1591
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6 Table I. Amount of KNOI, Y 2 5 a
Reproducibility and Accuracy of Method
Range of Photometric Mean Photometric Readings, % T Density 59-61 41-43.6 24.5-26 11.5
0.2212 0.379 0.598 0.939
Standard Deviation As As photometric KiYOs, density Y 0.00625 0.0069 0.0071 0.0000~
0.105 0.115 0.119 0.0
coefficient of Variation,
%
2.83 1.82 1.19
10 ... 20 Same approximate error is not apparent, a s i t is read to nearest 0.5%
inhibit the organism or the extracellular enzyme. In order to obtain valid results, a given amount of nitrate is added to decreasing concentrations of test substance. The dilution necessary to allow desired activity is found in the tube giving a 1 0 0 ~recovery o of the added nitrate. The test can be performed in the presence of nitrite, provided the combined amounts of nitrite present a t the end of the incubation period are not greater than the highest level in the standard series.
T. ACKNOWLEDGMENT
the nitrite by the organism is extremely small. It is therefore possible to determine nitrate in the presence of nitrite.
The authors are grateful to E. E . Pickett for the instrumcnt,ction and to Laura &I.Flynn for technical advice. LITERATURE CITED
Assoc. Offic. Agr. Chemists, “Official Methods of Analysis,” 7th
LIMITATION
ed., p. 14, 1950.
Timing is important A standard curve must be obtained with each series of determinations to correct for variation in temperature, time, and enzyme activity. As in most microbiological tests, the greatest accuracy is attained in the central portion of the test range. The test has been run in the presence of 2 mg. of glucose per tube without affecting the quantitative relationship of nitrate conversion to nitrite, although the turbidity is increased. When the turbidity is increased, it may be either corrected for by a blank or eliminated by centrifugation. The organism is large and is easily spun out after treatment with the color reagent. Urea in concentrations as high as 8 mg. per ml. exhibited no detrimental effect. With rumen fluid a fivefold dilution is necessary to prevent inhibition. Nitrate content of urine has been successfully determined in a tenfold dilution. K i t h some samples of natural materials there may be substances present which
Ibid., p. 15.
Balks, R., Reekers, I., Landwirtsch. Forsch. 6, 121-6 (1954). Barnett, A . J. G., “Silage Fermentation,” pp. 157-9, Academic Press, New York, 1954. Dickinson, W. E., AXAL.CHEM.26, 777-9 (1954). Hamm, R. E., Withrow, C. D., Ibid., 27, 1913-15 (1955). Jones, B. G., Underdown, R. E., Ibid.,25, 806-8 (1953). Muhrer, M. E., Case, A. A., Garner, G. B., Pfander, W. H., J . A n i m a l Sci. 14, 1251 (1955). Kason, A,, Evans, I€. J., J . BioZ. Chem. 202, 655-73 (1953). Kelson, J. L., Kurta, L. T., Bray, R. H., ANAL.CHEM.26, 1081-2 (1954).
Nicholas, D. J. D., Kason, Alvin, hIcElroy, W. D., J. Biol. Chem. 207, 341-51 (1954).
Saltaman, B. E., ANAL.CHEM.26, 1949-54 (1954). RECEIVED for review April 19, 1956.
Accepted June 15, 1956. Contribution from the Missouri Agricultural Experiment Station, Journal Series No 1617. Publication approved b y the director.
Detection and Identification of Clinically Important Barbiturates LEO LEVI Food and Drug Laboratories, Department o f National Health a n d w e l f d r e , Ottawa, O n t , Canada CHARLES
E. HUBLEY
Defence Research Chemical Laboratories, Defence Research Board, Ottawa, Ont., Canada Clinically important barbiturates in commercial preparations and biological materials can be made to react with aqueous copper sulfate-pyridine solutions to give characteristic dark purple-colored derivatives of the composition (NCHCHCHCHCH), L
Cu(OCNHCOCR’R“CON),. Chemical I
A
evidence
pre-
I
sented suggests that the metathetical reaction proceeds via the interaction of a negatively charged, enolized barbituric acid ion with a positively charged copperpyridine complex ion. In accordance with this mechanism the dissociation constants of the barbituric acids are a major factor governing product yields. The reaction becomes more sensitive as the copper sulfatepyridine ratio in the reagent is increased or the pyridine-water ratio of the system is decreased. Infrared absorption data suggest that in the complex the barbiturate is bonded to the central metal atom through the carbonyl oxygen in the 2 position. The compounds show unique features throughout the region studied, 4000 to 650 cm.-’, and hence, the method affords a high degree of specificity for detecting and characterizing these drugs.
T
HE toxicologist and forensic chemist are often concerned with the detection and estimation of barbituric acid derivatives, which “are the organic poisons most frequently encountered in the toxicological examination of organs and body fluids from cases autopsied in the Medical Examiner’s Office’’ ( 2 6 ) . The barbiturates can be identified by inspection of the infrared absorption spectra of their copper-pyridine complexes, as well as the infrared absorption spectra of the free barbituric acids recovered from the complexes. Because of the ease with which the derivatives can be prepared and the barbiturates recovered, the method should prove a valuable tool for the microchemical identification and characterization of clinically important barbiturates. MICROCHEMICAL REACTION WITH AQUEOUS COPPER SULFATE-PYRIDINE SOLUTIONS
I n 1931 the Dutch chemist Zwikker ( 2 7 ) prepared a crystalline derivative of the commonly used sedative Verona1 (5,5-diethyl barbituric acid) by reaction of the barbiturate with an aqueous solution of copper sulfate in pyridine. He assigned to the compound the formula (barbital)&u(pyridine)z. The metal was determined by the Bruhns method after wet ashing the complex in a sulfuric-nitric acid system. The barbiturate was recovered by extraction with a chloroform-ethyl acetate mixture following
ANALYTICAL CHEMISTRY
1592 treatment of the compound with dilute mineral acid, and the pyridine was estimated titrimetrically after decomposing the derivative in excess 0.lN hydrochloric acid. No further physicochemical properties of the compound were described, however, nor was there any attempt made to advance a mechanism by means of which the process of complex formation could be explained. The nature of this reaction is discussed here.
