ANALYTICAL CHEMISTRY
802 two portions of distillate be collected, each having a volume of about 45 ml. Since distillation is rapid, the second distillate can be obtained in less than 7 minutes. Samples having less than 20 micrograms of pCA (31 micrograms CMJ’) have been found in practice to give an insignificant amount of color in the second distillate. The collection of two separate small volumes of distillate rather than one large volume has the advantage that inasmuch as unknown concentrations of CMU are present in all samples, dilution of distillates containing small amounts of p C A is avoided, and complete recovery of large amounts is assured.
Table 11. Hydrolysis and Recovery of CJIU Hydrolysis Time, Hours 0.5 0.5 1.0 2.5 2.5
Fruit Homogenate, Grams 300 300 300 300 300
C31U Added. y 50 50 50 50 50
C3IL-n Recoyered. 44 7 48.9 49 1 48.5 40.6
y
sb
Recovery 89 98 98 97 99
a Corrected for a blank value on 300 grams of hoxogenate of 3.2 parent C3IU.
y
of ap-
The selection of a distillation rate of 7 nil. per minute Tyas an arbitrary one and mas merely the maximum attxined with the electric heater employed (Precision Scientific Co., Chicago, Ill., iiFul-Control’J). A lower rate is likely to require a greater volume of distillate for complete recovery. Since precipitation of pCA on the cold wall of the condenser is possible, it is specified that the condenser be flushed with hot condensate near the end of each collection of distillate.
Hydrolysis and Recovery of CMU. The rate of hydrolysis and recovery of CMU were tested by adding 50 microgram quantities t o pineapple fruit tissue and hydrolyzing with 15% sodium hydroxide for 0.5 to 2.5 hours. Distillation and color development were performed as specified in the above method. Table I1 presents the results which indicate that a 1-hour hydrolysis period is adequate for complete hydrolysis x i t h recoveries near 100%. Sensitivity. When the color measurement is made on the Evelyn photoelectric colorimeter with its ?/B-inch diameter absorption test tube, 3 micrograms (95% transmission) of CMU can be significantly determined. Thus the method is sensitive to 0.01 p.p.m. of CMU when 300 grams of sample are analyzed. Significant differences of this order of magnitude can be determined among various samples containing traces of C3IP in comparison with blanks giving an apparent reading of 0.01 p,p.ni., such as is obtained with pineapple fruit tissue. ACKNOWLEDGMENT
The authors wish to thank W.K. Lon-en, H. 11.Baker, and D. E. Wolf of The Grasselli Chemicals Department of E. I. du Pont de Nemours & Co. for making available their report on the determination of ChlU in adiance of publication, and R. W.Leeper for helpful suggestions. LITERATURE CITED
(1) Avorill, P. R., and Korris, X I . V., ANAL Cmar., 20, 763 (1948). (2) Bucha, H. C., andTodd, C. W., Science, 11,493 (1951). (3) International Critical Tables, 1st ed., Vol. 3, p. 221, Sew York,
McGraw-Hill Book Co., 1928. (4) Lowen, W. K., and Baker, H. X I . , ANAL.CHEM.,24, l l i 5 (1952). RECEIVED for review August 14, 1952 Accepted December 11, 1952. Published with the approval of t h e Director of the Pineapple Research Institute of Hawaii as Technical Paper No. 209.
Amperometric Method for Determining the Sorption of Iodine by Starch BRUCE L. LARSON, Laboratory of Biochemistry, Department of Dairy Science, University of Illinois, Urbana, Ill., AND KENNETH A. GILLES AND ROBERT JENNESS, Division of Agricultural Biochemistry, University of Minnesota, S t . Paul, Minn. activity of iodine in a starch-iodine solution has been T shown to be chiefly a function of the amount and physical condition of the amylose present in the starch. Various techniques HE
which have been employed for evaluating the equilibrium in such systems include spectrophotometric (1, 6, 10) and electrophoretic ( I f ) determinations of the complex and potentiometric (2, 16, 18) measurements of the activity of iodine. The potentiometric titration technique has been widely employed for determining the iodine uptake and hence the amylose content of starch samples. It was considered that titration of starch with iodine could he simplified by an amperometric adaptation of the dead-stop titration described by Foulk and Bawden (4, 7, 15) similar to that employed for iodometric determination of protein sulfhydryl groups (9). METHOD
