Accepted Manuscript

Title: Synthesis and Characterisation of Chitin from

Periwinkle (Tympanotonus fusatus (L.)) and Snail
(Lissachatina fulica (Bowdich)) shells

Authors: E.I. Akpan, O.P. Gbenebor, S.O. Adeosun

PII: S0141-8130(17)30585-8
DOI: http://dx.doi.org/10.1016/j.ijbiomac.2017.08.106
Reference: BIOMAC 8098

To appear in: International Journal of Biological Macromolecules

Received date: 15-2-2017

Revised date: 12-8-2017
Accepted date: 17-8-2017

Please cite this article as: E.I.Akpan, O.P.Gbenebor, S.O.Adeosun, Synthesis

and Characterisation of Chitin from Periwinkle (Tympanotonus fusatus (L.)) and
Snail (Lissachatina fulica (Bowdich)) shells, International Journal of Biological
Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.106

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 Synthesis and Characterisation of Chitin from Periwinkle (Tympanotonus fusatus
(L.)) and Snail (Lissachatina fulica (Bowdich)) shells
1Akpan E. I., 2Gbenebor O. P. and 2Adeosun S. O.
1Institut für Verbundwerkstoffe GmbH, 67663, Kaiserslautern, Germany
2Department of Metallurgical and Materials Engineering, University of Lagos, Nigeria

Abstract
This study characterizes chitin extracted from bio-sources of snail and periwinkle using
varied combinations of acid and alkali concentrations. A three level factorial design of
experiment with alkali and acid concentrations was used. FTIR, XRD and SEM were used
to investigate the structural changes after treatments. Results reveal that both alkali and
acid concentrations significantly affect the development of the functional groups and their
intensities in the extracted chitin. A certain combination of concentration of acid and alkali
can be used to obtain chitin with high degree of order (Crystallinity Index (CrI) > 0.9) and a
degree of de-acetylation (DD > 50 %). This results in combined high crystallinity and
degree of de-acetylation. The study also established that certain combination of acid and
alkali concentrations could lead to alpha to beta transformation in chitin structure.

Key words: De-acetylation; Chitin; Snail; Periwinkle

Introduction
Chitin, which is a major polysaccharide of the exoskeletons of insects and shells of
crustaceans, is an important polymer with beta (1-4)-linked N-acetylglucosamine repetitive
units. The antibacterial activity, biocompatibility, biodegradability, low toxicity and sorption
ability of chitin and chitin derivatives make them of huge commercial relevance [1–4]. Due
to these inherent important qualities, they have been synthesized and utilized in medical
industries, cosmetics, agriculture, biotechnology, biocomposites, papermaking, and water
purification. Specific applications of chitin and chitosan include;, wound dressings [5],
pharmacology [6], biotechnology [7], metal uptake from wastewater [8,9], solid-state
batteries [10], biodegradable pressure-sensitive adhesive tape [11], chelating agents
[12,13], wound-healing agents [14,15], drug carriers [16], food preservation [17], drug
development [18], tissue engineering [19], and membranes [20,21]. This broad application
profile of chitin and chitosan is basically tied to their bio-renewability, biocompatibility,
environmental friendliness, biodegradability and bio-functionality.

1
Despite its enormous abundance and wide anticipated applications, chitin is limited in use
because of its poor solubility characteristics. It is insoluble in dilute aqueous solvents and
some organic solvents owing to its strong intra- and inter-hydrogen bonds. Chitin
supramolecular structure is ordered in three different orientations of microfibrils giving rise
to three types of chitin including: α- chitin (anti-parallel chains), β-chitin (parallel chains),
and γ-chitin (the combination of parallel and anti-parallel chains) [22]. α - chitin have been
extracted from arthropods, fungi, and cysts of Entamoea [23]. β - chitin have been found in
pen of the Loligo squid, extracellular spines of the euryhaline diatoms [24,25],
pogonophore tubes [26], and the tube of vestimentiferan tube-worms [27] in well-defined
highly crystalline microfibrils. They are also found in extracellular fibers of diatoms, the
pens of squid [28] and the spines and chaetae of some annelids. γ-chitin have been found
in cocoon fibers of Ptinus beetle, the stomach of Loligo, stomachs of squid and in the
cocoons of beetles [4].

Studies have shown that α-chitin is the most stable and the most abundant form of chitin. It
is what contributes to the structural resistance in insect cuticles, shells of crabs, lobsters
and shrimp. α - chitin has strong inter-sheet and intra-sheet hydrogen bonding. β -chitin is
characterized by a weak intermolecular force [29] and has been shown to exhibit higher
reactivity and higher affinity for solvents than α-chitin [30–32]. β-chitin is soluble in formic
and some mineral acids. It is characterized by loose packing of molecules, which gives it
high affinity for various solvents and possibly enhanced reactivity compared to α-chitin.
They are also susceptible to swelling than α-chitin. It is expected that β –chitin will open a
new way to develop chitin chemistry and its applications. Most chitin related research has
been focused on α - chitin and its conversion to chitosan, very limited attention has been
paid to β-chitin. Most chitin related research has been focused on α - chitin and its
conversion to chitosan, very limited attention has been paid to β-chitin. Abdou et al. [33]
showed that XRD can be used to determine the change in structure from α to β chitin.
Brunner et al. [34] also showed that additional bands in FTIR spectra of β-chitin can be
used to differentiate between α and β chitin.

This study aims at investigating the variation in structure of chitin arising from variations in
treatment by examining structural and physico-chemical characteristics of the extracted
chitin. A three-level factorial design with acid and alkaline concentrations as factors is used
for the study. XRD, FTIR and SEM techniques are used for characterization of the
extracted chitin.

