V O L U M E 21, NO. 5, M A Y 1 9 4 9 contained chlorine in excess of the amount required for the precipitation of all the fluorine as lead chlorofluoride is illustrated by the fact that analyses indicated considerable amounts of fluorine present, when none was introduced. As the determination is indirect and the calculation of the fluorine prcsent is derived from the amount of chloride found in the precipitate, it is possible to calculate greater amounts of fluorine than are present. DISCUSSION
I t is concluded that the loner satisfactory limit of the method lies someahere betvieen 0.01 and 0.02 gram of fluorine. The pH of the precipitation medium must be controlled within limits narrower than previously supposed, especially with the quantities of fluorine studied here. Rigorous execution of the directions of Hoffman and Lundell (3) will lead to a precipitation medium in the safe pH range. However, perhaps more than the usual degree of precision in buffering is required, since the tolerable variation of the pH is so slight. Variations in strength, purity, or measurement of reagents may lead to pH values outside the safe pH range. Conse-
583 quently, when conducting an analysi., one should determine the pH of the mother liquor, exclusive of washings. The pH should fall b e h e e n 4.60 and 4.70. The validity of the result should be questioned if the pH is outside this range, especially if i t is high. Readjustment of the pH, if high, will not correct matters once the precipitation has taken place. When a sample containing less than 0.020 gram of fluorine must be used, it is recommended that enough fluorine be added as standard sodium fluoride solution to bring the total fluorine content t o a t least 0.0200 gram. This should be introduced into the filtrate after the separation of the silica. The quantity thus added should be subtracted in calculating the result. LITERATURE ClTED (1) Hammond, L. D., IND.ENG.CHEM.,16, 938-9 (1924).
(2) Hillebrand, W. F., and Lundell, G. E. F., “Applied Iiioiganic Analysis,” p. 601, New York, John Wiley & Sons, 1929. (3) Hoffman, J. I., and Lundell, G. E. F., Bur. Staridards J . Research, 3, 581-95 (1929). RECEIVED January 28, 1948.
Determination of levulose Colorimetric Determination in Presence of Dextrose D. T. ENGLIS .&ND J.-I?IES W. \IILES’, C‘nicersity
of
Illinois, Urbana, I l l .
T h e e s t a b l i s h m e n t of t h e conditions u n d e r which levulose h a s a pronounced reducing a c t i o n u p o n the Folin-Denis phosphotungstate-phosphomolybdate reagent while dextrose h a s a very slight a c t i o n h a s been m a d e the basis for a rapid colorimetric e s t i m a t i o n of t h e f o r m e r i n t h e presence of the l a t t e r .
I
Y T H E course of an examination of the official method ( I )
for the determination of vanillin, an interesting observation was made concerning the conduct of dextrose and levulose n-ith the Folin-Denis phosphotungstic-phosphomolybdatereagent used in this connection. A slight modification of the official procedure involved the substitution of trisodium phosphate for sodium carbonate as the basic reagent. However, the reaction \vas so slow a t room temperature that it was necessary to heat thr, mixture for 10 t,o 15 minutes to develop the desired color. Furthermore, when the procedure v a s applied t o commercial extracts, the results for vanillin n-ere always high ( 6 ) . In order t o seek out the sources of error, two probable interfering suhstances, dextrose and levulose, were treated with the reagent under the prescribed conditions. I t was then noted that destrow gave practically no color, even Lvhen present in concentrations up to 2%, whereas small quantities of levulose produced considerable color under these conditions. These ohservations suggested a method whereby levulose might be determined in t,he presence of dextrose. Some of the methods and factors concerned with the selective oxidation of levulose with other reagents have been discussed ( 4 ) . I n many instances the available procedures are involved and timeconsuming. I t is believed a quicker colorimetric procedure has advantages for certain types of work. EXPERIMENTAL
Equipment for Colorimetric Analysis. First, it was necessary to determine the nature of the color developed by the reagent. Accordingly, the reduction product was examined (6) ___ 1 Present address, College of Pharmacy, Cniversity of Kentucky, Louisville, Ky.