EXPERIMENTAL
Materials. The barbiturates investigated in this study \?-ere obtained from the folloving sources of supply: Amytal, Seconal, AlPhenal, s. E.Massengill Eli LillY and C0.j Indianapolis, Co., Bristol, Term.; Alurate, Hoffmann-LaRoche Inc., N u t l e ~ , N. J.; Dial, Ciba Co., Ltd., Montreal, Que.; Ipral, E. R. Squibb & sons, N~~ Brunswick, ?J. J.; Luminal, hlebaral, WinthropSteams, Inc., Windsor, Ont.; Sembutal, Neonal, Abbott Labora-
Table I. Physicochemical Characterization Compound
Chemical name
Trade-name
5-Ethyl-5-phenylbarbituric acid
Luminal
Structural formula
S a m e in standard textsa Phenobarbital (U.S.P.) Phenobarbitone (B.P.)
’
CHz-CH3
’
CH?-CH=CHz
HN-CO
o=c
Molecular Formula ( C u H i i N z 0 ~ ) z (CsHIWI C~
\C/ CH-CH \ / \ // NCH HN-CO C
‘C H=C/H
5-.4llyl-5-phenylbarbituric
acid
HN-CO
Alphenal O=C
’C\
yCH
/ \ I
\
(CI~HI~SZO~)ZCU(C~H~S)?
CH-CH
HS-CO
C
‘\ c n=c H /,
5,5-Diall~~lbarbituric acid
Dial
Diallylbarbituric acid (N.S.R.) Bllobarbitone (B.P.C.)
HN-GO
/
O=C
Fh-CO 5-Methyl-5-phenylbarbituric acid
Rutonal
.....
/ \
\
Amobarbital (N.F.) Amylobarbitone (B.P.C.)
5-Ethyl- I-methyl-5-phenylbarbituric acid
Mebaral
Mephobarbital (N.N.R.) hfethylphenobarbitone (B.P.C.) Phemitone (B.P.)
CHI \C/
HS-CO
Amytal
CH?CH=CH?
HX-CO
O=d
5-Ethyl-5-isoamylbarbiturio acid
CH?CH=CH?
\c/
/\/
Verona1
Barbital (U.S.P.) Barbitone (B.P.)
HN-CO
o=c / \
HS-CO 5-Allyl-5-( I-methylbutyl) barbituric acid
Seconal
Secobarbital (N.N.R.) Quinalbarbitone (B.P. Add. ’51)
bH
/
CH3 / 5,5-Diethylbarbituric acid
CH-CH
\CH=CH
\c/
CHz-CH3
(CsHii~z03)zCu(CaHsN)1
/ \
CHz-CHI
H X-CO
C Hz-C
o=c/
H=C H?
(CizHi;S?Oa)zCu(CsHb5)2
\C/
&I-&
‘cH-cn?-c
H2-cHI
dH3 5-Ethyl-5-butylbarbituric acid
Neonal
Butethal (N.N.R.) Butobarbitone (B.P.C.)
HN-CO O=C
/ \
CHz-CHa
/ \
HN-CO 5-Ethyl-5-( I-methylbutyl) barbituric acid
Nembutal
Pentobarbital (U.S.P.) Pentobarbitone (B.P.)
(CioHisN?03)zCU(CaHbN)1
\C/
HN-GO
CH?-CHz-CH?-CHa
CH?-CHs
o=d C‘ / 2y-d
c\
H-c
H?--CH2-c
(CiiHi-NzOa)zCu(CsH~N)z
H3
CH3 5-.4llyl-5-iaopropylbarbituric acid
5-Ethyl-5-isopropylbarbituric acid
Alurate
Ipral
hprobarbital ( N . S . R . )
Probarbital ( S . S . R . ) O=C
H=C Hz
(CiaHi3N?03)zCu(CsHsN)z
C ‘/
/ \
HS-CO
CH(CHd2
HS-CO
CHZ-CHI
/
\C/
\
/ \
HS-CO a
C Hz-C
H N-GO
o=d\
(CsHiaSzOa)zCu(CaHsN)z
CH(CHs)z
U.S.P., U. S.Pharmacopeia; B.P., British Pharmacopeia; N.X.R., New and Nonofficial Remedies; B.P.C., British Pharmaceutical Codex
V O L U M E 2 8 , NO. 10, O C T O B E R 1 9 5 6
1593
tories, Ltd., Montreal, Que.; Rutonal, Poulenc Ltd., Montreal, Que.; Veronal, Merck & Co., Ltd., hIontrea1, Que. Chemical names and structural formulas of the compounds are recorded in Table I. Procedures. To 50 ml. of a 0.005Jf solution of the substituted barbituric acid in 1 to 1 aqueous pyridine (except Mebaral, which n a s dissolved in 3 to 1 pyridine-water solution) was added slowly and a-ith constant stirring an equal volume of a 0.086f aqueous copper sulfate solution containing 25y0by volume
of pyridine. After letting the reaction mixture stand for 72 hours with intermittent stirring, the purple-colored precipitate which formed was filtered off, washed successively with small portions of distilled water, ethyl alcohol, and ether, and dried in vacuo over phosphorus pentoxide. The roducts were analyzed for copper by igniting accurately weighel samples of about 0.1 gram to constant weight (copper oxide). The pyridine content was determined by titration with 0.05A’ acetous perchloric acid follonjng the procedure described
of Barbiturate-Copper-Pyridine Complexes Barbiturate-Copper-Pyridine Complex
Chemical name
Rlol. wt.
