air mixtures is evidence that there were no serious losses in the latter case. The complete analyses were based on the gasoline-air mixtures. Gasoline concentrations of 20 to 25 mg. per liter of air at 100' C. at atmospheric pressure were used, giving total hydrocarbon concentrations of approximately 0.5 mole %. These concentrations were purposely made somewhat higher than those found in actual automobile exhaust gas samples to reduce the uncertainty in experimental measurements. The hydrocarbon types in these gasoline-air mixtures were determined on the basis of Equations 1 through 9. For comparison with the results on the liquid gasoline by the FIA method, the paraffins and cycloparaffins were assumed to
represent saturates; the olefins and coda compounds were assumed to represent unsaturates, and the aromatics were compared directly. The agreement in Table VI11 between the mass spectral results and FI-4 results is satisfactory considering the approximations involved. No attempt was made to introduce a mole to volume factor, nor were the mass spectral coefficients for the various compound types adjusted to fit gasoline type compounds rather than those predominant in automobile exhaust gases.
were collected by R. TV. Gates and I. S. Yaffe under the direction of W. C. Thuman. The author is indebted to Harold Eding for assistance and advice in interpretation of the mass spectra. LITERATURE CITED
ACKNOWLEDGMENT
(1) Brown, R. A., ANAL. CHEM. 23, 430 (1951). (2) Criddle, D. W., LeTourneau, R. L., Ibzd., 23,1620 (1951). (3) Taylor, R. C., Young, W. S., Ibid., 17,811 (1945). (4) Walker, J. K., O'Hara, C. L., Ibid., 27,825 (1955). (5) Young, R. E., Pratt, H. K., Biale, J. B., I b i d . , 24, 551 (1952).
The mass spectral determinations were performed by Lydia Peters and W. C. Crawford. The exhaust gas samples
RECEIVED for review February 15, 1958. Accepted December 11, 1958. Work supported by The Western Oil and Gas Association.
Identification of Position Isomers of Some Stearic
Acid Derivatives by Infrared Spectroscopy Study of trans 6- through 11-Octadecenoic Acids and Corresponding Contiguously Substituted Dihydroxystearic Acids in the Crystalline State HEINO SUSl Eastern Regional Research Laboratory, Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture, Philadelphia 7 8, Pa.
,The position of the double bond in trans 6- through 1 1 -octadecenoic acids can be determined by studying the equally spaced absorption bands in the 1180- to 1350-cm.-1 region. This band progression, which in the case of n-saturated fatty acids depends on the total chain length, is related to the chain segment next to the carboxyl group. The corresponding dihydroxy acids can b e distinguished by studying the 800- to 1200-cm.-1 region. The high and low- melting series show distinct differences. Spectral variations within the low- or highmelting series are small, but in many cases sufficient to permit positive identification.