2
Materials and Methods
Chitin Extraction
Lissachatina fulica (Bowdich) and Tympanotonus fusatus var fusatus shells were scraped
free of loose tissues, washed in water, dried and ground to 250 μm in a ball mill.
Demineralization was carried out at room temperature (32 °C) by soaking 100 g of ground
samples in 1.5, 1.7 and 1.9 M HCl. For each concentration, the process was repeated
several times until evolution of gaseous effluent ceased and the number of baths (in terms
of acid volume) use was noted. The demineralized samples were washed with distilled
water to neutral (pH7.0), filtered and dried in the oven at 70 °C for 4 hours to constant
weights. Deproteinization was carried out by heating mineral - devoid samples in
concentrations of 0.4, 0.8 and 1.2 M NaOH solutions in a beaker at 100 °C for 1 hour with
constant stirring. At the end of this period, samples were filtered and soaked in fresh sets
of alkali solutions (0.4, 0.8 and 1.2M) for 18 hours at room temperature (32 °C). The
samples were washed with distilled water to pH 7.0, filtered and oven dried at 70 °C.
Depigmentation and bleaching was carried out by soaking extracted chitin in 1M H 2O2 for
24 hours at 32 °C. The chitin was then washed in distilled water and dried for 4 hours in an
oven at 70 °C before characterizations.

X-Ray Diffraction (XRD)

XRD was conducted using PAN analytical X’ Pert PRO MPD X-ray diffraction system
PW3040/60 machine at Soochow University, Suzhou, China. Samples were exposed to a
monochromatic Cu Kα radiation (λ = 1.5406 Ȧ ), operating at 40 kV and 40 mA. The
samples were registered in a zero background sample holder to avoid external
background interferences. The diffractograms were registered in the range of 7° - 90° (2θ)
in a step scan mode of 0.026261 at a counting time of 17.34 seconds per step. To
calculate structural parameters, the diffractograms were first normalized on the basis of
constant total intensity. The best fitted curves to the original diffraction pattern were
obtained using the Gaussian functions and then separated into each component peaks.
The ratio of area under the crystalline diffraction peaks to the total area under the
diffraction pattern is used as a measure of crystallinity. Crystallinity Index (CrI) was
calculated using Equation 1 [35,36], where Ac and At are the sum of the areas under the
crystalline diffraction peaks and the total area under the diffraction pattern, respectively.
𝐴𝑐
𝐶𝑟𝑦𝑡𝑎𝑙𝑙𝑖𝑛𝑖𝑡𝑦 𝐼𝑛𝑑𝑒𝑥 (𝐶𝑟𝐼) = ⁄𝐴 [1]
𝑡

3
Apparent size of crystallites (Dap[hkl]) was determined according to Scherrer equation (eq.
[2]) after the mathematical treatment of the peaks corresponding to its deconvolution and
application of the Gaussian function.
Κ𝜆
𝐷𝑎𝑝[ℎ𝑘𝑙] = [2]
𝛽𝑜 cos 𝜃
where K is a constant; 𝜆 (𝐴̇) is the wave length of the incident radiation; 𝛽𝑜 (rad) is the
width of the crystalline peak at half height and 𝜃 (rad) is half the Bragg angle
corresponding to the crystalline peak.

Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray Analysis (EDS)
An ASPEX 3020 model variable pressure SEM at Soochow University, Suzhou, China
operated with an electron intensity beam of 15kV was used to observe the morphological
features of samples. Samples to be observed were mounted on a conductive carbon
imprint and coated for 5 minutes to enable it conduct electricity.

Fourier Transform Infrared Spectroscopy (FTIR)

A Nicolet 6700 M spectrometer from Redeemers University, Ede, Nigeria in transmission
mode was used in carrying out FTIR spectra of samples. Ten milligram of fine samples
were dispersed in a matrix of KBr (500 mg), followed by compression at 22-30 MPa to
form pellets. The transmittance measurements were carried out in the 400-4000 cm-1
range at a resolution of 4 cm-1.

Degree of Deacetylation (DD)

The degree of deacetylation (DD) of the chitin samples was calculated using the baselines
proposed by Domszy and Roberts [37] in Equation 3.
(𝐴1655 /𝐴3450 ) × 100
𝐷𝐷 = 100 − [ ] ⋯ ⋯ ⋯ ⋯ [3]
1.33
A1655 and A3450 were the absorbance at 1655 cm -1 of the amide-I band as a measure of
the N-acetyl group content and 3450 cm-1 of the hydroxyl band as an internal standard.
The factor ‘1.33’ is the ratio of A1655 /A3450 for fully N acetylated chitin.

Results and Discussion

Demineralisation and Deproteinisation
Demineralisation was carried out until evolution of gaseous effluent stopped and the
number of acid baths were measured and recorded for each case. Results show that 3.1,

4
2.95 and 2.88 litres/100 g of acid was used for complete demineralisation of Periwinkle
shell powder with 1.5, 1.7 and 1.9 M HCl respectively. However, 2.8, 2.6 and 2.5 litres/100
g of acid was used for complete demineralisation of Snail shell powder with 1.5, 1.7 and
1.9 M HCl respectively. This indicates that a higher amount of acid was needed to
completely demineralise Periwinkle shell than Snail shell. This may be attributed to the
difference in structure of the shells. It is also clear that lower acid concentration requires
higher volumes and vice versa.

