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
the washings failed to show traces of HC1 and then dried over sodium sulfate. The esterification of the nitrobenzoic acids was performed with an excess of diazomethane in a freshly prepared ether solution a t 0" C. The ether was then removed by evaporation a t room temperature and the esters were dissolved in a small volume of diethyl ketone. The resulting solution was used for the gas chromatographic analysis. The same procedure was employed for the isotope dilution analysis, except that radioactive benzoic acid was used and the reaction mixture was poured into an alkaline solution containing suitable amounts of inactive 0-, m-, and p-nitrobenzoic acids as carriers. GAS CHROMBTOGRAPHIC ,1NALYSIS. Figure 1. Modification of chromatoThe resolution of the three isomeric methyl nitrobenzoates was obtained graph outlet for collection of methyl with a 2-meter long, 6-mm. i.d. stainless nitrobenzoates steel column, packed with sodium dodecylbenzenesulfonate (2570 w.,'w.) on 60-mesh Celite C-22. The column phase was dried for several hours at temperature was 205" C. with nitrogen 130" C. Procedure. NITRATIONREACTION. as carrier gas, at' a flow rate of 2 liters per hour. The percentage of a given The nitration was performed accordisomer was determined by the internal ing to Hollemann (4) by slow addition normalization method, dividing the of pulverized benzoic acid to a fivearea of the corresponding peak by the fold excess of nitric acid, d = 1.52, sum of the areas of the methyl nitroMerck reagent grade, cooled at 0" C. benzoate peaks. Since preliminary The temperature of :he acid was kept analyses of synthetic mixt'ures having a in the range of = t l C. by a careful known composition showed that the control of the addition rate and occalibration factor was the same for casional stirring. The reaction mixture the three methyl nitrobenzoates, the was allowed to stand for 15 minutes peak areas were used for the calculation after the addition of benzoic acid had of the percentage without any correcbeen completed, then poured into an tion. excess of 5y0 sodium hydroxide soluISOTOPEDILUTION ANALPSIS. In tion a t 0" C. The solution was view of the similar properties of the acidified with dilute HC1 and extracted isomeric methyl nitrobenzoates-the with ether. The combined extracts 0- and m-isomers boil a t 275" C. and were washed with chilled water until
Table I.
Gas Chromatographic Analysis of Isomeric Methylnitrobenzoates"
No. of detns. o-Nitrobenzoic, yo m-Nitrobenzoic, % 15.4 f 0.3b 83 5 f 0 . 3 5 15.6 f 0 . 5 83.3 i 0 . 5 11 6 15.5 f 0 . 5 83.4 f 0 . 5 Av. value a Because of the small percentage of the ara isomer, its determination could not be accomplished with satisfactory accuracy a n g its percentage is therefore omitted. b Percentage of each isomer has been obtained from area of corresponding peak divided by sum of areas of the three isomers. Preliminary runs showed that the calibration factor for the 0- and m-benzoates is 1.00 f 0.01. Reaction I
Table II.
Isotope Dilution Analysis of Mononitrobenzoic Acids Formed on Mononitration of Benzoic Acid
Yield* of Isomers percentage" mononitrobenzoic Reaction o-Nitrobenzoic m-Nitrobenzoic p-Nitrobenzoic acid, YG 1 1 6 . 8 rfr 0 . 5 82.0 f 0 . 1 1.06 f 0.02 88.3 f 1 . 0 2 17.5 f 0 . 2 81.6 f 1 . 0 1.01 f 0.02 87.4 f 1 . 0 16.6 f 0 . 2 82.0 f 0.8 0.99 f 0.05 87.8 f 1 . 0 3 Mean valuesc 1 7 . 0 f 0 . 7 81.9 f 0 . 8 1.02 f 0.05 87.8 f 1 . 4 a Values represent percentage of a given isomer referred to total amount of mononitrobenzoic acids. Standard deviation listed refers to measurement,s performed on each reaction mixt'ure. b Nitration yield has been obtained by dividing the act,ivity contained in the mononitrobenzoic acids by the total activity of the starting material. c List,ed deviation of mean values indicates overall precision of t,he method, including reproducibilit,y of nitration conditions.
