Reaction between Chlorous Acid and Glucose Quantitative Stoichiometry and Evaluation of Reagent Decomposition HERBERT F. LAUNER and YOSHIO TOMIMATSU Western Regional Research Laboratory, Albany, Calif.
Sodium chlorite in w-eak acid is a potentially important reagent for quantitative carbohydrate chemistry because it is specific for aldoses or aldehyde groups and because it cannot cause the side reactions usually undergone by alkali-sensitive carbohydrates in the presence of the alkaline media of present reagents. Therefore, the reaction between chlorite and glucose, as a model substance, was studied. The stoichiometry of the reaction was shown to involve 3 moles of chlorous acid per mole of glucose. As the reagent not only oxidizes glucose but also simultaneously decomposes, it was necessary to account quantitatively for decomposition by assuming that the rate of the latter is proportional to the geometric mean of the chlorite concentration. This led to a simple analytical expression found to hold over the ranges 0.000004 to 0.0003-Vglucose, 0.0005 to 0.0032-Mchlorite, pH 2.4 to 3.4, at 50' C. Thus, glucose down to 0.67 per ml. can be determined by this method.
and Isbell (11) of the reactions o- sodium chlorite, SaC102 iTith sugars in weakly acidic solution. They found that a t pH 2.2, glucose, arabinose, and xylose were oxidized much more rapidly than fructose, sucrose, or methyl glucoside, the small changes in the last two resulting mainly from hydrolysis. Their results indicated that chlorous acid, HClO?, rather than chlorite ion, !?as the oxidizing agent and that the products, chlorine dioxide and chlorate, react s l o ~ l yor not a t all with aldoses. Exploratory experiments indicated that approximately 1 mole of glucose reacted with 3 moles of chlorite. Their study of the oxidation was rendered difficult by the simultaneous decomposition of the reagent. This decomposition Tvas studied by Barnett (1) and by Taylor, Khite, Vincent, and Cunningham (21, 2 2 ) .
c
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al c 0
T
HE methods available for the microdetermination of alde-
hyde end groups in polymeric carbohydrates have been mainly those used for simple sugars (IO), employing copper, ferricyanide, or hypoiodite reagent. -411 of these methods employ alkaline media, known to cause side reactions in oxystarches, oxycelluloses, and polyuronides (2, 3, S, 6 , 8, 9. 14, 16, 17, 18, 20, 23) and in the few dialdeh?.de type disaccharides that have been studied (8). The presence of dialdehyde groups in simple molecules, or of mono and dialdeh?.de groups nithin a polysaccharide chain, or of carboxyl groups on the number 6 carbon atoms, appears to promote alkaline hydrolysis a t acetal linkages ( 8 ) , leading to depolymerization and creation of more aldehyde groups (6, 8, 23). The removal of the aldehyde group by oxidation to carboxyl (20) or by methylation (16, 19) lessens alkali-sensitivity. Of the reagents named, neither copper nor ferricyanide is considered by some (10) t o be specific or stoichiometric with regard to aldoses. Hypoiodite, under proper conditions, appears to oxidize glucose and other simple aldoses quantitatively and stoichiometrically, although a 4% error in the glucose value is caused by the presence of an equal amount of fructose (13). This is apparently due to the typical ketose-aldose equilibrium in alkaline media, and represents another type of error caused by alkali-sensitivity. However, even substances regarded as stable in alkaline media undergo unknown reactions with hvpoiodite. This reagent was found to oxidize cellulose and hydrocelluloses indefinitely, a t rates which became constant and similar after the aldehyde end groups had been oxidized (14). Results with dextrins ( 4 ) also have indicated extensive overoxidation, increasing with degree of polymerization, when hypoiodite is used under conditions giving theoretical results for glucose. Thus, side reactions of this reagent with polysaccharides in general appear probable. I n apparent conflict with the foregoing, alkaline ferricyanide, under a given set of conditions of time, temperature, and concentration, has been reported (16) to give results in agreement with osmotic pressure data for amylodextrins, amyloses, and amylopectins, over a wide range of glucose units per chain. Because of these uncertainties inherent in alkaline methods, considerable interest attaches to the pioneer studies by Jeanes
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HOURS
Figure 1. Rate of Disappearance of Sodium Chlorite in Dark A l l curves.
