THE USE OF IODINE IN THE DETERMINATION OF GLUCOSE, FRUCTOSE, SUCROSE, AND MALTOSE. BY F. A. CAJORI. (From the Department of Chemistry, Stanford Universit2/, Calijoornia.) (Received for publication, October2,1922.) INTRODUCTION. Almost without exception, the available methods for the quantitative determination of individual sugars, in solutions where several of them are present together, involve the use of the polariscope. With sugar solutions of low concentration, the ob- served rotation may be only a few angular degrees, and results based on such slight differences are far from accurate. Again, highly colored solutions, such as are often obtained from plant material, must be clarified before they can be used in the polari- scope and not inconsiderable loss. of sugar by adsorption occurs during the clarification process. Moreover, solutions of plant origin often contain other optically active substances. Prerequisite to an understanding of the manner of carbohy- drate formation in the photosynthetic process is an accurate picture of the quantitative relations that exist between the various sugars of the leaf. Advance in this field is largely dependent upon the development of analytical methods, as the analysis of plant material presents many extraordinary difficulties not encountered in material of animal origin. Romijn (1) and Bougault (2) observed that glucose in alka- line solution is completely oxidized to gluconic acid by an excess of iodine and that under the same conditions fructose and sucrose are unchanged. This would seem to offer the basis for a method of distinguishing between the individual sugars without recourse to the use of the polariscope. These two authors recognized the possibilities of the use of iodine as a reagent for differentiating between aldose and ketose sugars, and they suggested methods 617 by guest on April 6, 2018 http://www.jbc.org/ Downloaded from
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THE USE OF IODINE IN THE DETERMINATION OF GLUCOSE, FRUCTOSE, SUCROSE, AND MALTOSE.
BY F. A. CAJORI.
(From the Department of Chemistry, Stanford Universit2/, Calijoornia.)
(Received for publication, October2,1922.)
INTRODUCTION.
Almost without exception, the available methods for the quantitative determination of individual sugars, in solutions where several of them are present together, involve the use of the polariscope. With sugar solutions of low concentration, the ob- served rotation may be only a few angular degrees, and results based on such slight differences are far from accurate. Again, highly colored solutions, such as are often obtained from plant material, must be clarified before they can be used in the polari- scope and not inconsiderable loss. of sugar by adsorption occurs during the clarification process. Moreover, solutions of plant origin often contain other optically active substances.
Prerequisite to an understanding of the manner of carbohy- drate formation in the photosynthetic process is an accurate picture of the quantitative relations that exist between the various sugars of the leaf. Advance in this field is largely dependent upon the development of analytical methods, as the analysis of plant material presents many extraordinary difficulties not encountered in material of animal origin.
Romijn (1) and Bougault (2) observed that glucose in alka- line solution is completely oxidized to gluconic acid by an excess of iodine and that under the same conditions fructose and sucrose are unchanged. This would seem to offer the basis for a method of distinguishing between the individual sugars without recourse to the use of the polariscope. These two authors recognized the possibilities of the use of iodine as a reagent for differentiating between aldose and ketose sugars, and they suggested methods
involving its use. Several other investigators have reported their experiences with iodine as an oxidizing agent for the quanti- tative determination of sugars with varying success.
In the following work we have endeavored to define more sharply the conditions under which sugars arc oxidized by iodine, and, on the basis of these observations, have developed a method for the determination of glucose, fructose, sucrose, and maltose when they occur together.
Romijn (1) was unable to obtain satisfactory quantitative results from the oxidation of glucose by iodine with sodium hydroxide or potassium hydroxide and suggested the use of sodium borate. With this weak alkali, 18 to 40 hours were required to complete the oxidation of glucose to gluconic acid. Willstatter and Schudel (3) pointed out the need of sufficient amounts of alkali to neutralize the acid formed in the reaction. With an excess of 0.1 N sodium hydroxide, and oxidation with iodine for 15 minutes, they were able to recover quantitatively the glucose present in invert sugar.
Bougault (2), using sodium carbonate, observed a quantitative oxidation of glucose by iodine in 30 minutes. Maltose, lactose, arabinose, and other aldose sugars were found by him to be oxidized by iodine. Colin and L&in (4) modified Bougault’s procedure, using disodium phosphate instead of sodium carbonate. They found that 1 hour was the time required for the oxidation of the glucose, with this modification.
