Table 11. Effect of Varying Activity of lSsWAdded Activity added (cpm) Activity extracted (cpm) 23,300 978 46,600 1516 69,900 2578 93,200 2975 117,000 3597 140,000 4732 163,000 543 1 186,000 5845 210,000 6219 233 ,ooO 7303 7835 256, 000 280, 000 8678 Slope = 0.0297 count extracted/count added.
ous butanol-ether mixtures (3, ethyl acetoacetate (6),ethyl acetate (7), butyl acetate (8), 2-methyl-1-propanol (9), 3methyl-1-butanol (IO), 1-butanol in chloroform ( I I ) , and 1octanol(I2). All of these were investigated and it was found that the most complete extraction, combined with the smallest extraction of interferences, and the smallest volume of organic reagent needed was obtained with 2-octanone, which has a very low solubility in water. (This consideration is important because it is desirable to count the entire organic phase.) With this extractant, efficiency of extraction as measured with a2Pwas about 85 %’ in the ppb phosphate range, using 3 ml of 2-octanone per 100 ml of water sample. Thus, the distribution coefficient is of the order of 200 for tungstomolybdophosphoric acid. (5) C. Wadelin and M. Mellon, ANAL. CHEM., 25, 1668 (1953). (6) K. Z. Stoll, ibid.,11, 81 (1938). (7) J. Hure and T. Ortis, Bull. SOC.Chim. Fr., 1949,834. (8) T.Koto, et a/., TecRiio/.Repts. Tohoku Unia. 15 (l), 70 (1950). (9) C. Sideris, ANAL. CHEM., 14,762 (1942). (10)C. Rainbow, Nature, 157,268 (1946). (1 1) K. T. H. Farrer and S. J . Muir, A m . Chem. Zizst. J. and Proc., 11, 222 (1944). (12) F. L. Schaffer et al., ANAL. CHEM., 25, 343 (1953).
The pH was kept in the range 0.9-1.2 which was found to be optimum for the formation and extraction of molybdophosphoric acid into 2-octanone. The exact nature of the tungstomolybdophosphoric complex is uncertain. In the procedure, the molybdophosphoric acid is formed first and 185Wis then added. A study of the effect of the amount of tracer added on the activity of the extracted complex was made and the data are presented in Table 11. These data show that the amount of la5W activity in the final sample varies with the amount of lSsW tracer added in a continuous, linear manner. The slope of the line is 0.0297. Evidently, molybdenum atoms (or molybdate ions) are replaced by tungsten (or tungstate) in a continuous manner. Considering the various steps involved in the deterrnination, several parameters are critical, The concentrations of molybdate and tungsten must be uniform for all samples because the extent to which the displacement reaction occurs is dependent on these concentrations. Another consideration is the contamination of reagents with phosphate, which affects the results even more markedly if the amounts of reagents are vaned. The extraction step must be carried out with care, and volumes of aqueous and organic phases must be kept uniform because a difference of 0.1 ml of organic extractant will affect the extraction efficiency by almost 2%’. The length of time that the phases are allowed to separate before they are drawn off must be kept uniform for good results. The method was used successfully for phosphate concentrations ranging from 10 ppb to 100 ppm. Substances which normally interfere in phosphate determination are atoms which form heteropoly complexes with molybdate similar to molybdophosphoric acid. These are Ge, As, Si, and Nb and, with the exception of Ge, it was found that they interfered in this method to about the same extent as they do in the common spectrophotometric methods. NO interference from Ge was observed. RECEIVED for review July 11, 1968. Accepted November 21, 1968.
