ANALYTICAL EDITION
334
Vol. 3, No. 3
‘Determination of Dextrose and Levulose in Honey by Use of Iodine-Oxidation Method’*2 R. E. Lothrop and R. L. Holmes BUREAUOF CHEMISTRY
AND SOILS,
WASRINGTON, D. c.
A study has been made of the effect of a number of have been used by Auerbach factors governing the oxidation by alkaline iodine and Bodlander (1)and others crystallization of honey solutions of dextrose, levulose, and sucrose, such as t o d e t e r m i n e d e x t r o s e in are not thoroughly untime, temperature, concentration, and rate of addition honey. derstood, but it is well known of reagents, etc., and a modification of the method During the past few years that certain floral types, such for determining dextrose and levulose in honey based considerable work has been as tupelo and sage, remain on the selective oxidation of dextrose is proposed. done on the iodimetric deterliquid for long periods of time, Under the given conditions, levulose and sucrose are mination of aldose sugars, whereas alfalfa and some of oxidized only to a very limited extent. The slight especially dextrose. Such the clover honeys soon beoxidation of levulose is apparently due largely to the m e t h o d s r e q u i r e only the come solid, owing to formaLobry de Bruyn rearrangement, and it has been shown most commonplace equiption of dextrose c r y s t a l s , to be influenced by time and temperature to a conment and can be carried out Browne (6) has pointed out siderable extent. The dewee of oxidation of sucrose when more elaborate polarit h a t the noncrystallizable is small and is not influenced by time to any extent. scopic e q u i p m e n t is n o t tupelo honey is characterized Results of determination of dextrose and levulose available. Romijn (23) first by an abnormally high perin a considerable number of honeys by the proposed used iodine in alkaline solucentage of levulose. iodimetric method indicate that levulose is prepondert i o n f o r determining aldeA better understanding of ant in normal floral honey, values for the levulosehydes. He found quantitathe factors involved in crysdextrose ratio ranging from 1.02 to 1.70. Results tive oxidation of aldehydes to tallization of honey is desirobtained by this method and the high- and low-temacids according to the equaable, since c r y s t a l l i z a t i o n perature polarization method show comparatively tion must be reckoned with in close agreement. the handling a n d s t o r a g e IZ 3NaOH RCHO+ RCOONa 2NaI 2H20 of honev. F o r i n s t a n c e , fermentation frequently results when separation of dextrose He also applied the method to estimating aldose sugars. in crystalline form increases the proportion of water in the Willstlitter and Schudel (29) also have reported that under remaining liquid portion, producing conditions more favor- suitable conditions dextrose is oxidized quantitatively to able to yeast activity. Crystallization is troublesome when gluconic acid by iodine in alkaline solution, whereas fructose honey which is packed in glass containers remains stored and sucrose are not affected. Judd (16), however, reported for any considerable period of time, or when honey is mixed incomplete oxidation of dextrose under the conditions of with peanut butter and similar substances for use as a spread. Willstatter and Schudel’s experiments, and also found levuI n the marketing of honey in solid form, the character of the lose to be oxidized to a considerable extent. Other workers crystalline mass is of importance, small evenly distributed (1-80) have reported on the effect of time, temperature, concrystals producing a smooth, creamy product suitable for centration of iodine and sugar, rate of addition of alkali, and spreading or for filling for layer cakes, etc., whereas coarse other factors involved in the oxidation, and although generally granulation produces a product unsuitable for such purposes. quantitative oxidation of the aldose sugars is shown to take Casual observation of a number of samples of granulated place under certain conditions, there is considerable conflict honeys will impress one with the great variety of the crystals regarding the influence of the various factors involved, as well as regards size and distribution, adhesiveness of the crystal- as the extent to which sugars other than those containing a lized masses, etc., no two samples possessing exactly the same free aldehyde group, such as levulose and sucrose, are oxidized. character. Auerbach and Bodlander (1) applied the iodimetric method With a view to obtaining information that would be of value to the estimation of dextrose in honey, and calculated levulose in dealing with problems of granulation, a study of some of from the difference between total reducing sugars (deterthe factors governing crystallization of honey has been under- mined by copper-reduction methods) and dextrose. They taken. In this connection a simple and accurate method for found that levulose was always preponderant in genuine. determining dextrose and levulose in honey is desirable, since honey, and fixed the dextrose-levulose ratio a t from 1:1.06 to the difference In tendency of honeys of different types to 1:1.19, although higher ratios were found for honeys that had crystallize is undoubtedly dependent to a considerable ex- been stored for some time. They used these values for distent upon the ratio in which these two sugars occur. Methods tinguishing between genuine and sophisticated honeys. of determining dextrose and levulose in the presence of each Gronover and Wohnlich (14) using Auerbach and Bodother as they occur in honey are of two types: polariscopic lander’s method to determine the levulose-dextrose ratio of (especially high- and low-temperature polarization), and the a number of genuine honeys, reported that the ratios deterso-called chemical methods, depending on the selective oxida- mined by Auerbach and Bodlander were too high for levulose, tion or destruction of one sugar in the presence of the other. some genuine samples showing a preponderance of dextrose. A selective reduction method for levulose has been developed Fiehe (IS) determined the dextrose-levulose ratios for apby Nyns (a$, and modifications of the iodimetric method proximately 50 samples of German honeys, using Auerbach Presented before the Division of Sugar 1 Received April 26, 1931. and Bodlander’s method, and found dextrose preponderant Chemistry at the 81st Meeting of the American Chemical Society, Inin more than 70 per cent of the samples examined. These dianapolis, Ind., March 30 t o April 3, 1931. * Contribution 108, Carbohydrate Division, Bureau of Chemistry and conflicting results leave some doubt as to the reliability of the methods used. Soils.
