High Resolution Infrared Spectra of Steroids in the Carbon-Hyd rogen Stretching Region ANNE M. SMITH Eastern Regional Research laboratory, Philadelphia 18, Pa.
C. ROLAND EDDY Temple University, Philadelphia 22, Pa. The infrared spectra of 40 steroidal sapogenins were investigated in the 3100- to 2 7 5 0 - ~ m . - ~region using a double monochromator equipped with lithium fluoride prisms, in order to correlate absorption in the C-H stretching region with structural features of the molecules. In addition to bands which can b e assigned to
I
stretching modes of =CH, -CHZ-, and -CH3 groups, absorption maxima and shoulders occur which cannot b e easily assigned to normal modes of isolated units. The majority of the observed bands are very sensitive to structural changes in the carbon skeleton. It is frequently pos4ble to distinguish between 2 5 ~ and 251 configurations, detect opening of E and F rings, and determine the configuration of the methyl group in the 20 position. A detailed study of the C-H region of steroids can thus serve as a useful supplement to investigations at lower frequencies.
I
in the carbon-hydrogen stretching region has increased with the advent of high resolution spectrophotometers. Fox and Martin (1-3) used a grating spectrometer to establish characteristic C-H stretching frequencies for a carefully selected group of hydrocarbons in which one or two functional groups predominated. A similar study on a series of deuterated ketones and esters was conducted by Kolin and Jones (9-11). The Fox and Martin assignments have been confirmed by work on fatty acids ( 4 ) , heterocyclic nitrogen compounds ( I 5 ) and oxygen- and sulfur-containing materials (12), and also have been applied to the analysis of mixtures (IS, 14). Jones and coworkers (5-7) have discussed the C-H stretching modes in steroids and used them to detect ethylenic double bonds; but no systematic study of the 3000-cm.-l region has been reported for steroids. A larpr number of reference grade steroidal sapogenins have been prepared at this laboratory by Wall and coworkrrs 7 7 - 1 9 ir, the course of a searcr NTEREYT
I
Typical skeleton structures of steroidal sapogenins A. 20a, 2 5 sapogenins ~ (3-deoxytigogenin) B. 20j3, 2% sapogenins
200, 251, sapogenins is less firmly established, but is believed (16) to involve ring inversion (Formula B). Other variations from the illustrated structures include acetylation at CS or at Cz and Cat unsaturation at CS, introduction of a carbonyl group at CIZ, cis or trans fusion of the A/B-rings, and rupture or cleavage of the *Eand F rings. Structural differences among the studied sapogenins are listed in Table I. The compounds fall into six general classifications: sapogenins, deoxysapogenins, 20cr sapogenin acetates, 2Op sapogenin acetates, open F-ring compounds, and cholestane derivatives. EXPERIMENTAL
for cortisone precursors. Lithium fluoride spectra were obtained for these compounds to correlate observed bands with molecular structure and to see if the Fox and Martin hydrocarbon assignments could be applied to steroids. The basic structure of the spirostane ring system of sapogenins has been well established by Marker and others (8). Convincing evidence has been presented (16) for the side-chain stereochemistry of 20a, 2 5 sapogenins, ~ illustrated by 3-deoxytigogenin (20a, 2 5 ~allospirostane, Formula A), and for the analo~ 20p, 2 5 compounds ~ gous 20a, 2 5 and
($0)* The side-chain structure
Table 1.
of
the
The infrared absorption studies were carried out on a Beckman Model IR-3 double-monochromator spectrophotometer equipped with lithium fluoride prisms. Spectra were recorded in carbon tetrachloride solution a t concentrations of 3 t o 10 grams per liter in a 1.02-mm. cell, using a tape-recorded blank of carbon tetrachloride in the same cell as a reference. The mechanical slit width was set at 0.18 mm. at 2750 cm.-l and attained a value of 0.15 The spectral mm. at 3100 cm.-l resolution of the instrument under these conditions is estimated to be around 3 cm.-1 (Sp!itting of HCl36 and HCI3' doublets with a separation of about 2 cm.-l can be well observed with 0.11mm. slits; the two bands are just
Structural Features of Steroidal Sapogenins Studied
Position
Sapogenin Sarsasapogenin Yamogenin Tigogenin Diosgenin Hecogenin Gentrogenin Correllogenin Gitogenin Yuccageniti Manogenio Kammogenii, Chlorogenir. Markogenii Smilagenin Rockogenir
A/B cis
...
