The equilibrium constant was determined for propionamide chlorination using Equation 7 with data from 10 different titrations. The titration volume was 120 ml and did not change appreciably during titration. The amounts of amide titrated varied from 0.01 to 0.5 mmole and HC1 concentrations from 0.5M to 3.OM. Both 0.420M and 0.042M hypochlorite titrants were included in the series of 10 titrations. The log,,K value for the chlorination of propionamide computed from these data is 5.0. Standard deviation is 0.2 logl& units. Activity effects have been ignored in these computations. No previous evaluation of this constant was found in the literature. Previous workers (5) have suggested the use of 20 dioxane as solvent for this titration. Our results showed that the dioxane reacts slowly with the chlorine causing unstable end
points and excessively large blanks. It was therefore eliminated in favor of an aqueous solvent system. The need for a closed system was suggested when a chlorine odor was detected above an open titration vessel. Therefore, the sealed system described above was employed. Comparison titrations confirmed that end points in an open vessel yield two to three per cent positive errors. The authors are of the opinion that the causes for most of the one per cent per minute loss of chlorine reported by Post and Reynolds (5) are accounted for in the results presented here. RECEIVED January 2, 1968. Accepted February 15, 1968. This work supported in part by National Science Foundation Undergraduate Research Participation Program (GY-960).
Separation and Identification of Aromatic Carbonyl Compounds as Their 4-Nitrophenylhydrazones by Paper and Thin-Layer Chromatography Ethel D. Barber and Eugene Sawicki U.S. Department of Health, Education, and Welfare, National Center for Air Pollution Control, Cincinnati, Ohio 45226 As A PORTION of a program for the study of aromatic carbonyl compounds, 4-nitrophenylhydrazones were used as suitable derivatives for the identification of such compounds in automobile exhaust by paper and thin-layer chromatography. Fracchia, Schuette, and Mueller (1) have shown the possibility of the presence of benzaldehyde in automobile exhaust by gas liquid chromatography. Although the 2,4dinitrophenylhydrazones have been widely used to identify carbonyl compounds, the 4-nitrophenylhydrazones show many more fluorescent and phosphorescent characteristics (2). In the work described here, the 4-nitrophenylhydrazones were prepared (3), and chromatographed on paper and silica gel in various systems. EXPERIMENTAL
Apparatus. Spectrophotometric measurements were made on a Cary Model 14 and Cary Model 15, manufactured by Applied Physics Corp. Standard chromatographic equipment including plates, applicator of fixed thickness, and developments tanks were obtained from Brinkman Instruments, Inc. Low-temperature fluorescence and phosphorescence of the compounds of glass-fiber paper were examin$d in a Chromato-vue cabinet, which is equipped with 3660A, 2537A, and white-light lamps (Kensington, Scientific Corp.). Reagents. Chemicals were reagent grade except where otherwise noted. Methanol was refluxed and distilled over silver nitrate made alkaline with sodium hydroxide (4). Chloroform was refluxed and distilled over 5 grams of 4(1) M. F. Fracchia, F. J. Schuette, and P. K. Mueller, Environ. Sci. Technol., 1, 915-22 (1967). ( 2 ) E. Sawicki and H. Johnson, Microchem. J., 8, 85-101 (1964).
(3) S. McElvain, “The Characterization of Organic Compounds,”
MacMillan, New York, 1949, p 199. (4) 0. Buyske, L. H. Wilder, P. Wilder, Jr., and M. Hobb, ANAL. CHEM., 28,911 (1956). 984
ANALYTICAL CHEMISTRY
nitrophenylhydrazine and 2.5 ml of glacial acetic acid per liter. The 4-nitrophenylhydrazine derivatives were prepared by the method described by McElvain (3). Schleichter and Schuell paper, 2043 B gl was used for the chromatograms. Silica gel, according to Stahl, manufactured by E. Merck, Darmstadt, Germany, was used for the thin-layer chromatography. Procedure, Paper Chromatography. The 4-nitrophenylhydrazones of the various carbonyl compounds were separated in the following systems: (A) papers impregnated with 35 formamide-65 ethanol by Crump’s system (5) of cyclohexane-benzene-dipropylene glycol in the ratio of 70 :30 :3 VjV; (B) papers impregnated with 50% N,N-dimethylformamide by Schnitt’s system (6) of dibutyl ether-N,Ndimethylformamide-tetrahydrofuran in the ratio of 85 :15 : 4 VjV; (C) papers impregnated in 25 N,N-dimethylformamide-75 % ethanol by Schnitt’s system (6); (D) papers impregnated with 2 0 x formamide-80Z ethanol with cyclohexane-cyclohexane-formamide in the ratio of 15 :12:7. Schulte and Storp’s system (7) of methanol-acetic acid in the ratio of 9.25-0.75 (VjV) was tried, but the results were unsatisfactory. Determination of RF Values. Solutions of pure 4-nitrophenylhydrazones of approximately 1 mg per ml of 9 5 z ethanol were chromatographed at 20” C by the procedure of ascending chromatography (8, 9). The spots were visible and could be detected very easily with white light. The results are recorded in Table I. Low-temperature fluorescence and phosphorescence studies were performed according to Sawicki and Johnson (2).
