Determination of Alpha, Beta-Unsaturated
Acids and Esters by Bromination FRANK
E. CRITCHFIELD
0
Development Department, Union Carbide Chemicals Co., Division o f Union Carbide Corp., South Charleston, W. Va. ,Under ordinary conditions alpha, beta-unsaturated acids and esters react slowly with bromine. However, when these compounds are converted to the corresponding sodium or potassium salts, the addition of bromine proceeds rapidly and smoothly. This principle has been used for the development of a bromination method for these conjugated unsaturated compounds. The alpha, beta-unsaturated acids are converted to sodium salts by neutralization with sodium hydroxide using phenolphthalein indicator. The salts are then brominated using a modification of the Kaufmann bromine-bromide reagent. In the case of unsaturated esters, potassium salts are formed by saponification. The excess potassium hydroxide is neutralized and the salt is then brominated. The method has been applied to the determination of the purity of 14 alpha, beta-unsaturated acids and esters. The standard deviation for the determination of the purity of ethyl acrylate for 15 degrees of freedom is 0.1 1. Compounds that interfere are discussed.
S
of the methods for the determination of alpha, beta-unsaturated acids and esters are based on the addition of a nucleophilic reagent to the unsaturation, such as morpholine (4, sodium sulfite (S), and dodecaneEVERAL
Table I.
thiol ( I ) procedures. Although alpha, beta-unsaturated acids and esters do not halogenate readily, several halogenation methods have been applied to some compounds of this class. These include the mercury-catalyzed bromate-bromide procedure (6) and the pyridine sulfate dibromide procedure ( 7 ) . Under ordinary conditions the bromine-bromide reagent of Kaufmann (6), or the modification of Byrne and Johnson (2) cannot be used for the quantitative determination of the unsaturation of acrylic-type acids and esters because the rate of addition of bromine is too slow. \T7illiams found that the rate of addition of bromine to the acrylate ion is approximately 300 times faster than to the relatively un-ionized acid (8). This principle has been used in these laboratories for several years for the determination of the unsaturation of acrylictype acids. The acid is first converted to the sodium or potassium salt by neutralization. The salts are then brominated using a modification of the Kaufmann reagent (6). In this investigation, this procedure has been extended to the bromination of alpha, beta-unsaturated esters and other alpha, beta-unsaturated acids and is particularly useful for determining maleic and fumaric esters. The unsaturation of these esters is difficult to obtain liy other methods. Although man! alpha, beta-unsaturated com-
Reaction Conditions for Alpha, Beta-Unsaturated Compounds by Bromination
Compound Acrylic acid
Reaction Conditions Saponification, Bromination, time, min. time, min." 5 to 60 5 to 60 5 to 60 15 to 60 5 t'o 60 5 to 20 45 to '60b 45 to 60"d 45 to 60b 30 to 6OC 30 to 60 45 to 60c.d 30 to 60 30 to 6OC 5 to 60 5 to 60 5 to 60 5 to 60 ... 45 to 60d ... 30 to 60
Butyl acrylate Cellosolve acrylate Crotonic acid Dibutyl fumarate Dibutyl maleate Diethyl fumarate Diethyl maleate Ethyl acrylate Ethyl crotonate Fumaric acid Maleic acid Methacrylic acid Methyl methacrylate 30 t o '60 a Use 10-ml. aliquot of sample dilution unless indicated by.c * Use 10 ml. of acetone as cosolvent. Use 15-ml. aliquot of sample dilution. Omit solid sodium bromide.
