NOTES

Utilization of a Prism Monochromator as a Sharp-Cut Bandpass Filter in Ultraviolet Raman Spectroscopy

S H I N J I H A S H I M O T O , T A K E S H I I K E D A , H I D E O T A K E U C H I , * AND ISSEI H A R A D A t Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980, Japan (S.H., H.T., I.H.); and Japan Spectroscopic Company, Ishikawa-cho, Hach- ioji, Tokyo 192, Japan (T.I.)

Index Headings: Ultraviolet Raman spectroscopy; Prism monochro- mator; Sharp-cut bandpass filter; Rayleigh line rejection.

INTRODUCTION

Ultraviolet resonance Raman (UVRR) spectroscopy is a useful technique for studying the structure and dy- namics of biological molecules. 1 Aromatic amino acid side chains in proteins and nucleic acid bases in DNA are examples of UV chromophores in biological systems, and the Raman scattering from such chromophores is reso- nance-enhanced with UV excitation. By tuning the UV excitation wavelength, one can observe the Raman scat- tering from a particular chromophore selectively and ob- tain structural information that is otherwise buried in the spectra. Another advantage of UVRR spectroscopy is that fluorescence from impurities in the sample or the sample itself appears in much longer wavelength regions than the Raman scattering, and the Raman spectrum becomes virtually fluorescence free. With these merits, UVRR spectroscopy has been increasingly applied to the structural studies of biomolecules and biomolecular as- semblies.

Several types of UVRR apparatuses have been devel- oped for biological applications. T M UV excitation is usu- ally made by the hydrogen-Raman-shifted harmonics of an Nd:YAG laser or a frequency-doubled dye laser pumped by an excimer laser. Frequency-doubled cw la- sers are also employed. Most of the apparatuses utilize

Received 23 March 1993. * Author to whom correspondence should be sent. t Deceased 10 August 1992.

an intensified diode array or a charge-coupled device (CCD) for the detector because of its multi-detection advantage over photomultipliers. In the spectrometer stage, three types of monochromators are currently used-- single, double, and triple--each of which has its own advantages. Generally, the optical throughput is highest in the single monochromator and the stray light level is lowest in the triple monochromator. As a compromise, the double monochromator is used to achieve reasonable levels of throughput and stray light rejection. Previously, we made a UVRR apparatus based on an [/6.8 80-cm double monochromator. 7 The monochromator was op- erated in second order and the middle slit was widely opened for detection with a doubly intensified diode ar- ray. Although our previous UVRR apparatus showed good performance in recording UVRR spectra of peptides, proteins, and nucleic acids, low-frequency Raman bands were sometimes covered by much stronger stray light, in particular for translucent samples that scattered inci- dent laser radiation strongly. The stray light is a common problem for single and double monochromators to be used with multi-channel detectors.

The best way to reduce the stray light level is to pre- vent the scattered laser light from entering the mono- chromator by using a filter. Sharp-cut filters suitable for such purposes are available in the visible region 12 but not in the UV region. We have made a small prism mono- chromator that works as a sharp-cut bandpass filter with a high throughput and negligible aberration. By install- ing the prism monochromator in front of the entrance slit of a single polychromator, we have succeeded in con- structing a high-throughput and low-stray-light UV Ra- man spectrometer. Here we describe the fore-prism UV Raman spectrometer and demonstrate its performance.

EXPERIMENTAL

Figure 1 shows the optical arrangement of the fore- prism UV Raman spectrometer. Basically the spectrom- eter consists of an f /6 200-mm prism monochromator and an f/6.3 575-mm grating polychromator (Jasco, TR- 600UV). The light coming through the entrance slit (S1) of the prism monochromator is collimated by a spherical concave mirror (M2) and weakly dispersed by a quartz Littrow dispersing prism (P) mounted on the rotation stage in a Czerny-Turner arrangement. The prism has an apex angle of 30 ° and aluminum coating on one side of the surface. The dispersed light is focused by another spherical mirror (M3) onto the entrance slit ($2) of the polychromator. The reciprocal dispersion of the prism

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D

---~"~----~:3 ~ s~ SI MI p

FIG. 1. Optical arrangement of the fore-prism UV Raman spectrom- eter: S! and 82, entrance slits of the prism monochromator and the grating polychromator, respectively; M1, flat mirror; M2-M5, spher- ical concave mirrors; P, Littrow dispersing prism with a 30 ° apex angle; G, MgF2-coated 4320-grooves/mm holographic grating with an effi- ciency of 35 % at 220 nm (another 2400-grooves/mm holographic grating blazed at 250 nm is also mounted); D, UV-enhanced CCD detector.

monochromator is ~ 1000 cm-1/mm at 240 nm. Light at the laser wavelength and fluorescence at much longer wavelengths are blocked by the slit $2, if the orientation of the prism and the width of $2 are properly adjusted. In the polychromator stage, the Raman scattered light is dispersed by an MgF2-coated 4320-grooves/mm ho- lographic grating (reciprocal dispersion, 59 cm-1/mm at 240 nm) and imaged onto a liquid nitrogen-cooled CCD detector with 1152 × 298 22.5-ttm-square pixels (Prince- ton Instruments LN/CCD-1152, UV-coated EEV Model 88130 chip). Since the prism and the grating are arranged in an additive dispersion mode, the reciprocal dispersion of the total system is 56 cm-i /mm at 240 nm.

