J. Phys. Chem. 1995, 99, 1834-1836
1834
A New Method for Circular Dichroism Detection Using Cross-Polarized Transient Grating Masahide Terazima Department of Chemistry, Faculty of Science, Kyoto University, Kyoto 606, Japan Received: September 23, 1994; In Final Form: November 22, 1994@
Circular dichroism detection based on the cross-polarized transient grating method is presented. This method is based on the spatial modulation of the left- and right-circular light, and the different absorption of these polarization is detected as a diffraction of a probe beam. It has a potentially high sensitivity with an excellent time resolution. The merits and limitations are discussed.
1. Introduction Circular dichroism (CD) is a very useful spectroscopic tool which can provide detailed information on the structures of molecular systems, such as the secondary structure of proteins. With increasing interest in the fast structural dynamics, the timeresolved studies are also highly desirable in the CD detection to characterize intermediates. In this Letter, we present a novel CD detection method which has potentially highly sensitivity and excellent time resolution. Several attempts have been developed to improve the sensitivity and time resolution of the CD detection. For that purpose, two strategies have been mainly used. One method is that the right- and left-circular polarizations are periodically changed temporally, and the difference in the absorbance is This method is currently widely used for measuring the CD spectrum with phase-sensitive detection.' For improvement of the sensitivity, a fluorescence detection? and a thermal lens detection method3 have also been proposed. Recently, a CD detection by a degenerate four-wave mixing method is demonstrated for a further sensitive method.? The other method is based on the optical effect of an optically active sample to the eccentric left- or right-elliptically polarized light.s By the interaction of light to the optically active matter, the ellipticity is changed, and the change is detected with a detection polarizer. Considering the increasing importance of the CD detection, it is desirable to develop another CD detection method based on a different concept. In particular, the photothermal method is promising with respect to the high sensitivity and fast time resolution. In this Letter, we demonstrate a new CD detection method based on a transient grating (TG) technique using a spatially modulated left- and right-circularly polarized light. Although the TG technique is one of the four-wave mixing spectroscopies, the concept of the measurement is different from the previous report of the application of the degenerate fourwave mixing method.J
where x and y axes are in the plane defined by the wave vector of the beams (kl and k2) and point into the direction of the bisectrix between the pump beams. The i, j, and k axes are the unit vectors along the x, y, and z axes, respectively. Although E2 does not exactly point to the i axis, the following results will be the same after an appropriate rotation of the coordinate.6 At the crossing region of the two beams, various polarization patterns are created (Figure l), and each polarization can be expressed by a sum of left- and right-circular polarization,6 which is expressed by
The intensity distributions of the ER and EL components (ZR and ZL) are calculated as
ZR = (E,2/2)(a2 + b2 -I-2ab sin(2kp))
+
1, = (EO2/2)(a2 b2 - 2ab sin(2k.p))
Since the phase difference of the modulation between the intensity of the left (ZL)-and right (I,)-circularly polarized light is 1 80°, any absorbance difference between these two polarizations induces a spatial modulation of the photoexcited state. Either the modulation of the excited state or the resultant heat by the radiationless transition from the excited state could be detected by a Bragg diffraction of another probe beam. Here we detect the heat as the thermal grating signal. Since the TG signal intensity (ZTG) is proportional to the square of the amplitude of the modulation,
ZTc
0~
{(eR- EL)abEo2}*
2. Principle To create the circularly-polarized light modulation in space, the interference pattern between two orthogonal linearlypolarized light (El and E2) is used.
This cross-linearly-polarized transient grating (polarization grating) technique has been used by Fayer and co-workers to investigate the dynamics of sodium vapor or molecular alignment process previously.' However, this is one of the first reports on an application to the CD detection to our knowledge.* 3. Experimental Section
@
Abstract published in Advance ACS Ahstracrs, February I , 1995
0022-365419512099-1834$09.00/0
The experimental setup is similar to that reported el~ewhere.~ An excimer laser pumped dye laser (Lumonics Hyper 400 and
0 1995 American Chemical Society
J. Phys. Chem., Vol. 99,No. 7,1995 1835
Letters
X
grating pulse
0
10
20
tips
10
20
tips
Figure 1. Schematic diagram of cross-polarization grating. Any absorbance difference between the left and right circular polarizations results in the population or thermal grating.
