i 75
V O L U M E 2 5 , NO. 1, J A N U A R Y 1 9 5 3 Table 1. -4ssay Data
Acid from
5‘% of Ester
Hydrolyzed
102 X Specific Activity,
Mean
“95Yo Interval Confidence of Mean” Interva! in yo of of Mean ’ Mean
rc./Mg.
Ethyl benzoate-acarbon-14 Ethyl-p-chlorobenaoate. u-carbon-14
100 3 100
3
6 Ethyl-p-toluate-acarbon-14
100
3 6
Ethyl-m-nitrobenzoatear-carbon-14 a
100
8.22, 8.20,8.22, 8.18, 8.21, 8.208 8.22 7.58, 7.59, 7.63, 7.63. 7.64, 7.615 7.62 7.00, 7.02, 7.00, 7.04, 7.03, 7.024 7.05, 7.03 6.47, 6.49, 6.48, 6.48, 6.47, 6.480 6.47, 6.50 6.49, 6.48, 6.47, 6.52, 6.47, 6.488 6.50 1.999, 1.996, 2.006, 2.006. 2.0060 2.010,2.012, 2.013, 2.006 1.872, 1.853, 1.865, 1.861, 1.8645 1.863,1.870.1.865, 1.867 1.845. 1.877, 1.852. 1.863, 1,8606 1.850,1.873,1.861.1.857, 1.868 1,530, 1.530, 1.545, 1.535. 1.5396 1.520,1.545,1.550,1.550,
10.013
=kO 16
f0.020
ztO.26
10.014
z t o , 20
10.009
10.14
iO.015
i0.23
&0.004
%0.20
10.002
i o , 22
10.007
10.38
%0.007
‘0.45
coYcLusIos
1 R.51
l.j20;-1.523, 1.530, 1.535. 1.5340 ztO.008 io.52 1.540. 1.545. 1.545 1.446, 1.432, 1.427, 1.428, 1.4399 3 =to.007 ztO.49 1.438, 1.448, 1.452. 1.448 Ethyl-p-aninate-rr1.414, 1.414, 1.414, 1.415, 1.4128 100 i.o.002 10.14 carbon-14 1.410, 1.410, 1.406, 1.412, 1.417 3 1.280, 1.276, 1.295, 1.295, 1.2926 10.007 50.54 1.305. 1.293, 1.295, 1.302 1.297, 1.303. 1.300, 1.300. 1.3010 6 i0.002 i.o.15 1.297 1.307, 1.300. 1.303, 1.302 Average “95% confidence interval of nieaii” = 1 0 . 2 9 % , a Prepared b y resublimation of sample of acid from 100‘7, hydrolysis of ethyl-m-nitmbenaoaterr-rarbon-14. loon
The corrected mean rate of drift values was multiplied by a factor to convert to the specific activity values listed in column three of Table I. The six to nine specific activity values n-ere averaged to obtain the mean values sholyn in CYJlUlllll four. Columns five and six list t,he “95% confidence intervals of the means” of the specific activities for the various acid samples assayed. Each of these confidence intervals was r:ilrulated as twire t,he “standard deviation of the mean.” The average of all the values of the confidence intervals in percentages (column six) is also indicated a t the bottom of the table.
Under thr conditions used in this work the average precision of the overall proceps of Van Slyke conibustion and radioassaving of the various carboxvl-labeled benzoic acids was mean ~ k 0 . 3 7for ~ the approximate 95”i, confidence interval of the mean of 6s t o nine assays. To obtain such precision it was necessary to perform all the assays on a given .ample of acid in one day. ACKNOWLEDG\IE\T
drnser to a given potential. For each 5-mL aliquot thc mran of ten observed “rates of drift” WHS recorded. T o correct for variations in the instruments, a series of “rates of drift” of a standard uranium chamber was recorded immediately after measuring each serie? of rates of drift resulting from a single wet combustion of a 5-ml. aliquot. The standard uranium chamber consisted of H 250-ml. gold-plated brass chamher containing a piece of uranium wrapped Kith aluminum to absorb alpha particles. Standardized against sodium carbonatecarbon-14 solution supplied by the Sational Bureau of Standards, it had a value equivalent to 0.1015 pc. of carbon-14. Mean ratee of drift readings were corrected to this stantlard by multiplication by a factor as follows: Correrkd mean rate of drift for 5-ml. aliquot = mean rate of drift from observed values X calculated rate of drift of standard mean rate of drift of standard from observed values
The modification to the xvet comhustion piocedure, used to prevent reaction of free chlorine I\ ith the mercury in contact with the frit valve, was developed by H. TI-, Davis of the University of South Carolina. 0. K. Neville and A. J . Weinberger prepared the standard used t o corrert Eo1 variation? in the instruments. LITERATURE CITED
(1) Xeville, 0 . K., J . Am. Chem. SOL..70, 3501 11948).
