Analysis and Comparison of Ultraviolet Spectrophotometric Curves of Steroids with Application of the Analog Computer EDWARD R. GARRETT,’ JAMES L. JOHNSON, and CLAYTON D. ALWAY
The Upjohn Co., Kalamazoo, Mich. ,The analog computer has been used to compare the effects of substituents on the ultraviolet absorption spectra of 4-ene-3-one steroids. This approach was applied systematically to provide a library of curves useful in characterizing substituents, even though the wavelength of the absorption maxima was unchanged. Typical comparisons gave the following results. Saturated substituents at the C-17 position did not alter the spectrum. A 1-ene function was bathochromic in the sense of skewing the absorption band. An 1 1-keto group was hypsochromic in the same sense. The configuration of an 1 1-hydroxyl had a pronounced effect when the A-ring chromophore was a lf4-diene-3-one.
displaced by the logarithm of ratio of the concentrations (6). Equipment is available to plot log A us. h directly ( 1 ) . A more valid test of identity could be effected, using a plot of the logarithm of the ratio of absorbances against wavelength. A straight line was proof of the superimposability of the spectra, and the displacement from log A J A , = 0 gave the logarithm of the ratio of
the concentrations represented by the two spectra. The preparation of such curves using the spectra measured today in most laboratories calls for timeconsuming arithmetic calculations and replotting by hand. This paper describes the use of function generators and an analog computer to establish the use of this inThe ultraterpretative approach.
I I
T
HE properties used classically in correlating electronic spectra with the structures of organic compounds include : wavelengths of maximal and minimal absorptions, absorption intensities a t such wavelengths, and changes in these properties-e.g., when they vary from one solvent to another or one p H to another (2,3,5-7,9). Numerical values for a few wavelengths are obtained readily and serve in cataloging spectral data and file searching. The most satisfactory characterizations or identifications by means of absorption properties, however, use complete spectra. It is not easy, therefore, to test identity when two curves showing absorbance as a function of wavelength represent different concentrations of the absorbing species. This is particularly true for the ultraviolet spectra of many steroids whose absorptions arise with a 4-ene-%one system. Subtle effects due to substituents a t other positions on the nucleus can be missed. A better procedure is to check the superimposability of curves showing the logarithm of the absorbance, log A , as a function of the wavelength, A. The curves for the same compound will have the same shape and will be 1 Present address, College of Pharmacy, University of Florida, Gainesville, Fla.
1472
ANALYTICAL CHEMISTRY
I
0.2
7
6
5
l-4 8
9
o.2bj
-0.2 0.0
u 260 2 5 0 2 4 0 230
260
250
260 250 240
2 4 0 230
12
II
IO
230
W A V E L E N G T H IN Figure 1, length 1. 2. 3. 4. 5. 6.
Plots of logarithms of ratio of molar absorptivities against waveIB/(lC,I’C), (lIC,ll’E)/II’C,II‘G/II”G (lB,lC,l’C)/lA IIA/IA ID/IA IID/IIA IID/ID
7. 8.
9. 10. 11. 12.
IID/IA IIC/IIA IIB/IIA IIC/IIB II’F/II’C (II‘G,II‘~G)/I~’c
13
Figure 2.
Computer setup for log ratio plots of ultraviolet absorbance curves
violet spectra of steroids are cases in point. I n many cases the band position for the 4-ene-3-one group in the A ring, I and 1', is only slightly, if at all, changed by structural variations at the 11, 17, or 21 positions ( 4 ) . This is also true for the 1,4-diene-3-one group, 11, II', and 11". The studies described below show that these changes introduce variations in the shapes of the absorption bands. THEORY
The absorbance, Ax, of a chromophore :tt a specific wavelength, A, in a cell of 1-em. length is proportional to the concentration of the absorbing substance, C', A A = QC
(1)
where Q is the molar absorptivity when C is in moles per liter. For a solution of concentration C1 Log
A I =
log
EX
+ log c1
(2)
Since log C1 is constant and independent of wavelength, the shape of a curve showing log AI plotted against wavelength, A, is determined solely by log EA. If a second solution is made u p at concentration Cz = RCl, then
+
Log A2 = log EX log Cz = log ex log c,
+
+ log R
(3)
This second curve of log A z us. X should have the same shape as the first log A 1 us. A. The two plots should be superimposable, although separated at all wavelengths by a distance of log R, where R is the ratio of the concentra-
tions. It also follows, for samples having identical spectra, that a plot of the logarithm of the ratio of the absorb&nces against the wavelength, A, is a straight line, displaced from zero by the logarithm of the ratio of the concentrations. This derives from Equations 2 and 3 using the properties of logarithms. Log A i / A z = log Q/EX log CI/C2
+ =
0
+ log R
(4)
K h e n the spectra of two samples differ, an expression similar t o Equation 4 is more suitable. Log AI/& = log
+ log CIICZ
~ 1 / ~ 2
(5)
It follows that a plot of log el/ez vs. A is obtained from a plot of log A1 ' A 2us. A by shifting the ordinate from log A1 /ii2= 0 to log C1IC2 as Log AI/Az
