310
INDUSTRIAL A N D ENGINEERING CHEMISTRY
feed mixture is apparently sufficient under these conditions to allow cleavage of the pantothenate contained therein. This appears as the most likely explanation, since pantothenate was found completely stable under even more drastic conditions when kept entirely free from moisture. Unfortunately, the natural stability of many of the vitamins-i. e., thiamine, pyridoxine, and ascorbic acid-is poor at the p H of optimal stability of pantothenate. This fact deserves careful consideration in the case of pharmaceutical preparations and foods where loss of pantothenate is to be avoided.
Summary Pantothenate destruction under ordinary conditions can be traced to hydrolysis of the molecule. A method is described for following the destruction of calcium d(+)-pantothenate by rapid polarimetric analysis. The rate of pantothenate destruction is a function of p H and temperature and is affected also by presence of other substances both in aqueous solution and in dry mixtures. Optimum stability of pantothenate lies in the approximate range, p H 5.5 to 7. The rate of destruction increases as the p H moves away from this range. Only traces of water are needed to cause significant destruction of pantothenate when other conditions favor hydrolysis. Special significance is attached to the apparent incom-
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patibility of pantothenate with certain other vitamins, notably thiamine.
Acknowledgment Acknowledgment is gratefully made to Eleanor Willerton, who conducted the microbiological assays. Thanks are expressed to Edmond E. Moore and Marjorie B. Moore for helpful discussion of many of the problems involved and for a supply of d( -)-a-hydroxy-P,P-dimethylbutyrolactone, and to Carl Nielsen and E. H. Volwiler for helpful advice and support of this project. Literature Cited (1) Grussner, A., Gatzi-Fichter, M., and Reichstein, T., Helv. Chim.
Acta, 23, 1276 (1940). (2) Strong, F. M., Feeney, R. E., and Earle, .4.,IND.ENQ.CEEM., ANAL.ED.. 13. 566 (1941).
(3) Waisman, H: A.; Mills, R. C., and Elvehjem, C. A., J. Nutrition, 24, 187 (1942). (4)
Weinstock, H. H., Mitchell, H. K., Pratt, E. F., and Williams,
R. J., J . Am. Chem. Soc., 61, 1421 (1939). ( 5 ) Williams, R. J., and Major, R. T., Science, 91. 246 (1940). (6) Williams, R. J., Weinstock, H. H., Rohrmann, E., Truesdail, S. A., Mitrhell, H . K., and Meyer, C. E., J . Am. Chem. Soc., 61, 454 (1939). (7) Woolley, D. W., Waisman, H. A., and Elvehjem, C. A., Ibid., 61, 977 (1939). (8) Woolley, D. W., Waisman, H. A., and Elvehjem, C. A., J . Biol. Chem., 129, 673 (1939).
Colorimetric Determination of Cobalt with o-Nitrosoresorcinol LYLE G . OVERHOLSER AND JOHN H. YOE, University of Virginia, Charlottesville, Va.
Y
OE and Barton ( 2 ) reported a study of the reaction of pnitroso-a-naphthol with cobalt, including spectrophotometric data, and found the reaction applicable to the colorimetric determination of small amounts of cobalt. The main disadvantage in using this reagent is due to the insolubility of the cobalt complex. The colored suspension tends to precipitate on standing, resulting in a limited stability. The authors observed that a similar organic compound, onitrosoresorcinol, also reacts with cobalt and may be advantageously employed as a colorimetric reagent for this element. o-h'itrosoresorcinol is slightly less sensitive for cobalt than is P-nitroso-a-naphthol, but solutions of the cobalt complex of onitrosoresorcinol are stable for several weeks. Cronheim (1) used o-nitrosophenol for the colorimetric determination of cobalt, extracting the cobalt complex with petroleum ether and measuring the intensity of the colored ether fraction. The authors were unable to extract the cobalt complex of o-nitrosoresorcinol with any immiscible solvent. Spectrophotometric data for solutions of o-nitrosoresorcinol, the cobalt complex, and the complexes of other metals are presented in this paper. A colorimetric method for the determination of cobalt in the presence of nickel is given. Apparatus and Materials Transmittancy measurements were made with a Beckman spectrophotometer, Model D, using a solution thickness of 1 cm. and distilled water as a standard. The visual observations were performed with 50-ml. (220-mm.) ATessler tubes. pH measurements were made with the glass electrode. 0-KITROSORESORCINOL (Eastman No. 2088). An aqueous 0.05 per cent solution of the sodium salt was employed. This reagent solution is stable for several weeks.