Lately, Judd (5) has published the results of a critical study of these methods. She was unable to confirm Romijn’s or Bougault’s findings that iodine oxidizes glucose to gluconic acid, and that under the same condi- tions fructose is not oxidized at all. She ascribed this failure, in part, to possible enolization of the sugars in the presence of alkalies. Judd believed, however, that valuable methods for the analysis of glucose-fruc- tose solutions could be developed by utilizing the reducing power of the sugar solution on iodine, as well as its reducing power on copper solutions. In contrast to the findings of Judd, Baker and Hulton (6), using Willstatter’s procedure, experienced no difficulty in recovering glucose as gluconic acid after 5 minutes oxidation with iodine.
EXPERIMENTAL PART.
Preliminary Experiments with Glucose and Fructose.-The reac- tion between glucose and an excess of iodine in a solution made alkaline with sodium carbonate was followed by determining the amount of iodine present in aliquot parts of the oxidation mixture at intervals during the course of the oxidation. The amount of iodine present was determined by titration with standard sodium
thiosulfate after having made the solution slightly acid, using soluble starch as an indicator. An approximately 0.1 N iodine solution, standardized with 0.05 N sodium thiosulfate was used. The 0.05 N sodium thiosulfate was prepared from a normal stock solution and standardized in the usual way with the aid of 0.1 N
potassium permanganate. The preliminary trials were carried out in a thermostat at 25°C. Later it was found that the velocity of the reaction was not noticeably changed at room temper- atures (18 -22”). Blank determinations were made, using distilled water instead of a sugar solution, but in all other respects the same conditions were maintained. Typical results of these trials, expressed in quantities of iodine present in the aliquot parts, are given in Table I.
These results show that iodine is rapidly reduced in the pres- ence of glucose, the reduction, apparently, being completed at the end of 20 minutes. On the other hand, there is no evidence of any reduction of iodine by fructose. These results confirm the findings of Romijn and Bougault. As indicated from the analysis of the blanks, there are always small losses of iodine from the solution during the course of the oxidation. That such losses do occur is not surprising, considering the volatile nature of iodine. Bougault noticed that on long standing more iodine disap- peared from his solutions than could be accounted for by the theo- retical amount required to oxidize the sugar to the mono-basic acid. He ascribed this to a secondary oxidation of the hydroxyl groups of the sugar. He reports no blank determinations, and it
seems much more reasonable to recognize the possibility of a mechanical loss of iodine than to assume oxidation of the hydroxyl groups of the sugars.
The apparent increase in the concentration of the iodine noted in the solutions during the second half hour of the oxidation is undoubtedly due to the liberation of iodine from the potassium iodide used in the iodine solution.
Bougault emphasized the necessity of having an excess of iodine present in order to insure complete oxidation of the glucose. . We also found this an important condition to be maintained. The amount of iodine to be used depends in part on the alkalinity of the solution. In Table II are given some of our experiments
TABLE II.
Oxidation of Glucose as InJluenced by Varying Amounts ofIodine and Sodium
where varying amounts of iodine and sodium carbonate were used.
It will be seen that with a sodium carbonate concentration of 1 or 2 per cent, three times the amount of iodine necessary to oxidize the sugar gives a complete reaction in 25 minutes. Twice the amount (1.8), as illustrated in Experiment 7, is not a large enough excess and incomplete oxidation resulted. In Experi- ment 1, the high concentration of sodium carbonate diminished the apparently large excess of iodine and again the glucose was incompletely oxidized.
We failed in attempts to oxidize glucose with iodine in neutral solution, but beyond having alkali present, and present in suffi-
cient quantities to neutralize the acid formed in the reaction, no advantage could be observed in increasing the concentration of the alkali. It is highly desirable to carry out the oxidations in solutions of low alkalinity because of the effect of alkalies on sugars. We finally chose for the oxidations, solutions with 1.0 to 1.5 per cent of sodium carbonate. Nef (7) reported that no enolization of glucose took place with this strength of sodium carbonate. The use of sodium carbonate instead of sodium hydroxide seemed desirable in order to avoid any possible enoli- zation of the sugars present.
Witzemann (8) found that disodium phosphate catalyzes the oxidation of glucose by hydrogen peroxide. In trials where we used 0.5 M NLHPO,, we were unable to note any increase in the velocity of the reaction between glucose and iodine that could
be ascribed to any catalytic influence of disodium phosphate. We also endeaGored to catalyze the oxidation of glucose by iodine by adding traces of ferrous chloride to the oxidation mix- ture, but obtained no evidence of any influence of this iron salt on the velocity of the reaction. Within limits, dilution of the iodine solution has no effect on the rate or completeness of the oxidation of glucose. Provided there is suf6cient iodine p&en& it.s concentration does not seem to be significant.