Solutions and reagents somewhat similar to those employed by Bates et al. ( 2 ) were used in conjunction with the titration apparatus and procedure of Larson and Jenness (9). The procedure (9) was altered by shunting resistances across the galvanometer to varv its sensitivity. Since standard solutions of iodine are rather unstable, iodine was liberated by titrating standard potassium iodate into an acid solution containing starch and potassium iodide. The detailed titratim procedure was as follows: Into a beaker (4 X 11 cm.) were introduced 10 ml. of a 0.5 N potassium hydroxide solution containing an amount of starch sample equivalent t o about 6 mg. of amylose, 75 ml. of distilled water, 10 ml. of normal hydrochloric acid, and 5 ml. of 0.4 N potassium iodide solution. I n the case of amylopectin and of starches very ]om- in
amylose content, about 50 mg. were titrated. The beaker was set in a water bath a t 25’ C., and the mixture was stirred a t a constant rate. An assembly carrying the electrodes and buret tip was next lowered into the solution, the galvanometer sensitivity was set a t 0.05 pa. per mm., and an initial reading was taken. With constant stirring, the mixture was then titrated with increments of a standard 0.005 A’ potassium iodate solution, 1.5 minutes being allowed for each increment before taking the galvanometer reading. Increments of 0.2 ml. were used except in regions exhibiting inflections in the plot of current us. milliliters of iodate wherein the increments were reduced to 0.1 ml. When the galvanometer deflection exceeded 50 mm. (2.5 pa.) the sensitivity was decreased to 0.20 pa. per millimeter by adjusting the shunt. Amylose and amylopectin were purified by Pentasol (Sharplee Chemicals, Inc.) fractionation according to the procedure of Lansky et al. (8),pentosans by the fractionation methods of O’Dwyer et a2. (18) and Wise (f?’), and raw defatted starches by the methanol extraction procedure of Doremus et al. (3) and Schoch (14). All samples were dried first a t room temperature in a vacuum oven for 9 hours, then in a vacuum oven at 90” C. for 6 hours, and finally stored in a desiccator over phosphorus pentoxide. RESULTS AYD DISCUSSION
Titration of Amylose and Amylopectin. Titration curves of a reagent blank, amylose, and amylopectin are shown in Figure 1. Over the range of 0 to 10 pa. (and even higher) the current flowing in the blank titration is a linear function of the free iodine present in the solution. From this curve it is possible to relate the free iodine concentration-Le,, that present free in the aqueous phase-to a given galvanometer reading. The curve for amylose shows that as iodine is added to the solution, after a small
803
V O L U M E 25, NO. 5, M A Y 1 9 5 3 initial increase, there is little change in the free iodine concentration until the amylose has been saturated with iodine. The slope then increases abruptly, and the curve becomes linear but with a smaller slope than the blank titration. The titration curve for amylopectin shows a small initial inflection similar to that of amylose and then a rapid change in slope with a linear portion whose slope is considerably less than that exhibited in the blank or amylose titrations.
0
f
P
Amylopectin,
1
corn-
c v
4
2
-
- - - - -0
.5
-
end point is obtained in Figure 2 by extrapolating to zero free iodine content. I n determining the end point in this manner, i t is assumed that the nonspecific binding is linearly related to free iodine concentration even a t very low iodine levels. It is difficult to prove or disprove the validity of this assumption. Amylopectin, which presumably does not form helices, exhibits a small initial sorption but this may be due to a small quantity of contaminating amylose. Consequently, the behavior of amylopectin cannot be used as an argument against the assumption of a linear relation at low iodine levels. Amylose depresses the slope slightly beyond the inflection point, a phenomenon which may he due either t o the presence of amylopectin as a contaminant or to an inherelit nonspecific binding by amylose itself. Since the true status of iodine binding by starches appears to be slightly indeterminate, all analyses reported in this paper have been expressed both as “total iodine binding” and as “amylose iodine binding.” Applicability. The reproducibility of the titration on a single sample of amylose v a s next sought. Although some variation in the slope of the curve beyond the inflection point n as sometimes observed, the end points were identical. A similar effect n a s noted in the titration of protein sulfhydryl groups (9). Volume changes during the titration would affect the slope of the curve, but the volunie of iodate required is so small relative to the total volume of solution as to make this source of error neg1igi’)le.