(2) Ropp, G. A , , and Raaen, V. F., J . Chetn. Phys. in press RECEIVED for review June 0, 1052. Accepted September 26, 10.52. Based o n work performed under contract W-7405-eng-26 for the I t o m i c Enern? Commission a t Oak Ridge National Laboratory.
Quantitative Estimation of the Carbonyl Groups in Saturated Ketocholanic Acids E. L. P R A T T , Winthrop-Steurns Inc., Rensseluer, ,V. Y.
S E of the accepted proceclurc~sused in large scalp conversion of cholic acid (3,7,12-trihydroq-rholanic acid) to dmowcholic arid (3,12-dih~droxycholanicarid) requires a selective oxidation of the 7-hydroxy group a i t h bromine to a monoketocholnnic arid. T h c carbonrl group is then rliminatcd through a WolffKishner rcduction. This paper proposes an analytical method for rapid control testing in the above process. The techniques described can be extended to the evaluation of certain saturated di- and triketorholanic acids. GENERAL
The literature contains various methods for estimating carbonyl compounds. Generally, these are procedures based on gravimetric assay of reaction products such as hydrazones ( d ) , semicarbazones (3), oximes ( 5 ) , hydantoins ( I ) , etc. The estimation of free acid after the oxime formation with hvdroxyl-
amine hydrochloride has been reported ( 6 ) . Very excellent polarographic techniques have been published, but these generally necessit,ite preliminary derivative prepzrcttion ( 7 , 8 ) . Little or nobhing has been repoi ted on the spc’ctrophotonietric estimation of ketones in the saturatcd rholanic acid series. T e t this approach presents a procedure \\hi( h is eltreniely rapid and. of even greater consequence, it is a nirLtwie of the kcto unit as such and not the measure of a rcaction product I n general, saturated slstenis do not ‘Lbsorb ultraviolet light and their spectra are studied in thr loxr frequency or infrared region. (In this discussion thr t e x t ni ultraviolet actually refers to that region of the ultraviolet spectrum which is easily studied and is limited to those wave lengths exceeding 220 nip.) Desoxycholic arid is an example of a saturated system in the bile acid series which shows no absorption in the ultraviolet region of the spectrum. If, however, a saturated cholanic acid possesses one or more keto groups, the carbonyl resonanre can br detected
AKALYTICAL CHEMISTRY
176 by ultraviolet spectroscopy. Furthermore, the absorbancy of incident light in terms of the molecular extinction coefficient ( e ) is proportional to the number of carbonyl groups in a given molecule if these groups are insulated from each other by three or more methylene units. For systems where the above condition is fulfilled, a definite maximum is observed a t 285 mp. The data obtained indicate that a synergistic absorption effectt will result if the insulating units (methylene linkage) between two keto groups are fewer than three in number. These carbonyl spectra are of weak intensity and would be obscured if observation were attempted on a parent polyene nucleus. I n the synthesis of desoxgcholic acid from cholic acid the ring and chain system remains saturated (except during the reduction step) and under these conditions a measurable carbonyl group is available.
40
r
4 u W
0
=
10
EXPERIMENTAL
All data reported in this study were obtained with 0.1 sodium hydroxide used as the solvent. Figure 1 represents the spectrum of dehydrocholic acid ( 3 , 7 , 12triketocholanic acid), melting point, 238.5' to 240' C. (corrected). The molecular extinction coefficient for this substance a t its 285 mp maximum has been found to be 106.8. Each carbonyl unit or -CH linkages and is insulated by more than two -CH*each may be considered to contribute 35.6 units to the total extinction value obtained. The spectrum of a diketocholanic acid is represented in Figure 1. The 3-succinoxy-7,12-diketocholanic acid used melted a t 230" C. (corrected). At the 285 mp maximum a molecular extinction value of 73.5 is apparent. .4gain, assuming equal contribution to the total extinction, each keto group . may be considered t o contribute 36.7 units. Figure 2 represents the spectrum of 7-keto-3,12-dihydroxycholanic acid [melting point, 171.2' to 172.8" C. (corrected)]. The molecular extinction value obtained at the 285 mp maximum is 34.1 units.