J
OKES, McKay, and Sinclair have pointed out that in the infrared absorption spectra of crystalline fatty acids a progression of uniformly spaced bands is observed in the 1180- to 1350cm.'I region and that the number of bands in the progression is related to chain length (4). For cis-unsaturated acids the progression becomes irregular,
910 *
ANALYTICAL CHEMISTRY
but for trans-9,lO-octadecenoic acid, the region is similar to one of the short chain saturated acids-e.g., lauric-suggesting independent behavior of the two chain sections (4). Corish and Chapman (2) found similar bands in the spectra of lower homologs (C, to Cu), although the regularity seems to decrease with decreasing chain length. Meiklejohn et al. (6) have found that in the higher members the number of bands in the progression is approximately equal to half the number of carbons in the chain. Jones, i\lcKay and Sinclair (4) suggested that the bands arise from the rocking and/or twisting vibrations of the CH:, groups. Ferguson (3) noticed that the single crystal spectra of eicosanoic acid obtained with polarized radiation by Cole and Jones (I) do not show parallel polarization of the progression bands, as would be expected for wagging or twisting modes. Meyer and Schuette (Y), who studied over 300 long-chain compounds, observed that all isomeric cis and trans forms of the octadecenoic acids gave fewer bands in the 1180- to
[email protected] region than was observed
for stearic acid. As the double bond approached the terminal methyl group, these bands increased in number. Five position isomers of trans-octadecenoic acid have been examined for correlations between the band progression and the position of the double bond. The results indicate that the number of bands in the progression series is related to the (total) length of the chain segment next to the carboxyl group. This offers a way to determine the location of the double bond in trans-monounsaturated fatty acids. In the particular series studied, the results are unambiguous. One sample (elaidic acid) was studied with polarized radiation to pinpoint the progression bands and locate the 1 3 0 0 - ~ m . -region ~ carboxyl band. The results indicate that the weak bands on the high frequency side of the progression region are of a different nature than the four main bands and that-contrary to saturated acids and particularly, eicosanoic acid (I)-no distinct carboxyl band is found in this region. Experimental. The fatty acids have been described ( 9 ) . The infra-
red spectra were obtained using a Beckman Model IR-3 instrument equipped with sodium chloride optics and a Perkin-Elmer Model 21 instrument equipped with sodium chloride optics and a silver chloride polarizer. The latter was used to study the sample of trans-9,lO-octadecenoic acid with polarized radiation. trans-6,7-; trans-7,8-; trans-8,9-; trans-g,lO-, and trans-11,12octadecenoic acids were studied as potassium bromide pellets. These were prepared by grinding about 1.5 mg. of the dry solid sample together with about 300 mg. of dry potassium bromide for 2 minutes in a vibrator-grinder and pressing the resulting mixture in a '/*-inch diameter die by applying a total force of 20,000 pounds for 5 minutes.
t- 6,7
t- ?,0
1-0,s
T
w 0
z a I-
4
t-9,10
2 Y,
z
a a
t-11,12
I-
An oriented sample of the trans9,lO-acid was prepared by melting the substance between two rock salt plates and subsequent cooling, using a temperature gradient. The resulting oriented film was studied with the electric vector first parallel and then perpendicular to the direction of crystal growth.
Results and Discussion. Figures 1 and 2 illustrate that in the region roughly between 1180 and 1320 em.-' there exists a series of relatively strong, equally spaced bands just as in the case of saturated acids. The "rule of two" relationship observed by Meiklejohn and coworkers (6) apparently applies to the first segment of the hydrocarbon chains in the trans-unsaturated acids studied. The number of these bands is equal to n/2 for the even members and t o (n - l ) / 2 for the odd members, if n is the number of carbon atoms in the chain segment between the carboxyl group and the double bond, the end atoms of the segment included. Some weaker, irregular bands are also observed, but the regular spacing and higher relative intensity of the progression bands makes their recognition relatively easy. Although in the saturated fatty acids the number of bands is indicative of the whole chain length, in unsaturated acids, the bands indicate the length of the chain segment next to the carboxyl group. This is illustrated by a comparison of Figures 2 and 3. Figure 3 shows schematically the approximate positions and relative intensities of the progression bands of the CS to C12 saturated acids. The data are computed from absorption curves published recently by Corish and Chapman ( 2 ) and obtained by them on solid samples using a low temperature cell. Data for the C1, saturated acid and trans-l0,11octadecenoic acid were not available.
