June IS, 1941
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ANALYTICAL EDITION
Insert a graduated tube, conveniently a part of a broken buret, open at both ends, in the neck of the separatory funnel by means of a cork which has a slit to permit air to escape. The tnhe FIGURE 2 should reach nearly t o the bottom of the funnel and sufficient water he added t o cover the bottom opening. The adaptor attached to the condenser shouldlead into the top of the graduated tube. Distill in the usual manner. As volatile oils lighter than water float on the surface the bil distilled over will always remain in the graduated tube and the water will rise in the funnel. As water is cullciwd, rcnwve ~t irom tlic iunnel by OpQUiUg the stopcork, t3king cnre thnt the wawr l w p l is never heloa the bottom of the midwtte.1 tubc. Continue r h r di4lsrion until thc oil is all dL+Tilled over. This point 1s easllv found. as the volume of oil can he
Hydrolysis and Catalytic Oxidation of Cellulosic Materials A Method for Continuous Estimation of Free Glucose R. F. NICKERSON Mellon Institute, Pittsburgh, Penna.
T .
HERE are few direct methods available for the investigation of the submicroscopic structure of cellulose and,
as a result, the elaboration of essential information on the natural fibers and industrial cellulosic products has been retarded. X-ray methods are restricted to the crystalloid fraction, and copper reduction tests indicate relative amounts of chemical degradation a t the molecular level. Dispersions of cellulose, such as in cuprammonium hydroxide, reflect the properties of the dispersed unit rather than the antecedent structure. I n this paper a new method of investigation is presented and discussed, based upon the observation that quantitative hydrolytic breakdown rates of cellulose in solutions of acid can be estimated continuously and interpreted in terms of structure. Several recent articles have dealt with the evolution of carbon dioxide from simple supam, polysaccharides. uronic acids. and ~~~~~~
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atmosphere of nitrogen, ogtaining yidds of about 0.38 per cent carbon dioxide from pure glucose and about 0.17 Der cent from purified, p+rrxle-frrc-cottoi ccllulosc. ‘l’lsy rhocrd, also, t i m t w i t h proprr mntrol tlic rntcs arc rcproduciblc. Sickcrson 311d l.caDe fiil uzrd nir as the mrrirr m s a d O ~ S F X V Qsrirall ~ urd fairiv u h i h n amounts of oarban dioxide from purified cottons of Various other cellulosic materials have been examined. According t o Whistler and eo-workers (7), glucose, viscose and cuprammonium rayons, and cellulose acetate corrected for its acetyl content yield carbon dioxide at similar, constant rates; purified cotton, however, is characterized by a much lower rate. These carbon dioxide data were reported as percentages based on the weight of starting material. Whistler’s data could have been calculated in terms of moles rather than unit weights. As the capacity of glucose and its polymers to yield carbon dioxide must depend rimarily on certain carbon-oxygen linkages of the glncose resiiuue, it is apparent that equal weights of glucose and anhydroglueose (cellulose) would not represent eompmrahle units;
a unit weight of cellulose contains about 1.11 weights of glucose. Thus Whistler’s figures for glucose, the regenerated celluloses, and the cellulose fraction of cellulose acetate appear to be similar, hut, on the basis of equivalent weights, glucose would exhibit a 10 per cent greater rate of evolution than the regenerated and substituted ceiluloses. The somewhat smaller, true evolution of carbon dioxide from cellulosic materials suggested that at least two reactions might be involved. The first reaction might be a rapid hydrolysis to glucose; the second, a slower oxidation of the glucose to an unstable intermediate product which decomposes and yields carbon dioxide. If, then, the second step could be accelerated and kept in pace with the hydrolysis, carbon dioxide evolution rates might provide data on the breakdown rates of celluloses. A method based upon this mechanism has been evolved. It was necessary as a first step to determine whether or not the carbon dioxide output from glucose could he increased and controlled. Preliminary experiments indicated that 9 per cent hydrochloric acid would be more satisfadory for the present investigation than 12 per cent. Other exploratory work led to the temporary selection of 0.5 molar as a suitable concentration of salt catalysts. The reasons for these choices are discussed in the subsequent sections.
