John P. Schelz and William c. Purdy
of Maryland College Park
University
An Experiment in Optical Rotatory Dispersion
The pheuomenon of optical rotatory dispersion-the change in optical rotation of a substance with wavelength-has proved in the last decade to be a valuable means of probing molecular structure and of solving problems in stereochemistry. Monochromatic polari~netrya t the sodium D-line (589 m ~ has ) been an important research tool, even though this wavelength represents an insensitive region of the spectrum for most colorless compounds. Prior to 1954, fewer than 100 rotatory dispersion curves had been recorded in the ultraviolet spectral region. Then came the development of the first commerciallyavailable manual spectropolarimeter, and the measurement of optical rotatory dispersion became a practicality. Djerassi (1) and his co-workers set out t o demonstrate the wide applicability of the method. Today it is possible to measure a rotatory dispersion curve with the use of an accessory which will convert a double-beam spectrophotometer into a recording polarimeter. The purpose of the present paper is to outline an experiment in which it is possible to study the optical rotatory dispersion of a substance in the ultraviolet region where it is anomalous, and to determine whether the rotatory dispersion curve of that substance might be put on a quantitative basis. The compound chosen is the 17-keto steroid androsterone (5~-androstan-3~-0117-one). Dioxane is the solvent. Foss (9) has recently given a concise explanation of the phenomena involved in the measurement of optical rotatory dispersion. Steroids appear structurally to be very complex molecules, yet stereochemically they are characterized by great simplicity. All are optically active and have frozen conformation. Then too, in the steroid field, there is an extensive relationship between structure and ultraviolet absorption (3). Androsterone is easily obtainable in sufficient purity so that there is no interference by optically active contaminants. It is a complex rotator, i.e., it shows both positive and negative rotations, depending on wavelength, and exhibits a single Cotton effect curve (see Fig. 1) with a peak at 312 mp and a trough at 275 mp ( I ) . Analytical uses are obvious for any property which is directly proportional to the concentration of a given substance in solution. Since the rotation of many compounds may be up to 100 times greater in the region of an absorption band than a t the sodium D-line, there is considerable gain in quantitative accuracy obtained by measuring rotation a t the wavelength of a peak or trough (4).
dispersion accessory (6) in a recording ultraviolet spectrophotometer (Perkin-Elmer Spectracord, Model 3000), with a hydrogen lamp source. The accessory consists of two units, one for the instrument sample chamher and one for the reference chamber. Each unit contains a polarizer prism assembly, space for our 22-mm round cells of 50-mm path length, and an analyzer prism assembly which can be rotated with respect to the polarizer. The units areidentical except that the analyzer prism scale of the one is marked off in positive angles (0-90°), while the scale of the other is marked off in negative angles. The analyzer prism settings may be set in increments of five degrees. The 90" position corresponds to crossed prisms, so that no light passes through the units. It is possible to record both the rotatory dispersion curve and the absorbance curve for a substance without having to remove the chart paper. This may be easily done by running the rotatory dispersion curve, then removing the units and transferring the sample solution in the same absorbance cell to a standard cell holder. To obtain the absorption spectrum, one sample solution must be replaced by the pure solvent. There are three major experimental parameters that can be varied in rotatory dispersion measurements. These are analyzer prism angle, source intensity, and cell path length. Use of these parameters enables one
The Instrument
I n our laboratory, we make rotatory dispersion measurements using the Perkin-Elmer optha1 rotatory
Figure 1. Optical rotatory dispersion curve for ondmrterone at a concentrotion of 0.05 g/l 00 ml.
Volume 41, Number 12, December 1964
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645
Table 1.
