A New Raman Microsam1 Glen F. Bailey, Saima Kint, : Western Regional Research Labor IT HAS BEEN LONG RECOGNIZED t..,.
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conjunction with infrared spectrometry, is a valuable tool for the characterization of molecular structure and for the analysis of molecular vibrations. In addition to the complementary vibrational frequency information provided by the two techniques, the depolarization ratios (I) of Raman lines give vital information regarding the natuie of the vibrational symmetry (2). In the past, sample size requirements have prevented general application of this technique to investigation of natural products. The introduction of continuous gas lasers has stimulated a renewed interest (3) primarily because of the high intensity, low beam divergence, and inherent polarization of the laser source. The He-Ne 6328-A source has been particularly useful in obtaining spectra of samples which fluoresce when exposed to shorter wavelength radiation. Damen, k i t e , and Porto (4) have demonstrated that the intensity of scattered Raman radiation is independent of scattering angle, 0, when the incident beam is polarized in a direction normal to the plane of 0. For isotropically scattered (in the plane of 0) Raman lines such as the 459 Acm-'
(1) Gerhard Herzberg, "Molecular Spectra and Molecular Structure 11. IR and Raman Spectra of Polyatomic Molecules," D. Van Nostrand, Princeton, N. J. 1947, p. 246-9. (2) E. B. Wilson, J. C. Decius, and P. C. Cross, "Molecular Vibrations," McGraw-Hill, New York, 1955, p. 43. (3) S. P. S. Porto. Ann. N . Y.Acad. Sei.. 122(6). ,. 643 (1965). (4j T. C. Damen,'R. C. C. Lelte, and S.'P. S Porto,'Phy; Rev. Letrers, W I ) , 9 (1965).
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accurately by measuring the intensity of the radiation which passes through an analyzer that has its electric vector in the plane of 0 and normal to the scattering direction. In order to obtain spectra of small samples, it is necessary that the cell volume be minimized, that the incident radiation I,ass through a large portion of ' t he sample, and that this 1mrtion fill the field of view of the m onochromator. We have > - ~ ~ . ~ > .~. n . . auopreo an excnarionjviewing geometry suggesreu 1uy ruriu (3) in which the laser radiation is passed down the axis of a cylinder and the Raman radiation is viewed 90' from this axis. In the following we describe a capillary cell technique (CLT) in which the capillaries themselves form an integral part of the optical system. Using the CLT and a IO-mW laser source we have been able to obtain good quality spectra without multipassing the laser beam. Furthermore, good SIN ratios are observed with reasonable resolution and scan rates, and with sample volumes of the order of 0.4-0.04 PI. Optical System. A Perkin-Elmer LR-1 spectrophotometer equipped with an 8- to 10-mW He-Ne 6328-A gas laser and . solid sampling accessory optics was used for a1II CLT measurements. The collecting optics for the scattei-ed radiation produce a 4:l magnified image of a 3.0 mm higlh hy 0.1 mm .n.Yb .:Al. -0--1--eo +hn I 7 hinh A .LI1l( r.dide entrance -,...p.C . . . 6 . . hu u, n v_-. .llr slit (IO-cm-1 spectral slit width). The borosilicate glass capillary cells are formed with a spherical lens on the tip and are oriented so that the top of the image of the entrance slit falls on the capillary about 0.5 mm helow the capillary lens. The incomine laser beam is focused hv means of a coated ~~
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Firmre 1. S tthematic dia(p m tranamrned and reflected ,,ar.. laser beam near the capil.,., tip vx
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Figure 2. Photograph of apparatus for positioning the capillary and the lens used to focus the laser beam above the capillary
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Figure 3. Photomicrograph of capillaries (a, 6,c, and d) used to obtain the spectra in Figures 4a, by and c, 5, and 6, respectively Lens diameters are indicated in mm. Cell volumes are 0.33,0.04,0.16, and 0.35 rl, respectively. The aluminizing was removed from the 0.58-mm and 0.41-mm capillaries after obtaining the spectra
converging lens (having 6.5-mm diameter and 12.4-mm focal length) to a point whost: location above the capillary tip corresponds to the focal length of the capillary lens (Figure 1). The laser beam is rendered parallel by the capillary lens and passes down the bore, through the volume viewed by the monochromator, and is eventually refracted at the liquid-air interface within the capillary. The parallelism is ultimately limited by diffraction effects which spread a 100-micron diameter beam of 6328-A wavelength about 40 as it travels for 5 rnm. Using simple thin lens formulae, it may be shown that the diameter, d, of the beam, within the capillary is given by:
where R is the radius of curvature of the capillary lens, p is the index of refraction of borosilicate glass (1.474), D is the diameter of the laser beam prior to focusing (3 mm), and f is the focal length of the converging lens. Using a 10-rnW laser (measured with a Spectra-Physics 401B power meter) and assuming no reflection losjes, the power density within a capillary tube with R = 0.24 nim is -90 W/cm2 compared to -0.14 W/cm2 in the incident beam. Because the Raman intensity is linearly dependent on tht: incident intensity (2), this represents a radiant emittance gain approaching 103 for an equivalent small volume element in the beam. This gain approximately compensates for the reduced number of emitters seen by the
