rnent of both reagents gives a higher color intensity. i\ eoncentration of 0.006% fructose results in a pinkpurple color m d when the concentration is increased to 0.1% or more a brown color is obtained. Cysteine a t concentrations of 3% and higher produces pink-purple and purple colors. A combination of 0.5yo cysteine and 0.002yo fructose as the reagent for carrying out the reaction proved to be most suitable. In an effort to decwase the reaction time to a minimum by raising the temperature, the effect of oxygen on the reaction was studied. When the reaction was carried nut at elevated tempcrat,ures to accelerate the rate and degree of the rca,ction, the color disappcarcd. The effect of osygcn on the reaction a t high temperatures is shown in Figure 1. 'I'riplicat,e samples were prepared at diffcrent concentrations and were treated as follows: The first set of samples was heated in ope11 test tubes, the sccond sct of snniplcs was stoppered with riibhrr stoppcrs, and the third set was heated under a stream of nit,rogcn. In thc second sct of samples a dwreasc in volume and, therefore, a dwreasc in prcxjure were noticed, for it was d i f f i d t to rcinovc the rubber stoppers from thc test tubes. This suggestcd that oxygm was used up during the reaction m d this resulted in a part,ial disappearance of the color.
II. Reproducibility of Results F ~ Concn. ~ of Unconverted ~ ~ ~ tation 9a-FluorohydrocortiTime, __ sone, pg./Ml. Hours Sample 1 Sample 2 Range 1 292 295 3 .O
Table
43
195
191
4 0
117 122
0 0
0 0
... .
I
.
Heating the samples under a stream of nitrogen protected the reaction mixture during the process. The most important factor influencing the velocity of the reaction was the temperature. An increase in temperature accelerates both the rate and degree of reaction. The reaction was carried out at room temperature (about 30" C.), 40°,70°, and 100" C. Figure 2 illustrates the relationship between temperature and time of heating necessary to reach equilibrium. The equilibrium state is reached in about 130 hours a t room temperature but in 1 1 minutes a t 100" C. At temperaturrs of 40" and 70" C.'a state of equilibrium was obtained in about 55 minutes but with a very low Golor development. The necessity for completing the test in a minimum of time led to the use of a high tcmperature to
increase the speed of the rraction. Substances used in the preparation of a -fermentation media contribut,c to the color developed in fermentation extracts. These substances can be eliminated from a sample by washing the methylene chloride sample with buffer solution. Washing the methylene chlorido extract with the buffer for 10 seconds resulted in a 0.5 to 1.0% loss of steroid into the buffer layer. The loss increased up to 12% after the 5minute washing. In samples taken during the latter stages of fermentation a brown color sometimes interferes in the determination of absorbance. This interference can be eliminated by extracting the steroid into the acid layer and washing off the impurities with methylene chloride. T o check the reproducibility of results, analyses of several fermentations were performed in duplicate. Results of a typical run are given in Table 11. The data indicate that results can be reproduced within *l'% at higher concentrations, and at lower conccntrations the results differ by *3.0y0from the average. LITERATURE CITED
(1) Rehm G . R., Slack, S. C., Mnder, W. J., ANAL.CHEM.31, 749-66 (1959). ( 2 ) Sih, C. J., Pan, S. C., Bennct.t, R. E., Ibid., 32, 669 (1960). RECEIVED for review January 81, 1961. Accepted May 9, 1961.
Microdetermination of Acetals of Acetaldehyde, Vinyl Ethers, and Other Compounds Containing Cornbined Acetaldehyde Groups MALCOLM C. BOWMAN,' MORTON BEROZA, and FRED ACREE, Jr. Enfomology Research Division, U. S. Department of Agriculture, Orlando, Ha., and Beltsville, Md.
b A method has been developed for determination of the pyrethrin and carbamate synergist, sesamex, which is also useful for determination of other compounds that liberate acetaldehyde on acid hydrolysis. The compound i s hydrolyzed with sulfuric acid, and the acetaldehyde, after steam-distilling, is treated with a solution of p-phenylphenol and cupric sulfate in concentrated sulfuric acid to give a violet color absorbing at 572 mp. No interference from the insecticides synergized by sesamex was encountered. The method is potentially useful for the structure determination of combined acetaldehyde groups in minute amounts of compound.
