Paper Chromatographic Identification of Carbonyl Compounds as Their 2,4-Dinitrophenylhydrazones in Automobile Exhaust ETHEL D. BARBER and J. P. LODGE, Jr. Division of Air Pollution, Robert A. Tuft Sanitary Engineering Center, Cincinnati 26, Ohio
b A method i s described for the separation and identification of carbonyl compounds in automobile exhaust by paper chromatography. Extended data are given for separations in a modified Meigh phase as well as for limited separations in DeJonge’s system. Although homologous aldehydes are completely separated, ketones show higher Rf values than aldehydes of the same carbon number. The results of analysis of several samples are presented.
A
the many components that have been cited as being present in automobile exhaust are aldehydes and ketones. Various methods have been employed for the purpose of identifying these materials. Because paper chromatographic methods h a r e been used to identify carbonyls in many types of mixtures as their 2,4-dinitrophenylhydrazones (6, 11, 12, 16-20, 23, 27), a study of oxygenated materials in automobile exhaust by this method was attempted. Various chromatographic systems were tried; the system that proved to be the most satisfactory was that first used by Rleigh (17, 18) and modified by Wallgren and Kordlund (27), and for our use adapted to ascending chromatography. MONG
EXPERIMENTAL
Apparatus. S p e c t r o p h o t o m e t r i c measurements were made on a Carp Model 11 manufactured b y iipplied Physics. Reagents. Chemicals used, except where otherwise noted, were reagent grade. Chloroform and ether were refluxed over 5 grams of 2,4-dinitrophenylhydrazine and 1 gram of trichloroacetic acid per liter and redistilled (9). Benzene was refluxed over 2,4-dinitrophenylhydrazine. Hydrochloric and sulfuric acids were diluted with distilled water. Known 2,4-dinitrophenylhydrazone derivatives were DreDared bv the method of Brady (4) or k l l i n (1). ” Schleicher and Schull paper 2043B and Whatman No. 1 paper were used for the paper chromatogi-aphs.
348
ANALYTICAL CHEMISTRY
Procedure. Meigh’s system of methanol-heptane as modified b y Wallgren and Nordlund (27) contained 10% of glacial acetic acid (methanol99% heptane in ratio of 1 : 4 v./v.), which was added after the papers were equilibrated in the two-phase system through a hole approximately ’/4 inch in diameter in the top of a center glass covering the chromatographic jar, so that the papers could be developed by the method of ascending chromatography (8). DeJonge’s system M ( 7 ) , consisting of 80% methanol-cvclohexane (in the ratio Of20:80) could‘be used to separate small quantities of the carbonyl derivatives ranging from formaldehyde to butyraldehyde. No separations could be achieved with this system for higher carbonyl derivatives. Larger quantities of materials could be separated when the papers were first impregnated with 10% propylene glycol in 20% acetone (12). Determination of R/ Values. Solutions of pure 2,4-dinitrophenylhydrazones of approximately 1 mg. per milliliter were chromatographed by the procedure of ascending chromatography at 20’ C. (7, 8). The spots, although visible, could be better detected with ultraviolet light. Although Rr values for a limited number of carbonyl derivatives in the modified Meigh phase (27) have been recorded for the method of descending chromatography, Table 1 records results obtained by the procedure employed in this work. Three to 5 pg. of derivatives could be separated by DeJonge’s system 31 ( 7 ) . On papers impregnated with propylene glycol, larger quantities could be separated by the same system. Preparation of Nonirradiated Dilute Automobile Exhaust Samples. Samples of dilute raw automobile exhaust ranging from 60 t o 90 liters in volume were obtained from a secondary sampling source from a n automobile on a chassis dynamometer operating under simulated driving conditions. T h e samples were collected a t a flow rate of 500 t o 1000 ml. per minute in two smog-type bubblers connected in series, each containing 15 ml. of a 15% sodium bisulfite solution a t icewater temperatures. These were stored in a refrigerator maintained a t 5’ C. The free carbonyl compounds from these combined solutions were released by the addition of sodium carbonate to
the solution in a closed flask with a side-arm dipping into a container of ether. Any vapors released during the neutralization were trapped in the ether. The aqueous layer was extracted with ether for 3 to 4 hours. The ether portion was then decreased to about 100 ml. and reacted with approximately 25 to 30 ml. of saturated 2,4-dinitrophenylhydrazinein 2N hydrochloric acid (4) or 2,4-dinitrophenylhydrazine in concentrated sulfuric acid and alcohol (1) by stirring the solution for 4 to 6 hours and allowing it to stand overnight in the refrigerator. The aqueous portion was distilled to about one third of its original volume and the distillate collected in ice water (13). The distillate was also reacted with a n equivalent amount of the 2,4dinitrophenylhydrazine reagent for 3 to 4 hours, and stored in the refrigerator
Table I.
