Ketose Formation in Alkaline Dextrose Systems J. B. GOTTFRIED AND D. G. BENJAMIN George M . M o f l e t t Research Laboratories, C o r n P r o d u c t s R e 3 n i n g Co., Argo, Ill.
D
ISCLOSURE of reactions involving the interconversion of
sugars with alkalies by Lobry de Bruyn and van Ekenstein (6) more than 50 years ago generated considerable interest and activity among carbohydrate chemists. Numerous workers have since conducted extended investigations of the chemical behavior of reducing carbohydrates in the presence of various bases. The principal interest of most investigators Beems to have been aimed at elucidation of the mechanism of the hransformation of glucose t o D-fructose, D-mannose, unfermentable substances, and organic acids, these being the principal products of the reaction. The basic concept of the reaction mechanism presented by Lobry de Bruyn and van Ekenstein was later expanded by Nef (6). The concept of intermediate enol formation, confirmed by Wolfrom and Lewis (10) in 1928, i s now the generally accepted mechanism through which the slugars are interconverted.
believed that the particular base used had a pronounced effect on the products of the reaction. For example, Kuzin ( 4 ) found that calcium hydroxide a t room temperature gave mannose but no fructose, whereas sodium hydroxide gave fructose but. practically no mannose.
1
1
1
0.692% NaOH
TIME -MINUTES. Figure 2. Interconversion of Dextrose to Ketose with Sodium Hydroxide at 102O C.
I5 20 TIME- MINUTES
25
30
Figure 1. Interconversion of Dextrose to Ketose with Various Bases at 102" C.
In carrying out work designed t o clarify the mechanism of the reaction, most investigators apparently paid small heed to the quantitative aspects of the reaction. This accounts for the fact that the final sugar mixtures of various authors were of widely different proportions. The "equilibrium" character of the transformation was recognized by the very earliest workers; likewise, it was generally recognized that true equilibrium is not achieved, owing t o secondary irreversible changes which occur. Numerous tentative conclusions were reached by various investigators. Among these, Spoehr and Strain ( 9 ) concluded that low hydroxyl ion concentration, coupled with relatively low temperatures, resulted in a low rate of interconversion. It was shown by Lobry de Bruyn and van Ekenstein ( 5 ) that many bases were capable of catalyzing the reaction, and most authors
Wolfrom and Lewie (10) established conditions for interconversion of glucose in lime water at 35" C. which resulted in the format#ion of negligible amounts of saccharinic acids. That essentially the same results can be achieved with other basea a t much higher temperatures appears not to have been recognized. It was the object of this work t o investigate the quantitative aspects of the Lobry de Bruyn-van Ekenstein transformation. The bases used were sodium hydroxide, sodium carbonate, calcium hydroxide, magnesium hydroxide, trisodium phosphate] and sodium sulfite. Most of the work was carried out at reflux temperatures (101 O to 103' C.) although the investigation was extended t o include experiments at 35', 70°, and 130' C. It was not within the scope of this work to investigate the numerous minor products of the reaction] but rather to determine quantitatively the factors governing the extent of formation of ketoses, acids, unfermentable substances, and color. No attempt was made t o determine the quantity of mannose formed. EXPERIMENTAL
Most experiments were performed under reflux conditions using pure dextrose solutions diluted with distilled water to the desired concentration. Percentage of dry substance was determined from the refractive index obtained on an Abbe refractometer by reference t o a table relating refractive index of dextrose solutions t o dry substance (9,11). Concentrations of dextrose were in the range of 10 to 60 grams per 100 ml. A11 percentage 141
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compositions referred to subsequently are on a dry substance basis. The solutions were brought to ;boiling in a three-necked, condenser-equipped flask, and the desired amount of alkali was added rapidly. One hundred-milliliter samples, removed by pipet a t intervals during continuous refluxing, were cooled rapidly in an ice-water bath prior to analysis. Experiments were similarly carried out a t 70" C. by keeping the reaction flask in a constant temperature bath. Experiments a t 130' C. were carried out by placing solutions of dextrose and the desired amount of base in 25-ml. tantalum bombs. The sealed bombs were placed for varying periods in an oil bath held constant a t 130" C. Determination of reaction velocity is probably inaccurate in this case because of the necessary time lag in bringing the solutions to the reaction temperature.