Copper, % Calcd. Found
Pyridine, % Calcd. Found
lfoles HClOi/mole
Decomposition range, e
c.
Melting Point of Recovered Barbituric Acid,
c.
j-Crhyl-5-phen3.lbarbituric acid
684.2
9.29
9.41
23.13
22.81
2,014
235-241
174.8-176.0
5-Allyl-5-phenylbarbituric acid
708.2
8.98
9.12
22.35
22.47
2.008
218-223
155.7-156.4
5&Diallylbarbituric acid
636.1
9.99
9.78
24.87
24.63
1.986
213-217
172.1-172.9
5-Methyl-5-phenylbarbituric acid
656.1
9.69
9.50
24.12
23.86
1.993
187-19 1
224.9-225.8
5-Ethyl-5-isoamylbarbituric acid
672.3
9.46
9.24
23.53
23.24
1.977
180-185
164.9-155. 7
5-Ethyl-1-methyl-5-phenyl barbituric acid
712.2
8.93
9.09
22.21
21.90
2.010
207-211
177.6-178.5
5,s-Diethylbarbituric acid
588.3
10.81
10.98
26.90
26.68
1.989
199-204
189.1-189.8
5-Allyl-5-( 1-methylbuty1)barbituric acid
696.3
9.13
8.94
22.73
22.40
1.978
177-183
87.4-88.2
5-Ethyl-5-butylbarbituric acid
644.2
9.87
10.05
24.53
24.37
2.014
179-184
126.9-128.2
5-Ethyl-5-(l-methylbutyl)barbituric 672.3 acid
9.46
9.63
23.53
23.36
2.009
160-163
127.8-128.6
5-.lllyl-5-isopropylbarbituric acid
640.2
9.93
9.68
24.72
24.45
1.970
189-193
140.2-141.4
5-Ethyl-5-isopropylbarbituric acid
616.2
10.32
10.04
25.68
25.33
1.983
188-192
201.2-201.0
ANALYTICAL CHEMISTRY
1594 by Levi and others (12). Melting points were taken on a FisherJohns apparatus using a heating rate of 5' C. per minute. The experimental data are shown in Table I. DISCUSSION
Reaction Mechanism. Under thc experimental conditions described, the over-all process was found to be in accord with the following sequence of intermediate reactions. Enolization and partial ionization of the barbituric acid derivative:
-
CO-NH
RH/
'CO-NH
RII'
PYRIDINE
CO-NH
R1\
\
c:
C O - N &'-OH
11
IONIZATION
CO-NH CO-N
Formation of a positively charged copper-pyridine complex ion, representing t,he reagent:
with the principles of Sidgwick ( a l ) , who explained the formation of complex compounds in terms of the electronic theory of valence I n accordance with the mechanism advanced, the pH of the barbituric acid solutions was found to decrease appreciably when the reagent was being added. Thus, during preparation of the Luminal-copper-pyridine complex, the pH of the system dropped from 7.01 to 6.48 on addition of the copper sulfate-pyridine solution (pH = 7.05). A pH depression of similar magnitude was observed in each of the reactions studied. Further evidence for the ionic nature of the process was secured by correlating the dissociation constants of the acids and product yields, as shown in Table 11. Under the experimental conditions, complex formation occurred instantaneously with Alphenal and Luminal ( K = 4.2 X 10-9 and 4.0 X 10-0, respectively), proceeded gradually with Dial, Rutonal, Amytal, Veronal, and Seconal ( K ranging from 1.9 X to 1.2 X 10-9), and occurred only sluggishly with Sembutal, Alurate, and Ipral ( K ranging from 1.4 X to 1.1 X lo-"). Also in accordance M ith these observations, complex formation failed to take place when the weak acid Evipal [5-(l-cyclohexen-l-yl)-3,5-dimethylbarbituric acid] ( K = 7.8 X was used for the reaction. However, the correlations do not rigorously apply to all compounds of the series-e.g., Alphenal, Neonal, and Alurate-and factors other than the degree of acidity of the barbituric acid derivative evidently codeterniine the course of the metathetical process.
Table 11. Dissociation C o n s t a n t s a n d P r o d u c t Yields
CH
\ , >N f
so:
CH=CH
CH=CH >N , CH-CH
CH
*
CH- CH
Interaction of the ionic species generated during these processes and simultaneous displacement of the equilibrium of the reaction toward the right:
Dissociation Constanto, Product Yields K x 109 % Theoretical' Substituted Barbituric Acid Luminal 4.0 4.2 Alphenal 1.9 Dial Rutonal 1.8 1.5 Amytal 1 4 Veronal 1.2 Seconal 1.5 Xeonal 1.1 Sembutal 1.4 Alurate Ipral 1.1 a Determined on 0.07.M solutious according to ( g o ) , using 50% aqueous ethyl alcohol a s solvent and 0.1.V aqueous sodium hydroxide as titrant.