XRD Analysis of Periwinkle Shell Chitin

X-ray diffraction results for chitin extracted from Periwinkle shell are presented in Figure 1.
The XRD patterns of all Periwinkle-chitin (see Figure 1) show two sharp crystalline
reflections at 20.6o and 26.4o with varying intensities after normalisation. These peaks are
typical reflections of partial de-acetylated chitin [38–43].

The XRD spectra for all the samples does not show peaks at 19 and 9 o that are prominent
crystalline peaks for α- chitin [33,44,45]. The peaks between 20.5 – 20.8 o are attributed to
(020) of chitosan whereas the peaks between 26.3 – 26.6 are attributed to (013) of α –
chitin. This confirms that the treated samples contain highly de-acetylated chitin. Table 1
shows the effect of processing parameters on the structure of the extracted chitin. The
(020) and (013) crystals are large in size and do not follow a particular pattern. However,
the values presented here are within the range of those presented by Lavall et al. [28] and
Cho et al. [41]. The CrI in Table 1 indicates that all de-acetylated samples have CrI values
above 0.9. The values of crystallite size are in line with those of the Crystallinity Index
(CrI). One most important finding in Table 1 is the fact that some chitin possesses a
combination of high CrI (>0.98) and DD (>55 %). The combination of high crystalline order
and high DD is important in the development of chitin that can be used in bio-structural
applications such as scaffolds and implants.

5
XRD results of chitin extracted from Snail shell are in Figure 2. All samples show
prominent peaks between 20 - 210 and 26 - 270. The presence of peaks between 20.5 –
20.8 o are attributed to (020) of de-acetylated chitin, whereas the peaks between 26.3 –
26.6 are attributed to (013) of α – chitin. Table 2 shows the effect of processing
parameters on the structure of the extracted chitin. The (020) and (013) crystals are larger
than (110). It is noted that all samples possess high degree of order (>0.95), which also
agrees with the crystallite sizes. It is also observed that some samples have high
crystalline and high degree of de-acetylation characteristics.

De-acetylation Mechanism Relative to Treatment

The progress of de-acetylation of chitin during treatment can be monitored effectively by
examining the changes in the functional groups in relation to the treatment parameters.
Since studies have shown that some treatments can lead to desired structural
transformations, it is important to identify which of the treatment would support this and
under what conditions. The process of de-acetylation involves the removal of acetyl groups
from the molecular chain of chitin, leaving behind the amino group (-NH2). Figure 3 shows
the effect of acid and alkali concentrations on the amide related bands during de-
acetylation.

Figure 3a shows the effect of treatment on the OH – NH band of periwinkle shell chitin.
The Figure reveals that during demineralisation the mineral compounds in the periwinkle
shell were removed, leading to the release of embedded chitin. Increase in acid
concentration resulted to increase in the OH - NH band intensity. The bands at 2853 and
2926 cm-1 attributed to CH2 bending due to pyranose ring [49] are found in demineralised
samples showing that the samples contain some amount of chitin [50]. Treatment with
alkali led to a reduction in OH-NH band indicating the occurrence of de-acetylation. The
bands at 2853 and 2926 cm-1 are found to disappear indicating that the chitin undergoes a
transformation yielding a highly de-acetylated chitin. The band is found to decrease
proportionately with alkali concentration. Other studies have shown that chitin possess
inter-sheet and intra-sheet hydrogen bonding, which decline during deacetylation. Figures
3b and c show other prominent amide peaks and their variation with treatment.

6
Another important peak is the C=O secondary stretch of amide I (1653 cm-1) which
possess high absorbance for demineralised samples (Figure 3c). The absorbance
however, decreased with alkali treatment. This band is a prominent chitin band and is
always used as an indicator for progress in de-acetylation. A decrease in the band after
treatment shows that de-acetylation occurred [51]. In this study, the decrease in the band
is noticed after treatment with alkali. It is also noted that, this band is dependent on alkali
concentration. As the concentration increases the absorbance reduces. The 1542 cm–1
band corresponding to -NH deformation and stretching C-H of amide II band is found to
have high absorbance for acid treated samples. Treatment with 1.9 M HCl/0.8 M NaOH
caused the band to increase but decreased with further increase in concentration to 1.9 M
HCl/1.2 M NaOH. Increase in this band is an indication of increase in the amount of NH 2
groups [51,52]. The decrease of 1542 cm–1 band in 1.9 M HCl/1.2 M NaOH observed in
relation to 1653 cm–1 band indicates that as 1543 cm-1 band is increasing 1653 cm-1 is
decreasing. This occurrence is a confirmation that increasing alkali concentration resulted
to increase in the amount of NH2 groups present.

The presence of a doublet amide I band is usually attributed to α-chitin structure [28].
However, it is also important to state that the presence of 1640 cm-1 OH bending which
relates to the presence of absorbed water sometimes interfere with the doublet causing it
to become a broad band [53]. Treatment with 1.9 M HCl/0.8 M NaOH shows a different
spectral pattern, indicating the development of a structure different from those obtained
with acid extracted chitin and the de-acetylated chitin from 1.9 M HCl/1.2 M NaOH
treatment. The spectrum of 1.9 M HCl/0.8 M NaOH shows many narrower bands in the C-
O-C and C-O stretching vibrations in 1200 – 950 cm-1 region. This difference in pattern is a
confirmation of structural difference.