Spectrophotometric Determination of Primary Aromatic Amines with 9-Chloroacridine James T. Stewart, Terry D. Shaw, and Anthony B. R a y Department of Medicinal Chemistry, School of Pharmacy, The University of Georgia, Athens, Ga. 30601 THEREACTION of organic amines with 9-chloroacridine to give highly colored 9-aminoacridine hydrochlorides has been reported by Burckhalter et al. ( I , 2). There are, however, no reported examples of the quantitative determination of amines with 9-chloroacridine. In this paper we describe a new spectrophotometric method for determining small quantities of some primary aromatic amines with 9-chloroacridine. The sensitivity of the method applied to certain amines rivals that of the commonly used diazotization-coupling procedures. EXPERIMENTAL
Apparatus. Spectra and absorbance measurements were made with a Perkin-Elmer Spectrophotometer, Model 202, (1) J. H. Burckhalter et a / . ,U. S. Patent 2428355 (1947). (2) J. H. Burckhalter et a / . ,U. S. Patent 2419200(1947). 360
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and a Beckman Spectrophotometer, Model DU. Matched cells with a 1-cm optical path were used. Reagents and Chemicals. Aniline, p-nitroaniline, p-bromoaniline, 2-nitro-4-methoxyaniline, o-aminophenol, o-phenylenediamine, p-aminophenol hydrochloride, and 9-chloroacridine, all Eastman grade, along with p-methoxyaniline and p-aminophenol, both practical grade, were obtained from Eastman Kodak Co. 2,4-Dimethoxy-5-chloroanilinewas obtained from Pfister Chemical Works, Ridgefield, N. J. and p-aminophenylmercaptoacetic acid was obtained from Evans Chemetics, Inc., Waterloo, N. Y . All other chemicals used were the highest grade of the commercially available materials. Fresh solutions (10-6 mole/ml) were prepared daily by dissolving weighed amounts of the amines in ethanol. Solutions of 9-chloroacridine (10-6 mole/ml) were prepared immediately before use by dissolving weighed amounts in ethanol.
The hydrochloride salts of 9-phenylaminoacridine, 9-panisylaminoacridine and 9-p-nitrophenylaminoacridinewere synthesized by means of the analytical procedure described herein for the purpose of monitoring the reaction quantitatively. Each compound was freed from its hydrochloride salt with aqueous sodium hydroxide solution and the melting points of the freed bases were verified from the literature (3). Procedure for Determining Primary Aromatic Amines. One milliliter of an ethanolic solution of 9-chloroacridine mole/ml) was placed in a 10-ml volumetric flask. To this was added 1 ml of an ethanolic solution of amine (10-6 mole/ml). If the amine was present as a hydrochloride salt, 1 ml of an aqueous solution of potassium bicarbonate mole/ml) was added to free the amine. Then the pH was adjusted to approximately 4 with 10% v/v aqueous hydrochloric acid. The solution was shaken for 5 minutes at room temperature, followed by the addition of ethanol to volume and the absorbance measured at 435 mp. Absorbance measurements were corrected for reagent blanks in the procedure. RESULTS AND DISCUSSION
Reaction Involved and Influence of Chemical Structure.
In this analytical procedure, 9-chloroacridine reacts with a primary aromatic amine to yield a highly-colored-orange product. Chemically this colored product is a 9-aminoacridine hydrochloride. The absorption curve in the visible spectrum for a typical sample of aniline shows an absorption maximum at 435 mp. Reagent blank readings at this wavelength are very low. In comparing absorption curves of the colored solutions obtained with equimolar concentrations of the various primary aromatic amines, it was noted that the curves were almost identical. Compounds such as o-aminophenol, o-phenylenediamine, p-methoxyaniline, p-aminophenol, p-aminophenol p-aminophenylhydrochloride, 2,4-dimethoxy-5-chloroaniline, mercaptoacetic acid, p-bromoaniline, and 2-nitro-4-methoxyaniline all produce color which absorbs at the same wavelength maximum and with essentially the same intensity as does aniline. Structurally these compounds contain various substituents in addition to a primary amino function. p-Nitroaniline showed somewhat less intense absorption than the other amines at 435 mp, presumably because the reaction between 9-chloroacridine and p-nitroaniline was shown to result jn only a 3 6 . 7 z yield of product under the conditions of the analytical determination (see Table 11). Other nucleophiles besides primary aromatic amines present in the solutions are probably reacting with 9-chloroacridine to affect the less-thanquantitative yields of the desired products. Standard curves can be prepared by plotting observed absorbance readings cs. the volumes taken of equimolar concentrations of various amines. In all cases Beer’s law holds for this system. Quantitative data from several systems shown in Table I reveal that use of this procedure permits the determination of primary aromatic amines in the presence of primary, secondary, and/or tertiary aliphatic amines, secondary and tertiary aromatic amines, heterocycles, and carbonyl-containing compounds. Generally, secondary aromatic amines will not give colored solutions. A few secondary amines have been found which react with 9-chloroacridine to yield a highlycolored solution. However, these solutions exhibit absorption maxima at wavelengths other than 435 mp and conse(3) A. Albert, “The Acridines,” 2nd ed., St. Martin’s Press, New
York, 1966, p 297.