t t
Behavior of Complexes in Aqueous and Nonaqueous Media. Zwikker found (37) that the barbital-copperpyridine complex decomposed in dilute mineral acid, a n d he estimated its pyridine content by dissolving the compound in excess 0.1N hydrochloric acid and back-titrating with standard alkali. Because 4 moles of the acid were consumed per mole of complex, it was assumed, in the absence of further data, that equimolar quantities Ti-ere used by the pyridine and the copper, respectively. Zwikker's experiment Tas confirmed, and the authors propose to interpret the mechanism of the reaction as follon~s:
t 2Ht t SO: R1'
CO-N
I,
L
R'
CO
- NH
p
CH - CH CH
\CH=CH/
t H,SO, R"\
/CO-N C
RI'
\CO-NH
CH=CH,
,,,/"\+
=' c-o-
N/ + CH- C
H
CH ~
(3)
CO-NH
R1\
CH- CH N+
\
CH= CH
CO-N
The enolization of barbituric acids in aqueous media, as illustrated in Equation 1, was investigated by Fox and Shugar (6), Loofbourow and Stimson (14), Stuckey (sa), and Wood (36). Studies on the coordination of nitrogenous bases with copper compounds, as shown in Equation 2, were made by Hoste (8), Ley and Hegge (IS), Pfeifferand Werner ( I @ , Pfeiffer and Glaser (16), and Taurins (34). Formation of the barbiturate-copperpyridine complex, Equation 3, follows interaction of the anion and cation released during the first two processes. The structures postulated for the derivatives are in accord
1
R' CU
CI,
'
2 HO ,
t2
\
Rll,~
N.HCI CH=CH (4)
V O L U M E 2 8 , NO. 10, O C T O B E R 1 9 5 6
1595
Two moles of the acid are used to generate 2 moles of the barbiturate and 1 mole of cupric chloride, while the other 2 moles are consumed in the conversion of the liberated base to its hydrochloride. Evidently, a molecular formula of ( C8H1103N2)2Cu(C6HbN)2, rather than [CBH1203( Nz?)]~CU( C&N)2 as proposed by Zwikker, is more in line with experimental observations. The formula shown in Pharmaceutisch Weekblad ( l 7 ) , ( C8H1203)2Cu(C&N)Z, actually lacks tn.0 nitrogen atoms and shows two excess hydrogen atoms. Yet the analytical data correspond to the composition (barbital)zCu(pyridine)z. Titrimetric analysis of the complexes in nonaqueous media proceeds as shown in Equations 5 and 6. CO
c'
- NH
CH-CH
/ , /N +
0 ,\ 4 \CO-N
c(
'
CO-N > c - 0
2CHc
CO - NH ,C=O 'CO-NH
CH - CH >N CH=CH
CH
CH=CH'
t
/"\
CO-NH
R"/
\
2 CH3COOH
1
CH=CH,
N' 3 CH- C
>
CH H ~
CH - CH
t 2 NQ
CH t CU(CH~COO),
CH = CH
t 2 HCIO, _* 2 CH4\
CH- CH >N.Ht CH= CH
+
2 ClO;
When the compounds are dissolved in glacial acetic acid, the metal is converted to copper acetate (greenish coloration occurs), while the barbituric acid and the nitrogenous base are simultaneously regenerated. Further evidence for the occurrence of this reaction stems from the finding that all but one of the barbituric acids precipitated on mere dilution of the system with water Melting points of the compounds, recovered by filtration and recrystallized from dilute ethyl alcohol, are shown in Table I. Only Seconal failed t o precipitate from the dilute glacial acetic acid solution of its complex, and this sedative was isolated by extraction with successive portions of chloroform. I n accordance with Equation 6 only 2 moles of perchloric acid were found to be consumed per mole of complex. No interference from the copper was encountered in these analyses because of its high degree of association with the solvent. Equivalence points, as indicated by distinct color changes of the systems from purple to blue-green, could always be readily detected. The observations made are, in some respects, reminiscent of those reported by Levi and Farmilo for the nonaqueous titration of amine-cadmium halide complexes in glacial acetic acid-dioxane solvent systems (9). Microchemical Value of Reaction. The products isolated from the reaction were found to be anhydrous and relatively stable compounds. They could be heated a t 100' C. for about 1 hour without suffering loss of weight or changing color. Decomposition, often accompanied by momentary melting to a greenish liquid, occurred over fairly narrow temperature ranges. With the exception of the Rutonal derivative, the complexes decomposed a t higher temperatures than the corresponding barbituric acids (Table I). The melting point of the Veronal complex was found to differ by about 35" C. from that of its Luminal analog, whereas the melting points of the pure drugs lie only 15' C. apart. Similarly, the Nembutal and Neonal complexes were found to decompose a t 160' and 180" C., respectively, whereas the pure compounds show practically identical melting points (127-9" C.).
