imposed by the requirement of water solubility for the sample. The use of elevated column temperatures might extend the usefulness of the technique to some acids which are poorly soluble in water a t room temperature. Presently under investigation is the use of nonaqueous and mixed solvents. ACKNOWLEDGMENT
The authors acknowledge the contribution of R. J. Cappell to the early development of the method. A. G.
Birdwell and D. A. Short obtained much of the data reported. LITERATURE CITED
(1) Bauman, W. C., Wheaton, R. M., Simpson, D. W., in “Ion Exchange
Technology,” F. C. Nachod and Jack Schubert, eds., p. 182, Academic Press, New York, 1956. ( 2 ) Buchanan, 11. L., Markiw, R. T., ANAL.CHEM.32, 1400 (196O). ( 3 ) Lingane, J. J., “Electroanalytical Chemistry” 2nd ed., p. 160, Interscience, New York, 1958. (4)Reichenberg, D., Chem. Ind. 1956, p. 958.
( 5 ) Reichenberg, D., Wall, W. F., J . Chem. SOC.,1956, p. 3364.
(6) Simpson, D. W., Bauman, W. C., Ind. Eng. Chem. 46, 1958 (1964). ( 7 ) Simpson, D. W., Wheaton, R . AI., Chem. Eng. Progr. 50, 45 (1954). ( 8 ) Thompson, J. F., Morris, C. J., Arch. Riochem. Biophys. 82, 380 (1959). (9) Wheaton, R. >I.) Chem. Eng. Frogr. 52, 428 (1956). (10) Wheaton, R. >I., Bauman, W. C., Ann. Y . Acad. Sci. 57, 159 (1953). (11) Wheaton, R. &I., Bauman, W. C., Ind. Eng. Chem. 45, 228 (1953). RECEIVEDfor review July 1 Accepted September 24, 1964.
, 1964.
Separation and Identification of Phenols in Automobile Exhaust by Paper and Gas Liquid Chromatography ETHEL D. BARBER, EUGENE SAWICKI, and SYLVESTER P. McPHERSON laboratory o f Engineering and Physical Sciences, Division of Air Pollution, Robert A. Taft Sanitary Engineering Center; Public Health Service, U. S. Department of Health, Education, and Welfare, Cincinnati, Ohio
b A method is described for the separation and identification of simple phenols in automobile exhaust b y paper and gas liquid chromatography. Extended data are given on the separations in Crump’s system on Schleicher and Schuell 2 0 4 0 A and 35 to 40% silica gel-impregnated 966 papers as well as separations on dimethylformamide-impregnated papers in dimethylformamide-hexane. The results of analysis of several samples by paper and gas liquid chromatography are presented.
H
(5)has shown that phenols comprise a significant portion of the compounds found in automobile exhaust. Stanley et al. (8) and Smith ( 7 ) also have shown the presence of phenols in automobile exhaust. Because paper chromatographic methods have been used to identify phenol8 in many types of mixtures, it was considered worth while to attempt a study of phenols in automobile exhaust by this procedure. Of the various procedures tried, the most satisfactory was that of Crump ( d ) , which uses the 0- and p-nitro azo derivatives of phenols for paper chromatography. This study was complemented by gas chromatography of the pure phenols. OFFMANN
EXPERIMENTAL
Spectrophotometric menqurements were made on a Cary ATodel 14 manufactured by Applied P h p i c s Corp. Gas chromatographic analyses were made with a PerkinElmer Model 800 equipped with a Apparatus.