Catalyst It appeared that the oxidative process would be simplified if complicating oxygendonator groups, such as chromates and sulfates, were avoided. Accordingly, the study of catalysts was restricted to the chlorides of metals, which were used at 0.5 M concentration in 9 per cent hydrochloric acid with pure glucose (Merck’s c. P. anhydrous dextrose) as the oxidizable substance. The catalytic activities of several salts are given in Table I.
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Vol. 13, No. 6
INDUSTRIAL AND ENGINEERING CHEMISTRY
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FIGURE1. CATALYTIC AXD CONCESTRATION EFFECTSOF FERRIC CHLORIDE ON EVOLUTIOK OF CARBON DIOXIDEFROM GLUCOSE 1 gram of glucose dissolved in 150 ml. of 9% hydroohlorio acid solution. HC1 concentration 9.0% throughout.
These data show clearly that ferric chloride increases the relative carbon dioxide output from glucose more than two hundred fold; consequently, ferric chloride was chosen as a suitable catalyst for the reaction. The catalytic effects of ferric chloride in 9 per cent hydrochloric acid on the oxidation of glucose were determined a t different concentration levels. The results, plotted in Figure 1, indicate that the carbon dioxide evolution curves are not independent of ferric chloride concentration. The approximate slopes of the curves-i. e., carbon dioxide evolution rates-appear to be almost directly proportional t o ferric chloride concentration (Figure 1, inset).
is added, the boiling ferric chloride-glucose solution is at first very turbid and rust colored but becomes clear again after a few hours of refluxing.) If the approximate slopes of these curves are plotted against the corresponding acid concentrations (Figure 2, inset), an inflection can be discerned in the region of 9 per cent acid. Below this concentration the curves, convex downward in the first few hours, gradually approach maximum slopes; a t higher concentrations the initial maximum slopes decrease and, after a few hours, the curves become convex upward. Furthermore, a t the high acid concentrations hot solutions of glucose become discolored and form a dark brown precipitate in increasing quantity. -4possible interpretation of these results is that, under the conditions indicated, glucose or its products become activated and polymerize. Khen the polymer precipitates, reactant is lost and the carbon dioxide evolution velocity decreases. Eight per cent hydrochloric acid was selected as the most consistent concentration for the purpose. A compromise between antagonistic effects, it is below the range of precipitate formation and, a t the same time, high enough to reduce the initial curvature. Thus this concentration of acid gives a carbon dioxide-time curve that is approximately linear for a considerable digestion interval. The dependence of carbon dioxide evolution rate on both ferric chloride and hydrochloric acid concentrations made a stock solution desirable. A sufficient volume, containing 8 per cent hydrochloric acid and 0.6 mole of ferric chloride per liter, was prepared for subsequent experiments. The reasons for this amount of acid have been discussed. The choice of the ferric chloride concentration was somewhat arbitrary; a rapid evolution of carbon dioxide would decrease experimental errors, but an extremely rapid rate might exceed the safe absorbing capacity of the apparatus.
Glucose Concentration The experiments outlined above mere conducted at a constant glucose concentration. Because the hydrolysis of cellulose would represent the continuous addition of free
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E TABLEI. EFFECTOF METALCHLORIDES IN 9 PER CENTHYDROCHLORIC ACID OK EVOLUTION OF CARBON DIOXIDEFROM GLUCOSE Salt FeCla cuc12 SnClr NiClz None
Salt Concentration
Carbon Dioxide per Mole of Glucose in 5 Hours
M
Mole
0.5 0.5 0.5 0.5 0
0.65 0.29 0.016
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Hydrochloric Acid The selection of 9 per cent hydrochloric acid for the work just described was based on indirect, preliminary experiments. Results of a more systematic investigation are shown in Figure 2; in the latter experiments ferric chloride and glucose concentrations were held constant while acid concentration was varied. With the exception of that for unsupplemented ferric chloride, the curves illustrate the dependence of reaction velocity upon acid concentration. (When no acid
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0 HOURS OF CONCENTRATION OF HYDROCHLORIC FIGURB 2. INFLUENCE ACID ON LIBERATION OF CARBON DIOXIDE FROM GLUCOSE
1 gram of glucose dissolved in 150 ml. of 0.5 M ferric ohloride aolution. FeCla ooncentration 0.5 molar throughout.