The reaction was found to become more sensitive when the copper sulfate-pyridine ratio of the system was increased. Accordingly, a microchemical reagent was made by dissolving 10 grams of copper sulfate in 100 ml. of water containing 50 ml. of pyridine, and a series of tests on minute amounts of drugs was performed in the follon-ing manner. Samples of 10 mg. of barbiturate were weighed out accurately, dissolved in 1 ml. of 50% aqueous pyridine, and diluted to 10 ml. with distilled water. To aliquots of 1.0, 0.5, 0.1, and 0.05 ml. of solution, volumes of 0.1, 0.05, 0.01, and 0.005 ml., respectively, of the reagent were added. The barbiturate-copper-pyridine complexes formed gradually on standing, positive tests being obtained on as little as 100 y of material. The increased sensitivity of the reaction afforded by the procedure described substantiates the reaction mechanism postulated earlier, for comparison of the experimental conditions governing the microchemical reaction with those previously reported shows that in the final system the copper sulfate-pyridine ratio was increased by a factor of 4 per unit volume, whereby formation of the copper-pyridine complex ions necessary for reaction (Equation l ) was facilitated. Also, a study of the experimental data illustrating the dependence of the microchemical reaction upon p H (Table 111) shows that the sensitivity of the reaction is progressively reduced as the pyridine concentration of the system is increased. This phenomenon is probably due to interaction of the barbituric acid with the relatively strong base, pyridine ( K = 2.3 X to form molecular complexes as reported by Raeth and Gebauer (18). Because of this reaction the enolization process (Equation 1) is retarded and, hence, the concentration of the anions necessary for reaction is depressed.
Table 111.
Dependence of Microchemical Reaction on p H
(100 ml. of 0.005M Veronal solution in aqueous pyridine)
a
Pyridine. Vol. %
p H of System
1 5 10 20 50 75
6.81 7.17 7.39 7.61 7.99 8.12
Time for Crystal Formation= 3 minutes 12 minutes 36 minutes 2 hours 1 week
...
On addition of 0.05 ml. of reagent t o 1 ml. of solution.
Because of these factors maximum sensitivity of the reaction can only be attained when the pyridine-water and the pyridinecopper sulfate ratios are minimal. I Y F R A R E D A N A L Y S I S O F COPPER S U L F A T E P Y R I D I N E DERIVATIVES
In order to enhance further the value of the reaction just discussed as a method for detecting and characterizing barbituric acid derivatives, a study of the infrared absorption spectra of both the barbituric acids and their copper sulfate-pyridine complexes was carried out. A collection of these spectra is given and some of their characteristic features are interpreted. EXPERIMENTAL PROCEDURES
The compounds were prepared in accordance with the procedures already described. A few milligrams of material, reduced to a fine powder after prolonged grinding in a small agate mortar, was triturated with a droplet of refined mineral oil (Kujol), The resulting smooth paste was spread as a thin even layer between two sodium chloride disks, and the infrared absorption was measured in a Perkin-Elmer Model 21 double-beam recording infrared spectrophotometer equipped with rock salt optics. A sodium
ANALYTICAL CHEMISTRY
1596 chloride compensating window was placed on the path of the reference beam. Four potassium bromide spectra are included in this survey. They were kindly supplied by the Perkin-Elmer Corp., Norwalk, Conn. The Hausdorff potassium bromide pelleting technique was used for their preparation (6). RESULTS AND DISCUSSION
Spectra of 5,5-Substituted Barbituric Acids. The usefulness of the infrared method of analysis for differentiating barbituric acid derivatives was first demonstrated by Umberger and Sdams (26),who recorded the absorption spectra of most of the commercially available barbiturates. However, they examined these compounds as chloroform solutions; therefore, relatively few bands were observed between 8 and 12 microns and no absorption of analytical value was detected beyond this region. The present investigation has shown that considerable structure exists throughout the entire fingerprint region in the null spectra of these compounds and that many of the bands observed can be assigned to specific functional groups.
Table IV. N-H and C=O Absorption Bands in Infrared Spectra of Barbiturates and Barbiturates-CopperPyridine Complexes Compound Alphenal
N-H Frequency, E Barbiturate Complex
C=O Frequency, Cm. -1 Barbiturate Complex
3240 3140
3200 3100
1757 1737 1710
1724 1670
Alurate
3219 3105
3190 3070
1750 1723 1695
1715 1665
Arnytal
3240 3120
3200 3070
1757 1725 1697
1720 1665
Dial
3220 3100
3200 3080
1750 1723 1695
1705 1678
Ipral
3210 3100
3180 3085
1745 1725 1689
1719 1662
Lumina
3200 3100
3180 3080
1723 1665
hlebaral
3212 3105
3160 3075
1772 1737 1710 1759 1713 1693
Nembutal
3250 3130
3195 3080
1758 1730 1696
1715 1666
Neonal
3222 3105
3200 3075
1757 1725 1696
1705 11'76
Rutonal
3240 3120
3170 3080
1751 1720 1700
1720 1679
Seconal
3228 3120
3180 3060
1760 1727 1697
1710 1665
Verona1
3248 3120
3180 3060
1750 1714 1700
1715 1678
1705 1650