Physico-Chemical Properties of Chitin Extracted from Periwinkle

Figure 4 shows the functional groups present in the extracted periwinkle shell chitin
samples relative to the difference in treatment. It is clear that the treatments brought about
varying changes in the structure of the extracted samples. Firstly, the virgin samples show
distinct peaks at 1475 cm-1 and 860 cm-1 (see Figures 4),which are for calcite [54–56]. Low
intensity peaks also appear at 1786 and 719 cm-1 which have also been associated with
the presence of C𝑂3−2 [55,57]. A short band exists at 700 cm-1 attributed to NH out plane
bending. These peaks are all absent in all the treated samples indicating effective calcite
removal from the shells.
7
Figure 4 also shows the presence of the amide I doublet at 1661 and 1631 cm-1 assigned
to the C=O secondary stretch of chitin [52,56,58,59] in 1.5 M HCl/0.8 M HCl. This doublet
mode of amide I band has been attributed to the occurrence of inter-molecular hydrogen
bonds from C=O…HN and intra-molecular bonds from C=O…HOCH2 [58,60]. The doublet
is only pronounced in samples treated with 1.5 M HCl/0.8 M HCl and overlapped to form a
broad peak in other samples. The presence of 1640 cm-1 OH bending linked to the
presence of absorbed water interfere with this band making it to become a broad band
[53]. Treatment with higher concentration did not show appreciable absorption of this band
relative to others, which is an indication that the resulting structure after treatment may not
be α-chitin as observed in other treatments. On the other hand it could be argued that
treatment with 1.5 M HCl/0.4 M NaOH and 1.5 M HCl/0.8 M NaOH brought about higher
degree of structural changes (higher CrI) than treatment with 1.5 M HCl/1.2 M NaOH [52].

The band at 1563 cm–1 corresponds to -NH deformation and stretching C-H of amide II
band. The -CO-NH deformation and the -CH2 wagging [61,62] appears at 1309 cm-1. This
band has been termed amide III band [62–64]. The CH2 ending and CH3 deformation band
is found at 1378 cm-1 and CH bend and CH3 symmetric. deformation at 1412 cm-1
[48,53,61,65,66]. The presence of these peaks is linked to α-chitin [48]. Figure 4 shows
that these two bands are only pronounced in 1.5 M HCl/0.4 M NaOH and 1.5 M HCl/0.8 M
NaOH treated samples but absent in 1.5 M HCl/1.2 M NaOH processed samples. Thus,
the later treatment produces structural transformation of most of the chitin. The 1563 cm–1
occurs with a second small band at 1553 cm –1 only in 1.5 M HCl/0.8 M NaOH treated
samples but absent in others. The 1563 and 1657 cm –1 bands have nearly the same
absorbance in the normalised spectra at 1.5 M HCl/0.8 M NaOH and 1.5 M HCl/0.4 M
NaOH treatments. These peaks are directly related to the degree of de-acetylation of chitin
and suggest that the two treatments produced similar structure.

For 1.5 M HCl/1.2 M NaOH treated sample, 1657 cm–1 band is higher than 1563 cm–1
band showing a structural difference with substantial de-acetylation as shown in Table 1. A
distinct band at 3263 cm-1 appears in samples treated with 1.5 M HCl/0.8 M NaOH
different from others. This band has been attributed to NH stretching vibrations [41,48,67].
Similarly a well resolved band appears at 3103 cm-1 in 1.5 M HCl/0.8 M NaOH treated
samples. This band is narrow in 1.5 M HCl/0.4 M NaOH treated samples and completely
absent in 1.5 M HCl/1.2 M NaOH treated samples. This band is recognized as H-bonded

8
NH groups, characteristic of secondary amides and is associated with α-chitin structure
and an overtone of amide I. Most prominent peaks in all treated samples are observed in
the range 1020 – 1160 cm-1. The peaks within this region are highly saturated which may
be attributed to the presence of C-O-C, C-OH and C-C vibration modes [53,68]. These
peaks are the highest peaks showing a large increase over virgin samples. It is believed
that these peaks only increase in response to reductions in de-acetylated fraction [53,68].
1156 cm-1 is attributed to COC bridge stretching [48], 1077 cm-1 to the C3-OH stretching of
hydroxyl groups and 1027 cm-1 to C6-OH stretching of hydroxyl groups [69–71].

Short intensity bands at 694 and 777 cm -1 attributed to OH and NH out of plane bending
are found in all samples but with differences in intensities. These bands are prevalent in α-
chitin [60,72]. Their decreasing absorbance with increase in concentration of alkali show
that the chitin fraction decreases with increase in alkali concentration during extraction. CH
bonds appearing at 2959 and 2934 cm -1 have been reported to be prevalent in chitin [50].
In this study, they show high absorbance for 1.5 M HCl/0.4 M NaOH and 1.5 M HCl/0.8 M
NaOH. However, they are almost absent in 1.5 M HCl/1.2 M NaOH, confirming that
increase in alkali concentration leads to increase in DD as revealed in Table 1. Other
peculiar bands are those at 2877 cm-1 attributed to methylene groups in chitin [50,
,65,70,73], 3449 cm-1 amide group peak [71], 1260 and 1208 cm-1 attributed to complex
variations of NHCO for secondary amides [52].

Generally, it is evident from these Figures that secondary amide peaks such as 3449,
3263, 3103, 1657, 1553, 1309 and 1261 cm -1 are very prominent in 1.5 M HCl/0.4 M
NaOH treated samples, moderate in 1.5 M HCl/0.8 M NaOH treated samples and nearly
non-existent in 1.5 M HCl/1.2 M NaOH treated samples. These peaks have been linked to
chitin [50]; their presence and high absorbance signifies that the samples are α-chitin. This
shows that 1.5 M HCl/0.4 M NaOH treatment condition does not impart full de-acetylation
and the samples contain considerable amount of chitin. Samples treated with 1.5 M
HCl/0.8 M NaOH on the other hand contain small amount of chitin than the later.
Therefore, increasing the concentration of alkali leads to increase in de-acetylation.
Similarly, C-H bonds related bands such as 2962, 2934, 2890, 1380 and 1205 cm -1
possess very high absorbance and broad peaks in 1.5 M HCl/0.4 M NaOH treated
samples, partially present in 1.5 M HCl/0.8 M NaOH treated. However, some of these
bands are absent in 1.5 M HCl/1.2 M NaOH treated samples while others possess very
low absorbance. This buttress the findings that samples treated with low alkali

9
concentration possess higher fraction of chitin compared to others. Moreover, it is evident
from Figure 4 that the OH band in 2600 - 3600 cm-1 range moved to lower frequency with
increase in alkali concentration pointing to increase in degree of de-acetylation (DD) and
the presence of disordered structure [74–76].