Figure 1. 1175- to 1350-cm.-1 region of trans-octadecenoic acids
I
I
I
I300
I
Go
I360
1250
1200
cm:'
Figure 2. Schematic presentation of absorption bands of frans-octadecenoic acids in 1 175- to 1350-cm.-I region
The estimated band positions for these compounds are indicated by broken lines in Figures 2 and 3. On the high frequency side of the band progression of the unsaturated acids there exists another progression of much weaker intensity (Figures 1 and 2). These bands are not influenced by the position of the double bond and appear a t about the same positions for all odd and even acids studied. They should not be included in the progression which is related to the length of the chain segments. The terminal chain segments (from the double bond to the methyl group) do not seem to give rise t o regularly spaced medium intensity bands. In this aspect they behave much like unsubstituted n-paraffins which show progression bands of considerably lower intensity and less regular spacings than the corresponding fatty acids. Some weak bands occurring between the main progression bands, and partially overlapping n-ith them, might be caused by the terminal segments, but no conclusions seem possible a t present, except that the double bond effectively interrupts the chains without having a noticeable activating effect. Complications may be encountered in determinations of the lengths of chain segments by the band progression method of which the first is the problem of polymorphism. Jones, hIcKay, and Sinclair (4) have shown that in the case of stearic acid, various polymorphic forms absorb differently in the infrared, including the progression region. In the case of trans-octadecenoic acids, no polymorphism has been reported ( 5 ) . No irregularities which might be caused by different crystal configurations have been found in this laboratory. Nevertheless, polymorphism is a potential source of trouble and should
I
I I \
c6
u
1250
1200 cm-'
CI2
Figure 3. Schematic presentation of absorption bands Csto CI2 saturated fatty acids in 1 175- to 1350-cm.-i region VS. Very strong SH. Shoulder
of
VOL. 31,
NO. 5 ,
M A Y 1959
91 1
HIGH
LOW
MELTING
MELTING
Figure 5. 1550- to 700cm.-1 region of high and 8,9-, low melting 6,7-, 9,lO-, 1 1 ,I 2-dihydroxystearic acids
W V
z
a c 1350
1300
1250
1200
1150 em-'
1 UI
Figure 4. 1150- to 1350-cm.-1 region of trans-9,lO-octadecenoic acid observed with polarized radiation
z
a a I-
- Electric
vector parallel to direction of crystal growth Electric vector perpendicular to direction of crystal growth
---
is00
be considered in attacking a new group of compounds. Also, in most fatty acid spectra there is a strong carboxyl band around 1300 cm.-' which overlaps with the progression and sometimes renders the counting of the significant bands difficult. Figure 3 shows the presence of such a band in several of the saturated acids. In the series of octadecenoic acids no band which could obviously be assigned t o the carboxyl group was found. Cole and Jones (1) have shown that in eicosanoic acid, the 1300-~m.-~ carboxyl band is polarized differently from the progression bands. In trans-9,lO-octadecenoic acid, on the other hand, all four major bands in the progression region show the same polarization, whereas the weak bands on the high frequency side of the region show no polarization under the same conditions (Figure 4). This seems to indicate that the carboxyl band is relatively weak in this series and does not overlap with the bands of the progression. A further problem is the setting of the frequency limits of the progression region. For the saturated acids, Meiklejohn et al. ( 6 ) have somewhat arbitrarily chosen the region between 7.43 and 8.47 microns (about 1346 to 1180 cm.-l). This is not appropriate in this series of unsaturated compounds. The weak bands in the 1300- to 1350-cn~'~region should be excluded because the spacings are irregular, the positions do not change with chain length, the intensity is considerably lower, and the polarization characteristics are different. These observations indicate that no universally valid progression region can be defined. 912
ANALYTICAL CHEMISTRY
1300
1100
900
roo
1500
1300
1100
For each group of compounds the bands belonging to the progression should be identified separately by applying the above criteria. Progression bands are usually aswagging signed to coupled -CH2and/or twisting vibrations (4, 6 ) . Ferguson (3) has recently pointed out that for eicosanoic acid the polarization data (1) are not in agreement with such an assignment. The results obtained by Meiklejohn et al. (6) on saturated acids and obtained in this laboratory on the octadecenoic acids indicate that thenumber of bands is closely related to the total length of the chain (or chain segment) rather than the number of CH2 units. The same conclusion is suggested by the data of Corish and Chapman (2). DISTINGUISHING BETWEEN POSITION ISOMERS OF CONTIGUOUSLY SUBSTITUTED DIHYDROXYSTEARIC ACID
Hydroxylation results in a different kind of modification of the basic structure than unsaturation. The chain of single-bonded carbon atoms is preserved, but the regular packing of the individual molecules is altered by the voluminous hydroxyl groups as well as by the new hydrogen bonds introduced. A different type of change in the band progression, which depends on the packing of the molecules as well as on the length of chain segments (8) would be expected. Specific COH absorption bands should occur around 3400 and 1000 cm.-l The exact positions of these bands would be expected to depend on the position of the hy-
900
700 Ern-'
droxyl groups and on the hydrogen bonds formed. Experimental. The preparation of the high and low melting dihydroxystearic acids has been described ( 9 ) . Infrared spectra of the 6,7-, 8,9-, 9,10-, and ll,l2-members of the high and low melting series a.ere obtained on a Perkin-Elmer Model 21 instrument by using the scanning and sampling techniques described. Some of the potassium bromide pellets were foggy and caused excessive scattering. Increasing the pressing time from 5 to 10 minutes eliminated this difficulty. Prolonged grinding (up to 10 minutes) was also tried, but resulted in loss of detail in the resulting spectra. probably because of a breakdown of crystallinity. Polymorphism is not ruled out in the dihydroxy acids, although care was taken to prepare all the samples by identical procedures (9).