2 442
ANALYTICAL CHEMISTRY
differential hydrogen flame detector. Columns were made of stainless steel. Reagents. Chemicals used, except where otherwise noted, were reagent grade. Phenols were freshly distilled or recryFtallized. Known o-nitroaniline derivatives were prepared according to Crump ( 2 ) . Schleicher and Schuell paper 2040 A g1. and Schleicher and Schuell 35 to 40yG silica gel-impregnated 966 were used for the paper chromatograms. Procedure. Crump’s system (2) of benzene-cyclohexane-dipropyleneglycol (in the ratio of 30:70:3, v./v.) with paper. impregnated with 20y0 formamide was used for separating phenols in which the 4 position is unsubstituted. Phenols in which the 4 position is suhqtituted were qeparated on papers impregnated with 75% acetone-25Yo N,AV-dimethylformamide (9) in the system N,N-dimethylformamidehexane ( 1 ) in the ratio 20:80 v./v. Determination of R , Values. Solution. of pure o-nitrophenylazo dyes of approximately 1 mg. per ml. were chromatographed a t 20’ C. by the procedure of ascending chromatography ( 3 ,4). The spots were visible and could be detected very easily with a white light. Some RI values have been recorded by Crump ( 2 ) for a limited number of o-nitrophenylazo dyes in benzene-cyclohexane-dipropylene glycol on Whatman KO. 1 paper. Table I records results for additional compounds and also presents values obtained for dyes in N,N-dimethylformamide-hexane. Preparation of Automobile Exhaust Samples. Samples of raw automobile
exhaust ranging from 3 to 5 cu. meters in volume were collected from an automobile mounted on a chassi. dynamometer and operated undel
simulated driving conditions. The samples were collected in four Greenburg-Smith gas-type impingers connected in series. Three of the impingers contained 250 ml. of 0 . 1 s to 1.0s sodium hydroxide solution a t ice-water temperature; the fourth was a cold trap. The free phenols were released from the three impingers by adding hydrochloric acid to the sodium hydroxide solution until the solution was acidic. The solution in each impinger was extracted with two 100-ml. portions of chloroform. The fourth impinger was washed with 50 ml. of chloroform. From 50 to 70 ml. of the chloroform extract from each of the three impingers were combined and reacted with 25 ml. of 0.0105.11 diazotized o-nitroaniline. .lfter thorough shaking, the mixture was allowed to stand for 2 minutes and was made alkaline by the addition of 25 ml. of sodium carbonate (2001, by weight) (3). After acidification with dilute hydrochloric acid, the azo dyes were extracted with ether until all color was removed from the aqueous solution. The ethereal chloroform phase was dried over anhydrous magnesium sulfate and evaporated to dryness with a cool stream of air. The azo dye extract was dissolved in chloroform and made to a specific volume. A blank was prepared in the same manner as the sample. Chromatography was conducted as described above. When the samples were collected in cold 1.ON sodium hydroxide solution, two impingers in series collected over 98% of the phenols with 95% in the first impinger. With 0.LV sodium hydroxide solution 9570 of the phenols were collected in the two impingers with approximately two thirds in the first impinger.
Procedure for Extracting Spots. After the samples were studied b y means of paper chromatography, the variouu spots were cut o u t a n d extracted in micro-Soxhlet extractors with 95YG ethanol for 4 t o 6 hours. T h e ultraviolet absorption spectra of the ethanol evtracts were obtained on the spectrophotometer. The component> present in automobile exhaust extracts were finally identified by comparison of their spectra with those of known derivatives. Quantitative results for each of the phenols found in automobile exhaust were calculated by direct comparison with known derivatives a t the wavelengths of their maximum absorption.
Paper Chromatographic Properties of o-Nitrophenylazo Dyes of Phenols Ry_ X 100_ ~ _ ~ Color of azo dyes Before AfF59Substance using Crump's Hexanetreatment treatment S & S 2040A paper system DMF - _ Pale yellow Yellow-orange Phenol 13 Yellow Reddish orange m-Cresol 24 Yellow Reddish orange o-Cresol 31 Orange Purple 97 19 p-Cresol Yellow Orange 69 1.2 o-Methoxyphenol Dk. orange Pale lilac 95 37 2,4-Xylenol Deep orange Pink 54 2.61 2,j-Xylenol Orange Deep pink so 3.6 2,6-Xyleno1 Orange Pink 51 3.1 2,3-Xylenol Orange Deep orange 3,5-Xylenol 37 Orange brown Lilac 3.4-Xvleno 1 98 26 Yellow ' Orange 2lEthi~lphenol 48 Yellow Orange 3-Ethylphenol 38 Yellow Bright pink 31 97 4-Ethylphenol Yellow Orange 51 2,3,5-Trimet hylphenol Orange 7.4 2,3,5,6-Tetramethylphenol Orange Lilac 32 97 2-n-But ylpheno 1 Orange Lilac 37 99 4-n-Butylphenol Brown Lilac 10 96 2-Hydroxydi benzofuran 93 Pink Fades 2-Naphthol 11 Pink Pink 93 1-Naphthol Yellow Lemon 2.0 Salicylaldehyde 85 3.0 p-Hydroxybenzaldehyde 12 2,5-Dichlorophenol Lemon Yellow m-Chlorophenol 18 Lilac 12 Orange 6-llethy1-2-lert-butylphenol Purple Brown 8 99 4-Phenylphenol Pale orange 10 Pale yellow 46 2-Phenylpheno 1 0 Catechol 5.0 Isoeugenol Orange Yellow 2.0 Salicylic acid Orange Yellow 0 m-H droxybenzoic acid 5.0 2,3-Jresotic acid Yellow 2.0 o-Mercaptobenzoic acid Yellow 2.0 1,3-Saphthalenediol Pink 2.0 1,5-Saphthalenediol Pale yellow Deep yellow 6.0 2-Naphthalenethiol
Table I.