Characteristic N-H (bonded) absorptions always occur in the region from 3250 to 3050 cm.-I. Generally, two distinct bands are observed (Figures 1 to 6, Table IV): a strong one near 3200 em.-' and a less intense one near 3100 cm.-l. Straight-chain iVsubstituted ureas also show such doublets, but invariably a t shorter n7avelengths (3450 to 3350 cm.-l) ( 3 ) . In solution, splitting of these bands into three or more components has often been noticed ( I , 25). With the exception of Luminal, which shows marked absorption a t 3335 cm.-' (free N-H stretching vibrations), none of the barbiturates examined absorbs a t frequencies higher than 3300 em.-'. This finding indicates that enolization
processes, such as those occurring in alkaline media and leading to the formation of unassociated -OH groups ( 2 3 ) , do not take place in the solid state. Strong absorptions are observed in the spectra of the compounds near 1700, 1730, and 1750 cm. -1. These reflect the vibrational characteristics of the carbonyl groups, as either individual or coupled oscillators ( 1 7 ) . The low-frequency band is associated with vibrations of the carbonyl bond in the 2 position, because this bond, flanked by two nitrogen atoms from which it can draw charge, may assume a character more like a single polar bond : I
I
(7) The band a t about 1730 cm.-1 corresponds to motions of the carbonyl bonds in the 4 and 6 positions, attenuated because of simultaneous coaxial out-of-phase oscillations of the carbonyl linkage in the 2 position. S o such interaction enters into formation of the third band, which is due to vibrations perpendicular to the axis of symmetry of the molecule, involving the 4 and 6 bond exclusively and occurring a t the highest frequency (about 1755 em.-'). No bands of analytical significance appear in the region between 3250 and 3050 cm.-' and 1750 to 1700 cm.-l. The absorptions a t 2920 and 2860 em.-' (C-H stretching vibrations) are due to the medium. Characteristic C-H deformation and C--S stretching vibrations are observed from about 1470 to 1250 cm.-1, thelatter being particularly marked in the spectrum of Mebaral because in this compound an imino hydrogen has been substituted by a methyl group. In the fingerprint region a fairly strong band is always observed between 850 and 800 cm.-l (usually a t 842 & 8 cm.-l). Barbituric acid, the parent substance of this series of compounds, also shows these spectral features. Thus, these bands alone represent valuable information because they indicate the presence of a barbituric acid-type compound ( 1 1 ) . Much more can be learned, hon.ever, from closer examination of the profiles of these spectra. For instance, all barbituric acids containing an allyl side chain-e.g., hlphenal, Blurate, Dial, and Seconal-show marked absorptions at 995 i: 5 cm.-' and 935 & 5 cm.-l, which distinguish them from all other members of the series. These vibrations are always slowed down (shifted toward higher wave lengths) by about 1 micron when the hydrogen atom attached to the central carbon of the unsaturated side chain is replaced by a heavy atom such as bromine ( I O ) . Those barbiturates containing a phenyl group attached to the carbon atom in the 5 position may be readily differentiated from those carrying trro alkyl substituents in this position, because the former show three or four distinct bands throughout the region from 800 to 700 cm.-', whereas the latter show only weak absorption in this portion of the spectrum. Some of the bands in the region from 800 to 700 cm.-' correspond to known ring vibrations in this portion of the spectrum. In addition to these spectral featurea which may serve to distinguish between various closely related groups of analogs within the series, each compound may be assigned several characteristic bands which differentiate it from all the others included in this survey. The position of these bands is given in Table V. Spectra of Barbiturate-Copper-Pyridine Complexes. The copper-pyridine derivatives show also characteristic N-H and C=O absorptions, but invariably a t lower frequencies than the corresponding free barbituric acids (Table IV). The IT-H frequency displacements are of the order of 20 to 40 wave num-
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
Figure 1.
Infrared absorption spectra of .4lphenal, Amytal, and their copper-pyridine complexes
1597
ANALYTICAL CHEMISTRY
1598
Figure 2.
Infrared absorption spectra of Alurate, Dial, and their copper-pyridine complexes
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
u w
z a m a
8 a m
W 0
z
2n
sm 4
W 0
z
2a
8 m 4
Lu
z 0
2 a
8 m a
Figure 3.
Infrared absorption spectra of Ipral, Luminal, and their copper-pvridine complexes
1599
ANALYTICAL CHEMISTRY
1600
Figure 4.
Infrared absorption spectra of Mebaral, Nembutal, and their copper-pyridine complexes
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
1601
W A X LENGTH, MICRONS 25
30
40
o=c
5.0
c
55
6.0
65
7.0
75
8.0
8S
90
9 5 10.0
11.0
120
L"--c*
FREOUENCY CM:'
Figure 5.
Infrared absorption spectra of Neonal, Rutonal, and their copper-pyridine complexes
13.0 140 150160
1602
ANALYTICAL CHEMISTRY
WAVE LENGTH, MICRONS 2.5
30
40
5.0
55
6.0
65
70
75
80
85
90
96 100
110
I20
130 I40 150160
00
01
w
0 O2
z a
03
p
a
04 0 5 0 6 07 0 8
Pt
I 5
01
y
02
z a
p
a
03
0.1 0 5
06 07
ii 1.5
01
w
0 O2
z a
03
0 04
a
0 5
06
07 08
98 I 5
01
y
02
z
m a K O' 0
g
01
a
0 5 06 07 0 8
9% a3 I 5
FREOUENCY CM:'
Figure 6.
