Vibrational Spectroscopy II:

 Raman Spectroscopy

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In a manner similar to Infrared (IR) spectroscopy, Raman spectroscopy is based on the existence of a spectrum of normal vibrational modes in a given molecular compound. Raman techniques differ, however, from traditional vibrational IR spectroscopy in that scattered radiation (vs. absorbed radiation) is studied (1-7).

When light is scattered from a molecule, most photons are “elastically” scattered. I.E. The scattered photons have the same energy (frequency) and, therefore, wavelength, as the incident photons. However, a small fraction of light (~ 1 in 10⁷ photons) is scattered at optical frequencies different from (usually lower than) the frequency of the incident photons. The difference in energy (or shift) between the incident photon and the Raman scattered photon is equal to the energy of vibration of the scatterer.

A Raman spectrum is generated by directing a laser onto the sample and observing the patterns of lightwaves which are scattered at higher and lower wavelengths relative to that of the incident laser beam. Most of the scattered photons will have the same wavelength as the incident radiation. This type of light scattering is called elastic scattering, or Rayleigh scattering, and contains no data of interest. Inelastic (Raman) scattering consists of scattering on either side of the primary frequency band. In inelastic scattering, photons are absorbed by the sample and then reemitted. The frequency of the reemitted photons is shifted up or down in comparison with original frequency, which is called the Raman Effect.

The shift is not dissimilar from what we have seen in fluorescent materials, where absorption results in a shift of the emitted frequency to lower energy or longer wavelengths. But in this case, the emitted wavelength could be either higher or lower, depending on the nature of the material. The shift provides information about vibrational, rotational and other low frequency transitions in molecules. Raman spectroscopy can be used to study solid, liquid and gaseous samples.

Furthermore, Raman interactions are dependent not on the existence of polar bonds, but instead on the existence of polarizable (or stretchable) bonds. This displacement

of atomic nuclei (nuclear displacement) will determine the polarizability and the maximum amplitude of any normal mode of vibration. Thus, in any normal vibrational mode, chemical bonds expand and contract to various degrees, depending on the magnitude of their polarizability. How polarizable a bond is will determine how it scatters incoming light. At the maximum amplitude (maximum expansion) the polarizability of the bond will not be the same as when the bond is at maximum compression (minimum amplitude). Thus, the polarizability fluctuates with the same frequency (or cycle) as the vibration. 

Thus, light scattering results from interactions with the polarizable bond that produce signals at the excitation wavelength (elastic) or at wavelengths plus or minus the vibrational mode of the bond (inelastic). An example of the latter is illustrated below, where the excitation frequency (9395 cm^-1) of the laser beam would be added (according to the principle of superposition of waves) to the vibrational frequency of the sample itself (2000 cm^-1) in order to obtain the final frequency of the composite waveform.

* Note: Whenever two distinct waveforms interact with each other, there will be some moment in space and time where the incoming lightwave will be perfectly in phase with the vibrational frequency of the molecular vibrations. This is the condition of coherence. Under these conditions, the two distinct waveforms will add together by constructive interference, and the composite wave will have a harmonic frequency which is an integer multiple of both individual frequencies. In the opposite case of perfectly destructive interference, the two waveforms will interact incoherently and cancel each other out. In all intermediate cases, the amplitude of the composite wave will be equal to the sum of the amplitudes of each individual waveform. This is the essence of the principle of superposition of all waveforms.    

If we now consider all inelastic scattering of lightwaves, we refer to the increased wavelength (expanded bond) as the Stokes signal, and the decreased wavelength (compressed bond) as the anti-Stokes signal.

There are two different quantum energy jumps which are important here. The first is the difference between the most excited energy state and the ground state. The second is the difference between the least excited state and the ground state. The latter is equivalent to what we will refer to as the Raman shift. Thus:  

Of these two signals, the Stokes signal is the stronger. But it is still several orders of magnitude weaker than the Rayleigh line (elastic scattering). The Raman spectrum can also be normalized and plotted as the shift in wavelength of the signal.

Thus, different chemical compounds and chemical bonds will be evidenced by different shifts in their Raman spectrum. 

Oscillating Dipoles

The Raman Effect is therefore based on molecular bond deformations in an electric field, E. The degree of deformation (or bond stretching) is determined by molecular polarizability, a. The laser beam can be considered as an oscillating electromagnetic wave with electrical vector, E. Upon interaction with the sample it induces electric dipole moment, P = aE, which deforms molecules. Because of periodical deformation, molecules start vibrating with characteristic frequency, m.

I.E. Monochromatic laser light with frequency f excites molecules and transforms them into oscillating dipoles. Such oscillating dipoles emit light of three different frequencies:

1) Elastic Scattering. A molecule with no Raman-active modes absorbs a photon with the frequency, f. The excited molecule returns back to the same basic vibrational state and emits light with the same frequency f as an excitation source. This type if interaction is called an elastic Rayleigh scattering.