I
GAS CHROMATOGRAPHY
Preparation of Column. Three hundred feet of stainless steel tubing (0.2-inch i.d.) were coated b y passing through it 40 ml. of a solution of the liquid phase (lOYo tricresyl phosphate5% orthophosphoric acid) in methanol under a helium pressure of 30 p.s.i. After all of the solution had cleared the end of the column, helium was passed through a t a rate of 10 ml. per minute for a t least 12 hours. The column was further conditioned by heating a t a rate of 0.5' per minute from 50' to 135" C. while purging with helium a t a rate of 10 ml. per minute. The temperature was maintained at 135' C. for several hours until a stable base line was obtained. Calibration of Column. The column mas calibrated by analyzing pure components and synthetic blends over a selected concentration range to establish reproducibility and linearity of detector response. Analysis of Automobile Exhaust Samples. A volume of 50 ml. of the chloroform extract was evaporated t o a final volume of 1 ml. by passing a n air stream across the container a t room temperature. Two-tenths microliter of the concentrate was injected directly onto the column with a Hamilton syringe (1-~1. capacity). The conditions of analysis are: column temperature 135' C; injection port temperature 280' C.; detector temperature 130' C.; carrier (helium) flow rate 30 nil. per minute; hydrogen flow rate 17.1 ml. per minute; air flow rate 400 ml. per minute. The contents of each impinger were analyzed separately in the same manner. The quantities of the various phenols found in automobile exhaust were calculated from the gas chromatograms by comparing their areas to the areas of the corresponding standards. DISCUSSION
AND RESULTS
The data in Table I give a survey of the mean R j values (18 to 20 runs) at 20' C. of the o-nitrophenylazo dyes of phenols in benzene-cyclohexane-dipropylene glycol (Crump's system) both on Schleicher and Schuell 2040 A paper
~~
S & S 966 (35407, silica gel-impregnated) paper 11 Phenol 19 m-Cresol 19 o-Cresol 95 13 4-Cresol o-llethoxyphenol 59 30 2,4-Xylenol 94 2,5-Xylenol 47 2,B-Xylenol 75 2,3-Xylenol 41 3,5-XyIenol 31 17 3,4-Xylenol 95 3-Ethylphenol 30 0 2-Et hylphenol 45 15 4-Ethylphenol 96 1-Naphthol 89 2-Saphthol 91 Salicylaldehyde 79 m-Chlorophenol 11 Deep yellow Pale yellow p-Xitrophenol 3 0 10 2-Hy droxydibenzofuran 91 Salicylic acid 3.0 2.0 m-Hydroxybenzoic acid a Tetraethylammonium hydroxide. and Schleicher and Schuell 35 to 4oY0 silica gel-impregnated 966. I n the presence of other materials the R, values of the smaller phenols are somewhat depressed, while those of the xylenols and larger phenols are not affected. The R, values on Schleicher and Schuell 2040 A are slightly lower than those recorded by Crump ( 2 ) on Whatman S o . 1 paper. The R , values on silica gelimpregnated papers are in general lower than those on Schleicher and Schuell 2040 .A. Silica gel-impregnated papers generally show sharper separations, with more vivid colors, of phenols (with the