1406
ANALYTICAL CHEMISTRY
15 t o 60 20 to 60
pounds can be brominated using the catalyzed procedures (6, 7), substitution reactions tend to occur when these methods are used. The procedure presented here is relatively free from such difficulties. REAGENTS
Bromine-bromide reagent, approsimately 0.2147 in bromine. Transfer 5.5 ml. of reagent grade bromine to a 1000ml. volumetric flask containing 500 ml. of methanol and 100 grams of reagent grade sodium bromide. Dilute to 1 liter with methanol and mix thoroughly. Transfer the reagent to a 1-quart screwcap bottle. Fit the bottle with a twohole rubber stopper and through one hole insert a 25-ml. pipet so that the tip extends belo\y the surface of the liquid; through the other insert a short piece of glass tubing to n.hich is attached an aspirator bulb. Sodium bromide, saturated aqueous solution. Transfer 500 grams of reagent grade sodium bromide and 500 ml. of distilled water to a 1-quart screwcap bottle, cap, and place on a mechanical shaker for at least 8 hours. PROCEDURE
Alpha, Beta-Unsaturated Esters. Transfer 2 5 . ml. of 1.ON potassium hydroxide into each of two 100-ml. volumetric flasks. Reserve one flask as a blank. Into the other introduce a n amount of sample containing not more than 35 meq. of unsaturated compound. Add the indicated amount of cosolvent t o each flask (Table I). Stopper the flasks and place on a mechanical shaker for the time specified in Table I. Remove the flasks from the shaker and add 2 or 3 drops of a 1.0% solution of phenolphthalein in methanol. Neutralize the contents of each flask with 0.5,V hydrochloric acid to the disappearance of the pink color. Carefully add 0.LY sodiuni hydroxide until the pink color reappears. Dilute to 100 ml., stopper, and mix. Continue with the bromination procedure described below. Alpha, Beta-Unsaturated Acids. Transfer 15 t o 20 nil. of distilled TTater t o each of t n o 100-ml. glassstoppered volumetric flasks. Reserve one flask for a blank. Into the othcr introduce a n amount of sample containing not more than 35 meq. of unsaturated compound. Add 2 or 3 drops cf phenolphthalein indicator t o each flask and neutralize with
0.5S sodium hydroxide just to the appearance of a pink color. Dilute the 100 ml. with distilled water, stopper, and mix. Continue with the bromination procedure. Bromination. Transfer 10.0- or 15.0-ml. aliquots (Table I) of the sample and blank dilutions to repective pairs of 250-ml. glassstoppered Erlenmeyer flasks. Add sufficient distilled water to make a total of 20 ml. Add 10 ml. of saturated sodium bromide to each flask and swirl to mix. Add 4 to 6 grams of reagent grade sodium bromide to each flask. Pipet 25.0 ml. of the brominebromide reagent into each flask, filling the pipet by pressure from the aspirator bulb. Allow the samples and blanks to stand a t room temperature for the time specified in Table I. Add 7 5 nil. of methanol and 10.0 ml. of 15% potassium iodide solution to each flask. Titrate immediately with standard 0.LY sodium thiosulfate to the disappearance of the yellow color. The difference between a blank and sample titration is a medsure of unsaturated coin pound.
Table 11. Effect of Saturated Sodium Bromide on the Bromination of Maleic Acid as the Disodium Salt
(Bromination time, 15 min.) Saturated Sodium Purity, 95.8 96.8 97.8 98.4 98.4
20
25 REACTION TIME, m8n a!
25L5'C
Figure 1 . Rate of bromination of maleic acid and its sodium salts 1. 2. 3.
Disodium salt Monosodium salt Free acid
DISCUSSION
Bromination of Sodium Salts. Vsing a modification of the Kaufmann bromine-bromide reagent, the rate of bromination of alpha, beta-unsaturated acids can be greatly accelerated by converting the acids to the sodium or potassium salts. This effect is shonn in Figure 1 for maleic acid. The reaction rate curves in this figure show t h a t the disodium salt reacts quantitatively with the reagent, while the monosodium salt and the free acid brominate too slowly for quantitative determination. This same effect is obtained N ith acrylic-type unsaturated acids that contain only one carboxylic acid group. For the bromination of alpha, betaunsaturated acids, the acids are converted to the corresponding sodium salts by titration with sodium hydroxide using phenolphthalein indicator. The neutralization must be performed carefully because excess sodium hydroxide consumes bromine; on the other hand, the free acid will not readiiv brominate. In the case of unsaturated acids that are stronger than acetic acid, the acids can be overtitrated with sodium hydroxide and acidified with a few drops of an acetic acid solution to prevent bromine consumption by sodium hydroxide. This modified procedure cannot be used for acids n-eaker than acetic because a portion of the sodium salt of the weak unsaturated acid Ivill be converted to the free acid and \\ill not brominate quantitatively. For this reason the modification is not specified in the Procedure Section, which is designed to be general in nature. Effect of Sodium Bromide. I n the original Kaufmann bromine-bromide
yo by mt.
Bromide, MI. 5 10 15
I
0
5
I
IO
I
IS 20 METHANOL, mi.
