Suggested Preliminary Standards for Calibration of Optical Rotatory Dispersion and Circular Dichroism Instruments DeLos F. DeTar Department of Chemistry and Institute of Molecular Biophysics, The Florida State University, Tallahassee, Fla. 32306 Two approaches have been used in the present study in an effort to define preliminary circular dichroism (CD) standards. The first involved a careful reexamination of 10-camphorsulfonic acid using the Kronig-Kramers transform to compare optical rotatory dispersion (ORD) and CD curves. The second approach utilized tris(ethy1enediamine) cobalt triiodide hydrate, a comound relatively easy to prepare and resolve and one or which three independent sets of absolute rotation values have been reported. Experience has shown that the angle measuring portion of a recording spectropolarimeter may not be in correct adjustment, and that the error may be a function of the full scale reading. This section of the instrument should therefore be checked against suitable ORD standards before attempting to use ORD values to establish a CD Calibration. It is clearly undesirable to use ordinary UV or visible spectrometric values as a substitute for polarimetric values in deciding the optical purity of an intended standard.
P
THISPAPER presents data on suggested standards for testing the ORD scale and for calibrating the C D scale of spectropolarimeters. In a paper which appeared after the present manuscript had been prepared (ZA), a similar study of the KronigKramers transform on 10-camphorsulfonic acid has led to a closely similar C D calibration factor. The use of 10-camphorsulfonic acid as a calibration standard was pioneered by the instrument manufacturers. Factors affecting accuracy in polarimetric measurements have been discussed ( I , Z), but a few salient points may be mentioned. In rough terms, a wavelength error of 1 nm leads to an error in rotation of about 1 % relative in positions remote from a Cotton effect. The temperature effect is variable, and often 1 or 2 “C control is adequate. Solvent effects and concentration effects are also variable. In most cases, specific rotation values are the same within 0.5% or better for concentration ranges of 1 % up to 3 or 4 %. Two sources of cell error must be checked. One is the presence of residual optical activity in the windows, and this may either be inherent or it may be introduced by strain. Its magnitude may readily be observed by running a solvent curve with the cell in two different rotational positions with respect to the axis of the light beam. Residual activity can be very important for ORD readings at low full scale angle and may also be significant for C D curves. The other source of cell error is reflection from the cell walls due to use of a cell of too small diameter. Reflection errors can be reduced by masking. For high polarimetric accuracy over a restricted spectral region, the best instrument is a manual polarimeter using sodium and mercury lines. The data may advantageously be averaged by application of the Drude equation (3). This is (1) F. J. Bates, “Polarimetry, Saccharimetry and the Sugars,” Natl. Bur. Standards, U. S. Circular No. 440 (1942).
(2) A. Heller in “Physical Methods of Organic Chemistry,” A. Weissberaer. Ed., John Wiley and Sons, Inc., New York, N. Y., 1960, VoL 1; Part 11, p 2147.(2A) J. Y.Cassim and J. T. Yaw, Biochem., 8, 1947 (1969). (3) D. F. DeTar, M. Gouge, W-Honsberg, and U. Honsberg, J. Amer. C.hem. SOC.,89, 988 (1967). 1406
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practical by computer techniques (4). The relationships are as follows: CY (observed angle) = [a] c I; concentration units are g/ml, the unit of length is the decimeter. [M = [a] WjlOO where W is the molecular weight. The Drude expression is [MI = a/(Xz - Xoz) where X is the wavelength; a and Xo are disposable constants. As an illustration of the results possible, we have been able to reproduce the rotation values of Bureau of Standards standard sucrose as follows: observed [ L Y ] ~ ~ &66.53, CY]^^^^ 78.23 (c 26,water); reported for the sample [ c Y ’ ] s66.53, ~.