-
Dye-300) was used for creating the polarization grating ( A = 510 nm, pulse width 20 ns). The linearly (vertical)-polarized laser pulse with a polarizer (extinction ratio having an energy -500 pJ/pulse was split into two equal intensity beams with a beam splitter. The polarization of one of the beams was converted to the horizontal polarization with a half-wave plate and polarizer pair. Both beams were crossed inside a 2 mm path quartz sample cell with an angle (0.05 rad) at an ambient temperature. The beam diameter at the crossing point was -50 pm. A He-Ne laser was used for probing the thermal grating. The polarization of the beam could be rotated by adjusting the A/2 polarizer pair. After passing through a glass filter, an interference filter, and a pinhole to remove the scattering light, the TG signal was detected by a photomultiplier (Hamamatsu R928) and averaged about 300 shots by a digital oscilloscope (Tektronix 2430A). A sample, A-(+)589-[Co(en)3]Br3,'O was kindly provided by Dr. M. Kojima of Okayama University. 4. Results and Discussion
Figure 2A shows the TG signal after the photoirradiation of M Co(en)3 aqueous solution with the parallel polarization. The signal rises instantaneously after the irradiation and decays back to the base line by the thermal diffusion process. When the polarization of one beam is rotated to make the crosspolarization, the signal becomes weak. We can, however, still detect a signal from the optically active sample (Figure 2B). To ensure that the observing signal is due to the different absorption for the right- and left-circularly polarized light, the same measurement was made for an optically nonactive sample under the same experimental conditions. We used methyl red solution for the reference. The absorbance of the reference (methyl red) solution at the excitation wavelength was adjusted to the same value as that of the Co(en)3 sample. Moreover, the photoexcited state of methyl red and Co(en)s deactivates nonradiatively within the laser pulse width. Therefore, the heat released after the photoexcitation of the methyl red solution should be the same as that of the Co(en)3 solution. Under the same conditions, there is no detectable signal from an optically nonactive sample (Figure 2B). On the basis of these facts, we conclude that the signal from the Co(en)3 sample under the cross-polarization conditions should originate from the different absorbance of the left- and right-circularly polarized light. The signal intensity under this condition is -7.8 x of that under the parallel polarization condition. Since the TG signal intensity is proportional to the square of the absorbance difference, A& = IER - ELI/E should be (2.8 f 0.9) x lo-* at this wavelength. This value agrees with the reported A& value (3.5 x 10-2)'o within the experimentaluncertainty. The largest part of the error comes from the estimation of the relative TG signal intensity
I 0
Figure 2. (A) Thermal grating signal of Co(en)3aqueous solution under the parallel polarization. (B) Thermal grating signal of Co(en)3aqueous solution under the cross-polarization (solid line) and that of methyl red (optically nonactive) solution (dotted line).
under the cross-polarization (Figure 2B) to that under the parallel polarization (Figure 2A). The CD detection by this method is sensitive to the orthogonality between the two linearly-polarized light. If weak light with the parallel polarization to one beam is contaminated in the other beam, the contaminated component and the other beam produces the intensity grating to give a nonnegligible TG signal. We have rotated one of the polarizers within &loaround the orthogonal angle and verified that the TG signal from any optical nonactive samples disappears at the orthogonal condition. This fact ensures that the effect of the finite extinction ratio of the polarizer as well as the possible depolarization of the light due to the lens and/or sample cell should be negligible under these experimental conditions; in other words, the polarization of the excitation light is pure enough for the CD detection. This TG detection of CD has several advantages over the other methods. Since the TG method is background-free detection, it is known to be highly sensitive. Therefore, only a small A6 or dilute sample is needed. At the same time the irradiation volume is quite small, about 5 x cm3 in our experiment. Therefore, the amount of sample we need could be very small L = 5 x lo-" mol). This M x5x merit will become important, especially for valuable samples such as proteins. Furthermore, since CD is detected as heat after the irradiation of the pulsed laser, a time-resolved experiment is easy to be performed; just photoexcite the sample by another pulsed laser (pump pulse) before the grating pulse. The time resolution is determined by the time delay between the pump and the grating pulses and limited by only the pulse width of the lasers. Nano- or picosecond time resolution of the two-step excitation TG experiments has been already demonstrated.'' There are several limitations and disadvantages at the same time. First of all, this method is difficult to use for measuring the CD spectrum, because it is necessary to readjust the Bragg
1836 J. Phys. Chem., Vol. 99, No. 7, 1995 angle of the probe beam with scanning the wavelength of the excitation light. Furthermore, a half-wave plate which works within the whole wavelength range is needed. Nevertheless, it could be possible to use a mechanically controlled optical element to automatically set the Bragg condition as the excitation wavelength is scanned. To control the polarization in a wide wavelength range, a pair of mirrors could be used to rotate the beam, although the adjustment for the exact orthogonal polarization condition could be difficult. Utilizing a Pockel cell would be better to rotate the polarization in a wide wavelength range, since the angle of the rotation can be adjusted by changing the voltage. These improvements for measuring the CD spectrum will be the subject of a future work. Second, although this cross-polarization condition produces the spatially modulated circularly polarized light, it produces the sinusoidal modulation of the direction of the linearly polarized light at the same time.6x7 Applying this method to the time-resolved CD detection by using a linearly-polarized preexcitation laser pulse, one should be careful with the linear dichroism. The photoselectively-inducedlinear dichroism can be prevented by detecting CD at a time long after the orientational relaxation time, which is about tens of picoseconds for a small molecule in nonviscous solution. Otherwise, the polarization of the preexcitation light should be randomly polarized. Extended discussions of this effect will be discussed in the future.6 It should be noted, however, that the directional modulation can be neglected as long as the molecules are oriented randomly such as the case in this work. Third, since, in this experiment, we detect the CD signal as heat, a solvent with a large dnldT is useful from a sensitivity point of view. Many organic solvents have large coefficients, and it is one of reasons for the high sensitivity of the photothermal techniques. Water is the worst medium in this respect, although it is frequently used in biological works. (Therefore, the example we show in this paper is performed under the worst sensitivity conditions.) To overcome this low sensitivity of the aqueous solution, the sensitivity can be increased by admixing an organic solvent to the aqueous solution if the sample does not mutate by the mixing. The sensitivity of this method also depends on the purity of the polarization of the excitation beam. Theoretically, the smallest g value (A€/€) is limited by the extinction ratio of the polarizers and the depolarization by optical elements. If the depolarization can
Letters be neglected, the smallest detectable g value should be the same order of the extinction ratio for our equipment). In spite of these limitations, this method will be used to study a conformational temporal development of biological macromolecules together with other traditional detection methods, because of the high sensitivity and no inherent time resolution. The detailed analysis of this CD detection method as well as a time-resolved CD study will be published in the near future.
