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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.53 no.3 Concepción Sept. 2008 


J. Chil. Chem. Soc, 53, N° 3 (2008) págs: 1565-1567






1Institute for Innovation and Development of Learning Process, Mahidol University, Rama 6 Rd., Rajchataywee, Bangkok 10400, Thailand
2Department of Chemistry (R3/1), Faculty of Science (Salaya Campus), Mahidol University, Phuttamonthon Sai 4 Rd., Salaya, Nakhon Pathom 73170, Thailand
3Center of Excellence for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University, Salaya Campus, Phuttamonthon Sai 4 Rd., Nakhon Pathom 73170, Thailand
4Department of Physics (BIOPHYSICS), Faculty of Science, Mahidol University, Rama 6 Rd, Rajchatavee, Bangkok 10400, Thailand


Analysis of the shift of wavelength máximum using a rapid colorimetric method was used to determine the ratio of amylose:amylopectin (Am:Ap) in acid-hydrolyzed tapioca starch. The absorbance máximum of 600 nm (Am:Ap of tapioca starch ≈ 22:78) moved to shorter wavelengths (590, 585, 570, 560, and 534 nm) as the decrease of the Am:Ap ratio due to hydrolysis of shorter chains that are not be able to form a complex with iodine. The amount of amylopectin itself may be unaltered or slightly decreased but the decrease in amylose caused a decrease in Am:Ap ratio.

Keywords: amylose, amylopectin, acid hydrolysis, colorimetric


Starch is the major carbohydrate reserve of plant tubers and seed endosperm.1 The largest source of starch is maize, wheat, potato, tapioca, and rice. Starch is widely used as thickener, water binder, emulsión stabilizer, and gelling agent. Each starch granule typically contains amylopectin, a linear chain of (1—>4)-α-D-glucose residues connected through branched (1—>6)-α-linkages, and a much larger number of the smaller amylose, α(1—>4) linearly linked D-glucopyranosyl residues.

Amylose is a hydrocolloid. Its extended conformation causes the high viscosity of water-soluble starch which varies relatively little with temperature. The extended loosely helical chains possess a relatively hydrophobic inner surface that is not able to hold water. Amylose forms useful gels and films. Its association and crystallization (retrogradation) on cooling and storage decrease storage stability, causing shrinkage and the reléase of water (syneresis). Increasing amylose concentration decreases gel stickiness, but increases gel firmness. Amylopectin interferes with the interaction between amylose chains (and retrogradation) and its solution can lead to an initial loss in viscosity and followed by a more slimy consistency.

The simplest and most common starch modification is by acid hydrolysis, which is widely used in food, paper, textile, and pharmaceutical industries.2,3 This is conducted mainly by soaking the starch in dilute acid.4

In normal starch, the amylose:amylopectin (Am:Ap) ratio is ≈ 1:4. Meals made with high amylose rice5 or cookies prepared from high amylose starches6 induce a lower postprandial glucose response in the blood. In addition, consumption of high amylose diet for several weeks results in lower postprandial concentration of insulin and lower fasting concentration of triacylglycerol and cholesterol.7 Starch films made from different Am:Ap ratio have different properties. Amylose in starch gives stronger film whereas amylopectin generally leads to lower mechanical properties.8,9 Physical and chemical properties of starch films can be tailored by adjusting Am:Ap ratio.10,11

The present work describes the use of a simple colorimetric method to analyze Am:Ap ratio via shift of wavelength máximum (λmax) seen at different stages of acid hydrolysis of tapioca starch.



Tapioca starch was obtained from a commercial source in Thailand. Hydrochloric acid and sodium hydroxide were purchased from Merck KG, Darmstadt, Germany. Potassium iodide, iodine, acetic acid, puré potato amylose, and ethanol were purchased from Sigma Co., Ltd., USA.

Acid hydrolysis of tapioca starch

Starch was suspended at 10% (w/v) in 0.7 M aqueous HC1 and 2.0 M aqueous HC1 for 0.5 h, 1.5 h, 2.5 h, and 3.5 h at 50°C with constant stirring and then the suspensions were neutralized with NaOH to a pH of 7.0 ± 0.5. The acid-modified starch was recovered by centrifugation and freeze-dried.