exception of 0- and m-cresols) found in samples containing complex mixtures. I n Crump's system ( 2 ) the results indicate a dependency of Rf values of the o-nitroazo phenolic dyes on such factors as steric hindrance around the hydroxyl group, and competition between intermolecular and intramolecular hydrogen bonding. The Rf values of the o-nitroazo phenolic dyes increase in the series phenol, o-cresol, 2-ethylphenol, and 2-n-butylphenol. While the strength of the intermolecular hydrogen bond of the azo phenolic hydroxyl group decreases with the size of the VOL. 36, NO. 13, DECEMBER 1964
a
2443
alkyl group, the steric effect increases. The R, value of the o-nitroazo dye from 2,3-uylenol is higher than that of the dye from o-cresol because of a steric reinforcement effect. The steric effect of the dye from 2,6-sylenol is greater than that found in the dye from ocresol, hence, the R, value is higher. Strong steric effects are found in oriitroazo phenolic dyes from 2-tert-butyl6-methylphenol and 2,3,5,6-tetramethylphenol. The intermolecular hydrogen bond is so weak that these compounds are found near the solvent front. All of the o-nitroazo phenolic dyes formed from the h u b s t i t u t e d phenols are found near the solvent front. This phenomenon is produced by the formation of the intramolecular hydrogen bond between the hydrosyl group and an azo nitrogen atom. Consequently the intermolecular hydrogen bond, which has a tendency to lower the R , value of the o-nitroazophenol, is not readily formed; thus the molecule advances across the paper rapidly. The steric effect a t the azo nitrogen ________
SAMPLE: AUTO E X I U L S T P m
Figure 1 . Gas chroma tog ra phic sepa ra tion of a standard mixtur e o f pure phenols a n d a phenolic fraction from automobile exhaust
-
I
ATTENUATION X 2
could also influence the mobility of the o-nitroazophenols as indicated by the increasing R j values of the azo dyes derived from m-cresol, 3-ethylphenol, and 3,5-sylenol. R , values in hexane-N,N-dimethylformamide also are recorded. Phenols ~
~~
~
(Paper chromatography) Sample no. 3.44 cu. m. of Standard Oil Extane fuel
Total VI1 4.78 cu. m. of Standard Oil Extane fuel
Azo dye, mg 12 0 18 0 11 0
Substance Pheno 1 m-Cresol o-Cresol p-Cresol 3,5-Xylenol 2,3-Xylenol 2.5-Xvlenol
5 5 3 2
Phenol m-Cresol o-Cresol p-Cresol 3,5-Xylenol 2,4-Xylenol 3,4-Xylenol
4 8 3 9
28.2 6.3 3.1 30.6 2.5 5.1
Trace Trace
Pure phenol, mg 4 7 7 5 4 8 2 3 2 6 1 5 1 3
10.9 2.6 1.3 12.9
1.1
Trace
Pure phenol, mg./cu. rn. 1.4 2.2
1.4
0.7
0.8 0.4 0.4 -
2.3
7.3 2.3 0.6 0.3 2.7 0.2
0.5 -
6.6
Total Table 111.