- log Cl/C* = log
(6)
~ i / ~ z
Such plots of the comparisons of the ultraviolet spectra of various steroids are given in Figure 1. APPARATUS AND PROCEDURE
All spectra were drawn from a library of spectra measured with a Cary Model 11 spectrometer using 1-em. quartz cells. The presentation showed absorbance as a function of wavelength. The solvent in all cases was 95% ethyl alcohol. Each spectrum was traced with conducting ink. In comparing the spectra of two steroids, each was placed in a curve-following function generator, Moseley Auto-
graph 4A, \\ith synchronized follower travel along the wavelength, A, axis. The height above the zero absorbance was proportional to the absorbance and to the potential generated from each generator. The quotient of these potentials was taken by the analog computer and simultaneously transferred to the logarithmic value on a third function generator fitted with a plot of log number vs. number in conducting ink. The potential was put into the third function generator on the abscissa as the log absorbance ratio. Simultaneously, this log absorbance ratio was plotted against A on an X - Y recoider; the pen travel along the wavelength axis was also synchronized with the tn-o other function generators. The resultant plot of log Al/=12 us. X was transformed into a log E ~ / E Z US. A plot by translating the ordinate by log Cl/C2. Referring to th'e computer setup diagram in Figure 2, amplifier 13 and its associated circuitry comprise a time base generator. The output of this generator is a potential increasing linearly with time. The rate of increase is proportional t o the setting of potentiometer 13B and is variable from 0 to 10 volt per second. A setting of one (1 volt per second) is satisfactory. This time base generator is connected directly to the X-axis inputs of function generators 1 and 2 and through potentiometer scale adjustment potentiometer 15A to the recorder. A scale adjustment potentiometer is used here because the particular recorder employed does not have built-in provision for adjustable scales, and its X-axis scale must be made to correspond closely to the X-axis scales of function generators 1 and 2. Initial condition VOL. 34, NO. 1 1 , OCTOBER 1962
1473
(1.C.) 4 makes i t possible to apply any desired potential to the three X axes simultaneously to facilitate scale adjustment and to start the operation at any desired point on the X axis. Switch A is the run-reset control. Switch B is the run-hold control. The curve following slide-wire of F.G. 2 is connected across F.G.power supply 2. Range and zero are adjusted to produce a potential of zero at the absorbance base line of the curve and about 100 volts a t the full scale point. This potential is measured at the slider by the null technique. This potential, labeled f ' (X), is connected to the input of amplifier 15 through a 10-megohm resistor. The use of a 10megohm resistor instead of the more usual 1-megohm resistor reduces the so-called loading error. The output of the amplifier is connected to the full scale end of the curve follower slidewire of function generator 1. The output of amplifier 15 is also connected through potentiometer 14A to inverting amplifier 14 and thence to the zero scale end of the curve follower slidewire of function generator I. The purpose of this circuit is to provide a zero adjustment for the function generator 1 slide-wire. The slider of F.G. 1 is connected through two parallel 10-megohm resistors to the input of amplifier 15. Thus a potential proportional to the height of the numerator curve (F.G.2) is applied to the input of amplifier 15. The output of 15 is fed back to its input through F.G. 1 so that the gain of amplifier 15 is in-
Table
I.
Substituted Steroids as Derivatives of Basic Structures,
Compound IA IB
R
Substituents R'
=O
IIA
-H
IIB
---OH
IIC
-OH
IID
=O
II'C
-OH
MATERIALS The steroids used for this work were
of the highest quality available. Solutions were prepared by dissolving accurately weighed portions of steroid in 95% ethyl alcohol. The steroid structures are given in Table I as derivatives of pregn-4ene-3,20-dione, I ; 17a-hydroxy-21-acetoxy-pregn-4-ene3,20 - dione, 1'; pregna - 1,4 diene3,20 - dione, 11; 11,9,17a - dihydroxy21 - acetoxy - pregna - 1, 4 - diene3,20 - dione, 11'; and 118,17a,21trihydroxypregna - 1,Cdiene - 3,20dione, 11'.
-
7H3
CHzOOCCHs I
c=o
I, 1', II, II', and IIH
Pregn-4-ene-3,20-dione (progesterone) 1101-Hydroxypregn-4ene-3,20-dione( 1lahydroxyprogesterone) 1lp-Hydroxypregn-Pene-3,20-dione(1lphydroxyprogesterone) 11p,l7a-Dihydroxy-21-acetoxypregn-4ene-3,20-dione (hydrocortisone-21-acetate) Pregn-Cene-3,11,20-trione(1l-ketoprogesterone)
I'C
ID
nected to scale adjusting potentiometer 10A and thence to the Y axis of the recorder. The apparent "hunting" of the computer system in Figure 1, is exaggerated to demonstrate the conformity of the log E ~ / E Z with the zero ordinate. The precision of the method for the determination of log ratios of the absorptivities with this equipment and computer program is given in the significant figures of Table 11.
