Determination of Color and Turbidity in Solutions of Granulated Sugar A. R. NEES The Great Western Sugar Company, Denver, Colo.
green and red light by an empirical formula and reported in terms of percentage absorption. The writer, using a Lange colorimeter, has not been able to substantiate the assumption that the absorption of red light is unaffected by the color of the solution or that the effect of turbidity is the same when using both filters. The figures tabulated in Table I are typical of numerous tests that were made to check the validity of this assumption. It is shown that the percentage absorption increases with increasing color, regardless of the type of color filter used. (The Lange red filter very closely resembles Corning signal red No. 245.) The turbidity, as calculated by the formula given below, is also included to show that the increased absorption shown by the red filters is not due to increasing turbidity. It is evident that readings obtained with the Lange colorimeter and calculated according to the method of Keane and Brice would give too high values for both turbidity and color.
A method has been devised for the determination of both color and turbidity in the same solution without filtration or other treatment. A photoelectric colorimeter is used and the readings are made using blue and yellow color filters. The apparatus is standardized by determining the relative percentage absorption of blue and yellow light by a given unit of color and turbidity. Knowing this relationship, it becomes a matter of simple calculation to express both color and turbidity as percentage of absorption of blue light, which in turn may be expressed in terms of the -log of the transmission if desired. The method is rapid and the results are reproducible. A colorimeter has not yet been devised which is suitable for routine control work.
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INCE the advent of photoelectric colorimeters of practical design, these instruments have been widely used in the sugar industry for the determination of color and turbidity of sugar solutions, sirups, etc. The results are obtained as percentage absorption or transmittancy of light through a column of solution of definite length. Since both color and turbidity influence the total light absorption, the value obtained is the sum of the two effects. I n some cases the total percentage absorption (or transmittancy) is determined. The solution is then filtered to remove turbidity and the color is determined on the filtered solution. The turbidity is calculated by difference. This method has nothing to recommend it, because such filtration, if it removes practically all of the turbidity, invariably removes some of the color, so that the determined color is too low and the calculated turbidity is too high. Other instruments have been designed to determine total color and turbidity in the usual way and to determine turbidity alone by the Tyndall 6eam method, thus permitting a calculation of color to be made. Numerous references to various methods are found in the literature (1, 3, 4, 6,7). Recently Keane and Brice (6) have described a method for determining both color and turbidity in granulated sugar solutions without filtration, using an instrument of their own design. Two light filters are employed, Corning Glass Company light blue green No. 428 and signal red No. 245. Their method is based on two assumptions: that a given unit of turbidity gives approximately the same percentage absorption with either filter; and that the spectral characteristics of the red filter are such that the absorption is practically unaffected by the color of the solution and may be considered as due to turbidity alone. The turbidity is measured directly as the percentage absorption of red light. Color is calculated from the relationship of the transmittancies of
FIGURE1. LANGECOLORIMETER 1. Lamp 2. Solution cells 3. Photoelectric cells 4. Color filters '
5 , 6. 7. 8, 9.
Shutter plate Galvanometer Rheostats
The idea of determining color and turbidity on the same solution is very attractive and further investigation of the possibilities resulted in the development of the method described here.
The Instrument The Lange colorimeter is simple and compact. Its chief disadvantage is that the column of solution under examination is only 34 mm. in depth. Nevertheless, results in the range of 1.0 t o 5.0 per cent absorption (the range covered by ordinary granulated sugar solutions) are reproducible to within 1 0 . 2 per cent. A higher degree of accuracy is desirable and can be had with instruments of equal sensitivity but designed to use a longer column of solution. A diagram of the instrument is shown in Figure 1. A series of color 142
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MARCH 15, 1939
143
effect of color and turbidity, it is a matter of simple calculation to TABLE I. EFFECTOF COLORON PERCENTAGE LIGHTABSORPTION determine the pro ortion of the total percentage absorption of any test solution wgich is due to color and turbidity. (Using various color filters) Caramel Added t o Filtered Sugar Solution M1./100ml. 0.5 1.0 1.5 2.0
Percentage Absorption BlueYel- OrangeBlueQ greenb lowQ redo Red“ 2.2 4.5 6.5 8.3
1.3 2.5
3.8 4.6
0.75 1.45 2.10 2.7
0.4 0.9
1.2 1.7
0.46 0.9 1.0 1.4
Calculated Turbidity 0.06 0.00 0.00 0.05
Color filters sup lied with Lange colorimeter. b Corning, light brue-green No. 428. C
Corning, lighthouse red No. 246.