trans
...
trans
... ...
trans
...
trans
...
trans c19
cis
trans
of -0COCHs c 2 6
and/or OH
L L D D D D L D D D D D L D
38 30 3P 3P 3P
11
3P
3P 2ffJ 3P 2% 38 2% 38 2a, 313 3P, 6a: 2P, 38 38 30, 128
Other Functional Groups A6
A5
12 c=o
As, 12 A6, 12 A6
12 Asr 12
c=O
c=o c=o c=o
3
VOL. 31, NO 9, SEPTEMBER
1950
1539
u
A I-
b 1 3
z
M
*
uA
+
I-
x
I
2100
3000
Cm"
28W
3000
2800
3000
Cm'l
cm-1
2100
3000 Cin.1
3000
2800
em-!
Figure 1 . Infrared fpectra of steroidal sapogenins and related material in the 31 00-to 2750- cm.-1 region. See Table II for identification of individual spectra barely distinguishable with 0.15-mm. slits.) The isolation and purification of the compounds have been described (17'19). Their high purity was confirmed by melting point, carbon and hydrogen determination, and optical rotation, in conjunction with sodium chloride spectra in the 1800- to 650-cm.-' region. Pure samples of cholestane and coprostane were supplied by Louis Fieser of Harvard University.
pounds with open E and F rings show only a weak band in this region. Addition of acetate groups increases the absorption of: the 2950-cm.-' band relative to 2930 cm.-', but the only compound in which CH3 absorption exceeds CH2 in this region are 20a tigogenin I assigned to the =CH stretching vibraderivatives, and some compounds withtion by Jones and coworkers (6-7). out a Cs methylene g r o u p i . e . , A6 The frequency is 10 to 20 cm.+ higher acetates and the 3,Gdiacetate of in the sapogenins than in unsaturated chlorogenin. aliphatic hydrocarbons (3). 2930-Cm.-' Region. Fox and 3000- to 2960-Cm. -l Region, Martin found both CH3 and CH2 RESULTS A N D DISCUSSION Most 208, 2 5 ~sapogenin acetates absorption near 2930 cm.-', but later exhibit an inffection around 2990 researchers were unable to resolve the Fox and Martin and subsequent incm.-'; the 20& 2 5 compounds ~ absorb two bands. I n the steroid series the vestigators found one band which they above 3000 cm.-l Compounds with a band found around 2930 cm.-' was I 12-ketone group do not show either assigned to asymmetrical CH, stretching associated with the =CH group and two of these bands. A shoulder around when comparison of sapogenin acetates bands associated with the symmetric 2975 cm.-' was found for most 200, 2 5 ~ with the corresponding deoxy comand asymmetric stretching of the CH2 sapogenins, but in none of the 200, 2 5 ~ pounds revealed a decrease in a b s o r p group. The CH3 group gives rise to compounds. In A5 compounds the tion due to substitution a t the 3 positwo main bands assigned to the symshoulder is hardly noticeable or is nontion. The similar effect of C3, Ca acetymetric and asymmetric stretching modes existent. Derivatives of yamogenin lation was mentioned above. Loss of an and to two weaker bands. A weak (a A5, 2 5 ~sapogenin) show a strong additional methylene group a t C2 by band, seldom observed, was associated shoulder a t 2964 cm.-' The origin of acetylation does not produce the same I effect. these shoulders is unknown. with -CH groups. Between seven and 2950-Cm.-' Region. All the com2920- to 2900-Cm.-1 Region. A pounds studied had a band close to shoulder near 2910 cm-1 occurring in nine bands were found for steroidal most sapogenins is more pronounced in sapogenins in the 3100- to 2 7 5 0 - ~ m . - ~ 2950 crn.-l, which was attributed to the asymmetrical CH3 stretching. The A5 compounds, and becomes a strong region. Assignments were made on band increases in intensity relative to band in A5 acetates. Its relative inthe basis of the observations of Fox the neighboring 2930-cm.-' band as tensity varies inversely with that of the and Martin and spectral differences the ratio of CH3 to CH, groups becomes 2860-cm.-' band in A5 acetates. Fox produced by changes in molecular strucgreater. This ratio is one to three in a and Martin associated a band in this ture. Frequencies mentioned in the typical sapogenin (Formula A). Comtext are only approximate. Precise region with the methyl group in
I
1540
ANALYTICAL CHEMISTRY
values are listed in Table 11. The spectra of the sapogenins in the 3100- to 2750-cm.-' region are shown in Figure 1. 3035-Cm. Region. A weak band was found around 3035 cm.-1 for all the A5 sapogenins. The band has been
Table II.
