study on amylose iodine complex from cassava starch by ...cassava starch using 1, 2, 3 and 4 ml of...
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Study on Amylose Iodine Complex from Cassava
Starch by Colorimetric Method
Sirinat Boonpo and Sukjit Kungwankunakorn Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
Email: {sirinat.yoy, sukjitk}@gmail.com
Abstract—The absorbance pattern of amylose iodine
complex from cassava starch sample was examined by
scanning of UV visible spectra between 200-800 nm. The
cassava starch sample was analyzed at the different
conditions to investigate maximum wavelength and
absorbance. To study about the elimination of lipid from
cassava starch, the defatting process was operated on 3
types of solvent (methanol, ethanol, and petroleum ether) by
stirring for 24 hours with solvent. The result indicated that
the maximum wavelength of amylose iodine complex of
cassava starch sample depended on the amount of iodine
that was used to form amylose iodine complex. Moreover,
the amount of iodine has the effect on absorbance of
amylose iodine complex. The analysis of amylose iodine
complex for defatted starch provided the higher absorbance
than that from nondefatted starch.
Index Terms—amylose, cassava, colorimetric method, starch
I. INTRODUCTION
Carbohydrate is a major staple food for people around
the world. It is one of biopolymer that contains a number
of glucose units. The combination of glucose units supply
two main structures as amylose and amylopectin [1] and
[2]. The ratio of amylose and amylopectin are the
significant factor to indicate quality of starch because it
controls starch physicochemical properties. Amylose is a
linear structure of carbohydrate whereas amylopectin is
branched structure. The ratio of amylose and amylopectin
in starches is depended on the source [3] and [4]. Cassava
(Manihot esculenta Crantzis) is one of the most
significant source of carbohydrate. The root of cassava is
the important part that contains numerous carbohydrate.
It composes of moisture, carbohydrate, fat, fiber, protein
and trace amount of vitamins and minerals [5].
For estimation of amylose content in starch sample, the
blue amylose iodine complex was applied. This method
was known as colorimetric method that has been widely
used in many laboratory of agriculture until nowadays.
The principle based on the forming of complex between
amylose and iodine that stains blue solution [6]-[8]. After
that quantitative analytical technics such as
potentiometric titration [9] and [10] or ultraviolet-visible
spectrophotometry [11]-[13] were operated to determine
the blue amylose iodine complex that related to amylose
Manuscript received January 19, 2017; revised June 24, 2017.
content. Hence, the forming process of amylose iodine
complex is very importance to investigate.
Commonly, starch from the different source not only
provide different amount but also a dissimilar property of
amylose that is the noteworthy indicator of amylose
content determination. Therefore, the suitable amount of
iodine should be studied for each type of starch sample to
acquire accurate data. However, the forming of amylose
iodine complex has a problem from natural interferences
which are the components of starch such as long
branched amylopectin, protein and lipid. These
components interfere the efficiency of amylose
determination by colorimetric method. As a result, the
improvement of sample preparation process is necessary
for increasing the efficience. Lipid is one of natural
interference in amylose determination. It binds with
amylose structure in nature that compete with iodine to
form complex. It should be eliminated from sample
before it analyzing by colorimetric method [14].
II. MATERIALS AND METHODS
A. Materials
All of cassava starch sample in this work was acquired
from the markets in Thailand. The sample was dried in
oven at 105°C for 24 hours and kept in desiccator. The
amylose standard from potato was purchased from Sigma
Chemical and was used for the investigation of amylose
iodine complex color. The 0.2% of iodine reagent was
prepared by mixing 0.2 g of iodine and 2.0 g of potassium
iodide. Then the volume of iodine solution was made up
to 100 ml with deionized water.
B. Starch Defatting
For study on the suitable condition to remove lipid
from starch sample, 3 types of solvent (methanol, ethanol,
and petroleum ether) were applied by solvent extraction.
The starch sample was stirred with each type of solvent
for 24 hours by magnetic stirrer. After that the mixed
solution was filtrated and air dried. The starch sample
was heated in oven at 105°C for 24 hours and kept in
desiccator.