HE factors governing
T
+
++ +
July 15, 1931
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Since the results of different investigators vary so widely as regards the conditions for quantitative oxidation of dextrose, and the extent to which sucrose and levulose are oxidized, a study was first made of some of the methods described by other workers in order to determine, if possible, the reasons for such discrepancies. I n addition, a careful study of the oxidation of dextrose, levulose, and sucrose under various conditions and mixed in various proportions has been made in order to devise a more satisfactory method for determination of these sugars as they occur in honey, based on oxidation of dextrose by alkaline iodine. Carefully prepared samples of these sugars were used for this purpose. Experimental Procedure MATERIALSUSED-The dextrose used had the following analysis: moisture 0.03 per cent, ash 0.02 per cent, and = 52.43", a t a concentration of 6.45 grams per 100 cc.; purity, determined by reduction of Fehling's solution (Munson and Walker method), almost exactly 100 per cent. The levulose used was prepared from 90 per cent levulose (Pfanstiehl brand) by recrystallizing 4 times from alcohol. It had the following analysis: ash 0.16 per cent, moisture 0.28 per cent, and CY'," = -92.49", a t a concentration of 9.0778 grams per 100 cc., corrected for ash and moisture. Vosburg's (28) rotation for levulose a t this concentration and temperature is -92.75'. Reducing power as determined by Munson and Walker's method was 0.9234, compared with that of dextrose. Soxhlet (26) gives the ratio for reducing power as 0.924. The thiosulfate used was standardized against c. P. potassium dichromate that had been twice recrystallized. The potassium iodide used had been recrystallized and gave no test for iodates. AUERBACH AND BODL~NDER METHOD(l)-This method employs a mixture of equal volumes of 0.2 M sodium carbonate and sodium acid carbonate as an alkaline medium with 0.1 N iodine, and allows ll/z to 2 hours to elapse for completion of the oxidation, With solutions of pure dextrose under the conditions stated in this method (2-hour periods of standing used) oxidation was not complete, although results as high as 99 per cent were obtained. Schuette (24) found that 3 to 4 hours were required to complete the oxidation of dextrose by this method. It would seem that the long time necessary for completion of the oxidation is objectionable, both from the standpoint of convenience and the chance of loss of iodine by volatilization, or to slow reactions of iodine with non-sugar substances in the honey. By the use of sodium hydroxide in place of the sodium carbonate-sodium acid carbonate mixture this long period of standing is avoided. It was also found difficult to secure good check results on duplicate analyses when this method was applied to several honey samples. No such difficulty was encountered when using sodium hydroxide and a shorter period of standing. SLATERAND ACREE'SMETHOD-slater and Acree (96)use the quantity of acid formed (as a result of the oxidation of the aldose sugar to acid), as well as the quantity of iodine consumed, as a measure of the degree of oxidation of the sugar. They report that the quantity of excess alkali (sodium hydroxide) used is one of the chief factors controlling the oxidation, and give figures showing a large over-oxidation of dextrose, as well as considerable oxidation of levulose (as much as 37 per cent) and sucrose when amounts of alkali in any considerable excess of that required for the reaction are used. These results are not in harmony with those of Willstjitter and Schudel (% Hinton I),and Macara (16), and others. These authors attempted to avoid the error due to excessive amounts of reagents by carefully controlling the excess of iodine and alkali used, and recommended titrating the excess iodine, partly a t least, while the solution is still alkaline. It has been shown by Chapin (9)that large errors are introduced
335
when iodine is titrated by thiosulfate in an alkaline solution, as a result of the reactions that take place between the tetrathionate formed and the alkali present, which ultimately involve loss of iodine. Mellor (21) gives the following reaction as taking place between tetrathionate and sodium hydroxide:
+
2NazSaO~ 6NaOH+3Na&Oa
+ 2NazSOs 4- 3H20
Since both the sodium thiosulfate and sodium sulfite formed in this reaction will reduce iodine, loss of iodine in this manner will cause the oxidation values for a sugar to appear higher than they actually are. This would explain their reported over-oxidation of dextrose with increasing excess of alkali, and the large apparent percentage oxidation of levulose and sucrose. By Slater and Acree's recommended method (very small excess of sodium hydroxide and iodine solutions were maintained) with pure dextrose solutions, complete oxidation was not obtained; values from 94 to 97 per cent oxidation resulted over periods of from 5 to 40 minutes. Titration of excess iodine was not carried out as recommended by them, as errors are introduced by titrating iodine with thiosulfate solution in the presence of alkali, but the entire content was made distinctly acid before addition of thiosulfate. I n order to determine the loss of iodine and sodium hydroxide resulting from alternate additions of acid and thiosulfate to an alkaline iodine solution, 20-cc. portions of 0.1 N iodine were treated with varying amounts of sodium hydroxide solution, and the iodine was titrated by adding acid and thiosulfate alternately until no more iodine was liberated. Table I gives the results. It will be seen that there is a considerable loss of iodine and sodium hydroxide, even in the total absence of a reducing sugar, and the ratio of iodine to sodium hydroxide consumed is nearly constant a t 0.690. This value agrees approximately with the theoretical ratio 0.7 calculated from the equation Na&Os
+ 20NaOH + 71a+4NazS04 + 14NaI 4- lOHtO
which represents the reaction that takes place between tetrathionate, iodine, and sodium hydroxide, and is very nearly the same as the value 2/3 representing the ratio in which iodine and sodium hydroxide are used up in the oxidation of an aldose sugar by alkaline iodine solution. Table I-Titration of Iodine in Alkaline Solution by Alternate Addition of Acid and Thiosulfate (All solutions 0.1 N ) RATIO AMOUNTUSED AMOUNT CONSUMED IODINE:SODIUM Sodium Sodium HYDROXIDE hydroxide Iodine hydroxide Iodine CONSUNED CC.
