Agricultural and Biological Chemistry

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Amperometric Titration of Starch with Iodine

Tsuneo Kobayashi & Noboru Kobayashi

To cite this article: Tsuneo Kobayashi & Noboru Kobayashi (1963) Amperometric
Titration of Starch with Iodine, Agricultural and Biological Chemistry, 27:6, 438-444, DOI:
10.1080/00021369.1963.10858120

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[Agr. Bioi. Chern., Vol. 27, No. 6, p. 438-444, 1963]

Arnperornetric Titration of Starch with Iodine

Part III. Titration of Various Starch-type Polysaccharides*

By Tsuneo KoBAYASHI and Noboru KoBAYASHI**

Department of Agricultural Chemistry, Tokyo University of

Agriculture and Technology, Fuchu, Tokyo
Received February 18, 1963

Amperometric titration with iodine, a method devised by Kobayashi and Yoshida1 l, was
applied to various starch-type polysaccharides in order to elucidate the relation between the
shape of titration curves and the structure of substances. Shapes of titration curves could be
divided into 2 types, provisionally named the curves of amylose-type, and amylopectin-type,
respectively.
Amyloses and linear dextrins with the degree of polymerization (DP) above 28 gave
curves of the former type, which exhibited segments with more or less low gradient in the
former half of the titration.
On the other hand, all of the branched polysaccharides, linear dextrins with DP below
25, Schardinger dextrins, and polyvinyl alcohol showed curves of the latter type, which rised
linearly with added iodine.
The mode of interaction with iodine was discussed for these polysaccharides together
with some observations on the starch-iodine coloration.

INTRODUCTION briefly reported in the present paper. Rela-

It was demonstrated previously 1 l that the
amperometric titration with iodine by means
of a rotating platinum electrode is a simple
and precise method for evaluating the iodine
affinity of starchy substances. The authors
have intended to apply the method to studies
on the nature of starch-iodine reaction and
on some practical problems related to starch
and its hydrolyzed products. In order to
obtain fundamental knowledges for these pur-
poses, the amperometric titration was made
on several starch-type polysaccharides which
vary in their molecular weights and degree
of branching. Results of titration will be
* Part II of the series : cj. I).
** Present address: Department of Agricultural Chemistry, Univer-
sity of Tokyo.
I) T. Kobayashi and S. Yoshida, Denpun KOgyOgaku Kaishi (J.
Techno!. Soc. Starch), 8, 48 (1960).
tionships between titration curves and the
structure of polysaccharides will be discussed
on the basis of these results together with
some observations on the starch-iodine reac-
tion.
MATERIAL AND METHOD
Materials. Materials used in this experiment were
amylose and amylopectin from potato starch, partially
hydrolyzed amylose, and linear dextrins prepared
from potato amylose, waxy rice starch, waxy maize
starch, phytoglycogen from sweet corn, Schardinger
dextrins, and polyvinyl alcohol.
Potato amylose and amylopectin were prepared by
the method of Wilson, Schoch and Hudson'>.
Partially hydrolyzed amylose and linear dextrins of
various chain lengths were prepared from potato
amylose by treating it with hydrochloric acid accord-
2) E.J. Wilson. Jr., T.J. Schoch and C.S. Hudson, J. Am. Chern.
Soc., 65, 1380 (1943).
 Amperometric Titation of Starch with Iodine 439

ing to the method of Ohashi"'. Some specimens of

linear dextrins were kindly supplied by K. Ohashi, pA

Gifu University. 3

Waxy rice starch was prepared by the alkali-soaking

method of Sato''·
Waxy maize starch and phytoglycogen from sweet
corn were generous gifts from T. J. Schoch, Com
Industries Co., Arago, Illinois, U.S.A.
Schardinger dextrins were prepared by using enzyme
solution from Bacillus macerans B-209-8 or B-209-9,
the Institute of Applied Microbiology, the University
of Tokyo. Some specimens of Schardinger dextrins
were kindly supplied by Z. Nikuni, Osaka University,
and T. Ando, the Institute of Physical and Chemical
Research.
Other reagents were commercial products.
Methods. The amperometric titration was made
at 25°C according to the procedure described in the
previous pa per1 ' . Average chain length of partially
hydrolyzed amylose and linear dextrins was estimated
by the method of Nussenbaum and Hassid5' or by
periodate oxidation according to the procedure de-
scribed by Kobayashi and Kadowaki"'· The coinci-
dence between results of the two alternate methods 10
was satisfactory. ml of 0.001 N Iodine

