Acrolein Determination by Means
A
OF
Tryptophane
Colorimetric Micromethod
SIDNEY J. CIRCLE, LEONARD STONE,
AND
C. S. BORUFF, Hiram Walker & Sons, Inc., Peoria 1, 111. are resorcinol in alkaline solution (1.9); ferric chloride, phenylhydrazine and hydrochloric acid (8); phenylhydrazine and strong acid (5); phloro lucinol in alkaline solution (11); and sodium nitroprusside a n f iperidine ( 4 ) . The colorimetric methods include the use of gloroglucinol, hydrogen peroxide, and hydrochloric acid (14); &&iff’s fuchsin reagent (10); pyrogallol and hydrochloric acid or phenol and sulfuric acid ( I ) ; and benzidine and acetic acid (1, 16). All these tests and methods suffer from one or more defects: nons ecificity, highly colored blanks, or no absorption maximum in t i e visible range on the spectrophotometer absorption curve.
A new colorimetric method of analysis for acrolein has the advantages over previous methods of a spectrally purer color with maximum in the absorption curve, a colorless blank, and greater specificity. The method is based on the color produced b y the condensation of acrolein with tryptophane induced b y concentrated hydrochloric acid, and i s capable of detecting quantitatively as little as 15 micrograms of acrolein. The method is suitable for quantitative work only in the absence of other aldehydes, unless their concentration i s relatively much less than that of the acrolein.
A
CROLEIN, a poisonous lachrymatory agent formed from glycerol by dehydration, is frequently found as an atmospheric contaminant in several industries. It has been reported &s a product of bacterial fermentation of glycerol (6, 15), and as an undesirable constituent of fruit brandy (12) and wine (15). Infrequently distillates from grain and molasses fermentations are noted to be “peppery”, owing to its presence. Although precipitation tests are available for the qualitative identification of acrolein, they are unsuitable for quantitative purposes. A recent polarographic method (Q),although specific, lacks the convenience of a colorimetric procedure. Many color testa and colorimetric methods for acrolein have been described in the literature. Among the qualitative reagents
I 30
Figure 2.
90 TIME IN MINUTES
bo
I I20
Effect of Time and Temperature on Development of Color
Extinction Is determined at 555 m r through a 13.06mm. path and PC-4 filter us. water. Acrolein, 190 micrograms, tryptophane, 0.005 millimole, 95% ethanol, 2.2 ml., 19 N HCI, 6.3 ml. In 10-ml. final volume. ACROLEIN-TRYPTOPHANE REACTION
Figure 1.
The color test presented in this paper was suggested by the protein-nitrite-hydrochloric acid test of Voisenet (15) for acrolein and formaldehyde. The Voisenet reaction has been reviewed by Block and Bolling ( 2 ) . Investigation of this reaction showed that tryptophane was a much more efficient reagent than the egg white solution recommended, and that the trace of nitrite ion was actually deleterious. By avoiding nitrite, the blank, which is highly colored in its presence, became colorless; in addition, the purity of color of the tryptophane-acrolein complex was improved considerably. This adverse effect of nitrites was also, observed by Brown (3) for the tryptophane-glyoxylic acid reaction. Concentrated sulfuric acid was found to give the bame color as concentrated hydrochloric acid, but the heat of dilution is greater. Glacial acetic acid gave no color.
Spectral Absorption Curves of AldehydeTryptophane Complexes
Extinction is determined through a 13.06-mm. path and PC-4 filter US. water. ’1 Formaldehyde. 9 Crotonaldehvde. @ Glvoxal. (3 Acetaldehyde. €IFurfural, aldehyde, 0.005 millimoler tryotophme 0.005 millimole. 9 5 % ethanol 2.0 ml.1 and 1 9 N H C I , 6.3 ml: in 10-ml. final vdlmne. Develob for 50 minutes at 4 0 ’ C. 0 Acrolein, 0.0035 millimoler no ethanol. Develop for 4 5 minuter
EXPERIMENTAL
.*
-.330 -- c-.
APPARATUS AND MATERIALS. Spectral extinction values were determined on a Coleman Model 11 spectrophotometer, using matched-square cuvettes with a path of 13.06 mm., the PC-4 filter, and water as the comparison standard.
0 Acrolein, 100 micrograms (0.0018 millimole), 9 5 % ethanol, 9.0 ml. Develop for 50 minuter et 40’ C. For the latter two curve% tryptophane, 0.005 millimole, 19 N HCI, 6.3 ml. in 10-ml. Rnal volume.
