Spot Test Detection and Colorimetric Determination of Aliphatic Aldehydes with 2-Hydrazinobenzothiazole Application to Air Pollution EUGENE SAWlCKl and THOMAS R. HAUSER Air Pollution Engineering Research, Roberf A. Tafi Sanifary Engineering Center, Public Healfh Service, U. S. Departmenf o f Health, Educafion, and Welfare, Cincinnafi 26, Ohio b A versatile new procedure for the detection and determination of aliphatic aldehydes i s introduced. All modifications of the new procedure have an especially high order of sensitivity for formaldehyde. On the spot plate 0.01 pg. of formaldehyde, 0.3 pg. of acetaldehyde, and 0.3 pg. of propionaldehyde can be detected; on paper 0.05 pg. of formaldehyde, 1 pg. of acetaldehyde, and 1 pg. of propionaldehyde can be detected. With proper standards, the amount of formaldehyde can be estimated. A tube containing silica gel impregnated with 2-hydrazinobenzothiazole solution can be used to detect or estimate formaldehyde in the air or in auto exhaust gases. A new colorimetric method for the determination of formaldehyde has also been evaluated. It i s reproducible and sensitive, and can be applied to the determination of formaldehyde in the air. In all these modifications an easily visible, brilliant blue color i s obtaina ble.
A
major offensive pollutants arising from combustion processes are the aliphatic aldehydes, of which formaldehyde appears to be the most prevalent in the air (6, '7) and the most reactive. A simple sensitive procedure is introduced for the analysis of aliphatic aldehydes (and especially formaldehyde). The all-around utility of the new procedure as a spot plate test, a spot paper test, a silica gel tube estimation, and in quantitative colorimetric analysis is especially gratifying. The new procedure can be applied to the analysis for aliphatic aldehydes in automotive exhaust, polluted air, and cigarette and trash fire smoke. It should be applicable in drug, milk, and food analysis, and in many other fields. Formaldehyde and other aldehydes will react with phenylhydrazine and then with potassium ferricyanide to give a red dye (4)which is reported to be unstable, although a reasonably improved modification has been proposed (6). Many modifications of this method
are available, some of which have been applied to the determination of formaldehyde in the air (2, 9). Since the hydrazine method showed promise of further development, a large number of hydrazines were investigated. 2 - Hydrazinobenzothiazole was found t o be the best of this group in respect to color intensity and stability while formation of the 1,5-di(2benzothiazolyl) formazan anion, D, was found t o give the most satisfactory analytical results. The mechanism of the 2-hydrazinobenzothiazole method as applied t o formaldehyde probably involves the following steps: formation of the hydrazone, A; oxidation of the benzothiazole hydrazine to a diazonium salt (or a hydrazine free radical), B; formation of 1,5-di-(2-benzothiazolyl) formazan, C ; and formation of the blue anion, D, in alkaline solution.
MONG THE
1434
ANALYTICAL CHEMISTRY
B
C
For the estimation of formaldehyde in the silica gel tube procedure, the redbrown color obtained after oxidation, the purple color obtained after treatment with alkali, or the intense blue color obtained after treatment with dimethylformamide can be used. The red-brown color is derived from the neutral formazan, C. The purple and intense blue colors are due to the formazan anion, D. This last color effect is based on the principle that anionic dyes absorb at longer wave length and with greater intensity in the more basic solvent ( 3 ) .
EXPERIMENTAL
Reagents and Apparatus. The following reagents were used: 2-Hydrazinobenzothiazole (Distillation Products Industries, Rochester, N. Y.), 0.5% solution in concentrated hydrochloric acid: H20 (1:9), The reagent solution is stable for a t least 2 weeks. Potassium ferricyanide, 0.2 and 1% aqueous solutions. Stock formaldehyde solution, 39.7%. Reagent, ACS reagent from Matheson, Coleman and Bell. The solution was analyzed by the gravimetric method of Yoe and Reid (8). Aliphatic aldehydes, Laboratory Services, Inc., Cincinnati, Ohio. The silica gel tubes (120 mm. long, 5-mm. inner diameter) were packed with 75 mm. of silica gel (desiccant grade, 100 to 200 mesh, Davidson Chemical Co., Baltimore, Md.) Twotenths of a milliliter of 0.5% 2-hydrazinobenzothiazole was dispersed in each gram of treated silica gel. A Cary recording quartz spectrophotometer, Model 11, was used for quantitative analysis Table 1.