with a Cenco spectrophotelonieter and the color curve was constructed. S o sharp change in absorption occurred at any point. There was a masirnum transmittance near 420 mp and a gradual reduction in transmittance down through 650 mp. Hence, it was evident that a simple filter photometer would be adequate for the colorimetric work and a red filter would be desirable to limit the source of illumination to a more effective region. -4Cenco-Sheard photelometer equipped with a red filter was selected as the instrument to be used. Determination of Optimum Time of Heating. The reagents used were the Folin-Denis phenol reagent ( 5 )and a 207, solution of trisodium phosphate in water. Six 5-mL portions of a 2% solution of levulose were placed in six 100-ml. volumetric flasks and 5 ml. of Fohn-Denis reagent were added to each. After 4 minutes, 10 nil. of the trisodium phosphate solution were added to each. Theflaskswereplacedin a water bath at 100°C. The first was removed after 2 minutes and made to volumr with v a t e r ; the second was a l l o ~ e dto heat for 5 minutes, the third for 10 minutes, etc. Each flask was made to volume and the colored solutions were compared in a cell of 1-cm. depth against water in the photelometer. The results are shown in Table I . From this experiment, it was determined that the color value was near its maximum after heating 10 minutes and this period n-as selected as thr optinium time of heating. Preparation of Working Curves. Solutions containing 0.1, 1.0, 2.0, 4.0, and 8.0% of levulose J-iere prepared; 5-ml. portions of each solution were placed in separate 100-ml. flasks, and a 5-ml. portion of the Folin-Denis reagent, was added t o each. After 4 minutes, each was nputralized with 10 ml. of 20% trisodium phosphate solution. All flasks were placed in boiling water and heatrd for 10 minutes, then made to volume with water, and the solutions were examined in the photelometer. The resulting colored solutions were compared with a blank treated in the same way. Color values are expressed as log 100/yo T or E for thr solution at 1-em. cell depth.
sa4
ANALYTICAL CHEMISTRY
To avoid the limitation of the volume of sugar solution taken for analysis to 5 ml. as in the previous experiments, the procedure was altered slightly in a second series of experiments to test the use of volumes up to 15 ml. The desired quantity of sugar solution was placed in the 100-ml. graduated flask, the volume was brought to a total of 15 ml. with water, then 5 ml. of Folin-Denis reagent were added. Beyond this point, all the steps were as in the previous experiments. The results are given in Table 11. The series of tests employed for the study of levulose was repeated, using solutions of dextrose instead of levulose (Table 111). I n all respects, the operations were identical. The data for the B series for both dextrose and levulose are plotted in Figure 1. These curves emphasize the differences i n the reducing ability of the two sugars for the reagent and furnish the informat,ion necessary for calculation of the amount of each in a mixture of the two. Although the E values obtained are not directly proportional to concentration of levulose, carrying out the reaction under carefully specified conditions gives data that make possible a satisfactory working curve.
Analysis of Mixtures of Dextrose and Levulose. A measured quantity of each dextrose and levulose solution was placed in a 100-ml. volumetric flask and enough water vias added to make the volume 15 ml. The reduction was then carried out in the prescribed manner. The quantities of dextrose and levulose contained in the mixtures taken for analysis are given in Table IV. The E values were calculated from the per cent transmittance of each solution. I n orclcr to use the n-orking curve (Figure 1) for the determination of levulose, the quantity of total sugars i n the mistuze must be known. For an unknown solution, the total reducing sugars may be determined by some other method-for example, the Lane and Eynon ( 2 )or Xunson and Kalker ( 3 ) procedures. The E value for a given solution is referred to the levulose curve and from this the approximate amount of levulose is deter-
Effect of Period of Heating on Color Value
Table I.
-4-3 . 5
59, .5 53 0 10 52.0 15 20 50.0 30 49.0 Folin-Denis reagent, and 10 ml. of 207, Final dilution t o 100 ml.