1
I
28
M
Figure 2. Effect of methanol on unsaturation of ethyl acrylate during saponification Saponification time a t 98' C., 25 min., bromination time, 15 min.
reagent the function of sodium bromide was t o stabilize the reagent and to inhibit substitution reactions. When a modification of this reagent is used for the bromination of the sodium salts of alpha, beta-unsaturated acids, the amount of sodium bromide in solution affects the rate of addition of bromine to the unsaturation (Table 11). Under the conditions of the method, 20 ml. of saturated sodium bromide are necessary for quantitatix-e bromination of disodiuni maleate in 15 minutes a t room temperature. Because sodium bromide accelerates the rate of bromination of the sodium salts of the acids, the sodium bromide solution must be saturated. In the procedure finally adopted, solid sodium bromide is added to the reaction flask t o ensure a saturated reaction solution and, therefore, a maximum reaction rate. In the case of the fumarates, solid sodium bromide cannot be used because disodium fumarate is not soluble in a reaction solution saturated with the bromide. This is not serious because disodium fumarate brominates within a reasonable length of time (Table I), without solid sodium bromide. When solid sodium bromide is present during the bromination of the sodium salts of unsaturated acids other than
fumaric, the addition of water to the reaction medium accelerates the rate of bromination. This occurs because water tends to dissolve the sodium bromide, increasing the bromide ion concentration of the reagent. Vhen solid sodium bromide is not present during the bromination (fumarates), moderate amounts of \\ ater have little effect upon the bromination rate. Saponification of Esters. Esters of alpha, beta-unsaturated acids can be brominated readily by first converting them to the corresponding sodium or potassium salts, by saponification with an excess of aqueous potassium hydroxide. Alcoholic potassium hydroxide cannot be used for the saponification because the alcohol will add to the unsaturation under the conditions of the saponification (Figure 2). The curve in Figure 2 vias obtained by saponifying ethyl acrylate with 1.ON potassium hydroxide in the presence of various amounts of methanol. The excess potassium hydroxide was neutralized and potassium acrylate was brominated using a modification of the Kaufmann reagent. Because the unsaturation available for bromination decreases as the methanol concentration increases, large amounts of primary alcohols cannot be used as cosolvents or be present in samples t o be analyzed. Aqueous potassium hydroxide is specified for the saponification. Many of the esters listed in Table I are not soluble in the saponification medium but this can be overcome in most cases by agitating the reaction mixture during the saponification. For those esters that cannot be reacted into solution, acetone can be used as a cosolvent. There should be the same amount of acetone in the blank, because a slight interference is obtained with this solvent during the bromination. The use of acetone as a cosolvent has been specified in Table I for the higher maleates and fumarates. RESULTS
The data in Table I11 compare the purity of several alpha, beta-unsaturated esters and acids by the procedure described with independent purity determinations. I n most cases the methods agree within 0.7%. The data VOL. 31, NO. 8, AUGUST 1959
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Table 111.
Determination of Purity
of Alpha, Beta-Unsaturated Compounds b y Bromination Average Purity, Wt. Bromination
Acrylic acid 98.4 f 0 . 2 (3) Butyl acrylate 98.0 i 0 . 1 (4) Cellosolve acrylate 94.5 i 0 . 1 (2) Crotonic acid 9 9 . 7 f 0 . 1 (4) Dibutyl fumarate 9 9 . 8 f 0 . 0 (2) Dibutyl maleate 96.5 i0 . 1 (2) Diethyl fumarate 99.4 i 0 . 1 (2) Diethyl maleate 9 8 . 3 i 0 . 1 (2) Ethyl acrylate 99. o e Ethyl crotonate 97.6 f 0 . 1 (2) Fumaric acid 9 9 . 2 i 0 . 1 (3) Maleic acid 98.6 It 0 . 1 (3) Methyl methacrylate 9 9 . 5 i 0 . 1 (3) a Figures in parentheses represent number of detns. Morpholine method (3). Sodium sulfite method (2). d Saponification. Std. dev. for 15 degrees of freedom is 0.11. f Total acidity.
in Table I11 show that for most of the compounds studied, a precision to *O.l% was obtained. I n the case of ethyl acrylate the standard deviation for 15 degrees of freedom was 0.11. The method has been used for the determination of the purity of maleic and fumaric esters. The unsaturation
Other 98. 5b 98.4b 95.46 99.9c 99.v 97. 2d 98. 7 c 98. 7c 99. O b 98. Ob 99.9f 99. Of 99.5b
of these compounds is difficult to obtain by other methods. The morpholine method (4) can be used if a conductometric titration is employed. This requires a special instrument and is timeconsuming. The sodium sulfite method (3) is applicable to only the lower esters because of solubility difficulties.