cc.
cc.
10 15 20 20 20 20 20 20
20 20 20 20 20 20 20 20
2.70 4.58 6.60 7.40 12.00 9.60 7.74 9.70
a Excluding
No. 1.
cc.
8::E
1.68 3.20 4.55 0.689 5.10 0.689 8.29 0.691 6.64 0.693 5.25 0.678 6.72 0.692 Av. ratio" 0.690
The reason for the fairly close agreement shown by values for percentage oxidation of a sugar when the calculations are based either on iodine or sodium hydroxide used up (given by Slater and Acree) lies in the fact that the values for the two ratios are so nearly the same. However, consistently higher results are obtained when the degree of oxidation is based on iodine consumed, which is to be expected, sihce a slightly greater proportion of iodine is consumed in reactions of iodine and sodium hydroxide involving tetrsthionate than is used in reactions involving oxidations of aldose sugars. With 0.08 gram of levulose, 40 cc. and 25 cc., respectively, of 0.05 N iodine and 0.1 N sodium hydroxide solutions, and the same period of standing (15 minutes), an apparent oxidation value
ANALYTICAL EDITION
336
of 27.47 per cent for the levulose was obtained when acid and the thiosulfate solutions were added alternately in back titration, whereas an oxidation value of only 2.70 per cent resulted when the solution was first made distinctly acid before titration with thiosulfate. These results show the large errors caused by titrating iodine in alkaline solution, and indicate that the large overoxidation of aldose sugars and the comparatively high oxidation of levulose and sucrose reported by Slater and Acree3 are due in large measure to the method of back titration. Large - over-oxidation of ,dextrose or any considerable oxidation of levulose or sucrose has not been found when reasonably large excesses of iodine and sodium hydroxide are present. Oxidation of Dextrose by Alkaline Iodine Hinton and Macara (16) found that dextrose could be oxidized quantitatively by addition of slightly more than twice the required amount of iodine and sufficient sodium hydroxide solution to combine with the acid formed, and with the excess iodine. By applying these conditions to pure dextrose, the results shown in Table I1 were obtained. Each titration was carried out on a different day. It appears from these results that 100 per cent oxidation of dextrose is readily obtained under the conditions of Hinton and Macara's experiments. T a b l e 11-Oxidation of Dextrose by Alkaline I o d i n e (Temp., approx. 25' C. (room), time of standing, 10 minutes) THIOSULFATE IODINEEQUIV. OF SODIUM HYTHIO- IODINE OXIDA. USED IODINE D R O X I D E ~ DEXTROSE SULFATE REDUCED TION
cc.
cc.
CC.
Gram
Cc.
CC.
%
20 40 40 40
20.20a 39.628 40.10b 40.18b
25 25 25 25
0,1000 0.0753 0.0762 0.0754
9.12a 22.85b 23.17b 23.39b
11.08" 16.67b 16.93b 16.79b
99.82 99.72 99.98 100.29
a
0.1
N.
Effect of Temperature on Oxidation of Dextrose and Levulose
For the oxidation of levulose it was found that temperature is an important factor, a change of temperature from 4" to 30" C. causing the percentage oxidation to increase fifteenfold. The results given in Table I11 show the effect of temperature on the degree of oxidation of dextrose and levulose. It is seen that whereas levulose is influenced considerably by temperature changes, dextrose is not materially affected. of T e m p e r a t u r e on Oxidation of Dextrose a n d Lev u 1ose IODINE SODIUMHYTHIOIODINE OXIDATBMP. UsEDb D R O X I D E ~ DEXTROSR SULFATEb REDUCEDb TION c. cc. cc. Gram CC. Cc. % T a b l e 111-Effect
DEXTROSE
40
'40
40 40
trose and levulose. It is seen from these results that dextrose is oxidized very rapidly, 5 minutes being sufficient for quantitative oxidation, and that longer periods of time (up to 30 minutes) do not increase the oxidation beyond that point. For levulose, however, the oxidation proceeds slowly and a t an almost uniform rate for periods up to 30 minutes. The oxidation of a mixture of the two sugars proceeds approximately the same as when they are oxidized singly, except that the oxidation of levulose is inhibited slightly by the presence of dextrose. Sucrose, on the other hand, is oxidized only very slightly by alkaline iodine solutions, and does not show any appreciable increase of oxidation with time, such as is shown by levulose. T a b l e fV-Effect of T i m e on Oxidation of Dextrose a n d of Levulose (Wt. dextrose for each titration, 0.0784 gram; wt. levulose, 0.0816 gram; temp., 20' C.) IODINESODIUM THIOIODINE NO. TIME USEDb HYDROXIDR" SULFATEb REDUCEDb OXIDATION Minutes Cc. CC. CC. CC. % DEXTROSR
1 2 3 4 5 6
Immediate 5 10 15 20 30
40c 400 40c 40c 40d 40d
25 25 25 25 26 25
1 2
5 10 20 30
20f 20f 200 20f
12.5 12.5 12.5 12.5
23.15 21.86 21.85 21.82 22.25 22.24
16.08 17.37 17.38 17.41 17.40 17.41
92.30 99.71 99.76 99.94 99.88 99.94
19.60 19.50 19.10 1s 95
0.22 0.30 0.72 0 87
1.21 1.78 3.97 4.80
LEVULOSE
36
4 a
0.1
N.