240
260
280
300
320
WAVE LENGTH,rnp
Figure 2. Spectrum of 7-Keto-3,12dihydroxycholanic Acid mary objective of this study was the development of an analytical procedure which would permit a rapid evaluation of the carbonyl group formed during the oxidation of cholic acid. Consequently, a compensating technique was desirable. The extraneous materials in the oxidation mixture of cholic acid contribute linearly to the gross absorption between 275 and 300 mp (see Figure 4). By the application of an equation, patterned after the work of Morton and Stubbs ( 4 ) , that fraction of thegross absorbancy which is due to the nonketonic components in a given oxidation mixture is corrected for. This equation is based on data obtained from a highly purified dehydrocholic acid (see Figure 1). The oxidation of cholic acid is normally carried out in alkaline solution. At any stage of this oxidation step a sample may be withdrawn and assayed for keto content. Results of the assay are obtained in a matter of minutes. PROCEDURE
Transfer an aliquot of the oxidation mixture approximately equivalent to 100 mg. of 7-keto-3,12-dihgdroxycholanicacid to a 25-ml. volumetric flask and make to volume with 0.1 S sodium hydroxide. Determine the absorbancy of this solution relative = 285 mp, A = 275 mp, and to 0.1 N sodium hydroxide a t
I
I
I
I
I
I
I
I
I
I
APPLICATION
The spectra previously discussed were obtained from relatively pure substances. The application of a spectrophotometric procedure of assay to a large-scale operation necessitates either a separation from or a compensation for the impurities which would be measured together with the carbonyl group. The pri-
"L 240
Figure 3.
260 WAVE LENGTH,mp 280
300
Spectrum of 3,6-Diketocholanic Acid
V O L U M E 25, N O . 1, J A N U A R Y 1 9 5 3 Table I.
177
L~~
torr.
1 3 ~ 14R 13 16 3-90
Observed Absorbancy, mp 285 275 300 0.337 0.312 0.229 0.295 0.267 0.194 0 334 0.309 0.222 0 337 0.306 0.229 0.261 0.228 0.163
0313 1204 1213
0 481 0,530 0..558
0,506
0.423 0.495
0.301 0.355 0.361
3-Si1ccinoxy-7.12-diketo-10 cholctnic acid
0.074
0.618
0.510
0.458 0.478 0.473 0.424
7-Keto-3.12-dihydroxvcholanic acid (impure)
0 174
0.126 0.329
0.103 0.228
0.169 0.354
Compound Dehydrocholic acid
SO.
Sodiuiiidehydrocholate
3-106 6530
0.371
.4b. 0.250 0.245
0.257 0.265 0.253
O , l o o r
l
275
280
of commercial samples of dehydrocholic acid and concentrated solutions of the sodium Wt. Wt. M x . Found, salt of dehydrocholic acid. Taken. f hIg./hIl. Mg./MI. I n the study of saturated 3.71 0.94 1.00 polyketosteroids the number of 3.77 0.93 1.00 3.77 0.97 1.00 keto groups as determined by 3.77 1.00 1.00 a typical hydrazone assay may 3.77 0.95 1.00 be compared u-ith the quality 3.98 1.82 2.00 3.98 1.91 2.00 and intensity of the spectrum 3.98 1.88 2.00 of the parent compound. Such 7.06 3.00 3.00 a comparison would present 11.34 1.92 2.12 added information as to the 11.34 4.01 5.00 r e l a t i v e p r o x i m i t y of the groups in question. This procedure, of course, should be cairicd out on pure substances tinti should not require baekground absorption correction. Typical data obtained from various commercial preparations of saturated ketocholanic acids appear in Table I. Molecular extinction coefficients used in all calculations are:
Assaj- of Typical Saturated Ketocholanic Acids [Me./nil. = corrected absorbancv X (M.W./c)]
l
,
l
285
,
290
l
,
295
l
Saturated monoketocholanic acid = 35.6 Saturated diketocholanic acid = i1.2 Saturated triketocholanic acid = 106.8
,
300
ACKNOW L E D G V E? T
W A V E LENGTH,m@
Typical Irrelevant ihsorption from Cholic Acid Oxidation Jlixture
Figure 4.