In general, compounds that interfere in the Kaufmann bromine-bromide method will interfere in this procedure. Large quantities of alcohols may interfere. Primary alcohols will interfere in the determination of esters but not in that of acids. Secondary alcohols and aldehydes are oxidized by bromine. Many of the interferences due to oxidation or substitution can be inhibited by conducting the bromination a t 0' C. LITERATURE CITED
(1) Beesing, D TV., Tyler, W. P., Kurta,
D. M . , Harrison, S. A., ANAL. CHEM.
21, 1073 (1949). (2) Byrne, R. E., Johnson, J. B., Zbid., 28, 126-9 (1956). (3) Critchfield, F. E., Johnson, J. B., Zbzd., 28,73 (1956). (4) Zbid., 28, 76 (1956). (5) Kaufmann, H. P., Z. Untersuch. Lebensna. 51, 3 (1926). (6) Lucas, H. J., Pressman, David, I N D . E N G . CHEhf., ANAL. ED. 10, 140 (1938). (7) Rosenmund, K. TV., Kuhnheim, W.,
Rosenherg-Gruszynski, D., Rosetti, H.,
Z. Unterszich. Nahr. u. Genussm. 46,
I54 119231. (8) Williams, G., Trans. Faraday SOC.37, 749 (1941).
RECEIVED for review November 13, 1958. Accepted April 24, 1959.
Dye-B ind ing Ca pacities of EIeven Electrophoretically Separated Serum Proteins R. D. STRICKLAND, T. R. PODLESKI, F. T. GURULE, M. L. FREEMAN, and W. A. CHILDSI Research Division, Veterans Administration Hospifal, Albuquerque,
b The use of dye-binding capacities for estimating serum proteins following their separation by electrophoresis in agar has been made possible by the availability of a gravimetric method suitable for measuring dye uptake of micro amounts of proteins. When 1 1 electrophoretically separated serum proteins were tested as to their abilities to bind Amido Black 1 OB, bromophenol blue, and Ponceau 2R, not only did the various fractions behave differently toward the different dyes, but corresponding protein fractions from different sera varied widely in their abilities to bind the same dyes. Large errors can result from dependence on this method for estimating electrophoretically separated serum proteins.
pipcr
rlectrophoresis, proteins of serum usually estiiimtcd by staining electrop1iorogr:Im and measuring &Y:mpmwiit,
1408
0
ANALYTICAL CHEMISTRY
the are the the
N. M.
amount of dye that associates with each fraction (2, 3, 6). This method would be satisfactory if all serum proteins had equal dye-binding capacities: actually, they differ in their affinities for dye, so that some workers (1, 3, 4) have determined proportionality factors to bring amounts of protein estimated by dyeing into agreement with those estimated by the classical methods. Staining has not been evaluated as a means for measuring the amounts of proteins separated by electrophoresis in agar. The availability of a gravimetric method (6) makes possible the direct measurement of the dye-binding capacities of serum protein fractions. This paper reports a n investigation of the staining properties of 11 serum proteins with respect to three commonly used dyes. METHODS
Sera Fere contributed by six healthy
Caucasian males whose ages ranged between 24 and 45 years. The serum proteins were separated by electrophoresis in agar using the apparatus and techniques as described (5). The protein fractions were located and prepared as described (5) and before staining, each protein precipitate was washed once with 5 ml. of 10% trichloroacetic acid. The proteins were stained by suspending the precipitates in 2 ml. of a dye solution. Three dyes were tested: Ponceau 2R (National Aniline Division, Allied Chemical Corp.), Amido Black 10R (Hartman-Leddon Co., Inc.), and bromophenol blue (Hartman-Leddon Co., Inc.). The dye solutions were prepared by dissolving I00 mg. of a dye in 100 ml. of a snlvent consisting of 10% acetic acid, 45% methanol, and 45y0 distilled water (v./v.). The proteins remained in contact with the dye solu1 Present address, Department of Surgwy, University of Colorado School of Medicine, Denver, Colo.
