Ultraviolet and visible refractive indices of spectroquality solvents

ANALYTICAL

43, 240-246 (1971)

BIOCHEMISTRY

Ultraviolet

and

Visible

Refractive

of Spectroquality II. Aqueous

Solutions

J. R. KRIVACIC Division of Molecular University of Alabama

Indices

Solvents of Polyhydroxy AND

Solutes

D. W. URRY

Biophysics/Laboratory of Molecular Biology, Medical Center, Birmingham, Alabama 36S

Received January 3, 1971 Due to the increasing intensity of interest in the optical rotatory properties of suspensions of optically active model systems (1,2) and the fact that distortions in the circular dichroism measurements on such suspensions have been demonstrated in this laboratory to arise from the absorption flattening of Duysens (3) and light scattering (1,2,4), which is further complicated by differential scatter (5), and due to the application of these principles to biological particulate systems (6lo), we report the refractive indices of aqueous solutions of ethylene glycol, glycerol, and sucrose. In the studies on particulate systems, numerous solvent systems are used, and in order to make comparative studies one must make Lorentz field corrections in the optical rotatory dispersion spectra. Furthermore, the construction of particle refractive indices (2,5,9) from particulate composition used in conjunction with the concept of molar refractivities has led to the understanding, in a semiquantitative nature, of the total scatter effect, which depends on the difference in particle and solvent refractive indices. All of these concepts lend credence to the statement that refractive indices as a function of wavelength of solvent systems are a necessary, indeed an integral, part of studies on particulate systems and of solvent studies on solubilized systems. The technique has been reported by this laboratory elsewhere (11). Some modifications have been made and are discussed here. In essence, the technique of variable angle, single reflection spectroscopy, and the commercially available attachments for UV-VIS work have made this work possible and the approach practical for laboratories desiring this kind of information. MATERIALS AND METHODS Ethylene glycol and sucrose are Baker Analyzed reagents. Glycerol was supplied as a spectroquality reagent by Matheson, Coleman & Bell. 240

241

REFRACTIVE INDEX OF POLYOLS

Aqueous ethylene glycol and aqueous glycerol solutions were constituted on a volume per cent basis, whereas sucrose solutions were made up on a weight per cent basis. The water used was deionized and then glassdistilled. As previously stated, some modifications have been introduced. The reflectance attachment was purchased from Harrick Scientific Corporation, Ossining, New York 10562. It is a retro-mirror variable-angle attachment incorporating UV-grade mirrors and an optical quality UVgrade sapphire hemicylinder as the internal reflecting element. The principles of the unit’s operation are discussed in references 12 and 13. The reason for choosing a sapphire hemicylinder over a Suprasil or Dynasil, which is more transparent in the far-ultraviolet, is that sapphire has a higher refractive index throughout the wavelength range studied. This allows one to study more concentrated solutions which have refractive indices higher than those of Suprasil or Dynasil elements. A second refiectance unit utilizes a Suprasil prism and was obtained from Wilkes Scientific Corporation, South Norwalk, Connecticut (see reference 11 for use and description). The refractive index of the sapphire was obtained from reflection scans at two angles using water as a sample. The refractive index of the sapphire hemicylinder is about 20/o lower at 4000 JI than that reported in the literature (14). This is consistent with a higher quality optical and UV-grade element than previously available. Typical values are 1.7200 at 6500 A, 1.7260 at 5000 A, 1.7352 at 4000 A, 1.7905 at 3000 W, and 1.8626 at 2000 A. As noted previously (II), with the refractive index of water known as a function of wavelength, the sapphire refractive index can be calculated using the data from spectra run above and below the sapphire-water critical angle. In general for slightly absorbing samples reasonably accurate calculations can be obtained if one keeps the optical density of the belowcritical angle scan between 0.8 and 1.0 (net reflectivity 16-157!, respectively) and the OD of the above-critical angle scan between 0.1 (80% R) ‘and 0.3 (50% R). The low-angle scan approximates t’he dispersion curve while the high-angle scan approximates an absorption curve. Actual experimental sapphire refractive indices were used to calculate solution refractive indices. For full theoretical details, see reference 12. All calculations were performed as outlined in reference 11 wit,h one additional equation. When K is below 0.005, equation 1 is used (15) : K = (1 - n2)(sin20 - n2)* loge R 4n2 cos 6 where K is the attenuation index, n is the relative

refractive

index

glycol

6500 If

* 1950 II.