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Vol. 17, No. 4
spectrophotometer. Ordinary diffuse daylight has little effect on the color, but bright light causes some fading. In the final method of analysis adopted, the procedure is identical to that above, except that in the case of unknown acrolein samples the acrolein must either be concentrated by distillation or other means, or diluted, as indicated by preliminary test, in order for its concentration to fall within the limits of the standard curve. It is possible to dispense with the cooling, if preferred, as the temperature does not rise above 40" C. on adding the acid a t room temperature, but the results are somewhat less consistent. SPECTRALABSORPTIOX OF ALDEHYDE-TRYPTOPHANE CoxIn Figure 1 are given absorption curves for the colors developed by reaction of tryptophane with acrolein, glyoxal, formaldehyde, acetaldehyde, crotonaldehyde, and furfural. Under the same conditions of development acrolein reacts faster and develops a deeper and purer color than any of the other aldehydes tested, with maximum sensitivity at 555 mp. If the formaldehydetryptophane-hydrochloric acid mixture is allowed to stand overnight, or if a higher temperature for development is used, a deep blue color does develop with maximum absorption a t 560 mp, in agreement with the observations of Komm ( 7 ) . A trace of oxidizing agent such as nitrite hastens the reaction with formaldehyde ( 7 ) and acetaldehyde, but is not necessary for acrolein. Under the conditions of development in Figure 1, formaldehyde develops a yellow color. EFFECTOF TIME AKD TEMPERATERE OF DEVELOPYENT. The curves in Figure 2 show that the higher the temperature of the developing bath, the greater the sensitivity and the shorter the time required to reach maximum extinction, but the less stable is the color. However, it is possible to stabilize the color a t its maximum development by immediate cooling in an ice bath. The color then remains fairly stable a t the maximum extinction value for several hours if the sample is kept refrigerated and away from light. The most suitable temperature from the standpoint of both convenience and stability is 40" C., a t which the time required for maximum development is about 50 minutes. EFFECT OF CONCENTRATION OF REACTANTS.There is an optimum concentration for the tryptophane and hydrochloric acid. Figure 3 shows the effect of tryptophane concentration on maximum extjnction, and Figure 4 the effect of hydrochloric acid concentration. In the case of ethanol, the mauim.im extinction PLEXES.
I .005 MlLLlMOLS
Figure 3.
Effect
I
.o I -0 I5 .02 .025 TRYPTOPHANE IN 10 M L . FINAL VOLUME
of Tryptophane Concentration on Extinction
Extinction 11 d e t m l n d at 555 mp through a 13.06mm. path and PC-4 Rlter u s . water. 0 Acrolein 0.0035 milllmole, no ethanol. Develop for 5 5 mlnutar at 39' C. 0 Acrolein: 0.009 millimolei 95% ethanol, 9.2 ml. Develop lor 50 minuter at 4 0 ' C. 0 Acrolein, 0.009. mlllimole~ 9 5 % ethanol, 2.9 ml. Develop tor EO mlnuter at 30' C. I 2 N HCI, 6.3 mi. in 10-ml. Rnal volume.
Two samples of acrolein were used, one supplied by Eastman Kodak Co. and the other by Shell Development Co., which resumabl contain hydroquinone as polymerization inhibitor. $art of the Eastman sample was redistilled and samples of distillate were collected with and without hydroquinone. Standard solutiom of the two original and two redistilled samples were prepared containing 100 micrograms of acrolein per ml. of 95% ethanol. No difference was detected in standard curves made up from these four samples. The standard solution in ethanol retains its strength for several weeks, but, if hydroquinone is absent, begins to lose its strength in a week or two. * A similar standard solution of acrolein in water was also made up, but found not to keep so well as the solution of acrolein in ethanol. The tryptophane solution was made up to 0.01 M by dissolving the requisite quantity of crystalline I( -)- or &tryptophane in 0.05 N hydrochloric acid. This solution keeps well when preserved with a few drops of toluene in an amber glass-stoppered bottle. The ethanol w&sessentially aldehyde-free. Hydrochloric acid was C.P. 37% or 12.0 N. Solutions of the aldehydes in Figure 1 other than acrolein were made up in water approximately 0.01 M each.