N: 40 cc. iodine == 39.23 d 40 cc. iodine 39.65 e Sample, 0.0758 gram. I 2 0 cc iodine e 19.82 I 20 cc iodine e 19.80 b 0.05 C
cc. 0.05 N thiosulfate. cc. 0.05 N thiosulfate. cc. 0.05 cc. 0.05
N thiosulfate. N thiosulfate.
of T i m e on Oxidation of Dextrose and Levulose Mixed in Approximately E q u a l P r o p o r t i o n s (Each sample contained 0.0784 gram dextrose, and 0.0816 gram levulose; 40 cc. iodine soh. 3 39.71 cc. 0.05 N thiosulfate; temp., 20' C.) SODIUM OXIDATIONOXIDATION IODINE HYDROX- THIOIODINE CALCD.AS OF TIME USEDb IDE" SULFATED R E D U C E D b DEXTROSE LEVULOSE Minutes Cc. Cc. CC. CC. % 9% ._ 5 40 25 22.19 17.52 106.57 0.56 10 40 25 21.99 17.72 101.72 1.65 17.87 102.32 2 23 20 40 25 21.84 18.01 103.38 3.25 30 40 25 21.70 a 0.1 N. b 0.05 N. T a b l e V-Effect
b 0.05 A'.
4 15 20 30
Vol. 3, No. 3
25 25 25 25
0.0747 0.0746 0,0745 0,0744
23.02 22.98 22.99 22.81
16.59 16.54 16.53 16.71
99.45 99.79 99.82 101.1OC
T a b l e VI-Effect of T i m e on Oxidation of Sucrose (Each sample contained 0.0760 gram sucrose; temp., 20' C.) IODINE SODIUM THIOIODINE OXIDATIME USEDc HYDROXIDE^ SULFATEb REDUCEDb TIONd
Minutes 10 20 30 a 0.
Cc.
cc.
CC.
CC.
%
20 20 20
12.5 12.6 12.6
20.23 20.21 20.20
0.09 0.11 0.12
0 54 0.66 0.72
1 N.
b 0.05
N.
20 cc. I?sol c 20.32 cc. of 0.05 N thiosulfate. d Calculated on basis of 1 mole sucrose t o 4 equivalents of iodine. c
LEVULOSEd
The results given in Tables IV, V, and VI show the effect of time on the degree of oxidation of dextrose, levulose, sucrose, and mixtures of approximately equal proportions of dex-
The results obtained for the influence of time and temperature on the oxidation of the various sugars given above by alkaline iodine are in virtual agreement with those obtained by Hinton and Macara (16) who made a somewhat similar study of these sugars. The fact that oxidation of levulose by alkaline iodine proceeds slowly and a t nearly a constant rate over a range of time periods up to 30 minutes, whereas sucrose does not behave in the same manner, might be explained on the basis of levulose undergoing the well-known Lobry de Bruyn rearrangement (7),which may be represented by the following equation : levulose Z N dextroseemannose
a Since completion of this work, an article b y Kline and Acree (17) has appeared, pointing out the error due t o the method of titration used by Slater and Acree mentioned above.
The resulting dextrose and mannose formed as a result of this rearrangement would be readily oxidized. The rate a t
THIOSULFATE EQUIV.20 cc. ' IODINEB CC.
19.86 19.80 20 12.5 19.65 19.80 20 12.5 19 50 12.5 19.80 20 18.93 12.5 19 74 20 a 0.1 N. b 0.05 N. 0 At 30' C. slight loss of iodine due t o volatilization d 0.0758 gram levulose for each titration. e Corrected for temperature.
4 15 22 30
0.06 0.15 0.30 0.81
0.35 0.89 1.78 4.S1C
probable.