Figure 5 shows the FTIR spectrum of Periwinkle samples treated with 1.7 M HCl with
different alkali concentrations in comparison with virgin Periwinkle shell. The spectrum is
closely related to that reported in Figure 4 but with certain differences in peaks. The
prominent amide I band attributed to C=O secondary amide stretch appears at 1654 cm -1
in 1.7 M HCl/0.4 M NaOH treated samples but are less prominent in 1.7 M HCl/0.8 M
NaOH and 1.7 M HCl/1.2 M NaOH treated samples [59]. This is an indication that
increasing alkali concentration produced increase in deacetylation. This scenario is
repeated for the peaks at 3387 and 3305 cm -1, which are OH and NH stretching of amine
groups [56,64,77]. Amide III bands also appear at 1253 and 1230 cm -1 [54], but are only
present in 1.7 M HCl/0.4 M NaOH treated samples and disappears in others. Other peaks
that are present in 1.7 M HCl/0.4 M NaOH treated samples but absent in others include
the CH and CH3 symmetric stretch at 1386 cm-1, C-N groups and CH2 wagging stretching
at 1338 cm-1 and CH2 ending and CH3 deformation at 1456 cm-1 [61,66]. The OH out of
plane bending at 777 cm-1 and NH out of plane bending at 694 cm -1 [56,60,72] are also
prominent in all samples. However, 1.7 M HCl/0.4 M NaOH treated sample possess higher
absorbance than others. Similarly, peaks at 3061 and 2932 cm-1 attributed to CH3
symmetric stretch and CH2 asymmetric stretch are also prominent in 1.7 M HCl/0.4 M
NaOH treated samples than in others.

The presence of 1654 cm-1 amide I peak in all 1.7 M HCl shows that all samples were not
fully de-acetylated. However, a difference in absorbance is noted. The absorbance is
higher in 1.7 M HCl/0.4 M NaOH than in others. This is an indication that 1.7 M HCl/0.4 M
NaOH treated samples possess higher chitin fraction than the others. This means that
increasing alkali concentration increases the degree of de-acetylation. It is also important
to note that this peak does not appear as doublet as seen in previous samples. A more
pronounced doublet is an indication of a higher degree of morphological arrangement
(higher CrI) [52]. On the other hand, it is observed that secondary amide related peaks
such as 3443, 3265, 3105, 1654, and 1253 cm -1 possess high absorbance in 1.7 M
HCl/0.4 M NaOH treated samples but are either very low or absent in 1.7 M HCl/0.8 M
NaOH treated and 1.7 M HCl/1.2 M NaOH treated samples respectively.

10
Figure 6 shows the effect of further increase in acid concentration (1.9 M HCl) on the FTIR
spectra of periwinkle chitin treated with different concentration of alkali. The spectra show
distinct chitin peaks in all treated samples but with differences in structure and
absorbance. C=O secondary amide stretch of amide I is shown at 1669 and 1638 cm -1
[66]. The peaks within this region are clustered in 1.9 M HCl/0.4 M NaOH and 1.9 M
HCl/0.8 M NaOH treated samples. However, characteristic peaks appear at 1669, 1648
and 1638 cm-1. These peaks are not prominent in 1.9 M HCl/1.2 M NaOH treated samples.
The peak at 1648 cm-1 may be attributed to the presence of bound water in the sample
[53]. The amide I peak also appear as a single peak and not as doublet as observed in 1.9
M HCl/0.4 M NaOH. The decrease in absorbance of the amide I peak with increase in
alkali concentration indicates increase in degree of de-acetylation. The range of 1557 -
1500 cm-1 show cluster of peaks in 1.9 M HCl/0.4 M NaOH and 1.9 M HCl/0.8 M NaOH
treated samples with distinct peaks appearing at 1557, 1539, 1520 and 1508 cm-1. The
peaks at 1557, 1539 cm-1 are attributed to the NH bend and CN stretch of amide II bands
[66]. In 1.9 M HCl/1.2 M NaOH these peaks are absent. The double band at 1438 and
1417 cm-1 in 1.9 M HCl/0.4 M NaOH samples are attributed to CH2 ending and CH3
deformation [56,78].

The bands at 1253 and 1231 cm-1 attributed to amide III [54] are distinct in 1.9 M HCl/0.4
M NaOH and 1.9 M HCl/0.8 M NaOH samples but not in 1.9 M HCl/1.2 M NaOH. These
absorbance bands are C-H bands which are peculiar to α-chitin. The broad OH and NH
stretch bands also appear between 3000 and 4000 cm-1 but with marginal absorbance.
The absorbance is found to decrease with increase in alkali concentration.

Deacetylation and physico-chemical properties of Snail shell chitin were also examined.
The results show the same trend with that of Periwinkle shell chitin. The data for Snail
shell chitin are given in supplementary data file.