Analysis of Glucose Solutions.-The preliminary experiments indicated that under suitable conditions iodine could be used as a reagent for the quantitative estimation of glucose. That this conclusion was justified is shown in Table III, where the results of the analysis of a number of glucose solutions are given. The protocol of a single experiment is given to illustrate the pro-
cedure that we adopted for the oxidation of glucose by iodine. The glucose used was a Bureau of Standards preparation and before using was dried in vacua over phosphorus pentoxide.
To 10 cc. of a glucose solution, containing 34.56 mg. of glucose, 2 cc. of a 15 per cent Na&Oa solution were added; 15 cc. of a 0.1 N iodine solu- tion were then added, and the flask was stoppered and placed in the dark at room temperature for 25 minutes. Slightly more 10 per cent H,SO# than that needed to neutralize the N&CO* was added and the iodine titrated immediately with 0.05 N Na2SIOI. From the titration, 3.82 cc. of 0.1 N iodine, or 48.5 mg., had been reduced. Calculated for glucose, 34.41 mg.
Within the experimental error, the molecular ratio of the iodine reduced to the glucose present is 2 to 1. In other words, the reaction proceeds to gluconic acid according to the equation:
CsHi,Os + 21 + HSO + CeHi,O, + 2HI
The amount of iodine reduced by the sugar may then be used as a measure of the amount of glucose present.
Determination of Glucose in the Presence of Fructose and Sucrose. -The oxidation of glucose by iodine proceeds in the presence of small or large amounts of fructose or sucrose, without being influenced by either sugar. In Table IV are given results of the determination of glucose when fructose or sucrose are also present.
Analysis of Glucose-Fructose-Sucrose Solutions.-Iodine oxidizea glucose but not fructose or sucrose. Cupric hydroxide oxidizes both glucose and fructose. The use of these two reagents, then, will enable a determination to be made of glucose and fructose when together in solution, or of glucose, fructose, and sucrose when these three sugars occur together. In the latter case, the determination of the reducing power of the sugar solution on both the iodine and copper reagents before hydrolysis of the sucrose, and the reducing power on either of the reagents after hydrolysis, will give data from which can be calculated the amounts of the individual sugars which are present.
We have successfully applied such a procedure for the deter- mination of the three sugars. Oxidation with iodine was carried out as described in the analysis of the glucose solutions. The copper reagent that we have used was Benedict’s reagent (9) and the technique followed was that lately described by Spoehr
(10) for the determination of small amounts of reducing sugars. Briefly stated, this technique consists in oxidizing the sugars by placing centrifuge tubes containing the sugar and copper reagent in a boiling water bath for 3 minutes, and removing the cuprous oxide by centrifuging; the residual cupric copper is determined by titrating the iodine, liberated from an excess of potassium iodide by the cupric copper, with sodium thiosulfate. Benedict’s solution was calibrated in terms of glucose and fructose by de- termining the reducing power of solutions of glucose and fructose made up from very pure and dry samples of these sugars.
One of the difficulties encountered was the establishing of suitable conditions for the inversion of the sucrose. A few hours heating on the water bath with sufficient hydrochloric acid to
TABLE IV.
Determination-of Glucose in the Presence of Fructose or Sucrose.
make a 1 per cent solution will completely invert the sucrose present. However, when fructose was also present a slight color- ing of the solution occurred during the heating, and the reduction of iodine or copper was greater than could be accounted for by the amounts of glucose present. Evidently, fructose in acid solution and at 100°C. is partly decomposed, yielding compounds which are oxidized by iodine and cupric hydroxide. We endeavored to hydrolyze the sucrose with 1 per cent hydrochloric acid at 25°C. Under these conditions, the inversion was incomplete even when allowed to stand over night.
It was finally found that 2 hours at 60°C. with 1 per cent hy- drochloric acid effected complete hydrolysis of the sucrose. The solutions remained water-clear and there was no evidence of any decomposition of fructose at this temperature. These conditions,
iodine and cupric hydroxide as oxidizing agents and carrying out the analysis according to the procedure and under the conditions that have been outlined above, the method would seem to be accurate for the determination of the individual sugars to within an error of 3 per cent.
Experiments with Maltose.-Maltose is oxidized by iodine, 2 molecules of iodine reacting with 1 molecule of the sugar. We have found that this oxidation proceeds somewhat more slowly than the oxidation of glucose by iodine. Where glucose was com- pletely oxidized by iodine in 25 minutes, maltose required 35 minutes to be completely oxidized to the mono-basic acid.