The unfermentable sugars probably consist of a mixture of 1,2anhydrofructopyranose, its nonreducing dimer and other substances ( 7 , 8 ) . Levulose, therefore, was calculated by subtracting total unfermentable substances from the sum of ketoses and saccharinic acids. Determinations of color were made on aliquots of interconverted solutions adjusted to pH 5.0 and diluted to contain exactly 9 grams of dry substance per 100 ml. Optical density was determined on a Coleman spectrophotometer and these values were converted to the corresponding Lovibond scale. DISCUSSION OF RESULTS
Systematic study of the factors governing the Lobry de Bruyn-van Ekenstein transformation involved investigation of the effects of the variables of base concentration, glucose concentration, time of reaction, and reaction temperature. Judging from results obtained with the several basic catalysts used, it appears that different bases affect the velocity of the reaction but not the nature or proportion of the products determined, a t least within the reaction temperature range of 70" to 130" C., and probably at any temperature. The chief factor governing the extent of formation of ketoses and saccharinic acids is the amount of base added. Formation of acids results in neutralization ef the added base, thereby essentially stopping the reaction a t a point determined by the amount of base added. Temperature is the chief factor governing the velocity of the transformation in the presence of a particular base, but this factor again does not appear t o affect the kind or proportion of products. Concentration of dextrose influences to some extent the amount of unfermentable sugars and color formed, but not the amounts of ketoses or acids produced.
/
44 -1.6
Vol. 44, No. 1
5
IO
15
20
25
30
% KETOSE(K)
Figure 3. Effect of Amount of Base on Extent of Ketose Formation
Immediately after the samples were cooled to room temperature they were titrated electrometrically with standard hydrochloric acid to pH 5.0, a value chosen because distilled water solutions of dextrose normally exhibit this pH value. The difference between equivalents of alkali initially added and equivalents of hydrochloric acid required to titrate to pH 5.0 was regarded as equivalents of organic acids formed. Calculation of organic acids as saccharinic, though perhaps not strictly valid, is not greatly in error. The figure so obtained is used as a measure of the percentage of hexose converted to acid on the assumption that principally monobasic acids are formed, mole for mole. Incorrectness of this assumption would not greatly alter the results, since less than 2% of the dextrose was converted to acids in most experiments performed. The Kline-Acree (1) modification of the Willstatter-Schudel method for the determination of aldoses in the presence of ketoses was used as a measure of extent of ketose formation. Amount of ketose was determined by deducting,from 100 the sum of aldoses and acids (as saccharinic). This figure thus includes levulose and unfermentable sugars. Kolthoff (3) found that Dfructose absorbs 1.2 ml. of 0.1 N iodine solution per gram. Since the samples analyzed contained only 0.18 gram of aldose and conversion to ketose was never more than 3070, they never contained more than 0.08 gram of ketose which, according to Kolthoff, would absorb no more than 0.1 ml. of 0.1 N iodine solution. This effect could cause a maximum error in the aldose determination of less than 0.5%. Unfermentable substances were determined by a standard fermentation procedure. Diluted samples, buffered with potassium acid phosphate and ammonium diacid phosphate, were fermented for 24 hours a t 30" C. with starch-free fresh yeast and dried yeast extract. Unfermentables were calculated from the specific gravity of the solutions before and after fermentation, after distilling off the alcohol formed. Unfermentable substances thus obtained include organic acids as well as unfermentable sugars.