RESULTS FIG. 1. Amperometric Titration Curves of Amylose

and Partially Hydrolyzed Amylose.
1. Titration of Amylose-type Polysaccharides (Linear
Curve 1 : Potato amylose 5 mg.
Polymers). 2: Partially hydrolyzed potato amylose (DP 150) 5 mg.
Titration curves of natural potato amylose 3: Blank.

were given in previous papers 1 ·7), and a typi-

cal one is reproduced in Fig. 1. Especially the
same curves were also shown by amyloses
slightly hydrolyzed with acid. But when
amyloses were hydrolyzed to have the degree
of polymerization (abbreviated as DP) below
250-300, their titration curves showed higher
values of electric current at their horizontal
segments, as illustrated in the curve No. 2 in
Fig. I for partially hydrolyzed amylose of DP
150. The length of their horizontal segments
was also shortened as the DP of the product
3) K. Ohashi, J, Agr. Chern. Soc. Japan (Nippon Ndgei-kagaku Kai-
shi), 33, 576 (1959).
4) S. Sato, " Kome oyobi Kome-denpun KenkyU (Studies on Rice and 0.001 N iodine (mll
Rice Starch)," TaigadO, Kyoto, 1944, p. 12. FIG. 2. Amperometric Titration Curves of Linear
5) S. Nussenhaum and W.Z. Hassid, Anal. Chern., 24, 501 (1952).
6) T. Kobayashi and M. Kadowaki, J. Agr. Chern. Soc. Japan (Nip-
Dextrins.
pon Ndgei-kagaku Kaishi), 27, 599 (1955). Each figures on curves shows the DP of dextrins.
7) T. Kobayashi and S. Yoahida, This journal, 24, 538 (1960). The amount of samples : 20 mg.
440 Tsuneo
was decreased, indicating that the iodine
affinity was decreased with decreasing DP.
Typical curves with linear dextrins were given
in Fig. 2, for examples with dextrins of DP
61, 41, 34, 28, 21 and 16. A linear dextrin
of DP 12 gave a curve, which was identical
with that of blank titration within experi-
mental errors, and it is omitted from the
Figure. Fig. 2 shows that the combination
with iodine was considerably weak for these
dextrins, and the inflexion point of the curve
can hardly be observed with linear dextrins
of DP below 28.
2. Titration of Amylopectin-type Polysaccharides
(Branched Polymers).
All of the polysaccharides of this type gave
linear curves, which rised from the beginning
pi\
3 4
0.001 N iodine (ml.)
FIG. 3. Amperometric Titration Curves of Several
Amylopectin-type Polysaccharides.
Curve 1 : Potato amylopectin 10 mg.
2 : Waxy maize starch 28 mg.
3 : Waxy rice starch 30 mg.
4: Corn phytoglycogen 30 mg.
5: Blank.
KOBAYASHI and Noboru KOBAYASHI
of the titration as shown in Fig. 3. These
curves were similar in nature, but showed
some variations in their gradients according
to the degree of branching in their glucosidic
chains. The gradient of the curves was in
the following decreasing order: phytoglyco-
gen, waxy rice starch, waxy maize starch, and
potato amylopectin. The order coincided
with that of their average chain lengths.
3. Titration of Schardinger Dextrins and Polyvinyl
Alcohol.
It was started 8 l that a helical configu-
ration is necessary for glucosidic chain to form
complex with iodine, and that non-starchy
pA
4 5
0.001 N iodine (ml.\
5
FIG. 4. Amperometric Titration Curves of Schar-
dinger Dextrins and Polyvinyl Alcohol.
Curve 1 : Schardinger a~dextrin (cyclohexaamylose) 5 mg.
2 : Schardinger a~dextrin (cyclohexaamylose) 10 mg.
3 : Polyvinyl alcohol 30 mg.
4 : Schardinger .8-dextrin (cycloheptaamylose) 25 mg.
5: Blank.
8) R.E. Rundle and D. French, J. Am. Ch1m. Soc., 65, 1707 (1943)·
 Amperometric Titation of Starch with Iodine
material such as polyvinyl alcohol is also
stained with iodine. Therefore, Schardinger
a-dextrin (cyclohexaamylose) was titrated as