Optical Rotatory Dispersion Curve f o ~ Androsterone
the same wavelength R
[a](degrees
R*
h(w)
a
(degrees)
x
10P)
=
recorded signal R,
A table of R versus a is used to determine a, using the appropriate value of 6, the analyzer prism setting. Optical rotatory dispersion curves are most frequently plotted with wavelength (in m ~ as) abscissa and specific rotation, [ a ] as , ordinate [a]=
'R
=
rotatory dispersion signal = reccoded signd/R,
to optimize the detectability of the instrument, size of sample, wavelength range, and accuracy. The Experiment
The optical rotatory dispersion curve for androsterone is to be calculated and plotted, and a calibration curve is also to be plotted for the optical rotation of androsterone a t its peak with respect to concentration, so that an unknown sample can be analyzed quantitatively. The procedure used with our instrument follows. With pure solvent in the cells in both of the rotatory dispersion units, the instrument is adjusted to 80% transmission, an arbitrary value. The analyzer prisms are set a t 75". These are always set a t the same value on both units. The positive unit is placed in the sample chamber and the negative unit in the reference chamber. The initial slit opening of the spectrophotometer should be the same for all curves. The R, curve for pure dioxane in both units is run down to the wavelength where the slits open fully, using a moderate scan speed setting. Solutions of androsterone in dioxane are prepared a t the following concentrations (g/100 ml): 0.02, 0.05, 0.10, 0.15, and 0.25. Both of the cells are emptied and refilled with a sample solution. The optical rotatory dispersion cnrve for each solution is run as above, noting the magnitude and sense of the rotation as the concentration increases. Pen deflections above the R, (pure solvent) line indicate negative rotations and those below the R, line, positive rotations. The optical rotatory dispersion curve for an unknown solution of androsterone in dioxane is recorded. The curve for androsterone a t a concentration of 0.05 g/100 ml in dioxane is calculated (Table 1) and plotted (Fig. 1). The trough of the curve was not obtained before the slits opened fully, the point a t which rneasurement stops. I n order to calculate or, the degree of rotation, transmittance values of R, and the recorded signal are measured at any wavelength a t which rotation is to be determined. The rotatory dispersion signal, R, is calculated by dividing the recorded signal by R, a t Table 2.
1000 -C1
where or = degrees of rotation, C = solution concentration (g/100 ml), and I = length of sample cell (in decimeters). The degrees of rotation a t the peak is calculated for each of the concentrations run, and a calibration curve of concentration (g/100 ml) versus ar is prepared (Fig. 2). and used to determine the concentration of the unknown sample. Table 2 shows the results obtained for the concentration of androsterone in some unknown solutions. The effect of solvent in rotatory dispersion measurements can be demonstrated by substituting methanol for dioxane. A hypsochromic shift is found in the rotatory dispersion curves in going from the non-polar to the polar solvent. The experiment serves to illustrate to the student a typical rotatory dispersion curve. It points out that it is possible to put optical rotation at the peak, the most. sensitive portion of the curve, on a quantitative basis. The authors are indebted to the National Science Found~tionfor partial support of this work. Literature Cited (1) DSERASSI, C., "Optical Rotatory Dispersion," McGraw-Hill
Book Company, Inc., New York, 1960. (2) Foss, J . G., J . CBEM.EDUC.,40,592 (1963). (3) DORFMAN, L., Chem. Reus., 53,47 (1953). (4) KLYNE,W. AND PARKER,A. C., in "Physical Methods in Editor, Vol. 1, Organic Chemistry," A. WEISSBERGER, Part 3, 3rd ed., Interscience Publishers, Inc., New York, 1960, p. 2335. R. E., Paper (5) SAVITZKY, A., SLAYIN,W., AND SALINGER, presented at the Pittsburgh Conference on AnalyticaL Chemistry and Applied Spectroscopy, march 3, 1959.
Data Testing Accuracy of Calibration Curve
Cone R
a (degrees)
(g/100 ml) (calib curve)
Known 01
,
,
,
,
,
,
,
,
, ,
,
1
0.2 0.4 0.6 0.8 ID 1.2 1.4 16 1.8 2.0 2.2 2.4 0 DEGREES OF ROTATION1 Figure 2. Calibration curve for androsterone .t the peak value of degrees of r o t a t i o ~
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Jaurnol of Chemical Education
