portion which should be combined with the chloroform effluent collected in a clean beaker. Evaporate off the solTable 1.
Determination of Ester Polymers in Oil
~
Polymer Ethylene-vinyl acetate
F’inyl acetatetallowfumarate-maleate Diethyl aminoethylmethacrylate-Iorol methacrylate
P.p.m. -
_
Added Found 4.0 4.5 1 2 . 0 11.2 20.0 21.3 40.0 40.0 100.0 102.5 8.6 8.0 16.0 16.6 32.0 3 0 . 5 100.0 9 6 . 3 50,O 4 7 . 2 100.0 9 5 . 5
Relative error, _
yo
+-6.6 12.5 +6.5 +2.5
vents from the beaker on a steam bath and dissolve the residue in exactly 2.00 ml. of carbon tetrachloride. Obtain the infrared spectrum of this solution against pure carbon tetrachloride in 1.0-mm. cells. Measure the absorbance of the carbonyl peak a t about 5.8 microns. The calibration curve is then used to obtain the polymer content of the oil. Calibration. Prepare oils containing 10, 20, 50, and 100 p.p.m., respectively, of the polymer additive that is being investigated. Treat each one in the same manner as described above and plot absorbances against the respective concentrations.
One of the advantages of this method is that changes in the composition of the oil will not affect results as they would if a cloud point technique were employed. In addition, nonpolymeric additiveseven ones containing carbonyl and absorbing strongly around 5.8 micronsdo not interfere. The polymer additives separated from oils by this method are recovered in a pure form. The method therefore can be useful also in the identification of polymer additives isolated from oils of unknown origin.
RESULTS AND DISCUSSION
(1) Gaynor, J., Skinner, 9. bl., Talanta 1, 105 (1958).
+7.5
LITERATURE CITED
+3.8 - 5.0
-3.7
-5 . 6 -4.5
Table I gives results obtained on oils containing known amounts of various ester polymers. The relative error in the concentration range of 10 to 100 p.p.m. is less than 8%.
RECEIVED for review August 16, 1963. Accepted November 4, 1963. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1963, Pittsburgh, Pa.
Modification of the Fuiiwara Reaction for Deter minutio n of Polyhalogenated Organic Compounds KENNETH C. LEIBMAN and JAMES D. HINDMAN Deparfment o f Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Fla.
b A sensitive method is described for the determination of some organic compounds containing more than one halogen atom attached to the same carbon atom, The method i s a rnodification of the Fujiwara alkaline pyridine reaction. The product of the Fujiwara reaction is treated with benzidine in formic acid and the resulting color estimated spectrophotomole of metrically. About 5 X trichloroacetic acid or chloral hydrate, or 1.5 X lo-* mole of trichloroethanol, may be assayed. Other compounds containing multiple halogen atoms attached to a carbon atom, except for perhalogenated hydrocarbons, give positive reactions under the conditions described.