a model for the helical configuration of gluco-
sidic chains. Results are shown in Fig. 4.
Curves for this dextrin showed no horizontal
segment as seen with those for amyloses, and
electric current was increased linearly with
added iodine. But the gradient of the curves
was smaller than that of the curves for amylo-
pectin-type polysaccharides mentioned above,
indicating that the affinity of cyclohexaamy-
lose for iodine is considerably higher than
that of the latter. Incidentally, blue color
was not produced with iodine under condi-
tions employed in this titration.
On the other hand, Schardinger ,8-dextrin
(cycloheptaamylose) gave a linear curve which
rised with much higher gradient than did
the curves for cyclohexaamylose.
Polyvinyl alcohol, which is stained with
iodine, was also titrated for the purpose of
comparison. It gave also a straight line
similar to that observed with branched poly-
mers, and the gradient of the curve was situ-
ated between those of corn phytoglycogen
and waxy rice starch. No blue color was
observed with polyvinyl alcohol under experi-
mental conditions.
DISCUSSION
Shapes of titration curves described above,
which obviously reflect the mode of inter-
action between polysaccharides and iodine,
will be divided into two types, provisionally
named here, titration curves of amylose-type,
and amylopectin-type, respectively.
Titration Curves of the Amylose-type.
Curves of this type are exhibited by amy-
loses and linear dextrins of DP above 28.
Polysaccharides of this group can form iodine-
complexes with measurable dissociation con-
stants. These complexes will have helical
configurations as insisted by Rundle et al. 8 >
441
The complex formation will be reversible in
general, but natural amyloses stand at the
extreme. The dissociation constant of the
iodine complex seems to be very near to zero
for these substances, as judged from very low
and constant electric currents at the horizon-
tal segments. Moreover, the constant current
maintained up to the end of complex forma-
tion suggests that the composition of the
iodine complex formed is essentially constant
during the titration, that is, iodine does not
enter into other amylose molecules until one
amylose molecule is saturated with iodine.
This is in accord with observations of Rundle
et al. 9 > Thus for natural amylases, the com-
position of the iodine complex is essentially
constant irrespective of the amount of free
iodine in the solutions. The complex of
these nature would be regarded as a "com-
pound" rather than a complex.
In the titration on amylose-type polymers
of DP below 250-300, the electric current at
the horizontal segment increased as DP was
decreased, indicating that they form reversible
complexes with iodine, the dissociation con-
stants of which being of the measurable order
of magnitude. In the present studies, how-
ever, little attempt was made for evaluating
accurate values of dissociation constants owing
to the lack in the amount of homogeneous
samples.
Combination with iodine became much
weaker for amylodextrins of DP below 100,
as observed from Fig. 2. Values of iodine
affinity and the concentration of iodine at the
end of complex formation are given in Table
I. Values in the fifth column of the Table
were calculated in the following way. The
electric current at the inflexion point of the
curve was read, and a point showing the same
electric curent was found on the blank titra-
tion curve. The amount of iodine solution
9) R.E. Rundle, J.F. Foster and R.R. Baldwin, J. Am. Chern. Soc.,
66, 2116 (1944).
442 Tsuneo KOBAYASHI and Noboru KOBAYASHI