T
analytical method described here stemmed from a desire to determine micro Rmounts of scsamex ( 4 ) [Sesoxane, 2-(2+thoxycthoxy)ethyl 3,4methylenrdioxyphrnyl acetal of acetaldehyde] in mi\tures with the insecticides it syncrgizes. This method appears to be gericrally useful for the determination of compounds containing combined acet:iltlchyde groups that can be liheratcd by trcntrnent with an aqueous mincrnl :~cicl. Examples are acetals of ac~tnldchydr,ncetaldrhydc polymers, and vinyl ethers. Vinyl cthcrs have recmtly betn made available ?ommercially and products, like sesamex, ' I'resent address, Campbell Soup Co., Camden, N J. HE
based on vinyl ethers are being developed and may be amenable to the prcsent method of analysis. The method is a modification of the one employed by Barker and Summerson (1) for lactic acid in biological materials, by Stotz (13) for acetaldehyde in blood, and by Giang and Smith (9) for metaldehyde (tetramer of acetaldehyde) in plant material. The compound is hydrolyzed with sulfuric acid; the acetaldehyde generated is distilled into aqueous sodium bisulfite and reacted with p-phenylphcnol in the presence of concentrated sulfuric acid and cupric ion. A violet color having an absorption maximum a t 572 mw (Figure 1) is produced. V O L . 33, NO. 8, JULY 1961
1053
'Jh, mnlysis is simple to run, it is iici,iir:ite to within 3y0in the microgram mngr, and rrsultR are reproduciblc. APPARATUS
Absorbance measurements were made with a Bcckman Model DU spectrophotometer in s uare Corex cells having a I-cm. light pat\. The reaction apparatus, described b Giang and Scheehter (8), is an alli s , systrm with two vertical conienscrs that permit refluxing and then distillation in the same apparatus without transfer. REAGENTS
The sulfuric acid-cupric sulfate p phenylphenol, and standard metaldehyde reagents were prepared as described previously (9). PROCEDURE
Preparation of Standard Curve. A test tube (15 X 150 mm.) containing 5 ml. of 29ib sodium bisulfite solution (freshly prepared) was attached to the reaction apparatus so t h a t the tip of the delivery tube extended to the bottom of the solution. The test tube was partly immersed in an ice bath during the reflux and distillation processes. Ten milliliters of 10% aqueous sulfuric acid (v./v.) in a 50-ml. distilling flask containing 1 t o 2 mg. of Carborundum powder waa chilled in an ice bath. The appropriate volume of standard metaldehyde solution was added to the flask, which was then attached to the apparatus. The mixture waa refluxed for 15 minutes while water maintained a t 5" C. was passed through both condensers. The water was then drained from the reflux condenser and the mixture was distilled until 3 to 4 ml. remained. The a p paratus was disconnected and both condensers and the delivery tube were washed with small portions of cold water, which were combined with the distillate. The chloroform was separated and extracted with three 10-ml. portions of cold water, which were combined with the aqueous distillate and diluted to 50 ml. in a volumetric flask. The solution was extracted with 5 ml. of redistilled hexane to remove traces of chloroform. One milliliter of the aqueous solution was mixed, by swirling, with 8 ml. of cold BUlfUriC acid-cupric sulfate reagent in a 15 X 150 mm. test tube partly immersed in an ice bath. The pphenylphenol reagent (0.2 ml.) was added in a similar manner, and then the tube was removed from the ice bath and allowed to stand in the dark a t room temperature for 1 hour. The tube wm heated in a water bath a t 100" C. for 90 seconds, then returned to the dark for about 30 minutes to adjust to room temperature. With distilled water in the reference cell, the absorbance was determined a t 572 mp. A standard curve was prepared by plotting concentration against the absorbance corrected for the blank of the reagents carried through the entire process. 1054
e
ANALYTICAL CHEMISTRY
--I
01
545
520
L---l--.L.570
595
~
.>
620
W A V E LENGTH IMILLIMICRONSI
Figure 1 .