"S 3
-0.6
-0.8
5
IO
15
20
25
30
% KETOSE
Figure 4. Relationship of Organic Acids to Ketose VARIATION OF BASE CATALYST. Figure 1 illustrates the equilibrium character of the reaction and shows that the type of base used has a limited effect on the time required t o reach equilibrium with respect to ketose formation. Figure 2 shows the effect of amount of sodium hydroxide used on extent of ketose formation from dextrose, in each case equilibrium being established substantially in 15 minutes. A large number of experiments carried out under the same conditions, but with different concentrations of dextrose, produced essentially identical results. These results indicate the inflexible relationship between amount of base used and amount of ketose produced. A simple logarithmic relationship exists between the propor-
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INDUSTRIAL AND ENGINEERING CHEMISTRY
143
RELATIONSHIP OF KETOSE TO ACIDFORMATION. It has already been shown that there is a fixed relationship between amount of base consumed and percentage of ketose formed. In order to obtain an equation which is independent of the kind of catalyst used, per cent base consumed may be expressed as per cent organic acid formed. This is possible because an equivalent of any base, as defined earlier, neutralizes an equivalent of organic acid, which is calculated on the basis of 180 molecular, or equivalent, weight. When all experimental ketose values are plotted against log per cent saccharinic acid, a straight line is obtained as shown in Figure 4. The equation of this line is
+
%Ketose = 16.4 X log % A 21.1 A = Organic acid (as saccharinic)
TIME- MINUTES Figure 5. Effect of Temperature on Rate of
Interconversion with Sodium Hydroxide
tions of ketose formed and base consumed. Since percentage base consumed is a straight-line function of percentage base added, an equation for the completed reaction may be expressed as follows % Ketose = C1 X log % base added f Cz (1) In the above equation, Cl is constant for all bases and Cz varies according t o the equivalent weight and baeicity of each base used. The basicity of the catalyst is defined as its acid neutralizing power above pH 5.0. It is evident, therefore, that in the case of a base substantially weaker than sodium hydroxide its acidneutralizing equivalence as here defined will normally be less than its stoichiometric equivalence. For example, trisodium phosphate titrated to p H 5.0 is essentially dibasic while sodium sulfite is essentially monobasic. Figure 3 shows that when the logarithm of the percentage base used is plotted against percentage ketose formed at equilibrium, a group of straight lines of equal slope is obtained. The equation then becomes % Ketose = 16.4 X log % base added CZ (2) in which Cz is 31.1 for sodium hydroxide, 31.7 for calcium hydroxide, 28.7 for sodium carbonate, 24.1 for trisodium phosphate, and 22.9 for sodium sulfite. This empirical equation, based on experimental results, is useful in predicting the quantity of dextrose converted to ketose solely on the basis of the amount of alkaline catalyst added. The equation holds for all dextrose concentrations and for all base concentrations which will not tend to carry the reaction beyond 32% ketose, the apparent maximum value. Comparison of calculated values with actual results obtained by analysis shows good agreement within the limits of experimental error. Table I gives a comparison of some of the results obtained with several of the common bases and basic salts.
+
TABLE I. ANALYTICALus. CALCULATED KETOSEVALUES % Ketose Base Sodium hydroxide
% Base"
Anal.
Calod.
0.118 0.214 0.372
14.8 20.0 23.9
15.9 20.0 24.1
Sodium carbonate
0.278 0.417 0.556
18.0 22.4 24.6
18.6 22.4 24.5
Calcium hydroxide
0.158 0.240 0.483
17.8 20.0 25.4
18.6 21.6 26.5
Trisodium phosphate
0.191 0.383 0.972
12.6 16.9 24.9
12.3 17.3 24.0
Sodium sulfite
0.350 0.700 1.400
16.1 20.0 25.8
15.2 20.4 25.3
Dry basis.
(3)
Equation 3 holds for all base catalysts, is entirely independent of the kind of catalyst used, and is valid at any stage of the reaction as well as a t equilibrium. This fact makes Equation 3 useful in that only a simple titration need be carried out to determine the amount of ketose formed, and therefore the need for determining the value of C2 of Equation 2 for each catalyst is eliminated. The fact that this relationship exists may be considered good evidence that the kind of base catalyst used has little effect on the course of the Lobry de Bruyn-van Ekenstein transformation, at least in the range of temperature investigated. That the calculated percentage of organic acid formed is in close agreement with the values determined by titratisil can be seen from results given in Table 11.
TABLE 11. BASEUSEDus. ACIDFORMATION % Acid, Dry Basiab B y titration Calcd.
% Base0
Base Sodium hydroxide
0.118 0.214 0.372 0.278 0.417 0.556
0.46 0.85 1.53 0.80 1.20 1.60
Calcium hydroxide
0.158 0.240 0.483
0.66 0.95 1.98
0.76 1.22 1.53 0.72 1.05 2.10
Trisodium phosphate
0.191 0.383 0.972 0.350 0.700 1.400
0.31 0.63 1.55 0.42 0.87 1.60
0.30 0.60 1.55 0.45 0.91 1.83
'
Sodium carbonate
Sodium sulfite a
b
0.48 0.88 1.52
Dry basis. As saooharinic.