0
compounds possessing more than one halogen atom attached to the same carbon atom, including trichloroethanol and its oxidation products and halogenated hydrocarbons such as chloroform and trichloroethylene, have been determined by various quantitative modifications of the reaction of Fujiwara (4). ill1 such methods involve treatment of the sample with pyridine and aqueous alkali. The color produced when this procedure is applied to trichloroacetic acid or chloral hydrate RGANIC
348
ANALYTICAL CHEMISTRY
shows a peak in absorption a t about 530 mp, and this wavelength has usually been employed for estimation of these compounds; such methods have been reviewed by Set0 and Schultze ( 7 ) . Friedman and Cooper (3) developed a more sensitive procedure for the determination of these two compounds by making use of the much higher absorptivity of the Fujiwara reaction product in the near ultraviolet. They were also able to measure trichloroethanol, whose Fujiwara reaction product shows no peak a t 530 mfi, a t its absorption maximum of 440 mp. In preliminary experiments it was found that high absorbance in the near ultraviolet caused by some tissue extracts made the estimation of small quantities of trichloro compounds quite difficult and imprecise in some cases. It was therefore of interest to find a method which could be used for the determination of trichloroethanol and its oxidation products, which would be highly sensitive and in which interference from tissue extracts would be minimal. Feigl (9) has described a spot test for polyhalogen compounds which is a micromodification of the Fujiwara reaction. He noted that the color of the Fujiwara reaction products is discharged by acetic acid, and that the
addition of benzidine then causes the formation of a violet-colored solution or precipitate. The present paper describes a quantitative procedure which has been developed from Feigl’s spot test, which satisfies the requirements outlined above. EXPERIMENTAL
Reagents. Solutions of trichloroacetic acid (reagent) and chloral hydrate (USP) were assayed as described in U. s. Pharmacopeia XVI, but by using more dilute acid and alkali. Certain test compounds (indicated in the text1 were distilled before use, where results with the undistilled material appeared anomalous. All other test compounds were used as supplied. Test solutions were made in water or in 95y0 ethanol. I n the latter case, no more than 0.1 ml. of the alcoholic solution was used, and 1.9 ml. of water was added to make up the test volume. Pyridine (reagent), formic acid (reagent, 8873, and benzidine (Matheson Coleman and Bell, m.p. 127-9’ C.) were used without further purification. Pyridine was purchased in 1-pint bottles, and the contents of each bottle tested for blank reaction upon opening. Pyridine which had been exposed to the atmosphere for long periods of time, and an occasional fresh batch of reagent pyridine, gave high blanks and was
w o v e Lmplh. m y
Figure 1 . Absorption spectra of products formed from 0.0925 Mmole of trichloroacetic acid in various modifications of the Fujiwara reaction 1. Method of Friedman and Cooper II. Procedure A, with substitution of 0.2 ml. of water per ml. of pyridine phase, in place o f benzidine-formic acid reagent. Spectrum determined immediotely thereafter 111. Procedure A, 30 min. after addition of benzidine-formic acid reagent
not used. A satisfiictory batch remains so for a month. Purification of benzidine by the procedure of Bing ( 1 ) resulted in only a very slight increase in sensitivity. For the benzidine-formic acid reagent used routinely, 3 grams of benzidine was dissolved in 887b aqueous formic acid and diluted to 100 ml. with 88% formic acid. This solution was prepared fresh weekly. Procedure. Two procedures are presented here. Procedure A is of general applicabilitj,, whereas Procedure B is useful in certain cases, to be discussed. Procedure A. To a test tube are added in the following order 2.0 ml. of the test sample, 2.0 ml. of pyridine, and 4.0 ml. of 10 molal potassium hydroxide solution, mixing well after each addition. A tube-vibrating device is convenient for intimate contact between the two phases. The tube is placed in a boiling water bath for 3 minutes, then transferred immediately to an ice bath When chilled, 1.0 ml. of the upper pyridine layer is placed in a 10 x 75 mm. round cuvette, 0.2 ml. of the benzidine-formic acid reagent is added, and the solution is well mixed. After 30 minutes standing a t room temperature the absorbance of the solution is retd in a spectrophotometer a t 530 mp against a blank prepared by the above procedure, substituting for the test solution 2.0 ml. of a medium of lJhe same composition, but not containing the test compound. Procedure B. I n a test tube, 2.0 ml. of the test solution is mixed with 4.0 ml. of 10m potassium hydroxide; the tube is placed in a boiling water bath for exactly 2 minutes, after which i t is chilled in ice. Two milliliters of pyridine is then added, the contents of the tube is well mixed, and the tube is replaced in the boiling water bath for 3 minutes. From this point, the procedure is identical with Procedure A above.