2446
ANALYTICAL CHEMISTRY
1 .
. .
-
U t i m a ( m i n u t e s ) Eo
60
I
Figure 2. Preparative scale gas chromatography of radioactive methyl nitrobenzoates
279 " C., respectively-preparative scale gas chromatography was chosen for the resolution of their mixture. Small aliquots of the esters solution mere injected into the sodium dodecylbenzenesulfonate column and the central portion of the elution peaks was collected. Considerable difficulty was experienced in this operation. I n the early runs, the melting point of the samples collected at the outlet of the chromatograph demonstrated that an extensive mixing of the different fractions occurred, despite the clean separation showed by the detector. Since it was clear that the mixing could only take place in the tubing from the detector to the outlet, unsuccessful attempts were made to avoid the mixing by increasing the temperature and reducing the bore of the tubing. -1 completely different technique had to be used for the collection of the fractions. The tubing was removed and the outlet of the detector block was machined to obtain a female conical joint, as shown in Figure 1. The gases from the column were allowed to escape through the outlet, 6, into the oven. When a peak emerged from the column, the glass trap, 5, consisting of 4-mm. 0.d. borosilicate glass tubing with a male ground joint, was fitted through the guide tube, 4, to the joint, 6. .A reasonably tight connection could be obtained and the base line was not affected by the trap insertion, provided that care had been taken to heat the trap a t the same temperature of the detector block. This could be accomplished easily by keeping the traps, before use, partially inserted in a suitable socket of the chromatograph oven. After the collection of the peak, the trap was withdrawn, different traps being used for different substances. With this technique, the mixing of the fractions was completely eliminated and the unavoidable Contamination of the trapped compounds by the stationary phase was substantially reduced. The isomeric nitrobenzoic esters, collected as described, were subjected to standard purification procedures, in order to remove traces of stationary phase. The purity of the final specimens used for the activity measurements was checked both by gas chromatography and by conventional chemical criteria. The melting points of the m- and p nitrobenzoic acid methyl eiters, for instance, as measured with a Kofler micro heating stage, were 78.5" and 96" C., respectively, in full agreement Kith the literature values.
RESULTS A N D DISCUSSION
An example of the separation of the methyl nitrobenzoates on the sodium dodecylbenzenesulfonate column is given in Figure 2. The dashed area of each peak indicates the portion actually collected in the isotope dilution experiments. The gas chromat'ographic analysis allows the determination of the ortho and meta isomers with reasonable precision, as shown by the results listed in Table I. The percentage of t h r para isomer, that is produced in a much lower yield, is not given in the table. sinre the results of the determinations carried out on different nitration mixtures, or even on the products of different esterifications of the same mixture, are affected by considerable deviations. This indicates that the recovery of the p-nitrobenzoic acid or its conversion to the methyl ester is not constant in the various runs. The combination of the isotope dilution analysis with a gas chromatographic separation overcomes this difficulty and allows determination of all the isomers with good precision, as summarized in Table 11. A comparison of the two tables shows that in the gas chromatographic analysis a lower percentage of o-nitrobenzoic acid and a higher percentage of the ineta isomer are obtained. The discrepancy with the results of the isotope dilution method is not large, while significantly higher than the standard deviation of both techniques. The difference is probably due to the fact