filters are supplied with it. After many trials the blue and yellow filters were selected as being most satisfactory. The spectral characteristics of the filters as given by the maker are shown in Figure 2. The blue filter was selected because it is very nearly complimentary to the color of the average sugar soluticn and consequently a relatively high percentage absorption is obtained for a given unit of color. The yellow filter gives a much lower percentage absorption for a given color intensity than the blue and the readings are more easily reproducible than those of the orange or red filters. The scale of the instrument contains 100 divisions and can be operated t o read directly in percentage absorption. The balancing rheostats, however, may be adjusted so 8s to increase the sensitivity, in which case a deflection of one scale division is equivalent to a fraction of 1 per cent absorption. I n this work the adjustment was made to maximum sensitivity and actual percentage absorption was obtained by dividing the net deflection by 2.3 when using the blue filter and 3.6 when using the yellow filter. These factors were determined by comparing the readings obtained a t norma!. sensitivity with those obtained for the same solution at maximum sensitivity. STANDARDIZATION OF THE INSTRUMENT. A clear, colorless sugar solution containing 50 grams of sugar per 100 ml. was first pre ared. This was made from highest purity sugar, treated witg Super-Norit or other high- rade carbon, then filtered through especially prepared Filter-bel. This solution serves as the standard and is assumed to have zero absorption. (Distilled water cannot be used as a primary reference standard because it has a lower transmissivity per unit depth than a sugar solution.) The glass solution cell which was to be used in future determinations was filled with this solution and another cell with distilled water and the difference in deflection of the instrument determined for both color filters, to give the “cell correction constant.” This constant is made up of the difference in optical characteristics of the cells, as well as the difference between water and sugar solutions. Its application permits the use of water as reference standard when making routine measurements. The next step in the standardization was to determine the relative percentage absorption for a given unit of color when using the blue and yellow filters. This was done by adding to a sugar solution successive small quantities of caramel solution which had been filtered through Filter-Cel, and determining the percentage absorption for each color filter. This o eration was repeated a number of times, starting with standarx sugar solutions as described above and with solutions of ordinary granulated sugar, An average figure was computed and it was found that the ratio of absorption with blue light to that with yellow light was 2.85 to 1.00-that is, if an absorption of 2.85 per cent is obtained with the blue light, an absorption of 1.00 per cent will be obtained with yellow light. In the same way by varying the turbidity and having the color remain constant, a relative effect of turbidity on the two types of light was determined and found to be in the ratio of 1.05 to 1.00 for the blue and yellow, respectively. This relation was established by using solutions of ordinary granulated sugar and comparing the percentage absorption of blue and yellow light before and after filtration through paper in the manner described below. The factor was arrived a t by averaging some 200 determinations. The data under “Total Percentage Absorption” in Table I1 will serve t o illustrate the method. It is evident that no significant error would be introduced if one assumed the factor to be 1.0 instead of 1.05. Having determined these ratios for the relative