Effect of Time on Oxidation of Dextrose, Levulose, and Sucrose
INDUSTRIAL A N D ENGINEERING CHEMISTRY
July 15, 1931
which levulose reduces alkaline iodine then would depend on the rate a t which the rearrangement takes place and on the rate a t which the resulting dextrose and mannose would be oxidized. Since both dextrose and mannose are readily oxidized by alkaline iodine, the rate of oxidation of levulose would depend primarily on the speed of the above-mentioned rearrangement, provided a definite concentration of hypoiodite be maintained to oxidize the dextrose and mannose formed. The effect of temperature and time on the oxidation of levulose previously pointed out supplies good evidence in support of this view. I n this event the percentage oxidation of levulose should be calculated on the basis of 1 molecular equivalent of levulose to 2 equivalents of iodine rather than 1 molecular equivalent of levulose to 4 equivalents of iodine (based on oxidation of a primary alcohol group) as pointed out by Kline and Acree (I?'). Effect of Rate of Addition of Alkali on Oxidation of Dextrose and Mannose by Alkaline Iodine Solution A number of oxidation experiments were conducted with mannose to determine whether or not it would undergo quantitative oxidation under the same conditions as dextrose. Under conditions previously determined as yielding 100 per cent oxidation of dextrose, it m7as found that mannose was oxidized only to the extent of about 80 per cent, even when it was allowed to stand for periods up to 30 minutes. However, 100 per cent oxidation of mannose resulted when the alkali was added slowly. It has been pointed out by Goebel (13)and others that the rate of addition of alkali to the iodine-sugar mixture is of considerable importance, rapid addition tending to produce conditions unfavorable for complete oxidation. When alkali is added to a mixture of iodine and aldose sugar, the following reactions are initiated:
++
+ + +
2NaOH Inz=*NaIO NaI 4 HzO NaIO RCHO NaOH-RCOONa 3NaIO+NaI03 2NaI
+ NaI + H1O (1) (2)
-
(3)
Too rapid addition of alkali produces iodate formation to such an extent that insufficient hypoiodite remains to oxidize the aldose sugar completely, whereas slower addition of alkali produces hypoiodite a t a rate more favorable for complete oxidation of the sugar and less formation of iodate. Table VI1 gives the results obtained when mannose was treated with alkaline iodine solution. It is seen that quantitative oxidation is obtained only when the reagents are added slowly to the mannose solution. of Rate of Addition of Alkali on t h e Oxidation of Mannose by Alkaline Iodine Solution (Each titration involved 0.0750 gram mannose; temp., 20" C . ) IODINE SODIUM H Y THIOIODINE No. TIME U S E D b DROXIDEa SULFATEb R E D U C E D b OXIDATION Minutes Cc Cc. cc. cc. 7" . . ~. 1 10 40 250 27.21 13.44 SO.68 2 30 40 250 27.24 13.41 80.50 3 10 40 25d 24.10 16.55 99.35 4 -30 40 25d 23.89 16.76 100.60 a 0.1 N. b 0.05 N. C Added to s o h at once. d Added at constant rate over period of 2 minutes.
Table VII-Effect
.I
With dextrose, quantitative oxidation was obtained whether the sodium hydroxide was added a t once or slowly over a period of time. However, when the amount of sodium hydroxide was increased and added at once, there was evidence of incomplete oxidation of dextrose. Table VI11 gives the results. It is seen that incomplete oxidation of dextrose results only upon addition a t once of a large excess of sodium hydroxide solution. The difference in behavior of dextrose and mannose in this respect apparently is due to differences in their rates of oxidation by alkaline iodine solution. Since the oxidhtion of levulose was shown to proceed a t an
337
almost uniform rate for periods up to 30 minutes, a study of the oxidation of levulose for longer periods of time was made in order to determine whether or not quantitative oxidation would result after a sufficient period of time had elapsed. A sample containing 0.075 gram of levulose, 40 cc. of 0.05 N iodine, and 25 cc. of 0.1 N sodium hydroxide showed 8.5 per cent oxidation after 20 hours' standing. This unexpectedly low result was undoubtedly due to the rapid conversion of hypoiodite into iodate, as has been pointed out by Goebel (IS). Another sample of levulose, when subjected to similar treatment except that the sodium hydroxide was added a t the rate of 2 to 3 cc. every 30 minutes, showed 18.92 per cent oxidation a t the end of 6 hours. These results indicate that it might be possible to bring about quantitative oxidation of levulose, provided a sufficient concentration of hypoiodite could be maintained over a long period of time. This is rendered difficult because of the rapid formation of iodate fram hypoiodite in-alkaline iodine solution. Table VIII-Effect of Rate of Addition of Alkali on Oxidation of Dextrose b y Alkaline Iodine Solution (Time of standing, 10 minutes; temp., 20' C . ) IODINE SODIUM IODINE NO. USEDa H Y D R O X l D E d DEXTROSE REDUCED& OXIDATION
cc. 40 40 40 40
1 2 3 4 a
0.05
cc.
Gram
25b 25C 45b 45c
0.0720 0.0745 0.0720 0,0720
N.
b Added at constant rate over period of
Added to soh. at once. d 0.10 N .
%
CC.
15.99 16.53 16.00 15.68
100.03 99.82 100.10 98.09
2 minutes.
0
Application of Iodimetric Method to Determination of Dextrose-Levulose Ratio of Honey
Since a method based on iodimetric determination of dextrose in the presence of levulose (as they occur in honey) would necessitate fixing the conditions of time and temperature for the reaction and correcting for the slight reduction of iodine by levulose under these conditions, the oxidation of dextrose and levulose in varying proportions was studied, time and temperature being fixed a t 10 minutes and 20" C., respectively. Results, given in Table IX, show that for a range of dextrose-levulose ratios a t least as wide as that encountered in honeys of different types, the oxidation of levulose is fairly constant a t approximately 1.2 per cent. of Levulose in Presence of Various Proportions of Dextrose (Temp., 20' C.; time, 10 minutes; 40 cc. iodine =a 40.53 cc. 0.05 N thiosulfate) APPROX.