ANALYTICAL EDITION
June 15, 1941
425
Apparatus and iMethod
HOURS
OF CARBON DIOXIDE FIGURE 3. RATESOF EVOLUTION F
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varying O ~ aniounts of glucose in 150 d.of 9% hydrochloric soid0.5 .M ferric chloride solution.
The apparatus of Dickson, Otterson, and Link (4) for uronide carbon dioxide was modified to facilitate rate measurements. A schematic diagram of the complete setup is shown in Figure 5 . The gravimetric type described by Whistler and co-workers (7) is robably also suitable for the purpose. !‘he present modification includes a flowmeter, A , a sulfuric acid trap, B, to remove volatile carbonaceous roducts (3,7) a thermometer in the reaction flask, parallef interchangeable carbon dioxide absorption columns, H, H ‘ , a bubble counter, C, and a soda-lime tower, D. The operation of this apparatus differs from the method of Dickson and co-workers only in the use of the parallel columns. The carrier air stream is diverted to either branch of the absorption system by means of a 3-way stopcock, E; the other stopcocks in the receiving branch, F and I , are turned to the proper positions. I and I’ are small needle valves. The substitution of columns involves some preliminary manipulations. The second column is assembled, set in place, and swept out for a few minutes with carbon dioxide-free air from the auxiliary scrubbing train, C, D. While this air current is still flowing, the barium hydroxide solution in the dropping funnel is admitted slowly to the column and the final volume is adjusted with successive rinses of boiled distilled water. The auxiliary air stream is then interrupted by a turn of stopcock F’ which leaves the branch ready to receive the carbon dioxide carrier gas. After the diversion of the carrier gas to the second branch, F’, G’, H’, any residual carbon dioxide in the tubing and flask, H , is drawn into the column by means of the auxiliary air input. This column is then isolated with stopcocks F and I removed
glucose to the system, it was necessary to determine the effects of varying the glucose concentration when the acid and 0) 4 I I I I 4 catalyst concentrations were held constant. The results, preW 1 J sented in Figure 3, are based on equal volumes of acid-catalyst 0 solution and the specified amounts of anhydrous, crystalline I2 glucose. 5 3z The curves are linear and, within a small error, the slopes are directly proportional to glucose concentration. But the f W evolution of carbon dioxide does not begin instantaneously. 0 2X Time is reckoned from the onset of boiling in the solution and Second Addition P 0 it is evident (Figure 3) that there is an induction period of t about 0.4 hour before carbon dioxide appears in quantity. 0 m 1This induction period may be the time required for apprea 4 0 ciable carboxyl formation and decarboxylation to occur. J That the same lag is present even after the digestion has been a Iin progress for some time is demonstrated in Figure 4, which g o I 2 3 4 5 6 HOURS shows the effect of a second addition of glucose. The lag OCcurs in both instances. This phenomenon suggests that the FIGURE 4. INDUCTION PERIOD BETWEEN ADDITION OF induction period is not a peculiarity associated with the comGLUCOSEAXD APPEARANCEOF CARBON DIOXIDE SHOWN mencement of the experiment but intervenes betreen any TO O C C C R BOTH.4T BEGINNING AND DURING A RUK addition of glucose and the appearance of carbon dioxide. Therefore the rate of carbon dioxide evolution at any time may be taken as a measure of the amount of glucose present in the system 0.4 h o u r previously. To summarize, hydrochloric acid and ferric chloride in the proper concentrations cause glucose to yield carbon dioxide at a rapid but fairly constant rate; the rate of ACUUM AIR evolution is roughly proportional to the glucose concentration; there is a uniform time lag between additions of glucose to the system and the appearance of carbon dioxide. DIAGRAM OF APPARATUSFOR CARBON DIOXIDE RATEMEASUREMENTS FIGURE 5. SCHEMATIC
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and the titration is made in the usual way. Hollow 3-ml. glass beads are satisfactory for the columns. A bulb-driven wash bottle tends to avoid error from the accumulation of respiratory carbon dioxide.
