Krishnaswamy, K. G.

Sreenivasan, A.

English

Publications of the IAS Fellows

SEPARATION AND DETERMINATION OF THE AMYLOSE 
AND AMYLOPECTIN FRACTIONS OF STARCH 
BY K. G. KRISHNASWAMY AND A. SREENIVASAN 
(From the Department of Chemical Technology, Bombay University, 
Bombay, India) 
(Received for publication, July 14, 1948) 
Most of the earlier procedures used for separating starch into amylose, 
the linear unbranched component, and amylopectin, the branched chain 
component (1, 2), have involved degradation and hydrolysis of the starch 
molecules, During the last few years, however, a number of methods have 
been proposed which fulfil, more or less, the necessary requirement of pro- 
tecting the starch constituents from degradation. The more important of 
these methods for the separation of amylose from starch is based on its 
selective diffusibility in water at 60” or 80” (1,3), precipitability with bu- 
tanol (4), thymol (5), or nitroparaffins (6), and adsorbability on cellulose 
(7). It is shown in this communication that these methods fail to effect 
clear-cut as well as quantitative separation of the two starch fractions, 
while the purity of the products obtained is also variable. It has been 
possible, by suitable combination of certain of these procedures, to pre- 
parelamylose and amylopectin, judged for their purity by the intensity of 
their iodine colorations under standard conditions, and to determine their 
exact percentages in any starch preparation by reference to a calibrated 
curve for intensity of iodine coloration with known mixtures of the pure 
fractions (3). 
EXPERIMENTAL 
Preparation of Starch-The major part of the studies reported here was 
carried out with a sample of starch prepared from a local variety of peas 
(Pisum sativum). The seeds, softened by soaking overnight in water, were 
ground to a not too fine consistency and the mash was extruded through a 
cloth bag into a sufficient volume of distilled water. The residual pulp 
was mashed and pressed out a second time. The combined extract was 
let stand and the sludge which separated was purified of proteinaceous 
material by repeated agitation and settling. The starch suspension was 
finally kneaded through muslin into water and centrifuged to separate the 
starch, which was washed successively with 20 and 80 per cent ethanol and 
allowed to dry at room temperature (28”). Analysis of the product gave 
0.88 per cent protein, 0.25 per cent ether extractive, and 12.75 per cent 
moisture. 
1253 
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1254 AMYLOSE AND AMYLOPECTIN STARCH FRACTIONS 
Determination of Iodine Coloration-The intensity of color developed in 
a 2 mg. per cent solution of starch or of the various starch fractions, on 
addition of a solution of iodine in potassium iodide to a final concentration 
of 4 mg. per cent of iodine, was measured in a 10 mm. cell by a Klett- 
Summerson photoelectric calorimeter with Filter Ks6 in position (3). The 
calorimeter was initially adjusted so that the blank, which had a light yellow 
color due to the iodine in solution, gave a zero reading; the color measure- 
ments recorded are in terms of scale readings in the instrument. 
Fractionation of Starch by Selective Extraction of Amylose with Hot Water 
-5 gm. of air-dry starch, mixed with water to avoid lump formation, were 
treated with about 300 ml. of water at 60” and the suspension maintained 
at this temperature for 4 hours with slow stirring. It was then centrifuged 
at 3000 R.P.M. and the supernatant passed through a sintered glass No. 4 
TABLE I 
Extraction of Starch with Water at 60’ 
Original starch .............. 
Fraction I. ................. 
“ II. ................ 
“ III. ............... 
On dry basis 
Yield 1 Yield 2 Yield 3 
ger cent per cent *cl cent 
14.4 14.7 15.8 
0.9 0.6 Trace 
84.5 85.3 83.5 
Intensity of iodine color (scale 
readings)* 
Yield 1 Yield 2 Yield 3 
143 149 148 
279 271 269 
181 180 
101 103 100 
* Klett-Summerson calorimeter. 
filter, which was found more convenient to use than filter paper coated with 
Hyflo Super-Cel, as recommended in the original procedure (3). The clear 
filtrate, after addition of methanol to a concentration of 20 per cent volume 
per volume, was let stand for 48 hours. At the end of this period, the pre- 
cipitated amylose (Fraction I) was filtered through a sintered glass No. 4 
crucible, washed with 95 per cent ethanol, and finally with absolute alcohol 
before drying in a vacuum oven. The filtrate was further treated with 
methanol to 50 per cent volume per volume strength and allowed to settle 
as before. The precipitate (Fraction II) was filtered, mashed, and dried to 
constant weight. 
