nickel L a radiation in iron or cobalt are not well known (8) and extrapolation from known values gave analyses which were in error by approximately 20%. Therefore the empirical method of Ziebold and Ogilvie (5) was used. As copper K radiation will cause fluorescence of iron K radiation, it is necessary to evaluate any possible effect on the iron determination by the copper in the beryllium-copper substrate and the copper plate. This evaluation was made by copper plating a bulk sample of nickel-iron and taking readings across the interface. At 50% of the iron intensity of the bulk sample, the result was identical to that obtained on the bulk sample. Even at 30% intensity, the iron value was high by only 6% of the amount present, giving 20.2% Fe instead of 19.1% Fe.
(8) K. F. J. Heinrich, “X-ray Absorption Uncertainty,” in “The Electron Microprobe,” T. D. McKinley, Ed., John Wiley and Sons, New York, 1966, p 296.
The a factors were tested by analyzing three bulk samples of trinary alloy and one sample of thin film, all of which had been chemically analyzed. These comparisons are shown in Table 11. The precision of measurement and accuracy compared to chemical analysis is excellent. The method provides a rapid technique for the determination of the composition of trinary films and could be extended to the analysis of any micron or submicron sized sample. ACKNOWLEDGMENT The author is grateful to F. B. Humphrey and T. Suzuki of Caltech for the preparation of the standards, to R. M. Shoho and P. Husman of the Autonetics Division of North American Rockwell for the reference samples and chemical analyses, and to A. E. Bence of Caltech for discussions of the correction procedures. RECEIVED for review March 11, 1968. Accepted April 10, 1968.
Spectrophotometric Determination of Pyrazolines and Some Acrylic Amides and Esters A. R. Mattocks Toxicology Research Unit,Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey, England A SENSITIVE COLOR reaction for acrylamide was required in connection with studies on the neurotoxicity of this compound. This was achieved as a result of the discovery that pyrazolines, which are readily formed from the reaction of diazomethane with many acrylic and related compounds, including acrylamide, give intensely colored derivatives with certain aldehydes. The reaction of diazoalkanes with a-unsaturated carbonyl compounds to form pyrazolines is well known, and has recently been reviewed ( I ) . The reaction times vary widely, depending on the stereochemistry of the unsaturated compound ( 2 ) . Thus, ethyl acrylate (I) reacts rapidly with diazomethane in ether to give 3-ethoxycarbonyl-1-pyrazoline(11) (3, 4). CHr=CH,COOEt
cHzN2:
~ ‘ “ “ “ - (-coo‘t H
I
I[
m
The latter readily tautomerizes, for example in the presence of alcohols, to the more stable 2-pyrazoline (111) (3). Extension of this reaction to acrylic amides has not previously been reported. Acrylamide (IV) reacted very rapidly with diazomethane in methanol-ether, t o give a stable crystalline product, mp 97’ C, formulated as (V) by analogy with
(1) C. H. Jarboe in “Chemistry of Heterocyclic Compounds,” A. Weissberger, Ed., Vol. 22, Interscience, New York, 1967, p 209. (2) A. Ledwith and Yang Shih-Linn,J. Chem. SOC.(B), 83 (1967). (3) D. S.Matteson, J. Org. Chem., 27,4294 (1962). (4) A. Ledwith and D. Parry, J. Chem. SOC.(C), 1408 (1966).