Carbonyl 2,4-Dinitrophenylhydrazones Rf Value X 100
Meigh’s Substance using modified System S & S 2043B phase M 2,4-Dinitrophenylhydrazine 0 Formaldehyde 11.10 38.18 Acetaldehyde 18.37 57.17 Propionaldehyde 25 73 74.86 Butyraldehyde 37.50 Isobutyraldehyde 38.02 Isovaleraldehy de 45.71 Hexaldehyde 52.39 Heptaldehyde 58.16 Octyl aldehyde 68,92 Nonyl aldehyde 75.74 Decyl aldehyde 76,35 -4crolein 20.13 22.22 smears Crotonaldehyde somewhat 30,31 -4cetone 45.85 Methyl ethyl ketone Methyl propyl 54,20 ketone Diethyl ketone 57.65 Fufural cz s
trans
Glyoxal (bis) Formaldehyde Acetaldehyde Propionaldehyde Butyraldehyde Acrolein
3.15 10,08
n
36.21 57.17 73.89 88.57 62.67
Table II.
Carbonyls Found in Automobile Exhaust Benzene extract Ether extract Car-. CarDNP,a bonyl, DSP, bonyl, mg. mg. mg. mg. 0.48 0.14 1.00 0.28
Benzene
+ ether extract
CarCarbonyl, DNP, bonyl, mg./l. Substance mg . mg. gas 1b 1450: 1 Methyl ethyl 1.48 0.42 4.68 ketone 90 Liters of gas Acetonec 0.90 0.22 0.64 0.16 1.54 0.38 4.17 WOGA Fuel 3 Propionaldehydec Acrolein ... ... 0.13 0.03 0.13 0.03 0.35 Acetaldehyde 1.37 0.27 ... ... 1.37 0.27 2.99 Formaldehyded trace trace 2e 1230: 1 Methyl ethyl 5.83 1.67 1.04 0.30 6.87 1.96 32.73 ketone 60 Liters of gas Acetone 1.97 0.48 40.72 9.93 42.69 10.41 178.47 WOGA Fuel 5 Crotonal 0.15 0.04 ... ... 0.15 0.04 0.72 Acrolein 1.73 0.41 0.80 0.19 "53 0.60 10.02 Acetaldehydee Formaldehyde 0.09 0.01 ... ... 0.09 0.01 0.20 a 2,4-Dinitrophenylhydrazone. Hydrazones were formed with D N P in 2N HCl(18). c Assumed to be present from R f and fading characteristics in basic solution. Not determined quantitatively, but identified by spectra. e Hydrazones formed with D N P in conc. HzS04 and ethanol (IS). Sample no.