The gelatinous residue remaining after the centrifuging of the aqueous 
starch suspension was ground well, dehydrated by repeated additions of 
alcohol, filtered, and dried in vacua (Fraction III). Table I gives a set of 
typical results obtained together with the iodine colorations of the starch 
and of the different fractions. 
In the set of experiments given in Table II, the t,emperature of fractiona- 
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K. G. KRISHNASWAMY AND A. SREENIVASAN 1255 
tion was kept at 80”, as recommended by Meyer (1) ; the procedure was 
otherwise the same as that described above. 
Fractionations of pea starch and of the crude amylopectin (Fraction III, 
Table I) were also attempted by treatment for 48 hours at room tempera- 
ture with 1: 2 chloral hydrate solution in water, as recommended by Meyer. 
The products obtained gave iodine coloration averaging 74 and 67 re- 
spectively; by using chloral hydrate solution at SO”, the corresponding color 
readings were 54 and 56. 
TABLE II 
Fractionation of Starch with Water at 80” 
Fraction I ........... 
‘I II. ......... 
“ III. ........ 
................. 
................. 
................. 
Yield 1 Yield 2 
per cent per cent 
16.0 16.3 
Trace Trace 
82.9 79.6 
Intensity of iodine color* 
Yield 1 Yield 2 
220 224 
114 104 
* See Table I. 
TABLE III 
Fractionation by Rutanol Extraction 
Fraction Yield 1 Yield 2 
Butanol-pptd. by autoclaving. 38.6 39.2 
“ “ Waring blendor. 39.5 39.4 
Butanol-non-pptd. by aut,oclaving.. 60.7 59.9 
‘I “ Waring blendor. 59.4 60.7 
per cent per cent 
Iodine coloration’ 
Yield 1 
243 
234 
52 
47 
Yield 2 
241 
230 
49 
50 
* See Table I. 
Fractionation by Selective Precipitation of Amylose with Butanol- 
Schoch’s butanol precipitation method (4) was closely followed, except for 
the purification of the separated amylose by recrystallization from the 
boiling water-butanol mixture. Since it was thought that some hydrolytic 
degradation of starch was likely to occur as a result of the high temperature 
treatment under pressure, an attempt was made to eliminate autoclaving 
in Schoch’s procedure by securing dispersion of starch with high speed stir- 
ring. A properly gelatinized paste of 5 gm. of starch in about 500 ml. of 
boiling water was treated in a Waring blendor in two lots for 5 minutes each. 
Subsequent separation of the starch fractions was effected as described by 
Schoch. Typical results, by both procedures, are given in Table III. 
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1256 AMYLOSE AND AMYLOPECTIN STARCH FRACTIONS 
Fractional Precipitation of Amylose with ThymoE-The high speed stirring 
recommended by Haworth et al. (5) did not result in suflkient dispersion 
and it was found more effective to use a Waring blendor for aiding dis- 
persion. The results obtained by this method are shown in Table IV. 
Fractionation by Preferential Adsorption of Amylose on Cotton-With a 
1 per cent starch paste, gelatinized and dispersed in a Waring blendor 
according to the procedure of Pacsu and Mullen (7), not more than 0.3 per 
cent of amylose (iodine coloration, average 223) was obtainable, therefore 
suggesting that preferential adsorption of amylose on the cotton used was 
far from satisfactory; somewhat similar results were obtained by using 
filter paper pulp as an adsorbent. 
TABLE IV 
Fractionation by Thymol Extraction 
Fraction Yield 1 Yield 2 
Iodine coloration* 
Yield 3 
Yield 1 Yield 2 Yield 3 
per ceni per cent par cenf 
Thymol-pptd.. . . . . . . . . . . . 39.7 39.9 40.7 222 220 208 
Thymol-non-pptd.. . . . . . . . 60.1 60.2 59.1 50 52 45 
* See Table I. 
