coefficient of approximately 5.6 aiid containing 10% sulfur, 5% nitrogen, 0.1% vanadium, and 0.01% copper would have a total mass absorption coefficient of less than 60. I n order to test for matrix effects, a standard containing 51.8 p.p.m. of nickel was prepared in a 50 weight yo chloroform-oil solution. Because the mass absorption coefficient of this oilchloroform mixture is calculated to be approximately 60, any matrix effects that are produced by differences in the mass absorption coefficients between the nickel standards prepared in oil and the various oils analyzed should be indicated. However, as shown in Table 11, the calculated and observed f ( R ) values are very nearly the same as the values of f ( R ) observed for the oil-chloroforni based standard and the f(R) predicted by the pure oil standards. This indicates that the method is independent of ordinary matrix effects. I n view of the usual range of iron concentrations in petroleum samples it \Todd be predicted that, although iron has an absorption edge between the nickel and cobalt K , peaks, it would produce little interference. To test this, 100 p.p.m. of iron Kere added to a nickel standard solution containing 57.8 p.p.m. of nickel and the usual amount of cobalt internal standard. As shown in Table 11,this concentration of iron would result in approximately a 3% error in f(R), and a 3% error in nickel concentration. However, the concentration of iron usually found in crude oil is less than 100 p.p.m. and the presence of iron should result in not more than about 1% error for a typical crude oil. However, if a crude oil sample contained 100 pap.m. or more of iron, the iron concentration could probably be determined by the internal
standard method using the cobalt peak as the internal standard peak, and a correction could be applied to the nickel concentration. As shown in Table 11, the change of operating conditions to 25 kv. and 25 ma. resulted in approximately the same R value for the 57.8 p.p.m. nickel standard, thus indicating that the long-term stability of x-ray emission is of little importance so long as shortterm stability is maintained. Indeed, after 1 month, the same calibration curve was found to apply. Thus, the method eliminates the frequent recalibrations required for external standard methods.
The close correIation between the internal standard method and other methods indicates that it is accurate, as well as precise and suitable for a large variety of oils. The precision for duplicate determinations was found to be within 0.6% for all determinations as compared with a theoretical precision of 0.4%. The accuracy was within 3%, with an accuracy approaching 1% for oils containing little iron. The method is rapid, and frequent recalibration is unnecessary. Precautions were taken to eliminate sources of error due to matrix effects, sample evaporation, density changes, and x-ray emission variability.
COMPARISON WITH OTHER METHODS
ACKNOWLEDGMENT
The nickel contents of various oils obtained by the internal standard method are compared to those obtained by other methods in Table 111. The results with Tatums crude oil and its fractions are in excellent agreement with spectrographic methods. With the other oils the two x-rays methods give results generally in good agreement, despite the difference in methods. The spectrographic methods in some cases give low results. This is particularly apparent in the N. Belridge oil. This oil contains the highest content of nickel-porphyrin complex of any oil studied in this laboratory (3). Nearly half of the nickel and vanadium of this oil is present as the porphyrin complexes. It appears that methods involving ashing may give low nickel contents because of the volatilization of nickel complexes during the ashing process. Such loss would be minimized by wet-ashing methods, but still may be sizable. The x-ray fluorescence methods, which do not require ashing, avoid this error.
The authors gratefully acknowledge the assistance of J. R. Lindley, of this station, who designed the special sample holder and adjustment mechanism. LITERATURE CITED
(1) Davis, E. N., Hoeck, B. C., ANAL. CHEM.27, 1880 (1955). (2) Dunning, H. N. Myers, A. T., Moore, J. W., Ink Eng. C h m . 46, 2000 (1954). (3) Dyroff, G. V., Skiba, P., ANAL. CHEW26, 1774-8 (1954). (4) Gamble, L. W., Jones, W. H., Zbid., 27, 1456 (1955).
(5) Hansen, John, Skiba, Paul Hodgkins, C. R., Zbid., 23, 1362 (1951j. (6) Horecay, J. T. Hill, B. N , Walters, A. E., Schutze, h. G., Bonner, W. H., Zbid., 27, 1899 (1955). ( 7 ) Klug, H. P., Alexander, L. E., “XRay Diffraction Procedures,” pp. 28190, John Wiley, New York, 1954. (8) Zbid., pp. 415-16. (9) Milner, 0. I., Glass, J. R., Kirchner, J. P., Yurick, A. N., ANAL. CHEV. 24, 1728 (1952). RECEIVED for review September 15, 1958. Accepted December 22, 1958. Divisioii of Analytical Chemistry, 135th Meeting, ACS, Boston, Mass., April 1959.