Infrared absorption spectra of Seconal, Veronal, and their copper-pyridine complexes
1603
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6 bers, indicating that a greater degree of intermolecular hydrogen bonding occurs in the derivatives than in the free barbituric acids. Of the three carbonyl bands seen in the spectra of the barbituric acid compounds, only two are retained in the spectra of the copper-pyridine complexes. This phenomenon is discussed below. No distinctive bands are found between the regions characterized by strong N-H and C=O absorptions. Beyond this range, however, fairly intense bands occur in all spectra a t 1575 f 5 -
Table V. Position of Key Bands in Infrared Spectra of Barbiturates and Barbiturate-Copper-Pyridine Complexes Characteristic Vibration Frequanciea, Cm.-'
Compound Alphenal Complex Alurate Complex -4my tal Complex Dial Complex Ipral Complex Luminal Complex Mebaral Complex Nembutal Complex Neonal Complex Rutonal Complex Seconal Complex Verona1 Complex
940 815 1280 1283 1240 1315 1217 995 1332 775 1301 790 771 875 1222 1295 1260 1265 1410 1270 1320 1252 1335 1185
765 777 1258 1257 1212 1285 1000 950 1312 758 1253 780 754 835 1153 1250 1241 1218 1235 760 1280 1237 1320 1170
710 767 1214 1235 1165 1253 950 935 1220 720 1238 770 735 797 1048 1222 1215 775 712 735 1213 1220 1310 1155
689 726 937 1223 670 1222 930 920 1152 695 1225 735 725 773 819 780 1170 763 687 695 925 700 1230 770
cm.-', 1500 i 10 cm.-', and 1420 f 7 cm.-l, while in the fingerprint region characteristic absorptions are always observed at 1218 f 5 em.-', 1160 & 12 cm.-lJ 775 f 5 cm.-l, 702 f 8 cm.-1, and 644 f 3 cm.-'. It is unlikely that the 1575-cm.-l band is due to N-H deformation frequencies, because it is not found in the spectra of the free barbituric acids. It may be identified with vibrations of the C=N group of the pyridine molecule (19) (Figure 7 ) in accord with the Raman data reported by Hibben (7). However, Blout and others have shown that this assignment cannot be made in conjugated systems involving both C=C and C=N bonds ( 2 ) . The authors agree with this interpretation and hesitate to associate this vibration with any specific group of the molecule. A characteristic triplet is alrays observed a t 1072 zk 3.cm.-l, 1045 =t3 cm.-l, and 1018 f 4 cm.-l. Pyridine exhibits three intense absorptions in this region (Figure 7), which appear to be displaced toward higher frequencies and compressed into a narrower range in the spectra of the complexes. These bands are very sharp and their intensities increase generally with their vibration frequency. Only in the Mebaral-copper-pyridine complex is this pattern somewhat modified, because the barbituric acid itself exhibits marked absorption throughout this region. The characteristic assembly of bands described is also observed in the spectrum of the copper-pyridine-cyanate complex (Figure 7), which compound was prepared in accordance with the procedure reported by Davis (4). I t is, therefore, probable that these absorptions are primarily associated with the copper-pyridine skeleton of the molecule. The intense band occurring in the spectrum of the copper-pyridine-cyanate complex a t 2200 ern.-' is clearly due to the presence of the nitrile linkage (C=N). The compound also shows the band a t 1575 em.-', which, as already shown, is observed in all the barbituric acid spectra, and it likewise exhibits marked absorptions near 775, 700, and 640 cm.-l, all of which cannot yet be explained in terms of molec-
WAVE LENGTH. MICRONS
FREOUENCY CM:'
Figure 7 .
Infrared absorption spectra of pyridine and pyridine-copper-cyanate complex
ANALYTICAL CHEMISTRY
1604
WAVE LENGTH, MmONS 25
30
4.0
5.0
NH-CO
o=c
1
*“-eo
5.5
N
6.5
7.0
7.5
8.0
8.5
9.0
9.5 10.0
110
12.0 13.0 140 I50 I60
CH~-CH,
c
CII--c*
\\
/ \ Y
c
C”
*il-co
C*
6.0
0-c
N
cH2-c*,
\ I \\
c
//
w-CH
FREQUENCY CM:‘
Figure 8. Potassium bromide spectra of barbiturates and barbiturate-copper-pyridinecomplexes
V O L U M E 28, NO. 1 0 , O C T O B E R 1 9 5 6 ular structure and are to be regarded primarily as skeletal vibrations of the copper-pyridine portion of the system. In spite of the many structural similarities thus brought out in the infrared spectra of these compounds, there exist many structural differences, particularly throughout the fingerprint region (1250 to 650 cm.-l). I n general, the spectra of the derivatives show more structure and a greater degree of dissimilarity from one another than the spectra of the corresponding barbiturates. Therefore, they should prove particularly valuable for identification purposes. Bands which may be used to differentiate the various members of this sefies are listed in Table V. Potassium Bromide Spectra of Barbiturates and CopperPyridine Derivatives. These spectra are shown in Figure 8. Their profiles are in most respects identical to those of the corresponding spectra measured as mineral oil suspensions, except for certain broad characteristic differences which should be noted. Thus, throughout the region from 3000 to 2800 cm.-1 the potassium bromide spectra show bands which are absent from the Nujol spectra because of masking by the intense absorption (C-H stretching frequency) of the medium. Similarly, structure is no longer hidden in these spectra throughout the C-H deformation region (1460 to 1380 cm. -1). The spectra of the complexes show a broad but intense band a t 3475 cm.