Determination of Total Amylose Content UNIT E2.

3
of Starch
Starches from various sources contain different amounts of amylose and amylopectin, BASIC
which influence physicochemical properties such as gelatinization, retrogradation, water PROTOCOL
absorption, and paste viscosity. For those reasons, the amylose content must be deter-
mined accurately. Previous studies have shown that colorimetric methods (based on
amylose-iodine complex formation) are influenced by amylopectin chain length (which
is known to vary in starches from different botanical sources). For example, the outer
branches of potato amylopectin, which are longer than those of cereal starches, can bind
iodine and cause an overestimation of the amylose content if not taken into consideration.
This Basic Protocol takes into account the iodine affinity of amylopectin and involves the
following steps (1) starch defatting; (2) dispersion of starch in dimethylsulfoxide
(DMSO); (3) determination of the absorbance at 600 nm of the dispersed starch; (4)
preparation of a standard curve over the range 0% to 100% amylose, using mixtures of
pure amylose and amylopectin; and (5) calculation of total amylose content from the
standard curve. In this protocol, potato is the source of amylose and amylopectin.
Materials
Starch sample
75% (v/v) n-propanol in water
Pure potato amylose type III and amylopectin (Sigma)
90% (v/v) dimethylsulfoxide (DMSO) in H2O
Iodine solution (see recipe)
Cellulose extraction thimble
Cotton wool plug
Heating mantle
Soxhlet apparatus
30°C forced air oven
Round-bottom screw-cap tubes with a Teflon-faced rubber liner in caps
Vortex mixer
85°C water bath
50-ml volumetric flask
Quartz cell (1-cm path-length)
UV/visible spectrophotometer

Defat starch sample

1. Weigh accurately 5.0 g of starch into a cellulose extraction thimble and cover the
mouth with a cotton wool plug.
2. Extract the lipids with 120 ml 75% n-propanol at 85°C with a heating mantle for 7
hr in a Soxhlet extractor.
Lipid-complexed amylose chains interfere with amylose-iodine (I3− and I5−) complex
formation. Therefore, extraction of complexed lipids with 75% n-propanol is necessary
prior to determination of total amylose content.
3. Air dry the thimble containing the lipid-free starch for 12 hr.
4. Remove the lipid-free starch from the thimble and oven dry 24 hr, 30°C.

Starch and Starch

Derivatives
Contributed by R. Hoover and W.S. Ratnayake E2.3.1
Current Protocols in Food Analytical Chemistry (2001) E2.3.1-E2.3.5
Copyright © 2001 by John Wiley & Sons, Inc.
 Disperse lipid-free starch (Solution I)
5. Weigh 20 mg of lipid-free starch (correct to 0.1 mg) into a round bottom screw-cap
tube fitted with a Teflon-faced rubber liner in the cap.
6. Prepare a series of mixtures of pure potato amylose and amylopectin (0%, 10%, 20%,
40%, 50%, 60%, 80%, 90%, and 100% amylose). Weigh 20 mg of each into
round-bottom tubes with caps.
7. Add 8 ml 90% DMSO to each round-bottom tube and mix vigorously for 2 min using
a vortex mixer.
8. Heat the tubes in a water bath at 85°C for 15 min with intermittent mixing.
9. Allow the tubes to cool to room temperature (∼45 min).
10. Check for the presence of clear gels in the cooled, dispersed solution.
If a gel is detected, repeat steps 5 to 9 with more vigorous mixing at shorter time intervals
in step 7.
11. Dilute the sample to 25 ml with water in a volumetric flask.

Determine absorbance of dispersed starch solution

12. Add 1.0 ml of the diluted solution (Solution I) and 40 ml of distilled water into a
50-ml volumetric flask.

1.8

1.6

1.4

1.2

1
A 600

0.8

0.6

y = 0.0168x + 0.2138
0.4 R2 = 0.9998

0.2

0
20 40 60 80 100
% Amylose

Determination of Figure E2.3.1 Plot of absorbance at 600 nm against percentage amylose (w/w) for mixtures of
Total Amylose
Content of potato amylose and amylopectin with iodine. The absorbance of 0% amylose is due to the I2 affinity
Starch of the long outer branches of amylopectin.

E2.3.2
Current Protocols in Food Analytical Chemistry
13. Add 5 ml of iodine solution and mix vigorously. Then, adjust the volume to 50 ml
with distilled water and mix vigorously. Allow color to develop for 15 min.
14. Mix the contents of the flask by hand.
15. Measure the absorbance of the sample and each of the standard mixtures at 600 nm
against a reagent blank as the reference.
The reagent blank contains all reagents in the same amounts without the sample containing
starch.