Table IX-Oxidation
LEVULOSE-
HY- THIO-IODINEOXIDATION OF
LEVU- TROSE IODINE DROXDEXTROSELOSE RATIOU S E D b l D E a Gram
0
Gram
0.0804 0.1608 0.1206 0.0804 0.0603
0.0720 0.0720 0.0720 0.0720 0.0720 0 a 0.1 A'. 6 0.05 N .
CG.
20 2:OO 40 1.75 40 1.00 40 0.75 40 , 40
.
OXIDATION
SODIUM
DEX-
SULREF A T E b DUCEDb
cc.
cc.
cc.
12.5 25.0 25.0 25.0 25.0 25.0
19.99 24.20 24.29 24.36 24.39 24.60
0.27 16.33 16.24 16.17 16.14 15.93
CALCD.AS LsvuDEXTROSELOSE
%
.., 102.15 101.59 101.15 100.96 100.03 ,
% 1.51 1.12 1.16 1.34 1.27 ,,
The dextrose content of different types of honey varies considerably, so that the quantity of sodium hydroxide and iodine remaining unused after the oxidation is complete would also pary, depending on the amount of dextrose present. In order to determine what effect this rariation in excess of the reagents might have on the degree of oxidation of dextrose and levulose, experiments were carried out in which the quantities of dextrose and levulose used were kept constant while the amounts of iodine and sodium hydroxide were varied so as to produce variations in the excess quantities of reagents comparing with conditions met in honey analysis. ,The results,
ANALYTICAL EDITION
338
given in Table X, show that over the range of dextrose content of honeys of different types the degree of oxidation of the two sugars is approximately constant. Table X-Effect of Variation of A m o u n t s of Iodine a n d S o d i u m Hydroxide on Oxidation of Dextrose-Levulose Mixture (Each sample contained 0.0754 gram dextrose and 0.0741 gram levulose; 40 cc. iodine 3 40.18 cc. 0.05 N thiosulfate: time, 10 minutes; temp., 200 C.) OXIDATION SODIUM THIOIODINE CALCD. AS IODINE USEDb HYDROXIDE^ SULFATE) REDUCED) DEXTROSE cc. CC. cc. cc. % 45 28 28.20 17.00 101.55 40 25 23.19 16.99 101.48 36 22 19.20 16.96 101.31 a 0.1 N . b 0.05 N .
Method In view of the foregoing results, the following method has been adoptedfor the analysis of honey: To 20 cc. of a solution containing 0.2 gram of honey in a 250-cc. Erlenmeyer flask, add 40 cc. of 0.05 N iodine solution. Run in 25 cc. of 0.1 N sodium hydroxide, stopper, and let stand 10 minutes a t a temperature of 20" C. Acidify with 5 cc. of 2 N sulfuric acid and titrate a t once with 0.05 N sodium thiosulfate, using starch as an indicator. The weight of dextrose in grams (not corrected for reduction of iodine by levulose) is found by multiplying the cubic centimeters of 0.05 N iodine reduced by 0.004502. The total reducing-sugar content of the honey (calculated as dextrose) is determined by Munson and WalkNote-Good agreement was obtained between results for reducing sugars determined by Lane and Eynon's (19) volumetric method and the Munson and Walker gravimetric method.
er's method, and the per cent dextrose and levulose calculated as follows: Approximate $7, levulose = % reducing sugars (calcd. as dextrose) - % dextrose (iodimetric method) 0.925 True % dextrose = % dextrose (iodimetric method) - 0.012% levulose True yo levulose = % R. S. (calcd. as dextrose) true % dextrose 0.925 True % ' reducing sugars = % dextrose 4-% ' levulose
-
'
(4)
(5) (6)
(7)
I n these equations, the factor 0,012used is taken from Table IX, and represents the reduction of iodine by levulose. The correction as carried out by using the approximate per cent levulose does not give an absolutely correct value, but the difference is small and represents a negligible quantity, well within the experimental error of the method. The ratio 0.925 used for the relative copper-reducing power of dextrose and levulose was determined by Munson and Walker's method with pure dextrose and levulose at concentrations approximately the same as those used in the method. Browne (6) has treated the subject of relative copper-reducing power of dextrose and levulose exhaustively, and gives the ratios determined by several different methods. These values are in rather close agreement with the ratio 0.925, although the ratio varies somewhat depending on the method used for its determination. The results obtained by applying this method to determining dextrose and levulose in a number of samples of representative honeys4 are given in Table XI. I n every instance the levulose is in excess of dextrose, the levulose-dextrose ratios ranging from 1.02 to 1.70 which is a much greater range than that set by Auerbach and Bodlander (1) for German honeys. The average resulting values for levulose and dextrose (39.99 4 The honeys used in this investigation were all fresh samples collected during the 1930 season. The importance of using fresh samples in studying the levulose-dextrose ratio of honeys of different floral types is brought out by the findings of Auerbach and Bodlander (I) that the proportion of levulose in honey apparently increases on storage.