~ 78.34. We have also found consistent reproducibility of readings on aspartic acid samples within a standard deviation of 0.2z for some dozen samples read at different times and varying widely in actual angle of rotation. While this latter result does not guarantee accuracy, it does show that the angle scale is accurately linear. The manual instrument has also been tested against quartz plates calibrated by the Bureau of Standards. We have used the readings obtained on the manual instrument in establishing secondary standards for use with the recording spectropolarimeter. Comparisons over the range of overlap of the manual and the recording instruments (365-590nm) using sucrose, aspartic acid, proline, 10-camphorsulfonic acid, and tris(ethy1enediamine) cobalt triiodide hydrate show that an absolute accuracy of about 1% is attainable on the recording instrument if it has been properly adjusted. This is more than adequate for the studies of ORD curves. Since the angle setting of the analyzer has been shown to be accurate over this range, we tentatively assume that the same accuracy is attained in the regions below 365 nm which are not accessible to the manual instrument. The principal source of error would be wavelength accuracy. This seems to be about 1 nm on the JASCO instrument. As far as we can ascertain, the C D calibration values now in tacit use are roughly correct although based on 10-camphorsulfonic acid of rather uncertain pedigree. During a service call one of our instruments was calibrated with a sample which was only about 90% pure. Various workers have reported use of the Kronig-Kramers transform to relate experimental ORD and experimental C D measurements (5). Published results to date are not useful for calibration purposes since reference standards were not included. We have now carried out an extensive study of the C D and ORD curves of 10-camphorsulfonic acid as obtained with the JASCO spectropolarimeter equipped with a C D accessory. These data have been combined with the highly reliable values obtained with the Rudolph high precision polarimeter at the mercury and sodium lines. The C D curve was empirically simulated by means of a summation of nine Gaussian curves. By use of the computer program CDORD we carried out (4) D. F. DeTar, Biophysical J.,6,505 (1966) (OPTROT).
(5) A. Moscowitz in C. Djerassi, “Optical Rotatory Dispersion,
Applications to Organic Chemistry,” McGraw-Hill, New York, N. Y . , 1960.
Table I. Comparison of Observed and Calculated ORD and CD Values for 10-Camphorsulfonic Acid in Water at 25 "C, 0.1M
Wavelength, nm 589.3 578.0 546.1 435.8 365.0 305.O 270. 289.6 WL IN NM
Figure 1. CD and ORD curves of 10-camphorsulfonic acid Dotted curves show eight of the nine Gaussians whose sum gives the solid line labeled CD. Solid lime labeled ORD was obtained from the CD curve Diu the Kronig-Kramers transform. Letters, numbers, and symbols represent the experimental points. Experimental results below 200 nm are of relatively low accuracy
weighted least squares adjustment of selected sets of the Gaussian parameters based both on the C D and on the ORD components. The extent of the resultant match is illustrated in Figure 1. A comparison of a selected set of calculated and observed values is presented in Table I. Further representative calculated and observed values is presented in Table I. Further representative calculated CD and ORD values are given in Table 11, and the Gaussian terms used to generate the calculated C D and ORD data are summarized in Table 111. It should be noted that the exact values of the Gaussian parameters are rather sensitive to small errors. In other words, many of the parameters are strongly correlated and must not individually be taken too seriously. Overall these curves reproduce the observed ORD and C D values very well. Computation of the data shown in Figure 1 and in Tables 1-111 did not utilize any correction for refractive index. As long as measurements and calculations refer consistently to a single medium and are made over a wavelength range where refractive index does not change too drastically, the Lorentz refractive index corrections are in fact trivial. We did actually perform a set of computations using the full refractive index corrections and found indeed that there are only negligible differences from the values reported. Although the present results should prove of considerable value in defining C D standards, what is clearly needed is a set of readily available standard compounds having C D maxima in several different spectral regions, and whose C D values have been obtained accurately by absolute measurements. With such standards it would be possible to make routine checks of instrument performance. Tris(ethy1enediamine)cobalt triiodide hydrate is one possible candidate. It is readily prepared (6), and the C D curve has been measured by rough absolute techniques in the presence of bromide anion in two different laboratories (7, 8) and for the iodide (9) in a third with relatively concordant results (about 8 spread). 10-Camphorsulfonic acid is another possible candidate. Although C D values have been reported (IO), they agree so (6) J. A. Broomhead, F. P. Dwyer, and J. W. Hogarth, Znorg. Syn., 6, 186 (1960). (7) J. P. Mathieu, J. Chim. Phys., 33, 78 (1936). (8) A. J. McCaffery and S. F. Mason, Mol. Phys., 6, 359 (1963). (9) P. F. Arvedson and E. M. Larsen, Znorg. Chem., 5,779 (1966). (10) T. M. Lowry and H. S. French, J . Chem. SOC.,2054 (1933).