X = 300 mp. (Use of a Beckman 1Iodel D C spectrophotometri equipped u-ith a n ultraviolet accessory unit or an equivalent spectrophotometer is recommended.) Calculate the concentration in milligrams pc’r milliliters of the i-keto acid intermrdiate in the original reaction mixture as follom: Corrected absorbancy = 4.29 X (observed absorbancy 285 mp) - 2.574 X (observed a h s o r h n c y 275 mp) - 1.716 X (observed absorbancy 300 niH) hIilligrams of keto acid milliliter
Thanks are extended to Hugh B. Corhitt for his hrlpful criticism and suggestions in the preparation of this paper, and to George Jacobs for his assistance in the preptrdtion of the graphs. Samples of 3,6-diketorholanic acid and 3-succinoxy-7,12diketocholanic acid were generouslv furnished by J. S. Buck and C. 11. Suter of the Sterling-Jl‘iiithrop Iteqearch Institute. LITERATURE C I T E D
(1) Henze and Soeer. J . A m . Chem. ,Sot. 64.522 (1942). ( 2 ) Klein, Keiner. and Gordon = ~ S I L CHEV., 20, 174
absorbanm ) (101.5) (25) - (:is 0(corrected ) (volumc of sample talipn 111 milliliters)
01
-
1Iillierams of keto acid - (corrected absorbancyi 1284.1) volume of sample taken in milliliters milliliter Further application of this procedure is found in the evaluation
(1948). (3) &ladigan, Zeniio, and Pheasant, Ibzd., 23, 1691 (1951). (4) Morton and Stubbs, A n a l y s t . 71, 348 (1946). (5) Shi.iiier and Fuson, “Identification of Organic Compounds,” 3rd ed., p. 202, New York, John JViley 6E Sons, 1948. (6) Troaaolo and Lieber. .-~N.LL. CHEM..22. 764 (1950). (7) Werthessen and Baker, Endocrinoloyi, 36, 351 (1945). (8) Wolfe, Hershberg, and Fieser, J Btol Chem., 136, 653 (1940). RECEIVED f o r review AIurcIi 12, 1952. .iccepted Beptemher 9 , 1 0 3 ~ .
Microdetermination of Carboxy Groups In .4romatic and Aromatic-Type Heterocyclic Acids AIORTON BER0Z.i Bureau of Entomology and Plun t Quarantine, U . S. D e p a r t m e n t of Agriculture, Beltsrille, M d . S STUDIES on the struetuie of four insecticidal alkaloids
I isolated from Trtpterygzrcrri todjordzz ( I ) , a small quantity of
Cl1H,aOJ and Cl,H,,O,S, was isolated. On the basis of experimental data. the d i b a w acids nere believed to have the following structuial formulas
h o dibasic acids,
fiCOO€€
,,-,CH,CHCH~COOH I
1 1
!
I
y
~
CH3
kH3
I
!
I1
Each of these compounds hai one carlios)- group attached to them and the ring, and it should be possible t o t1ecarho~:~~l:ite
obtain 1 mole of carbon dioxide from a c h mole of dibasic acid. The hydroxy-dibasic acid (11) should yield an additional mole of carbon dioside offing to the a-hydroxy carboxylic acid structure of the side chain. Hubacher (2’) has described both a gravimetric and a volumetric method suitable for the quantitative estimation of carboxy groups attached to an aromatic ring, but these methods require a large amount, of sample, generally 1 gram or more. T h e ~ ~ l ~ ~ ~ ~ ~ following micromethod, which requires only 5 t.0 10 mg. of sample, is a modification of Hubacher’s gravimetric method. Analyses on seven known acids were in good agreement with those obtained by Hubacher on a macro scale. The method has also been found useful for decarboxylation of aromatic-type heterocyclic acids, such as pyridine, pyrazine, and furan acids. With the present
~
~
