Catalog of Infrared Absorption Spectra of Steroidal Sapogenin Acetates C. ROLAND EDDY, MONROE E. Wa4LL, AND MARY KLUMPP SCOTT Eastern Regional Research Laboratory, Philadelphia 18, Pa.
A very convenient tool in the identification of a steroid is its infrared spectrum. The infrared absorption spectra between 700 and 1400 cm.-' are presented for the acetates of twelve of the most common sapogenins derived from plFnt saponins. The spectra are highly characteristic and make possible a ready identification of an unknown sapogenin by comparison of its spectrum with these reference curves.
T
HIS laboratory is engaged in a large scale investigation of frared absorption curves were obtained with a Becknian IR-3 the steroidal sapogenins that occur in the plant kingdom. spectrophotometer, using sodium chloride prisms. ConcentraPure sapogenins have been isolated and identified by methods tions were approximately 10 grains per liter, in cells approximately described in a previous paper (IO). A4sa means of characteriz1 mm. thick, ing these compounds, their infrared absorption spectra have Since the earlier manuscripts of this series were first written, been obtained. The spectra so obtained are highly characteristic the wave-length scale of the infrared instrument has been recaliof the individual sapogenins and constit'ute a more positive brated. -4 slight shift from the earlier calibration was found, identification of unknown sapogenins than any other combinaprobably due to a sniall temperat'ure difference resulting from an tion of physical constants. improvement in temperat,ure control equipment.. Wave numbers A sapogenin can be identified either by means of its spectrum reported in this paper and in the first of the series (IO) are corin chloroform or by means of the spectrum of its acetate in carbon rect,. Some of the wave numbers appearing in the second (9) disulfide. -4lthough hot,h of these methods are used routinely and t,hird (8) papers of the series are in error hl- approximately in this laboratory, the acetate spectra are found to differ more from one an___~ other than do the spectra of the free sapogenins. Hence an unknown can be Table I. Physical Constants of Sapogenins identified with greater certaint,y by Specific Kotationb, Estimated Melting Pointu, C . Degrees Purity, means of its acetate spectrum, especially Conipound ' plant Source Genin .\cetate Genin Acetate yo if it not' 1.54-155 -75 -40 98-100 269-273 pure* In this Chlorogenin Chlorogalurn p o m e r i d i a n u m 196-190 -121 -121 95-100 200 paper, reference absorption spectra are Diosgenin Dioscorea composiia -75 -103 98-100 271-271 244-145 Yucca filamentova presnted for the most common sapogenin ~ ~ ~ AgaueC $ ~ ~ ~ 268-268 n 247-250 +6.G -1.2 98-100 260-263 -50 8 1 98-100 243-244 Xamnmgenin Yucrafilameutosa acetatcs. Kryptogenin Dinscoread 174-177 146-150 -18; -175 90
'
.
+-
- 50 245-247 218-250 Yucca gloriosa , Yucca carnerosana 198-203 190-192 -74 -84 -89 -7.5 199-202 133-141 I.ucca c f . thornberi -7.5 -50 180-184 150-152 Agave lecheguilla Yucca gloriosa 207-211 210-212 -6.3 -72 yuccasp, 239-241 178-180 -86 -140 Kofler melting points. dllrotations at 2 5 O C. in CHCla, 589 m p , concentrations of 8 = 1 w'ams per liter. Repurified from mixed batches from different species. Repurified from a sample obtained from Syntex, S.A., Mexico, D.1'.
hlanogenin Samogenin Sarsasapogcnin Smilagenin
EXPERIMENTAL
Details of the methods of separation and purification of the sapogenins are given in a previous paper (IO). Table I gives the plant source and physical constants of each sapogenin. The in-
90 80 90-100 90-100 98-100 98-100
in
a b c
d
WAVELENGTH, M I C R O N S
8
100
9
10
I
I
I
I
II I
I
12
13
14 I
1
" I
7k6 783
I00
747 80
60
40
20 CHLOROGENIN
ACETAT E
I 800 W A V E NUMBER. c m . '
Figure 1.
Chlorogenin Acetate (22-Isoallospirostan-3~,6a-diol3,6-Diacetate) 9.9 grams per liter in CS?. 1.1-mm. cell
266
1
0 700
V O L U M E 25, NO, 2, F E B R U A R Y 1 9 5 3
267
W A V E N U M B E R , cm-'
Figure 2.
Diosgenin Acetate (A6-22-Isospirosten-3p-ol 3-Acetate) 10.0 grams per liter in CS2. 1.1-mm. cell WAVELENGTH,
9
8 I
I1
12
I
I
I
WAVE
Figure 3.