DISCUSSION OF RESULTS 
The pronounced difference in the affinity of amylose and amylopectin 
for iodine has formed the basis for the determination of their relative pro- 
portions in starches potentiometrically (S), absorptiometrically (3), or 
spectrophotometrically (9, 10). On the assumption that the intensity of 
iodine coloration with amylose or amylopectin fractions will be determined 
by their respective freedom from each other, it becomes apparent that the 
higher the scale reading, the purer will be the fraction in respect to its amy- 
lose content, and that, conversely, purity with regard to amylopectin will 
be connoted by the lowest scale reading. Based on this criterion, it would 
follow from the data presented here (Tables I to IV) that no single method ef- 
fects simultaneously efficient and quantitative separation of the two starch 
fractions. The procedure of McCready and Hassid (Table I) gives, in one 
step, the purest amylose fraction, judging from its iodine-staining property; 
its solubility is, however, only of the order of 15 per cent, which is very 
low. That the amylopectin fraction obtained here is admixed with a high 
amount of amylose is evident from the values for iodine coloration as com- 
pared to the amylopectin fraction obtained by the procedures of Schoch 
(Table III) and of Haworth et al. (Table IV). The amylose fraction yielded 
by Meyer’s extraction procedure (Table II) is only about 80 per cent as 
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K. G. KRISHNASWAMY AND A. SREENIVASAN 1257 
pure as the corresponding fraction obtained according to the method of 
McCready and Hassid; this is no doubt due to the fractionation tempera- 
ture being near the gelatinization point of the starch and consequent con- 
-60 _ 
I 
!  
1 
I 
t 
I 
1 I 
40 60 80 100 
I Amylose 
100 80 60 40 20 a 
$ Amylopectin 
FIG. 1. Color intensities of mixtures of amylose and amylopectin from pea starch 
with iodine. 
tamination with amylopectin by the disintegration and rupture of the 
granules. The use of chloral hydrate solution to purify amylopectin from 
admixed amylose resulted in a product still containing about G to 10 per 
cent of amylose. 
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1258 AMYLOSE AND AMYLOPECTIN STARCH FRACTIONS 
Both butanol and thymol undoubtedly effect very much better fractiona- 
tion of the starch components than does extraction with hot water, and, 
indeed, the yields of amylose and amylopectin correspond more nearly to the 
correct values deduced below than do those obtained by hot water extrac- 
tion. However, it is clear from a comparison of the iodine coloration of 
amylose fractions (Tables III and IV) that they are respectively only about 
85 and 77 per cent as pure as that obtained by the McCready and Hassid 
method. It has been possible to obtain pure amylose by successive re- 
crystallizations from boiling water-butanol mixtures as recommended by 
Schoch (4, ll), but, the yields being no longer quantitative, it appeared 
preferable to do so in a single extraction with hot water at 60”. 
Although the amylopectin fractions obtained by selective precipitation 
with butanol or thymol have given the lowest intensity of iodine coloration 
TABLE V 
Fractionation of Mung Starch 
Starch analysis 
Moisture, ye.. . . . . . . . . . . . . 
Proteins,~~................................................... 
Ether extractives, %.......................................... 
Iodine coloration of starch*. . . . . . . . 
“ ‘I “ amylose fraction*. . 
“ ‘I “ amylopectin fraction*. 
Amylose (from Fig. 2), %. . . . 
Amylopectin (from Fig. 2), To.. . . 
* Scale readings, Klett-Summerson calorimeter. 
13.50 
0.69 
0.23 
127 
287 
34 
36.8 
63.2 
of all the methods studied, it was felt that, since it is always the residue in 
the mother liquor after the amylose had been precipitated, amylopectin 
may not be easily obtainable in as pure a form as the amylose component. 
We therefore attempted to ascertain whether by butanol fractionation of 
the residue from the hot water treatment of starch at 60” (Fraction III, 
Table I) a purer preparation of amylopectin could be obtained than by 
Schoch’s method from the original starch. By this procedure, a product 
was secured which gave an iodine coloration of only 43 or 44 units. This 
was the purest amylopectin obtainable; a product with similar purity could 
also be prepared by thymol fractionation of crude amylopectin. 