0
CHpCH.CONH2 cH2N2
H
IP
P
I
p PI
(111). Infrared spectra and microanalysis supported this structure. Acidic Ehrlich reagent (4-dimethylaminobenzaldehyde) and 4-dimethylaminocinnamaldehydeare known (5) to react with some amines to give colored Schiff‘s bases. It was found that the pyrazolines (II), (111), and (V) gave strongly yellow colored derivatives with Ehrlich reagent, and with 4-dimethylaminocinnamaldehyde even stronger and more stable purple colors were formed. The derivatives are presumably Schiff‘s bases of the form (VI). The pyrazolines (11) and (111) gave identical 450 mp), with Ehrlich reagent. colored derivatives (A,, This is not surprising in view of the ease with which (11) isomerizes to (111). The colors from the above reagents are formed very rapidly at room temperature. They are stable for many hours in solution, and the intensities decrease linearly with dilution if absolute ethanol is used. The acid used in the coupling reaction is apparently not critical: HCl, HC104 and BFI are equally effective. The above color reactions have been made the basis of a spectrophotometric method for the determination of these pyrazolines, and of acrylamide and related acrylic compounds. The procedure will be described for acrylamide, but it is ( 5 ) F. Feigl, “Spot Tests in Organic Analysis,” 7th ed., Elsevier, New York, 1966, p 243. VOL. 40, NO. 8, JULY 1968
1347
Compound
Table I. Calibration Data for Some Pyrazolines and Acrylic Compounds Absorbance (for 1 l i g in 10 ml final Reagent= , , A, (mp) volume) Mol wt
Acrylamide (IV) Acrylamide (IV) Pyrazoline from acrylamide (V). Pyrazoline from acrylamide (V). Ethyl acrylate (I) Ethyl acrylate (I) Pyrazoline from ethyl acrylate (111)~ Pyrazoline from ethyl acrylate (1II)C N-Methyl acrylamide N-Hydroxymethyl acrylamide N,N-Diethyl acrylamide Methyl methacrylate a
71 71 113 113 100
i
538 440 538
11
440
1
545 450 545 450 442 44 1 433 430
i 11
100
11
142 142 85
i
101
11
127
ii
100
11
11
11
0.130
0.067 0.10
0.047 0,094 0.042 0.08
0.033 0.050 0.040 0.037
0,042
Molar absorptivity 92,200 47,600 113,000 53,100 94,000 42,000 113,500 46,800 42,500 40,400 47,000 42,000
Reagents: (i) 4-dimethylaminocinnamaldehyde (ii) 4-dimethylaminobenzaldehyde
* From slope of calibration curve Diazomethane stage omitted
applicable to other amides and esters, including those listed in Table I, if allowance is made for differences in molecular weight and. , ,A, The pyrazolines themselves are estimated by omitting the diazomethane stage. EXPERIhlENTAL
Reagents. DIAZOMETHANE. This is a solution in ether, best prepared by the method of D e Boer and Backer (6). This solution may be stored for many weeks in a deep freeze, in a plastic-stoppered flask. I t should be redistilled shortly before use to eliminate impurities formed during the preparation, o r on keeping, which would otherwise react with the color reagent and form strongly colored blanks. (The diazomethane sometimes contains an impurity which gives strong colors with the aldehyde reagents. The majority of this impurity can be removed by distilling the ethereal diazomethane solution, preferably after adding about one third its volume of ethanol, which ensures that a substantial residue remains in the distilling flask after almost all the diazomethane has distilled. However, even when freshly redistilled diazomethane solution is evaporated to dryness and 4-dimethylaminocinnamaldehyde reagent added, a slight pink color is formed. This is presumably either caused by a decomposition product of the diazomethane, or to a product from the reaction of diazomethane with trace impurities in the solvent.) The final strength of the diazomethane solution is not important, because a large excess is used in the estimation procedure. WARNING.Diazomethane is highly toxic and carcinogenic (7). All operations with it must be conducted in a well ventilated fume cupboard. ‘TRIS’SOLUTION.A solution of tris(hydroxymethy1)aminomethane, 0.1 w,’v in methanol. This solution is stable. 4-DIMETHYLAMINOCINNAhlALDEHYDE REAGENT.The COmpound (0.1 gram) recrystallized from benzene-light petroleum, is dissolved in absolute ethanol (99 ml) and concentrated hydrochloric acid (1.0 ml). EHRLICHREAGENT.4-Dimethylaminobenzaldehyde (Analytical Reagent grade) (2 grams), is dissolved in concentrated
