Anal. Chem. 1988, 60, 2658-2661
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Near-Infrared Fourier Transform Raman Spectroscopy Using Fiber-optic Assemblies E. Neil Lewis, V. F.Kalasinsky,' a n d Ira W.Levin* Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
An optical arrangement is described for coliectlng Fourler transform (Fl) Raman spectra by using near-infrared laser excitation at 1064 nm and optical flber assemblies. The advantages of this approach lie particularly In the ease of achieving precise optical alignment with nonvlslble laser exdtatlon radlatlon. I n addltlon, spectra may be Owahred within a closed system, and applications to biomolecular samples sensltlve to high temperatures and temperature instabliities are simplified. Highqualtty spectra are easily obtained from strong scatterers either by using a fiber-optlc scheme for canylng only the Incident radlatlon or by uslng a second fiber bundle to collect the Raman scattered radlation. For the fiber-optlc systems, Raman spectra of lipid bilayer and micellar aggregates demonstrate both the level of detection sensltlvtty and the effects of inddent laser power densities on sample temperatures of both fragile and weak biological scatterers. With a fiber-optic carrler, lncldent NdYAG laser power densities are estimated to be a factor of 300 less at the sample than those obtained with conventional, focusing optlcal schemes.
Although the feasibility of Fourier transform (FT)Raman spectrmcopy has been amply demonstrated (1-7), applications extending the technique particularly to more demanding chemical identifications and to biomolecular characterizations require several additional experimental considerations for preserving the ease and flexibility of the method. Our interest in the development of this spectroscopic technique arose from studies within our laboratory involving various biochemically oriented systems. Since many intact cellular biochemical preparations, as well as specifically prepared model systems, exhibit highly fluorescent properties, biomolecular aggregates have generally been considered intractable to conventional dispersive b a n technqiues employing visible laser radiation. In order to avoid or reduce these sometimes overwhelming fluorescence problems, interferometers were coupled to near-infrared laser sources (1-7'). Because of the decrease, however, in scattering cross section a t longer excitation wavelengths, it was frequently necessary to employ high laser power densities to achieve adequate spectral signal/noise characteristics for dilute molecular suspensions. Thus, as we assembled our near-infrared F T - b a n instrumentation,two major factors limiting the usefulness of this relatively new approach were quickly identified; namely, problems associated particularly with imprecise sample alignment procedures and the generation of excessive temperatures and temperature instabilities within the sample. In the present communication we address the utility of fiber-optic assemblies as a means not only of overcoming these difficulties, but of significantly enhancing the attractiveness of the technique as a more general spectroscopic tool. A set of fiber optics provides an efficient means of transporting the exciting radiation to the sample and of carrying 'Permanent address: Department of Chemistry, Mississippi State University, Mississippi State, MS 39762. This article not subject to
the Raman scattered radiation to the spectrometer, as demonstrated in visible Raman spectroscopic instrumentation (8-10). Interest in this approach arises primarily from the ease in making repetitive optical alignments, a property particularly desirable when one is dealing with near-infrared Nd:YAG laser radiation, from the desirability of minimizing the number of external optical components, and from the ability to sample, when needed, in remote or hostile environments. Thus, in our FT-Raman applications fiber-optic components have dramatically simplified our current optical designs, first, by virtually eliminating optical alignment problems associated both with nonvisible radiation and necessary sample interchanges and, second, by significantly reducing the necessity for high laser power densities to obtain meaningful spectral signal/noise ratios. Further, we are able to maintain temperature stability within biological samples to approximately fl "C over a wide range of temperature conditions. EXPERIMENTAL SECTION The optical arrangement for determining FT-Raman spectra consists of a Spectron Model SL501T NdYAG laser operating at 1064 nm coupled to a Bomem DA3.02 interferometer. Optical fiber bundles with numerical apertures of approximately0.54 were obtained from Dolan-Jenner Industries, Inc. The exciting laser radiation was passed through a beam expander in order to fill the 3-mm diameter of the transmitting quartz fiber bundle. Typical power levels ranged from 300 mW to 1.2 W of unpolarized radiation at the exit of a 2-m fiber bundle. The terminus of the fiber was then placed within close proximity of the sample, which was usually encased in a thermostated jacket. Conventional melting point capillary tubes (2-mm 0.d.) were used for aqueous dispersions, liquid and solid samples, while capillary tubes with a 4-mm spherical end were also used for liquid samples. Since the latter tubes more closely matched the diameter of the excitation beam exiting the fiber bundle, Raman bands from the glass sample cell were totally absent from the collected data. For an optical configuration in which a fiber bundle was used only for excitation, the Raman scattered light was collected by a 50-mmfocal-length lens (f/ 1)and focused into the spectrometer emission port with a 200-mm-focal-length,f/4 lens (7). The distance between the excitation fiber bundle and the collection lens precluded the possibility of Raman scattering from the fiber bundle itself entering the interferometer. For depolarization measurements in this mode, the unpolarized light from the terminus of the fiber was used to excite the sample, and a Polaroid analyzer was placed between the collection optics and the entrance aperture of the interferometer. For this measurement scheme, depolarization ratios are 617 and 5 6 f 7 for depolarizedand polsrized lines, respectively (11). For the system using a second fiber-opticbundle, the collection fiber was placed between the sample and the infrared source compartment. Thus, the scattered Raman emission emanating from the end of the collection fiber replaces the interferometer's infrared source. For our system, conversion between the infrared and Raman modes of operation becomes extremely simplified. Phase spectra for processing the generated interferograms were obtained by directing a white light source through the fiber to the sample area and collecting the light through the emission port of the interferometer or the second fiber. For even slight or minor changes in the arrangement of the optical components or sampling accessories, it is necessary to obtain a new phase spectrum.