COBALT. A stock solution containing 1 mg. of cobalt per ml. was prcpared from c. P. cobalt nitrate hexahydrate. Solutions containing 20 or 100 p. p. m. of cobalt, prepared by dilution of the
stock solution, were used in the experiments. NICKEL.c. P. nickel nitrate hexahydrate was purified by precipitating, as potassium cobaltinitrite, any cobalt present and filtering. The nickel was precipitated as the hydroxide, filtered, washed thoroughly, and dissolved in hydrochloric acid. The nickel content was determined gravimetrically with dimethylglyoxime. BUFFER. A buffer solution having a pH of 6.0 was prepared bv addine 363 ml. of 0.5 M sodium hvdroxide to 500 ml. of 0.4 M phassium biphthalate and di1uting"to 1 liter with water. The pH of the buffer is practically unchanged when diluted from 25 to 100 ml. All other reagents used were of the highest purity obtainable.
Experimental The usual procedure followed in this work was to transfer the desired quantity of cobalt to a 100-ml. volumetric flask, add 25 ml. of buffer and 5 ml. of the reagent, and dilute to the mark with water. After thorough mixing, color comparisons were made in Kessler tiibes and transmittancg measurements made on another portion of the solution. Using Kessler tubes, 1 part of cobalt in 20,000,000 parts of solution may be determined at cobalt concentrations of 0 to 0.08 mg. per 100 ml.; 1 in 10,000,000at 0.08 to 0.2 mg.; 1 in 5,000,000 a t 0.2 to 0.25 mg. These are also the approximate increments to be employed in making up a standard series. The spectrophotometer is slightly more sensitive; 1 part of cobalt in 50,000,000 of solution may be detected. Transmittancy curves for solutions of the reagent and of the cobalt complex are given in Figure 1. The concentration of the reagent must be kept relatively low to prevent a decrease
ANALYTICAL EDITION
May 15, 1943
WAVE
LENGTH
OF COBALT CONCENTRATION FIGURE1. EFFECT
1. 2. 3. 4. 5.
25 p. p. m. of reagent, no Co 0.1 D. D. m. of Co 0.5 b. b. rn. of Co 1 p. p. m. of Co 2.5 p. p. m. of Co
in the sensitivity for cobalt, because the reagent is highly colored and absorbs strongly in the same spectral region as the cobalt complex. The reaction rate, however, is prohibitively slow a t very lorn reagent concentrations. The concentration of reagent recommended, 2.5 mg. per 100 ml., results in immediate color formation and also permits the determination of small amounts of cobalt. The maximum amount of cobalt determinable is limited by the relatively small amount of reagent employed. No increase in intensity occurs if the quantity of cobalt present exceeds 0.26 mg. with 2.5 mg. of reagent. These quantities correspond to a molar ratio of reagent to cobalt of slightly greater than 3 to 1. This indicates that the compound formed is a n inner complex of nitrosoresorcinol and the cobaltic ion, similar to that obtained with P-nitroso-a-naphthol. The maximum difference between the transmittancy of solutions of the reagent and of the cobalt complex occurs a t a wave band of 420 to 430 mp a t low cobalt concentrations. Most of the measurements were made in this region. A wave length of 450 m p may be advantageously used for cobalt concentrations above 0.15 mg. per 100 ml. The validity of Beer’s law at 430 and 450 m p is shown in Figure 2. EFFECT OF pH. The color of the reagent varies markedly with the pH of the solution. Below pH 2.5 it has a pale greenish-yellow color which increases in intensity and becomes orange as the pH increases from 2.5 to 5.6. From 5.6 to 6.5 little change in the intensity occurs, but it increases slightly a t 7.0. A further increase in pH results in a slight decrease in intensity. The transmitt)ancy curves of solutions of the reagent a t various pH’s are given in Figure 3. KO break in the curve a t a wave length of 440 mp is observable a t a pH of 4.0. The pH also influences the intensity of the red color of the cobalt complex. No visible reaction occurs below a pH of 2.0. The intensity of the color increases from 2.0 to 5.6, is practi-
31 1