(vol. %) 1.3392 1.3501 1.3576 1.3599 1.3720 1.3841 1.3976 (vol. %) 1.3409 1.3462 1.3507 20% 1.3552 25% 1.3602 40% 1.3873 60% 1.4178 80% 1.4446 Sucrose (wwt. yo) 4.71% 1.3395 9.57% 1.3478 14.67% 1.3538 19.57% 1.3618 24.32% 1.3692 29.87% 1.3772 40.6OoJ, 1.3945 45.80yo 1.4071 50.63% 1.4192 61.02% 1.4430

Ethylene 5% 10% 20 % 30% 40% 50% 70% Glycerol 5% 10% 15%

Solution

1.3490 1.3544 1.3590 1.3638 1.3689 1.3934 1.4250 1.4520

1.3389 1.3442 1.3495 1.3546 1.3603 1.3921 1.4217 1.4489

1.3398 1.3471 1.3551 1.3631 1.3710 1.3808 1.3999 1.4114 1.4214 1.4443

1.3423 1.3479 1.3525 1.3570 1.3622 1.3835 1.4188 1.4458

1.3411 1.3494 1.3554 1.3636 1.3708 1.3790 1.3957 1.4083 1.4206 1.4442

1.3475 1.3561 I.3616 1.3702 1.3772 1.3860 1.4016 1.4150 1.4260 1.4599

1.3485 1.3596 1.3670 1.3686 1.3800 1.3928 1.4060

4500

1.3384 1.3436 1.3553 1.3646 1.3754 1.3850 1.4046

21” Na D

1. Refractive

1.3415 1.3528 1.3605 1.3618 1.3734 1.3864 1.3996

5900 B

TABLE a

1.3515 1.3601 1.3656 1.3744 1.3815 1.3907 1.4059 1.4189 1.4306 1.4558

1.3515 1.3576 1.3629 1.3675 1.3730 1.3978 1.4291 1.4564

4

1.3579 1.3664 1.3716 1.3807 1.3877 1.3975 1.4124 1.4323 1.4465 1.4500

1.3577 1.3636 1.3689 1.3738 1.3796 1.4198 1.4332 1.4635

1.3576 1.3590 I.3736 1.3802 1.3914 1.3973 1.4190

1.3680 1.3762 1.3810 1.3911 1.3980 1.4087 1.4391 1.4613 1.4761 1.4931

1.3677 1.3737 I.3791 1.3840 I.3898 1.4464 1.4607 1.5038

1.4068 1.3965 1.4133 1.4053 1.4158 1.4249 1.4481

3000

Polyhydroxy

3500 A

of Aqueous

1.3520 1.3628 1.3706 1.3731 1.3844 1.3967 1.4093

4000

Indices A

Solutes

1.3869 1.3947 1.3986 1.4094 1.4168 1.4301 1.4460 1.4661 1.4854 1.5089

1.3864 1.3925 1.3980 1.4031 1.4090 1.4514 1.4688 1.5213

1.3952 1.3936 1.4256 1.4304 1.4380 1.4448 1.4795

2500

II.