monochromator across the illuminated region of the capillary, compared with the number viewed in the multipassed 2.5-ml cell supplied by the manufacturer. In order to utilize the backscattered radiation, we aluminize the back side of the capillary (-I/* mm below the capillary lens and for a length of 4 to 8 mm) using standard vacuum coating methods. While the achieved intensity gain is only -1.75 as opposed to the theoretical factor of 2, the system is inherently simpler to align and less expensive than one equipped with conventional backing mirrors. Capillary Support System. Figure 2 shows a photograph of the assembly used to hold the capillary and converging lens. A dual universal stage was constructed from microscope mechanical stages to allow independent positioning of both the capillary cell and the converging lens. The capillary, surrounded by Teflon (Du Pont) sleeving to prevent crushing, is held in a removable 0.0- to 1.38-mm pin vise. The pin vise rests on the surface of the adjustable platform. This arrangement allows precise adjustment of the orientation of the capillary along the axis of the image of the entrance slit. A hole is drilled in the top of the monochromator case behind the entrance slit and fiber optics are used to back-illuminate with 6328-A light to make the capillary coincident with the entrance slit image. The weak beam reflection from the tip of the capillary is viewed with the aid of a mirror placed on the platform next to the pin vise. When the focus of the upper lens is at the tip of the capillary, the reflected image on the underside of the lens mount is -3-4 mm in diameter and VOL 39, NO. 8, JULY 1967
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becomes larger as the converging lens is raised or lowered. Maximum Raman intensity is achieved when the converging and capillary lenses are separated by the sum of their focal lengths. This alignment of their common focal point is verified by measurement of the diameter of the reflected light beam (dashed line in Figure 1). Capillary Construction. Capillaries are drawn from clean borosilicate glass tubing having 10-mm 0.d. and 8-mm i.d. The ratio of outer diameter to inner diameter is important in achieving the desired capillary lens geometry-viz, a spherical exterior and a flat inside surface. Lengths of -30 cm of uniformly-round capillaries with 0.025- to 0.05-mm wall thickness and with 0.15- to 0.5-mm 0.d. are placed in a pin vise and rotated about a horizontal axis with a 600-rpm motor. The motor is mounted on a hand-driven cross-slide which allows horizontal movement perpendicular to the rotation axis. The flame from a gas-oxygen microtorch is positioned vertically near the rotating capillary so that, on operation of the crossslide, the capillary passes through the upper 1 1 4 of the 3 mm high blue inner cone of the flame. The capillary is driven through the flame at the rate of -2 mm per second and as the capillary traverses the flame, the excess tubing is drawn off. ANALYTICAL CHEMISTRY
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Figure 4a. Spectra of CCL with analyzer [/ and to the incident electric vector Cell volume is 0.33 pl. Both spectra were run on the same section of chart without shift of the ordinate zero. The features at about 385 Acm-l and 545 Acm-1 are spurious lines from Rayleigh scattering. 0.58-mm capillary lens (Figure 34; 10-cm-l spectral slit width; 0.8-second time constant; scan rate 0.9 to 0.8 cm-'/second; laser power is 10 mW. Observed pa for 459 Acrn-' line = 0.008 (peak height measurement)
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b. Spectra of CC14 with analyzer dent electric vector
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Cell volume is 0.04 pl. Laser power is 6.7 mW. Instrumental conditions same as in a The flame size and traverse rates are adjusted to yield capillary lenses having dimensions typical of those shown in Figure 3a,b, c, and d. Each capillary lens is inspected under a microscope using oil immersion to detect any defects or bubbles which may have formed in the fusion process. A large fraction of the capillaries passed visual inspection and gave surprisingly uniform results. Typical aluminized capillaries (oil immersed, filled with CC14 and indene) are shown in Figures 3b and c, respectively. Volumes of the capillaries are determined by measuring the inside diameter of the tube with a calibrated ocular scale and assuming a 5-mm filled length. Because of the ease of capillary construction, the cells are not reused. Sampling Techniques. Clean, low-viscosity samples are transferred to the capillary cells with smaller (open ended) capillaries of -10-cm length which are filled by capillary action. The transfer tube is emptied by gravity or air pressure as required by the viscosity of the sample. Occasional trapped air bubbles are spun out of the sample with a centrifuge. Viscous samples are transferred to the tube by evacuating the capillary, dropping its open end into the sample, and returning the system to atmospheric pressure. Samples containing particulate matter are purified by micro distillation, We have also obtained successful spectra of
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Figure 5. Spectra of benzene with analyzer 11 and 1 to the incident electric vector