cally constant from 5.6 to 6.3, and decreases slightly with increasing pH above 6.5. A p H of 6.0 was employed in all experiments, although the method is applicable a t a pH range of 5.6 to 6.5. Figure 4 shows the transmittancy curves of solutions of the cobalt complex a t various pH’s. To afford increased buffer capacity, the concentration of the buffer may be increased without any effect on the intensity of the color. A biphthalate-sodium hydroxide buffer was employed in preference to a phosphate or citrate buffer because the presence of phosphate or citrate causes a decrease in the intensity of the color of the cobalt complex. The rate of reaction is dependent on the pH of the solution, being slower a t a pH of less than 5.6 than above this value. The reaction occurs practically instantaneously a t a p H of 6.0 and above. STABILITY.Solutions of the cobalt complex are stable for a t least a month. The transmittancy, a t 450 mp, of a solution containing 0.15 mg. of cobalt was 0.128 when prepared and remained unchanged for a month. The transmittancy of solutions of the reagent changes slightly on long agingfor example, the transmittancy a t 450 mp increased from 0.488 to 0.510 after a month. The transmittancy increases slightly at wave lengths less than 500 mp but decreases at longer wave lengths on aging. Visually, solutions of the reagent fade slightly on long aging in the buffer or aqueous medium. INTERFERENCE. I n these studies the usual procedure was followed but the ion in question was added prior to the addition of the reagent. Observations were made in the absence of cobalt and a t a cobalt concentration of 1 p. p. m. The presence of 5 grams of sodium nitrate, potassium chloride, ammonium nitrate, sodium chloride, or ammonium
l-----T J
1.50
030K
I
I
1.0
I
1.5
I
I
2.0 2.5 WRTS PER MILLION OF COBALT 0.5
Z 0
FIGURE 2. TEST OF BEER’S LAWWITH COBALT-NITROSORESORCINOL SOLUTION 1. 430 mp
2. 3. 4.
430 rnp, 50 p. p. m. of Ni 450 mp 450 mp, 50 p. p. m. of Ni
312
Vol. 15, No. 5
INDUSTRIAL AND ENGINEERING CHEMISTRY
Y
I
I
I
350
I
I
I
550
450 WAVE
650
LENGTH
SOLD
FIGURE 3. EFFECTOF pH
ON NITROSORESORCINOL TIONS
1.
2. 3. 4. 5.
p H a 4.0 p H = 4.6 p H = 5.2 p H = 5.6-6.3 pH = 7.0 (25 p. p. m. of reagent)
chloride had no effect on the transmittancy of the solutions at 430 mp. Visually, the presence of more than 1 gram of ammonium chloride produced a green tint. All the other salts cause a similar off shade of color on standing. However, if the color comparisons are made within an hour the salts cause no interference. The concentration of various ions that may be present without interference is given in Table I.
Cadmium. A solution of the cadmium complex gives a transmittancy curve similar to that for zinc, as seen in Figure 5. Visually, a cadmium concentration of 100 p. p. m. causes no interference; 1000 p. p. m. of cadmium cause no interference if the transmittancy measurements are made a t a wave band of 425 mp. The measurements may be made immediately, since this high cadmium concentration does not decrease the reaction rate of cobalt with the reagent. The cadmium concentration must be limited to approximately 1000 p. p. m. to avoid possible precipitation of the hydroxide. Palladium. The transmittancy curve for a solution of the palladous complex given in Figure 5 is similar to that for the cobalt complex. The intensity of the colored palladous complex a t low concentrations is almost as great as that of the cobalt compound. The reagent is not recommended for palladium, however, because the complex is slow in forming and the colored solutions are not stable after the complex has formed. Palladium must be absent to avoid interference. Iron. The reagent reacts with ferric ions, giving a green solution at a concentration as low as 0.1 p. p. m. The use of citrate or tartrate does not com letely eliminate this interference. The intensity of the colorel solution increases and the transmittancy decreases slowly on aging. The interference due to iron can be eliminated by precipitating the ferric iron with potassium fluoride and filtering. Unfortunately, some of the ferric salts, from different sources, contained an unidentified interfering substance. Solutions prepared from ferric nitrate nonahydrate or ferric ammonium sulfate dodecahydrate caused no interference, visually or spectrophotometrically. Those prepared from ferric chloride hexahydrate or iron wire did interfere, producing slightly darker colored solutions than obtained in the absence of iron. This interference could be eliminated by purifying the iron salts before use. The reagent is not recommended for cobalt if iron is present, because it is impossible to know whether or not the interfering substance accompanies the iron. The results obtained, however, do show that a clean-cut separation of cobalt from iron is possible by precipitating the iron as the complex fluoridefor example, no cobalt is lost when the iron is precipitated from a solution containing 500 mg. of iron and 0.05 mg. of cobalt, a ratio of 10,000 to 1.