Maltose is much more resistant to acid hydrolysis than is sucrose. We could observe no evidence of hydrolysis of maltose by 1 per cent hydrochloric acid at 60°C. at the end of 2 hours and even at the end of 24 hours at 60X, the hydrolysis was incomplete. 3 hours heating at 100°C. with 1 per cent acid effects complete hydrolysis of maltose but such vigorous treatment has been found partly to decompose fructose if present in the solution. In order, then, to extend the iodine-copper method that has been outlined above to solutions that contain maltose in addition to the other sugars, other means than acid hydrolysis of the maltose would seem to be necessary. For this purpose we have used maltase prepared from fresh yeast by the method of Willstatter, Oppenheimer, and Steibelt (11). We found that maltose is com- pletely hydrolyzed to glucose with yeast maltase after 3 hours digestion at 30°C. in a solution whose reaction has been adjusted to a pH of 6.1 to 6.8 with acid phosphate, according to the direc- tions of Willstatter. The increase in the reducing power of the solution, as a result of the glucose formed in the reaction, may then be taken as a measure of the amount of maltose present.
Freshly prepared enzyme solutions must be used. After 2 or 3 days, the activity of the maltase was greatly diminished and proved to be unsatisfactory for a quantitative hydrolyis of mal- tose. Yeast maltase solutions contain substances that react with iodine, and in the calculations, based on the amount of iodine reduced as the result of the enzyme hydrolysis, it was found necessary to apply rather large corrections.
The results of the experiments with maltose are given in Table VII.
By the use of maltase solutions and adequate control experi- ments, planned to determine the activity of the maltase prepara- tion, as well as the reducing power of this preparation on iodine and cupric hydroxide, it would seem possible to extend the iodine- copper method to include maltose in addition to the other sugars. With a mixture of sugars, the maltose, unchanged by acid hydroly- sis of sucrose at 60°C would be measured by the increase in glucose content of the solution after digestion with maltase.
TABLE VII.
Determinations of Maltose with Iodine under Various Conditions.
Oxidation with I for 35 min. ‘I “ “ I‘ 35 “ “ “ “ “ 35 “ “ “ I‘ “ 35 ‘C “ “ ‘I ‘I 35 “ “ “ “ “ 35 “
1 per cent HCl, 2 hrs. at 60’. Hydrolysis with maltase.
“ “ “ I‘ “ “ “ “ “
The results reported in this paper are based on the analysis of pure sugars. It is well to point out that in applying this method to sugar-containing fluids such as plant extracts that contain, in addition to the sugars, other compounds, the accuracy of the results will depend on the freedom of the solution analyzed from reducing substances other than sugars, or compounds that will react with iodine.
SUMMARY.
The oxidation of glucose by iodine has been investigated and the conditions whereby a quantitative oxidation of glucose to gluconic acid may be effected have been established.
Using iodine and cupric hydroxide as oxidizing agents, a method has been presented for the determination of glucose, fructose, and sucrose where these sugars occur together, and in small quantities.
Results suggesting that the method may be extended to include maltose are given.
The larger part of the experimental work reported in this paper was done in the Coastal Laboratory of the Carnegie Institution of Washington, at the suggestion and under the direction of Dr. H. A. Spoehr. The author is greatly indebted to Dr. Spoehr for his helpful advice and criticism, and to the Carnegie Institution for the privilege of working in the Coastal Laboratory.
BIBLIOGRAPHY.
1. Romijn, G., 2. anal. Chem., 1897, xxxvi, 349. 2. Bougault, J., J. pharm. et chim., 1917, series 7, xvi, 97; Compt. rend.
Acad., 1917, clxiv, 1008. 3. Willstlitter, R., and Schudel, G., Ber. them. Ges., 1918, li, 780. 4. Colin, H., and L&in, O., Bull. Sot. chim., 1918, series 4, xxiii, 403. 5. Judd, H. M., Biochem. J., 1920, xiv, 255. 6. Baker, J. L., and Hulton, H. F. E., Biochem. J., 1920, xiv, 754. 7. Nef, J. U., Ann. Chem., 1914, cdiii, 204. 8. Witzemann, E. J., J. Biol. Chem., 1929-21, xiv, 1. 9. Benedict, S. R., J. BioZ. Chem., 1908-09, v, 485.
10. Spoehr, H. A., Carnegie Inst. Washington, Pub. 687, 1919, 31. 11. Willstiitter, R., Oppenheimer, T., and Steibelt, W., Z. physiol. Chem.,