3035 Cm.-1 Compound Sapogenins Tigogenin
I
=CH
20a 3-deoxysapogenins Tigogenin Hecogenin Srnilagenin Sarsasapogenin Rockogenin 2Oa A6 3-deoxysapogenins Diosgenin Yamogenin
Absorption Bands of Steroidal Sapogenins and Related Compounds
2950 Cm.-l 3000-2960 As-m. -8H8 Cm.-l 2978"
2952
2930
2973" 2973"
2952 2953 2952 2951 2953
2928 2929 2928 2932 2929
2964"
2952 2953
2929 2933
20a sapogenin acetates Tigogenin Gitogenin Hecogenin Manogenin Chlorogenin Smilagenin Sarsasapogenin Markogenin
2952 2952 2953 2954 2954 2954 2953 2954
t.riim -.
2850 Cm.-l
2830 Cm.-l
No., Concn., Fig. 1 G./L.
2861
2849
1
7.5
2872 2874 28704 2870" 2874
2857 2859 2862 2862 2861
2846 28505 2855" 2854a 2851"
2 3 4 5 6
4.0 4.3 3.6 3.7 4.2
2907 2909"
2873 2874
2861 2864
2847 2849
7
4.1 4.0
2930 2929 2930 2931 2930 2930 2935 2936
2909= 2909" 2908" 2909" 29090 2909O 2910" 2909"
2873 2873 2874 2874 2874 2873 2873 2875
2861 2862 2861 2863 2861 2863 2864 2866
2846 2848
9 10 12 13 14 15 16
4.6 5 2 10 10 10 4.1 4 9 10
2953 2953 2955 2957 2951 2958
2931 2929 2930 2931 2936' 2937
2908 2907 2908 2907 2909 2909
2873 2873 2873 2875 2874 2873
28610 2861" 2860 2863 2863a
2847O 2852 2853"
2992"
2953
2932
2872
2860
2992"
2954
2932
2872
3002" 2993" 3005"
2953 2953 2953 2953 2953 2954
2932 2932 2934 2933 2934 2931
2920" 2908" 2921" 29070 2907" 2907" 2919 290g5 2909" 2911"
2872 2872 2872 2874 2873 2874
2992a 3008
2953 2954
2933 2936
2907 2908
2873 2873
2951 2953
2927 2934 2934
29035 2902 2900
2867O 2868 2864
2856
2950"
2935 2935
2915 (2892)
2867"
2849 2858
2929 2933 2932
2907O (2885)
2867 2868 2862
2975" 2972a 297g5 2975" 2972'
2907"
Spec-
2873
2968"
3033 3032 3019
2860 2930 2870 Cm.-' Cm.-1 Cm.-1 - .... A s b . 2920-2910 Sym. Sym. -CH*Cm.-l -CHa -CH2--
28542851" 2848 2848" 2852"
2829a 282ga
8
11
2839"
2 0 a A s sapogenin acetates
Diosgenin Yuccagenin Gentrogenin Kammogenin Yamogenin Correllogenin
3034 3034 3034 3038 3032 3033
208 sapogenin acetates Tigogenin Gitogenin Hecogenin Manogenin Chlorogenin Sarsasapogenin Smila.genin Markogenin 208 Aa-sapogenin acetates Diosgenin Y amogenin 20a-dihydrosapogenin acetates (open F ring) 3-Deoxytigogenin Tigogenin Gitogenin 20P-dihydrosapogenin acetates Tigogenin Hecogenin
3033 3032
2972" 2962O
2962
29905 2966"
Cholestane and related compounds (open E and F rings) Cholestane 2950" Cholestanol 2951" Coprostane 2950 a Bands appearing as inflections on more intense bands. branched-chain or unsaturated hydrocarbons. Pozefsky and Coggeshall (1.2) observed a 2910-cm.-' band for a number of compounds, mostly aldehydes and ketones. It would seem that double bonds and carbonyl groups increase the absorption of nearby methyl
groups around 2910 cm.-l A few 208 sapogenin acetates have an additional shoulder at 2920 cm.-l, and open Fring steroids have one at 2900 cm.-1 The origin of these inflections is unknown. 2880- to 2830-Cm.-l Region. The
17
10
18
10
19 20 21 22
7.5 10 7 5 40
2851
23
4.2
2863
2848
24
10
2861 2863 2859 2865 2864 2865
2851 2852
25 26 27 28 30 29
10 10 10 4.0 10 4.2
