Fresenius J Anal Chem (1991) 339:271-275 Fresenius' Journal of

© Springer-Verlag 1991

Fourier Transform Raman spectrometry Norman Wright, Phil Stout, and K. Krishnan

Bio-Rad, Digilab Division, 237 Putnam Avenue, Cambridge, MA 02]39, USA

Introduction

Raman spectrometry has traditionally been limited to the ultraviolet (UV) and visible regions of the spectrum because scattering intensity is proportional to the fourth power of the frequency of the incident radiation (I ~ re). Thus, the efficiency of scatter is diminished as the excitation source moves to longer wavelengths. However, the possibility of extending the Raman technique with excitation in the near- infrared (NIR) region has always been attractive. Excitation at the customary visible frequencies can often lead to fluores- cence, and, as a resonance, rather than scattering phenome- non, fluorescence is much stronger than Raman scattering and can obscure any Raman signal. Few species have electronic absorption bands in the near-infrared, thus, ex- citation in this region is less likely to produce fluorescence.

The 1.064 btm line of the Nd : YAG laser has been used for the development of a nearqnfrared FT-Raman system. A laser line filter is used to reject all emission from the laser except the 1.064 gm line. Once the radiation from the laser passes through the line filter, the beam is focused onto the sample. The scattered radiation from the sample is collected at 90 ° or ~80 °, and is directed into the near-infrared in- terferometer of the Bio-Rad FTS spectrometer. Prior to reaching the interferometer, the scattered radiation passes through a Rayleigh rejection filter.

The combination of a continuous wave (CW) Nd: YAG laser, an extremely sensitive detector specifically configured for emission spectrometry, custom optics and filters to allow observation of Raman scatter, and the advantages available with a Fourier Transform spectrometer have produced a powerful analytical tool.

Theory

Raman spectrometry is a complementary technique to in- frared spectroscopy, and provides information about the vibrational energy levels of molecules [1-4] . In Raman spectrometry, the radiation from a powerful monochromatic source, such as a laser, is focussed on the sample under study. The radiation scattered by the sample consists of the wavelength of the incident radiation (known as the Rayleigh

Offprint requests to: N. Wright

line) plus a number of other lines characteristic of the molecular vibrations (Raman lines). The Raman lines at frequencies higher than that of the Raleigh line are known as anti-Stokes lines, and those at lower frequencies are known as the Stokes lines. The frequency shifts of the Raman lines from the Rayleigh line correspond to the molecular vibrational energies. Figure 1 illustrates the Raman scattering mechanism.

The molecule in its lowest vibrational state is excited to a virtual state by the monochromatic radiation of energy by0. The transition back to the ground state produces the Rayleigh line. The transition to the higher vibrational levels will produce the Stokes Raman lines of energies less than hv0. The excitation to the virtual state of molecules in higher vibrational levels will produce transitions back to the vibrational ground state, and lead to anti-Stokes Raman lines of frequencies higher than hv0. Since the anti-Stokes Raman lines originate from the excited vibrational states of the molecule, the intensity of the anti-Stokes lines will be much less than those of the Stokes Raman lines. The Rayleigh scattering is very intense, and the Rayleigh line is usually two or three orders of magnitude more intense than the Raman lines.

An FT-Raman spectrum is presented in Fig. 2 which shows the Stokes and anti-Stokes bands for sulfur. Note that the bands are symmetrical about the Rayleigh line centered at zero Raman shift, but that the anti-Stokes bands are considerably weaker. A notch filter was used with the FT-

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Fig. 2. FT-Raman spectrum showing the Stokes and anti-Stokes lines for sulfur

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Fig. 3. Schematic diagram of the Bio-Rad FT-Raman system. A: Beam Blocker; B: Attenuators; C: Line Filters; D: Focusing Lens; E: Turning Mirror; F: Sample Mounts; G: Rejection Filters; H: Aperture; I: Detector; J: Optional, He-Ne Alignement Laser; K: Optional Notch Rejection Filter

Raman system for the data acquisition in order to remove most of the very intense Rayleigh line. It allows Stokes bands above 70 cm -1 and anti-Stokes bands above 100 cm -1 to be observed, That is why the small band at about 85 cm-1 is not seen in the anti-Stokes spectral region.