Pure preparations of amylose and amylopectin can thus be obtained in 
one and two operations, respectively, by a combination of the features of 
McCready and Hassid’s method for amylose and that of Schoch or of 
Haworth el al. for amylopectin. By using various proportions of the starch 
components prepared as above, the color intensities of the mixtures in 
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K. Cl. KRISHNASWAMY AND A. SREENIVASAN 1259 
solution (2 mg. per 100 ml.) with iodine can be plotted against per cent 
concentration of the two fractions when a linear relationship similar to that 
reported by McCready and Hassid (3) is obtained (Fig. l), and from which, 
I  
I  
8 
8 
1 I  L 
0 20 
% 
4ilnylose 60 80 100 
100 a0 
/$ 
61mylopect:: 20 0 
FIG. 2. Color intensities of mixtures of amylose and amylopectin from mung 
starch with iodine. 
after ascertaining the color intensity of the original starch with iodine under 
identical conditions, its proportions of the two constituents can be read; 
the latter can also be deduced by simple extrapolation, as there is strict 
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1260 AMYLOSE AND AMYLOPECTIN STARCH FRACTIONS 
proportionality between color intensity with iodine and amylose or amylo- 
pectin content. In this way, the preparation of pure starch used in these 
studies, with its iodine coloration of 147 (Table I), can be observed to con- 
sist of 45.0 per cent amylose and 55.0 per cent amylopectin. 
By the foregoing procedures for the preparation of pure amylose and 
amylopectin fractions, and by using a preparation of mung (Phaseolus 
radiatzts) starch, the resulting observations are given in Table V and in 
Fig. 2. 
Differences such as are recorded here in intensities of iodine coloration 
with pure amylose or amylopectin preparations from natural starches are 
bound to exist because of possible heterogeneity as to molecular size as well 
as, with amylopectin, to variations in the degree of branching (cf. (8) ). 
Although the various methods for the fractionation of starch examined 
here do not effect a clear-cut and quantitative separation of the unbranched 
and branched components in their pure state, fractionation by selective 
precipitation of amylose with butanol or with thymol, as recommended by 
Schoch (4) and by Haworth et al. (5), gives an approximate idea of the rela- 
tive proportions of the two constituents. However, to obtain them in a 
pure state for examination of their individual properties or for a precise eval- 
uation of their percentages in any starch sample by reference to a calibrated 
curve or by extrapolation, as described here, it would appear necessary to 
resort to a combination of procedures involving the properties of amylose 
for selective diffusibility in water at 60” and precipitability with butanol- 
or thymol-saturated water. 
SUMMARY 
1. A comparative study has been made of the procedures for the fraction- 
ation of starch based on the differential solubilities of amylose and amylo- 
pectin in hot water and in butanol- or thymol-saturated water. 
2. It is shown that the method of extraction with hot water at 60” yields 
an amylose fraction which is the purest obtainable, judged from the in- 
tensity of its coloration with iodine; amylose separation is not, however, 
quantitative. 
3. ‘Fractionation of starch by selective precipitation of amylose with 
butanol or thymol gives only a rough indication of the relat,ive proportions 
of the linear and branched components; besides, separation, as judged by 
the iodine-staining properties of the products obtained, is not clear-cut. 
4. A procedure is outlined for obtaining highly pure preparations of 
amylose and amylopectin from a starch sample. By quantitatively deter- 
mining the color intensities of the starch and of known mixtures of its amy- 
lose and amylopectin fractions with iodine, their proportions in the former 
can be precisely estimated. 
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K. G. KRISHNASWAbtY AND A. SREENIVASAN 1261 
BIBLIOGRAPHY 
1. Meyer, K. H., in Kraemer, E. O., Advances in colloid science, New York 1, 
143 (1942). 
2. Peat, S., in Annual review of biochemistry, Stanford University, 16,76 (1946). 
3. McCready, R. M., and Hassid, W. Z., J. Am. Chem. Sot., 66,1154 (1943). 
4. Schoch, T. J., J. Am. Chem. Sot., 64,2957 (1942). 
6. Haworth, W. N., Peat, S., and Sagrott, P. E., Nature, 167, 19 (1946). 
6. Whistler, R. L., and Hilbert, G. E., J. Am. Chem. Sot., 67, 1161 (1945). 
7. Pacsu, E., and Mullen, J. W., 2nd, J. Am. Chem. Sot., 63,116s (1941). 
8. Bates, F. L., French, D., and Rundle, R. E., J. Am. Chem. Sot., 66,142 (1943). 
9. Kerr, R. W., and Trubell, 0. R., Paper Trade J., 117, No. 15,25 (1940). 
10. Baldwin, R. R., Bear, R. S., and Rundle, R. E., J. Am. Chem. Sot., 66,111 (1944). 
11. Wilson, E. J., Jr., Schoch, T. J., and Hudson, C. S., J. Am. Chem. Sot., 66,138O 
(1943). 
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Separation and determination of the amylose and amylopectin fractions of starch

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Separation and determination of the amylose and amylopectin fractions of starch

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