C. Preparation of Amylose Iodine Complex
The analysis of blue amylose iodine complex of
cassava starch sample was adapted from the method of
McCready (1943) [15]. A 100 mg of cassava starch
Journal of Advanced Agricultural Technologies Vol. 4, No. 4, December 2017
©2017 Journal of Advanced Agricultural Technologies 345doi: 10.18178/joaat.4.4.345-349
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sample was dissolved with 1 ml of 95% ethanol and 10
ml of 1 M sodium hydroxide. After that the sample was
refrigerated at 4°C for 24 hours then volume of sample
was adjusted to 100 ml with deionized water at the same
temperature. The prepared solution was allowed to stand
for 16-18 hours at 4°C. The 5 ml of the prepared solution
was used to develop the blue amylose iodine complex.
The pH of the prepared solution was adjusted by 0.05 M
of hydrochloric acid. After that 0.2% iodine reagent was
added into the solution and the volume was made up to
100 ml with deionized water.
D. Characterization of Defatted Starch
The UV visible spectrometer (Perkin Elmer, model
Lambda 25) was operated to study the spectra of amylose
iodine complex from cassava starch sample. To
investigate the effect of iodine amount, a 0.2% iodine was
varied from 1 to 4 ml to form amylose iodine complex.
Then the complexed solutions were analyzed by UV
visible spectrometer in scan mode. The solution of
amylose iodine complex from nondefatted and defatted
cassava starches using 2 ml of 0.2% iodine were analyzed
III. RESULTS AND DISCUSSION
A. The Effect of Iodine Volume for the Characteristic of
Amylose Iodine Complex Eferences
The amylose iodine complex using different amount of
iodine reagent was investigated by the adapted method of
McCready using 0.2% iodine as a reagent. The 3 types of
cassava starch from the markets were used as studied
samples. A 5 ml of the solution including 100 mg of
cassava starch sample in total volume of 100 ml, was
used for developing the blue complex. After that the
prepared solution was analyzed by UV visible
spectrometer. The spectra of amylose iodine complex of
cassava starch using 1, 2, 3 and 4 ml of 0.2% iodine
volume were investigated by UV visible spectrometer in
scan mode between 200-800 nm. The spectra are shown
in Fig. 1-Fig. 3.
The spectra show that the maximum wavelengths of
amylose iodine complex shift to shorter wavelength when
increase amount of 0.2% iodine reagent. The range of
maximum wavelength and absorbance at the maximum
wavelength of amylose iodine complex using different
0.2% iodine reagent volume from 3 types of cassava
starch samples are shown in Table I.
Figure 1. The spectra of amylose iodine complex of cassava starch sample 1, ((a), (b), (c), (d) are 1, 2, 3 and 4 ml of 0.2% iodine volume to
form the blue complex, respectively).
Figure 2. The spectra of amylose iodine complex of cassava starch sample 2, ((e), (f), (g), (h) are 1, 2, 3 and 4 ml of 0.2% iodine volume to
form the blue complex, respectively.
Figure 3. The spectra of amylose iodine complex of cassava starch sample 3, ((i) ,(j), (k), (l) are 1, 2, 3 and 4 ml of 0.2% iodine volume to
form the blue complex, respectively.
TABLE I. THE MAXIMUM WAVELENGTH AND ABSORBANCE AT MAXIMUM WAVELENGTH OF AMYLOSE IODINE COMPLEX
Sample I2 1.00 ml* I2 2.00 ml* I2 3.00 ml* I2 4.00 ml*
Abs at λmax λmax (nm) Abs at λmax λmax (nm) Abs at λmax λmax (nm) Abs at λmax λmax (nm)
1 X̅
SD 0.7406 0.0065
617-621 0.8591 0.0040
607-609 0.9064 0.0129
597-603 0.9545 0.0081
596-599
Journal of Advanced Agricultural Technologies Vol. 4, No. 4, December 2017
©2017 Journal of Advanced Agricultural Technologies 346
in fixed mode for study about absorbance. The surface
morphology of the nondefatted and defatted cassava
starch samples were investigated by Scanning Electron
Microscope (SEM; JOEL, model JSM-5910). The sample
was dried in oven at 105℃ for 24 hours and kept in
desiccator before it was examined by SEM.