M IC RONS
10
13 I
14 1
N U M B E R , cm"
Gitogenin Acetate (22-Isoallospirostan-Za,3~-diol 2,S-Diacetate) 10.0 grams per liter in CSa.
all of the spectra presented here possess these four bands, with the exception of kryptogenin, the structure of which lacks the E and F rings. The acetate bands in the 1240 cm.-' region are characteristic of the steric relationship between the CS hydrogen and the C, acetate, as sholvn by Jones et al. (4). hlolecules with a cis relationship have a more complex system of bands (Figures 8, 9, and 10) than molecules with either a trans relationship or a 5,6 douhle hond (Figures 1 to 7, 11: and 12). In the steroidal sapogenins, this makes it possible to determine the type of 4 / R ring fusion, since the configuration a t C, is always p in the naturally occurring steroidal sapogenins. Thus a complex band near 1240 c m - l indicates cis ring fusion, whereas a single band indicates trans (allo) ring fusion or A s uusatwation. With the sapogenins there is also not much interference from a second acetate group, as the 1240 cm.-l band is merely broadened or doubled by a diacetate (Figures 1, 3, 5 , 6, 7 , and 12) with a shape clearly distinguishable from the greater complexity of the cis bands (Figures 8, 9, and 10). These spectra also agree with the other generalimtions of Jones et d. for t.he simpler steroids ( 2 , .3,
1 crn. -1; correct values of some of these can be obtained from the current paper or froni the first paper. DISCUSSION OF SPECTRA
A41thoughthe complete spectra from 670 to 5000 cm.-l have been obtained, the portions useful for differentiating sapogenins are the fingerprint region from i o 0 to 1400 cm.-l and the carbonyl region from 1600 to 1800 cm.-l Figures 1 to 12 give the fingerprint spectra from i o 0 to 1400 cm.? Figure 4 also shons the carbonyl region of hecogenin acetate, a typical 12-keto sapogenin. The 1736 cm.-' band is present in all the sapogenin acetates (1i35 and 1736 in the monoacetates, stronger in the diacetates band is present ant1 shiftcd t o 1736 to 1744). The 1712 only in the keto sapogenins (1714 in kainmogenin and manogenin, 1720 in kryptogenin). As curves of all the sapogenin acetates of a given carbonyl claqs are essential13 the same between 1600 and 1800 cm.-l, this region of the spectrum is not reproduced in this publication for any except hecogenin acetate. It may be noted that, although the acetate hand and the 12-keto band are I\ (,I1 resolved in carlion disulbclr, they are not well resolved in c hloioform. ;1 previous pappi (9) inentioned the usefulness of the four ahsorption bands near 860, 900, 920, and 980 cm -l for identifying steroidal sapogenins as a class and for differentiating between is0 and normal configurations of the F ring. I t can be seen that
1.1-mm. cell
.
6, 6 ) . Except for krl-ptogeriin, these spectra show absorption bands in the regions 1038 to 1056, 1063 to 1098, 1124 to 1143, and 1158 to 1190 cm.-l. as found in ketals bv BerEmann and Pinchas ( I ) . However, unlike their open-chain ketals, the 1063 to 1098
268
5.6
IO0
ANALYTICAL CHEMISTRY W A V E L E N G T H , U IC R O N S
0
60
10
9
I
I
12
II
I
I
13
I
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eo 60
40
- 20
20 HECOGENIN ACETATE
0
le00
1700
I' 1 0 I601
I200
1300
I100
1000
eo0
900
750
WAVE N U M B E R , c d '
Figure 4.
Hecogenin Acetate (22-Isoallospirostan-3@-ol-l2-one 3-4cetate)
10.0 grams per liter in CSz.
This sample has no absorption bands between 700 and 750 e m . - 1
1.1-mm. cell.
W A V E N U M B E R , cm.'
Figure 5.
Kammogenin Acetate (A~-22-Isospirosten-2a,3@-diol-l2-one 2,3-Diacetate) 1.1-mm. eel1
10.0 grams per liter in CSz.
I
100
I
I
I
I
I
I I
*
BO
0 I4
#
60 c 4
t z
40
1
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a I-
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1
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-
H I C-CH,-CH,-C-CH,OAC
- eo
do CHI
-60
- 40
Ac 0
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0
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H-C
f
LII
CHI
u 1032
I
KRYPTOGENIN
I
ACETATE
I
20
-0
roo
V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3
269 WAVELENGTH, MICRONS
e
9
10
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13 I
I2 I
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14 I
100
I '
7i2
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-00
- 60
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H
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MANOGENIN ACETATE
8
WAVELENGTH, MICRONS IO II
9 I
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I2 I
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I
15
20
14
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H
- 20 SAMOGENIN
I
I 1400
I100
1200
I300
1000
ACE TATE
-0 700
800
900
W A V E N U M B E R , ern-'
Figure 8.