U S . Copyright. Published 1988 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
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1000 1250 1500 1750 WAVE NUMBER DISPLACEMENT (cm-') Figure 1. FTRaman spectra of benzene at k m - ' resolution collected with the single-flberoptic arrangement and 500 mW of laser excitatlon. Collection times were (A) 6 min and (e) 15 s.
RESULTS Both 180" and 90" sample scattering geometries can be adapted to FT-Raman instrumentation in a manner analogous to that used in dispersive Raman instruments (2,3,6). The need for bulky sampling accessories dictates that the 90" arrangement be used in our experiments. However, the nonvisible character of the Nd:YAG excitation, even with an infrared viewer, increases the difficulties of aligning samples, in contrast to the problems encountered either with a 180" scattering arrangement or with visible laser radiation. One protocol, established for 90" scattering, has employed a second low-power visible laser (HeNe) aligned collinearly with the Nd:YAG laser by means of a dichroic beamsplitter (6). The HeNe laser, however, is only used for sample alignment. With the fiber-optic arrangement, the HeNe alignment laser and ancillary optics are unnecessary, since alignment simply involves mounting the fiber bundle adjacent to the sample cell. It is quite easy to position the fiber rapidly to ensure an optimized signal and a reproducible angular relationship between the excitation and collection axes. Figure 1 displays Raman spectra of benzene collected with the single-fiber-optic arrangement and with a capillary tube having a spherical bulb at the end. The spectra were collected at 4-cm-' resolution by using 500 mW of laser excitation radiation. Data collection times were 6 min and 15 s for parts A and B of Figure 1, respectively. The signal-to-noise ratio in Figure 1A is almost 200, and spectra of comparable quality are readily attained for other strong Raman scatterers by using this scheme. Depolarization data were also recorded for benzene and yielded ratios in close agreement ( f l % ) with the theoretical values expected from this type of excitation/ collection scheme. Using 1.2 W of laser excitation and 3-min collection time, we obtain, as shown in Figure 8, a spectrum of indene collected with the dual-fiber-optic scheme outlined
previously. Although this optical arrangement provides a convenient means of collecting Raman radiation, the inherent losses of signal intensity along the length of the collection fiber bundle lower the signal/noise characteristics of this scheme compared to those of a single-fiber-optic assembly. Previous attempts to obtain FT-Raman spectra of turbid aqueous lipid bilayer dispersions by using the standard optical arrangement of a focusing lens before the sample often resulted in spectra of limited quality. The heating effects of moderately focused laser radiation induce the lipid aggregates to migrate out of the exciting laser beam, resulting in Raman signals from a highly dilute dispersion. Spectra of polycrystalline lipid samples present less of a challenge, but with focused laser radiation may still easily reach temperatures in excess of 100 "C in the volume sampled by the laser beam. A cooling arrangement whereby the sample was bathed in a stream of cold nitrogen gas gives more reasonable spectra (7), but it is often difficult to determine the temperature of the sample a t the point of the laser probe. With the fiber-optic arrangement, laser power densities at the sample are estimated from cross sectional areas to be as much as a factor of 300 less than with the conventionally focused system; thus, heating effects are significantly reduced. Figure 3A shows spectra of the C-H stretching mode region of an aqueous dispersion (20% (w/w)) of dipalmitoylphosphatidylcholine (DPPC) in both the gel phase a t 20 "C (lower trace) and the liquid crystalline phase at 45 "C (upper trace) collected by using 500-mW and 1-W laser excitation power levels, respectively. These spectra were obtained with the optical fiber in place and with the sample capillary positioned in a stream of nitrogen gas a t ambient temperatures. A comparison between the upper and lower traces in Figure 3A clearly demonstrates the substantial heating effects possible with routine Nd:YAG laser power densities, even with fiber-optic carriers. Figure 3B displays spectra of the same spectral region of polycrystalline DPPC at 25 "C, obtained with 500-mW laser power (lower trace), and 80 "C, obtained with 1W of power (upper trace). Sample temperatures are monitored by the relative intensities of the symmetric and asymmetric methylene stretching modes a t approximately 2850 and 2880 cm-', respectively (12).The observed I ~ / I m ratio is compared to calibration spectra recorded on a dispersive Raman instrument using low power density levels (100-200 mW) from a visible argon ion laser and a thermoelectric device for carefully regulating the sample temperature (13). We conclude that the fiber-optic assembly provides a significant improvement over the conventional, direct focusing method of excitation, since one can easily maintain sample
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
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