Results and Discussion. All hydroxylated acids show strong absorption around 3400 cm.-l and a series of medium strength bands between 1000 and 1200 cm.-1, corresponding to the 0-H stretching vibrations and the C-0stretching and/or deformation vibrations, respectirely. The 1180- to 1350-crn.-l region shows only weak bands with no apparent regularity. The infrared spectra are extremely similar above 1560 cm.-l, showing a broad, unresolved 0-H band around 3400 cm.-l, C-€I stretching bands around 2900 cni.-' and the carboxyl band a t 1710 cm.-1 Figure 5 gives the observed spectra in the 700- to 1550cm." region. The most obvious differences between the various isomers occur between 1000 and 1150 em.-',
in a region commonly associated with COH vibrations. Members of the high melting series are clearly distinguishable from the corresponding low melting substances by a comparison of the absorption patterns in this region. The low melting members have a more complex structure, as might be expected from the complicated interactions and hydrogen bonds, which are possible if both groups are on the same side of the chain. (Similar complexities might be expected in the OH stxetching region if investigated under higher resolution. Current studies with sodium chloride optics showed merely the presence of a broad unresolved band which was sirnilar for all compounds.) The differences between the individual members within one series are small, but sufficient t o allow identification and differentiation in a number of instances. KO regularities comparable nith the band progression of unsaturated acids could be detected. Aside from the bands which could be associated with the hydroxyl groups, puzzling phenomena appear on bands commonly associated with the carboxyl group. Thus, the strong band usually appearing around 1300 mi.-’ is oc-
casionally shifted to 1225 cm.-I and the band around 925 ern.'' is split into a number of weaker bands, giving rise to a pattern which appears to be different for each compound. Together region this with the 1000- to 1200-~m.”~ furnishes sufficiently distinctive fingerprint patterns to allow the identification of each individual isomer by infrared spectra. The position isomers of contiguously substituted dihydroxystearic acid could not be identified by clear-cut regularities like the trans-monounsaturated acids, but in the region roughly between 800 and 1200 cm.-’, there is enough spectral detail to allow identification on a fingerprint basis. ACKNOWLEDGMENT
The author is indebted to H. R. Knight and Daniel Swern of this laboratory, and to J. B. Brown, Ohio State University, for the samples of hydroxylated and unsaturated acids. Thanks are also due to G. C. Nutting for useful suggestions and t o Anne Smith and Carl T. Leander, Jr., for help in the experimental work.