Saniogenin icetate (22-Isospirostan-2~,3~-diol2,3-Diacetate) i.0 grams per liter i n CSz.
e
9
1.1-mm. cell
WAVELENGTH, M I C R O N S IO II
12
14
13
U
a b-
S ARS AS AP O G E NI N ACE TATE
1400
1300
1200
I100
1 1000 WAVE
Figure 9.
900 NUMBER,
I
I eo0
cm-'
Sarsasapogenin Acetate (Spirostan-30-01 3-Acetate) 10.0 g r a m s per liter in CSz.
1.1-mm. cell
I
I
lo 700
ANALYTICAL CHEMISTRY
270 cm. - I band is not exceptionally strong in the closed-ring sapogenin ketals. IDENTlFICATION OF A SAPOGENIN
An unknown sapogenin acetate can be readily identified by means of these curves by the folloming procedure.
A. First examine the spectrum for the presence of the four absorption bands near 860, 900, 920, and 980 em.-' If any one of the four IS missing, the substance (like kryptogenin) is not a true steroidal sapogenin with an F ring. If the bands occur a t 850, 900, 920, and 986 cm.-l, Kith the 920 band stronger than the 900 band, it is a sapogenin with "normal" configuration of the F ring. Compare n i t h the rest of the bands of Figure 9 to determine TT hether it is sarsasapogenin (the most common "normal" plant sapogenin). If the four bands occur a t 865, 900, 920, and 981 em.-', with the 900 band stronger than the 920 band, the substance is a sapogenin with "iso" configuration of the F ring. B. Determine whether the unknown is the acetate of a monoor of a dihydroxy sapogenin by examining the acetate bands near 1240 and Ii10 cm.-l Figures 2, 4, 9, 10, and 11 show the strength of these bands typical of monohydroxy sapogenin acetates. Figures 1, 3, 5, 6, 7 , 8, and 12 show the much stronger acetate bands typical of sapogenin &acetates. C. If the preceding examination indicates a monohydroxy sapogenin, distinguish among diosgenin, hecogenin, smilagenin, and tigogenin by examining details of the fingerprint. Diosgenin is identified by prominent bands a t 813 and 838 crn.-l (possibly associated with a A; ethylenic bond). Hecogenin is identified
Smilagenin is identified by by its carbonyl band a t 1712 the multiple nature of the band b e h e e n 1200 arid 1260 em.-' and by its unusually high transmittance a t 1031 em. -l Tigogenin is identified by the band a t 995 cm.-l arid by the fact that it has strong bands a t both 960 and 1025 em. -I D. If the examination under B indicates a dihydrouy sapogenin, di$inguish among chlorogenin, gitogenin, kammogenin, manogenin, samogenin, and yuccagenin by the following details: Chlorogenin is identified by the band a t 1198 cm.-' and by the two strong bands of nearly equal strength a t 1028 and 1055, with a high tranqmittance maximum between them a t 1041 mi.-1 Gitogenin is identified by the general appearance of the band betxeen 1030 and 1060 cm.-' and by the t x o weak bands a t 913 and 935 em.-' Kammogenin is identified by its carbon>-l band a t l i 1 4 cm.-' and the svstem of bands a t 810, 826, and 845 em.-' Rfanogenin is identihed by its carbonyl band a t 1714 ern.-' and absence of bands between 800 and 850 cm.-' Samogenin is identified by the resolved acetate band a t 1221 em.-' and hy bands a t 940 and 1145 c m - ' Yuccagenin is identified by the strong band a t 911 ern.-' and the bands a t 825 and 843 cm.-l E. Confirm the identification by comparing the spectrum of the unknown with the entire fingerpiint spectrum of the corresponding reference conipound. The two must agree a t every absorption band, not only in n'ave number but also in relative intensity. Occasionally other snpogeniiis arc found in plant e\tr;xts besides those listed here but have not as j e t heen isolated in iufficiently pure form by the author5 to warrant publication of
3
w' z a
I-
-
P
VI
z
U 6 I-
WAVE
Figure 10.
N U M B E R , cm."
Smilagenin Acetate (22-1sospirostan-3~-013-lcetate) 10.0 grams per liter i n CSs.
1.1-mm. cell
WAV E LE NG TH, M I C R O N S I
1
I
I
I
I
I
100
100
EO
BO
z 60
60
8 W U
c a
t z z
40
40
U k
20
20 TICOGENIN ArFT4TE
0 1400
1300
1200
1100
1000
900
800
WAVE N U M B E R , em-'
Figure 11. Tigogenin Acetate (22-Isoallospirostan-3~-ol3-Acetate) 10.0 grams per liter in CSI.