Determination of Cobalt i n the Presence of Nickel Nickel also forms a colored complex with o-nitrosoresorcinol under the conditions used for the determination of cobalt. The transmittancy curve for the nickel complex (Figure 5 )
TABLE I. CONCENTRATIOKS SOT INTERFERING Ions
Concentration
P. p . B a t + , Ca++, M g + + , P b + + + + , T h + r i +p b + + + y t + + +
g++ +
Al+++,Hg+, Hg++ T i + + + +Z r + + + bg+,
m.
100
1; 1 0.5
The interference of the other common metals is considered in more detail in the following sections. Copper. The cupric ion reacts with o-nitrosoresorcinol, giving a complex that is nearly as intense in color as that, with cobalt. From the transmittancy curve for a solution of the copper complex (Figure 5) it is evident that no wave band may be employed that will eliminate the interference of the cupric ion. The technique used for determining cobalt in the presence of nickel, described below, is not applicable for copper. Apparently, the complex formed with the cupric ion is more stable than that with nickel. The quantity of copper present must not exceed 0.01 to 0.02 mg. in either the visual or spectrophotometric methods. Zinc. The transmittancy curve for the zinc complex is given in Figure 5. The reagent is not sensitive for zinc, a concentration of 20 p. p. m. not interfering in the visual method. A zinc concentration of 1000 p. p. m. causes no interference, if the transmittancy measurements are made at a wave band of 420 mp. The high zinc concentration does cause a decrease in the rate of formation of the cobalt complex. Thus, a t a zinc concentration of 1000 p. p. m. it is necessary to allow the solutions to stand 3 t o 4 hours before making the measurements if the cobalt concentration is 1 p. p. m. At lower cobalt or zinc concentrations a shorter period of time is re uired. No evidence of the precipitation of zinc hydroxide a-as %served, even at a zinc concentration of 5000 p. p. m.
WAVE
LENGTH
FIGURE 4. EFFECTOF pH CINOL
1. 2. 3. 4.
pH pH
-a
4.0
ON COBALT-NITROSORESORSOLUTIONS
4.6 5.2 and 7.0 p H = 6.0 (0.5 p. p. m. of Co)
pH
ANALYTICAL EDITION
May 15, 1943
313
with increasing temperature. Results of experimentation are given in the following procedure: PROCEDURE FOR DETERMINING COBALT IN THE PRESENCE OF NICKEL. Transfer the test solution to a 100-ml. Erlenmeyer flask, and add 25 ml. of buffer and 5 ml. of reagent. Heat on a steam or water bath for a period of time depending upon the cobalt and nickel concentration present. Cool, transfer quantitatively to a 100-ml. volumetric flask, and dilute to the mark with water.
Using 5 mg. of nickel 2 hours of heating are required for a fmal cobalt concentration of 1 p. p. m: 3 hours for 1.5 p. p. m. With 10 mg. of nickel 4 hours are reqdred for 1 p, p. m. of cobalt; 6 hours for 1.5 p. p. m. For 15 rag. of nickel 6 hours are required for 1 p. p. m.; 10 hours for 1.5 p. p. m. of cobalt. A longer time is required if the volume of the solution is greater than 40 ml. during the heating eriod. It is impractical for the nickel concentration t o exceex 150 p. p. m. and the cobalt concentration 1.5 p. p. m. The use of a spectrophotometer is recommended. With this instrument it is possible to detect 0.002 mg. of cobalt in the presence of 10 mg. of nickel-i. e., 0.02 per cent of cobalt.