(6) Th. J. De Boer and H. J. Backer, Organic Syntheses, 36, 16 (1956). (7) R. Schoental, Nature, 188, 420 (1960). 1348
0
ANALYTICAL CHEMISTRY
hydrochloric acid ( 2 ml) and absolute ethanol, and made u p to 100 ml with absolute ethanol. This reagent is stable if kept in the dark. Procedure. Samples for estimation should contain acrylamide, preferably in the range 2-20 pg, in a dry form or in not more than 0.1 ml aqueous solution or 1 ml of ethanol or methanol, in test tubes (15 x 120 mm is a suitable size). 1. Add ‘tris’ solution (1 ml), shaking to dissolve the acrylamide. (If the ‘tris’ is omitted, the results are often very variable. The use of ‘tris’ was arrived at empirically, and its mode of action is uncertain. Sodium pyrophosphate has a similar effect.) 2. In a fume cupboard, add 2 ml of diazomethane solution. Shake, and allow to stand at room temperature for 10-20 minutes. Should the solution become colorless, more diazomethane must be added. With acrylamide, 10 minutes is sufficient for completion of the diazomethane reaction, but a longer time (up to 30 minutes) is not detrimental. 3. Gently boil off the ether and excess diazomethane by shaking the tube in a hot water bath. The solution must not be taken to dryness or blown with air or nitrogen (this may cause evaporation of some of the pyrazoline derivative). When it is colorless (no diazomethane left), it may be removed from the fume cupboard. Colored derivatives may now be formed by either of the following methods. 4a. Cool to room temperature, add 1 ml of 4-dimethylaminocinnamaldehyde reagent, and allow to stand for at least 5 minutes. Dilute with ethanol to 10.0 nil, and measure the absorbance at 538 mp against a reagent blank prepared as above but without acrylamide. Very weak samples may be diluted to 5.0 ml instead of 10.0 ml, and strong samples may be further diluted with ethanol, and measured against a similarly diluted blank. The color is stable for at least 24 hours, provided the sample tubes are stoppered and kept in the dark. 4b. The procedure is the same as 4a, but with Ehrlich reagent in place of 4-dimethylaminocinnamaldehyde. The absorbance should be measured a t 440 mp, preferably within one hour, The alcohol used for dilution must be free from aldehydes or ketones. Compounds likely to interfere by forming strong colors with the reagents include pyrroles, indoles and related compounds, aromatic amines, and hydrazine. These can be detected and a blank prepared, if necessary, by omitting the diazomethane stage. Larger amounts of carbonyl compounds may also interfere by reducing the strength of color.
Detection of Acrylic and Related Compounds on Chromatograms. A modification of the foregoing procedure provided a sensitive means of detecting some of these compounds on paper and thin-layer chromatograms. Reagents. Diazomethane solution, prepared as already described, was diluted with three times its volume of light petroleum (bp 80-100 "C). The 4-dimethylaminocinnamaldehyde reagent, described above, was diluted with one volume of ethanol and two volumes of light petroleum (bp 80-100 "C). Procedure. The method was tested by applying methanol solutions of various compounds (10 pg in 1 pl) to Whatman No. 1 paper and t o Kieselgel G (Merck) plates (0.25 mm layers), and allowing them to dry. What ,follows was carried out in a fume cupboard and extreme precautions were taken to prevent the exposure of personnel to diazotnerltane. Papers were placed in a shallow enamel tray and just covered with the diazomethane solution (about 40 ml to 100 sq in. or 650 sq cm), and were left until dry (about 5 minutes). They were then sprayed with the diluted aldehyde reagent. Compounds which appeared within a few minutes, as purple spots against a white ground, are indicated in Table 11. The same procedure was less successful with thin-layer plates, probably because much diazomethane decomposed on contact with the adsorbant, and conversion of the compounds t o pyrazolines was less efficient (Table 11). Chromatographic methods were not investigated in detail but acrylamide, run on Whatman No. 1 paper with tolueneethanol (equal volumes) as eluant, had RF 0.67. On a Keiselgel G plate, with toluene-ethanol (3 1, volume), it had RF 0.37.