EFFECTOF TEMPERATURE. It was stated earlier that temperature in the range of 70" to 130" C. affects appreciably only the velocity of the transformation reaction. Several experiments carried out at 70"and 130" C. with sodium hydroxide as catalyst produced essentially the same results as those carried out a t reflux temperatures. A comparison of rate of approach to equilibrium a t three different temperatures isghown in Figure 5, which shows the same equilibrium ketose value for all three cases. The curve representing the course of reaction a t 130 C. is probably inaccurate because of the unavoidable time lag in bringing the solutions to reaction temperature. From the limited amount of work done on the effect of temperature on rate of reaction, it is not possible to establish a precise temperature rate relationship. However, the results at 70" and a t 100" C. are sufficiently accurate t o permit the following approximate expression to be established O
100- T
t = tiw X
2.5
lo
where t = time in minutes tlm = time in minutes to equilibrium a t 100" C. T = temperature, " C.
(4)
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Equation 4 merely states that the reaction rate is multiplied by a Factor of 2.5 for every loo C. rise in temperature. More experimental work is required to establish the exact value of the constant, but even in its present form it is useful in estimating the time required for carrying out the transformation reaction at any desired temperature.
as well as other substances, as reported by sattier and Zerban (7, 8). Although precise analysis for unfermentables proved somewhat difficult, a relation between total ketoses and unfermentable substances was observed. This relationship, shown in 'Figure 7, is approximated by the following equation: log G = 0.0645 K
+ 0.007 D - 0.94
(5)
where G = per cent total unfermentables K = per cent ketose D = per cent dry substance The initial concentration of dextrose does influence the extent of formation of unfermentables but only up to a concentration of 40% dextrose. At higher concentrations the term 0.007 D in Equation 5 appears t o become constant a t a value of 0.28. Table I11 shows quite good agreement between unfermentable values calculated from Equation 5 and those determined by analysis.
40'
2&
4k
81, I d 0 0
I
I
I200
1-
I
I
1600
1800
TABLE111. EFFECT OF DEXTROSE CONCENTRATION ON FORMATION OF UNFERXENTABLES
TIME- MINUTES (t)
0.214Y0sodium hydroxide
Figure 6. Relationship of Temperature to Reaction Time
The d u e for tla is 15 minutes for interconversion with sodium bydroxide, but it is necessary t o establish this value (reaction time a t 100 C.)for other bases t o permit use of Equation 4 in such cases. Figure 6 shows a time-temperature curve for sodium hydroxide in which tlw is assigned a value of 15 minutes. On this basis it should be possible t o carry out the reaction in 1 minute at 130" C. If is assigned a value of 40 minutes for calcium hydroxide, the reaction a t 35" C. should reach equilibrium in 258 hours. This figure closely approximates that reported by Wolfrom and Lewis (IO)as that required for a D-glucose solution in 0.035 N calcium hydroxide to reach optical equilibrium a t 35" C. FORMATION OF UNFERMENTABLF. SUBSTANCES. Interconversion of dextrose t o levulose with alkaline catalysts is always accompanied by formation of certain amounts of unfermentable substances. A portion of the unfermentable material consists of the saccharinic acids and the remainder probably consists of fructopyranose anhydride and difructopyranose anhydride,
LOG G,=0.06?5 K + p.007
Concentration, Dextrose Grama per 100 M1. 15 30 45 60 Dry basia.
% Ketosea 19.6
19.8 19.9 20.0
% Acid@ 0.83 0.85 0.86 0.87
% ' Unfermentablea* Anal. Calod. 2.9 2.64 3.5 3 36 4.1 4 09 4.2 4.26
FORMATION OF COLOR.I t is well known that the interconversion reaction is accompanied by pronounced color formation. It is also generally agreed that the mechanism of color formation is quite complex and is influenced by numerous factors, One of these factors undoubtedly is inter- rather than intramolecular oxidation, because color formation can be very substantially reduced by reducing the possibility of such oxidation t o a minimum. This is shown by the fact that when dextrose is interconverted with sodium sulfite the amount of color formed is less than 10% of that formed when using sodium hydroxide or other bases.