LITERATURE CITED
(1) Cole, A. R. H., Jones, R. N., J . Opt. SOC.Am. 42,348 (1952). (2) Corish, P. J., Chapman, D., J . Chern. SOC.1957,1746. ( 3 ) Ferguson, E. F., J . Chem. Phys. 24, 1115 (1956). ( 4 ) Jones, R. N., McKay, A. F., Sinclair R. G.. J . Am. Chem. SOC.74, 2575 (1952): (5) Lutton; E. S., Kolp D. G., Ibid., 73, 2733 (1951). (6) Meiklejohn, R. 4., Meyer, R. J., Aronovic, S. M., Schuette, H. A., MelANAL.C~EM.29,329(1957). oche, v. ( 7 ) .Meyer, R,. J., Schuette, H. A., .Division of Paint, Plastics, and Printing Ink Chemistry, 128th Meeting ACS, Minneapolis, Minn., 1955. (8) Susi, Heino, Koenig, N. H., Parker, W. E., Swern, Daniel, ANAL. CIIEM. 30, 443 (1958). (9) Swern, Daniel, Witnauer, L. P., Fusari, 8. A,, Brown, J. B., J . Bm. Oil Chemists’ SOC.32, 539 (1955).
w.,
RECEIVED for review February 28, 1958. lccepted December 18, 1958. Presented in part at the Kinth Bnnual Conference of Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1958. Mention of specific products does not imply endorsement by the department to the possible detriment of others not mentioned.
Spectrophotometric Determination of Residua I A‘-3-Ketosteroids in Bulk A”‘-3-Ketosteroids Differential Rates of Thiosemicarbazone Formation ARLINGTON A. FORIST Department o f Physical and Analytical Chemisfry, The Upjohn Co., Kalamazoo, Mich.
b Chemical or microbiological production of A1*4-3-ketosteroidsfrom A43-ketosteroids requires a method for measuring small amounts of residual A4-3-ketosteroids in A1~4-3-ketosteroids. A spectrophotometric procedure i s based on the difference in rates of thiosemicarbazone formation. Analysis of standard mixtures of representative steroid pairs indicates a mean deviation for per cent A4-3-ketosteroid of f0.04 over the range 0 to 6%.
R
A114-3-ketosteroids, such as prednisolone and 60-methylprednisolone, have assumed commercial importance because of their high glucocorticoid and anti-inflammatory activities (1, 6, 8, 11). Their production involves chemical (5) or microbiological ( 7 ) dehydrogenation of appropriate A4-3-ketosteroids and a method for the ECENTLY,
determination of residual A4-3-ketosteroids in A1,4-3-ketosteroidsis needed. The reaction of thiosemicarbazide with A1g4-3-ketosteroids proceeds very slowly compared to Ad-3-ketosteroids. The determination of small amounts of A4-3-ketosteroids in Al~4-3-ketosteroids is based on these differences in rates of thiosemicarbazone formation. The procedure utilizes a modification of the spectrophotometric technique of Talbot, Ulick, Koupreianow, and Zygmuntowicz (10) based on the observation of Evans and Gillam (4) that excess thiosemicarbazide does not interfere in the determination of thiosemicarbazones of a-P unsaturated ketones,
solution is diluted to 100 ml. with water. This reagent is stable a t room temperature for several months. Standard St,eroid Mixtures. A standard solution of the appropriate A1f4-3ketosteroid in 95% ethyl alcohol is prepared to contain about 0.10 mg. per ml. Standard mixtures are also prepared which contain, in addition to the A1t4-3ketosteroid a t the above concentration, approximately 1, 2, 3, 4, and 5% of the corresponding A4-3-ketosteroid. APPARATUS
Glass-Stoppered Test Tubes. Prepared by sealing the ends of T 19/38 outside ground joints. Constant Temperature Water Bath, 25’ C.
REAGENTS
Thioseinicarbazide Hydrochloride, 0.1M. A sample of 0.9114 gram of thiosemicarbazide is dissolved in 50 ml. of 0.2N hydrochloric acid and the resulting
Spectrophotometer. A Beckman Model DU spectrophotometer equipped with a jacketed cell compartment maintained a t 25’ C. was used. Such thermostatic control should not be necessary in an air-conditioned laboratory. Spectra VOL. 31, NO. 5, MAY 1959
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