Preparation for colorimetric absorption analysis

A fraction (0.10 g) of the acid-modified starch was dissolved in a mixture of 1 mi of 95% ethanol and 9 mi of 2 M NaOH at 95°C. When the starch was dissolved completely, this solution was diluted with deionized water in a 100 mi volumetric flask. An aliquot of 5.0 mi was mixed with 2.0 mi of 1 M acetic acid, 2.0 mi of freshly prepared iodine reagent and 91.0 mi of deionized water. The iodine reagent was prepared by dissolving 0.20 g of iodine and 2.0 g of potassium iodide with deionized water in a 100 mi volumetric flask.

Colorimetric absorbance analysis

Absorbance spectra from 400 to 800 nm were recorded for all samples with a 1 cm path-length cuvette in a Cary 300Bio UV/Visible spectrophotometer.

A standard curve was plotted for various concentrations of puré potato amylose. Potato starch was used as the standard because potato and tapioca starch have approximately the same amylose and amylopectin content1,12 Spectra for blanks of the amylose solutions and the iodine reagent were also recorded.

Figure 1 shows a typical absorbance spectrum. Data analysis of the spectra was conducted by smoothing of the spectra. Two separate pieces of data were then extracted: the absorbance at 610 nm (λ610, (a) in Figure 1), which was used to determine the Am:Ap ratio, and the wavelength with máximum absorbance (λmax) within a given range of 400-800 nm obtained using a mathematical algorithm (b in Figure 1).13


The Am:Ap ratio in native tapioca starch was approximately 22:78. Figure 2 shows that the Am:Ap ratio obtained by an iodometry method at a single wavelength (λ610) measurement decreased with hydrolysis time using 2 M HC1. but not with 0.7 M HC1. This change was seen after 30 min of incubation.

There was a shift of λmax to shorter wavelengths with hydrolysis time (Figure 3). The change was more pronounced using 2 M HC1. A good correlation was obtained between λmax and amylose content for percent amylose content between 10 - 20 (Figure 4).

Starch is hydrolyzed by acid into shorter glucose chains. With the iodometry method, approximately 6 glucose units are required to form a complex with iodine that will give a measurable absorption at 610 nm.14 With 0.7 M HC1, hydrolysis does not change the Am:Ap ratio because there is competition between hydrolysis of the amorphous part of the amylopectin into shorter glucose chains capable of forming a complex with iodine resulting in an increase in the Am:Ap ratio and hydrolysis of the original amylose into shorter chains that are not able to form a complex with iodine (resulting in decrease inthe Am:Ap ratio). With 2.0 M HC1, this effect was observed during the first 30 min of hydrolysis. From 30 - 210 min of hydrolysis by 2.0 M HC1, the observation of competition between hydrolysis of the amorphous part of amylopectin and hydrolysis of the original amylose is no longer reflected in the analysis, perhaps as all the amorphous part of amylopectin has been hydrolyzed. What was observed was the decrease of the Am:Ap ratio due to hydrolysis of shorter chains that are not be able to form a complex with iodine. The amount of amylopectin itself may be unaltered or slightly decreased but the decrease in amylose caused decrease in Am:Ap ratio.


In summary, it has been demonstrated that λmax shifts to shorter wavelength with decreasing Am:Ap ratio, and provides a simple method of determining Am:Ap ratio following acid hydrolysis of tapioca starch.


This work has been supported in part by Postgraduate Education and Research Program in Chemistry (PERCH), The Thai Center of Excellence for Physics (Integrated Physics Cluster), Coordinating Center for Thai Government Science and Technology Scholarship Students of the National Science and Technology Development Agency (CSTS, NSTDA), Thailand Research Funding Agency (TRF), and Thailand National Center for Engineering and Biotechnology (BIOTEC).



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(Received: May 28, 2007- Accepted: May 2, 2008)

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