Prepare standard curve for mixtures of amylose and amylopectin

16. Plot a standard curve (Fig. E2.3.1) for mixtures of pure potato amylose and amy-
lopectin.
17. Determine the regression equation for the standard curve and use this equation to
calculate the total amylose content of the sample.
The regression equation for the standard curve shown in Figure E2.3.1 is y = 0.0168x +
0.2138 (R2 = 0.9998); where x = % amylose and y = absorbance at 600 nm.

absorbance − 0.2138
= % amylose
0.0168

The standard deviations of all the data points in Figure E2.3.1 were less than 0.01 (not
shown), and the data points are means of three individual determinations against a reagent
blank.

REAGENTS AND SOLUTIONS

Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Iodine solution
Prepare a 0.0025 M I2/0.0065 M KI mixture. For a 100-ml solution: Dissolve 0.1079
g KI in 5 ml of water in a 100-ml volumetric flask. Add 0.0315 g I2, shake well (by
hand) until all I2 is dissolved in KI. Adjust the volume to 100 ml. The solution should
be stored in a dark bottle, 4°C, until used.

COMMENTARY
Background Information
The formation of a helical complex between
amylose and iodine gives rise to the typical deep
blue color of starch dispersions stained with
iodine and forms the basis for quantitative de-
termination of amylose content. In I2/KI solu-
tions, polyiodide ions such as I3− and/or I5−
(Teitelbaum et al., 1978, McGrance et al., 1998)
interact with amylose forming single left-
handed V-type helices. The helix consists of six
anhydrous glucose residues per turn with a
pitch of 0.8 nm and a hydrophobic helical cavity
diameter of 0.5 nm. McGrance et al. (1998)
have shown that an I5− is present within almost
every turn of the amylose helix, parallel to its
long axis. Sen et al. (1997) have shown that the
iodine affinity of amylose varies between 17
and 22 g I2 per 100 g amylose in maize, accord-
ing to the genotype, while the affinity of amy-
lopectin for iodine is only 1.05 to 1.25 g I2 per
100 g amylopectin. Amylopectin and the iodine
complex exhibits a purple color with λmax at
530 nm. Morrison and Karkalas (1990) have
shown that there are atypical types of amy-
lopectin, (1) high molecular weight amy-
lopectin with A and B chains 5 to 15 glucose
residues longer than normal; (2) amorphous
amylopectin with similarly extended A and B
chains that elute from GPC columns with amy-
lose; and (3) normal amylopectin which con-
tain very long chains (CL 85-180) with infre-
quent branching. Atypical types of amylopectin
bind more iodine and give higher λmax than
Starch and Starch
normal amylopectin, so that they give an in- Derivatives