Conclusion
Physico-chemical properties and de-acetylation progress during extraction of chitin from
Periwinkle and Snail shell have been presented in the study. It was realised that;
1. Certain combinations of acid and base concentrations produces chitin derivatives
with a combination of high CrI (>0.98) and DD (>50 %).

11
 2. The structures of chitin extracted are in close relation to the concentration of acid
and alkali used in the extraction.
3. The XRD shows that all treated chitin contain a combination of chitosan and α -
chitin.
4. All Snail shell chitin extracted from this study possess Crystallinity Index (CrI) > 0.9.
5. It is also noted that Snail shell chitin are easily amendable to de-acetylation than
Periwinkle shell chitin.

Acknowledgement
The research group acknowledge with grateful thanks the University of Lagos for providing
the University Research Grant (CRC NO.2015/19) used for this study. We are also grateful
for the assistance provided by Department of Metallurgical and Materials Engineering,
University of Lagos to use the Metallurgy Laboratory.

References
[1] M.H. Struszczyk, Polimery 47 (2002) 396–403.
[2] P.K. Dutta, J. Dutta, V.S. Tripathi, Journal of Scientific and Industrial Research 63
(2004) 20–31.
[3] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras,
Current Chemical Biology 3 (2009) 203–230.
[4] M. Rinaudo, Progress in Polymer Science 31 (2006) 603–632.
[5] A. Laaraibi, I. Charhouf, A. Bennamara, A. Abourriche, M. Berrada, J. Mater. Environ.
Sci. 6 (2015) 3511–3516.
[6] T. Chandy, C.P. Sharma, Biomaterials, Artificial Cells and Artificial Organs 18 (2009)
1–24.
[7] S. Hirano, in: M.R. El-Gewely (Ed.), Biotechnology Annual Review, Elsevier, 1996, pp.
237–258.
[8] C. Peniche-Covas, L.W. Alvarez, W. Argüelles-Monal, J. Appl. Polym. Sci. 46 (1992)
1147–1150.
[9] K. Nair, P. Madhavan, Fishery Technology 21 (1984) 109–112.
[10] L. Arof, R. Subban, S. Radhakrishna (Eds.), Polymer and Other Advanced Materials:
Emerging Technologies and Business, Plenum Press, New York, 1995.
[11] J. Xu, S.P. McCarthy, R.A. Gross, D.L. Kaplan, Macromolecules 29 (1996) 3436–
3440.
[12] Y. Koyama, A. Taniguchi, J. Appl. Polym. Sci. 31 (1986) 1951–1954.
[13] K. Kurita, Y. Koyama, A. Taniguchi, J. Appl. Polym. Sci. 31 (1986) 1169–1176.
[14] H. Kaneko, A. Shirai, K. Takahasi, Y. Miura, K. Nitta, S.-I. Nishimura, N. Nishi, S.
Tokura, A. Tsutsumi, Polym. Int. 36 (1995) 365–372.
[15] D. Horton, J. Lehmann, Carbohydrate research 61 (1978) 553–556.
[16] S. MIYAZAKI, K. Ishii, T. NADAI, CHEMICAL & PHARMACEUTICAL BULLETIN 29
(1981) 3067–3069.
12
[17] E.I. Rabea, M.E.-T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut,
Biomacromolecules 4 (2003) 1457–1465.
[18] C.R. Fontana, D.S. dos Santos Junior, J.M. Bosco, D.M. Spolidorio, R.A. Chiérici
Marcantonio, Drug Delivery 15 (2008) 417–422.
[19] X.H. Wang, D.P. Li, W.J. Wang, Q.L. Feng, F.Z. Cui, Y.X. Xu, X.H. Song, M. van der
Werf, Biomaterials 24 (2003) 3213–3220.
[20] H.S. Blair, J. Guthrie, T.-K. Law, P. Turkington, J. Appl. Polym. Sci. 33 (1987) 641–
656.
[21] S. Nakatsuka, A.L. Andrady, J. Appl. Polym. Sci. 44 (1992) 17–28.
[22] E. Khor, L.Y. Lim, Biomaterials 24 (2003) 2339–2349.
[23] R. Minke, J. Blackwell, Journal of Molecular Biology 120 (1978) 167–181.
[24] W. Herth, W. Barthlott, Journal of Ultrastructure Research 68 (1979) 6–15.
[25] J. Blackwell, K.D. Parker, K.M. RUDALL, Journal of Molecular Biology 28 (1967) 383–
385.
[26] J. Blackwell, Biopolymers 7 (1969) 281–298.
[27] F. Gaill, J. Persson, J. Sugiyama, R. Vuong, H. Chanzy, Journal of Structural Biology
109 (1992) 116–128.
[28] R.L. Lavall, O.B. Assis, S.P. Campana-Filho, Bioresource technology 98 (2007) 2465–
2472.
[29] K.M. RUDALL, W. KENCHINGTON, Biological Reviews 48 (1973) 597–633.
[30] K. Kurita, K. Tomita, T. Tada, S. Ishii, S.-I. Nishimura, K. Shimoda, J. Polym. Sci. A
Polym. Chem. 31 (1993) 485–491.
[31] K. Kurita, K. Tomita, S. Ishii, S.-I. Nishimura, K. Shimoda, J. Polym. Sci. A Polym.
Chem. 31 (1993) 2393–2395.
[32] K. Kurita, S. Ishii, K. Tomita, S.-I. Nishimura, K. Shimoda, J. Polym. Sci. A Polym.