10
2856 2858 2853O
2851 2848
2829 2830 2832 2833 2831 2831
2853 2854
2829 2831
31
2847" 2847 2847
2831a 2834s
33 34 35
32
10
7.5 7.5 7.5
36 37
28480
10
10
38
4.2 5.0
39
40
5.8
symmetrical vibrations of both CHa and CH2 groups appear in this region, around 2870 and 2860 cm.-l, respectively, for the sapogenins examined. The 2870-cm.-l CH1 band is weak in 3deoxysapogenins compared with the 2860-crn.-' CH2 band. A third band VOL 31, NO. 9, SEPTEMBER 1959
0
1541
appears around 2850 cm.-' in tigogenin derivatives and A6 compounds, but is only an inflection in most other steroidal sapogenins. This band was also assigned to the CHZ group because it decreases with increase in ring substitution. The band is almost undetectable in ketonic sapogenins. The 2860-cm.-" CH1 band almost disappears in A5 acetates, except for carbonyl compounds. I n compounds with open E and F rings, the bands in this region appear a t lower frequencies. A band near 2830 cm.-' is associated with A6 unsaturation, and is stronger in C=O compounds, but no definite assignment can be made. CONCLUSIONS
Structural features of various steroidal sapogenins can be identified from details of their C-H stretching spectra. Of the seven to nine bands observed, five can be associated with specific
I
modes of =CH, CH,, and CH, groups. All observed bands seem to show a strong dependence on the over-all structure of the molecule. Easiest to characterize are &deoxysapogenins, sapogenin acetates, Ab compounds, and open-ring compounds. I n 3-deoxysapogenins, the CH2groups absorb so much more than the CH3 groups that CHs bands a t 2950 and 2870 cm.-l are perceptible only as shoulders on the slopes of strong CH, bands. Acetylation increases the relative intensity of these
CH, bands, making them nearly equal to the CHZbands. The presence of two additional bands around 3035 and 2830 ern.'' is sufficient to characterize A5 unsaturation. In addition, A5 acetates show increased absorption around 2910 cm.-', paralleled by decreases near 2930 and 2860 cm.-' Open-ring compounds have broader and fewer bands, and those below 2880 cm.-' are shifted to lower frequencies. The 208 compounds are readily recognized by inflections around 3000 cm.-l, and in some cases also a t 2920 cm.-l The CH2 bands of 208, 2 5 acetates ~ are slightly stronger, and the CHS bands weaker than those of the corresponding 20a compounds. cis-A/B sapogenins with 2 5 ~methyl groups have a greater absorption area and broader individual bands than their trans-A/B, 2 5 counterparts. ~ N o transA/B, 2 5 compounds ~ and only one cisA/B, 2 5 ~compound were available. The cis- and trans-25~compounds were similar, suggesting that the band-broadening and increased area are mainly caused by D, L isomerism in the 25 position. ACKNOWLEDGMENT
The authors acknowledge many helpful discussions with Heino Susi of the Eastern Regional Research Laboratory. LITERATURE CITED
(1) Fox, J. J., Martin, A. E., Proc. Roy. SOC.(London )A162,419 (1937). (2) Ibid., A167, 257 (1938).