Absorption curve of color
The color reaction conformed to Beer's law, and 1 p . of acetaldehyde produced an absortance of about 0.150. Analysis of Samples. Chloroform solutions of the samples for analysis were prepared by diluting 200 pl. of each to 50 ml. in volumetric flasks. The weights of the samples in these solutions were calculated from the volumes and the specific gravities of the samples. This procedure waa used in order to minimize any error resulting from the high volatility of some of the compounds. Each solution was then diluted with chloroform to contain 100 to 200 pg. of combined acetaldehyde per milliliter. Both 1-and Zml. aliquots of each solution were then analyzed by the method used for the preparation of the standard curve. Residue Analysis. Solutions containing 1.75y0 of Sevin (1-naphthyl methylcarbamate) and 17.5y0 of sesamex were made up in chloroform and in deodorized kerosine, a small quantity of chloroform being added to the latter t o facilitate solution. Two-tenths milliliter of the chloroform solution was applied to the inner surface of each of five glass dishes (90-mm. diameter) for residual analysis. The quantity of sesnmex was determined either immediately or after the dishes had stood a t room temperature for 3, 7, 10, or 14 days. Portions of the kerosine solution mere applied to another group of dishes, then aged and analyzed in the same manner. RESULTS AND DISCUSSION
Determinations of acetals and vinyl ethers were first attempted by the method of Giang and Smith (9) for the determination of metaldehyde, but the low and erratic yields of acetaldehyde indicated that a more vigorous hydroly-
Table
I.
Hours
Stability of Color a t
Absorbance
after
First
Reading 2 6 18 26 50
I n dark 0.6 6.9 13.4 17.0 30.7
25' C. %
LOSS,
In artificial light 3.7 10.0 26.5 33.2 49.2
si!: was necessary. Yields were inrrcased by using 10 yo sulfuric acid for hydrolysis instead of the 0.4 % concentration employed by Giang and Smith. Even with the higher acid eoncentration, quantitative yields were obtained only after the reaction mixture had been reflused for 15 minutes prior to distillation. The introduction of cold water in the condenser during the reflux period was especially necessary with highly volatile materials to prevent thcir loss by distillation before the hydrolysis could be effected. Like Stotz (IS), we found that 8 parts of acid to 1 part of water is probably the most practical proportion for color development, no color being obtaincd with acid-water ratios of 2 to 1 or less. No better substitute for p-phenylphenol was found, although 2,4-dinitro-O-phenylphenol, p-hydroxyazobenzene, o-phenylphenol, 2-hydrosy-5-(a-mcthylbenzyl)biphenyl, 2-chloro4-phenylpheno1, m-nitrophenol, 4-phenylazo-m-cresol, 1-naphthol, 2-naphthol, and p-nitrosophenol were tried. Only 2-ehloro-4phenylghenol produced color, but it waa about 60% LW strong as that obtained with p-phenylphenol. The data given in Table I show that the color fades after development, but dny error in fading is insignificant when absorbance readings are made a t the interval designated in the procedure. The absorbance blank differs with each preparation, but is usually 0.030 to 0.040. It is therefore advisable to run a blank with each set of analyses. The results of determining a number of vinyl ethers and acetals of acetaldehyde are shown in Table 11. In all instances the values found for aeetaldehyde were within 3% of the theoretical. The method has been used to determine the persistence of sesamrx residues on glass (Table 111). The presence of Sevin (1.75%) or pyrethrins (1.75%), the insecticides that sesamex synergizes (6, 7), did not interfere with the determination of the synergist in ehloroform or in deodorized kerosine solution. The method is potrntially valuable for the structure detemmination of compounds with combined acetaldehyde groups, but little work has been done on this problem. This potential may be realized when the present analysis of combined acetaldehyde groups is compared with that of combined formaldehyde groups, a method which has been more thoroughly studied (9, 9, 6). In the formaldehyde analysis, mineral acids liberate formaldehyde from methylene groups attached to oxygen, nitrogen, and sulfur--e.g., -OCH20-, -NCHzN-, SCH*S-, SCHZN--, -N=CH, (e). Acetals of acetaldehyde behave in a like manner when OR CHsCH' hydrolyzes with acid to
'OR
0H give CHsCH(
,
which is hydrated
OH acetaldehyde. Vinyl ethers hydrolyze to hemiacetals, which cleave further to give acetaldehyde: CHFCH-0-R
HOH
~Hf
CH,--CH< OH OR
-
HOH
-___
Table 11.