Chem. 32 (1994) 1027–1032.
[33] E.S. Abdou, K.S.A. Nagy, M.Z. Elsabee, Bioresource technology 99 (2008) 1359–
1367.
[34] E. Brunner, H. Ehrlich, P. Schupp, R. Hedrich, S. Hunoldt, M. Kammer, S. Machill, S.
Paasch, V.V. Bazhenov, D.V. Kurek, T. Arnold, S. Brockmann, M. Ruhnow, R. Born,
Journal of Structural Biology 168 (2009) 539–547.
[35] A.K. Gupta, R.P. Singhal, J. Polym. Sci. Polym. Phys. Ed. 21 (1983) 2243–2262.
[36] G. Nansé, E. Papirer, P. Fioux, F. Moguet, A. Tressaud, Carbon 35 (1997) 175–194.
[37] J.G. Domszy, G.A.F. Roberts, Makromol. Chem. 186 (1985) 1671–1677.
[38] W. Sajomsang, P. Gonil, Materials Science and Engineering: C 30 (2010) 357–363.
[39] P. Gonil, W. Sajomsang, Int J Biol Macromol 51 (2012) 514–522.
[40] B.A. La Juárez-de Rosa, P. Quintana, P.-L. Ardisson, J.M. Yáñez-Limón, J.J.
Alvarado-Gil, Journal of Materials Science 47 (2012) 990–998.
[41] Y.-W. Cho, J. Jang, C.R. Park, S.-W. Ko, Biomacromolecules 1 (2000) 609–614.
[42] M.-K. Jang, B.-G. Kong, Y.-I. Jeong, C.H. Lee, J.-W. Nah, J. Polym. Sci. A Polym.
Chem. 42 (2004) 3423–3432.
[43] M.-T. Yen, J.-H. Yang, J.-L. Mau, Carbohydrate Polymers 75 (2009) 15–21.
[44] F.A. Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Carbohydrate Polymers 77
(2009) 410–419.
[45] M. Kaya, I. Sargin, K.Ö. Tozak, T. Baran, S. Erdogan, G. Sezen, Int J Biol Macromol
61 (2013) 459–464.
13
[46] T. Liu, B. Li, X. Zheng, S. Liang, X. Song, B. Zhu, J.F. Kennedy, J. Xia, Carbohydrate
Polymers 82 (2010) 753–760.
[47] S. Liu, J. Sun, L. Yu, C. Zhang, J. Bi, F. Zhu, M. Qu, C. Jiang, Q. Yang, Molecules
(Basel, Switzerland) 17 (2012) 4604–4611.
[48] Y. Wang, Y. Chang, L. Yu, C. Zhang, X. Xu, Y. Xue, Z. Li, C. Xue, Carbohydrate
Polymers 92 (2013) 90–97.
[49] P. Praveen, V. Rao, Procedia Materials Science 5 (2014) 1155–1159.
[50] K. Velde, P. Keikens, Carbohydrate Polymers 58 (2004) 409–416.
[51] A.T. Paulino, J.I. Simionato, J.C. Garcia, J. Nozaki, Carbohydrate Polymers 64 (2006)
98–103.
[52] D. Zvezdova, НАУЧНИ ТРУДОВЕ НА РУСЕНСКИЯ УНИВЕРСИТЕТ 49 (2010) 65–
69.
[53] M. Duarte, M. Ferreira, M. Marvão, J. Rocha, Int J Biol Macromol 31 (2002) 1–8.
[54] M.A. Rahman, J. Halfar, Scientific Reports 4 (2014) 1-11.
[55] N. Udomkan, P. Limsuwan, Materials Science and Engineering: C 28 (2008) 316–319.
[56] O.P. Gbenebor, S.O. Adeosun, G.I. Lawal, S. Jun, Materials Chemistry and Physics
184 (2016) 203–209.
[57] S.Y. Aku, D.S. Yawas, P.B. Madakson, S.G. and Amaren, The Pacific Journal of
Science and Technology 13 (2012) 57–63.
[58] J. Brugnerotto, J. Lizardi, F. Goycoolea, W. Argüelles-Monal, J. Desbrières, M.
Rinaudo, Polymer 42 (2001) 3569–3580.
[59] M. Kaya, T. Baran, A. Mentes, M. Asaroglu, G. Sezen, K.O. Tozak, Food Biophysics 9
(2014) 145–157.
[60] Y. Shigemasa, H. Matsuura, H. Sashiwa, H. Saimoto, Int J Biol Macromol 18 (1996)
237–242.
[61] M. Kaya, T. Baran, Int J Biol Macromol 75 (2015) 7–12.
[62] S.M.B.d. Andrade, R. Ladchumananandasivam, B.G.d. Rocha, D.D. Belarmino, A.O.
Galvão, MSA 03 (2012) 495–508.
[63] M. Kaya, S. Erdogan, A. Mol, T. Baran, Int J Biol Macromol 72 (2015) 797–805.
[64] M. Kaya, O. Seyyar, T. Baran, S. Erdogan, M. Kar, Int J Biol Macromol 65 (2014) 553–
558.
[65] T.S. Trung, H.N.D. Bao, International Journal of Carbohydrate Chemistry 2015 (2015)
1–6.
[66] M. Kaya, T. Baran, S. Erdogan, A. Mentes, M.A. Ozusaglam, Y.S. Cakmak, Materials
science & engineering. C, Materials for biological applications 45 (2014) 72–81.
[67] X. Hu, Y. Du, Y. Tang, Q. Wang, T. Feng, J. Yang, J.F. Kennedy, Carbohydrate
Polymers 70 (2007) 451–458.
[68] T. Wanjun, W. Cunxin, C. Donghua, Polymer Degradation and Stability 87 (2005)
389–394.
[69] P. Kolhe, R.M. Kannan, Biomacromolecules 4 (2003) 173–180.
[70] A.-J. Zhang, Q.-L. Qin, H. Zhang, H.-T. Wang, X. Li, Czech J. Food Sci. 29 (2011)
616–623.
[71] Z. Zakaria, Z. Izzah, M. Jawaid, A. Hassan, BioResources 7 (2012).
[72] I. Rumengan, E. Suryanto, R. Modaso, S. Wullur, T.E. Tallei, D. Limbong,
International Journal of Fisheries and Aquatic Sciences 3 (2014) 12–18.