WAVE
LENGTH
FIGURE 5. TRANSMITTANCY CURVESFOR SOLUTIONS OF METALCOMPLEXES OF NITROSORESORCINOL 25 p p m of rea ent 2006 p.' p. 'in. of 8 d 2000 p. p. m. of ,Zn 4. 50 p. p. m. of Ni 6. 5 p; p. m. of Cu 6. 3 p. p. m. of Co 7 . 10 p. p. m. of Pd 1
2: 3.
shows that a t no wave band may the interference of nickel be effectively eliminated. The nickel concentration would have to be limited to 0.5 p. p. m., if this interference could not be eliminated by some method. Using a constant cobalt concentration, the intensity of the color increases with increasing nickel concentration up to approximately 10 p. p. m. The intensity is less immediately after adding the reagent at higher nickel concentrations but increases on standing until the intensity is the same as at 10 p. p. m. This is understandable, if it is remembered that the total intensity of the color is the sum of that of the cobalt and nickel complexes and that the intensity of the cobalt complex is greater than that of the nickel at the same concentration. At the lower nickel concentrations, the cobalt complex forms immediately and any reagent in excess of that required for the cobalt forms the nickel complex simultaneously. At the higher nickel concentrations, however, the formation of the nickel complex is favored, but since the cobalt complex is more stable than the nickel complex a slow shift from the nickel to the cobalt complex occurs, resulting in the increase in the color intensity on standing. The transmittancy of solutions of the nickel complex is practically independent of the nickel concentration, if the latter is above 10 p. p. m. This indicates that the concentration of nickel does not have to be controlled carefully, p'oviding the concentration is above 10 p. p. m. The reaction rate, between the cobalt and reagent, however, decreases with increasing nickel concentration, thereby setting a practical limit to the amount of nickel that may be present. An increase in the concentration of the reagent increases the reaction rate but results in a decrease in the sensitivity. This difficulty is avoided if the volume of the solution is kept below 50 ml. during the aging period and diluted to 100 ml. before making the measurements. The aging is carried out a t approximately 100 C., because the reaction rate also increases O
Some transmittancy values for mixtures of cobalt and nickel are given in Table 11. Additional results are shown in Figure 2 for various cobalt concentrations in the presence of 5 mg. of nickel. Beer's law is valid, although the lines have origins different from those obtained in the absence of nickel. The visual method is applicable if appropriate amounts of nickel are present in the standards and the standards are heated for the proper periods of time. Visually, the method is slightly less sensitive for cobalt in the presence of nickel, owing to the increased intensity of the colored solutions. The effect of potassium fluoride on the method was studied briefly in connection with the possible determination of cobalt in solutions also containing nickel and iron. The presence of potassium fluoride decreases the color intensity of the nickel complex, especially on heating. The aging can be carried out a t room temperature, however, since the presence of potassium fluoride increases the reaction rate of the cobalt and reagent. The visual method is not applicable to mixtures of cobalt and nickel if potassium fluoride is present, but spectrophotometric measurements made a t 450 mp are satisfactory.
TABLE11. TRASSMITTASCY VALUES Sickel Added ."io.
Cobalt Added
Transmittancy at 430 mp
0
0.05 0.10 0.15 0 0 0 0.05 0.05 0.05 0.10 0.10 0.10 0.15 0.16
0,246 0.130 0.071 0.403 0.401 0.402 0.218 0.213 0.213 0,121 0.120 0.122 0.063 0.068
0
0
L 10
1 5 10 1 5 10 1
5
M g.
Summary A study of the reaction between cobalt and o-nitrosoresorcinol has shown that the reagent may be used for the colorimetric determination of cobalt. Spectrophotometric data for solutions of the reagent, for the cobalt complex, and for other metal complexes are presented. Procedures are given for the determination of cobalt alone and for cobalt in mixtures of nickel and cobalt.
Literature Cited (1) Cronheim, G., IND. ENG.CHEM.,ANAL.ED.,14,445 (1942)
(2) Yoe, J. H., and Barton, C. J., Ibid., 12,405 (1940).