PROCEDURE AND METHOD OF ANALYSIS. Preliminary investigation showed that in order to obtain consistent data, it was necessary to pay meticulous attention to the variables involvedLe., time and temperature of development, and concentrat:ons of reactants. I n studying the effect of variation of a particular constituent on the development of maximum color, the concentration of every other constituent must be held constant. For example, in determining one of the standard curves in Figure 6, a series of samples w&s made up in 10-ml. volumetric flasks by pipetting into each 0.5 ml. of 0.01 M tryptophane, amounts of acrolein in 95% ethanol (100 micrograms per mL) varying from 0 to 2 ml., enough 95% ethanol to make its total quantity in each flask 2 ml., and 1.2 ml. of water. The flasks were cooled in an ice bath and made up to volume with ice-co!d 12.0 N hydrochloric acid, to avoid premature heating on mixing. If desired, the order of addition of water and hydrochloric acid may be reversed to give the same results, provided all ingredients are ice cold-that is, 6.3 ml. of 12 N hydrochloric acid may be added to each flask, and the dilution to 10 ml. made with water. The color is then developed in subdued li ht in a bath a t 40" C. for 50 minutes, and the extinction read in the
I
I
I
I
I
I
I
t X w
I 5 6 7 6 9 M L . I 2 N H U IN 10 M L . F I N A L VOLUME
Figure 4.
Effect of Hydrochloric A c i d Concentration on Extinction
Extinction ir determined at 555 mil through a 13.06-mm. path and PC-4 Rlter us. watrr. 0 Acrolein, 0.0035 millimolw no ethanol. Develop lor 70 minuter at 9 . 1 0 c. 0 Acrolein, 0.009 millimole, 9 5 % ethanol, 2.1 mi. Develop for 50 mlnuter at 4 0 ° C. 0 Acrolein, 0.002 millimole, 9 5 % ethanol, 2.9 mi. Develop lor EO minuter at 30' C. Tryptophane, 0.005 millimole in 10-ml. Rnal concentration.
April, 1945 Table
1.
ANALYTICAL EDITION Effect of Acrolein-Tryptophane Ratio on Extinction
Acrolein in 10-511. Final Volume
Tryptophane in 10-MI. Final Volume
Extinction at 555 mu
0.005 0.0038 0.005 0.0025 0.005 0.0018
0.595 0.610 0.410 0.418 0.277 0.277
Y
146 146 97 97 88 88
0.0026 0.0026 0.0017 0,0017 0.0012 0.0012
Table
11.
26 1
Effect of Formaldehyde and Acetaldehyde on Extinction Reached by Acrolein-Tryptophane Complex
Acrolein in '10-511. Final Volume Millimole 0.002
Formaldehyde in 10-M1. Final Volume Millimole
...
...
0.005 0.194 49 0.0009 0.194 49 0.0009 0.0013 0.081 27 0.0005 0.005 0,0005 0.055 27 0.0005 0,006 0.024 10 0.0002 0,0003 0.022 10 0.0002 For each sample, n o ethanol and 8 . 3 ml. of 12 N HC1 in 10-ml. final volume. developed a t 30' C. for 80 minutes. Extin'otion determined through a 13.06-mm. path and PC-4 filter u8. water.
...
...
0.002
0.002 0.002 0.002
0.002
0.001
0.002
..
..
0 :001
0.001
0.001
0.001 0 001
0 .'001
0.0005
...
.4cetaldehyde in 10-5fl. Final Volume Millimole
...
Extinction a t 555 mp 0.770
0.080
0.002
0,005
0,002 0.002
0.590 0.880 0.550
... 0.001
0.355 0,038 0.002
0 ,001 0,001
0.298 0.301 0.298
... 0,0005
0.140 0.022 0.001
0 .0005
0 .'do05 0,0005
0.123 0.123 0.123
...
...
0.001'
...
...
0.0005
0 0005 0.0005 0.0005
0.0005
...
...