Table I. Analysis of Known Primary Aromatic Amine Mixtures for Primary Aromatic Amine
Primary aromatic amine Components, concn of Found, mole of Mixture 2.500 X 10-8 mole ml-l ml-1, X 10-8 theory 1 Aniline 2.495 99.8 n-Butylamine N-Methylaniline Morpholine 2 p-Methoxyaniline 2.500 100.0 Di-n-butylamine Salicylic acid p-Dimethylaminobenzaldehyde 3 p-Nitroaniline 2.500 100.0 Quinoline Triethylamine N,N-Dimethylaniline Table 11. Absorbance Readings of Reaction Products Formed during the Analytical Procedures before and after Heating for 15 Minutes Compared to Pure Reaction Products at 435 mp
Absorbance Concn,
Pure Analytical reaction syntheml-l, Before After sized X 10-8 heating heating product mole
Amine Aniline p-Methoxyaniline p-Nitroaniline
2.5 2.5 2.5
0,230 0.240 0.165
0.230 0.240 0.165
0.260 0.300 0.450
Yield 88.5 80.0 36.7
quently do not interfere with the determination of primary aromatic amines. Any tertiary amine formed from the reaction between 9-chloroacridine and a secondary aromatic amine should be readily hydrolyzed to acridone because of loss of resonance stabilization occasioned by the steric effect of hydrogen atoms in the 1 and 8 positions of the acridine nucleus (4). Acridone should then be the only source of interference from secondary aromatic amines, and it has been shown in this laboratory to have very low absorbance at 435 mp. Any problem concerning overlap in absorption curves for a solution of a primary and secondary aromatic amine at 435 mp can be overcome by the use of simultaneous spectrophotometric analysis because there is a significant variation in absorption maxima for the two species. Further work on the color reaction and identification of the product formed between these secondary amines and 9-chloroacridine is underway in our laboratory. Color Development and Stability, The analytical method is essentially a micro procedure, and sensitivity is in the range of to lo-* mole ml-1 of amine, which makes it comparable to other amine determinations, particularly the popular diazotization-coupling procedures. In the original synthetic procedure it was suggested that the amine and acridine be heated on a steam bath for 15 minutes to ensure a more complete reaction. This did not prove to be necessary in the analytical procedure because maximum color development, and hence yield of product, could be obtained upon shaking the solution at room temperature for 5 minutes. A comparison of absorbance readings of analytical solutions containing aniline, p-methoxyaniline, and p-nitroaniline, before and after heating on a steam bath for 15 minutes, to the respective aminoacridine hydrochlorides at pH 4 showed that the yield of product was not improved sig(4) Ibid., p 287. VOL. 41, NO. 2, FEBRUARY 1969
361
nificantly by heating. These results can be seen in Table 11. Repeated readings on a series of different samples indicated good color stability for periods up to one week. Solutions of 9-chloroacridine must be prepared immediately before use because the acridine undergoes rapid ethanolysis in ethanol (5). Velocity constants of the nucleophilic replacement of the chlorine atom by ethoxide ion in 9-chloroacridine and other chloroheterocycles have been studied by Chapman and Russell-Hill (6). These solutions are permissible to use for approximately one-half hour after preparation. Decomposition can be seen by the appearance of a brownish-colored precipitate. Two batches of powdered reagent, both obtained (5) A. Albert, “The Acridines,” 2nd ed., St. Martin’s Press, New York, 1966, p 254. (6) N. B. Chapman and D. Q. Russell-Hill,J . Chem. Soc., 1956, 1563.
from Eastman Kodak, were utilized during the course of the research and behaved the same in their reactions with primary aromatic amines. No special precautions had to be taken in the storage of 9-chloroacridine powder even though the literature states that decomposition will occur unless stored over potassium hydroxide at 4 “C (5). In summation, spectrophotometric measurements with 9-chloroacridine provide a relatively simple and rapid means of determining primary aromatic amines in the presence of aliphatic amines and secondary and tertiary aromatic amines. It is comparable in its sensitivity to diazotization-coupling techniques and possesses the advantage of simplified reagent preparation. RECEIVED for review September 23, 1968. Accepted November 18, 1968. Investigation supported in part by the Office of General Research, University of Georgia.
Identification of Alkyl Aryl Sulfides in Wasson, Texas, Crude Oil R. L. Hopkins, R. F. Kendall, C. J. Thompson, and H. J. Coleman Bartlesville Petroleum Research Center, Bureau of Mines, U.S. Department of the Interior, Bartlesville, Okla. THERE is ample evidence that thiols (1-3), sulfides (1, 4 4 9 , thiophenes ( I , 9-12), benzothiophenes (13,14), and thiaindans (15) are present in crude oils. However, prior to this investigation no alkyl aryl sulfide had been identified in any crude oil. This paper describes the isolation and the positive identification of (2-methyl-1-thiabutyl)benzene (phenyl sec-butyl sulfide), the tentative identification of three other alkyl aryl sulfides, and establishes for the first time the presence of this class of sulfur compounds in petroleum.
