Study on Amylose Iodine Complex from Cassava Starch by ...cassava starch using 1, 2, 3 and 4 ml of...

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

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


©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.

% 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.



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.



Study on Amylose Iodine Complex from Cassava Starch by ...cassava starch using 1, 2, 3 and 4 ml of...

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