Sodium chlorite was 0.0008M, pH 2.4, temperature 50' C.
T. Glucose, a t 0.0001M
Control without glucose T - D . Difference between curves T and D G. Calculated from Equation 8 Dotted line corresponds t o glucose added as calculated from chemical Equation 1 D.
Strong support of the specificity of chlorous acid for oxidizing aldehyde groups in alkali-sensitive substances was rendered by the results of Rutherford et al. (80). They showed, within experimental error, that the dialdehyde groups, as measured by the periodate consumed by the cellulose, were quantitatively oxidized to carboxyl groups by chlorite. Launer, Wilson, and Flynn (12) studied the reaction between
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V O L U M E 2 6 , NO. 2, F E B R U A R Y 1 9 5 4
383
chlorous acid and glucose, cellobiose, and several other aldoses, and developed both volumetric-iodine, liberated from potassium iodide, titrated with 0.025.1- thiosulfate-and photometricdetermination of the chlorine dioxide evolved-methods of measuring the extent of oxidation. A procedure for determining glucose was recommended which specified the use of 5 millimolar chlorite a t 40" C. for 20 hours in an acetate buffer a t pH 3.5, this reagent being standardized against glucose and used empirically. I t n a s evidrnt that the reagent could not be used in a stoichiometric manner unless concurrent decomposition were quantitatively evaluated. The present work \vas undertaken t o test severely, over a wide range of conditions, the theoretical treatment and evaluation of the concurrent decomposition of the reagent during oxidation of glucose. By eliminating light effects, precision has been considerably improved: the results are believed to be adequate to establish the stoichiometry of the oxidation reaction sufficiently so that this reaction can be used in a versatile and relatively quantitative manner. Although glucose was used as the model aldose, i t is hoped that the results will lead to the development of a reliable end group method for aldose polymers. PRELIMINARY DISCUSSION
I n the presence of glucose, sodium chlorite disappeared in a typical experiment, Experiment 1, as shown by curve T,Figure 1, greatly exceeding the dotted ordinate corresponding t o the amount of glucose added. This ordinate was calculated from Equation 1 suggested by Jeanes and Isbell RCHO aldose
+ 3HCI02 = RCOOH + 2C102 + HCI + H2O aldonic acid
(1)
HOURS Figure 2. Rate of Oxidation of Glucose at Various Initial Concentrations, COof Chlorite, a n d G Oof Glucose
trasted with C T , which refers t o the test solution with glucose. Equation 4 states that the decomposition of the reagent in any solution is proportional t o C2 during an infinitesimal interval, during a finite interval in which is the and proportional t o mean concentration. The problem of evaluating DT is solved if ET during the finite interval can be evaluated. This cannot be done rigorously by integrating the rate function for oxidation plus decomposition because this is not knom-n, so a less rigorous procedure is necessary. Now for the control can be evaluated by integrating Equation 4 to give:
c2
Solely on the basis of chemical Equation 1, the amount of glucose oxidized would be expressed in terms of chlorite as folhws:
'/a(Co - C T )
(2)
where Go and Co are initial concentrations, and G and CT are the concentrations a t a later time of glucose and chlorite, respectively. Figure 1 shows, however, that oxidation is not the only reaction and that the total chlorite decrease must be lessened by DT, the concurrent amount of decomposition of reagent, giving: Go
-G
= '/~(CO-
CT - D T )
2,o
c,
DERIVATION OF AN4LYTIC4L EXPRESSION
Go - G
$5
IO
and was exceeded because sodium chlorite also disappeared in the absence of glucose, as s h o w by the decomposition curve, D. The kinetics of the decomposition of chlorous acid were studied by Barnett ( 1 ) but its stoichiometry is of no concern here. JYhen D is used as a decomposition ccrrection for T by simple subtraction of ordinates, the net curve, 2'-D, is far too low and furthermore, recedes from the calculated ordinate. Obviouslv, D overcorrects. This is explained by the fact that actual decomposition v a s greater in D than in 1' because in the latter, the chlorite concentration, 11hich governs the decomposition, was being concurrently decreased by oyidation of the glucose. Curve G represents the rate of disappearance of chlorite owing to glucose, after concurrent decomposition has been properly evaluated, as discussed below.