1,4276 1.4350 1.4442 1.4544 1.4662 1.4944 1.4816 1.5148 1.5241 1,5723

1.4318 1.4371 1.4439 1.4590 1.4574 1.4958 1.5083 1.5851

1.4388 1.4290 1.4563 1.4636 1.4744 1.4801 1.5169

2000

A

1.4454 1.4534 1.4744 1.4706 1.4832 1.5194 1.4960* 1.5256* 1.5292* 1.5791*

1.4506 1.4559 1.4629 1.4722 1.4853 1.5040* 1.5159* 1.5918*

1.4456* 1.4396* 1.4624* 1.4683* 1.4774* 1.4849* 1.5244*

1900

IL

REFRACTIVE

INDEX

OF

POLYOLS

243

(sample/sapphire), f3 is the angle of incidence, and R is reflectance. On nonabsorbing samples, the error analysis computer program TABLE, which is designed to find optimum angles of incidence, is not really necessary because it searches for angles yielding net optical densities of 0.8 (16% R) and 0.2 (63% R), respectively. For absorbing samples this program should be used. All the optical densities are relative to the sapphire-air baseline at the corresponding angles. Since the gross optical densities of the sapphire hemicylinder are greater than one below 32OOA, the signal-to-noise ratio is lower; therefore, greater errors are expected when solutions are run and their respective refractive indices and attenuation constants are calculated. This is found to be so. RESULTS

The refractive index data are presented in Table 1. The the sodium D line at 21°C are included for comparison with at 25”. Data presented with an asterisk are for 1950 A and solution runs using the Harrick Scientific reflectance attachment, the remainder of the solutions were run using the Wilkes reflectance attachment. Table 2 presents the data in terms of the least squares fitted equation

values at the data represent whereas Scientific dispersion

?b = 1 + 4X2/(X” - C) where x is in centimeters, and A and C are the regression coefficients. The accuracy of the equation is included for wavelength regions where there is maximum error in the fit. All other regions have maximal errors of +0.3% or less. The errors are the smallest for solutions run on the Suprasil prism attachment because of the inherently lower gross absorption of the fused quartz. When the dispersion becomes anomalous, i.e., when local absorption band contributions to the dispersion become significant, large errors may occur in the fitted dispersion equation. Discrepancies in the experimental refractive indices, e.g., 10% ethylene glyco1 between 3500 and 2500 A, occur because of changes in incident angle and errors involved in reading this angle, in this case +_O.l’, and will cause errors in the least squares fitting of the dispersion equation. Such errors manifest themselves by displaying low errors of fit in the anomalous regions. Typical attenuation indices of the solutions run between 0.003 and 0.01, as in the case for 61.02% sucrose and 80% glycerol at 1950 A. Thus, we are dealing with low absorbing samples. The greatest errors are in the 3200 to 2650 k range, because the absorption of the sapphire plus the

244

KRIVACIC TABLE Coefficients

2. Analysis

Ethylene 5% 10%

c x

eq.

Equation

Wavelength of greatest

A

10”

range error, Max.

error

of fit

glycol

20% 30% 40% 50% 70% Glycerol 5% 10% 15% 20%

25% 40%

60% 80% Sucrose 4.71% 9.57yo 14.67% 19.57% 24.32% 29.87% 40.60%

0.329196 .343701 .348654

12.4459 8.67282 10.8894

.350575 .362881

10.6085 9.93212

.376560 .386291

8.61648 10.8309

0.334391 .339456 .343700 .348100 .352898 .375672

8.37651 8.43956 8.57039 8.61235 8.70385 10.9292

.408786 .431442

8.19537 10.5027

0.332403 .340792 .346884 .354651 .361773 .368813 .385540

8.81533 8.57537 8.26765 8.45837 8.37045 9.11112 8.70649

45.80%

.396559

9 86207

50.63%

.407657

10 1628

61.02%

.431826

9.25112

(+)

URRY

of Dispersion

of dispersion

A

Solution

AND

Indicates

equation

multiple UV mirrors greater than 1.0.

less than

3300-1950 3150-2650 3200-2650 2200-1950 2100-1950 3250-2650 2150-1950 4000-2400 2100-1950

+1.0-3.0% +1.0-1.7% +1.0-1.5% -l.O-1.8% -0.9-1.2%

2000-1900 2000-1900 2000-1900 2000-1900 2000-1900 3300-2650 2300-1950 3250-2650 3250-2650

+0.7-1.0% +0.5-0.9% +0.5-0.9% +0.4-1.0% +0.4-1.4% +1.0-1.4% -l.O-1.6% +0.7-0.9% +0.8-1.0%

2100-1900 1950-1900 2100-1900 2000-1900 2100-1900 2100-1900 3250-2650 2200-1950 3250-2650 2300-1950 3250-2650 2200-1950 35503400 2800-2650