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Figure is a composite of separate scans with zeros displaced for clarity, but with identical instrumental conditions. 0.41-mm capillary lens (Figure 36); lO-cm-’ spectral slit width; 2.8-second time constant; scan rate 0.90-0.72 cm.-l/second; laser power 10 mW. Observed p a for 992 Acm-l line = 0.019 (peak height measurement)
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c. Spectrum of CCla with filters inserted at exit slit to attenuate scattered 6328 light
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organic solids by filling the capillary with molten sample using the vacuum technique described above. After crystallization the capillary is mountei in the pin vise and the solid state spectrum measured. h4easurements in the molten state are made by placing a hemicylindrical heater made from No. 20 Nichrome resistance wire (5 mm in diameter by -10 mm long) behind the sample. RESULTS
By using the CLT we have been able to obtain consistently good Rarnan spectra with as little as 0.4- to 0.04-pl samples. Comparisons of the Rarnan peak heights show that the sensitivity using the 0.3-pl capillary cell is -0.5 that obtained with the 2-ml cell supplied by the manufacturer. In Figure 4a are spectra of CCI4,obtained in the capillary cell shown in Figure 3a, with the analyzer 11 and Ito the polarization of the incident radiation. The aluniinixing was removed for the photomicrograph after the spectrum was scanned. The measured value of the depolarization ratio, ps, for the 459 Acm-1 line is 0.008 which compares favorab y with the value obtained by Douglas and Rank ( 5 ) of 0.0065 and by Porto (6)of 0.005. The value
E. Douglas and El. H. Rank, J . Opt. SOC.Am., 38, 281 (1948). (6) S. P. S. Porto, J . Opt. SOC.Am., 56(11), 1585 (1966). (5) A.
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Figure 6. Spectra of indene with analyzer incident electric vector
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we find using the manufacturer’s 2-ml cell is 0.006 0.001, The lines at 218 and 314 Acm-1 have a measured ps = 0.75 + 0.01. In Figure 46 are spectra of CCla obtained with the capillary shown in Figure 36. The sample volume is -0.04 pl. The instrumental conditions are the same as in Figure 4a except that laser power is only 6.7 mW. We note that reducing the sample size increases the intensity of the spurious VOL. 39,
NO. 8, JULY 1967
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Figure 7. Spectrum of longifolene No analyzer. 0.40-mm capillary lens (not shown); 10 cm-l spectral slit width; 2.8-second time constant; scan rate 0.90-0.48 cm-l/second; laser power 10 mW, Filters inserted to remove spurious features at 385 and 545 Acm-l peaks and the background noise, and only slightly reduces the Raman intensity. The depolarization measurements are not as good as those obtained with the larger capillaries, particularly for the depolarized lines at 218 and 314 Acm-l. These two lines have a measured p s of 0.5 instead of 0.75. These results lead us to conclude that relative depolarization measurements made with the smallest volume cells may be corrected provided that some lines may confidently be assigned as being depolarized. This will be possible for those cases where the molecule is known to possess at least two elements of symmetry. Where absolute depolarization measurements are required the larger volume capillaries should be used. Figure 4c shows a spectrum of CCla in the 0.04-pl cell without analyzer and with filters inserted to attenuate stray 6328-A scattering. The 180' backscattering technique developed by Hawes et al. (7) can potentially collect a higher fraction of the total Raman scattered radiation than the CLT described here, but gives a high value of p (0.40) which appears to result from skew ray reflections. In Figure 5 are spectra of benzene (capillary c in Figure 3) obtained with / / and I orientations of the analyzer. The 992 Acm-l line has a measured ps = 0.019 in the capillary cell as compared with 0.017 in the 2-ml cell. Douglas and Rank (5)report a value of 0.020 for p s , and Porto (6) reports
(7) R. C. Hawes, K. P. George, D. C. Nelson, and R. Beckwith, ANAL.CHEM.,38, 1842 (1966).
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0.065. In this instance our measurements support the Douglas-Rank value. The measured values of ps for the lines at 1595, 1178, and 606 Acm-1 are 0.72, 0.72, and 0.73, respectively. The uncertainty in these values due to noise level is -0.03. In Figure 6 are spectra of indene (capillary d in Figure 3), and I orientations of the analyzer, and obtained with measured without zero suppression. These spectra may be compared with those given in reference (7). Finally, in Figure 7 we show a spectrum of longifolene (ClsHzr) which is obtained from Pinus longifolia Roxb. in quantities typical of natural products. We find that the S/N ratio is quite adequate to characterize the spectrum of this substance.
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ACKNOWLEDGMENTS
We thank F. T. Jones for the photomicrographs in Figure 3 and for discussions of the microscopical work, N. T. Mirov for the sample of longifolene, R. S. Thomas for use of the aluminizing apparatus, and R. C. Hawes for stimulating discussions. RECEIVED for review February 17, 1967. Accepted April 19, 1967. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