E2.3.3
Current Protocols in Food Analytical Chemistry
 Table E2.3.1 Determination of Total Amylose Content
Starch sourcea
Potato
Field pea
Oat
aStarches were defatted prior to amylose estimation using the method
outlined in the Basic Protocol.
bTotal amylose content was determined using the Basic Protocol. The values
represent the means ± standard deviations of three determinations.
flated value in the iodometric determination of
amylose. Banks and Greenwood (1975) have
shown that atypical types (1) and (2) amy-
lopectins occur in several legume and tuber
starches; and type (3) has been reported in
sweet potato starches (Takeda et al., 1986). The
authors have used the methods cited in the
literature (McCready and Hassid, 1943; Wil-
liams et al., 1970; Juliano, 1971; Morrison and
Laignelet, 1983; Knutson, 1986; Yun and
Matheson, 1990; Sen at al., 1997; Moham-
madkhani et al., 1999) for the iodometric esti-
mation of amylose content of cereal, tuber, and
legume starches. The authors’ experience has
shown that none of the above methods yield
reproducible results. The method reported in
this unit for total amylose content is a modifi-
cation of the procedure of McGrance et al.
(1998). The authors have found the 75% n-
propanol solvent system used to defat the starch
to be more efficient than the water-saturated
butanol used by McGrance et al. The iodine
solution has also been optimized. This method
is reproducible, can be applied to low and very
high amylose containing starches, and also
takes into account the contribution from amy-
lopectin to λmax. Furthermore, the defatting step
in this procedure minimizes interference of
amylose-lipid complexes in amylose determi-
nation.
Critical Parameters and
Troubleshooting
The defatted starch should not be removed
from the thimble until contents have been air-
dried for at least 12 hr. Removal prior to this
would result in contamination of starch with
cellulose from the thimble. The dried defatted
starch should be in the form of a free flowing
powder. The presence of aggregated starch
clumps would result in the formation of insol-
uble gels during step 9.
Determination of
Total Amylose In step 9, a clear gel may develop during the
Content of determination of high amylose containing
Starch
E2.3.4
Total amylose content (%)b
24.03 ± 0.04
43.73 ± 0.03
23.07 ± 0.04
starches (this can be confirmed by centrifuging
the dispersed sample at 10,000 × g for 15 min.
The presence of a gel would be indicated by the
formation of a precipitate at the bottom of the
centrifuge tube). If a gel is detected, repeat steps
5 to 9 with more vigorous mixing at shorter time
intervals during the heating cycle in step 8.
A quartz cell (1-cm path length) must be
used for measuring absorbance. The use of
disposable plastic cells will introduce a signifi-
cant experimental error due to the variations in
the absorbances resulting in non-reproducible
results.
The color development (step 13) should be
conducted within the temperature range 23° to
25°C. Temperatures outside this range will sig-
nificantly alter the absorbance.
Anticipated Results
Table E2.3.1 illustrates the total amylose
content of field pea, oat, and potato starches.
The values (which are means of triplicate meas-
urements) are reproducible and the method can
be used for all types of starches. Very high
amylose containing starches (i.e., >50% amy-
lose) may not yield consistent values if suitable
precautions (see Critical Parameters and Trou-
bleshooting) are not taken during step 9. The
method can easily accommodate small samples
for microdeterminations, if necessary.
Time Considerations
For analysis of amylose content of oat, field
pea, and potato starches (in triplicate) may take
∼53 hr.
Literature Cited
Banks, W. and Greenwood, C.T. 1975. Starch and
Its Components. Edinburgh University Press,
Edinburgh.
Juliano, B.O. 1971. A simplified assay for milled
rice amylose. Cereal Sci. Today 16:334-338.
Knutson, C.A. 1986. A simplified colorimetric pro-
cedure for determination of amylose in maize
starches. Cereal Chem. 63:89-92.
Current Protocols in Food Analytical Chemistry
McCready, R.M. and Hassid, W.Z. 1943. The sepa-
ration and quantitative estimation of amylose
and amylopectin in potato starch. J. Am. Chem.
Soc. 65:1154-1157.
McGrance, S.J., Cornell, H.J., and Rix, C.J. 1998. A
simple and rapid colorimetric method for the
determination of amylose in starch products.
Starch 50:158-163.
Mohammadkhani, A., Stoddard, F.L., Marshall,
D.R., Uddin, M.N., and Zhao, X. 1999. Starch
extraction and amylose analysis from half seeds.
Starch 51:62-68.
Morrison, W.R. and Karkalas, J. 1990. Starch. In
Methods in Plant Biochemistry, Vol. 2. Academic
Press, Orlando, Fla.
Morrison, W.R. and Laignelet, B. 1983. An im-
proved colorimetric procedure for determining
apparent and total amylose in cereal and other
starches. J. Cereal Sci. 1:9-20.
Sen, M., Thevanat, C., and Prioul, J.L. 1997. Simul-
taneous spectrophotometric determination of
amylose and amylopectin in starch from maize
kernel by multiwavelength analysis. J. Cereal
Sci. 26:211-221.
Takeda, Y., Tokunaga, N., Takeda, C., and Hizukuri,
S. 1986. Physicochemical properties of sweet
potato starches. Starch 38:345-350.
Current Protocols in Food Analytical Chemistry
Teitelbaum, R.C., Ruby, S.L., and Marks, T.J. 1978.
Amylose-iodine complex formation. J. Am.
Chem. Soc. 100:3215-3218.
Williams, P.C., Kuzina, F.D., and Hlynka, I. 1970.
A rapid colorimetric procedure for estimating
the amylose content of starches and flours. Ce-
real Chem. 47:411-420.
Yun, S.H. and Matheson, N.K. 1990. Structural
changes during development in the amylose and
amylopectin fraction separated by precipitation
with concanavalin A of starches from maize
genotypes. Carbohyd. Res. 270:85-101.
Key Reference
McGrance et al., 1998. See above.
Gives detailed experimental information about the
selection of the wavelength and the calculations for
the determination of the amount of iodine that
should be used.
Contributed by R. Hoover and
W.S. Ratnayake
Memorial University of Newfoundland
St. John’s, Canada
Starch and Starch
Derivatives
E2.3.5