Recovery of Acetaldehyde from Compounds Containing Combined Acetaldehyde Groups
Compound Ether 2-Butoxyethvl vinyl
-L
Butyl vinyl Isobutyl vinyl 2-Ethylhexyl vinyl Ethyl vinyl
Similarly, one would expect CHaCH< attached like the above-mentioned methylene to nitrogrn or sulfur to yield acetaldehyde on acid hydrolysis. I t is not anticipated that all compounds with combined acetaldehyde groups should always be determined by the procedure reported here. Determination of combined formaldehyde groups ( 2 ) showed that more color could often be produced by varying such conditions as heating time and amounts of reagents. Such differences would be expected to apply also to the determination of combined acetaldehyde groups. An increasing demand exists for methods to determine the structurc of minute amounts of compounds that are being made available by some of the newer techniques--e.g., paper and gas chromatography. The present method, requiring only micro amounts of compound, may be of value for such determinations. Another potential use is for the determination of end ethylidene (CH8CH=) groups in a manner analogous to the ingriiious procedure of Bricker and Roberts ( 4 ) for determining end methylene groups. The procedure would involve osidation of the double bond to a vicinal pair of hydroxyl groups, which could then be split by periodic arid and the resultant acetaldehyde distilled and determined :
This type of analysis has already been carried out by Huggins and Miller (II), who determined l,%propanediol by oxidizing it with periodic acid to get acetaldehyde and formaldehyde; the acetaldehyde was selectively aerated, collected in bisulfite, and determined colorimetrically. To use the method for structural determinations would require full knowledge of interferences, and some of this information is already available. Barker and Summerson (1) listed 71 compounds that do not interfere when present in 50 to 10 times the amount of the
Methoxyethyl vinyl Acetaldehyde Diisobutyl acetal 2 4 2-Ethoxvethoxvl-
ethyl 3,~-meth~hencdioxyphenyl acetal (sesamex)
Amount, Added,
Acetaldehyde Equiv.,
rg.
rg.
rg.
%
343 686 312 624 306 612 644 1288 300 600 358 716
105 210 137 274 134 268 181 363 183 366 154 308
106 207 136 274 138 265 181 364 181 363 151 30 1
101 99 99 100 103 99 100 100 99 99 98 98
656 1312
166 332
168 331
101 100
904 1808
134 267
135 275
101 103
lactic acid for which they were analyzing. They also listed a number that do, and gave means of overcoming interferences. Their analysis ia similar to ours. Ethanol, acetylmethylcarbinol, acetone, and 2,3-butylene glycol do not interfere (I, IS). Diacetyl develops a green color in the analysis, which may be destroyed by preliminary treatment with periodic acid (Is). Lactic and pyruvic acid interfere and undoubtedly can be removed by preliminary treatment with an ion exchange resin in a manner similar to that of Markus ( l a ) , who removed interfering cations. Henry et al. (IO) reported that acetic acid gives a color with pphenylphenol; i t could also be removed with an ion exchange resin. The distillation step lends specificity to the analysis by separating the highly volatile acetaldehyde (b.p. 21’ C . ) from the nonvolatile interferences. Thus, Barker and Summerson (I), Stotz (Is),Westerfield (14, and Giang and Smith (9) were able to determine acetaldehyde in biological materials without interferences. However, in determining metaldehyde in plant material, Giang and Smith washed the chloroform extract of the plant with a 2% sodium bisulfite solution to remove free acetaldehyde and related materials. By the exercise of this precaution, their blank was kept low. The separation of acetaldehyde from formaldehyde (gives green color in test) by aeration has been mentioned (IO). As in most highly sensitive methods, care must be taken to avoid contamination. Lubricating grease, chromic acid (from cleaning solution) , perspiration, even the analyst’s breath in blowing out a pipet have been reported to interfere
111.
Table
Persistence of Sesamex Residues on Glass
Days after Application 0
3
7 10 14
Acetaldehyde Recovered
Recovery, Mg./Sq. Cm. Amlied in A plied in deb;lorised ch7oroform kerosine 0.65 0.60 0.50 0.47 0.47
0.66
0.60
0.60 0.47 0.44
with the acetaldehyde determination (I,Q,
18). LITERATURE CITED
(1) Barker S. B., Summerson, W. H., J . Biol. dhem. 138,535 (1941). (2) Berosa, M., ANAL. CHEM.26, 1970 (1954). (3) Bricker, C. E., Johnaon, H. R. IND. ENQ.CHEM..ANAL. ED. 17, 40d
’ 159 (1950): (13) Stotz, Elmer, J . Biol. C h a . 148, 585 (1943). (14) Westerfeld, W. W., J . Lab. Ctin. Mcd. 30,1076 (1945).
RECEIWD for review January 11, 1961. Accepted April 28,1961. VOL. 33, NO. 8, JULY 1961
* 1055
