Spectrophotometric Determination of Microgram Quaatities of Ethyl Acetoacetate FRANCIS
N. McMlLLAN
Biochemical Research Section, Armament Test Equipment Laboratory, Air force Armament Center, Eglin Air force Base, Fla.
-4 rapid, simple, and accurate method for determining microgram quantities of ethyl acetoacetate depends upon the ultraviolet absorption of the ethyl acetoacetate carbanion. The carbanion is formed by adding 1 ml. of approximately Q.1N aqueous sodium h>-droxide to 5 ml. of the ethyl acetoacetate in water. Beer's law is obeyed in the concentration range of 1 to 7 p.p.m. at 272.5 mfi. The determinations in this range had a calculated average probable error of zt0.9%.
k = 3.2 X sec.-1 (ti/* = 38 minutes a t 24" C.). Once enough sodium hydroxide is added to obtain the maximum absorbance, the addition of more sodium hydroxide has no effect on the rate a t which the absorbing species disappears. As the absorbing species is not stable, the best analytical results are obtained by reading its absorbance a t a predetermined time after the addition of sodium hydroside. Three minutes was chosen as a convenient time in this investigation. METHOD
ETHODS for the deterrniiiation of small concentrations of ethyl acetoacetate are numerous. Most of the methods can be classified into three groups: methods depending on the purple ferric-ethyl aceto acetate complex (1, 9, 7 ) ; methods depending on the condfnsation of ethyl acetoacetate with salicylaldehyde ( 4 ) ; and methods depending upon the Japp-Klingemann reaction (3, 6). Hon-ever. none of these methods met the requirements of this laboratory. The method developed here depends upon the strong ultraviolet absorption of ethyl acetoacetate in aqueous sodium hydroxide (Figure 1) a t a wave length of 272.5 mp ( 5 ) .
Reagent. Use approximately 0 . 1 S sodium hydroxide. Standards. Use vacuum-distilled commercial grade ethyl acetoacetate. Weigh about 0.5 gram of ethyl acetoacetate into a 50-ml. volumetric flask containing approximately 20 ml. of distilled water. Pipet 10 ml, of this solution into a I-liter flask, make to volume with distilled water, and designate it as stock solution. Prepare standards containing about 0.2 to 7 p.p.m. from the stock solution by diluting the proper quantities of gtock solution in volumetric flasks with approsimately 1% et,hyl alcohol (by volume) in distilled water.
Table I. No. of Detns.
9 9 9 9 Q
Ethyl Acetoacetate,
P.P.M. 0.20 0.51
Probable Error". P.P.M. +0.03
+ O 04 +0.01 &0.05 3~0.03 =to.o4 +o. 02 +0.02
1.02 2.04 3.06 4.08 5.10 6.08
7.14
f0.03
Absorbance 0.027 0.069 0.138 0.277
0.416 0,554 0.692
0.825 0.969
Standard deviation X 0.67.
Procedure. Pipet 5 ml. of standard or unknown into a test tulle. Add 1 ml. of approximately 0.1A7sodium hydroxide from a pipet and mix. Read the absorbance at 272.5 mp against a reference blank prepared from 5 ml. of distilled water and 1 ml. of the same sodium hydroside solution. Plot the absorbance against concentration to get the standard curve. Typical data, obtained a t room temperature (22" to 24' C.), are shown in Table I.
L
230
a44
250
W
Figure 1.
Ethyl Acetoacetate Determination
L
V
*bo I L
210
L
U
G
I
Y
,
?so m p
2%
:
Ultraviolet absorption spectrum of ethyl acetoacetate carbanion
When sodium hydrokick is added to ethyl acetoacetate in large excess, the carbanion of ethyl acetoacetate is formed immediately. With a sufficient excess of sodium hydroxide, the maximum absorbance is obtained. Based on maximum absorbance of the ethyl acetoacetate carbanion, the molar estinetion coefficient was calculatrd to he 21,900. The absorbing species disappears with a pseudo first-order specific rate constant
DISCUSSION
Ethyl acetoacetate reacts rapidly ll-ith excess sodium hydroside to give a carbanion which absorbs strongly in the ultraviolet. This absorbance furnishes the basis for a fast, easy, and accurate method for assaying ethyl acetoacetate in microgram quantities. The absorbance of the ethyl acetoacetate carbanion can also be used to determine the ionization constant of ethyl acetoacetate when the hydronium ion concentration is known. In addition, the absorbance permits the determination of the reactive intermediate a t any desired time. Therefore, the kinet,ics of the base-catalyzed decomposition of ethyl acetoacetate can be determined without making the steady-state approsimation. Furthermore, it seem? probable that this technique can 1532
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
1533
bc applied to other pseudo acids Khich form resonancestabilized carbanions. Other studies are n o v being conducted and will be rrported 1at)er. LITERATURE CITED
Jonas, J . Lab. Clin. M e d . 2 4 , 20W3 (1938). (2) Kleeberg, J., Biochem. 2. 219, 381-4 (1930).
( 3 ) lioblin, A., U. S. Army Chelnical Corps, CRLR 195 ,t933) 19349 450-4. (4) Le Fevrei R.J. w.r J. Chem. (5) Morton, R. A., Rosny, W.C. I-.,Ibid.,1926, 706-13. (6) Rosenthal, S. AI., J . Bid. Chem. 179, 1233 (1949) ( 7 ) Zwarenstein, Harry, J . Lab. Clin. M e d . 30, 172 (1945).
(1) Kamlet,
RECEIVED for review March 17, 1956. Accepted J u n e 20,
19Si.
Near-Infrared Spectra of Fatty Acids and Some Related Substances RALPH T. HOLMAN Hormel lnstitute and Department of Physiological Chemistry, University of Minnesota, Austin, M i n n .
PAGE R. EDMONDSON Department o f Medicine, University of Minnesota M e d i c a l School, Minneapolis, M i n n .
The spectral absorption of a series of fatty acids and other lipides has been measured betw-een 0.9 and 3.0 microns. By means of these spectra, band assignments have been made for many organic structures. I t is possible to distinguish cis double bonds, terminal double bonds, hydroxyl groups, amine groups, acyloin, hjdroperoxide, methyl and ethyl esters, acids, CH2, and CHI groups. Near-infrared spectra should simplify characterization of many common chemical structures, and may be valuable in the solution of many problems in lipide chemistry.
3500
3750
4000
5000
4500
6000
ACETIC ACID
7000
T
HE study of the structui,c and composition of many natural
and synthetic fatty substances has been facilitated by the use of ultraviolet and infrared spectra. The ultraviolet absorption spectrum is generally useful for detection and measurement of conjugated unsaturated systems, and the infrared spectrum indicates the types of interatomic linkages and structural groups present. Until recently the conventional spectrophotometric equipment did not allow the measurement of spectra in the near-infrared range. The practical limits of ultraviolet and visible spectrophotometers are about 0.2 to 1.1 microns, and thcse are rarely
7
FREQUENCY, CM.-' 9000 3750
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6000 7OOC ~
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BUTYRIC ACID
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VALERIC ACID
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16
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28
WAVE LENGTH M I C R O N S
26
24
22
20
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