corresponding to the latter point was read on stances tested here. After the formation of
the abscissa, and converted into the normality loosely-bound complex, the iodine added com-
of iodine in the solution. Values were cor- bines increasingly tightly with glucosidic
rected for the increase in volume due to the chain, giving a segment with more or less
addition of 0.001 N iodine solution. These lower gradient in titration curves. And after
values seemed to be related to the nature of the inflexion point has been reached, the
combining site in glucosidic chains for iodine, iodine added is again combined more and
and results in Table I suggest that the com- more loosely, which gives a rapid rise in
bination with iodine is made looser as DP of electric current.
dextrins decreases. Therefore, an unbranched glucosidic chain
would be divided into two segments, which
TABLE I. AMPEROMETRIC TITRATION OF PAR-
TIALLY HYDROLYZED AMYLOSE AND LINEAR combine iodine loosely, and tightly, respec-
DEXTRINS tively. The loosely-binding segments would
DPof Amount of Iodine Iodine con- probably be at the both ends of an un-
the the sample consumed Iodine centration
(ml. of affinity at the end branched chain, and their length is presumed
subs- titrated
tance (mg.)
0.001 II of complex to be of about 25 glucose units. This pre-
N I2) formation
150 5 6.65 13.30 6.95x I0- 6 N sumption is made by considering that linear
61 20 20.15 12.78 1.67 X I0- 5 N dextrins of this size seemed to be consisted of
41 20 18.4 11.63 2.9x10- 5 N only these segment, as judged from their
34 20 11.4 7.23 4.1 X 10- 5 N linear titration curves.
28 20 5.3* 3.36* 5.1 X I0- 5 N* Thus, the interaction of various polysac-
* Only approximate values could be obtained.
charides with iodine would be regulated by
Values of iodine affinity II (see previous the ratio of loosely- to tightly-binding seg-
paper 1 ) for definition) also decreased rapidly ments in their glucosidic chains. In natural
as the DP of dextrin decreased to below 100, amyloses the proportion of the former is neg-
and no iodine affinity could be estimated with ligible, but much larger proportions are con-
dextrins of DP below 25. Therefore both tained in dextrins of smaller sizes. This
the iodine affinity and the nature of combin- assumption may be extended and justified by
ing site are evidently affected by DP. the use of amperometric titration, and fur-
As indicated by results mentioned above, ther studies are in progress.
the amperogram of starchy substances is very Titration Curves of the Amylopectin-type.
useful in characterization of starch and its All of the branched polymers tested here,
products. Moreover, the results allow a de- Schardinger a- and ,8-dextrins, polyvinyl al-
piction on the starch-iodine interaction as cohol, as well as linear dextrins of DP below
described below. 25 showed curves of this type. The gradient
When the first few drops of iodine solution of curves varied according to the kind and
are added to a glucosidic chain, they form a amount of substances. It is not clear from
complex with very loosely bound iodine. The these results whether these substances combine
iodine in such a complex would hardly be more or less specifically with iodine. It would
discriminated from free iodine in ampero- safely be stated, however, that the results
metric titration. This results in a rapid were not solely ascribed to non-specific ad-
rise in electnc current at the very beginning sorption due to high-molecular-weight nature
of titration, which is common for all sub- of substances, since dextran, a high-molecular
 Amperometric Titation of Starch with Iodine
polysaccharide composed of n-glucose, gave
exactly the same result as blank titration.
In order to compare affinities of these sub-
stances for iodine, the amount of each sub-
stance was calculated, which would be re-
quired to give the same gradient of the curve
as that for 10 mg of potato amylopectin.
Values were expressed by taking the value for
potato amylopectin to be unity, and given in
Table II. Thus, values which were inversely
proportional to the affinity towards iodine
were obtained.
TABLE II. RELATIVE VALUES OF THE GRADIENT
