTIR spectra of the chitin formed. Absorption band of the chitin produced was
similar to standard chitins. Several vibration bands were present between 3425-2881 cm-1
ranges which relate to primary amines of v (N-H) in v (NH2) [22]. The presence of v (N-H), v
(O-H) and v (NH2) band groups in the chitin was noted at 3425-3422 cm-1 wavelength.
Methyl group available in NHCOCH3, Methylene group presence in CH2OH and pyranose
ring in methyne group were observed at the relevant stretching vibrations of 2921 – 2879 cm-1
(Fig. 8). The absence of bands at 1540 cm-1 implies protein contaminants were absent in chitin
samples analyzed. This was also observed by Wasko et al. [23]. Furthermore, other bands
identified in the spectral analysis are; 3618 cm-1 (O-H stretching), 2862 cm-1 (asymmetric CH stretching), 1465 cm-1 (CH3 symmetric deformation, CH bend), 1172 cm-1 (amides C-N
vibrations), 1095 cm-1, 1026 cm-1 (C-O-C asymmetric stretching) [24, 25].
Degree of acetylation was determined to be 120%, this was higher than that reported by
Majtan et al. [26]. Higher DA values was reported for bumblebee and crude crab chitin of
132.5% and 151.7% respectively. It was deduced that the presence of some inorganic
materials that were not totally removed in the production process were responsible for high
DA values. Although, DA values should be lower than 100% since protein removal was by
alkali treatment, nevertheless higher DA values might be noted using various methods to
determine the DA [27]. In contrast, the use of C CP/MAS-NMR spectroscopy has been noted
to be the most suitable method for determining the DA due to its sensitivity to local structure
changes [26].
Figure 8 FTIR analysis of chitin extracted
4.1.2. SEM analysis
Fig. 9 illustrates the SEM of chitin prepared from snail shells. The chitin structure appears to
have smooth surfaces of different particle sizes with void spaces. Similar structures of chitins
were previously isolated from periwinkle shells reported by Gbenebor et al. [28] and Akpan et
al. [29].
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Figure 9 Structural morphologies of the chitin extracted at 500x (left) and 1000x (right) magnification
5. CONCLUSION
Chitin was successfully isolated from snail shells at the different range of particle sizes using
chemical methods: deproteinization and demineralization methods. In this study, kinetics of
snail shells demineralization of different particle sizes was carried out using 1.2 M HCl. The
reaction was modelled based on the shrinking core models. It was observed that for all
particle sizes in the following ranges of 6.3 – 4.75 mm, 4.75 -2 mm, 2 - 1 mm, and 600 -300
μm, the demineralization was largely chemical reaction controlled, with an R2 values of about
0.9. Chitins extracted was characterized using FTIR and SEM analysis which shows that the
surface morphologies and FT-IR bands of the chitin analyzed were typical of earlier studies.
ACKNOWLEDGEMENT
The authors appreciate the financial support of Covenant University for the publication of this
manuscript.
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