The absorbances obtained are compared with those produced in simultaneous experiments made with standard solutions of the test compound. Absorption Spectra. I n Figure 1 are shown the spectra of the pyridine phases produced after subjecting trichloroacetic acid to the Fujiwara pyridine alkali reaction under the conditions of Friedman and Cooper (I) and under the conditions described here (11), and after the addition of benzidine-formic acid reagent to the latter (111). The absorbance at 530 mp of the product of the benzidine reaction is five to eight times that of the Fujiwara reaction product at the same wavelength, and about five times that of the product of the Friedman and Cooper method at 370 mp. Study of the Reaction. Except where noted, the following experiments were performed with trichloroacetic acid as the test substance. Chloral hydrate in general behaved identically in Procedure A. Composition of Alkaline Pyridine Reaction Mixture. The concentration of potassium hydroxide used in the method is not optimal. The sensitivity of Procedure A increased as higher concentrations of alkali were used. However, when concentrations greater than 10m were employed in experiments where tungstic acidtreated tissue extracts mere present, turbidity developed in the pyridine layer which either rexained or caused precipitation when benzidine reagent was added. This did not occur on using 1Om potassium hydroxide, which yielded 92y0 of the absorbance obtained by the use of 20m (near-saturated) potassium hydroxide, when trichloroacetic acid was the test substance. With trichloroethanol, however, the loss in sensitivity was greater; the reaction with 10m alkali yielded only about half the color intensity as did that with 20m potassium hydroxide. Sensitivity of the method increased markedly with decreasing ratio of pyridine volume to sample solution volume. Practical considerations, however, dictated that this ratio not be brought below 1. With this ratio fixed, the optimum volume proportions of sample: pyridine :alkali were found to lie between 1:1:1 and 1:1:3.5. Reaction Times. Heating the sample in alkaline pyridine for 2 to 4 minutes was found t o give the greatest intensity of color after the benzidine reaction. The absorbances a t 530 mp after heating in the first part of Procedure A for 2 or 4 minutes were less than 3% lower than those after a 3-minute reaction time. On heating the sample with alkali and pjrridine for periods of greater than 4 minutes, however, the sensitivity of the method dropped sharply. After chilling, the product of the alkaline pyridine reaction could be kept at room temperature for 30 minutes, or in an ice bath for a t least 2 hours, before adding benzidine reagent to the pyridine layer, without affecting the results.
Concentration of Benzidine (%I
Figure 2. Effect of benzidine concentration in Procedure A with trichloroacetic acid Variables in the Benzidine-Formate Reaction. Figure 2 shows the results of applying Procedurc -1 to aliquots of a solution of trichloroacctic acid using reagents in the final reaction containing various concentrations of benzidine in 887, formic acid. Optimum absorbance was obtained when the reagent contained between 2 and 13% benzidine. Solutions of benzidine in 99+% formic acid yielded lower absorbance in the procedure than when 88% formic acid was used. The solubility of benzidine declines rapidly as 88% formic acid is diluted with water, and saturated solutions of benzidine in more dilute formic acid gave suboptimal results. When glacial acetic acid was substituted for 88% formic acid, the maximum color obtained was almost as intense as when formic acid was used, but faded rapidly. Benzidine is relatively insoluble in hydrochloric acid; a saturated solution in 1M hydrochloric acid gave a suboptimal color. Other amines and related compounds tested gave less satisfactory results. Ammonium acetate, glycine, t-butylamine, diphenylamine, p-aminobenzoic acid, sulfanilamide, p-aminohippuric acid, phenylhydrazine, 1-amino-2-naphthol-4-sulfonic acid, and p-(p-aminophenylazo) benzenesulfonic acid yielded very slight or no color when employed under similar conditions as was benzidine. Aniline, p-toluidine, and ophenylenediamine yielded relatively high absorbances a t their maximum of 495 mp, but did not afford the sensitivity obtained with benzidine. The time course of changes in absorbance at 530 mp after mixing th? pyridine layer from the alkaline pyridine reaction of trichloroacetic acid with the benzidine reagent is shown in Figure 3. The color develops rapidly, 80% of the maximum absorbance being found 30 seconds after mixing. Full development of color, however, requires 30 minutes a t room temperature. The color is stable for about 4 hours after reaching its maximum. After 18 hours a t room temperature, the absorbance had declined to 85% of its maximal value. Effect of Tissue Extracts upon the Method. The presence of tissue extracts in the sample may cause a VOL. 36, NO. 2, FEBRUARY 1964
349
Table 1.