A 150-ml. volume of acid-catalyst solution appears to be a satisfactory amount for a single experiment. This volume of the stock solution was used in the blank on the apparatus and reagents and in the series of runs plotted in Figure 3. From the latter results it is evident that the rates of carbon dioxide evolution are not dependent upon the ratio of glucose to solution volume in the range specified. A rough measurement of the volume of acid-catalyst solution is, therefore, sufficiently accurate. The weight of sample is chosen so that the total amount of free glucose acquired by the system is less than 1 gram. The rates of evolution then fall within the range mentioned above and the safe absorbing capacity of the apparatus is not exceeded. Two-gram samples of the natural celluloses and 1-gram samples of the regenerated and substituted cellu1os.s have been found to be within the suggested limit.
BEHAVIOR OF GLUCOSE A N D FRUCFIGURE7. DIFFERENTIAL TOSE IN ACID-CATALYST SOLUTION in which G is the slope for 1 gram of glucose under the same conditions. Carbon dioxide lags behind the glucose content by the induction period, a ; hence, the calculated percentage breakdown has occurred a t T - a.
Discussion The method as described is applicable to glucose and to substances, such as cellulose, which yield practically pure glucose on hydrolysis. Impure materials may require correction factors-for example, there is no carbon dioxide evolved from acetic acid under these experimental conditions. It is possible, therefore, to examine cellulose acetate if a simple weight correction is made for the acetyl content. The response of fructose, a ketose, to the catalytic oxidation is very different from that of glucose. This can be seen in Figure 7 where curves for sucrose and its components are reproduced. The curve for fructose was obtained by subtraction of the glucose component. Dissimilar amounts of carbon dioxide from different sugars in the uncatalyzed system have been reported by Norman (6) and by Campbell and collaborators (3). It appears, therefore, that the present method cannot be applied indiscriminately. The study by this method of the breakdown rates of various cellulosic materials will be reported in a subsequent paper. While this article was in press, a ferricyanide method for the FIGURE6. COMPARISON OF CARBON DIOXIDEEVOLUTION determination of fructose in the presence of glucose was pubRATESOF CORNSTARCH, GLUCOSE, AND MERCERIZED COTTON lished by Becker and Englis (1). Their curves for glucose UNDER SIMILAR CONDITIONS and fructose are strikingly similar to those in Figure 7 above. The conversion of carbon dioxide evolution rates to apSummary parent glucose content can be made in several ways-for A method is described by which quantitative, hydrolytic instance, starch, like glucose, yields an approximately linear decomposition rates of glucose polymers, particularly cellurate curve (Figure 6). The ratio of the slopes for equal lose, can be determined. The basis of the method is the fact weights of the two substances is 1.10 and corresponds to 110 that the normally slow evolution of carbon dioxide from gluper cent of glucose from starch on acid hydrolysis. I n the cose in acid solutions can be increased a t will by the use of a case of cellulosic materials, however, the type of curve obsuitable catalyst; the accelerated evolution rate is directly tained (Figure 6) requires different treatment. If the times of proportional to the free glucose in the system. Glucose set the observations in hours, T , are corrected for the induction free by hydrolysis can be estimated continuously. Experiperiod, a, then a graph on log-log paper of the observed mental conditions and methods of calculation are presented. amounts of carbon dioxide, C, from 1 gram of the material and the corrected time values, T - CY,is approximately linear. Literature Cited c = A(T - a ) B (1) Becker and Englis, IND. ENQ.CHEM.,Anal. Ed., 13, 15 (1941). (2) Birtwell, C., Clibbens, D. A., and Geake, A., J. Teztile Inst., 15, where A and B are constants. The first derivative of this T161 (1924). (3) Campbell, W. G., Hirst, E. L., and Young, G. T., Nature, 142, equation gives the slope a t any time. [A similar equation 912 (1938). has been used by Birtwell, Clibbens, and Geake (2) to describe (4) Dickson, A. D., Otterson, H., and Link, K. P., J . Am. C h a . the variation of copper number with time when cotton is SOC.,52, 775 (1930). immersed in acids.] Since a unit weight of cellulose yields (5) Nickerson, R. F., and Leape, C. B., IND. ENG.CHEM.,33,83 (1941). ( 6 ) Norman, A. G., Nature, 143, 284 (1939). 1.11weights of glucose on complete hydrolysis, the percentage (7) Whistler, R. L., Martin, A. R., and Harris, M., J. Research breakdown of the cellulose is given by the expression Natl. Bur. Standards, 24, 13 (1940).
CONTRIBUTION from the Cotton Research Foundation Fellowship a t Mellon Institute.
