Food Chemistry 130 (2012) 11421145

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Food Chemistry
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Short communication

Potentiometric titration for determination of amylose content of starch A

comparison with colorimetric method
Derek X. Duan a,1, Elizabeth Donner a, Qiang Liu a,, Damon C. Smith b, Franois Ravenelle b,2
a
b

Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9
Labopharm Inc., 480 Boul. Armand-Frappier, Laval, Qubec, Canada H7V 4B4

a r t i c l e

i n f o

Article history:
Received 18 October 2010
Received in revised form 4 May 2011
Accepted 31 July 2011
Available online 17 August 2011
Keywords:
Amylose
Auto-titrator
Iodine
Potentiometric
Starch

a b s t r a c t
Amylose, the amount of which varies signicantly depending on the source, is one of the key components
of starch. The proportion of the essentially linear amylose and the highly branched amylopectin greatly
inuences the physico-chemical properties of starch. In this study, potentiometric titration using an autotitrator and colorimetric methods were compared for determining amylose contents of a variety of starch
samples. Potentiometric titration results for starches from a variety of botanical sources were within the
reported literature ranges while the colorimetric method seemed to overestimate amylose content.
Crown Copyright 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Starch is a natural polysaccharide that has been widely studied
for many years in the food industry as well as for use in non-food
applications (Ao et al., 2007; Dumoulin, Alex, Szabo, Cartilier, &
Mateescu, 1998; Liu, Weber, Currie, & Yada, 2003; Ravenelle &
Rahmouni, 2006). It is well known that starch is composed of
two types of macromolecules: the essentially linear amylose and
the highly branched amylopectin. Many of the physicochemical
properties of starch, such as solubility, swelling ability, crystallinity, retrogradation and texture, which impact its intended use,
are dependent on the ratio of amylose and amylopectin (Jane
et al., 1999). More importantly, amylose-to-amylopectin ratio is a
major factor affecting the formation of resistant starch (Xie, Liu,
& Cui, 2006). A number of techniques, including potentiometric
titration, iodine colorimetric determination, concanavalin A precipitation, high performance size exclusion chromatography
(HPSEC) and differential scanning calorimetry (DSC) have been
used for amylose determination (Zhu, Jackson, Wehling, & Geera,
2008). Iodine colorimetric determination method is the most frequently used method due to its simplicity and long history of usage

Corresponding author. Tel.: +1 519 780 8030; fax: +1 519 829 2600.
E-mail address: qiang.liu@agr.gc.ca (Q. Liu).
Present address: Eyegenix LLC, 1946 Young Street, Suite 288, Honolulu, HI 96826,
USA.
2
Present address: Theratechnologies Inc., 2310, Boul. Alfred-Nobel, Montral,
Qubec, Canada H4S 2B4.
1

in the food industry. However, this method requires a reliable pure

amylose from different sources to construct a standard curve,
which is problematic at times. Moreover, iodine also binds to long
amylopectin chains and intermediate size polymers, albeit with a
much lower afnity, which would affect the accuracy of the results
(Himmelsbach, Barton, McClung, & Champagne, 2001). It has been
found in our laboratory that iodine colorimetric determination
gave higher amylose content values than expected. In this study,
potentiometric titration using an auto-titrator and colorimetric
methods were compared for determining amylose content of a
variety of starch samples in order to nd a more precise and efcient way for amylose content determination.
2. Materials and methods
2.1. Materials
Potato starch was isolated from potatoes received from Agriculture and Agri-Food Canada Research Centres in Charlottetown,
Prince Edward Island, Fredericton, New Brunswick, and Lethbridge,
Alberta, following the isolation procedure of Liu et al. (2003). Dried
starch was passed through a 125 lm sieve. All other starch samples including rice, wheat, normal corn, waxy corn starch, and potato amylose were obtained from Sigma Aldrich (St. Louis, MO,
USA); tapioca and high amylose corn starch (Hylon V and VII) were
obtained from National Starch and Chemical Company (Bridgewater, NJ, USA). Other reagents and chemicals were obtained from
Sigma Aldrich (St. Louis, MO, USA).

0308-8146/$ - see front matter Crown Copyright 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.07.138

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D.X. Duan et al. / Food Chemistry 130 (2012) 11421145

2.3.3. Apparatus
An auto-titrator (Mettler Toledo DL50) equipped with a DM140
platinum electrode, DV1010 burette and LabX light titration software (version 2.1) (MettlerToledo Inc., Columbus, OH) was used
for the titration of dispersed starch solutions at room temperature
(21 C). Titrant addition was based on the kinetics of the titration
curve (dynamic addition), and measurement of potential was equilibrium controlled. Recognition of the equivalence point (EQP) was
set by adjusting the threshold to appropriate values. Termination
of the titration occurred after one equivalence point was reached,
or the maximum volume of the burette (10.0 mL) was dispensed.
The software reported the volume of titrant at EQP, iodine afnity
(IA, %), and the content of amylose (%).