+0.2-0.4% +0.3-0.45% +0.2-1.6% +0.3-0.5% +0.35-0.8% +0.5-1.7% .+0.7-0.9% -0.7-1.0% +0.9-1.2% -0.9-1.3% +1.0-1.2% -O.l-1.8% -0.9-1.2% +l.o%

+O.S% -0.7-0.9% +0.70/, -O.S-1.0%

experimental.

rises rapidly

and then plateaus at optical

densities

DISCUSSION

While there are many errors involved in the calculation of optical constants such as repsrting the optical densities to three decimals with

REFRAmIVE

INDEX

OF

245

POLYOLS

some error in that third decimal place (16), assuming a constant I,/I_L for the wavelength region studied, and reading the angle of incidence, etc. (17,18), we wish to stress here that, the results are in surprisingly reasonable agreement with sodium D line data and other more sparsely available dispersion data (19). The technique can be made quite routine and such data are required by those using optical rotatory dispersion or those studying particulate systems with optical rotatory dispersion or circular dichroism. The approach, as outlined, is relatively quick and inexpensive but does require computer facilities. The reflection method is the only approach presently available for absorbing samples. The critical angle method does not work in absorbing media. Anomalous dispersion can be obtained. In this connection we have duplicated the work of Hansen (20) on eosin Y solutions and independently on 2,5diphenyl-3,4-dimethyloxazolidine in methanol (0.1 gm/ml) as a check on the method. SUMMARY

The refractive indices in the wavelength 6500 to 1900 A are reported as a function of concentration for several aqueous solutions of polyhydroxy solutes. The technique of variable-angle single-reflection spectroscopy is used. Typical values for the attenuation index, K, are also included. ACKNOWLEDGMENT The authors wish to thank support.

the Mental

Health

Board

of Alabama

for fipancial

REFERENCES URRY, D. W., HINNERS, T. A., AND MASOTTI, L., Arch. Biochem. Biophys. 137, 214 (1970). 2. URRY, D. W., HINNERS, T. A., AND KRIVACIC, J.: Anal. Biochsm. 37, 85 (1970). 3. DUYSENS, L. N. M., Biochim. Biophys. Acta 19, 1 (1956). 4. URRY, D. W., in “Spectroscopic Approaches to Biomolecular Conformation” (D. W. Urry, ed.), p. 105, American Medical Association Press, Chicago, Illinois, 1970. 5. URRY, D. W., AND KRIVACIC, J., Proc. Nat. Acad. fki. u. s. 65, 845 (1970). 6. URRY, D. W., AND JI, T. H., Arch. Biochem. Biophys. 128, 802 (1968). 7. Jr, T. H., AND URRY, D. W., Bio&m. Biophys. Res. Commun. 34, 404 (1969). 8. URBY, D. W., MASOTTI, L., AND KRIVACIC, J., ibid. 41, 521 (1970). 9. MASOTTI, L., URRY, D. W., AND KRIVACIC, J., Biochim. Biophys. Acta, in press. 10. URRY, D. W., MASOTTI, L., AND KRIVACIC, J. submitted for publication. 11. KRIVACIC, J. R., AND URFGY, D. W., Anal. Chem. part I of series. 42, 596 (1970). 12. HARRICK, N. J. ibid. 3’7, 1445 (1965). 13. HARRICK, N. J., in “Internal Reflection Spectroscopy,” p. 182. Interscience, New York, 1967. 14. JEPPESEN, M. A., J. Opt. Soe. Amer. 48, 629 (1958). 1.

246

KRIVACIC

AND

URRY

15. HANSEN, W. N., Spectrochim. Acta 21, 209 (1965). 16. FAHRENFORT, J., AND VISSER, W. M., Spectrochim. Acta 18, 1103 (1962). 17. GILBY, A. C., BURR, J., JR., AND CRAWFORD, B., JR., J. Phys. Chem. 70, 1520 (1966). 18. GILBY, A. C., BURR, J., JR., KRUGER, W., AND CRAWFORD, B., JR., ibid. p. 1525. 19. FASMAN, G. D., Methods Enzymol. 6, Chap. 126, pp. 955-957 (1963). 20. HANSEN, W. N., Anal. Chem. 37, 1142 (1965).