(3) Zbid., A175,208 (1940). (4) Guertin, D. L., Wiberley, S. E., Bauer, W. H., J . Am. Oil Chemists' SOC. 33, 172 (1956). (5) Jones, R. N., Herling, F., J . Org. Chem. 19, 1252 (1954). (6) Jones, R. N., Humphries, P., Packard, E., Dobriner, K., J . Am. Chem. SOC. 72,86 (1950). (7) Jones, R. N., Williams, V. Z., Whalen, H. J., Dobriner, K., Zbid., 70, 2024 (1948). (81 Miiker, R. E., Wagner, R. B., Ulshafer, P. R., Wittbecker, E. L., Goldsmith, D. P. J., Ruof, C. H., Ibid., 69, 2167 (1947). (9) Nolin, B., Jones, R. N., Can. J . Chem. 34,1382 (1956). (10) Ibid., p. 1392. (11) Nolin, B., Jones, R. N., J . A m . Chem. SOC.75,5626 (1953). (12) Pozefsky, A., Coggeshall, N. D., ANAL.CHEM. 23,1611 (1951). (13) Saier, E. L., Coggeshall, N. D., Zbid., 20, 812 (1948). (14) Saunders, R. A., Smith, D. C., J . Appl. Phys. 20, 953 (1949). (15) Tallent, W. H., Siewers, I. J., ANAL. CHEM.28,953 (1956). (16) Wall, M. E., Ezperientia 11, 340 (1955). (17) Wall, M. E. Krider, M. M., Rothman, E. S., Eddy, C. R., J . Biol. Chem. 198, 533 (1952). (18) Wall. M. E.. Serota., S.., J . Am. Chem. ' doc. 78,'1747 (1956). (19) Wall, M. E., Walens, H. A., Ibid., 77, 5661 (1955). (20) Zbid., 80, 1984 (1958). RECEIVED for review January 1, 1959. Accepted May 25, 1959. Abstracted from a thesis in chemistry submitted by Anne M. Smith in partial fulfillment of the requirements for the master of arts degree at Temple University, Philadelphia, Pa., June 1958.
X-Ray Powder Diffraction Data of Azoic Coupling Components ISIDORE SCHNOPPER, JOSEPH 0.BROUSSARD, and COSMO K. LaFORGlA U. S. Customs laboratory, New York, N. Y. ,X-ray powder diffraction data are presented for 15 organic compounds. The data permit identification of the azoic coupling components of commercially available Naphthol AS, some of its derivatives, and related compounds.
A
of coal tar products provided for under the United States Tariff Act as amended (8) N IMPORTANT CLASS
Naphthol AS 3-Hydroxy-2-naphthanilide
1542
ANALYTICAL CHEMISTRY
includes Naphthol AS, its derivatives, and related compounds (1, 3-5). These products are azoic coupling components extensively used in dyeing and printing fabrics, and thus of great commercial importance. The Tariff Act specifies that the ad valorem rate of duty applicable to importations of dutiable coal tar products shall be based upon the American selling price of any similar competitive article manufactured or produced in the United States. To that end, one function of the U. S. Customs Laboratories is to advise customs officers on the identity and comparability status of imported coal tar products. To improve this service, an effort has been directed towards an increasingly greater use of x-ray dif-
fraction as an adjunct to the conventional methods of chemical analysis. This paper is the result of a part of that effort. EXPERIMENTAL
The Naphthol AS types used for this work are commercially available and have an indicated purity of 950/, or better. Each compound was recrystallized from xylene (ACS) and the melting point determined to obtain an indication of its purity. The compounds were identified by mixed melting point determinations using identical compounds obtained from widely different sources. The recrystallized compounds were prepared for x-ray work by grinding in an automatic agate mortar for 15 minutes. The powders were then packed