Dil.
overnight. Both the aqueous portion and the ether portions were extracted separately with 7.5 to 100 ml. of benzene 8 to 10 times. The extracts were washed Kith 75 to 100 ml. of either 2N hydrochloric acid or 4iv sulfuric acid (depending upon how the 2,4-dinitrophenylhydrazine reagent mas prepared) some 10 to 12 times to remove the excess 2,4-dinitrophenylhydrazine (20). The e\tracts mere evaporated and made to a specific volume. Blanks prepared in the same manner as the sample were prepared for the ether extracts and the distillates which are labeled the benzene extracts. Chromatography proceeded as described above. Procedure for Extracting Spots. After t h e samples were studied b y means of paper chromatography, t h e various spots were c u t out and extracted in Soxhlet extractors with 95% ethanol for 4 to 6 hours, followed b y similar extractions with chloroform. T h e chloroform was evaporated t o dryness; t h e resulting extract was taken u p in the ethanol fraction and reduced b y evaporation to an appropriate volume for study of the ultraviolet spectra on the spectrophotometer ( 5 , 9 ,l4,15,%$,26). So that the estracted solutions could be studied in a basic medium, appropriate quantities of the neutral solutions were evaporated t o dryness by means of suction or heat or both. The extracts were taken u p in 0.5 ml. of benzene and 4.5 ml. of alcoholic KOH (0.25.V) ( 9 ) . The spectra of the resulting solutions were studied on the spectrophotometer from 350 to 800 mu for fading characteristics (9, 14). The quantities of the hydrazones were ralculated by direct comparison with kiion n derivatives at their malimum intensities both in neutral and basic solutions. DISCUSSION AND RESULTS
The data in Table I give a survey of the mean R, values of the 2,4-dinitrophenylhydrazones in methanolheptane-acetic acid, as determined by
the method of ascending chromatography at 20' C. As in most paper chromatographic determinations, these values are depressed somewhat in the presence of foreign unidentified material. Values in DeJonge's system iLI ( 7 ) are also recorded for a number of derivatives. Although the 2,4-dinitrophenylhydrazones of saturated aldehydes of a homologous series are separated satisfactorily in the modified Meigh system, methyl ketones of the same carbon number show higher R f values. These R f values, with the possible exception of acetone, are approximately equivalent to that of a saturated aldehyde whose carbon number has been increased by a value of 1. Alkyl 2-enals show R, values similar to those shown by saturated aldehydes of one carbon atom less. In DeJonge's system ( 7 ) a n alkyl-2enal has a slightly higher Rfvalue than that of a saturated aldehyde of one carbon atom less. Table I1 lists the carbonyls that were positively identified in dilute irradiated automobile exhaust from R, values and from the spectra of these compounds in basic and neutral solution (5, 14, 15,9&, 26). Several other materials that produced spots could not be identified. For example, in Sample 1 in both the benzene and ether extracts there appeared spots that showed a blue fluorescence in ultraviolet light at Rf 84 to 86 and R, 63 to 70. The ultraviolet spectra of the former exhibited maximum intensities from 260 t o 275 mp in ethanol; the latter exhibited bands at 275, 283, and 333 mp in ethanol. A spot that was prominent a t the origin and thought to be excess 2,4-dinitrophenylhydrazine in both the ether and benzene extracts of sample 1 produced a slight blue color when mixed with alcoholic potassium hydroxide and showed bands of equal intensities a t 425 to 445 mp and 613 to 540 mp. A
yellow spot, which appeared in the ether extracts of samples 1 and 2 with a n R f value of 95 t o 98, mas also present in a larger quantity in a third sample. When portions of the pure carbonyls were oxidized with freshly precipitated silver oxide ( I S ) , the spot did not appear in the oxidized fraction, an indication of an easily oxidizable material. Ethanol extracts of thi.; yellow material showed bands a t 256 and 238 mp. A chromatograph of the ether extract of sample 2 showed the presence of a substance that exhibited a blue fluorescence in ultraviolet light and showed a broad absorption maximum from 254 to 300 mp in ethanol. No bands appeared in basic solution. I n the benzene extract of sample 2 a substance that did not migrate from the origin showed a broad band from 562 to 566 mp in alcoholic potassium hydroxide and produced a blue color. Several substances are known to remain at the origin in the modified Meigh system. Among them are glyoxal (bis), methyl glyoxal (bis), and dimethylglyoxal (bis). Although qualitatively dicarbonyls produce a blue color in basic solution, this is not necessarily an indication of bis derivatives, since all deril atives giving particularly stable anions may show similar colors (22).