Acknowledgment. The author is indebted to Prof. M. Kojima in Okayama University for kindly providing Co(en)3 sample and to Prof. N. Hirota in Kyoto University for helpful discussion and encouragement. References and Notes (1) Velluz, L.; Legrand, M.; Grosjean, M. In Optical Circular Dichroism, Principles, Measurements and Applications; Academic Press: New York, 1961. (2) (a) Turner, D. H.; Maestre, M. F.; Tinoco, I., Jr. J . Am. Chem. SOC. 1974, 96, 4340. (b) Tinoco, I., Jr.; Turner, D. M. J. Am. Chem. SOC. 1983,83, 535. (c) Thomas, M.; Patonay, G.; Warner, I. Rev. Sci. lnsrrum. 1986, 57, 1308. (d) Xie, X.; Simon, J. D. Rev. Sci. Instrum. 1989, 60, 2614. (e) Xie, X.; Simon, J. D. J. Phys. Chem. 1990, 94, 8014. (3) (a) Tran, C. D.; Xu, M. Rev. Sci. Instrum. 1989, 60, 3207. (b) Xu, M.; Tran, C. D. Appl. Spectrosc. 1990, 44, 962. (4) Nunes, J. A,; Tong, W. G. Anal. Chem. 1993, 65, 2990. ( 5 ) (a) Lewis, J. W.; Tilton, R. F.; Einterz, C. M.; Milder, S. J.; Kuntz, I.D.; Kliger, D. S. J. Phys. Chem. 1985, 89, 289. (b) Einterz, C. M.; Lewis, J. W.; Milder, S. J.; Kliger, D. S. J . Phys. Chem. 1985, 89, 3845. (c) Bjorling, S. C.; Goldbeck, R. A,; Milder, S. J.; Randall, C. E.; Lewis, J. W.; Kliger, D. S. J . Phys. Chem. 1991,95,4685. (d) Bjorling, S. C.; Zhang, C.-F.; Farrens, D. L.; Song, P.-S.; Kliger, D. S. J. Am. Chem. SOC.1992, 114,4581. (e) Lewis, J. W.; Goldbeck, R. A.; Kliger, D. S.; Xie, X.; Dunn, R. C.; Simon, J. D. J . Phys. Chem. 1992, 96, 5243. (6) Terazima, M. To be published. (7) (a) Rose, T. S.; Wilson, W. L.; Wackerle, G.; Fayer, M. D. J. Phys. Chem. 1987, 91, 1704. (b) Deeg, F. W.; Greenfield, S. R.; Stankus, J. J.; Newell, V. J.; Fayer, M. D. J. Chem. Phys. 1990, 93, 3503. (c) Fourkas, J. T.; Trebino, R.; Fayer, M. D. J . Chem. Phys. 1992, 97,69. (d) Stankus, J. J.; Tone, R.; Fayer, M. D. J. Phys. Chem. 1993,97,9478. (e) Sengupta, A.; Terazima, M.; Fayer, M. J . Phys. Chem. 1992, 96, 8619. (8) A reviewer pointed out that the CD detection based on a similar idea was presented in the 207th American Chemical Society National Meeting in March 1994 by J. A. Nunes and W. G. Tong. (9) Terazima, M.; Hirota, N. J . Chem. Phys. 1991, 95, 6490; J. Appl. Phys. 1993, 73, 7672; J . Chem. Phys. 1993, 98, 6257. (10) McCaffery, A. J.; Mason, S. F. Mol. Phys. 1962, 6, 359. (1 1) (a) Miller, R. J. D.; Pierre, M.; Rose, T. S.; Fayer, M. D. J . Phys. Chem. 1984, 88, 3021. (b) Terazima, M.; Hirota, N. Chem. Phys. Lett. 1993, 214, 541. JP942575M