14
[73] R.M. Abdel-Rahman, R. Hrdina, A.M. Abdel-Mohsen, M.M.G. Fouda, A.Y. Soliman,
F.K. Mohamed, K. Mohsin, T.D. Pinto, Int J Biol Macromol 80 (2015) 107–120.
[74] H. Gocho, Carbohydrate Polymers 41 (2000) 87–90.
[75] B. Focher, P.L. Beltrame, A. Naggi, G. Torri, Carbohydrate Polymers 12 (1990) 405–
418.
[76] Y. Zhang, C. Xue, Y. Xue, R. Gao, X. Zhang, Carbohydrate research 340 (2005)
1914–1917.
[77] L.O. AHMAD, D. PERMANA, S.H. WAHAB, L.O. SABARWATI, A.N. RAMADHAN, U.
RIANSE, Advances in Environmental and Geological Science and Engineering 38
(2015) 373–378.
[78] M. Kaya, V. Baublys, I. Satkauskiene, B. Akyuz, E. Bulut, V. Tubelyte, Int J Biol
Macromol 79 (2015) 126–132.

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Figure 1: X-ray diffraction spectrum of Periwinkle shell chitin

16
Figure 2: X-ray diffraction spectrum of Snail shell chitin

Figure 3a: Effect of acid and alkali concentration on amide specific bands in 2600 – 3600
cm-1 range

17
Figure 3b: Effect of acid and alkali concentration on amide specific bands in 850 – 1250
cm-1 range

Figure 3c: Effect of acid and alkali concentration on amide specific bands in 1400 – 1800
cm-1 range

18
Figure 4: FTIR of treated Periwinkle chitin within the range of (a) Virgin sample (b) 1.5
MHCl/0.4 MNaOH (c) 1.5 MHCl/0.8 MNaOH (d) 1.5 MHCl/1.2 MNaOH treated samples

19
Figure 5: FTIR of treated Periwinkle chitin within the range of (a) Virgin sample (b) 1.7
MHCl/0.4 MNaOH (c) 1.7 MHCl/0.8 MNaOH (d) 1.7 MHCl/1.2 MNaOH treated samples

20
Figure 6: FTIR of treated Periwinkle chitin within the range of (a) Virgin sample (b) 1.9
MHCl/0.4 MNaOH (c) 1.9 MHCl/0.8 MNaOH (d) 1.9 MHCl/1.2 MNaOH treated samples

Table 1: Structural Parameters Calculated from XRD patterns of Periwinkle shell Chitins
Sample 2𝜃 (020 of da (101) 2𝜃 (013 CrI DD (%)
a
chitosan) (Å) of chitin) d (013)
1.5 MHCl+0.4 MNaOH 20.64 48.8 26.41 39.6 0.971 52
1.5 MHCl+0.8 MNaOH 20.70 42.5 26.49 42.6 0.987 61
1.5 MHCl+1.2 MNaOH 20.59 38.9 26.41 51.6 0.996 67
1.7 MHCl+0.4 MNaOH 20.81 41.9 26.58 32.4 0.989 53
1.7 MHCl+0.8 MNaOH 20.70 53.6 26.49 52.6 0.997 64
1.7 MHCl+1.2 MNaOH 20.66 53.2 26.46 50.6 0.895 77
1.9 MHCl+0.4 MNaOH 20.73 27.2 26.51 29.6 0.879 64
1.9 MHCl+0.8 MNaOH 20.62 49.3 26.40 46.1 0.994 55
1.9 MHCl+1.2 MNaOH 20.66 64.6 26.45 63.1 0.936 63
*da= crystallite size, DD = Degree of De-acetylation, CrI: Crystallinity Index

Table 2: Structural Parameters Calculated from XRD patterns of Snail shell Chitins

21
Sample 2𝜃 (020 CrI DD
2𝜃 (013 da (013) of da (020) da (110) (%)
of chitin) (Å) chitosan) (Å) 2𝜃 (110) (Å)
1.5 MHCl+0.4 MNaOH 26.34 44.3 20.58 81.1 18.91 70.9 0.979 53
1.5 MHCl+0.8 MNaOH 26.30 51.8 20.52 46.3 18.95 65.7 0.957 51
1.5 MHCl+1.2 MNaOH 26.29 36.0 20.51 34.1 18.99 72.6 0.930 60
1.7 MHCl+0.4 MNaOH 26.44 38.1 20.66 43.0 19.10 72.0 0.976 52
1.7 MHCl+0.8 MNaOH 26.28 44.7 20.50 44.2 18.90 72.2 0.983 58
1.7 MHCl+1.2 MNaOH 26.46 35.0 20.69 34.6 19.07 64.1 0.976 56
1.9 MHCl+0.4 MNaOH 26.46 42.7 20.71 46.8 19.05 69.7 0.952 50
1.9 MHCl+0.8 MNaOH 26.30 47.8 20.51 49.2 18.92 75.7 0.969 46
1.9 MHCl+1.2 MNaOH 26.34 47.0 20.56 48.3 19.03 88.2 0.976 56

22