Detection of Aliphatic Aldehydes
Identification Compound Limits, fig. Spot Plate Formaldehyde" 0 01 Acetaldehyde" 0.3 Glvosale 2 0 Propionaldehyde. 0 . 55 1.5 Butanal 7 . .i5 2-Methylpropanal Hep tanal 1.5 1.5 Heptanal Octanal 3.0 Ocfanal 6.0 Konanal Decanal 3.0 Undecanal 3.0 Lauraldehyde 3.0 Hydrocinnamaldehyde 15.0 Hydrotropicaldehyde 30.0 Phenylacetaldehyde 30.0 Spot Paper Formaldehyden 0.05 Acetaldehyde" 1.0 Propionaldehyde" 1.0 Butanal 5.0 I n aqueous solution. Remainder of aldehydes in dimethylformamide solution. 0
Spot Plate Procedure. METHOD. One drop (0.03 ml.) of the 2-hydrazinobenzothiazole reagent is placed on a white porcelain spot plate followed by 1 drop of the aqueous test solution. The homogeneous mixture is allowed to stand for 2 minutes. Then 1 drop of the 1% ferricyanide solution is added and the mixture is allowed to stand for 2 minutes. One drop of 10% aqueous potassium hydroxide is then added. A positive test is indicated by the formation of a deep blue color. The blank is pale yellow. The limits of identification for different aliphatic aldehydes are given in Table I. DISCUSSION. The new method is apparently more sensitive than any of the procedures reported in the literature (1) for the detection of formaldehyde. For example, in the spot plate modification, an identification limit of 0.01 pg. and a concentration limit of 1 to 12,000,000 are obtained for formaldehyde. The other aliphatic aldehydes are anywhere from 30 to several thousand times less sensitive. Color stability in all cases is satisfactory. Nitromethane gave a blue color; the identification limit was 10 pg. Acrolein gave a green color, with an identification limit of approximately 300 pg. The following compounds gave negative results in all procedures: acetone, ethyl methyl ketone, acetophenone, benzophenone, biacetyl, chloral, benzaldehyde, piperonal, 1-naphthaldehyde, 9-anthraldehyde, 1-pyrenealdehyde, furfural, cinnamaldehyde, methanol, 2methoxyethanol, anisyl alcohol, safrole, glycolic acid, phenol, 1-naphthol, glucose, 1-octene, 2,3-dimethyl-l,3-butadiene, and ferrous sulfate. The waterinsoluble compounds were tested in dimethylformamide solution. Some of these compounds interfered in the chromotropic acid procedure. Spot Paper Procedure. METHOD FOR SOLUTIONS. Ten microliters of the benzothiazole hydrazine solutions are placed on the filter paper, immediately followed by 1 p1. of the test solution in the center of the hydrazinic spot. After standing 1 minute, 10 p1. of 0.2% potassium ferricyanide are added. Two minutes later, 30 pl. of 10% aqueous disodium phosphate are added so that the spot on the paper is entirely covered. An immediate blue stain indicates the presence of aliphatic aldehydes. The blue color is stable. Occasionally a very pale blue color forms in the blank after several minutes of standing, but this does not interfere with the identification of aldehydes. Limits of identification are given in Table I. METHOD FOR GASES. One drop (0.03 ml.) of the benzothiazole hydrazine solution is placed in the middle of an 11-cm. diameter filter paper. The paper is then exposed for a few seconds (for auto exhaust fumes) to a few
minutes (for bus terminals, heavily traveled roads, garages, etc.). The drop should not be allowed to dry. After 1 minute, 1 drop of 1% potassium ferricyanide solution is added. After a 2-minute interval, 1 drop of the potassium hydroxide solution is added. DISCUSSION. On paper, approximately 0.05 pg. of formaldehyde can be detected while other aliphatic aldehydes are detected in the microgram range. A blank must be run concurrently with the test because a pale blue ring can be obtained after the paper stands for a while. The test of automotive exhaust fumes dramatically demonstrates the quantity of aldehydes expelled into the air by automobiles. Since an afterburner or some other device may become standard automotive equipment for the elimination of exhaust pollution, the spot paper or silica gel test can then be of value as a rapid check of its continuing efficiency. I n the spot paper procedure for solutions, nitromethane gave a negative test. However, when 10% potassium hydroxide was substituted for the phosphate, an identification limit of several hundred micrograms was obtained. Silica Gel Tube Procedure. A definite volume of polluted air is pulled through two tubes set up in series. For auto exhaust, 50 to 200 ml. will suffice. T o the top of each tube enough 1% ferricyanide solution is added so t h a t the wet front &-ill reach the halfway point in the tube. The tubes are allowed to stand for 18 minutes. I n the tube through which the air first passes, a red-brown color in the top 5 mm. of the silica gel layer indicates the presence of formaldehyde. The amount of formaldehyde is proportional to the intensity and extent of the