Table 11. Relation of Color Value and Concentration of Sugar Concn. of Levulose,
%
Levulose, Gram
T,%
E
A
1.0 2.0 4.0 8.0
0.05 0.10 0.20 0.40
63.0 54.0 40.8 24.8
0,201 0.268 0.389 0.605
B
1.0 2.0 4.0 6.0 8.0
0.207 0.319 0,444 0.569 0.648 A. 5 ml. of sugar solution 5 ml. of Folin-Denis reagent and 10 ml. of 100 ml., after heating 10 mid. 20% NaaPOd. Final dilution B. Same as A except 10 ml. of water added with 5 ml. of sugar solution. 0.06 0.10 0.20 0.30 0.40
61.9 48.0 36.0 27.0 22.5
60
Table 111. Relation of Color Values and Concentration Concn. of Dextrose,
%
A
1.0 2 0 4 0 8 0 1.0 2.0 4.0 6.0 8.0
B
A and B
83
Dextrose, Gram 05 10 20 40 0.0.5 0 0 0 0
0.10 0.20
0.30 0.40
I
I
I
'I/ . 5 c
L
'1.4
1 Figure 1. Relation of Color Value and Sugar Concentration
Table IV,
Determination of Levulose in Presence of Dextrose
Sugar Taken, G r a z _~ Levulose Devtrose
~ ~ t ~ Sugar, Grain T. %
0.20
0.20
0.40
0.10
0.10
0.20
0.20
0.10
0.30
0.10
0.05
0.15
0.10
0.20
0.30
0.20
0.40
0.60
E
Levulose Found, Gram
0.488 0.484 0.347 0.345 0.488 0.481 0.471 0.337 0.337 0.342 0.347 0.347 0.527 0.523
0.200 0,197 0.106 0.105 0.222 0.212 0,205 0.105 0.105 0,090 0.094 0,094 0.203 0.202
l
32.5 32.8 45.0 45.2 32.5 33.0 33.8 46.0 46.0 45.5 45.0 45.0 29.7 30.0
Transniittance
3 4 5 6 5 ml. of 2% levulose, 5 ml. of
NasPO4 solution.
I
/c
1 2
I
u?
Time of Heating, Minutes
Solution
L
I
T,%
E
97.5 96.0 87.0 83.5 96.5 94.5 89.8 85.5 79.5
0.011 0.016 0.060 0.078 0.015 0.025 0,047 0.068 0.100
in Table I1 except t h a t dextrose is sugar present.
mined (here it is assumed that all the color is due to the levulose). This quantity is subtracted from the quantity of total sugars to obtain the approximate quantity of dextrose present. The quantity of dextrose found by this method is referred to the dextrose curve to obtain the E value for the dextrose; thus, the amount of color resulting from the dextrose is estimated. The dextrose E value so obtained is subtracted from the experimental E value to obtain the corrected E value for the levulose. More exact calculation could be made, using this result as a first approximation and proceeding further, as is indicated in the previous article ( 4 ) . The amount of levulose present is determined finally by referring this levulose E value to the levulose curve. The results calculated by the above method are given in the last column in Table IV. The method is convenient and rapid. Deviations from the curves that are given may characterize the use of equipment, reagents, and conditions not exactly identical to those employed here. It is recommended that each observer prepare similar Ivorking curves for conditions under which the operations are to be performed. Slight changes in temperature and time of heating may make possible a better utilization of the reaction for determination in the low concentration range for levulose. LITERATURE CITED (1) Assoc. Offic. Agr. Chemists, "Official and Tentative Methods of Analysis," 6th ed., p. 366, 1945. (2) I b i d . , p. 570. (3) I b i d . , p. 571. (4) Englis, D. T., and Becker, H. C., IND.ENG. CHEM.,ANAL.ED., 11,145 (1939). ( 5 ) Folin, O., and Denis, W. J., J . Bid. Chem., 12, 239-43 (1912). (6) Miles, J. W., M.S. thesis, University of Illinois, 1947. RECEIVED .July 23. 1948.