-0.9
% KETOSE (K) Figure 7. Relationship of Unferrnentables to Ketose
Figure 8. Effect of Sodium Hydroxide and Dextrose Concentrathnson Color Formation
January 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
% NoOH 6i.b.I
145
% NaOH (daw
Figure 9. 10” Be’. Dextrose Solutions Refluxed with Sodium Hydroxide for 10 Minutes
Figure 10. 21” Be’. Dextrose Solutions Refluxed with Sodium Hydroxide for 10 Minutes
Under specific experimental conditions with a given catalyst extent of color formation can be successfully related t o time, solution normality with respect to base, and dextrose concentration. For instance, when sodium hydroxide is used as catalypt at reflux temperatures under atmospheric conditions, extent of color formation may be expressed according to the equation
of actual fructose present is decreased by the quantity of unfermentable substances present. Unfermentable content was not reported by Wolfrom and Lewis, but an experiment conducted by the authors under the exact conditions used by Wolfrom and Lewis indicated Equation 5 t o be valid. Thus, the indicated lLIevulose”value of 30.9 includes 9 to 10% unfermentable sugars. The net levulose content, therefore, is of the order of 21%. If correction is made for the unfermentable content of interconverted sugar solutions, it is found that the maximum amount of levulose it is possible to produce by the Lobry de Bruyn-van Ekenstein transformation is of the order of 21% and not 31%. As the reaction is pushed beyond this equilibrium point by use of larger amounts of alkali, more unfermentables are formed a t the expense of levulose, thereby preventing the levulose content from exceeding this maximum. This effect is illustrated in Figures 9 and 10. Figure 9 shows the distribution of products when loo BB. dextrose solutions are interconverted with various amounts of sodium hydroxide, and Figure 10 shows a sinfilar distribution in the case of 21” BB. solutions. I n the latter case the levulose content does not exceed 18% because of the greater formation of unfermentables owing to the higher dextrose concentration used.
log C
0
whereC
0.418 log t
+ 1.795 log N - 1.349 log BC. + 5.711
(6)
color (Lovibond units) for solutions containing 9 grams per 100 ml. at pH 5.0 1 = time in minutes N = initial sodium hydroxide normality of solution Be. -- dextrose concentration in O Be.at 60”F. 5
This equation is valid only fort values no greater than the equilibrium time of the interconversion reaction. Color and dextrose concentration may be expressed in units other than those shown if the constants are changed appropriately. The factors given in Equation 6 are shown graphically in Figure 8, which indicates the effect of dextrose concentration on color formation. It is evident that the amount of color formed at any given point of ketose formation increases logarithmically with dextrose concentration. In order to test the validity of the empirically derived equations when applied to conditions used by other investigators, comparison of computed values was made with those reported by Wolfrom and Lewis (IO). Calculated results are in fair agreement with those of Wolfrom and Lewis, as shown in Table IV. The authors took the liberty of substituting the term iiketose” for fructose used by Wolfrom and Lewis, because it is probable that the 30.9% “fructose” reported by them consists of both fructose and unfermentable sugars. Since these unfermentable substances cannot be fructose, it is reasonable that the amount
OF CALCULATED DATA WITH THOSE OF TABLE IV. COMPARISON
WOLFROM AND LEWIS
1 M glucose in 0.035 N calcium hydroxide at 35’ C. for 10 days Calcd. Authors Wolfrom and Lewis 67.4 68.0 65.8 Aldose 29.4 29.2 30.9 Ketose 3.2 2.8 3.3 Nonsu am (saccharinic acids) T o t 3 unfermentables 11.9 11.2 ?
LITERATURE CITED
(1)Browne, C . A., and Zerban, I?. W., “Physical and Chemical Methods of Sugar Analysis,” 3rd ed., p. 898,London, John Wiley & Sone, 1941. (2)Cleland, J. E., Evans, J. W., Fauser, E. E., and Fetzer, W. R., IND. ENG.CHSM.,ANAL.ED.,16, 163 (1944). (3) Kolthoff, I. M., 2. Untersuch. Naht. u. cienuasm., 45, 131 (1923). (4)Kuzin, A.,Ber., 69, 1041-49 (1936). (5) Lobry de Bruyn, C. A., and van Ekenstein, Alberda, Rec. trav. chim., 14.204-16 (1895);I5,93-6 (1896);16,257-61 (1897). (6)Nef, J. U.,Ann., 357,294-312 (1907);376, 1-119 (1910);403, 204-383 (1913). (7) Sattler, Louis, and Zerban,F.W., IND.ENG.CBM.,37, 1133-42 (1945). (8)Ssttler, Louis, and Zerban, F . W., S w a t , 39, 28-9 (1944). (9)Spoehr, H.A., and Strain, H. H., J . Biol. Chem., 85,365 (1929). (IO) Wolfrom, M. L.,and Lewis, W. Lee, J . Am. Chem. Soc., 50, 837-54 (1928). (11)Zerban, F. W.,and Martin, J., J . Assoc. Ofic. Agr. Chemists, 27, 295 (1944). RECEXVED July 31, 1950. Presented before the Division of Sugar Chemistry CEEMICAL 8001E%Y, Houston,Tex. at the 117th Meeting of the AMEXUCAN