red-brown stain. -4few drops of 10% potassium hydroxide solution are added slowly, dropwise, until the redbrown area turns purple. Gentle suction can be used here. Here again the intensity and extent of the purple color are an indication of the amount of formaldehyde in the gas sample. A few drops of dimethylformamide are then added to the column so that the purple color will turn bright blue. The second tube (blank) is treated similarly from beginning to end. The silica gel tubes can be fused a t both ends a t any stage of the operation and saved for future comparisons. The colors are stable. The steps given here should be checked for positive results with the vapors over a 38% aqueous formaldehyde solution or with auto exhaust fumes. Through the use of appropriate standards the amount of formaldehyde present in auto exhaust fumes or polluted air can be estimated. Colorimetric Procedures. METHOD. For the analysis of an impinger-collected (7') air sample, 10 ml. of the
collecting solution (equal volumes of 0.5% 2-hydrazinobenzothiazole and distilled water) and 10 ml. of the same solution (obtained from a second impinger connected in series and following the first impinger) are pipetted into two 100-ml. volumetric flasks, one used as a blgnk. Following the addition of 5 ml. of 1% ferricyanide solution to each flask, both flasks are shaken and allowed to stand for 18 minutes. If formaldehyde is present, a red precipitate is usually seen a t this stage. To each flask are added 10 ml. of dimethylformamide followed by 10 ml. of 10% aqueous potassium hydroxide solution. Both mixtures are diluted to the mark with water. The molar absorptivity of the unknown compared to the blank is then determined a t the wave-length maximum of 582 mp within 15 minutes. I n the analysis of an aqueous test solution, 5 ml. of the solution are treated with 5 ml. of 0.5% 2-hydrazinobenzothiazole in a 100-ml. volumetric flask. In this case, the blank would contain 5 ml. of water and 5 ml. of the 0.570 hydrazine solution. After a 5-minute waiting period, both mixtures are treated in the same fashion as with the 10 ml. of collecting solution. The blank is usually pale yellow-green. Spectral data for some aliphatic aldehydes analyzed in this fashion are given in Table 11.
Table 11. Colorimetric Determination of Aliphatic Aldehydes Molar 3fiaY.,
Compound Formaldehyde Acetaldehyde Propionaldehyde
?*IM
Absorptivity, €
582 610s
48,000 42,000
576 600s 5i7 600s
1,815 1,560 1,581 1,470
DISCLS~XON. I t ivould appear that formaldehyde is the principal aldehyde in the air (6, 7 ) , although it is possible that near some pollution sources or under heavy smog conditions relatively large amounts of other aldehydes might be present. I n the new method, the color obtained with formaldehyde is at least 25 times as intense as with other aliphatic aldehydes, while heterocyclic and aromatic aldehydes give negative results. Other factors being constant, the color obtained in the quantitative procedure is approximately 5 times as intense as in the phenylhydrazine method (6). I n the colorimetric procedure the absorbance does not change for a t least 20 minutes after the final dilution is made. After 16 hours' standing, the blank becomes pale blue and the absorbances of VOL. 32, NO. 11, OCTOBER 1960
1435
the standard solutions are - 10% of the initial values. Thus it is evident that the colors are stable enough for analytical work. The absorption spectrum of the blank as compared to 10% aqueous dimethylformamide is practically negligibleabsorbance of 0.1 a t 582 mp. Reproducibility in absorbance obtained with standard formaldehyde solutions is excellent. For example, over a period of a month 20 determinations of solutions containing 50 pg. of formaldehyde gave absorbance values of 0.79 i 0.02. Beer’s law also was found to hold over a range of 10 to 150 pg. per 100 ml. of final solution. Variables in the colorimetric procedure were investigated. An increase in the concentration of the hydrazinobenzothiazole caused an increase in the color of the blank; a decrease in concentration caused a decrease in the intensity of the blue color in the standard solution. At least 5 minutes’ standing was necessary for the formation of the hydrazone; a shorter reaction time caused a decrease in the intensity of the blue color. The smaller the concentration of potassium ferricyanide, the longer the oxidation period to obtain maximum color development. Using a 2% solution gave good results, but a bluish colored blank. A 3y0 solution of potassium ferricyanide produced a precipitate. The most satisfactory results were obtained with the 1% solution and a 17- to 25-minute oxidation