Let a = total percentage absorption with the blue filter b = total percentage absorption with the yellow filter 2 = percentage absorption of blue light due to color y = percentage absorption of blue light due to turbidity Then
x + y = a z + Y = b 2.85 1.05 1.052 2 . 8 5 ~= 2.953
(1)
+
or (2) Solving for z and y 2.85 b - a = percentage absorption due to turbidity (3) = 1.70 x = a - y = percentage absorption due to color (4) Both color and turbidity are thus expressed in terms of percentage absorption of blue light. 90,
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20
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IO: 0 400
600
500
700
“P
FIGURE 2. TRANSMISSION OF COLOR FILTERS
It is evident that the relation between the blue and yellow absorption will vary with change in the spectral character of the color to be measured. The color in refined cane sugars is probably nearly all due to caramel or the decomposition products of invert sugar, while in beet sugars the color is only slightly influenced by the presence of these substances. Therefore to make the method applicable to beet sugars, it became necessary to determine the blue-yellow absorption ratio for the type of color found in them. This was done by using high raw sugars as the source of color. The solutions were filtered with Filter-Cel to remove, as nearly as possible, all turbidity. Portions of these solutions were mixed with granulated sugar solution which had likewise been treated for the removal of turbidity. The ratio between the blue and yellow absorption as determined by this method was 3.1 to 1.0. The eauation becomes
Description of Method Test solutions are made up containing 50 grams of sugar per 100 ml., brought to the boiling point to expel the air, then cooled t o room temperature. (Solutions containing invert sugar should be de-aired without heating.) The net deflection is obtained for both color filters by comparison with
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TABLE 11. Total Percentage Unfiltered Blue Yellow 1.33 2.18 1.62 2.70 1.33 2.59 1.89 3.26 0.82 1.74 2.19 3.48 2.72 4.35 3.25 4.45 1.33 2.52 1.25 2.17 1.31 2.35 1.01 1.83 1.56 2.87 1.47 2.63 1.29 2.52 1.05 2.09 2.06 3.09 1.64 3.14 1.17 2.52 2.08 3.52
Sample 1 2 3 4 5
! i3
9 10 11 12 13 14 15 16 17 18 19 20
PERCENTAGE
Absorption Filtered Blue Yellow 1.74 0.82 2.18 1.14 1.14 2.28 1.33 2.61 0.78 1.61 2.61 1.36 2.17 3.83 1.92 3.13 1.05 2.17 0.00 1.87
AND TURBIDITY TESTS TABLE 111. COLOR
Color
Turbidity
Color
Turbidity Color Turbidity Color Turbidity
Color
Turbidity Color Turbidity Color Turbidity Color Turbidity Color Turbidity
Caramel Added Original 1.0 ml. 2 ml. 3 ml. 4 ml. 5 ml. Sugar No. 1, Unfiltered Solution 5.1 4.0 4.4 2.4 3.2 1.6 1.5 1.3 1.1 1.1 1.3 1.3 Sugar No. 1, Filtered through Paper 1.7 2.1 3.3 3.7 4.7 5.2 0.8 0.9 0.7 0.8 0.7 0.9 Sugar No. 1, Filtered through Filter-Cel 5.1 4.1 3.6 2.1 3.0 1.3 0.6 0.6 0.4 0.5 0.3 0.4 Sugar No. 2, Unfiltered Solation 4.0 3.5 2.3 3.1 1.2 2.5 2.5 2.6 2.5 2.8 Suear No. 2, Filtered through Paper 2.6 315 - 3 . 8 1:3 2.3 1.4 1.5 1.3 1.3 1.1 Sugar No. 2, Filtered through Filter-Cel 0.7 1.5 2.3 2.9 3.7 0.3 0.4 0.3 0.3 0.3 Sugar No. 3. Unfiltered Solution 1.41.8’ 3.0 3.8 4.0 1.3 1.4 1.2 1.2 1.4 Sugar No. 4, Unfiltered 3.3 4.0 3.0 1.5 2.0 3.0 3.0 3.0 3.0 2.9 , 1.6 1.5
Sugar No. 5, Unfiltered 3.3 3.1 2.3 1.4 1.5 1.7
4.3 1.4
6 ml. 5.5 1.5 5.6 0.9 5.7 0.6
4.9 2.4
5.4 2.4
4.7 1.3
5.2 1.3
4.3 0.2
5.1 0.2
5.0 1.3
6.5 1.3
4.7 2.9
5.4 2.8
5.0 1.4
5.5 1.5
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water and applying the cell correction described above. The percentage absorption is calculated by dividing the net deflection by 2.3 for the blue filter and 3.6 for the yellow filter. The color and turbidity are then calculated according to the formula.
