I S D C S T R I A L ,450 ESGIXEERISG CHEMISTRY
332
of dilution, mould be desirable, however, in many cases. Further work is being done with this possibility in view. It appears that the colloids which are flocculated a t the isoelectric point and by addition of bentonite are those which owe their stability in honey predominantly to their electric charge. On the other hand, honey colloids which are stabilized primarily by hydration remain unflocculated and are not eliminated by the procedures described. The proportion of honey colloids which can be flocculated by the methods described and the chemical nature, origin, properties, and function in honey of colloids of different types will be discussed in a subsequent publication. Summary and Conclusions
Data obtained in this investigation indicate that a number of important properties of honey are influenced to a considerable extent by the presenceof colloidalmaterial. It has been ascertained that the colloids of honey usually have a positive electric charge, but that in some honeydew and tree honeys with a high ash content the charge may be negative. In such cases the pH value of the honey solution is invariably greater than pH 4.3. Honey colloids exhibit a definite isoelectric point which,
Vol. 23, S o . 3
in the honey samples so far examined, is nearly constant a t pH 4.3. On the basis of the foregoing observations a mutual flocculation method has been devised for elimination of a large proport,ion of colloids from honey by use of bentonite. This procedure gives promise of being useful for improving the quality of some low-grade American honeys and also possibly for improving the clarity and general appearance of the better grades of table honeys, which is desirable in view of the demand for clarity and brilliancy of appearance of products of this character. Many phases of the subject of honey colloids are being investigated, and the results of this continued investigation promise to throw considerable light on the significant part that colloids play in various properties of honey, and in the general chemistry of this product of insect life. Literature Cited Anderson, T r a n s . Faraday Soc., 19, 635 (1924). Badollet and Paine, I n t e r n . S u g a r J . , 28, 23-8, 97-103 (1926); Planter Sugar hgfr.,79, 121 (1927). (3) Badollet a n d Paine, Intern. Sugar J . , 28, 137-40 (1926). (4) Marvin, J. Econ. Enlomol., 23,431-8 (1930). ( 5 ) Paine and Badollet, Facts About Sugar, 21, 1212 (1926): I N D . Eac. C H B H . , 19, 1246 (1927).
(1) (2)
-
Hydrolysis of Inulin under Pressure' E. C. Kleiderer and D. T. Englis C H E M I C A L L A B O R A T O R Y , U X I V E R S I T Y O F 1 1 L I S O I S , V R B A S A , ILL.
I
N T H E preparation of an edible sugar sirup any method that reduces the ash content and simplifies the procedure is desirable. In most instances where purified polysaccharide materials are used as the source of the simple sugar, the ash content is due primarily to the neutralization of the acids employed to catalyze the hydrolysis. One well-known example is the sodium chloride in the glucose sirups prepared from c o r n s t a r c h . Similar salts would characterize the prepar a t i o n of a levulose sirup froin inulin by h y d r o l y s i s w i t h the aid of m i n e r a l acids.
Attempts have been made to hydrolyze pure inulin by heating with water a t 100" C. in a sealed tube and under a reflux condenser for 40 hours. In the first case the extent of hydrolysis as indicated by copper reduction method was about 12 per cent, and in the second 15 per cent. With carbon dioxide at a pressure of 70-80 atmospheres conversion of the inulin is complete in 1 hour a t 160" C. and practically so in 21/2 hours a t 130" C. When nitrogen i s substituted for carbon dioxide the rate is lowered slightly, and apparently the effect upon hydrolysis due to the acidity of the carbonic acid is not large. On the basis of reducing sugar produced, hydrolysis under pressure, with or without carbon dioxide, has proved practicable for the conversion of pure inulin and will avoid the introduction of reagents contributing to the ash content of the sirup. The sirups obtained have a very satisfactory appearance and taste. Sulfur dioxide a t a pressure of 3 atmospheres gives practically complete conversion of the inulin in 30 minutes at 145" C.