x
z
( 5 ) B. G. Crump, J. Chromatog., 10,21-8 (1963). (6) W. Schnitt, ANAL.CHEM., 28, 249 (1956).
(7) K. E. Schulte and C. B. Storp, Fette Seifen Anstrichmittel, 57, No. 1, 36-42 (1955). (8) A. P. DeJonge, Rec. Truu. Chim., 74, 260 (1955). (9) A. P. DeJonge and A. Verhage, Ibid., 76,221 (1957).
Thin-Layer Chromatography. Chromatoplates, 20 by 20 cm, were prepared by the method described by Stahl (10) with silica gel G as absorbent using the thickness of 0.25 mm. The plates were activated in 110-120' C for one hour and stored in a desiccator oven indiccating silica gel until used. The chromatographic tanks were lined with filter paper, and equilibrated 4 to 5 hours before the plates were chromatographed. The following systems were employed: (A) dichloromethane, (B) benzene-methanol in the ratio 95 : 5 (VjV), (C) benzene, and (D) ethylacetate-hexane in the ratio of 1 :1 VjV. Determination of Rp Values on Thin-layer Plates. A solution (10-15 pl) of the 4-nitrophenylhydrazones of approximately 1 mg per ml was placed at intervals 1.5 cm from the base of the chromatoplate. The solvent was allowed to travel to a height of 10 to 11 cm. The chromatoplates were examined under white light. The results are recorded in Table 11. Preparation of Automobile Exhaust Samples. Samples of raw automobile exhaust were collected from an automobile mounted on a chassis dynamometer and operated under simulated driving conditions. The samples were collected in 1.ON sodium hydroxide at ice water temperature in three Greenburg-Smith gas-type impingers connected in series. The contents of each impinger were acidified with hydrochloric acid to a pH of approximately 4.0 and extracted with two 100-ml portions of carbonyl-free chloroform (11). From 70 to 80 ml of the chloroform extract from each impinger was combined and refluxed with 0.16 of a gram of 4-nitrophenylhydrazine dissolved in 10 ml of methanol and 7.5 ml of glacial acetic acid for 3 to 4 hours. The resulting solution was evaporated to dryness with a cool stream of air. A blank was prepared in the same manner. Chromatography was conducted as described above. Procedure for Extracting Spots. After the samples were studied by means of paper or thin-layer chromatography, the various spots were either cut out of the paper and extracted in micro-Soxhlet extractors with 9 5 x ethanol for 4 to 6 hours or they were scraped from the plates and extracted with hot ethanol. The extracts were evaporated to an appropriate volume for study of the ultraviolet absorption spectra. The quantities of the 4-nitrophenylhydrazones identified were calculated by direct comparison with known derivatives at their wavelength of maximum absorption. DISCUSSION AND RESULTS
The results in Table I summarize the mean RF values (1020 runs) of 4-nitrophenylhydrazones of aromatic carbonyls and a few aliphatic carbonyls in (A) cyclohexane-benzenedipropylene glycol, in comparison with values found in other systems at 20" C. With system A in the benzaldehyde series, hydroxyl substituents lower the RF values considerably; the hydroxyl substituent in the ortho position shows the least effect. Methoxy substituents likewise lower the RF values, but the effect is less than with hydroxyl substituents. Two methoxy groups show a similar lowering of RF values equivalent to hydroxyl groups. Although anisaldehyde or 4methoxybenzaldehyde cannot be separated from 2-methoxybenzaldehyde in system A, these can be separated in system B. The combination of a hydroxyl and a methoxy group as in the 4-nitrophenylhydrazone of 2-hydroxy-3-methoxybenzaldehyde also results in a lowered Rp value. The 4-nitrophenylhydrazone of acetophenone shows a higher RF value in system A than does the benzaldehyde series, (IO) E. Stahl, "Thin-layer Chromatography," Springer-Verlag, Berlin, Heidelberg, Germany, 1962. (1 1) E. D. Barber, E. Sawicki, and S. P. McPherson, ANAL.CHEM., 36, 2492 (1964).