(1) S. F. Birch, J. Ins!. Petrol., 39, 185-205 (1953). (2) H. J. Coleman, C. J. Thompson, R. L. Hopkins, and H. T. Rall, J . Chem. Eng. Data, 10,80-4 (1965). (3) D. Haresnape, F. A. Fidler, and T. A. Lowry, Ind. Eng. Chem., 41, 2691-7 (1949). (4) J. S. Ball and H. T. Rall, Proc. Am. Petrol. Inst., Sect. III, 42, 128-45 (1962). (5) H. J. Coleman, N. G. Adams, B. H. Eccleston, R. L. Hopkins,
Louis Mikkelsen, H. T. Rall, Dorothy Richardson, C. J. Thompson, and H. M. Smith, ANAL.CHEM., 28, 1380-4 (1956) (6) C. F. Mabery and W. 0. Quayle, J . SOC. Chem. Ind., 19, 505-6 (1900); Am. Chem. J., 35,404-32 (1906). (7) C. J. Thompson, H. J. Coleman, R. L. Hopkins, and H. T. Rall, J . Chem. Eng. Data, 9, 473-9 (1965). (8) Ibid., 10, 279-82 (1965). (9) S. F. Birch, T. V. Cullum, R. A. Dean, and R. L. Denyer, Ind. Eng. Chem., 47,240-9 (1955). (10) C. J. Thompson, H. J. Coleman, Louis Mikkelsen, Don Yee, C. C. Ward, and H. T. Rall, ANAL.CHEM., 28, 1384-7 (1956). (11) C. J. Thompson, H. J. Coleman, C. C. Ward, and H. T. Rall, J . Chem. Eng. Data, 4, 347-8 (1959). (12) H. J. Coleman, C. J. Thompson, R. L. Hopkins, and H. T. Rall, J . Chromatog., 20, 240-9 (1965). (13) H. J. Coleman, C. J. Thompson, R. L. Hopkins, N. G. Foster, M. L. Whisman, and D. M. Richardson, J . Chem. Eng. Data, 6,464-8 (1961). (14) F. P. Richter, A. L. Williams, and S. E. Meisel, J. Am. Chem. Soc., 78, 2166-7 (1956). (15) C. J. Thompson, H. J. Coleman, R. L. Hopkins, and H. T. Rall, ANAL.CHEM., 38,1562-6 (1966).
362
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EXPERIMENTAL PROCEDURES Preparation of Concentrate. The origin of the 200 to 250 “C distillate and of the thiaindan concentrate, in which the alkyl aryl sulfides were found, has been described in a previous publication (15). Referring to the chromatogram of Figure 4 of that publication (15), material producing the chromatographic peak at about 28 minutes retention time was the object of this investigation. Identification of Alkyl Aryl Sulfides. Most of the material producing the peak at 28 minutes was identified using data from mass and infrared spectrometry, gas-liquid chromatography (GLC), and microdesulfurization. The mass spectrum of the material under investigation indicated an intense molecular ion at m/e 166, two units higher than the 164 ion for the 2,2-dimethyI-l-thiaindan, which had been identified (15) as the principal component of the material emerging from the GLC column at 36 minutes. A parent mass of 166 could result from several classes of sulfur compounds-namely, cycloalkylthiophenes, dithienyls, alkyltetrahydrobenzothiophenes, alkylbenzenethiols, and alkyl aryl sulfides. The cycloalkylthiophenes were eliminated by GLC retention time data and/or spectral evidence. The 2- and 3-cyclohexylthiophenes have retention times 4 to 10 minutes beyond the trapped area on both the polar and nonpolar columns. The disubstituted thiophenes (methyl cyclopentyl) of molecular weight 166 were eliminated by infrared spectral data. 2,5-Disubstituted thiophenes have a very strong band at 12.6 p (16). All other disubstituted thiophenes have a medium to strong band between 11 and 12 p (17). Absorp(16) Dorothy M. Richardson, Norman G . Foster, Barton H. Eccleston, and Cecil C. Ward, U. S. Bur. Mihes Rept. Imest. 5816,22 pp (1961). (17) John F. Zack, Jr., I. Synthesis of Compounds Containing
Condensed Thiophene and Pyrrole Rings. 11. Spectra Studies of Thiophene Derivatives. Ph.D. Thesis, Univ. of Illinois, Champaign-Urbana, Ill., 1956.