Tlrn.
Inwr,)
Figure 3. Time course of color development after addition of benridineformic acid reagent decrease in the sensitivity of the method. The extent of the change depends upon the tissue present. Thus, a water extract from 200 mg. of rabbit liver acetone powder, deproteinized with tungstic acid, caused a 30% decrease in absorbance in the estimation of trichloroacetic acid, whereas a similarly deproteinized 9000 X gravity supernatant fraction from 500 mg. of rat liver caused a 15% decrease. In the case of trichloroethanol, the decrease was only 5% in each experiment. When quantitative analyses are to be made in solutions containing biological material, therefore, standard solutions made in the same medium should be assayed simultaneously. In addition, the colorimetric blank should be prepared from a solution of the same composition as the assay sample, with the exception of the test compound. Tungstic acid appeared to be the most satisfactory protein precipitant for use in conjunction with this procedure. In most cases, use of perchloric or sulfosalicylic acid resulted in greater lowering of sensitivity. Routinely, protein-containing fluids or homogenates were treated with 0.1 volume each of 10% sodium tungstate and 2N sulfuric acid. Aliquots of the supernatant fluid after centrifugation were then assayed. Specificity and Sensitivity of the Method. I n Table I are listed a series of compounds tested by using Procedures A and B, with the absorbances obtained in each case. I n general, compounds bearing more than one halogen atom linked to a carbon atom give strong positive reactions in Procedure ,4, with the absorbance a t 530 mp greater than 5 per pmole. The chief exception to this rule is the class of perhalogenated aliphatic hydrocarbons. Thus, carbon tetrachloride, hexachloroethane, 1,1,2trichlorotrifluoroethane, and tetrachloroethylene show very little response in this test. Chloralose gives no color, but chloral betaine yields approximately the same color intensity as does chloral hydrate. All compounds bearing but one halogen atom per carbon atom show very little response in this method, except for cis-l,2dichloroethylene and 2-chloroethanol, which yield relatively high (but still less than 1.0) absorbances per pmole. The only compound of those tested which yields as high an absorbance in Procedure B as it does in Procedure A is trichloroacetic acid. Procedure B may therefore be used for 350
ANALYTICAL CHEMISTRY
Compound Ali hatic halides 8hloroform Iodofornib Carbon tetrachloride 1,2-Dichloroethane s-Tetrachloroethane Hexachloroethane 1,1,2-Trichlorotrifluoroethane Vinyl chloride cis-l,2-Dichloroethylened trans-1 ,2-Dichloroethylene6 Trichloroethylene Tribromoethylene Tetrachloroethylene Heptachlor5 Aromatic halides p-Chlorotoluene 2,4-Dichlorophenol Acids Monochloroacetic Trichloroacetic Aldehydes and derivatives Chloral hydrate Chloralose Chloral betaine Alcohols, etc. 2-Chl~roethanol~ 2,2,2Trichloroethanold 2-Chloroethylamine
Specificity and Sensitivity of the Method Maximum Absorbance at 530 mp amount per pmole tested, Sourcea pmoles Proc. A Proc. B R E R R R E
0.05 20.0
C M
0.05
11.1
8.1 0,001
0.12 0.0 0.0
0,002
5.0 0.05 5.0
0.06
7.7 0.006
0.008
36.6~
1 .o
0.014 0.001
0.0
E
2.0
0.69
0.016
E R E E V
20.0 0.1
0.003
0.05 5.0
0.05 9.0 5.8
0.005
3.14
0.24 0.002 1.07
M E
1.0 I .o
0.0 0.0
0.0 0.0
R R
1.0 0.05
14.Of
0.0
0.0 13. If
U F
0.075 20.0
13.OJ 0.002
I
0.03
0.0 0.033 0.003
E
1.o
0.70
0.0
A A
20.0
0.25
4.0f 0.017
0.0 0.0
0.1
14.8
0.0
0.016
0.0
R, reagent grade chemical; U, USP chemical; A, Aldrich Chemical Co.; C, California Cor . for Biochemical Research; E, Eastman Organic Chemicals; F, Fisher Scientific s o . ; M, Matheson Corp. (vinyl chloride) or Matheson CoIeman & Bell; VI Velsicol Chemical Co. * Practical grade. Ethanol solution assayed by method of Maemoto et al. (6). * Freshly distilled. e 1,4,5,6,7,8,8-He tachloro-3a,4,7,7a-teetrahydro-4,7-methanoindene.The sample contained about 90% Reptachlor. J Standard curve6 are linear a t least to absorbance of 1.0, and pass through the origin. Standard deviations: trichloroacetic acid, procedure A; 0.22; procedure B, 2.5; chloral hydrate, 0.12; trichloroethanol, 0.13. 0 Solution prepared by dissolving tablet of Beta-Chlor (Mead Johnson Labs).