Table I. Paper Chromatographic Properties of 4Nitrophenylhydrazones Red = reddish, dr = dark, ye1 = yellowish, and, It = light Substance Benzaldehyde Salicylaldehyde rn-Hydroxybenzaldehyde 4Hydroxybenzaldehyde o-Methoxybenzaldehyde Anisaldehyde 2-Hydroxy-3-methoxy benzaldehyde Van i11in Veratraldehyde Piperonal Acetophenone 2-Hydroxyacetophenone 3-Hydroxyacetophenone 4Hydroxyacetophenone 3,4Dimethylacetophenone 2,4Dimethylacetophenone 2,5-Dimethylacetophenone Benzalacetophenone Propiophenone Butyrophenone Benzophenone o-Hydroxybenzophenone m-Hydroxybenzophenone 4Hydroxybenzophenone 2-Hydroxy-5-methyl benzophenone fi-Naphthaldehyde Cinnamaldehyde Furfural 5-Methyl-2-furfural a Smears
A 43 7 4 3
29 27 4 3 8 17 56
RF X 100
B 44 34 24 23 30 26
24 45 35
24
72 86 81
53 50 50 54
76 84 94 89 22 93 6
44 60 58
3
14 47 54 14 34 8 43
8
33
14
40
Color withKOH rust rust
L
2
deeppurple It purple pink purple
3 11
5
9
D 37 6
20
5 5
2 60
C 41 27 12
purple orange purple-redpurple purple
45
43
38 15 13 50 20
7 2 2 lavender 29" deep pink 41 ..
37" 46 50
27
26" rust 46 rust 94 brown 15 72 83 3 deep pink
7
brown
and thus permits the satisfactory separation of benzaldehyde and acetophenone. Hydroxyl substituents produce similar lowering of the RF values as with the benzaldehyde series. In system A, 2-hydroxyacetophenone, 3-hydroxyacetophenone, and 4-hydroxyacetophenone cannot be separated, although they can be separated in system B. Although salicylaldehyde and 2-hydroxyacetophenone display similar Rp values in system B, this pair can be separated in system C. The two compounds when present alone give different colors with fresh alcoholic potassium hydroxide. The substitution of methyl groups on the acetophenone increases the RF values in system A. The position of the methyl group seems to have little effect on the RF values in system B, although it does affect the Rp value in system A. Butyrophenone, with the same number of carbon atoms as 2,4dimethylacetophenone, displays about the same Rp value in system A but shows a different RF in system C. Benzophenone shows higher R p values in system A than do the other ketones studied. o-Hydroxybenzophenone gives two spots in system A. The substitution of hydroxyl groups on the benzophenone lowers the RF value by a considerable amount only when the substitution takes place in the 4-position. All of the 4-nitrophenylhydrazones can be detected with visible light as yellow areas, but they take on characteristic hues from pink to purple when sprayed with fresh alcoholic potassium hydroxide. These colors are listed in the table, The results in Table I1 give a survey of the mean RP values (8-10 runs) of the 4-nitrophenylhydrazones on silica gel in VOL 40, NO. 6, MAY 1968
985
Table 11. Thin-Layer Chromatographic Properties of 4Nitrophenylhydrazones of Aromatic Carbonyls Red = reddish, dk = dark, ye1 = yellowish, and It = light
Substance Benzaldehyde Salicylaldehyde 3-hydroxy benzaldehyde
4Hydroxybenzaldehyde 2-Methoxybenzaldehyde Anisaldehyde 2-Hydroxy-3-methoxybenzaldehyde Vanillin Veratraldehyde Piperonal Acetophenone 2-hydroxy acetophenone
3-Hydroxyacetophenone 4Hydroxyacetophenone 2,4Dimethylacetophenone 3,4Dimethylacetophenone 2,5-Dimethylacetophenone
Propiophenone Butyrophenone Benzophenone o-Hydroxybenzophenone rn-Hydroxybenzophenone 4Hydroxybenzophenone 2-Hydroxy-5-methylbenzophenone (3-Naphthaldehyde Cinnamaldehyde Furfural 5-Methyl-2-furfural
RF X A 58 48 38 7 81
53 33
36 33
55 67 59 14 26 70 72
85 67 73 81
49 89 59 64 24 47 47 19 56 72 63 64
100
B 60 43 28 24 63 72
39 34 54 71 67 46 25 31 78
86 81 67 77 88 53
Silica gelG C D 30 63 23 51 6 39 2 47 21
57
9 2 2
65 41 41 42
34
red-purple purple-red purple-brown red
80
ye1 red
24
red orange red bright red bright red
26
purple
47
85
91
red
50
63
5
33
2
59 67
11
a Colors vary from wet to dry areas. Different solvent on the plates may cause a change in color.