F T - R a m a n sys tem

An FT-Raman system consists of a near-infrared FT-IR spectrometer bench, a source of monochromatic radiation to focus on the sample, and optics for collecting the Raman scatter. Dispersive Raman systems require a double or triple monochromator arrangement to separate the frequencies and for rejection of the Rayleigh line. Since all of the scattered radiation from the sample is collected and Fourier transformed in the FT-Raman experiment, a Rayleigh line rejection filter is used here. The detector is a liquid nitrogen- cooled indium gallium arsenide (InGaAs) detector, with a specially configured preamplifier required for the low levels of collected radiation typical in the Raman experiment. The sample is mounted on an XYZ stage to allow for optimal positioning in the laser beam.

Position for 180 ° Collection

Position for 90~'Collection

Fig. 4. a 180 ° backscattering position used for FT-Raman sampling. b 90 ° position used for FT-Raman sampling

Figure 3 shows a schematic diagram of the Bio-Rad FT- Raman system. The 1.064 btm line ofa Nd: YAG laser is used to produce the Raman scatter. The heart of the FT-Raman accessory consists of Raman collection and pre-sample optics, including filters for the elimination of unwanted laser plasma lines [5]. A focusing lens is inserted in the accessory to provide a laser spot size at the sample approximately equal to the output beam waist of the laser. The backscattered (90 ° or 180 ° collection) Raman radiation is collected by a gold coated mirror providing a large solid angle. Dielectric filters, or the optional notch filter, eliminate the strong Rayleigh line before it enters the interferometer. The resulting radiation is detected by the high-sensitivity cooled InGaAs detector.

Beam blockers physically prevent the laser beam from entering the accessory when either the sample compartment cover or purge cover are open. The use of attenuators may be necessary where the sample of interest is a very strong scatterer, or for a sample which has a tendency to heat up with the incident laser energy.

The physical properties of samples require that both 180 ° (backscattering) and 90 ° collection positions be available in Raman spectrometry. The movable turning mirror, shown in Fig. 4a, is configured for 180 ° collection. It is plate mounted and uses easily loosened fasteners so that it can be positioned for the alternate 90 ° collection path to the sample, as shown in Fig. 4b. The incoming laser beam is designated

273

by the solid line, the sample as a solid ball, and the scattered radiation path, directed to the detector, by the shaded area.

A variety of sample holders are provided that fit onto the XYZ movable stage. The sample holders have been de- signed so that they can be interchanged without losing sample alignment. Optimization of sample position in the X- and Y-axis directions is best performed when the laser beam is in contact with the sample. Therefore, these controls have been placed outside the compartment to allow for operation of the FT-Raman instrument during positioning.

The diagram in Fig. 3 b also shows the suggested location for mounting a polarizer when polarization measurements are desired. Raman spectra can be obtained in two polariza- tions. Molecules with small size [4] or an element of symmetry will show different spectra with the different polarizations, as will oriented materials. By rotating the polarizer between horizontal and vertical orientations, the depolarization ratios of bands can be measured. Measurement of the depolarization ratio of a Raman line gives information re- lated directly to the symmetry of that molecular vibration. A non-symmetrical vibration gives rise to very little polariza- tion and is said to be depolarized, while a totally symmetric mode will give a band that is polarized. Therefore, the depolarization ratio provides useful information when assigning frequencies to particular modes of vibrations within a molecole [6].

C o n f i g u r a t i o n

Bio-Rad offers three options for an FT-Raman system, described below.