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% RSD 0.8842 0.4647 1.4282 0.8522
2
X̅
SD % RSD
0.8035
0.0069 0.8640
609-613 0.9161
0.0053 0.5739
601-605 0.9727
0.0139 1.4320
595-600 1.0048
0.0072 0.7175
591-597
3
X̅ 0.7826 616-621 0.8960 603-608 0.9475 598-603 0.9821 594-598
SD 0.0092 0.0097 0.0159 0.0178
% RSD 1.1731 1.0866 1.6777 1.8170 *N=7
The 1 ml of iodine reagent provided the maximum
wavelength in the range of 617-621 nm, 609-613 nm and
616-621 nm for sample 1, 2 and 3, respectively. The 2 ml
of iodine reagent provided the maximum wavelength in
the range of 607-609 nm, 601-605 nm and 603-608 nm
for sample 1, 2 and 3, respectively. The 3 ml of iodine
reagent provided the maximum wavelength in the range
of 597-603 nm, 595-600 nm and 598-603 nm for sample
1, 2 and 3, respectively and 4 ml of iodine reagent
provided the maximum wavelength in the range of 596-
599 nm, 591-597 nm and 594-598 nm for sample 1, 2 and
3, respectively. The absorbance of the blue complex from
3 types of cassava samples with 1, 2, 3 and 4 ml of 0.2%
iodine were specific detected at 610 nm and the average
(X̅) of the absorbance at 610 nm was compared by
statistics method (Mann-Whitney). The result in Table II
indicated that the adding of different 0.2% iodine volume
from 1- 4 ml in the sample that contain the same amount
of amylose, provided statistically significant difference in
absorbance.
TABLE II. THE ABSORBANCE OF AMYLOSE IODINE COMPLEX AT 610
NM USING DIFFERENT VOLUME OF 0.2% IODINE
Sample
Absorbance at 610 nm
I2 1.00 ml* 2.00 ml* 3.00 ml* 4.00 ml*
X̅ 0.7375a 0.8591b 0.9009c 0.9486d
1 SD 0.0068 0.0038 0.0133 0.0080
%RSD 0.9287 0.4464 1.4738 0.8432
X̅ 0.8034d 0.9138e 0.9667f 0.9959g
2 SD 0.0069 0.0054 0.0138 0.0079
%RSD 0.8538 0.5902 1.4279 0.7927
X̅ 0.7817h 0.8949i 0.9437j 0.9744k
3 SD 0.0093 0.0098 0.0160 0.0174
%RSD 1.1863 1.0937 1.6908 1.7901
*N=7
An alphabet is the different data of the average absorbance that were analyzed by Mann-Whitney method: a difference of alphabet mean that
average absorbance is difference.
However, the observation of amylose iodine complex
solution color showed a green tone when used 0.2%
iodine more than 2 ml. In some cases, the solution
changed to green color by adding only 2 ml of 0.2%
iodine. The scanning of UV visible spectra of amylose
iodine complex, as shown in Fig. 1-Fig. 3, illustrated the
increasing of disturbance in ultraviolet region (200-400
nm) when increase 0.2% iodine reagent volume. The
using of iodine reagent more than 2 ml provided spectra
that interfered the characteristic peak of amylose iodine
complex in the ultraviolet region. For study about the
effect of amylose iodine solution color, the amylose
standard from Sigma Chemical was used as
representative amylose substrate. The stock standard
amylose was prepared by McCready method using the
solution of a 100 mg of amylose standard in total volume
of 100 ml. The amount of stock standard amylose was
varied from 1 ml to 6 ml by using 1 or 2 ml of 0.2%
iodine as showed in Table III. By adding 1 ml of 0.2%
iodine, the amylose complex solutions showed blue color.
A light blue tone presented with the adding of 1-2 ml
amylose standard as well as a dark blue tone presented
with the adding of 3-6 ml. By adding 2 ml of 0.2% iodine,
the amylose complex solutions showed blue and green
color. A green tone presented with the adding of 1-3 ml
amylose standard and a dark blue presented with the
adding of 4-6 ml. The result indicated that using small
amount of amylose standard and large amount of iodine
reagent provided a green tone solution. The green tone
solution may occur by the excess volume of iodine and it
also effected spectra in ultraviolet region. From the
statistics (Mann-Whitney) result of the amylose iodine
complex absorbance at 610 nm (Table II), it showed
significant difference absorbance of the blue and green
tone solution. Therefore, the adding of the same 0.2%
iodine volume for every sample types, might cause the
error for amylose analysis.
B. The Defatting Process of Cassava Starch Sample
To study the elimination of lipid from cassava starch
sample, the 3 types of solvent (methanol, ethanol, and
petroleum ether) were applied by solvent extraction
method for 24 hours. The defatted and nondefatted
starches from sample 1 were used to develop the amylose
iodine complex using 2 ml of 0.2% iodine reagent. Then
the amylose iodine complex solution was analyzed by
UV visible spectrometer in fixed mode at 610 nm, as
showed in Table IV.