+
RESULTS
Calibration curves from the compounds listed in Table I were linear through the origin for amounts up to a t least 90 pg, with the exception of N-methyl acrylamide (low results above 70 pg) and methyl methacrylate (points rather scattered). Low results were due to incomplete conversion to the pyrazoline, and could probably be improved by increasing the time of reaction with diazomethane. The reaction of diazomethane with acrylic amides was not greatly slowed by substituents o n the nitrogen, just as its reaction with acrylic esters is not much influenced by the nature of the alcohol moiety ( 2 ) . The reaction rate is much slower, however, when substituents are attached t o the unsaturated carbon atoms (2), and satisfactory calibration curves could not be obtained for methacrylamide, crotonamide, senecioic amide, or methyl tiglate. Table TI shows the absorption maxima for the 4-dimethylaminocinnamaldehyde derivatives of pyrazolines from a number of compounds. In addition t o those listed in Table I, the colorimetric method could probably be adapted for estimating acrylonitrile, acrolein, maleic and fumaric acids and their esters, and methyl vinyl ketone. Estimations in Urine. The procedure using 4-dimethylaminocinnamaldehyde was satisfactory for estimating acrylamide in urine. For preparation of a calibration curve, solutions of acrylamide were made in rat urine. Samples (0.10 ml) of these solutions were diluted with 1 ml of methanolic 'tris', and estimations were carried out exactly as described above. A normal urine sample was used for a blank, giving a pale pink solution. The calibration curve was a straight line, for samples containing between 50 and 2000 pg of acrylamide per milliliter of urine. The slope was 0.12
Table 11. Colors Formed from 4-Dimethylaminocinnamaldehyde Reagent and the Pyrazoline Derivatives from Some Unsaturated Carbonyl Compounds, in Solution and on Chromatographic Paper and Thin-Layer (Kieselgel G ) Plates Strength of purple spot on chromatogram (10 pg compound applied in 1 p l methanol)a Paper Thin-layer Compound , , ,A, mp S S 538 Acrylamide S S N-Methyl acrylamide 537 N-Hydroxymethyl acrylamide 540 S S N,N-Diethyl acrylamide S S 528 510; 389w W 0 Methacrylamide W 0 Crotonamide 533 W 0 Senecioic amide 510 S M 545 Acrylic acid W 0 Crotonic acid 546 T 0 Tiglic acid 512 T 0 505 Senecioic acid S M Maleic acid 553 S S Fumaric acid 553 Acrylonitrile 556 5 15 ; 400w M Acrolein 516; 4 0 0 ~ W T Crotonaldehyde 547 Methyl vinyl ketone a Key: S, strong; M , medium; W , weak; T, trace; 0, no spot visible; w, weaker. per microgram--i.e., almost the same as that for pure acrylamide (Table I). The method has been applied to the urine from rats dosed with acrylamide. Reactions of Pyrazolines with Other Carbonyl Compounds. Besides the two reagents already mentioned, other aldehydes which gave yellow colors with the pyrazolines (111) and (V) in acid solutions included vanillin, salicaldehyde, 4-hydroxybenzaldehyde, and 2-hydroxy-l-naphthaldehyde, but not benzaldehyde. An interesting feature of the reaction is that it is reversible, F o r example, the color from vanillin and (111) was rapidly discharged by adding acetone. The color was partly restored by adding excess of vanillin, or by boiling off the excess acetone. The color from Ehrlich reagent and (111) was also discharged by excess of acetone, but more slowly. Other carbonyl compounds, such as ethyl methyl ketone, formaldehyde, acetaldehyde, and benzaldehyde, had the same effect. The color from 4-dimethylaminocinnamaldehyde was more stable, and was able to displace those already formed by using Ehrlich reagent or vanillin. The yellow Schiff's base from aniline and vanillin was also decolorized by acetone in acid solution, but more slowly. The kinetics of these competitive reactions would make an interesting study. It is evident that carbonyl compounds can interfere with the color reactions which form the basis of the quantitative spectrophotometric method described in this paper. ACKNOWLEDGMENT
The author thanks B. A. J. Alexander for technical assistance, and K . Hashimoto for collaboration with the chromatography. RECEIVED for review January 29, 1968. Accepted March 25,
1968.
VOL. 40, NO. 8, JULY 1968
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