Previous Work
The first attempt to prepare lerulose from inulin without the use of mineral acids was that of Crookemit (I), who in 1843 claimed conversion of inulin by heating a solution of it a t 100" C . for 15 hours. Dubrunfaut (3) in 1856, Prantl ( I O ) in 1870, and Kiliani (7) in 1880 were unable to confirm the observations of Crookewit. Dubrunfaut claims to have accomplished practically complete conversion by heating a solution of inulin to 100' C. in a closed tube for a long period of time. I n 1869 Dragendorf ( 2 ) in general confirmed the work of Dubrunfaut and specified the optimum conditions for the reaction as follows: 1
Received November 8. 1930.
"Heat 1 part of inulin with 4 parts of water in a closed tube to 100" C. for 40 to 45 hours." In 1880 Kiliani repeated the work and r e p o r t s a f a i r l y complete conversion, with a small a m o u n t of inulin or inulin intermediate remaining. Conclusions as to the extent of the hydrolysis appear to be based on (1) solubility of the reaction products in Tvater, dilute alcohol, and a b s o l u t e a l c o h o l ; (2) ultimate analysis for carbon and hydrogen, and (3) specific rotatory ralues. Although the presence of fructose in considerable amount is probable, the percentage conversion by the procedure outlined is a matter of some question. Harding (4)states that he is inclined to doubt if these results can be-repeated. However, when the inulin was treated with dilute hydrochloric acid, a 96.i per cent conrersion was indicated by copper reduction methods. It is difficult to understand why heating in a sealed tube a t 100" C. would be more favorable to increasing the rate of hydrolysis than heating in an open tube at the same temperature, unless the presence of abundant oxygen in the open tube might tend to cause destruction of the sugar so that the apparent amount produced is diminished. Hydrolysis with water alone would of course be the simplest treatment which could be accorded the polysaccharide material and, if satisfactory, would eliminate the objectionable salt formed by
March, 1931
I,VDUSTRIAL AAID EXGINEERIXG CHEMISTRY
neutralization of the hydrolytic agent. However, in view of the questionable methods used by Kiliani and others for determining the completeness of hydrolysis, it seemed necessary to repeat some of the experiments to see if the results could be confirmed and then proceed to the other methods of treatment. Hydrolysis Experiments o n Inulin-Water Mixtures
Kiliani's directions were closely followed in this experiment and in the one following for the purpose of confirming his results if possible. Fifteen grams of inulin and 75 cc. of water were placed in a Carius tube and sealed. The tube was heated in a bomb furnace a t 100" C. for 40 hours. At the end of this time the bomb was removed and the contents analyzed for reducing sugars by the method of RIunson and Walker. Sole-The amount of cuprous oxide precipitated was calculated as invert sugar, which would indicate values somewhat too low. However, i t is doubtful if t h e experimental error from other sources was of lesser magnitude, so n o extrapolation t o t h e equivalent of pure fructose was made I n t h e later experiments on presslire hydrolysis, since t h e control was expressed a s invert sugar, t h e percentage relations are practxally identical t o what they would have been if calculated with fructose as t h e reducing sagar
It mas found that there were present 1.9 grams of reducing sugar or 12.6 per cent hydrolysis of the inulin to fructose. By taking the remainder of the solution and freezing it, 1.1 grams of insoluble substance separated out. The expcriment was repeated with similar results. A second 15 grams of inulin and 90 cc. of water were heated on the steam bath under a reflux condenser for 40 hours. The solution soon darkened and at the end of the experiment was dark brown. By freezing and keeping the solution frozen for a week no inulin separated upon thawing. Reducingsugar determinations showed 15.4 per cent conversion to fructose. It is probable that Kiliani has been misquoted by a number of writers and his reported conversion of more than 96 per cent of the inulin to fructose accomplished by acid hydrolysis and estimated by reduction methods has been inferred for the sealed-tube treatment. If one adopts the failure to recover more than a small fraction of the inulin with its original solubility as a criterion for completeness of conversion, his observations as to the effect of treatment with water a t 100' C. are confirmed. However, although the bulk of the material is altered, the change is to some product non-reducing in character and intermediate between inulin and fructose. Some of the lack of agreement in the results of the previous workers may be due to differences in the character of their initial inulin material. Hydrolysis under Pressure of Carbon Dioxide