Spectrophotometric Determination of Cycloheximide ARLINGTON A. FORIST and SUSAN THEAL Department of Physical and Analytical Chemistry, The Upjohn Co., Kalamazoo, Mich.
b The antibiotic cycloheximide (actidione) hus been determined routinely b y a microbiological assay utilizing the inhibition of the growth of Saccharomyces pastorianos. A chemical method has been developed based on the reaction with alkaline hydroxylamine to produce a hydroxamic acid, followed by conversion to the highly colored ferric hydroxamate. Analysis of standard samples indicates a mean recovery =k standard deviation of 1042
ANALYTICAL CHEMISTRY
n
100.2 & 1.7%. Analysis of typical bulk cycloheximide preparations b y the chemical method gives results in excellent agreement with those obtained by solubility analysis and by bioassay, CH3
T
antibiotic cycloheximide (I) (5) isolated from Streptomyces griseus (3, 6) is highly active against a large number of yeasts, but has HE
I no marked antibacterial activity (10). Recently, cycloheximide has received
Table 1. Determination of Standard Cycloheximide Samples
Figure 1. Production of hydroxamic acid from cycloheximide
Taken,
Found,
Mg./Ml. 0.22 0.66 0.90 0.90 0.95 0.98 1.12 1.34 1.31 1.34 1.59
Mg./hfl. 0.23 0.67 0.88 0.90 0.96 0.97 1.10 1.33 1.37 1.34 1.58 1.65 1.78 2.21
1.64 1.78 2.23
Recovery,
%
104.5 101.5
97.8 100.0 101.1 99.0
98 2
99.3 102.2 100.0 99.4 100.6 100.0 99.1
Rlean recovery =k standard deviation
Table II.
0
IO
20
30
40
50
wide usage in the treatment of fungal diseases in plants ( 2 ) . As a result, a chemical procedure is needed which is more precise than the microbiological assay involving inhibition of the grovth of Saccharomyces pastorianus ( l o ) . A procedure based on hydroxamic acid formation from the imide group, utilizes a modification of the method of Goddu, LeBlanc, and Wright (4) for the determination of esters. REAGENTS
Hydroxylamine Hydrochloride, 12.5% in methanol (12.5 grams in 100 ml. of solution). Sodium Hydroxide, 12.575, reagent grade, in 85% methanol. A sample of 12.5 grams of sodium hydroxide is dissolved in 15 ml. of water and, on cooling, the resulting solution is diluted to 100 nil. with methanol. Ferric Perchlorate, stock solution. An 800-mg. portion of iron powder is mixed with 3 ml. of water in a 50-ml. beaker. Ten milliliters of 7Oyo perchloric acid and 7 ml. of water are then slowly added dropwise. If small particles of iron are not dissolved, the process may be hastened by gcntle heating. The resu'ting solution is quantitatively transferred to a 100-ml. volumetric flask with anhydrous 2B ethyl alcohol and, with cooling under running tap n.ater, gradually diluted t o 100 ml. with anhydrous 2B ethyl alcohol (nondenatured ethyl alcohol may be used equally well in this and the subsequent reagents). Ferric Perchlorate, reagent solution. A 40-ml. aliquot of the stock solution is transferred to a 1-liter volumetric flask followed by 12 ml. of 7Oy0 perchloric acid. The resulting solution is gradually diluted to 1 liter with anhydrous 2B ethyl alcohol with cooling under running tap water.