R"
IC
-0
I1 CH200CCH3
c=o
R" @-OH
CHzOH I
Pregna-1,4-diene-3,2O-dione( A' prc-
gesterone)
11a-Hydroxypregna-lJ4-diene-3, 20-dione ( 1101 hydroxy-A'-progesterone) 118-H ydroxypregna-l ,4-diene-3,20dione (1lp-hydroxy-A'-progesterone) Pregna-l,4-diene-3,11,2O-trione ( 11-
H
-H
II'E
-OH
- - CHI
-H
II'F
-OH
--F
-H
II'G
-OH
-H
-CHs
1I"G
-OH
-H
--C!H3
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versely proportional to the height of the denominator curve (F.G.1). Thus the output of amplifier 15 is a potential proportional to the ratio of the height of the curves at the point along the X axis determined by the potential of the time base (X axis) generator. The proper adjustment of potentiometer 14A is achieved by temporarily connecting a 10-megohm resistor between input and output of amplifier 15 and manually positioning the F.G.2 follower near full scale. The F.G.1 follower is manually positioned at the base line. The potential of the F.G.l slider is measured and then adjusted to zero by means of 14A. After the adjustment has been made, the 10-megohm resistor must be removed. The capacitor connected between input and output of amplifier 15 stabilizes the circuit. Its value is not critical but should not be so large as to produce sluggishness in the X axis motion of function generator 3, or so small as to allow jitter. The output of amplifier 15 is connected to the X axis input of function generator 3. Function generator 3 is fitted with a logarithmic curve, so that the height of the follower is proportional to the logarithm of the X axis input. The full scale end of the follower slide-wire is connected to the f100-volt reference supply; the zero scale end is grounded. The potential of the F.G. 3 slider is connected to the input of summing amplifier 8. A zero adjustment potential is derived from potentiometer 8A and connected to the input of amplifier 8. The output of amplifier 8 is con-
ANALYTICAL CHEMISTRY
keto-A'-progesterone)
1lp, 1701-Dihydroxy-21-acetoxypregna1,4- diene- 3,20- dione ( A'- hydrocorti-
0
11"
I
R
11' RESULTS AND DISCUSSION
The detailed plots of the logarithms of the ratios of ultraviolet spectrophotometric absorbances against wavemethylpregna-1,4-diene-3,20-dione length are given as log el/* vs. X in ( 6a-methyl- A1-hydrocortisone-21Figure 1 for the pairs of steroids listed acetate) 11p,17~-Dihydroxy-21-acetoxy-6ain Table I. The pertinent data from fluoropregna-l,4-diene-3,20-dione (6athese plots obtained by application of fluoro-A1-hydrocortisone-21-acetate) 1lp, 1701-Dihydroxy-21-acetoxy-2-methyl- the analog computer techniques are given in Table 11. pregna-1,4-diene-3,2O-dione (2-methylA1-hydrocortisone-21-acetate) These techniques permit a charllp,l7~,21-Trihydroxy-2-methylpregna- acterization of such curves a,nd the 1,4-diene-3,20-dione (2-methyl-A'specific data of Table I1 detail the hydrocortisone) specific characteristics of such spectro-
sone-21-acetate) 1lp,l7a-Dihydroxy-21, acetoxy-6a-
photometric comparisons with the wavelengths of the minima and maxima of such logarithmic ratios of spectrophotometric absorbances and the isobestic wavelengths a t equivalent molar absorptivities for the two steroids. The studies on these series of steroids permit some general conclusions. The nature of a saturated 17-substituent on the D ring does not affect the shape or absorbance values of the ultraviolet spectrophotometric absorption due to the A ring chromophores, 4-ene-3-one or lj4-diene-3-one. This is specifically shown by curve 1 of Figure 1, obtained for the spectral comparisons of a 17,-hydroxy21-acetoxy-20-one side chain at the C-17 position with a 20-one pregnane side chain (items 1 and 8, Table 11) and with a 17a-21-dihydroxy-20-one (item 13, Table 11) side chain. The introduction of an 11-hydroxy substituent when the basic chromophore is a 4-ene-3-one skews the basic absorption band toward the red (bathochromic) and decreases the absorbance a t the retained maximum (hypochromic). The steric positioning of the 11-hydroxy has no apparent effect (items 1 and 2, Table 11, and pertinent figures). As modification of the spectral absorbance is not dependent on whether the 11hydroxyl is O( or 8, the weighting a t the 11 position rather than any steric interaction appears responsible. Solvation of the 11-hydroxyl by the ethanolic solvent could enhance this weighting. A general result of the addition of a 1-ene t o a 4-ene-3-one chromophore is to skew the absorption band to the red-Le., increase the absorptivities a t wavelengths higher than the maxima. The net result i s a slight bathochromic shift with no significant hyperchromic effects. For example, the addition of a 1-ene to a 4-ene-3,20-dione as in progesterone (item 3, Table 11) produces a shift to
Table II.
the red of the wavelength of the maximum absorbance (bathochromic), Ah = +5 mp, as would be expected from previous information in the literature ( 4 ) , which also implied that cross conjugation does not introduce hyperchromic effects. Inspection of Figure 1, 3, shows that two energy levels of the activated chromophores may be considered separately. Although the activated 4ene-3-one has less absorbance