Blank. For each sample, 2 . 2 ml. of 95% ethanol, 0 . 0 0 5 millimole of tryptophane and 8 . 3 ml. of 12 N HCl in 10-ml. final volume; developed a t 40' C. for 50 minutes. Extinction determined through a 13.06-mm. path and PC-4 filter 88. water. a
tLA
I.o
2.8
P IV
fx *b W
.4
.2
value increases directly proportionally to the increase in ethanol concentration, as shown in Figure 5. In the absence of ethanol maximum development is reached in 40 minutes a t 27' C., but .I the level reached is lower for a given concentration of acrolein I 2 3 (see Figure 6). Thus ethanol has an augmenting and stabilizing M L . 95% ETHANOL I N 10 M L . FINAL VOLUME effect on the color but slows up the reaction. The most favorable ratio of tryptophane to acrolein is roughly Figure 5. Effect of Ethanol Concentration on 2 moles per mole, but this is not critical. For the concentration Extinction of tryptophane adopted in the method of analysis (0.005 milliExtinction is determined at 5 5 5 mu through I 13.06-mm. mole in 10 ml. of final volume), practically no error is introduced path and PC.4 Rlter u.. waier. Acrolein 0 009 millimole^ tryptophane 0.005 millimole; by the excess of tryptophane in the lower range of the standard 1 2 N HCI, 6.3 ml. in 10-ml. final volime. Develop lor curve, where the rntio may be as high as 25 moles per mole, as 50 minutes at 40 C. shown in Table I. EFFECTOF OTHERA,LDEHYDES ON DEvEx.OmfENT OF COLOR. In Table 11 the presence of formaldehyde or acetaldehyde in concentration equal to acrolein during the development of the tryptophane-acrolein reaction is seen to depress the maximum extinction value reached, even though in the absence of acrolein the other aldehydes produce little color with the tryptophane for the time and temperature of development chosen. Since this antagonistic effect of other aldehydes applies even a t an acrolein concentration of 28 micrograms (0.0005 millimole), this precludes the possibility of determining acrolein in the presence of other aldehydes by the present method, unless their concentration is relatively much less than that of the acrolein. CONFORMITY OF CALIBRATION CURVES TO BEER'SLAW. Calibration curves for 20 40 eo 80 100 I20 140 160 180 the tryptophane-acrolein reaction are MICROGRAMS ACROLEiN IN I O - M L . FINAL VOLUME given in Figure 6, one without ethanol and two with ethanol under two different Figure 6. Relation of Acrolein Concentration to Extinction development conditions. It is evident Exlinction is determined a 5 5 5 m r through a 13.06mm. path and PC-4 Iltn na. web. that in the range 15 to 150 micrograms 8 N o ethanol. Develop$or 40 minutes a1 27' C. Beer's law is obeyed. These curves are 95% ethanol 2.0 mI. Develop for 50 mlnules 11 40' C. 95% ethanol: 2.0 mI. Develop elther lor 90 minutes at 3 0 ° C. or lot 150 rnlnukr 11 95' C. readily duplicated and hence need be Twplophane, 0.005 mlllimolr~ IP N HCI, 6.3 nl. i n 10-mi. Rnal volume.
8
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determined but once for a given set of conditions. However, it is a simple operation to redetermine one or two points on the curve each time a determination on an unknown sample is made. LITERATURE CITED
(1) Berezova, M. K., Hig. d. Sanit. (U.S.S.R.), 1940, No. 10,31. (2) Block, R. J., and Bolling, D., “Amino Acid Composition of Proteins and Foods”, pp. 91-5, Springfield, Ill., Charles C. Thomas, 1945. (3) Brown, W. L.,J. Biot. Chem., 154, 57 (1944). (4) Feigl, F., “Qualitative Analysis by Spot Tests”, p. 308, New York, Nordemann Publishing Co.. 1937. (5) Ganassini, D., Boll. 8oc. med. chir., Pa& (May, 1912). (6) Humphreys, F.B.,J . Infectious Diseases, 35, 282 (1924).
Vol. 17, No. 4
(7) Komm, E.,2. physiol. C h m . , 156, 35 (1926). (8) Meth, C h . - Z t g . , 30,666 (1906); C h . Z&r., 1906,11, 822. ENQ.CH~M., ANAL.ED., 15, 107 (1943). (9) Moshier, R. W.,IND. (IO) Moureu, C., and Boismenu, E., ,J. pharm. Aim., 27, 49, 89 (1923). (11) Nierenstein, M.,Collegium, 1905, 158; C h m . Zentr., 1905, 11, 169. (12) Reindel, F., and Sichert. K., 2. Spiritmind., 63, 281 (1940). (13) Tsalapatanis, L.,Anales SOC.qufm. argenlina, 5, 244 (1917). (14) Ucdina, I. L., H i g . Truda Tekh. Bezopasnosti (U.S.S.R.), 15, 63 (1937). (15) Voisenet, E., Ann. inst. Pasteur, 32,476 (1918). (16) Zhitkova, A.S., and Kaplun, S. I., tr. by Ficklen, 3. B., “Some Methods for Detection and Estimation of PoisonousGases and Vapors in the Air”, pp. 133-6, Service to Industry, Box 133, West Hartford, Conn., 1936.