-‘ which does not appear in the Nujol spectra of the compounds. No specific structural group has been assigned to this absorption, which may be due to free or associated N-H, or/and associated 0-H stretching frequencies arising from hydroxyl groups which may form as a result of enolization during the pelleting process. Relationships between Spectral Data and Structural Features of Derivatives. The structures postulated for the barbituratecopper-pyridine complexes require: ( a ) enolization of the barbituric acid, followed by reaction of the enolate ion with the linkage; and ( b ) establishment of reagent to form a (3-0-Cu this bond through the carbonyl group in the 2 position of the barbituric acid molecule. Chemical evidence for the first of these requirements has been presented above, but no data regarding the nature of the carbonyl-metal linkage were given. The present investigation has shown that such information may be obtained from an examination of the infrared spectra of the compounds. Considering the spectral characteristics of the three carbonyl groups of the barbituric acid molecule discussed earlier in this paper, it would be reasonable to conclude that, as a result oE complex formation through the carbonyl linkage in the 2 position, the band a t 1700 em.-’ should no longer exist in the spectra of the derivatives, nor should the 1730-cm.-l band, which is a band for a combination of carbonyls in the 2,4, and 6 positions. Also, because the symmetry of the barbituric acid molecule incorporated into the complex is destroyed with the formation of a C=N bond during the enolization process, the remaining carbonyls in the 4 and 6 positions will no longer be equivalent and will, therefore, give rise to separate bands. Accordingly, only two carbonyl bands are observed in the spectra of the complexes, and they no longer occur a t the original positions. The direction of the shifts observed may also be explained on the basis of the structure assigned to the complexes. Because the oxygen of the carbonyl bond in the 2 position is linked to a copper atom, no drain of negative charge from the nitrogen atoms in the 1 and 3 positions to the 2 position occurs. Hence, this charge can add single bond character to the carbonyls in the 4 and 6 positions. As a result of such charge transfers the corresponding carbonyl vibrations are displaced toward lower frequencies, the magnitude of the shifts being about 30 to 45 wave numbers, as shown in Table IV. The position occupied by the strong absorptions of pyridine (1580 to 1595 crn.-l) in the complexes is illustrated in the spectrum of copper-pyridine-cyanate, which compound may be considered as the simplest derivative of pyridine containing a C-O-
1605
Cu linkage (Figure 7). I n the complex this absorption occurs as a doublet, comprising a strong band at 1605 cm.-l and a sharp but less intense band at 1575 cm.-l. Both bands persist in the barbiturate complexes, the position of the 1575-cm.-l band being constant within a few wave numbers. The high frequency band is broader, more intense, and not quite so constant (1600 to 1625 cm.-l) because of variable contributions afforded by the C=N bonds formed in the barbituric acids prior to complex formation. The N-H stretching frequencies of the complexes are always considerably less intense than those of the corresponding free barbituric acids, which is in accord with the disappearance of an N-H group from the barbituric acid ring as a result of the enolization process. Also in agreement with this interpretation is the observation that the intensity of the N-H absorption is most drastically reduced in the Mebaral complex, because in this compound two of the four imino hydrogens of the barbituric acid moiety of the molecule have been replaced by methyl groups (Figure 4, spectrum 14). ACKNOWLEDGMENT
The authors wish to thank 1., I. Pugsley, Department of National Health and Welfare, and H. Sheffer, Defence Research Chemical Laboratories, Defence Research Board of Canada, for permission to publish the results of this investigation. They are also indebted to Martin Butler, Department of National Health and Welfare, for drawing the spectral curves; to E. C. Kerr, Department of National Health and Welfare, for final p r e p aration of the illustrative material; to the Perkin-Elmer Gorp. for kindly supplying the potassium bromide spectra, and to Modeste Pernarowski of this laboratory for technical assistance.
LITERATURE CITED
Angyal, C. L., Werner, R. L., J . Chem. SOC.1952, 2911. Blout, E. R., Fields, hl., Karplus, R., J . Am. Chem. SOC.70, 194 (1948).
Boivin, J. L., Boivin, P. A . , Can. J . Chem. 32, 561 (1954). Davis, T. L., Logan, A. V., J . Am. Chem. SOC.50,2493 (1928). Fox, J. J., Shugar, D., BulE. soc. chim. Belges 6 1 , 4 4 (1952). Hausdorff, H., A p p l . Spectroscopy 7, 75 (1953). Hibben, J. H., “The Raman Effect and Its Chemical hpplications,” p. 294, Fbinhold, New York, 1939. Hoste, J.,Research (London) 1, 713 (1948).
Levi, L., Farmilo, C. G., ANAL.CHEM.25, 909 (1953). Levi, L., Hubley, C. E., Instrument News 5, (2), 1 (1954). Levi, I,., Hubley, C. E., Farmilo, C. G., Proc. Roy. Can. Mounted Police Crime Detection Seminar 1, 91 (1954). Levi, L., Oestreicher, P. &I., Farmilo, C. G., Bull. Sarcotics Li. Y. Dept. Sociol A f f a i r s ,5, 15 (1953). Ley, H., Hegge, H., Ber. derrt. chem. Ges. 48, 70 (1915). Loofbourow, J. R., Stimson, &I. M., J . Chem. SOC.1940, 1276. Pfeiffer, P., Glaser, H., J . pi-akt. Chem. 151, 134, 145 (1938). Pfeiffer, P., Werner, H., Hoppe-Seyler’s Z. physiol. Chem. 246, 212 (1937). Price, W. C., Bradley, J. E. S.,Fraser, R. D. B., Quilliam, J. P., J . Pharm. and Pharmaeol. 6, 522 (1954). Iiaeth, C., Gebauer, R., U. S.Patent 2,134,672 (Oct. 25, 1938). Randall, H. AT., Fowler, R. G., Fuson, N., Dangl, J. R., “Infrared Determination of Organic Structures,” p. 32, Van Nostrand, Kew York, 1949. Saunders, L., Srivastava, R. S., J . Pharm. and Pharinacol. 3, 78 (1951). Sidgwick, N. V., J . Chem. SOC.1941,433. Stuckey, R. E., J. Pharm. and Pharmacal. 13, 312 (1940). Ibid., 14,217 (1941). Taurins, A., Can. J . Research 28B, 762 (1950). Umberger, C. J., Adams, G., ANAL.CHEM.24, 1309 (1952). Wood, J. K., J . C h a . SOC.89, 1838 (1906);95,979 (1909). Zwikker,J. J. L., Pharm. Weekblad 68, 975 (1931).
RECEIVED for review April 29, 1955. Accepted May 29, 1956. Presented in part, Division of Analytical Chemistry, 128th meeting, ACS, Minneapolis, Minn., September 1955.