2.2. Amylose/iodine complex colorimetric method

The apparent amylose content was measured based on the colorimetric method of Williams, Kuzina, and Hlynka (1970). Briey,
the starch samples were rst dispersed in 0.5 N KOH, and after
dilution and neutralisation with 0.1 N HCl, an iodine reagent was
added and the absorbance of the blue complex was measured at
625 nm with a spectrophotometer. A standard curve was constructed using mixtures of potato amylose and waxy corn starch
(100% amylopectin), maintaining the level of amylose at 20%.
The reported values are means of duplicate measurements.

2.3. Potentiometric titration method

2.3.4. Calculation
The iodine, which binds to amylose, was produced by the reaction of the titrant KIO3 with potassium iodide and hydrochloric
acid, as shown in the following scheme:

Potentiometric titration method was adapted from the method

of Schoch (1964).

KIO3 5KI 6H ! 3I2 6K 3H2 O

2.3.1. Reagents
i.
ii.
iii.
iv.
v.

Iodine afnity was calculated from the volume of titrant (VEQ,

mL) at EQP using the equation:

Potassium iodate solution for titration (0.0005 N)

HCl (1 N)
KOH (0.5 N)
KI (0.4 N)
Ascorbic acid (as primary standard to check the titer of
potassium iodate solution)

IA%

T  VEQ  c  z  M  100
m

where T is the titer of the titrant, c is the nominal concentration of

the titrant (0.0005 N), z is the stoichiometric factor of the I2 production reaction (3), M is the molecular weight of I2 (254 g/mol), and m
is the dry mass of the sample (mg).
Titer of the titrant was determined using ascorbic acid as a primary standard. Briey, 12 mg of ascorbic acid was added to a
100 mL titration beaker, in addition to 5 mL of 1 N HCl, 5 mL of
0.4 N KI and 50 mL of distiled water. The mixture was titrated with
0.0005 N potassium iodate until the equivalence point was
reached. Titer was calculated using the following equation:

2.3.2. Sample Preparation

A starch sample (2050 mg, equivalent to 510 mg of pure
amylose depending on the amylose content of the starch) was
weighed into a 50 mL beaker. Ten millilitres of 0.5 N KOH was
added, and the mixture was stirred magnetically at room temperature for 20 min or until complete dispersion of the starch. The dispersed starch solution was transferred to a 100 mL titrator beaker,
using 50 mL of distilled water to quantitatively transfer the solution. 10 mL of 1 N HCl and 5 mL of 0.4 N potassium iodide solution
were added to the beaker, which was installed on the auto-titrator
for titration with 0.0005 N potassium iodate.

m  1000
titer
VEQ  c  M  z

where m is the mass of ascorbic acid (g), VEQ is the volume of titrant
at the equivalence point (consumption, mL), c is the nominal con-

60

370
Equivalence Point (EQP)

50

360

40

30
340
20
330
10
320

1st derivative
0

310

-10

inflection point
signal
300

-20
0

Volume of titrant (mL)

Fig. 1. Titration prole of ascorbic acid with potassium iodate.

10

1st derivative (mV/mL)

Potential (mV)

350

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D.X. Duan et al. / Food Chemistry 130 (2012) 11421145

value for potato amylose is 19.9%, corn 19%, tapioca 18.6% and
wheat 19.9% (Schoch, 1964). Rice amylose has been reported to
have an iodine afnity of 16.7% (Hamori & Kallay, 1972).

Equivalence Volume (VEQ, mL)

10
y = 0.112x + 0.243
R2 = 0.9995

9
Amylose
8
y = 0.3203x + 0.2534
R2 = 0.997

7
6

Amylose%

Potato Starch

IAstarch
 100
IAamylose

5
4

2.3.5. Validation of the method

A series of validation experiments on the precision, linearity,
and determination limit of the method were carried out.

3
2
1
0
0

10

20

30

40

50

60

70

80

3. Results and Discussion

Sample mass (mg)

Fig. 2. Linearity of the titration method with potato amylose and potato starch.

A typical titration prole of ascorbic acid with potassium iodate

is shown in Fig. 1. The sharp peak of the 1st derivative of the potential indicated the equivalence point of the reaction. It should be
noted that, unlike the potentiometric titration method by Schoch
(1964), which determined the amount of bound iodine by extrapolation of bound iodine vs. free iodine, the consumed/bound iodine
is directly calculated from the volume of titrant at the EQP (equivalence point).
The titer of potassium iodate solution was found to be independent of the mass of the ascorbic acid standard used, and had a value of 1.01 0.03, n = 18.