Attempts to hydrolyze inulin with water alone a t 100" C. having proved impractical, attention was next directed to the use of carbon dioxide under pressure. Krase and Goodman (8) have recently pointed out some interesting industrial applications and theoretical considerations relative to the use of carbon dioxide urider pressure as a means of increasing the hydrogen-ion concentration of a solution and have made preliminary studies of the acidities attainable. Initial experiments looking toward the hydrolysis of starch by such a treatment have also been made by Jenkinson (6) with some success. Since inulin is much more siisceptible to hydrolysis than starch. the outcome seemed promising. The apparatus used was that of Jenkinson and was made available through the courtesy of N. JI7.Krase. It consisted of a pressure bomb made of manganese bronze which was surrounded by a heating coil as shown in the illustration. The operation of the apparatus is as follows: The solution of inulin
333
is introduced into the supply tank, A , through the plug 7 . Pressure is placed on this tank by closing valves 1, 6, and 5 and opening valve 3. A pressure of 2 atmospheres is sufficient. Valves 2 and 3 are then closed and valve 5 is opened to release the pressure on bomb B. Valve 5 is then closed and valve 6 is opened. Valves 3 and 4 are allowed to remain closed. Then by opening valve 2 the solution is forced into the bomb B. Valve 2 is then closed and valve 6 is also closed. Then valve 3 is opened and the full tank presbure is exerted upon the solution in the bomb B. By careful regulation of valve 6 a small amomt of carbon dioxide can escape and hence agitate the solution. The current :s now turned on and the temperature a t which the experiment is to be run is maintained by means of the heating coil and is determined by the thermocouple. At the end of the experiments valve 3 is closed, and upon opening valve 5 the converted solution of fructose is blown out. The bomb is then rinsed with water and is ready for a second experiment.
1
J
The rate of hydrolysis of the inulin was determined by analyzing for total reducing sugar by the method of Munson and Kalker ( 9 ) . h sample of inulin solution was made up and a blank run for free reducing sugar. A second sample was hydrolyzed with 0.4 N hydrochloric acid for 1 hour a t 100" C. The difference of these t v o solutions represents the maximum hydrolysis possible and was called 100 per cent. The solution coming from the bomb was analyzed by the same method, and the difference of this sample and the blank, divided by the 100 per cent value, is the percentage conversion given in the accompanying tables. In all cases approximately a 10 per cent solution of inulin was used. The inulin used was Pfanstiels c. P. special grade, made from dahlia tubers. The pressure of carbon dioxide in the bomb was that furnished from a cylinder containing liquid carbon dioxide and varied between 70 and 80 atmospheres, depending upon the temperature. Owing to the small capacity of the bomb, it was necessary to utilize the whole sample for the establishment of each individual value in the table; consequently the study required
INDUSTRIAL AiVD ENGINEERILVGCHEMISTRY
334
a much longer time than if several samples could have been taken at different intervals from the same run. The results are given in Table I. Table I-Effect of Pressure of Carbon Dioxide u p o n t h e Rate of Hydrolysis of Inulin a t Different Temperatures CONVERSION
TIME
100' C.
1
Hours
1
1/1
1
Cot pressure, atm.
I
130' C.
% '0
70
150' C.
130' C.
%
5%
%
41 65 82 92 96
83.6 105 O a 101 00
5k'6
..
.. ..
...
...
to
80
20
Since some steam is lost as the sample is withdrawn the values are probably a little high throughout the table owing to a slight concentration of the solution in the operations of hydrolysis and sampling. a
An examination of this table shows that the rate of hydrolysis is not so much dependent on the concentration of carbon dioxide as upon the temperature, since changing the pressure of carbon dioxide from about 70 to 20 atmospheres a t 130" C. only reduced the apparent conversion from 65 to 55 per cent in 1 hour. At 150" C. and with the higher pressure of carbon dioxide the reaction is complete within an hour. The velocity constants, assuming a monomolecular reaction, have been calculated for the series a t 130" C. (Table
11)
!
TIME Minules 30 60 90 120 150
k
=
2.3 log
SUGAR
VELOCITY CONSTANT'
For interval
Complqte conversion
M g . fler cc. 37.1 58.7 74.2 83.3 86.8
i'dg. p e r cc.