Option 1. This is a complete FT-Raman system. It consists of a Nd: YAG laser, Raman accessory, a NIR FT-IR optical bench, and an SPC workstation based on the Motorola 68020 or 68030 series microprocessor. The data system consists of a 100 Mbyte hard disk upgradable to a 380 Mbyte disk, a 1.44 Mbyte 3.5 inch floppy drive, a six-color single page plotter, a 14 inch color monitor, a special keyboard with twelve function keys and four user definable keys, and a joystick for cursor control and display zoom and roll. An 80 column dot matrix printer is optional.

Option 2. This option is designed for customers who already possess an FTS system. It consists of a NIR FT-IR optical bench, Nd:YAG laser and Raman accessory. This second optical bench would be under the control of the 3200 series workstation already possessed by the customer.

Option 3. This option is designed for customers who already possess an FTS system with a NIR optical head, and consists of a Nd:YAG laser and Raman accessory to be integrated with the existing NIR system.

In all cases, the recommended Nd:YAG laser can be purchased separately.

R e s u l t s a n d d i s c u s s i o n

Since the mechanism of Raman scattering is quite different from that of absorption, the selection rules for the two kinds of processes are different. Vibrational transitions that are forbidden in infrared absorption may be permitted in the Raman effect, and vice versa. The two experimental methods

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Fig. 5. FT-IR (top) and FT-Raman (bottom) spectra for para-xylene

are, thus, essentially complementary in character. Figure 5 shows the FT-IR (top) and FT-Raman (bottom) spectra for para-xylene, which has a center of symmetry. Note that although some bands do appear in both spectra, for the most part the absorptions are mutually exclusive.

FT-Raman spectrometry enjoys several distinct ad- vantages over dispersive Raman spectroscopy. First, since this Raman system is based on a Fourier transform spectrometer, it takes advantage of the higher optical throughput, the wavelength accuracy, and reproducibility, due to fringe counting using the internal spectrometer helium-neon laser. One of the many benefits afforded by the latter two advantages is the ability to perform spectral subtractions. A full spectrum can be collected at 8 cm-1 resolution in under 2 s. Computer control of the mirror stroke allows the acquisition of spectra with differing re- solutions. The problem of fluorescence is minimized by using a near-infrared, rather than a visible, laser for excitation [6-81.

Many of the same samples which lend themselves for examination by infrared spectroscopy can also be examined by Raman spectrometry. Surface studies are easy, as Raman spectroscopy is a scattering technique. Examination of films, liquids, and powders require no special sampling prepara- tion. Additionally, the study of aqueous samples in glass or quartz vessels, which is difficult in the mid-infrared region due to the strong infrared absorption bands of water and quartz, is facilitated by Raman spectrometry because there do not exist any significant bands which would cause in- terference in the solution spectrum. Figure 6 illustrates an application of Raman spectroscopy to the study of aqueous solutions. Note that no bands due to presence of water can be seen in the aqueous sugar solutions. Rather, the two spectra reflect the broadening of spectral features due to the loss of structure upon dissolution of the highly crystalline, solid sugar sample in water.

Powdered inorganic materials, such as lanthanum vanadate, can be easily examined, either pressed into a holder, or contained in a glass capillary tube. Acquisition of Raman spectra for this material (Fig. 7) would have been quite difficult in the past using visible Raman spectrometers due to its highly fluorescent background.

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Raman Shif t (cm-1)

Fig. 6. FT-Raman spectra ~rcrystalline, solid sugar(bottom) and itsaqueoussolution(top)

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F i g . 8 . FT-Raman spectra for the pharmaceutical compounds aspirin (bottom) and phenacetin (top)

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Fig. 9. FT-Raman spectra showing the effect ofsignal averaging. 8scans @ottom)and 128scans(top)

Specificity of compounds is retained, as in infrared spectroscopy, making identification possible. Figure 8 shows the fingerprint region from both aspirin (bottom) and phenacetin (top). The crystalline bands are sharp and well defined, with easy differentiation between the two com- pounds.