10
9
8
7

RSD (%)

3.1. Titration of ascorbic acid with potassium iodate

6
5
4
3
2
1

3.2. Validation of the titration method

0
0

10

20

30

40

50

60

70

80

Sample mass (mg)

Fig. 3. Relative standard deviation of amylose content of potato starch as a function
of sample mass. The determination limit was 3 mg of amylose (or 15 mg of potato
starch) which could be titrated with good precision (RSD 61%).

centration of the titrant (0.0005 N), M is the molecular weight of

ascorbic acid (176.12 g/mol), and z is the equivalent number (3)
of the I2 production reaction.
Amylose content was calculated from the ratio of iodine afnity
of the starch to the iodine afnity of pure amylose. The literature

Linearity of the method was tested using potato amylose (1

16 mg) and potato starch (675 mg). The method had excellent linearity as shown in Fig. 2, with r2 values of 0.9970 and 0.9995 for
amylose and potato starch, respectively.
The determination limit of the method, i.e. the smallest amount
of amylose that could be titrated with good precision, was found to
be around 3 mg of amylose (equivalent to 15 mg of potato starch,
as shown in Fig. 3). When the amount of amylose was lower than
the determination limit, 3 mg, relative standard deviation increased to above 1%, possibly due to the detection limit of the electrode. On the other hand, it should be noted that as sample mass

Table 1
Amylose content of potato starch by potentiometric and colorimetric methods.a

a
b

Potato growing location

Potato variety

Amylose content of
starch by potentiometric
methodb (%, w/w)

Apparent amylose content

of starch by colorimetric
method (%, w/w)

Charlottetown, PEI

Goldrush
Norland
Yukon Gold
Organic Goldrush
Organic Norland
Organic Yukon Gold

21.3 0.1
19.2 0.0
20.9 0.3
21.0 0.1
18.7 0.3
20.3 0.3

33.0 0.1
31.4 0.0
33.1 0.2
32.5 0.3
30.8 0.3
31.7 0.0

Fredericton, NB

1505815
1506003
F01013
F04012
F04054
F04056
F62008
H0867521

19.1 0.0
22.1 0.2
20.2 0.2
19.1 0.3
20.2 0.1
23.5 0.1
21.4 0.1
22.6 0.0

29.8 0.1
34.9 1.5
31.3 0.6
34.9 0.1
32.6 0.3
36.6 0.0
33.7 0.1
36.4 0.4

Lethbridge, AB

AC Stampede Russet
CV920281
CV920564
FV124862
V11021

21.4 0.1
23.2 0.6
24.9 0.0
21.3 0.1
23.1 0.2

31.9 0.5
35.2 0.3
38.9 0.3
32.5 0.3
34.8 0.2

Values denote means standard deviations, n = 2.

Literature value of amylose content of potato starch is 2124% (Williams et al., 1970).

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D.X. Duan et al. / Food Chemistry 130 (2012) 11421145

Table 2
Amylose content of commercial starches by potentiometric titration and colorimetric method, with a comparison to literature values.
Starch

Amylose content by
potentiometric methoda (%, w/w)

Amylose content by
colorimetric methoda (%, w/w)

Literature valuesb

Corn, normal
Corn, Hylon V
Corn, Hylon VII
Corn, waxy
Potato
Rice
Tapioca
Wheat

22.4 1.2
55.1 0.6
66.5 0.6
1.0 0.2
26.1 0.5
15.2 0.4
21.3 0.3
22.7 0.1

25.7 0.3
41.3 5.6
54.3 0.6
6.3 1.7
30.3 0.2
18.0 1.2
26.1 0.1
30.4 0.5

1924%
50%
70%
<2%
2124%
1621%
1521%
2226%

a
b

Values denote means standard deviations, n = 2.

Amylose content ranges reported by Williams et al., 1970; values for Hylon V, VII and waxy maize are commonly reported theoretical values.

was increased from 50 to 75 mg, relative standard deviation also

increased. This could be caused by the higher volume of titrant required for a larger amount of starch, which may increase the experimental error due to imperfect mixing in the titrator beaker. It is
recommended to perform the titration with 2050 mg of starch
(equivalent to about 510 mg of amylose based on different amylose contents in starch samples) using the current conditions to ensure satisfactory precision.

that one of the advantages of potentiometric titration is that it does

not require the starch solution to be clear and colourless, which
would otherwise compromise the colorimetric method if colour
and precipitates exist in the sample solutions. With the possibility
of using an autosampler, potentiometric titration with an autotitrator could potentially be used in industry to determine amylose
contents of samples in the process of automation.
Acknowledgements