Calculated Valuesa [MI [a1 50.4 21.9 54.2 23.6 67.3 29.1 184.0 78.6 546.1 234. 4709. -5819. 8193 , e
Observed :a1 22. lb 23. 5b 29.03 78.4b 236 . b 4654d -5810d (8216e*f)
a Molar rotation, specific rotation, calculated from the simulated Gaussian Curve (Tables I1 and 111). Rudolph high precision polarimeter, averages of 5 runs, sodium and mercury lines. c Wavelength f 2 nm absolute, kO.1 relative. Molar rotation, average of four runs on JASCO; precision (scatter) is about 1 and accuracy is about k l S % relative. E Molar ellipticity. f Average of four runs on JASCO. This is the calibration value.
Table 11. Calculated ORD and CD Values of 10-Camphorsulfonic Acid, Water, 25OC
Wavelength, nm 600 550 500
450 400 350 310 306 300 296 290 280 276 270 266 260 250 240 230 220 210 200
[MI 47.2 65.4 96.6 156.6 295.9 768.8 4107. 4642 4189. 2925. -23. -4338. - 5239. - 5819. - 5745. -5320. -4702. -4317. -4541. -5781. - 8477. - 8448.
PI
0. 0.
1317. 2704. 5369. 6950, 8176. 6253. 5307. 3445. 2377. 1306. 364. 32. -1. -185. - 2484. -10556.
Table 111. Arbitrary Gaussian Peaks Simulating CD Curve of 10-Camphorsulfonic Acid. Band center Amplitude wl, nm Band width, nm PI 10. 130.b -2870 188.1 - 3279 11.0 194.0 9.49 -11880 9.189 -2222 205.4 261.2 11.65 898 273.2 9.987 1748 13.03 286.0 6069 9.789 291.3 1499 10.26 2778 300.0 a The solid lines in Figure 1 and the calculated values in Tables I and I1 were computed from the summed curve based on the Gaussian peaks (CD) or from the Kronig-Kramers transform
(ORD). b Arbitrary remote peak to trim up ORD (Drude term).
VOL. 41, NO. 11, SEPTEMBER 1969
1407
poorly with current recommendations that they must be ignored. Further rotation and CD data are given in the Experimental. EXPERIMENTAL
Rotation Measurements. Procedures for mechanical and electrical adjustments of the JASCO were from Durrum Instruments. The most direct check is to measure the distance of travel of the analyzer arm by using a dial gauge. We have made an extensive evaluation of the angle measuring unit and have recommended to Durrum procedures which they have adopted to eliminate a potential source of large systematic error. Our instrument as originally adjusted had a 10% error. Procedures used with the Rudolph polarimeter have been described elsewhere (3). Calculations. The AE = FS.SR/(C.C) where FS is the nominal full scale C D setting, SR is the scale reading (fraction of full scale), C is the molar concentration, 1 is the path length in centimeters. [SI = 3300 A€ (5). To set the full scale readings it is recommended that A E , ~ . 2.49 be used for 10-camphorsulfonic acid which has [MI&,., 51.0. If the only impurity in the 10-camphorsulfonic acid is water, then the is correct, or somewhat less relationship A€ = 0.0488 [M]5~P.3 precisely AE = 2.38 X The latter can be taken directly from the standard test curve if one is certain that the ORD values are correct. This is the general approach usually followed. 10-Camphorsulfonic Acid. The commercial acid is about 94% pure, but most of the impurity is water: the compound is quite hygroscopic. It is reported that air dried material forms a sesquihydrate (11). There is also some confusion in this paper about rotation values: they accord better with anhydrous material than with the hydrate. A dried commercial sample had 98 % of the expected maximum rotation. For calibration purposes we used a sample twice recrystallized from acetic acid and dried until there was no odor of acetic acid in a stored sample. Before use the material was redried at 50 OC over Pz05 under reduced pressure; map. 195-7 o d (reported 191-2) (12). Anal. Calcd. for CloH~04S: C, 51.7; H, 6.94; M.W. 232.3. Found: C, 51.6; H, 7.25. Rotations using the Rudolph: b 1 % . 