Analysis of Bulk Cycloheximide Preparations
Cycloheximide, yo SpectrophotoSolumetric bility method Bioassay analysis
60
MINUTES Alkaline Hydroxylamine Reagent. Equal volumes of the 12.5% liydroxylamine hydrochloride and 12.5% sodium hydroxide solutions are mixed and the insoluble sodium chloride is removed by filtration through Khatman KO. 40 paper. This reagent is usable for four hours. Standard Cycloheximide Solution. An approximately 0.008M solution of cycloheximide is prepared by transferring an accurately weighed sample of 55 to 60 mg. of standard material to a 25-ml. volumetric flask, dissolving the sample in anhydrous 2B ethyl alcohol, and diluting the resulting solution to 25 ml. 11-ith this solvent. APPARATUS
Constant temperature water bath, 50" C. A Beckman Model B spectrophotometer with 1-cm. cells is used for absorbance measurements. PROCEDURE
A sample of approximately 40 mg. of the material to be analyzed is accurately weighed into a 25-ml. volumetric flask, dissolved in anhydrous 2B ethyl alcohol, and diluted to 25 ml. with this solvent. A 5-ml. aliquot of the sample solution is transferred to a 50-ml. volumetric flask followed by 3 ml. of the alkaline hydroxylamine reagent. The solution is mixed and the tightly stoppered flask is immersed in a 50' C. constant temperature water bath for 1 hour. The flask is then removed, shaken under running tap water while the solution is diluted to about 40 ml. with the ferric perchlorate reagent, and stored in the absence of light for about 10 minutes. (Usually several samples are run simultaneously and each is brought to this stage before proceeding further). The solution is then diluted to 50 ml. with
100 2 &1.7CT,
Sample 1 2 3 4 5 6
1000 100.3 99.8 94.0 99.6 77.9
102 105 102 101,98
...
72
99.8 99
i4-i5 100
...
Standard for hydroxamic acid procedure. a
the ferric perchlorate reagent and thoroughly mixed. The absorbance of thc resulting solution is measured within 1 hour a t 530 mp in a 1-em. cell us. a reagent blank similarly prepared. The sample should be stored in the dark between the final dilution and the absorbance determination. Two standard samples are run in parallel with the unknown. Five milliliters of the standard cyclohexiniidc solution are added to one 50-ml. voliimetric flask; 2 nil. of the standard solution and 3 ml. of anhydrous 2B ethyl alcohol are added to a second 50-ml. volumetric flask. Each sample is carried through the procedure described above. The per cent cycloheximide, % C, in the sample is calculated from the equation :
where
Asso= observed absorbance a t 530 a530
=
UI
=
mp for the sample absorbance per mg. per ml. for the standard a t 530 mp (about 6.0) mg. of sample per nil. of solution
RESULTS AND DISCUSSION
Imides react with alkaline hydroxylamine to produce hydroxamic acids which in turn form highly colored ferric VOL. 3 1 , NO. 6, JUNE 1959
1043
complexes (1, 8). This reaction has received little use as a means of determining imides. A method for the determination of a-phenyl-a-ethyl glutarimide has been reported ( 7 ) , but under the conditions employed, the color was stable for only 5 minutes. Recently, Goddu, LeBlanc, and Wright (4) have made a complete study of the factors involved in the hydroxamic acid procedure for the determination of esters and have described concentrations of acid and of ferric ion necessary for a stable color. These conditions have h e n incorporated into the present method for cycloheximide. Formation of hydroxamic acid from cycloheximide a t 25" and 50" C. is shown in Figure 1. Reaction a t 25" C. is incomplete after 60 minutes whereas a t 50' C. hydroxamic acid production is maximal within 50 minutes. .4 1hour reaction period a t 50" C. has been adopted for routine use. The ferric hydroxamate from cycloheximide has a n absorption maximum a t 530 mp typical of such compounds (4). Under the conditions employed the color is stable for a t least an liour in the absence of light. To avoid photo-
chemical instability, samples should be protected from direct light as much as possible. Absorbance a t 530 nip follows Beer's law over the range 0.2 to 2.2 mg. of cycloheximide per milliliter of sample solution. The response varies slightly from day to day, however, and for maximum accuracy standards should be run in parallel with unknown samples, Representative data obtained in the analysis of standard cycloheximide solutions (Table I) indicate a mean recovery of 100.27, vith a standard deviation of =k1.77,, Results of the analysis of a group of bulk cycloheximide preparations are shown in Table 11. Agreement n ith the microbiological assay ( I O ) and with solubility analysis (9) is excellent. The method is rapid, accurate, and precise. Other functional groups capable of yielding hydroxamic acids under the conditions employed (such as anhydrides, acid chlorides, lactones, and esters) constitute positive interferences. High concentrations of carbonyls, transition elements, and ions capable of forming complexes with ferric iron will affect the intensity of the color and should be avoided.