Excitation of Raman scattering was effected by the first anti-Stokes stimulated Raman radiation (240 nm) from hydrogen gas (790 kl~a) pumped by the fourth har- monics of an Nd : YAG laser (Quanta Ray, DCR-3G) op- erating at 30 Hz. 7 The beam spot size at the sample point was ~ 500 #m × 1 mm, and the typical pulse energy was 15 #J. The scattered light was collected in a 180 ° back- scattering geometry with an [/2 UV achromatic lens (the doublet lens of CaF2 and synthetic quartz) and focused onto the entrance slit S1 with an f-matching achromatic lens (f/6). A quartz-wedge polarization scrambler was inserted between the f-matching lens and the slit S1.

The acetonitrile and carbon tetrachloride used were spectroscopic grade, and a mixture of these components was put into a quartz rectangular cell for Raman mea- surement. Purple membrane was prepared from Halo- bacterium halobium strain $9.13 About 3 mL of mem- brane suspension was recirculated by a peristaltic pump through a 1.5-mm-inner-diameter quartz capillary tube. For solid tyrosine, a pellet made of a 1:1 (w/w) mixture of tyrosine and KBr was spun. The reflection of laser light from the quartz or pellet surface was prevented from entering the slit S! by tilting the cell or the pellet. All the spectra were recorded with an SI slit width of 200 #m (spectral slit width, 11.2 cm ~). Wavenumber calibration was made with the Raman bands of cyclohex- anone/acetonitrile (1:1, v/v). Spike noises due to cosmic ray events were eliminated from the spectra by using the method reported previously. ~4

RESULTS AND DISCUSSION

Figure 2 shows 240-nm Raman spectra of a 4:1 (v/v) mixture of acetonitrile and carbon tetrachloride recorded on the fore-prism UV Raman spectrometer at varied

I I I ! I I I 1 I

I000 800 600 400 200 WAVENUMBER/cm-'

FIG. 2. UV Raman spectra of a clear sample, a mixture (4:1, v/v) of acetonitrile and carbon tetrachloride. The polychromator slit ($2) width was (A) 1.7, (B) 1.6, (C) 1.5, (D) 1.4, (E) 1.3, (F) 1.2, (G) 1.1, or (H) 1.0 mm. All the spectra were excited at 240 nm (15 ttJ/pulse, 30 Hz) and recorded with a 1-min exposure time. The entrance slit (S1) width was 200 #m and the spectral slit width was 11.2 cm -1. Scattering from the quartz cell wall was subtracted.

widths of slit $2. The slit width was 1.7 mm for the spectrum in Fig. 2A and narrowed stepwise by 0.1 mm each for the spectra in Figs. 2B-2H. When the slit is opened too wide (Fig. 2A), Rayleigh scattered light comes into the polychromator, and the stray light gives rise to a strong background in the low-wavenumber region and to a ghost band around 660 cm -1. This spectrum dem- onstrates that stray light is a serious problem even for clear samples when one is recording Raman spectra with a single polychromator, particularly in low-wavenumber regions. By a procedure of narrowing the slit gradually, the low-wavenumber background and the ghost band weaken rapidly, as shown in Fig. 2B-2D. The rapid de- crease of stray light indicates that the aberration of the prism monochromator is negligibly small, and the image of S1 is formed sharply at the slit $2. Thus, the prism monochromator combined with the slit $2 works as a sharp-cut filter. Further narrowing of the slit, of course, rejects the Raman scattered light as well. A band at 218 cm -1 due to CC14 suddenly weakens in Fig. 2G ($2 width, 1.1 mm) and disappears in Fig. 2H (1.0 mm). The sudden disappearance of the 218-cm -1 band again shows the sharpness of the prism filter.

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1500 I000 500 WAVENUMBER / cm-'

FIG. 3. UV Raman spectra of translucent and solid samples: A and B, purple membrane (150 #M in bacteriorhodopsin); C, solid tyrosine in a KBr pellet (tyrosine:KBr = 1:1, w/w). The polychromator slit ($2) was opened 5 mm in A or 1.5 mm in B and C. The spectra were excited at 240 nm (15 ~J/pulse, 30 Hz) and accumulated for (A) 1, (B) 40, or (C) 15 min. The entrance slit (S1) width was 200 #m (spectral slit width, 11.2 cm -l). Raman bands arising from the quartz capillary tube were subtracted in spectrum B.