90.15 90.15 90.15 90.15 90.15
0.0177 0.0174 0.0190 0.0212 0.0367
L, a-x
An examination of the data in Table I1 shows that the reaction is probably not monomolecular. This observation is in agreement with those of Hibbert and Percival ( 5 ) ,who hydrolyzed both inulin and another fructose anhydride, levan, with 0.1 N oxalic acid a t 65" C. However, if the various apparent degradation products of inulin reported by Tanret (11) have a real identity, one might not expect even such uniform values for the velocity constant as are here observed. Hydrolysis under Pressure of Nitrogen
I n order to determine just how much the conversion was due to the carbonic acid, experiments were run in an inert atmosphere of nitrogen but using the same procedure of manipulation and analysis. The results are given in Table 111. Table 111-Effect of Pressure of Nitrogen upon Rate of Hydrolysis of Inulin a t Different TemDeratures
Hours 1/2
1 11/2 2
Pressure, atm.
I
146' C.
70
%
1i:o
31.4 48.4
9.0
..
37:O 46
160' C.
ture increase is the principal factor affecting the rate of hydrolysis and the acidity due to the carbonic acid is of lesser significance. It will be recalled that with the carbon dioxide a t a pressure of about 70 atmospheres the hydrolysis was complete a t the end of 1 hour. When nitrogen was substituted for carbon dioxide the conversion a t the end of an hour was 86 per cent. As was expected, varying the pressure of nitrogen from 46 to 93 atmospheres made practically no difference when the temperature was maintained constant. It was quite surprising that there was almost no caramelization in any of the experiments. The product of the 160" C. temperature hydrolysis under an atmosphere of nitrogen showed a slight darkening but the destruction of sugar was very slight. It was not possible with the limited quantities of material available to conduct experiments as to the physiological effects of feeding the sirups. One cannot, therefore, be absolutely certain that no by-products of unfavorable character had been formed in the apparent drastic hydrolysis treatment. Based on appearance and taste alone, the sirups after a single norite treatment were very satisfactory and similar to the better samples produced by hydrolysis with mineral acid a t lower temperatures. No effort was made to obtain crystalline levulose from the solutions, and additional study will be necessary to determine whether the sirups produced by this procedure will yield the crystalline sugar with greater or less ease than those prepared by other methods. Having observed the effect of the anhydride of a very weak acid and that of a neutral solution upon the rate of conversion of inulin a t different temperatures, experiments were planned using an anhydride which would form a somewhat stronger arid solution than carbon dioxide. This was accomplished by using sulfur dioxide. Hydrolysis under Pressure of Sulfur Dioxide
I n this series of experiments the manipulation was identical with those previously described but the analysis required a slight deviation. Most of the sulfur dioxide was removed from the hydrolyzed solution by bubbling air through it and the remainder was removed by titrating with iodine to oxidize the sulfur dioxide to sulfuric acid. The slight iodine excess was removed with sodium thiosulfate. From this point the regular Munson-Walker procedure was followed. Table IV-Conversion
of I n u l i n b y T r e a t m e n t under Pressure w i t h Sulfur Dioxide 3 atmospheres 1/2 hour
Pressure of SOz Time . Conversion: 1000 C 130' C. 145' C.
35 4% 59.3% .QS.8%
As can be noted in Table IV, the hydrolysis using only 3 atmospheres of sulfur dioxide is almost complete in hour a t 145" C. Since the sulfur dioxide had considerable corrosive action on the bomb, extensive experiments were not carried out. Literature Cited
CONVERSION 130' C.
Vol. 23, No. 3
130' C.
70
R
86:5
14:6
9e:o
39:7 93
An examination of Table I11 confirms the conclusions of the experiments given in Table I-namely, that the tempera-
Crookewit, A n n . , 46, 184 (1843). Dragendorf, Jahresber. Chem., 22, 747 (1869). Dubrunfaut, Compl. rend., 42, 803 (1856). Harding, Sugar, 26, 406 (1923). Hibbert and Percival, J . A m . Chem. Soc., 62, 3998 (1930). Jenkinson, University of Illinois, B. S. Thesis, 1930. Kiliani, A n n . , 206, 168 (1880). Krase and Goodman, Chem. Met. Eng., 36, 162 (1929). Munson and Walker, J . 4 m . Chem. SOC.,28, 663 (1906); 29, 541 (1907). Prantl, Jahresber. Chem., 23, 848 (1870). Tanret, Bull. SOC. chim , [3] 9, 227, 622 (1893).