Sample VI11
3.5 cu. m. of
Standard Oil Extane fuel
Phenols Found in Automobile Exhaust
(Gas liquid chromatography) Impinger Impinger I Pure Irnpinger I, 11, plus 11, phenol, Substance mg. mg. mg. mg./cu. m. Salicylaldehydea 0.22 0.22 0.06 2,6-Xylenol 3.69 1.05 Phenol 2.89 0.80 2.49 0.67 0.70 o-Cresol 1.82 1.07 0.31 0.16 p-Cresol 0.91 0.45 2.05 0.59 m-Cresol 1.60 2-Et hylphenolb 0.66 0.19 0.66 2,5-Xylenol 2,4-Xylenol 1.84 0.74 0.53 1.10 2,3-Xylenol 2.62 0.96 0.75 1.66 3,4-Xylenol 1.70 0.49 1.01 0.69 3,5-Xylenol __ __ ___ 16.3 4.7 11.6 4.8 ~
Total Not determined; acetophenone has same retention time, two emerging as one peak. * 2,4-Xylenol, 2,5-xyleno12and 2-ethylphenol emerge as one peak. 5
2444
ANALYTICAL CHEMISTRY
$ OETOClDRTEMP: I W C 6 SAMPLE: SmTHETlC BLEND I SAMPLE S I Z E : 0 2 1LI
Table II. Phenols Found in Automobile Exhaust
V
r)
that show a high R, value and are not separated in Crump’s system are separated satisfactorily in this system. The R, values of p-cresol, 2,4-xylenol, and 3,4-sylenol are slightly higher in mistures of foreign materials than in mistures of pure compounds. The colors of the various dyes also are shown before and after treatment with 5y0 aqueous tetraethylammonium hydroxide. The colors produced with tetraethylammonium hydroside are permanent and become more intense with standing. Such colors are more characteristic of the various compounds than are the color changes produced when the compounds are esposed to ammonia vapors (2). Table I1 lists the phenols in automobile exhaust that were positively identified by the R , values and the spectra of the compounds in neutral solution. Several other materials that produced distinct spots could not be identified. For esaml)le, when larger quantities of sample V were used, spots appeared a t R , 79.0, 84.50, and 87.0. The second and third spots were thought to be 2,6-sylenol and salicylaldehyde, respectively, but could not be identified by spectra. I n sample VI1 unknowns appeared a t R , 45 to 50, 55 to 60, 60 to 66, and 84. These were not identified by color change with tetraethylammonium hydroside or spectra. Table I11 shows the results obtained from the analysis of a similar sample of auto eshaust by gas liquid chromatography. The contents of each inipinger were analyzed for phenols, and the total phenol concentration for the two impingers is given in the fifth column of the table. S o phenols were found in inipinger 3 or 4 by this method. The values have been corrected for loss of phenols by evaporation. Figure 1 (top) shows a typical gas chromatogram of the phenols found in automobile eshaust. The peaks in automobile exhaust are compared with
similar peaks of a synthetic misture of pure phenols (bottom). Several prominent peaks in the automobile exhaust fraction have not been identified. The over-all recovery of the method is from 7 5 to 80%. The total phenol concentrations of the identified phenols are lower than values obtained by Stanley et al. (8). The differences arise because in determination of total phenols the estimations of quantities are based on the absorption of phenol itself rather than on individual components. Other differences occur because this method does not estimate the quantity of unidentified components nor the huge quantity of material left a t the origin. When several automobile samples were pooled and concentrated, the presence of acetophenone was indicated,
first detected from t,he characteristic odor of the exhaust concentrate. Gas chromatographic determinations with either diisodecyl phthalate or tricresyl phosphate colunins showed the presence of a peak that had the same retention time as pure acetophenone. Spectrophotometric studies on the sample in chloroforni or aqueous alkali showed an absorption band a t 253 mp. In concentrated sulfuric acid the band was at 300 nip. Pure acetophenone shows identical properties. Positive results for a compound containing a ---CO---CH9group were obtained with 2,2'-dinitrobiphenyl in S,S-diiiiethylforniaiiiide (6). ACKNOWLEDGMENT
The authors are indebted to Henry Johnson and Myrna Morgan for colecting the samples.
LITERATURE CITED
(1) Borecky, J., Mikrochim. -1cta 5 , 824-9
(1962). (2) Crump, G. B., J . Chromatog. IO, 21-8 (1963). (3) DeJonge,'A. P., Rec. Trav. Chini. 74, 760 (1955). (4) DeJonge, A. P., Verhage, .4.,Ibzd., 76, 221 (1957). ( 5 ) Hoffmann, D., Wynder, E. L., National Cancer Institute, Monograph 9. 91-116 119621. (6) 'Sawicki, ~ E i. o>e~, J., Stanley, T. W,,, Mzlcrochim. Actu 2 , 286-90 (1960). (7) Smith, R. G., lIacEwen, J. E., Barrow, R. E., J . A m . Ind. H y g . iissoc. 20, No. 2, 119 (1959). (8) Stanley, T. Mi., Yawicki, E., Johnson, H., Pfaff, J. D., 145th meeting, American Chemical Society, New York, September 1963. ( 9 ) Sundt, E., J . Chronmtog. 6, 475-80 (1961). RECEIVEDfor review June 8, 1964. Accepted October 5 , 1964.