3 22.1, [aI%.o 23.5, [a1%3.1 29.0, [&!5.8 78.4, [a]&, 236.3 (c 1, water); precision of the averages is 0.5% relative. Rotations using the JASCO: [a]:& 2003, - [a]$&= 4504 (c 0.2, water); precision [CY]&- 2501, [OL];!~ of the averages is 1 % relative. The CD value AB& 2.49 is assumed on three bases. First, the instrument as set by Durrum service gave a value of 2.28, and this is presumably comparable to settings used elsewhere. (However, the Durrum camphorsulfonic acid was only about 90% pure, and the rotation value at 589.3 would not check the values reported
here or in the literature.) Further, the 2.49 figure gives good concordance between the observed CD of Co(en)aIa~HzO and the absolute values reported by three independent groups. And most importantly it is in agreement with Kronig-Kramers transform results. Reported rotations for 10-camphorsulfonic acid are as fOllOWS: [0!]&,3 21.63 ( I I ) , [~~]$?g,3 23.11, [ 0 1 I i ! ~ .30.44 ~ (C 29, water) (13); CY]',",?^ 22.4 (c 5.4, water); [a]32&.1982, [ c Y ] ~ o , [a]272 4450 (c 0.5, water) (10): We note that the rotations decrease by about l % relative per 5 O C decrease in temperature. The only reported AE 1.54 (IO)is in poor agreement with the recommended values. We cannot recommend the use of the UV spectrum of camphorsulfonic acid for ORD-CD calibration since this may be strongly influenced by factors having no relationship to optical activity. Tris(ethy1enediamine)cobalt Triiodide Hydrate (6). After recrystallization from water, the observed rotations were (Rudolph): [a]i8e.3 91.9, CY]^.!^.^ 262.7 (c 0.3, water); reported [a]D 85.6, (9), 89 (0, 91 (11). Rotations on the JASCO: if'^ - [a]4611260; Ae;ig 1.99 reported A€ 1.71 (9); for the bromide 1.76 (7), and for unspecified salt 1.89 (8). Anal. Calcd. for C6HZ4CoI3N6:C, 11.63; H, 3.90; N, 13.56; M.W. 619.9. Calcd. for C6Hz4CoI3N6.H20:C , 11.30; H,4.10; N, 13.18; M.W. 637.9. Found: Cy 11.12; H, 3.37; N, 13.3. While our sample appears to be the hydrate as specified, there is an ambiguity in the reported AE calculation (9). Larsen apparently used a MW of 620. A -60 is reported by McCaffery and Mason (8). We are interested in the band as a potentially very useful check point. Unfortunately the absorption band virtually obscured the CD measurement. We estimate A6215 to be -22 + 4 in poor agreement. Other Possible ORD Standards. Aspartic acid (c 1, water): [M]i&.3 33.02 (reported 32.97) ( I ) , [wi.!6.139.40 (reported 39.36) (1); sucrose (c 26, water): 66.5; [cY]%:B.I 78.7; proline (c 1, water): [M]i&.3- 61.8, [M]&.l - 72.7. These may be used to check the gross performance of the ORD calibration. ACKNOWLEDGMENT
Analyses and Rudolph readings were made by Mrs. L. Ross; ORD and C D readings on the JASCO, by W. Rodewald and K. Cooper. RECEIVED for review November 4, 1968. Accepted June 9, 1969. Work supported by the Public Health Service, Department of Health, Education and Welfare under Grant GM12666. (13) E. M. Richards and J. M. Lowry, J. Chem. Soc., 127, 1503
(1925).
(11) W. J. Pope and C. S . Gibson, J. Chem. SOC.,97,2211 (1910). (12) P. Lipp and H. Knapp, Chem. Ber., 73B, 915 (1940).
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(14) H. Burgess and C. S. Gibson, J. SOC.Chem. Znd., 44, 496T (1926).