ACKNOWLEDGMENT
The authors are indebted to Thomas Chulski and L. M. Humphrey for the solubility analyses and to J. W. Snyder and associates for the bioassay results. LITERATURE CITED
(1) Bergmann, Felix, ASAL. CHEM.24, 1367-9 (1952). (2) Ford, J. H., Xiomparens, Killiam, Hamner. C. L.. Plant Diseuse Reatr. 42, 680i95 (195s). (3) Ford, J. H., Leach, B. E., J . Am. Chem. SOC.70, 1223-5 (1948). (4) Gpddu, R. F., LeBlanc, N. F., Wright, C. M., AKAL. CHEAI. 27, 1251-5 (1955). (5) Kornfeld, E. C., Jones, R. G., Parke, T. V., J . Am. Chem. SOC.71, 150-9 (1949). --, \ - -
(6) Leach, B. E., Ford, J. H., Whiffen, A. J., Ibid.,69, 474 (1947). (7) Sheppard, Herbert, D'Asaro, B. S., Plumnier. A. J.. J . Am. Pharm. Assoc.. Sci. Ed. 45, 681-4 (1956).. (8) Solowav, Saul, Lipschitz, Abraham, AXAL.CHEM.24, 898-900 (1952). (9) Tarpley, William, Yudie, Milton, Ibid.,25, 121-7 (1953). (10) Whiffen, il. J., J . Bacterid. 56, 28391 (1948).
RECEIVEDfor review October 9, 1958. Accepted January 19, 1959.
S pect rophoto met ric Investigat io n of the Ana Iyt ica I Reagent 1-(2-Pyridylazo)-2-naphthol and Its Copper Chelate BURTON F. PEASE' and MAX 8. WILLIAMS Deportment of Chemistry, Oregon State College, Corvallis, Ore. The dye, 1 -(2-pyridylazo)-2-naphthol, PAN, has been used as a valuable indicator for the titration of a variety of metals. It was suggested as a colorimetric reagent for the determination of zinc, copper, nickel, and cobalt. More information i s needed concerning the dye and some of its metal chelates, because of its rapidly expanding use. The dye i s an acid-base indicator which may exist in three different forms. Absorption spectra of these forms have been obtained at various pH values in water and 2070 dioxane. The dissociation constants have been determined to be 1.26 X and 6 X lO-'3 in 20% dioxane. The absorption spectrum of the copper-dye chelate was obtained at pH 5.0 in 20% dioxane and the existence of only a 1 to 1 complex below pH 8 was verified by Job's method and spectrophotometric titrations. The equilibrium constant for the reaction HKE Cu++ $ CUKE+ H f is 6.4 X l o 3 and the
+
1044
+
ANALYTICAL CHEMISTRY
stability constant for the chelate i s approximately 1 OI6.
of which are important in the analytical chemistry encompassing the use of PAN'. REAGENTS
A
1-(2-pyridylaxo)3-naphtholl PAN, has been introduced by Cheng and coworkers (3-7) as a valuable indicator in complexometric titrations of copper, ziiic, cadmium, and indium solutions with (ethylenedinitri1o)tetraacetic acid (EDTA). The use of this reagent has been extended with good results, by Flaschka and Abdine (10, 11) and others, to a variety of different metals in the micro- and macrotitration range. Cheng and Bray (6) suggested its use as a color-forming agent for the spectrophotometric determination of several metals. The dye is now commercially avaihble. There has been, however, no study of its equilibrium forms, dissociation constants, behavior a t various pH, effect of various solvents, metal chelate ratios, or stabilities, all N ORAKGE-RED
DYE,
1- (2-Pyridylazo)-2-naphthol. The
solid reagent was prepared in this laboratory according t o the procedure of Chichibabin (8, 9). An analysis showed the dye to contain 71.3% carbon, 4.46% hydrogen, and 16.7% nitrogen. The calculated values are 72.4% carbon, 4,45y0 hydrogen, and 16.9% nitrogen. A stock solution of the dye u-as prepared by dissolving 37.4 mg. in 100 ml. of methanol, and later standardized according to the spectrophotometric procedure indicated. Standard Copper Solution. This solution was prepared by dissolving 1.6113 grams of electrolytic copper in 25 ml. of 1 t o 3 nitric acid. The solution 'sas boiled for 20 minutes to eliminate oxides of nitrogen, cooled, and diluted t o 500 ml. with distilled 1 Present address, Shell Oil Co., Martinez, Calif.