Microscopical Identification of Ultramarine Blue in Complex Pigment Mixtures I. M. BERNSTEIN, H. D. Roosen Company, Brooklyn, N. Y. A microscopical method at 1940 magnification has been developed b y which ultramarine blue in low concentrations can be positively and quickly identified. The ashing of a thin film of printing ink or paint in situ on a microscope slide for direct examination avoids obscuring the tinctorially weak ultramarine blue when opaque whites or colored ash pigment components are present. The presence or absence of the cobalt blues or violets must be determined, since these pigments, being also heat-resistant, interfere with the ultramarine blue identification. A microscopical procedure based on the differential retistance of the cobalt blues to cold dilute mineral acid has been developed. Bocsuse of the greater analytical difficulty of identifyiig ultramarine blue in the presence of the cobalt blues and violets three micromethods have been devised: a method for identifyiny small amounts of hydrogen sulfide using as reagent an acidified qel, a differential method for destruction of ultramarine blue, and a method for identifying ultramarine blue b y microscopic identification of cubic sodium chloride crystals.
I
T IS often of analytical interest to identify ultramarine blue in
pigment mixtures, which may be in the form of wet printing ink or paint, dry color, or even printed matter or dried coating. The identification of this pigment by heretofore used methods may be simple or of considerable difficulty, depending on the composition of the pigment mixture as well as on the percentage of ultramarine blue contained therein. What little there is in the literature (I, 6, 7, 8, 10) deals with simple cases, and is therefore of minor analytical value. Refractive index measurements (6)can be used, but the procedure is questionable when dealing with small percentages of ultramarine blue in complex pigment mixtures. Since it is formed as a result of calcination, ultramarine blue is heat-resistant. Therefore when present by itself or in substantial proportions in admixture with opaque whites, transparent extenders, or organic colors, ‘asimple ashing in a crucible with resulting blue coloration may be regarded as a presumptive test for ultramarine blue. The cobalt blues and violets, however, are also heat-resistant, and the appearance of a blue ash cannot be taken as proof of ultramarine blue unless it can be shown that the cobalt pigments are absent. The cobalt blues consist of Thenard’s blue (cobaltous aluminate), Coeruleum blue (cobaltous stannate), and smalt (potassium cobaltous silicate) ; and the cobalt violets, of cobaltous arsenate and phosphate. Other heat-resistant blue pigments are oil blue (cupric sulfide), Colour Index No. 1289, and Egyptian blue (calcium copper silicate), Colour Index KO.1284, but these latter are not commercially used and are not considered in this paper.
From the analytical viewpoint the identification of the cobalt blues in the presence of ultramarine blue can, in certain cases, be established on the basis of their high cobalt content (18 to 30%), but. more generally the identification rests on the differential resistance of the cobalt blues to cold, dilute mineral acids (9). The identification of ultramarine blue in the presence of cobalt blues is, however, a more difficult problem. It has been suggested (4, 6-8) that the destruction of the blue color of an ash by dilute mineral acid with accompanying evolution of hydrogen sulfide may be considered further proof of ultramarine blue. While the absence of hydrogen sulfide on acidification would be proof of the absence of ultramarine blue, the presence of hydrogen sulfide might be due to other sulfides in the pigment mixture (a), or to barium sulfide which could be formed during the ashing as a result of the reduction of barium sulfate. Destruction of the blue color of the ash on acidification would be proof of ultramarine blue, provided the initial blue color of the ash were of sufficient intensity to enable one to note its disappearance. This presumes relatively high percentages of ultramarine blue, the absence of the cobalt blues, and the absence of a differently colored ash from some other component of the pigment mixture. If iron blue were present, the reddish brown iron oxide resulting from the ashing of the iron blue would obscure the ultramarine blue. Other colored ash pigments include ochre and synthetic iron oxides, chromium oxide and tetrahydroxide, lead and zinc chromates, cadmium sulfide, selenium sulfide, etc., I n both noncolored and colored ash pigment mixture components in which ultramarine blue is present in such small amounts aa to be unobservable to the unaided eye, microscopic examination of the ash will, to some extent, show the blue particles. There is, however, considerable danger in transferring the ash from the crucible to the slide, that the tinctorially weak ultramarine blue particles will be coated by the finer opaque white or colored ash particles and thereby obscured (3). This difficulty suggested performing the ashing on the microscope slide itself, so that no disturbance of the ashed pigment particles and therefore no obscuration by coating could occur. This method worked very well and has been used by the writer over a period of years. ASHING THE FILM
PREPARATION OF FILMFOR ASHING. The film is prepared on the slide for subsequent ashing, in the case of a wet printing ink or paint, by tapping out with one’s finger a very thin film in the csnter of and covering about one fourth the area of the slide. The ink or paint must be soft in body, t o allow the trans-