3.3. Comparison of amylose content determined with potentiometric

titration and the colorimetric method
The results of the amylose contents of potato starches determined with potentiometric titration and colorimetric method are
listed in Table 1. The literature value for potato starch is 2124%
(Williams et al., 1970). Potentiometric titration results for potato
starches were within the reported literature range while the colorimetric method seemed to overestimate amylose content. Colorimetric method requires pure amylose for construction of a
standard curve, in which the results are apparently related to the
purity of the amylose used. We tested the iodine afnity of the
pure potato amylose using potentiometric titration and found
its IA value was as low as 16% to 17%, while the theoretical value
is 19.9%. This could explain why the colorimetric method overestimated the amylose content of these samples. On the other hand,
the structure of amylose and amylopectin used in the standard is
another factor for the result since there are differences in the
molecular characteristics of amylose and amylopectin between
the standards and test samples (Chung & Liu, 2009).
The amylose content of other starch samples determined with
potentiometric titration also agreed with previously reported literature values (Table 2). High amylose corn starches (Hylon V and
VII) and waxy corn starch (02% amylose content) were also included for comparison showing similar results as in other starch
samples.
4. Conclusion
When potentiometric titration was rst introduced to determine amylose content, the titration and curve plotting was done
manually, which was claimed to be time consuming (Schoch,
1964). However, with the help of an auto-titrator, potentiometric
titration proved to be a quick, precise, and robust method for amylose determination. The determination limit for the current potentiometric titration method was 3 mg of amylose. It should be noted

The authors want to thank Sam Sakher from Mettler Toledo for
technical support on the auto-titrator, Kristina Humphreys for colorimetric results, and Labopharm Inc. and AAFC-MII for nancial
support.
References
Ao, Z., Simsek, S., Zhang, G., Venkatachalam, M., Reuhs, B. L., & Hamaker, B. R. (2007).
Starch with a slow digestion property produced by altering its chain length,
branch density, and crystalline structure. Journal of Agriculture and Food
Chemistry, 55, 45404547.
Chung, H., & Liu, Q. (2009). Impact of molecular structure of amylopectin and
amylose on amylose chain association during cooling. Carbohydrate Polymers,
77, 807815.
Dumoulin, Y., Alex, S., Szabo, P., Cartilier, L., & Mateescu, M. A. (1998). Cross-linked
amylose as matrix for drug controlled release. X-ray and FT-IR structural
analysis. Carbohydrate Polymers, 37, 361370.
Hamori, E., & Kallay, M. C. (1972). Rate studies of the amylose-iodine reaction in
unfractionated starch samples of various plant origin. Biopolymers, 11, 475482.
Himmelsbach, D. S., Barton, F. E., II, McClung, A. M., & Champagne, E. T. (2001).
Protein and apparent amylose contents of milled rice by NIR-FT/Raman
spectroscopy. Cereal Chemistry, 78, 488492.
Jane, J., Chen, Y. Y., Lee, L. F., McPherson, A. E., Wong, K. S., Radonsavljevic, M., et al.
(1999). Effects of amylopectin branch chain length and amylose content on the
gelatinization and pasting properties of starch. Cereal Chemistry, 76, 629637.
Liu, Q., Weber, E., Currie, V., & Yada, R. (2003). Physicochemical properties of
starches during potato growth. Carbohydrate Polymers, 51, 213221.
Ravenelle, F., & Rahmouni, M. (2006). Contramid: High-amylose starch for
controlled drug delivery. In R. H. Marchessault, F. Ravenelle, & X. X. Zhu
(Eds.), Polysaccharides for drug delivery and pharmaceutical applications
(pp. 79104). Washington, DC: American Chemical Society.
Schoch, T. J. (1964). Iodimetric determination of amylose. Potentiometric titration:
Standard method. In R. L. Whistler (Ed.). Methods in carbohydrate chemistry (vol.
IV, pp. 157160). New York: Academic Press Inc..
Williams, P. C., Kuzina, F. D., & Hlynka, I. (1970). A rapid colorimetric procedure for
estimating the amylose content of starches and ours. Cereal Chemistry, 47,
411420.
Xie, X., Liu, Q., & Cui, S. (2006). Studies on the granular structure of resistant
starches (type 4) from normal, high amylose and waxy corn starch citrates. Food
Research International, 39, 332341.
Zhu, T., Jackson, D. S., Wehling, R. L., & Geera, B. (2008). Comparison of amylose
determination methods and the development of a dual wavelength iodine
binding technique. Cereal Chemistry, 85, 5158.