Gas Chromatographic and Isotope Dilution Analysis of isomers Formed on Mononitration of Benzoic Acid BIANCA ALIPRANDI, FULVIO CACACE, and GIOVANNA ClRANNl Centro Nazionale di Chimica delle Radiazioni e Radioelementi del C.N.R., lstituto di Chimica Farmaceutica e Tossicologica dell 'Universit; di Roma, Italy A technique involving the combination of the isotope dilution method with preparative-scale gas chromatography for the analysis of complex reaction mixtures i s described. An example of application to the analysis of the isomers formed on mononitration of benzoic acid i s given. The results are compared with those from a conventional gas chromatographic analysis.
G
CHROMATOGRAPHY is largely employed for the analysis of complex mixtures of products which often occur in the study of organic reactions, since it offers advantages of sensitivity and convenience that cannot be matched by alternative methods. I n those cases, however, where a preliminary isolation procedure or the preparation of volatile derivatives is required before the mixture can be chrornatographed, the accuracy of the analysis can be impaired by losses affecting to a different extent the recovery of each reaction Iiroduct. The errors may be particularly serious when products formed in ve to be determined T. accuracy, as in the study of many electrophilic substitutions. Another technique frequently employed is the isotope dilution method, whose accuracy deliends only on the purity of the compounds isolated from the reaction mixture, n-hile conililetely indeiwndent of their recovery. In view of AS
these characteristics, it seems that a combination of the isotope dilution method with the fast and efficient separation obtained by gas chromatography should afford a convenient and precise analytical tool. With this technique, the reaction is carried out using a labeled reactive. The radioactive products are diluted with a known amount of inactive carriers, separated by gas chroniatography, and their yields are determined by measuring the specific activity of each fraction. There are obviously two ways to assay the radioactivity of each product after the gas chromatographic seliaration. One is to collect, the fractions leaving the chromatograph and measure their specific activity with a static detector. The second is to assay continuously (concurrently with the mass anal the activity of the effluent vapor>. For the specific liroblein under consideration which requires the highest accuracy in the specific activity determination, the collection of fractions, while tinic consuming, is more convenient, since both the mass and the activity of each saniplc can be assayed with a much higher precikm with a static mea+uremcnt, As an exanilde of alJl,lication of this technique the ]iresc>nt paper tiescribes the analy& of the i ~ ~ n i (f was checked by a radio gas chromatographic apiiaratus already described ( I ) . The inactive samples of benzoic and nitrobenzoic acids were llerck and C. Erba reagent grade, respectively. Their puritywas checked by gas chroliiatography. The benzoic acid carbosyl-Cl' \ v u 1)repared by rarbonating a solrition of phenyl magncsiuin hroniide \vi t h C 1 4 0 2 a t -20" C. ( 3 ) . T h e c.i.iidi8 i'atiioac*tive acid was piiiified hy ~ , q ) c a t t dc,t,y>tallizations and mbliinations, diluted to the required sIiec+ic, activity. a n d its purity was chcvkcd hy radio ga" chroniatogi.alihy. 'I'he statio~laryIJll:L eiii1)loyed for t h P .;rparatioil o f tlic, isoinc,ric nitlobcrizoatc-.)at~~.;I\ 3' olitaincd froiii a commercial soditilii c 1 ~ ~ d c c y l ~ ~ I i t ~ n z c ~ n ~ su If ( 111at e '31n iIC. 'I'hi> c I ' I I d i' prod t i i v t \\-LIS (,Xtl'a('t?d \ V l t h ( ' h ~ ~ J ~ U f ~ J I ' t ti h 1e , estravt dt,ic.tl o v c r . s o ( l i i i i i i + r t l f : i t e , a n d evapoixtcd. Th(7 i , c i t l r t c > wis tii,icd at 130" C. undw vacuwi1 I)oforc l)c,ing adiorlied on ('c,litr. 'l'lic, -t:iti~ii:iry VOL. 36,
NO. 13,
DECEMBER 1964
2445