The quantitative results are only estimates. The values obtained are between 60 to 65y0 of those obtained employing the 4-hexylresorcinol ( 2 ) method for analyzing automobile exhaust. Errors arise possibly because of the collection efficiency in the bubblers, ( 2 ) loss of the free carbonyls Then released from the bisulfite addition compounds. incomplete formation of the 2,4-dinitrophenylhydrazone under the conditions employed (Rf ), incomplete extraction of the spots from the paper (RO), and decomposition of the hydrazones in solution upon standing VOL. 35, NO. 3, MARCH 1963
a
349
or upon prolonged evaporation (25). The latter was indicated in the case of methyl ethyl ketone in sample 2 ; earlier determinations showed a value some 50% higher than determinations made on the same solution after it had stood for some time. Although the quantitative results for all of the carbonyl compounds determined are lorn, those for formaldehyde are the most striking. It is apparent that much of the formaldehyde is lost before the sample is processed ( 3 ) . A third sample collected a t room temperature failed to show the presence of methyl ethyl ketone. I t is generally known that higher aldehydes and ketones react to only a limited extent to form bisulfite addition compounds ( I O ) , and Wilson (28) has shown that the rate of trapping methyl ethyl ketone as a bisulfite compound is considerably less at room temperature than in ice water. ACKNOWLEDGMENT
pure compounds and for helpful suggestions. We are also grateful to Lois Lage Leng for the collection of the samples. LITERATURE CITED
( 1 ) Allen, C. F. H., 53, 2955 (1930).
J. Am. Chem. SOC.
(2) Altshuller, A. P., Cohen, I. R., ANAL. CHEM.33, 726 (1961). (3) Altshuller, A. P., Cohen, I. R., Meyer, M. E., Wartburg, A. F., Jr., Bnd. Chim. Acta 25, 101-17 (1961). (4) Bradg, 0. L., Analyst 51, 77 (1926), J . Chem. SOC.1931, p. 756. (5) Braude, E. A., Jones, E. R. H., J . Chem. SOC.,1945, p. 498. (6) Buske, D. A., Owen, L. H., Wilder, P., Jr., Hobbs, M. E., ANAL.CHEM. 28. 910-15 (1956). (7) DeJonge, ’A. P.; Rec. Trav. Chim. 74, 760 (195.5). \ - - - - I .
(8) DeJonge, A. P., Verhage, A,, Rec. Trav. Chim. 76, 221 (1957). (9) Ellis. R.. Gaddis. A. M.. Currie. G. T.. ANAL.’CHEM. 30. 475-9 (1958). ’
.,
(107 Fieser, L. F.,’ Fieser,’ M., ’“Organic
Chemistry,” Heath, Boston, 1957. (11) Gasparic, J., Vecera, M., Collection Czech. Chem. Commun. 22, 1426 (1957). (12) Homer. L.. Kirmse. W.. Ann. Chem. ’ 597, 48-68 (1955). (13) Huelin, F. E., Australian J. Sci. Res. B., 5, No. 3, 328-34 (1952). I
The authors are indebted to Eugene Sawicki for furnishing some of the
(14) Jones, L. A,, Holmes, J. C., Seligman, R. B., ANAL. CHEM. 28, 191-4 (1956). (15) Jones, L. A., Kinney, C., Hancock, J., J . Am. Chem. SOC.82, 105 (1960). (16) Klein, F., DeJonge, K., Rec. Trav. Chim. 75, 1285-8 (1956). (17) Meigh, D. F., Nature 169,706 (1952). (18) Ibid., 170, 579 (1952). (19) Miller, J. M., Kirchner, J. G., ANAL. CHEM.25, 1107 (1953). (20) Mold, J. D., McRae, M. T., Tobacco Sci. 1, 40-6 (1957). (21) Neuberg, C., Grauer, A., Pisha, R. V., Anal. Chim. Acta 7, 238 (1952). (22) Neuberg, C., Strauss, E., Arch. Biochem. 7, 211 (1945). (23) Rice, R. G., Keller, G. J., Kirchner, J. S., ANAL. CHEM.23, 194 (1951). (24) Roberts, J. D., Green, C., J . Am. Chem. SOC.68, 214 (1946). (25) Smith, Ivor, “Chromatographie and Electrophoretic Techniques, Vol. 11,” pp. 264-5, Interscience, New York, 1960. (261 Timmons, C. J., J . Chem. Soc., 1957, . p. 2613. ‘ (27) Wallgren, H., Nordlund, E., Acta Chem. Scand. 10, 1671-3 (1956). (28) Wilson, K. W., ANAL. CHEM. 30, 1127 (1958).