All the advantages of a computerized FT-IR system are available to the FT-Raman system. Figure 9 shows the effect of co-addition. The signal-to-noise improved as expected, by a factor of four, when the number of scans-co-added was increased from 8 to 128. Small bands, buried in the noise of the bottom spectrum, are now clearly seen in the top spectrum.

Spectral subtraction is also possible with Raman spectra, as the intensity of bands is linearly proportional to concen- tration and the frequency scale is reproducible. Figure 10 shows the application of spectral subtraction when a

spectrum of pure ortho-xylene (Fig. 10b) was removed from a mixture (Fig. 10a) of ortho-xylene and meta-xylene. The difference spectrum (Fig. i0c), obtained following the sub- traction, corresponds to that of pure meta-xylene (Fig. 10 d) verifying that spectral stripping is indeed valid.

Software manipulation, involving such procedure as Fourier self-deconvolution, are also applicable to FT- Raman spectra. Since co-addition can yield relatively noise free Raman spectra, deconvolution can be used to resolve overlapping bands. Figure 11 shows an example of an EDTA solution before (bottom) and after (top) Fourier self- deconvolution to achieve separation and baseline resolution of the overlapping bands. Some of the bands exhibit negative lobes as the bandwith used for deconvolution was not appropriate for all the bands. Normally, smaller regions, where the bands all have similar widths, would be examined for optimal application of deconvolution.

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Fig. 10a-d . FT-Raman spectra illustrating spectra subtraction. a Mixture of ortho- and meta-xylene; b pure ortho-xylene; c difference spectrum; d pure meta-xylene

Conclusions

FT-Raman spectrometry has advanced significantly through the combination of CW N d : Y A G lasers, high sensitivity near-infrared detectors, and state-of-the-art filter tech- nology. Obtaining fluorescence-free spectra through near- infrared excitation has now become practical.

Raman spectrometry provides data which is comple- mentary in nature to infrared spectra and often helps provide unambiguous structural determination. Sample preparation, if necessary, is often easier than for the mid-infrared analysis. Sample categories for which Raman spectrometry can pro- vide significant data include: aqueous solutions, catalysts [9], food products, pharmaceuticals, polymers [9], thin films, minerals and inorganics [10], and biological [9, 11] compounds. Typical scan times are I min or less. FT-Raman has truly become a powerful, general purpose analytical tool.

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Fig. 11. Ft-Raman spectra showing the original EDTA solution spectrum (bottom) and the result (top) following Fourier self- deconvolution

References

1. Herzberg G (1945) Infrared and Raman spectra. Van Nostrand Reinhold Company, New York

2. Colthrup NB, Daly LH, Wiberly SE (1975) Introduction to infrared and Raman spectroscopy. Academic Press, New York

3. Long DA (1977) Raman spectroscopy. McGraw Hill, New York 4. Durig JR (ed) (1981) Vibrational spectra and structure, vol 9.

Elsevier, New York 5. Hirschfeld T, Chase B (1986) FT-Raman spectroscopy:

Development and justification. Appl Spectrosc 40 : 133 - 137 6. Hallmark VM, Zimba CG, Swalen JD, Rabolt JF (1987)

Fourier Transform Raman spectroscopy: Scattering in the near- infrared. Spectrosc. 2: 40 - 47

7. Chase DB (1987) Fourier transform Raman spectroscopy. Anal Chem 59:881A-889A

8. Zimba CG, Hallmark VM, Swalen JD, Rabolt JF (1987) Fourier transform spectroscopy of long-chain molecules con- taining strongly absorbing chromophores. Appl Spectrosc 41 : 721 - 726

9. Durig JR (ed) (1985) Chemical, biological and industrial ap- plications of infrared spectroscopy. Wiley, New York

10. Ross SD (1972) Inorganic infrared and Raman spectra. McGraw-Hill, London

11. Schmidt ED, Schneider FW, Siebert F (eds) (1988) Spectroscopy of biological molecules - New advances. Wiley, New York