Co
S o w , DT cannot be directly measured, but can be expected to obey the decomposition rate law, regardless of simultaneous oxidation. This law was found by Barnett ( 1 ) to be
where C, is the chlorite concentration of a solution containing no glucose, which is referred t o in this paper as a control, as con-
(5)
DT
(6)
kDCoCTt
and Equation 3 becomes
-G
= ' / ~ ( C O-
CT - k o C o C ~ t )
(7)
By combining Equations 5 and 7 to eliminate k ~ t a, n expression is obtained for glucose concentration in terms of measurable quantities: Go - G
(4)
Co = Dc = koCcCct
.\/a
Go
(3)
-
e,
which shows that equals thegeometric mean, . \ / C ? , of the initial and final chlorite concentrations in the control, By assuming that ET likewise equals the geometric mean, in the test, then
l/g(co
- CT)X
C CC
(8)
When the oxidation is complete G becomes zero and Equation 8 affords a means of determining glucose from chlorite titers of test and control.
ANALYTICAL CHEMISTRY
384 TEST OF EQUATION 8 AND ACCURACY OF METHOD
Equation 8 was tested with pure glucose over a wide range of conditions: 0.000003 to 0.0003M glucose (0.6 to 507 per ml.), 0.0005 to 0.0032M sodium chlorite, and p H 2.4 to 3.4, all a t 50' C. in the absence of light. Details of analysis are given belop-. I n a series of rate experiments, chlorite was determined a t various times in test and control pairs, glucose was calculated from Equation 8, and the percentage yield of glucose was plotted against time. Typical results are shown in Figures 2 and 3, the yield values reaching maxima a few per cent over 100.
certain time.
A decline over long periods can be shown t o be in
co
accord with the behavior of the function (C, - C T ) E with resprct to time. I n another series of experiments, accuracy and reproducibility were investigated, usually at two time periods. Yields were calculated by substituting in Equation 8 the mean values of si.; determinations each of C, and of CT for each experiment. The results given in Table I confirm the curves of Figures 2 and 3 in showing that glucose can be determined over a mide range to a n accuracy of a few per cent by substituting chlorite values i n Equation 8. The agreement of results a t two time periods for thr same sets of conditions-Experiments 11, 11, 15, and 17-also confirms the absence of continued, indefinite oxidation of glucose beyond the gluconic acid stage by the reagent. The decomposition factor Co/C, distinguishes, in Figure 1. curve Gfrom curve 2' - D (mathematically equivalent to C, C T ) , whose ordinates, multiplied by CO/C'~,gave curve G. The values for C,/C, usually exceeded 2 a t the 22-hour point and approached 5 for the latter parts of Experiment 1, Figure 3. Since this corresponds to "corrections" of 200 to 500%, it was intentionally a very severe test of Equation 8. It is of interest to note that curve T - D, Figure 1, can never be expected to reach the theoretical glucose value, regardless of experimental conditions, unless Co/C, = 1-Le., unless there is no decomposition of reagent during the test. Finally, it may be noted that the maximum on curve T - D corresponds to a point a t which considerable glucose remains. Since the slope is zero a t the maximum, the two rates on T and D must be equal a t this time. However, because of glucose, CT must be lower than in the control, and therefore, the rate of decompofiition must be lower in the test than in the control a t this point. Therefore, reagent decrease by glucose must be a factor in helping to equalize the disappearance rates.
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50
2.4 2.8 3.1 3.4
Co
8 0 0 m