Vol. 3, No. 3
and 33.72, respectively) are approximately the same as those obtained by Browne (6) (levulose 40.50, dextrose 34.02) for 92 samples of American honeys in which a method based on high- and low-temperature polarization was used. The levulose content of a number of the samples determined by highand low-temperature polarization are given in Table XII. The values do not differ materially from results obtained by the iodimetric method, the average difference in values for the two methods being 1.33 per cent. Table XI-Dextrose a n d Levulose C o n t e n t of Honeys of Different Floral Types Determined b y Iodimetry RATTO PREDOMINANT LEVULOSE: SAMPLE FLORAL SOURCE LEVULOSE DEXTROSEDEXTROSE 1184 1189 1195 1200 1201 1202 1203 1206 1207 1208 1209 1210 1213 1214 1215 1216 1228 1229 1234 1237 1239 1244 1248 1251 1252 1255 1256 1257 1279 1285 1287 1337 1377
Buckwheat Sumac Dandelion Honeydew Catclaw Chinquapin Gallberry Catclaw Orange Tarweed Holly Manzanita Eucalyptus Fruit bloom
. .
. .
Mangrove Sourwood Fireweed White mesquite Wild flowers Horsechestnut Wild flowers White clover Fireweed Bitterweed Palmetto Cotton
Av.
%
%
37.55 39.21 38.72 32.96 40.12 41.80 41.72 39.76 41.03 35.91 38.76 38.43 40.58 38.12 39.95 46.12 37.74 39.78 41.69 42.19 38.86 41.20 43.17 39.46 41.56 42.20 42.26 37.82 40.74 41.38 37.67 40.56 40.25
36.74 32.82 35.64 29.45 35.92 30.20 32.70 36.50 34.11 31.24 31.08 34.07 34.61 33 BO 29.32 27.21 33.00 35.40 33.45 38.22 36.51 36.19 29.70 31.54 38.01 32.93 33.31 32.98 36.09 32.22 36.58 32.95 38.40
1.02 1.19 1.09 1.12 1.12 1.39 1.28 1.09 1.20 1.15 1.25 1.13 1.17 1.13 1.36 1.70 1.14 1.12 1.25 1.08 1.06 1.14 1.45 1.26 1.09 1.28 1.27 1.15 1 13 1 28 1.03 1.23 1.05
33.72
1.19
39.99
.
Table XII-Comparison of Levulose C o n t e n t of Honeys Determined b y Iodimetry a n d High- a n d Low-Temperature Polarization PR EDoM I N A N T LEVULOSE SAMPLE FLORAL SOURCE Iodimetrically By polarization 1189 1195 1200 1201 1202 1203 1207 1216 1229 1239 Av.
%
%
40.03
38.70
Sumac Dandelion Honeydew Catclaw Chinquapin Gallberry Orange Tupelo Sweet clover Alfalfa
Further experimentation is now being conducted to determine the influence that substances other than sugars present in honey have on the determination of dextrose and levulose by this method. I n view of the close agreement between levulose values obtained by this method and the high- and low-temperature polarization method, it would appear that errors from this source are not excessive. Acknowledgment Grateful acknowledgment is made of the valuable suggestions offered by H. S. Paine, chemist in charge of this division, during the course of this work. Literature Cited Auerbach and Bodlander, 2. angew. Chem., 86, 602 (1923); 2. Nuhr. Genussm., 47, 233 (1924). Baker and Hulton, Biochem. J., 14, 754 (1920). Bland and Lloyd, J. Soc. Chen. Znd.. 93, 948 (1914). (4) Bougault, Compt. re?& 164, 1008 (1917). (5) Browne, Bur. Chem., Bull. 110 (1908). (6) Browne, J. Am. Chen. Soc., 28, 439 (1906); Intern. Sugar J., 13. 35 (1921). (7) Bruyn, Rec. Irar. chin.. 14, 156 (1895).
INDUSTRIAL AND ENGINEERING CHEMISTRY
July 15, 1931
Cajori, J . Biol. Chem., 64, 617 (1922). Chapin, J . A m . Chem. SOC.,38, 625 (1916). Colin and Lievin, Bull. SOC. chim., 23, 403 (1918). Englis and Byer, IND. END. CHEM.,Anal. Ed., 2, 121 (1930). Fiehe, Z. Nahr. Genussm., 52, 244 (1926). Goebel, J . Biol. Chem., 7 2 , 801 (1927). Gronover and Wohnlich, Z. Nahr. Genussm., 48, 405 (1924). Hinton and Macara, Analysl, 49, 2 (1924). Judd, Biochem. J . , 14, 265 (1920). Kline and Acree, IND. END. CHBM.,Anal. Ed., 2, 413 (1930); Bur. Standards J . Res., 6, No. 5 , 1060 (1930). Kolthoff, Pharm. WeekbZad, 60, 394 (1923); Z . Nahr. Genussm., 46, 141 (1923).