(A) dichloromethane, (B) benzene-methanol, (C) benzene, and (D) ethylacetate-hexane. With system A in the benzaldehyde series, hydroxyl substitutents lower RF value; the hydroxyl substituent in the 2-position shows the least effect. Methoxy substituents attached t o the benzaldehyde produce an elevation of RF values, as is produced with 2methoxy-benzaldehyde, but a slight depression of RF values with the 4- or p-substituent. The combination of hydroxyl and methoxy groups results in a similar lowering of RF values. With the acetophenone group in all systems, a hydroxyl substituent produces a decrease in RE value. The smallest decrease is evident with the ortho substituent in systems A, B, and C, whereas the lowering effect is the same for all hydroxyacetophenones in system D. Only in systems A and B can the ortho, meta, and para hydroxyacetophenones be separated. Substitution of methyl groups elevates the RF value in systems A and B. Thus, in system A, 2,4-dimethylacetophenone and 3,4-dimethylacetophenone cannot be completely separated, while in system B they can be separated. p-Hydroxybenzophenone, o-hydroxybenzophenone, and m-hydroxybenzophenone give multiple spots in system A ; o-hydroxybenzophenone gives two spots in system B. Some aliphatic 4-nitrophenylhydrazones were run for comparison of 4, vaIues. Colors with 10% potassium hy986
ANALYTICAL CHEMISTRY
APPLICATION
pink red-brown red-brown
7 12 27
Color with KOHa red-brown red-brown brown dk purple
droxide were recorded. All spots gave blue colors with 1 ferricyanide-1 % ferric chloride solutions. When aromatic aldehydes were chromatographed on Kieselguhr G impregnated with 10% formamide solution (12) and developed in the system A that was used for paper separations similar to those obtained by chromatography (3, paper chromatography in system A resulted. When basic aluminum oxide and cellulose were tried as absorbents, all R F values were identical.
Benzaldehyde and acetophenone were positively identified by RF value, by ultraviolet spectra, and by fluorescence, and phosphorescence characteristics as components in automobile exhaust. When 2.83 m 3 of indolene gasoline were used, the quantity of benzaldehyde that was found was 1.34 mg. This is equivalent to 0.47 mg/m3. The quantity of acetophenone found was 1.66 mg or 0.59 mg/m3. The quantity of orrho and meta hydroxybenzophenone present (calculated as o-hydroxybenzophenone) was 2.70 mg or 0.95 mg/m3. Propionaldehyde and acetone were present but these were not determined quantitatively. Several other materials that produced spots were not positively identified. For example, a spot that appeared at Rp 84-86 exhibiting maxima in the ultraviolet region at 410361 mp in ethanol and a peak at 276 mp was thought to be 2 4 dimethylacetophenone, but the fluorescence and phosphorescence characteristics were not conclusive enough to positively identify this spot. A spot at an RF value of 79-82 exhibiting maxima in the ultraviolet range at 374, 295, and 275 mp was probably of an aliphatic nature because it exhibited phosphorescence in the solvents employed ( I , 13). Other unidentified spots appeared at Rp 60-65 and RF 34-36. The ultraviolet spectra of the former showed bands at 320 mp and 312 mp. A second automobile exhaust sample, in which 3.5 m3 of Standard Oil extane fuel was used, showed 3.76 mg of benzaldehyde to be present. This is equivalent to 1.07 mg/m3. The quantity of acetophenone found was 3.56 mg or 1.02 mg/m3. Propionaldehyde and acetone were identified qualitatively. Some six other spots, thought to be aromatic in nature, were visible by paper chromatography, but these have not been identified. Although 1.ON sodium hydroxide may not be the most desirable medium for collecting aromatic carbonyls, this investigation shows that some carbonyls may be collected in it. 4-Nitrophenylhydrazine with small quantities of acetic acid added, maintained at ice water temperatures, and processed immediately may prove to be a better collecting system, but care must be taken to prevent decomposition of the 4-nitrophenylhydrazine. ACKNOWLEDGMENT
The authors are indebted to Donald L. Klosterman for collecting the automobile exhaust samples. RECEIVED for review October 16, 1967. Accepted February 15, 1968. Mention of commercial products does not constitute endorsement by the Public Health Service.
(12) G. A. L. Smith and P. J. Sullivan, Analyst, 89, 312-18 (1964). (13) E. Sawicki and J. Pfaff, Microchem. J.,12, 7-25 (1967).