that the solubility in ether of the oand m-nitrobenzoic acids is comparable, while the latter has a much lower solubility in the cold water employed to wash the ether extracts containing the mixture of acids. A crude calculation shows that the lower percentage found in the gas chromatographic analysis can be explained with the higher loss of the o-nitrobenzoic acid in the extraction and washing operations performed. Since the isotope dilution method, aside from its higher sensitivity, is not affected by this kind of error, the results listed in Table I1 should be regarded as more reliable. The results of Table I1 are in surprisingly good agreement with those reported by Hollemann and by Cooper and Ingold ( 2 ) . I n fact, the only significant (30y0)discrepancy with the Hollemann values (0-nitrobenzoic acid, 18.2%; m-nitrobenzoic acid, 80.2%; p-nitrobenzoic acid, 1.3%) refers to the isomer produced in a very low yield. I n addition, the isotope dilution method allows the determination of the total yield of the mononitrobenzoic acids from the nitration reaction (last column of Table 11). X yield of ca. 9Og-/, was obtained in the three reactions carried out. Because in the gas chromatographie and isotope dilution analysis no unreacted benzoic acid was detected, this would suggest that a fraction of the benzoic acid is converted to polvnitrobenzoic acids and/or oxidized by the concentrated nitric acid. Finally, the good. agreement among the results of different runs shows that a satisfactory
reproducibility in the control of the nitration conditions has been achieved. I n conclusion, it seems that the coiiibination of the isotope dilution technique tvit'h a preparative scale gas chromatographic separation offers in many cases substantial advantages over a direct gas chromatographic analysis. I n fact, the results are not affected by the losses suffered by each component in the isolat,ion procedure-losses difficult to predict with the necessary accuracy in many cases. On the other hand, the use of a preparative column makes it possible to collect samples which can be weighed and counted with a highly precise static detector. Therefore, the amount of each coniponent can be determined with a much higher accuracy than could be achieved with a flow activity detector. ACKNOWLEDGMENT
The authors are indebted to Giordano Giaconiello for helpful suggestions and to Elena Ciranni for skilled assistance. LITERATURE CITED
(1) Cacace, F., Cipollim, R., Perez, G., AXAL.CHEM.35, 1384 (1983). ( 2 ) Cooper, K . E., Ingold, C. K., J . C'hem. Soc. 1927: p. 836.
( 3 ) Dauber, %'. G., Reid, J. C., Yankwich, P. E , ASAL. CHEM.19,829 (1947) ( 4 ) Hollemann, A . F., Rec. Trav. Chzm 18, 267 (1899). ( 5 ) Wilebach, K. E., Sykes, I T . K., Sczence 1 2 0 , 494 (1954).
RECEIVED for review January 6, 1964 ilccepted August 4, 1964.
Thin Layer Chromatographic Analysis of Simple AI kyl Phenols G. 8. CRUMP Central laboratories, Shell Research Itd., Whitehall lane, Egham, Surrey, England
b Mixtures of 20 simple alkyl phenols coupled as p-nitrophenylazo dyes have been largely separated into single components on thin layers of silica gel impregnated with alkali. For the best separation of the phenols the two dimensional chromatographic technique was used. The solvent systems were acetone/chloroform followed by di-n-propylamine/benzene. The identification of the phenols was facilitated by the relative positions and characteristic colors of the chromatographic zones. The method has been applied to identification of the phenolic constituents of kerosines. Attention is drawn to the close similarity of the separations of the simple
phenols effected by the above method and by chromatographic separation on papers impregnated with formamide.
S
of phenols have been analyzed by gas liquid chromatography (GLC) but some isomers are not separated ( 4 ) . The paper chromatographic analysis of phenols (uncombined, or as azo dyes formed by reacting them with diazotized amines) is well established, particularly on papers impregnated with amines (2, 6, 6, 10). To date the most successful method uses papers impregnated with formamide and phenols coupled as the MALL QUANTITIES
0- or
p-nitrophenylazo dyes. However, in this instance, the phenols coupled in the ortho position (that is, having alkyl substituents in the 4 position) were not separated. The use of thin layer chromatography for t,he separation and identification of phenolic materials is recorded in the literature (3, 7 : S,22) but does not apply to the separation of mixtures of simple alkyl phenols. This paper describes a method in which mixtures of 20 alkyl phenols, coupled to form 0- and p-nitrophenylazo dyes, are largely separated into single components by two-dimensional chromatography on thin layers of alkali treated silica gel. These dyes were V OL. 36, NO. 13, DECEMBER 1964
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