The stray light problem is much more serious for trans- lucent, turbid, and solid samples that scatter the laser beam strongly. Figures 3A and 3B show 240-nm Raman spectra of purple membrane recorded with $2 slit widths of 5 and 1.5 mm, respectively. The purple membrane is a translucent suspension of protein-embedded mem- brane fragments whose diameters are 100-500 nm. From such a large molecular assembly, strong Mie scattering arises at the wavelength of the incident laser radiation as well as the Rayleigh scattering. In Fig. 3A, a very strong background covers Raman bands in the whole spectral range. The prism filter does not work properly with the 5-mm setting of 82, and the spectrum in Fig. 3A corresponds to what would be recorded with the poly- chromator only. By narrowing the slit width to 1.5 mm, however, one ensures that light below 300 cm -1 is blocked out, and the prism filter becomes very effective. The strong background disappears completely and many Ra- man bands due to protein aromatics in purple membrane are clearly disclosed in Fig. 3B. The effectiveness of the prism monochromator as a sharp-cut bandpass filter is further demonstrated in Fig. 3C, which shows a 240-nm

Raman spectrum of solid tyrosine in a KBr pellet. Com- pared to a spectrum excited with visible radiation, phe- nol ring modes are selectively enhanced in the 240-nm excited spectrum. This spectrum may be the first ex- ample of UVRR spectra recorded for solid samples down to 300 cm -1.

As described above, the prism monochromator works successfully as a sharp-cut bandpass filter. The remain- ing property that is important for an element of a spec- trometer is the optical throughput. We have calculated the throughput of the prism monochromator at 240 nm by taking into account the reflection loss at the prism surface and by assuming a 90% reflection efficiency at the aluminum surfaces of the prism and mirrors. The calculated throughput is 78 % for the prism and 73 % for the three mirrors. The total throughput is estimated to be 57%, which is close to a value (~65%) measured at 488 nm. The prism filter retains the high throughput above 200 nm and may be usable down to 180 nm.

Similar filters can be made by use of a grating. How- ever, the reflection efficiency of a grating varies with wavelength and is not higher than the throughput of the prism even at the maximum efficiency wavelength in the UV region. In some commercial Raman spectrometers, zero-dispersion double-grating monochromators are em- ployed for Rayleigh line rejection. Although such a dou- ble-grating filter can eliminate the Rayleigh line very well, its optical throughput is seriously low, particularly in the UV region, and most of the Raman scattered pho- tons are lost in the filter stage2 The loss of Raman signals could be minimized by installing a prism filter instead of the double-grating filter without significant increase in stray light level. It is interesting to note that the prism filter can be utilized not only in the UV region but also in the visible region.

ACKNOWLEDGMENT

This work was partly supported by a research grant (No. 02554018) from the Ministry of Education, Science, and Culture of Japan.

1. I. Harada and H. Takeuchi, in Spectroscopy of Biological Systems, R. J. H. Clark and R. E. Hester, Eds. (John Wiley & Sons, New York, 1986), pp. 113-175.

2. L. D. Ziegler and B. Hudson, J. Chem. Phys. 74, 982 (1981). 3. S. A. Asher, C. R. Johnson, and J. Murtaugh, Rev. Sci. Instrum.

54, 1657 (1983). 4. R. P. Rava and T. G. Spiro, J. Am. Chem. Soc. 106, 4062 (1984). 5. S. P. A. Fodor, R. P. Rava, T. R. Copeland, and T. G. Spiro, J.

Raman Spectrosc. 17, 471 (1986). 6. C. M. Jones, V. L. Devito, P. A. Harmon, and S. A. Asher, Appl.

Spectrosc. 41, 1268 (1987). 7. H. Takeuchi and I. Harada, J. Raman Spectrosc. 21, 509 (1990). 8. F. Sureau, L. Chinsky, C. Amirand, J. P. Ballini, M. Duquesne, A.

Laigle, P. Y. Turpin, and P. Vigny, Appl. Spectrosc. 44,1047 (1990). 9. J. B. Ames, M. Ros, J. Raap, J. Lugtenburg, and R. A. Mathies,

Biochemistry 31, 5328 (1992). 10. R. L. Benson, K. Iwata, W. L. Weaver, and T. L. Gustafson, Appl.

Spectrosc. 46, 240 (1992). 11. S. Kaminaka and T. Kitagawa, Appl. Spectrosc. 46, 1804 (1992). 12. M. M. Carrabba, K. M. Spencer, C. Rich, and D. Rauh, Appl.

Spectrosc. 44, 1558 (1990). 13. D. Oesterhelt and W. Stoeckenius, Methods Enzymol. 31,667 (1974). 14. H. Takeuchi, S. Hashimoto, and I. Harada, Appl. Spectrosc. 47,

129 (1993).

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