the estimation of trichloroacetic acid in the presence of alkali-labile polyhalides. Procedure B, however, is less precise than is Procedure A. In the case of heptachlor, the result of Procedure B is about one third that of Procedure A. Absorbances between 0.06 and 0.07 at 530 mp are obtained in Procedure A with 5 mpmoles of trichloroacetic acid or chloral hydrate or with 15 mpmoles of trichloroethanol. DISCUSSION
The procedures described here afford quite sensitive means of estimating several polyhalogenated compounds. Optimal conditions were worked out for trichloroacetic acid in biological ma-
terial. For the other compounds shown in Table I, modifications of the method may well yield more sensitive estimations. Indeed, the compounds which gave negative results by this procedure might be assayed by suitable modifications of the method; thus, the Fujiwara reaction has been used for the estimation of carbon tetrachloride (6). Feigl (2) suggested that the products of the Fujiwara reaction are Schiff bases of glutaconic aldehyde, formed by ring opening of the pyridinium adducts formed between pyridine and the polyhalogenated compounds. On hydrolysis of the original Schiff bases and treatment with benzidine, the same derivative should be formed in all cases. The colors of the products formed from
.
various polyhalides in the standard Fujiwara alkaline-pyridine reaction differ greatly; chloroform, chloral hydrate, and trichloroacetic acid yield a magenta color in the pyridine phase, and the polyhaloethylenes aff 3rd an orange to salmon color, while in the case of the haloethanols, the pyridine layer is colored a bright yellow. After treatment with the benzidine-formic acid reagent, however, the color is a clear red and appears the same in all cases. Although the spectra of the Fujiwara reaction products of trichloroacetic acid and trichloroethanol are quite different, those of the red compound formed after treatment with benzidine are identical. In some cases, the color of the Fujiwara
reaction product may be very pale, yet an intense red color will be developed after benzidine reagent is added; thus, with some compounds, the increase in optical density is much greater than in the case of trichloroacetic acid as shown in Figure 1. The results therefore favor a hypothesis calling for different intermediate products from different halogenated compounds in the first part of the reaction, and a common product in the second step. ACKNOWLEDGMENT
The authors thank Elsa Ortiz and Richard Julius for technical assistance in various phases of this work.
LITERATURE CITED
(1) Bing, F. C . , J. Bid. Chem. 9 5 , 387 (1932). (2) Feigl, F., "Spot Tests in Organic Analysis," 5th ed., p. 313, Elsevier, Amsterdam, 1956. (3) Friedman, P. J., Cooper, J. R., ANAL. CHEM.30, 1674 (1958). (4) Fujiwara, K., Sitz. Nut. Ges. Rostock 6, 33 (1916); C . A . 1 1 , 3201 (1917). (5) Habgood, S., Powell, J. F., Brit. J. Ind. Med. 2, 39 (1945). (6) Maemoto, K., Seike, N., Hirata, M., Kobunshi Kagaku 1 5 , 660 (1958); C.A. 5 4 , 14775 (1960). (7) Seto, T. A., Schultze, M. O., ANAL. CHEM.28, 1625 (1956).