1.1-mm. cell
0 700
V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3
27 1
WAVELENGTH, MICRONS
their spectra. Two of them can be readily identified by the following characteristics: 9-Dehydrohecogenin can be identified by is0 configuration from A, monohydroxy from B, a,@-unsaturated ketonic band a t 1680 cni.-’ 9-Dehydromanogenin can he identified by is0 configuration from A, dihydrosy froin B: a,& unsaturated ketonic band a t 1681 cm.-‘ similar systematic identification procedure can be set up for the unncetylated sapogenins in chloroform solution. The spectral tests for F ring, normal us. is0 configuration, carbonyl, and A s unsaturation are the s:tme as for the acetat,es. The distinction hetween mono- and dihydrosy sapogenins is not a3 clear cut as with the acetates, hut can usually lie made in the 1000 to I100 cm. -1 region. I n general, monohydroxy sapogenins have a single hand near 1050 cni. -1, Tvhercas dihydroxy sapogenins have a band that is stronger, broader, or more complex. If these preliminary tests point to a known genin whose spectrum turns out to be substantially identical to that of the unknown, and if the spectrum of the unknown is rlearly different from any of the other known spectra, the identification is considered satisfactory. On the other hand, if the sample is not pure enough to allo~rn. decision among the known spectra, it is best to acetylate and compare with the acetate curves. ACKKOWLEDGRIENT
The authors gratefully acknowledge the assistance of MaryAnne Morris and A4udrryE. Jones in various phases of this in-
vestigation. The sapogenins vere isolated by Arthur Finchler, H. TT’. Jones, If. E. Kenney, R. F. Mininger, and Samuel Serota. LITERATURE CITED
Bergmann, E. D., and Pinchas, S., Rec. trav. chim.,71, 161 (1952). Jones, R. X’., Humphries, P., and Dobriner, K., J . Am. Chem. Soc., 71, 241 (1949). Jones, R. N., Humphries, P., and Dobriner, K., I b i d . , 72, 956 (1950). Jones, R. N., Humphries, P., Herling, F., and Dobriner, K., I b i d . , 73, 3215 (1951). Jones, R. N., Humphries, P., Packard, E., and Dobriner, K., Ibid.,72,86 (1950). Jones, R. N., Williams, V. Z., Vihalen, 11.J., and Dobriner, K., Ibid., 70,2024 (1948). Krider, hI. M., and Wall, 11.E., I b i d . , 74, 3201 (1952). Rothman, E. S., Wall, h4. E., and Eddy, C. R., I b i d . , 74, 4013 (1952). Wall, M. E., Eddy, C. R., hIcClennan, M. L., and Klumpp, h1. E., h A L . CHE?,f., 24, 1337 (1952). Wall, hI. E., Krider, hI. SI.,Rothman, E. S., and Eddy, C. R., J . Biol. Chem., in press. RECEIVED for review September
4 , 1952. Accepted October 2 2 , 1Q52 Sixth in a series on steroidal sapogenins; for uaper V see (7). Work done as part of a cooperative arrangement between t h e Bureau of P l a n t Industry, Soils, a n d Agricultural Engineering and Bureau of Agricultural a n d Industrial Chemistry (U. S. Department of Agriculture) a n d the ?;ational Institutes of Health (Federal Security Administration).
Induced Reaction Method for Determination of Fluoride Ion JACK L. LARIBERT Kansas State College, Manhattan, Kan.
T
HIS study was undertaken to investigate a fundamentally new and sensitive method for quantitatively determining fluoride ion in solution. The increasing use of fluoride ion in drinking water has heightened interest in methods capable of producing good precision a t concentrations of 1 p.p.m. Methods reported in the literature, if sufficiently sensitive, are usually colorimetric procedures which involve the reaction of fluoride ion with complexes of zirconium, iron, or titanium. The standard method of the American Water Works iissociation ( 1 ) involves the reaction of fluoride ion to displace alizarin sodium monosulfonate, sodium 1,2-dihydroxyanthraquinone-3-sulfonate, from its pink zirconyl complex in arid solution to produce grada-
tions of color through yellow in proportion to the fluoride ion concentration. Xumerous variations of this method utilize other polyhydroxyanthraquinone derivatives. Other proposed methods (3) involve the decolorization of ferric ion complexes, such as the thiocyanate, or pertitanates by reaction with fluoride ion. The method described in this study differs from others in being made quantitative by timing the appearance of color produced by a reaction whose rate is proportional to the fluoride ion concentration in the sample. Although visual color comparisons were used, little practice is needed to obtain precision and the time measurements can be made with any watch having