339
(19) Lane and Eynon, J . SOC.Chem. Ind., 42, 32T (1923). (20) Levy and Doisy, J . Bdol. Chem., 77, 733 (1928). (21) Mellor, “Comprehensive Treatise on Inorganic Chemistry,” Vol. p. 616, Longmans, Green, 1930. (22) Nyns, Bull. assocn. &ole sup. brasserie Louuain, 25, 63 (1925). (23) Romijn, Z. anal. Chem., 36, 18 (1897): 36, 349 (1897). (24) Schuette, J . A m . Oficial Agr. Chem., 11, 164 (1928). (25) Slater and Acree, IND. END. CHEM.,Anal. Ed., 2, 274 (1930). (26) Soxhlet, J . prakt. Chem., (2) 21, 227 (1880). (27) Voorhies and Alvarado, IND. ENG. CHEM, 19, 848 (1927). (28) Vosburg, J . A m . Chem. Soc., 42, 1696 (1920). (29) Willstatter and Schudel, Ber., 51, 780 (1918). (30) Zablinsky, Z . V n . deut. Zuckerind., 69 (N.S . 56), 159 (1919).
Impurities in White Sugars
x,
-
I I-De termination of Sulfates, Sulfites, and Aldehyde Sulfites112 J. A. Ambler, J.. B. Snider, and S. Byall BUREAU O F CHEMISTRY AND SOILS,WASHINGTON, D. C.
The quantity of sulfates in white sugars may be results shown in Table I, i t accurately determined by direct precipitation with present in the juices of is evident that sucrose does barium chloride from the acidified aqueous solutions the sugar cane (22) and not interfere in the precipitaof the sugars. By titration of acidified sugar solutions of the sugar beet (21) in tion of the barium sulfate and with standard iodine solution both before and after amounts varying annually that there is practically no treatment with alkali, the quantities of sulfur dioxide and also varying with the choice in the two methods. in the sugar as inorganic sulfites and as aldehydetype of soil and the climatic The cold method was adopted sulfite addition compounds may be determined. Some for the rest of the work on conditions. The quantity in sugars contain small quantities of other iodine-absorbcommercial sugars because of the factory juices is further ing impurities which it is suggested may be polyoften augmented by sulfates its simplicity and also because phenolic. in the lime (10, 28) and the it produced only very slight water (11) used in the manudiscoloration of th8 solutions, facturing processes, and in those factories which use sulfur whereas those which had been heated were badly discolored dioxide, by oxidation of sulfurous acid by the oxygen of the and turbid from the decomposition of the invert sugar formed air. Liming and carbonation do not completely remove the by the action of the acid on the sucrose. sulfates (19),a portion of which passes on into the evaporators Table I-Precipitation of Barium Sulfate in Presence of Sucrose and vacuum pans where the calcium sulfate frequently conSOa RECOVERED tributes to the formation of scale on the heating surfaces. SO3 TAKEN Hot precipitation Cold precipitation Gram Gram Gram The soluble alkali sulfates, unless their concentration is ex0.0049 0.0050 0.0056 tremely high, do not separate before graining of sugar and 0.0096 0.0101 0.0099 0.0502 0.0493 0.0500 are occluded in the growing sugar crystals. 0.0986 0.0989 0.0974
HE s u l f a t e i o n i s
T
Determination of Sulfates
The true quantity of sulfates in white sugar cannot be determined from an analysis of the ash of the sugar because during incineration at least a portion of the sulfates is reduced to sulfide by the carbon formed from the sugar, as is shown . by the stain of sulfide formed on the dish when the ashing is done in platinum, and by a distinct odor of hydrogen sulfide when the ash is acidified. However, the sulfates may be accurately and easily determined in acidified sugar solutions by precipitation with barium chloride (9). Two series of solutions containing known quantities of potassium sulfate in 200 grams of 50’ Brix solutions of cube sugar were prepared and acidified with 5 cc. of concentrated hydrochloric acid. One series was heated to boiling and treated with 10 cc. of 10 per cent barium chloride solution. The other series was treated a t room temperature with the same quantity of barium chloride. After the solutions had been thoroughly stirred, they were allowed to stand overnight, the first series on the steam bath, the other a t room temperature. The precipitated barium sulfate was collected on tared Gooch crucibles fitted with thick asbestos pads, washed thoroughly with hot water, dried, and heated to 550’ C. in a muffle furnace, cooled, and weighed. From the 1 2
Soils.
Received April 20, 1931. Contribution 109, Carbohydrate Division, Bureau of Chemistry and
0.1965
0.1972
0.1967
For the determination of sulfates in white sugars, 5 cc. of concentrated hydrochloric acid and 10 cc. of 10 per cent barium chloride solution were added to 100 grams of the sugar dissolved in 100 cc. of water. The mixtures were stirred thoroughly and allowed to stand overnight. After the precipitated barium sulfate had been collected and weighed as before, it was calculated to the equivalent weight of SOs. Some of the results on typical white sugars are recorded in Table 11,column 3. The second column gives the percentage of ash in the various sugars as determined by direct charring and incineration a t 550 C. While no direct relationship of ash and sulfate content was to be expected, high sulfates generally accompany high ash contents, especially in the beet sugars. O
Determination of Sulfites
The presence of sulfites in sugar is attributed solely to the use of sulfur dioxide in the process of manufacture. The methods of determining sulfites in food products, including sugar, have received extensive study in England as a consequence of the establishment by the government (8) of a maximum sulfur dioxide content permissible in foodstuffs. The official method finally adopted (7, 17) consists of distillation under prescribed conditions from acid solution in an inert atmosphere and absorption and oxidation of the sulfur dioxide to sulfuric acid, which is determined by titration. Various