RECEIVED for review September 6, 1963. Accepted October 28, 1963. Supported in part by a Public Health Service grant (OH-00124).
Compositiion of the DinucIear Aromatics, C12 to in the Light Gas Oil Fraction of Petroleum
CIA,
BEVERIDGE J. MAlR and T. JACK MAYER Petroleum Research laboratory, Carnegie Institute o f Technology, Pittsburgh 7 3, Pa.
b A substantially complete analysis in terms of the amounts of the individual components in the dinuclear aromatic portion of petroleum boiling in the range from 2-55' to 275" C. was achieved. Nine of the ten possible dimethylnaphthalenes, two ethylnaphthalenes, two methylethylnaphthalenes, two n-propylnaphthalenes, biphenyl, three methylbiphenyls, a methylacenaphthene, and some polymethylbiphenyls were isolated or identified. Small amounts of benzothiophenes were also present. The compounds were isolated principally by preparative scale gas liquid chromatography and identified by mass, nuclear magnetic resonance, and infrared spectrometry.
this research illustrates the application of modern physical separation processes and spectroscopic methods of identification to the analysis of a very complex mixture of hydrocarbons. PREPARATION
Figure 1 shows schematically the procedure used to separate the dinuclear aromatics, 230" to 305' C., from the original petroleum. The entire light gas oil fraction comprising 17y0 of the original petroleum was separated by adsorp-
AMERICAN PETROLEUM INSTITUTE
T
research is part of a continuing investigation of the composition of the reference petroleurn of the American Petroleum Institute Research Project 6. Analysis in terms of the amounts of the individual components in the dinuclear aromatic portion of petroleum boiling in the range from 2515' to 275'C. was substantially complete. Nine dimethylnaphthalenes, two ethylnaphthalenes, two methylethylnaphthalenes, two npropylnaphthalenes, biphenyl, three methylbiphenyls, a methylacenaphthene, and some polymethylbiphenyls were isolated or identified, as well as small amounts of benzothiophenes. In addition to yielding basic information concerning the compositionof petroleum,
OF STARTING MATERIAL
HIS
tion to give an aromatic portion and a paraffin-cycloparaffin portion (11). The aromatic fractions from this operation were blended according to their refractive indices; those with refractive indices, n'D" in the range from 1.590 to 1.604 (lot D, Figure 1) comprised the dinuclear aromatics. Results of the distillation of this material, in columns of high separating efficiency, a t 30 mm., are given in Figure 2. The fractions from this distillation were blended into seven portions as shown. This investigation is concerned with the composition of part of portion 1and of portions 2,3, and 4. Except for portion 2, the first step consisted of an azeotropic distillation. Portion 2 contained solid material which was separated prior to the aaeotropic distillation. PROCEDURE
C,I TO Ctrl2XT-305%.
PARAFFINS AND OYCLOPARAFfIN8
AROMATICS
19.6% ~
BLENDED ACCORDIN0
INDEX
-ACTIVE
KWON
1
RIITKN
2
p a m c ~WRTION
3
4
R~ON mnw
5
6
Figure 1. Schematic diagram showing origin of dinuclear aromatic material used in this investigation
Selected azeotropic distillate fractions were separated by preparative scale gas liquid chromatography using polar and nonpolar columns, supplemented in some instances by crystallization, to give individual compounds or relatively simple mixtures. The individual compounds and the components of the simple mixtures were identified by low voltage mass, infrared, and nuclear magnetic resonance spectrometry. The amounts of the individual components were determined by analytical gas liquid chromatography supplemented, where necessary, by mass, nuclear magnetic resonance, and infrared spectrometry. VOL. 36, NO. 2, FEBRUARY 1964
351