period. For example, in a final solution containing 100 pg. of formaldehyde, oxidation times of 5, 10, 15, 20, 25, and 30 minutes gave absorbance values of 1 .OO, 1.25, 1.57, 1.59, 1.58, and 1.56, respectively. Heat applied durmg the oxidation step in an attempt to shorten the time resulted in decomposition. The reddish brown precipitate of the formazan obtained after the oxidation step is so insoluble that formaldehyde could probably be determined gravimetrically by this method. This precipitate can also be extracted with chloroform or o-dichlorobenzene and then made alkaline t o give a purple (chloroform) to blue (0-dichlorobenzene) color. Once the blue dye mas formed, some dimethylformamide was necessary to keep it in solution. The presence of 10% dimethylformamide gave satisfactory results. A somewhat greater concentration gave a more highly colored blank. With a further increase in concentration of dimethylformamide, the inorganic salts tended to precipitate. The concentration of the alkali was not too critical. Doubling the alkali concentration had no effect on the color intensity. The procedure of Wilson (7) was used for preparing and collecting air-aldehyde mixtures for analysis by the impinger method. However, in 7itTilson’s method the gas is collected in aqueous sodium bisulfite, while in the present method 0.25% 2-hydrazinobenzothiazole solution is used. One hundred and ninety-
eight micrograms of formaldehyde were driven over into 20 ml. of the collecting solution six times, with analyses of 10-ml. portions each time. Absorbance values of 1.56 0.02 were obtained. Because a theoretical absorbance value of 1.59 should have been obtained, efficiency in the collection of aldehyde is in the neighborhood of 97 to 99%. Some interferences were noted. Nitromethane gives a blue color in very high concentrations and an absorbance of about 10. The larger aliphatic aldehydes were somewhat water-insoluble and gave essentially negative results in the colorimetric procedure.
*
LITERATURE CITED
(1) Fe,ig!! F., “Spot Tests in Organic Analym, 5th ed., p. 348, Elsevier, New
York, 1956. (2) Kersey, R. W., hladdocks, J. R., Johnson, T. E., Analyst 65, 203 (1940). (3) Sanicki, E., Stanley, T. W., Hauser, T. R., ANAL.CHEM.31, 2063 (1959). (4) Schryver, S. B., Proc. Roy. SOC. (London) 82B, 226 (1910). (5) Tanenbaum, hl., Bricker, C. E., ANAL. CHEM.23, 354 (1951). (6) Thomas, J. F., Sanborne, E. N., Mukoi, M., Tebbens, B. O., A . M . A . Arch. Znd. Health 20, 420 (1959). ( 7 ) \viISOn, K. Nr., A N A L . CHEM. 30, 1127 (1958). (8) Yoe, J. A,, Reid, L. C., IND.ENCI. CHEM.,ANAL.ED., 13, 238 (1941). (9) Zurlo, N., Griffini, A. M., M e d . laooro 45, 692 (1964). RECEIVEDfor review February 15, 1960. Accepted June 8, 1960. Air Pollution S mposium, Division of Water and Waste &emistry, 138th Meeting, ACS, New York, N. Y., September 1960.
The Fluorescence Spectra of Aromatic Hydrocarbons and HeterocycIic Aromatic Compounds BENJAMIN 1. VAN DUUREN Institute o f lndustrial Medicine, New York Universily Medical Center, New York, N. Y.
b The application of fluorescence spectroscopy in the qualitative and quantitative analysis of aromatic compounds was investigated. The replacement of hydrogen atoms of aromatic hydrocarbons by alkyl groups in some instances gives noticeable shifts in fluorescence spectra. The replacement of carbon atoms of aromatic hydrocarbons by nitrogen and by oxygen was also examined. Certain compounds show markedly different fluorescence behavior in different solvents or in their ionized forms. This relationship between changes in fluorescence spectra with changes in solvents was compared with the effects of the same solvents on the ultraviolet absorption spectra. Fluorescence excita1436
e
ANALYTICAL CHEMISTRY
tion spectra obtained with an automatic recording spectrofluorometer augment identifications of compounds made on the basis of ultraviolet and fluorescence emission spectra. The effect of concentration on fluorescence intensity was also examined.
R
in instrumentation and the availability of commercial automatic recording spectrofluorometers have made possible the more extensive use of fluorescence spectroscopy for analytical purposes. It has served as a valuable tool in this laboratory in the identification of cigarette tar aromatic hydrocarbons (13, 14) and heterocyclics (16). The aromatic compounds were separated ECENT ADVAKCES
and identified by their R , values, ultraviolet spectra, and fluorescence spectra. In the course of the author’s work on the cigarette tar aromatics, a number of aromatic compounds were purified and their fluorescence spectra examined. Some of the observations made, new data, and conclusions on the fluorescence of these compounds are described in this report. EXPERIMENTAL
Purification of Aromatic Compounds, All compounds used in this work were subjected t o purification procedures, including column chromatography, crystallization, and/or vacuum sublimation. The criteria of