IN THE TITRATION CURVES
Schardinger S-dextrin
(cycloheptaamylose)
Corn phytoglycogen
Polyvinyl alcohol
Waxy rice starch
Waxy maize starch
Potato amylopectin
Schardinger a-dextrin
(cyclohexaamylose)
Linear dextrin (DP 21)
Linear dextrin (DP 16)
It is evident from Table II, that the com-
bination with iodine is strongest for cyclohexa-
amylose among these substances. It would be
further suggested from results above, that
these substances except cyclohexaamylose are
not able to form helical complex with iodine,
because, if any helical complex would be
formed, a gradient lower than that for cyclo-
hexaamylose would be expected. This point
will be discussed in the following section
together with the mechanism of coloration
with iodine.
The Combination with Iodine and the Coloration
of Iodine Reaction.
The relationship between the color of
iodine reaction and the chain length of amylo-
dextrins was reported by several workers,
whose results differed slightly each other. Re-
sults of Mould and Synge1 0>, and Ohashi3'> are
10) D.L. Mould:and R.L.M. Synge, Biochem. J., 58, 585. (1954).
443
compared in Table III for example. The
minimum chain length required for an amylo-
dextrin to produce red color with iodine was
reported to be 21 by Ohashi, and 25 by Mould
and Synge. These results were essentially in
accord with our results that linear dextrins
of DP below 25 hardly combine nor produce
red color with iodine. A slight discrepancy
between results of Ohashi and ours seems
due to the difference in iodine concentration
at observation.
TABLE III. RELATIONSHIP BETWEEN THE COL-
ORATION OF IODINE REACTION AND THE
CHAIN LENGTH OF AMYLODEXTRIN
Results of Mould and
12.5
Results of Ohashisl SyngelOl
12.5 ~ ~~
DP Color DP Color
10.0
8.6 16 Faint reddish yellow 10> Colorless
5.4 21 Red 10-25 Orange
1.0 28 Reddish purple 25-40 Red
34 Purple 40-130 Blue
0.34
41 Blue
3.94
4.11 61 Blue
The relationship seems to be somewhat
different for amylopectin-type polysaccharides.
Potato amylopectin and waxy rice starch em-
ployed in this experiment were of the aver-
age chain length 26 and 18, respectively 6 >.
Though the value for waxy maize starch used
here was not estimated, it seemed to be similar
to that of waxy rice starch. If average chain
length at least 25 would be required for
developing visible red color with iodine as
presumed from results on linear dextrins,
these amylopectin-type polysaccharides would
not be stained with iodine, or would be
stained only very faintly. But interesting
enough, they gave distinct red or purple color
with iodine even in very low concentrations
used in the amperometric titration, in spite
of the fact that they hardly showed a specific
combination with iodine in the titration.
Their color intensities were too high to be
444 Tsuneo KOBAYASHI
ascribed to possible contamination with com-
ponents of longer chain lengths. On the
other hand, cyclohexaamylose which did not
develop color with iodine under experimen-
tal conditions, showed considerable affinity for
iodine in the titration. It would, then, be
presumed that a helix composed of six gluco-
sidic units are necessary to develop consider-
able affinity for iodine. Thus the complex
formation and the coloration with iodine did
not run parallel with these substances.
Observations described above led the au-
thors to consider that the formation of helical
complex with iodine will not be the sole
mechanism of iodine reaction of amylaceous
substances. Of course, helical complex will
make the major contribution to the colora·
and Noboru KOBAYASHI
tion. But additional mechanism which is
able to explain these observations is neces-
sary. Moreover, some other mechanism will
be required for explaining iodine coloration
of non-starchy substances such as polyvinyl
alcohol.
Acknowledgement. The authors express their
sincere thanks to Dr. Thomas J. Schoch, Corn
Products Co., Arago, Illinois, U.S.A., Prof. Z.
Nikuni, Osaka University, Dr. K. Ohashi,
Gifu University, and Dr. T. Ando, The Insti-
tute of Physical and Chemical Research,
Tokyo, for their kind supply of samples used
here. Thanks are also due to Mr. S. Naka-
yama, and Mr. H. Hiramoto for their tech-
nical assistance.