Discussion of Results This method of color and turbidity analysis has been applied to several hundred samples of granulated sugar. I n most cases determinations were made on the original solution and on the same solution after double filtration through filter paper (B & A, Grade A) on a Buchner funnel. This sort of filtration removes coarse suspended matter, but not color or colloidal matter. The total percentage absorption for both color filters, for filtered and unfiltered solutions, as well as the calculated values for color and turbidity, are given in Table I1 for twenty different sugars. The results tabulated here were selected to represent the various types of sugar ordinarily encountered. For example, Nos. 1, 5, and 12 are low in both color and turbidity; No. 7 is high in both color and turbidity; No. 8,
VOL. 11, NO. 3
ABSORPTION Calculated Values Percentage Absorption Percentage Absorption Due t o Turbidity Due t o Color Unfiltered Filtered Unfiltered Filtered 1.21 1.34 0.40 0.97 1.16 0.68 1.54 1.50 1.82 0.77 0.63 1.65 1.30 0.76 1.96 1.85 1.34 0.40 0.40 1.21 1.65 1.83 0.81 1.80 2.31 2.04 1.45 2.38 1.62 2.83 1.41 1.72 1.72 0.80 0.54 1.63 1.31 0.86 0.53 1.34
which shows a high total absorption, is relatively low in color and high in turbidity; and No. 18 is high in color and low in turbidity. There is a wide variation in the amount of turbidity removed by filtration, This merely means that a variable percentage of the total turbidity is made up of suspended particles which are filterable on paper. The color of the filtered and unfiltered solutions agrees within the limits of error of the method (about *0.2 per cent) regardless of the variation in turbidity. It is, therefore, possible to measure variations in turbidity while the color remains constant. Tests were also made in which the color varied while the turbidity remained constant. This was done by adding successive portions of caramel to sugar solutions, and calculating the color and turbidity for each increment of color. The details of the tests and the results are shown in Table 111. It will be noted that the turbidity remains practically constant throughout any given series. There are occasional discrepancies in both color and turbidity which are probably due to errors in reading the instrument. The method has been tested under two sets of conditions: constant color and variable turbidity, and constant turbidity and variable color. It has been found adequate in defining both conditions. The method as herein described is empirical in that the results are expressed in terms of percentage absorption of a particular blue light by a column of solution 34 mm. deep, the light source being a 25-watt tungsten-filament lamp a t 110 volts. It is applicable for measuring color intensity and turbidity in solutions in which the spectral characteristics of the color remain practically constant, and where the total absorption does not exceed 8 to 10 per cent. Beyond this point the deviation from the assumed direct proportionality between the percentage absorption and the concentration of light-absorbing substances exceeds the limits of error of the method. It is possible, however, to extend the application to juices, sirups, and other solutions of high percentage absorption by substituting the value of -log T,where T is the percentage transmission, for the percentage absorption in the equation. If the measurements are carried out a t constant concentration of sugar and a t constant depth of solution, -log T is proportional to the concentration of light-absorbing substances. By the application of Lambert and Beer’s law the results may be reduced to terms of unit concentration and unit depth. For details of the theoretical considerations involved the reader is referred to Eggert and Gregg (2). The principles of the method can also be applied to colorimetric analysis where turbidity is an interfering factor, or for the determination of precipitates in terms of turbidity in
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ANALYTICAL EDITION
145
(3) Gillett, T. R., and Holven, A. L., IND.ENQ.CAEM., 28,391 (1936). (4) Holven, A. L., and Gillett, T. R., Facts About Sugar, 30, 169
colored solutions. Any suitable pair of color filters may be used. In any case the absorption ratios of the filters must be determined for the color and turbidity in question and these ratios must remain approximately constant for the range of conditions under investigation.
(1935).
(5) Keane, J. C., and Brice, B. A., IND. ENO.CHEM.,Anal.
Ed., 9.258
(1937).
(6) Zerban, F. W., and Sattler, L., Ibid., 3, 226 (1931); 8, 168 (1936). (7) Zerban, F. W., Sattler, L., and Lorge, I., Ibid., 6, 178 (1934); 7, 157 (1935).
Literature Cited (1) Baloh, R. T., IND.ENO.CHEM.,Anal. Ed., 3, 124 (1931). (2) Eggert, John, and Gregg, S. J., "Physical Chemistry," p. 550, New York, D. Van Nostrand Co., 1933.
RECEIVED Ootober 10, 1938. Presented before the Division of Sugar Chemistry at the 98th Meeting of the Amerioan Chemioal Society, Milwaukee, Wis.. September 5 to 9, 1938.