.
RECEIVED for review September 13, 1962. Accepted January 14, 1963.
Thin Layer Chromatography of Some Methylated Glycosides MILDRED GEE Wesfern Regional Research Laborafory,’ Albany, Calif.
b Thin layer chromatography on silica gel serves to separate anomers and isomers of methylated sugar glycosides. The area on the developed and dried plates occupied by the sugar derivative was revealed by spraying with dilute sulfuric acid and charring at 110” C. The entire procedure requires only about 1 hour.
T
EXPERIMENTAL
chromatography on silica gel is versatile in resolving many complex mixtures of compounds into single components. A number of review articles have appeared (1, 6-8), and recent studies in the field of carbohydrates include resolutions of free sugars (16-18) and sugar esters (9, 19). I n our studies of the structure and composition of carbohydrates, i t became necessary to separate mixtures of methylated sugars. Methylated sugar mixtures have been separated by chromatography on columns (15), paper (5, I d ) , reversed-phase paper ( W I ) , and most successfully by gas chromatography (2-4, 10, 11, 13). Gas HIN
350
chromatography requires expensive apparatus, b u t its resolving power is sufficient t o separate anomers and isomers of methylated methyl glycosides. Thin layer chromatography has now been applied to the separations of some methylated sugar glycosides on silica gel G into alpha and beta anomers and pyranose and furanose isomers.
LAYER
ANALYTICAL CHEMISTRY
Preparation of Chromatoplates. Double strength window glass (20 cm. x 20 cm. x 0.3 cm.) was coated with a layer of silica gel G (Merck, Darmstadt, Germany) at a thickness of 250 microns, using an R. S. Co. Model 200 Spreader (Research Specialities, Inc., Richmond, Calif.). The coating mixture was prepared by mixing one part by weight of silica gel G with two parts distilled water and stirring t o a uniform consistency. If the gypsum binder should begin to set during the preparation, water must be added to maintain a consistency of thick cream. After the glass plates were coated, they were dried at room temperature for 1 hour and then in an oven at 100” C.
for 30 minutes. ThP coated chromatoplates mere cooled and used for chromatography as described. Compounds Examined. Mixtures of completely methylated a and p anomers of furanose and pyranose sugar glycosides were made b y direct methylation of sugars (20) b y Kuhn’s procedure (14). They will be referred to hereafter as methylated sugars. The configurations of the various sugars resolved by silica gel chromatography n‘ere assigned by comparing RIfs of methylated sugars of known identity, separated, and collected by gas chromatography. Comparisons were made by addition of a known standard to a mixture and noting enhanced concentration of a particular spot. Gas chromatographic standards were prepared by methylation of glycosides of known configuration. Acetone solutions of methylated sugars were applied a t a distance of 2 em. from the bottom edge of the chromatoplates in 1 to 2 1 A laboratory of the Western Utilization Research and Development Division, Agricultural Research Service, U. S. Department of A4griculture.