Table IV shows the slight increase of amylose iodine
complex absorbance when the defatting process was
applied with methanol, ethanol, and petroleum ether as
solvent. The statistics method (CRD) was operated to
compare the average absorbance between the blue
complex solutions from nondefatted and the defatted
starches. It presents that the nondefatted and the defatted
starch by 3 types of solvent provided nonsignificant
difference of absorbance. The surface morphology of the
nondefatted and defatted cassava starch were analyzed by
scanning electron microscope as shown in Fig. 4. The
result showed the dissimilar texture of the nondefatted
and defatted starch. The surface of nondefatted starch
sample showed rather smooth area. The surface of the
defatted starch by the 3 type of solvents showed the
bumpy area that due to the disappearance of lipid.
Journal of Advanced Agricultural Technologies Vol. 4, No. 4, December 2017
©2017 Journal of Advanced Agricultural Technologies 347
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TABLE III. THE SOLUTION COLOR OF AMYLOSE IODINE COMPLEX FROM AMYLOSE SUBSTRATE USING DIFFERENT VOLUME OF 0.2% IODINE
0.2%
Iodine reagent
Volume of amylose standard solution
1.00 ml 2.00 ml 3.00 ml 4.00 ml 5.00 ml 6.00 ml
1.00 ml
2.00 ml
TABLE IV. THE ABSORBANCE OF AMYLOSE IODINE COMPLEX AT 610
NM
Starch sample
Average
absorbance at
610 nm*
SD* % RSD*
nondefatted 0.8396 0.0159 1.8958
defatted by methanol 0.8526 0.0193 2.2634
defatted by ethanol 0.8538 0.0077 0.9080
defatted by petroleum ether 0.8513 0.0093 1.0866
*N=3
Figure 4. The SEM image of surface morphology from the nondefatted and defatted cassava starch sample, (a) is the image from nondefatted
starch and (b), (c), (d) are the image from the defatted starch using methanol, ethanol, and petroleum ether as solvent, respectively.
IV. CONCLUSION
In this study, the result of UV visible spectra of
amylose iodine complex indicated that the adding of
different iodine amount showed the maximum
wavelength at different wavelength. And the effect of
iodine amount also related to the absorbance of amylose
iodine complex that absorbance increase when increasing
of iodine volume. However, the adding of excess iodine
amount presented the green tone solution that provided
the significant different absorbance of amylose iodine
complex by comparing with the blue tone solution using
the statistics method (Mann-Whitney). Hence, the adding
of excess iodine volume may cause the error of amylose
iodine complex analysis when the wavelength was fixed
at 610 nm. Thus, the suitable amount of iodine should be
considered for analysis amylose in each type of sample.
The result of defatting indicates that the defatted cassava
starch samples by solvent extraction using 3 types of
solvent for 24 hours provided higher absorbance of
amylose iodine complex. However, these absorbances are
not significant different when test by CRD method.
ACKNOWLEDGMENT
The author would like to acknowledge Science
Achievement Scholarship of Thailand, SAST and
Department of Chemistry, Faculty of Science, Chiang
Mai University for financial support and facilities.
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Sirinat Boonpo was born in Mahasarakham,
Thailand on March 20, 1987. She received her
B.Sc degree in the field of chemistry from Mahasarakham University, Mahasarakham,
Thailand in 2008 and M.Sc. degree in the field of chemistry from Chiang Mai University,
Chiang Mai, Thailand in 2011. She is now
studying for her Ph.D. degree at Chiang Mai University, Chiang Mai, Thailand. Her
research interests focus on the determination of amylose content in starch sample.
Sukjit Kungwankunakorn is a lecturer in the Department of Chemistry at Chiang Mai
University, and has served as the Head of
Forensic Science Program in Graduate School, Chiang Mai University since 2010. Her
researches have focused on method developments for the analysis of heavy metals
using spectrometric methods, the analysis of
food and environmental samples and the analysis of forensic samples with advanced
analytical techniques. She earned her PhD in Chemistry from The University of Manchester, Manchester.
Journal of Advanced Agricultural Technologies Vol. 4, No. 4, December 2017
©2017 Journal of Advanced Agricultural Technologies 349