2-2
I.O 1.5 2.0 m I . 0.005N K IO,
2.5
Figure 1. Titration Curves for a Reagent Blank, 45.3 hlg. of Corn Amylopectin, and 6.28 Rlg. of Corn Amylose Showing correction for free iodine and for nonspecifically bound iodine
Computation of Iodine Binding. The total iodine bound up t o the point of inflection can be calculated readily from the difference between total and free iodine in the system a t that point. The free iodine is equivalent to the amount of iodate required in the blank titration to produce a current flow equal to that a t the inflection point in the case of the starch sample. Thus (Figure 1) the total bound iodine i4 computed from the difference A -B. For the pa1 ticular titration shown, the difference amounts to 1.82 - 0.02 = 1.80 ml. of 0.005 S potassium iodate or 18.1 grams of iodine per 100 grams of amylose. The titration data can also lie plotted in the form employed by Bates et al. (Z), bound iodine us. free iodine, to yield a plot analogous to theirs (Figure 2). In making such plots, the free iodine is computed from the blank titration and the bound iodine from the differences betTreen the blank and samples curves a t various current values. I t is unnecessary, however, to plot the data for amperometric titrations in this manner because the same end point is given by the direct plot of current t s . iodine added. I n this respect the amperometric method differs from the potentiometric procedure in nhich the plot of bound zs. free iodine must be made to ascertain the end point. The iodine binding computed as outlined above represents the total iodine bound by the sample. Most of it is doubtless bound specihcallv 111 the helices of the amylose molecule hut a small portion apparentlv is bound or adsorbed nonspecifically as evidenced by the fact that the slopes of the plots beyond the inflection point are smaller than the slope of the blank titration. Amylopectin exhibits this apparent nonspecific binding t o a marhed degrer; amylose only to a small extent. In employing iodine IJinding as a measure of amylose content, it would be desiraiJle to determine only the iodine bound by the amylose helices. This value can be approximated by extrapolating the linear ascending portion of the plot back to the level of residual current (about 0.3 Ma.) which flowed before any iodate was added. In Figure 1 this end point is illustrated by point C. The same
, .I 2 .3 .4 .5 Free Iodine (os ml. .005N K I O , )
Figure 2.
Titration Curve for Corn Amylose
Same titration shown in Figure 1 plotted in form employed by Bates et ai. ( 2 )
Also the changes in pH and the iodide concentration during the titration due to the liberation of iodine from the iodate are very small. The reproducibility attained in six titrations of a sample of amylose is shown in Table I (ser‘es a). The effect of amylose concentration on the amperometric titration is shovn in Table I (series b). From a relatively concentrated solution of amylose in 0.5 S potassium hydroxide aliquots were withdrawn which represented 6.3 to 41.8 mg. of am>-lose. These were found to bind t n the average a total of 18.1 grams of iodine per 100 gramq of amylose. It appears that iodine binding is not greatly depenuext on amylose concentration in the range studied. If this method is to be employed for the analysis of raw starches, it must be independent of other iodine binding materials such as amylopectin and some of the pentosans which give a blue color
804
ANALYTICAL CHEMISTRY
wit’h iodine. I t x a s s h o m in Figure 1 that amylopectin caused a depression of the slope nith a binding of iodine, but apparently not a t any specific iodine level. Thus, it does not interfere in the end point. To ascertain whether pentosans interfere with the amylose analysis, samples of purified xylans were titrated. Aspen bark xylan and flax straw xylan failed to bind iodine or to form a blue color. Furthermore, a reduced TTheat straiv xylan which gave a blue color with iodine showed no iodine uptake by the amperometric titration. These results, of course, do not exclude the possibility that other pentosans may bind iodine in appreciable amounts. Titration of Various Starches. The titration procedure was applied to a series of ran- defatted starches of various botanical origin which represented loa to very high amylose contents. The results of this study (Table 11) are expressed as “total” and “amylose” iodine binding. Amylose contents were calculated on the basis of the iodine binding capacity of corn amylose. This assumption is not strictly correct since “pure” amyloses isolated from various sources differ somewhat in iodine sorption (2,16,18).
Table 1.
Titration of Corn Amylose
samplerTeight, Series
Mg.
Iodine Binding, Grams per 100 Grams Total “.hnylose“
KO systematic study has been made of the extent to 15 hich results obtained by this method agree with those obtained by the potentiometric method. The sample of corn amylose x a s reported to bind 18.9 grams of iodine per 100 grams as determined potentiometrically (13). >lost of our data on this sample fall slightly below this value. The iodine binding capacity (total) and the calculated amylose contents of the starches reported in Table I1 agree well with the currently accepted values as determined by the potentiometric method (2, 5, 16, 18). Consequently, it seems likely that the method described herein measures essentially the same property as the potentiometric method. The slightly lower values reported for the iodine sorption of the sample of corn amylose cannot be attributed to volatilization of iodine during the titration since this would have an effect in the opposite direction, Furthermore, titrations performed in a closed system yielded the same results as those in the open beaker, doubtless because the time necessary to conduct an amperometric titration is considerably shorter than for the potentiometric titration. The temperature of the solution also did not affect the amount of iodine bound as long as it did not vary by more than 1” C. Table I1 s h o w that the method of calculation has a large effect on the calculated amylose contents, especially with the samples of low amylose contents. Since these samples contain ’ considerable amylopectin, the apparent amylose content will be too high if not corrected for the nonspecific binding of amylopectin. Advantages. The method described in this paper for determining the iodine sorption of starch has a number of advantages over the potentiometric procedure. The end point is sharp, and the iodine uptake is readily and quickly calculated from a plotted curve. With the time schedule employed, it is convenient to plot the current us. milliliters of iodate while the titration is proceeding. I n addition, the method utilizes as a primary standard potassium iodate with all of its advantages over the use of iodine which is unstable and must be frequently standardized against
Table 11.