Selective Oxidation of Levulose with Potassium Ferricvanide J
D. T. ENGLIS AND H. C. BECKER University of Illinois, Urbana, Ill.
T
HE alkaline copper sulfate solution of Fehling (3) was probably the first chemical reagent used for the determination of sugars and even today the copper methods still outnumber all others. In 1859, however, Gentele (4) made use of an alkaline potassium ferricyanide solution for the determination of reducing sugars in the presence of sucrose and dextrins. Apparently not much work was done with this reagent until the advent of the Hagedorn-Jensen method (6) for blood sugar. Since that time its scope has been widened until today it is a very valuable reagent for the biochemist and is becoming increasingly more so for the sugar analyst. Since 1929 Hanes (6), Callow ( I ) , Hulme and Narin (8),Cole (3), Miller and Van Slyke ( l a ) , and several others have modified and expanded the Hagedorn-Jensen method to include other sugars of biochemical interest, while Whitmoyer (18), Hassid (Y),and Strepkov (16) have utilized it for the analysis of plant extracts and sugar solutions. Its reported freedom from atmospheric oxidation, lack of filtration necessity, and the ease and accuracy with which its reduced form can be determined are factors which recommend it very highly. Within the last two years, another property of this reagent has been discovered-its ability to oxidize levulose selectively in the presence of dextrose. Strepkov (14) has developed a micromethod for this determination using an alkaline ferricyanide solution as the oxidizing agent. Two cubic centimeters of the alkaline potassium ferricyanide reagent [1.65 grams of potassium ferricyanide and 80 grams of sodium monohydrogen phosphate dodecahydrate per liter 1 and 1 cc. of sugar solution containing up to 1.5 mg. of levulose are heated in a closed tube a t 60" C. for 2.5 hours. After this time the solution is cooled, and acidified with 10 per cent acetic acid, 2 cc. of an iodine solution are added, and the excess is titrated with 0.005 N thiosulfate. Dextrose in amounts up to 0.5 mg. is reported to have no reducing action. In the experimental work reported below it is shown, however, that dextrose does have a definite reducing action, but only a small fraction of that of levulose, and an attempt was made to find conditions which would decrease its action even more. One of the conditions studied was that caused by the addition of phosphate. It has been known for many years that phosphate has a definite effect upon the oxidation of sugars, but the question as to what the effect is remains a point of controversy. Kappanna (9) reports that the iodometric oxidation of dextrose to gluconic acid in a solution buffered to pH 7 with
phosphate does not occur a t all, and that a t a constant alkalinity the phosphate concentration has no effect. Theriault, Butterfield, and McNamee (16),working with pH values in the physiological range and temperatures of 20" to 50" C., found that the oxidation of dextrose by atmospheric oxygen was not catalyzed by phosphates. According to Malkov and Zwetkova (11) the oxidation of dextrose by hydrogen peroxide in the presence of ferrous sulfate is inhibited by phosphate, while in the absence of hydrogen peroxide a catalytic action is observed. Work by Kuen (10) shows that a t a pH of 7 the oxidation of dextrose by hydrogen peroxide is increased fourfold in the presence of phosphate, and Witzemann (19) states that sodium hydrogen phosphate is a specific catalyst for this reaction. Warburg and I'abusoe (17) and Nicloux and Nebenzahl (IS) found that phosphate catalyzes the oxidation of levulose. In this paper, experimental results indicate that under the conditions used the rate of oxidation of levulose by potassium ferricyanide in alkaline solution is affected very little by phosphate, while the oxidation of dextrose is inhibited. The catalytic action of traces of iron is well known and undoubtedly the ferricyanide reagent furnishes enough ferric ions to bring this factor into operation.
Experimental The levulose used in these experiments was a pure-white crystalline product with a specific rotation of -91 and the dextrose was a crystalline product with a specific rotation of $52.5; the inorganic reagents were of analytical grade. During all the determinations, the constant-temperature water bath was maintained within *0.1" C. of the specified temperature. The concentration of both the dexkrose and the levulose solutions was 1 mg. per cc. and 10-cc. portions were used for each analysis. Throughout all the work the following procedure was used for each analysis. A 10-cc. portion of the sugar solution was placed in a 125-cc. Erlenmeyer flask and 25 cc. of the alkaline potassium ferricyanide reagent were added from a graduated cylinder. A strip of sheet lead made into a collar and fitted over the neck of the flask prevented its tipping or floating when placed in water deep enough t o cover it several centimeters above the level of the solution inside. As soon as the solution reached the water-bath temperature, the flasks were stoppered to prevent any oxidation of potassium ferrocyanide by atmospheric oxygen. Tests were carried out by heating a standard ferrocyanide solution in both stoppered and unstoppered flasks for 2 hours at 50" C. At the end of this period of heating both showed a slight oxidation, but the unstoppered flask about 1.2 per cent more than the stoppered. At specific intervals of time the flasks were removed from the water bath, cooled under a tap, and acidified with 6 N sulfuric