Titration of Starches and Starch Fractions
Iodine Binding, Grams per 100 Grams .4mylose Content (W)on Basis of Starch or starch Fraction Total “Amylose“ Total iodine “Amylose” iodine Waxy sorghum 0.149 0.05 0.80 0.29 Corn amylopectin 0.266 0.224 1.5 1.3 Yautia 1.73 1.58 9.6 9.4 Tapioca 3.48 19.4 3.30 18 9 Sago 4.47 24.8 4.19 23.9 Corn 4.53 25.2 4.18 23.9 Kheat I 4.59 25.5 4.25 24.3 Wheat I1 4.74 26.4 4.39 25.1 Buckwheat 4.78 26.6 4.35 24.9 Flowering canna 6.45 35,8 5.26 30.1 Field pea 6.45 35.8 6.18 35.4 Wrinkled pea 11.9 11.3 66.2 64.7 Potato amylose I 18.2 101.1 18.1 103.3 Potato amylose I 1 18.8 104.5 18.3 104.5 Corn amylose (mean) 1 8 0 100.0 17.5 100,o
some other standard. Finally, the simplicity of the apparatus and procedure recommend it for rapid and routine determinations. For routine analysis it would not be necessary to determine the whole titration curve as shown in Figure 1. Only the slope of the straight line after the amylose has been saturated with iodine and a blank titration are required. For this purpose, it is convenient to adjust the galvanometer sensitivity to 0.20 pa. per mm., and to add the iodate solution cautiously until a permanent galvanometer reading of about 8 mm. (1.5 pa.) is obtained. Further increments of the iodate solution are added, and the galvanometer readings are recorded until a current of about 8 pa. is reached. The end point may then be obtained from these data by extrapolating to the level of residual current flow xhere no iodate was added in the blank; a perpendicular dropped from this point to the abscissa reads directly in the milliliters of iodate equivalent to the iodine that the sample has bound. Such a titration and calculation can easily be accomplished in a period of 10 minutes. ACKNOW LEDGMEh-T
The authors n-ish to thank C. E. Rist of the Sorthern Regional Research Laboratories, Peoria, Ill., and Fred Smith and F. A. Loewus of the Division of Agricultural Biochemistry, University of Minnesota, St. Paul, Minn., for furnishing some of the starches and starch fractions. LITERATURE C I T E D
Baldxin, R. R., Bear, R. S., and Rundle, R. E., J . Am. Chcm. Soc., 66, 111 (1944). Bates, L. F., French, D., and Rundle, R. E., Ihid., 65, 142 (1943). Doremus. G. L.. Creshaw, F. -1..and Thurber. F. H..Cereal Chem., 28, 308 (1951). Foulk. C. IT., and Bawden, h.T., J . -4m. Chem. Soc., 48, 2045 (1928).
Kerr, R. W.,“Chemistry and Industry of Starch,” 2nd ed., p. 882, New P o r k , Academic Press, Inc., 1950. Kerr, R. W., and Trubell, 0. R., Paper T m d e J . , 117, 25 (1943). Laitinen, H. -4., ANAL.CHEX, 24, 48 (1952). Lansky, S., Kooi, M., and Schoch, T. J., J . Am. Chem. Soc., 71, 4068 (1949).
Larson, B. L., and Jenness, R., J . Dairy Sei., 33, 891 (1950). hIcCready, R. M.,and Hassid, W.Z., J . Am. Chem. Soc., 65, 1154 (1943).
Mould, D. L., and Synge, R. L. M., Biochem. J . , 50, xi (1952). O’Dwyer, M. H., Ibid., 28, 2118 (1934). Rist, C. E., private communication, 1951. Schoch, T. J., J . Bm. Chem. Soc., 64,2954 (1942). Stock, J. T., Metallurgia, 37, 220 (1948). \\%on, E. J., Schoch, T. J., and Hudson, C. S., J . Am. Chem. Soc.. 65. 1380 (1943). (17) Wise, L. E., “Wood Chemistry,” p. 257, New York, Reinhold Publishing Corp., 1948. (18) Wolff, I. A,, Gundrum, L. J., arid Rist, C. E., J. A m . C h m . Soc., 72, 5188 (1950).
RECEIVEDfor review November 3, 1952.
Accepted January 10, 1953. Paper 2879, Scientific Journal Series, Minnesota Agricultural Experiment Station, Published with the approval of the Director of the Illinois Agricultural Experiment Station.