2) Stokes Scattering. A photon with frequency f is absorbed by Raman-active molecule which at the time of interaction is in the basic vibrational state. Part of the photon’s energy is transferred to the Raman-active mode with frequency, m. The resulting frequency of scattered light is reduced to the frequency: f – m. This Raman frequency is called Stokes frequency, or just “Stokes”.

At room temperature the thermal population of vibrational excited states is low, although not zero. Therefore, the initial state is the ground state, and the scattered photon will have lower energy (longer wavelength) than the exciting photon. This Stokes shifted scatter is what is usually observed in Raman spectroscopy.

*Note: The vibrational energy is ultimately dissipated as heat. Because of the low intensity of Raman scattering, the heat dissipation does not cause a measurable temperature rise in a material.

3) Anti-Stokes Scattering. A photon with frequency f is absorbed by a Raman-active molecule, which, at the time of interaction, is already in the excited vibrational state. Excessive energy of excited Raman active mode is released, molecule returns to the basic vibrational state and the resulting frequency of scattered light is increased to f + m. This Raman frequency is called Anti-Stokes frequency, or just “Anti-Stokes”.

Thus, a small fraction of the molecules are in vibrationally excited states. Raman scattering from vibrationally excited molecules leaves the molecule in the ground state. The scattered photon appears at higher energy, as shown in Figure 1.1b. This anti-Stokes-shifted Raman spectrum is always weaker than the Stokes-shifted spectrum, but at room temperature it is strong enough to be useful for vibrational frequencies less than about 1500 cm^-1.

The Stokes and anti-Stokes spectra contain the same frequency information. The ratio of anti-Stokes to Stokes intensity at any vibrational frequency is a measure of temperature. Anti-Stokes Raman scattering is used for thermometry with no physcial contact. The anti-Stokes spectrum is also used when the Stokes spectrum is not directly observable, for example because of poor detector response or spectrograph efficiency.

Vibrational Energies  

The energy of a vibrational mode depends on molecular structure and environment. Atomic mass, bond order, molecular substituents, molecular geometry and hydrogen bonding all effect the vibrational force constant (like a spring constant) which, in turn dictates the vibrational energy. For example, the stretching frequency of a phosphorus-phosphorus bond ranges from 460 to 610 to 775 cm^-1 for the single, double and triple bonded moieties, respectively. Much effort has been devoted to estimation or measurement of force constants. For small molecules, and even for some extended structures such as peptides, reasonably accurate calculations of vibrational frequencies are possible with commercially available software.

Vibrational Raman spectroscopy is not limited to intramolecular vibrations. Crystal lattice vibrations and other motions of extended solids are Raman-active. Their spectra are important in such fields as polymers and semiconductors. In the gas phase, rotational structure is resolvable on vibrational transitions. The resulting vibration/rotation spectra are widely used to study combustion and gas phase reactions generally. Vibrational Raman spectroscopy in this broad sense is an extraordinarily versatile probe into a wide range of phenomena ranging across disciplines from physical biochemistry to materials science to forensic science.

Instrumentation

About 99.999% of all incident photons in spontaneous Raman undergo elastic Rayleigh scattering. This type of signal is useless for practical purposes of molecular characterization. Only about 0.001% of the incident light produces inelastic Raman signal with frequencies f ± m. Spontaneous Raman scattering is very weak and special measures should be taken to distinguish it from the predominant Rayleigh scattering. Instruments such as notch filters, tunable filters, laser stop apertures, double and triple spectrometric systems are used to reduce Rayleigh scattering and obtain high-quality Raman spectra.

 A Raman system typically consists of four major components:

1. Excitation source (Laser).
2. Sample illumination system and light collection optics.
3. Wavelength selector (Filter or Spectrophotometer).
4. Detector (Photodiode array, CCD or PMT).

A sample is normally illuminated with a laser beam in the ultraviolet (UV), visible (Vis) or near infrared (NIR) range. Scattered light is collected with a lens and is sent through interference filter or spectrophotometer to obtain Raman spectrum of a sample.

Since spontaneous Raman scattering is very weak the main difficulty of Raman spectroscopy is separating it from the intense Rayleigh scattering. More precisely, the major problem here is not the Rayleigh scattering itself, but the fact that the intensity of stray light from the Rayleigh scattering may greatly exceed the intensity of the useful Raman signal in the close proximity to the laser wavelength. In many cases the problem is resolved by simply cutting off the spectral range close to the laser line where the stray light has the most prominent effect. People use commercially available interference (notch) filters which cut-off spectral range of ± 80-120 cm-1 from the laser line. This method is efficient in stray light elimination but it does not allow detection of low-frequency Raman modes in the range below 100 cm-1.

Stray light is generated in the spectrometer mainly upon light dispersion on gratings and strongly depends on grating quality. Raman spectrometers typically use holographic gratings which normally have much less manufacturing defects in their structure then the ruled once. Stray light produced by holographic gratings is about an order of magnitude less intense then from ruled gratings of the same groove density. Using multiple dispersion stages is another way of reducing stray light. Double and triple spectrometers allow taking Raman spectra without use of notch filters. In such systems, Raman-active modes with frequencies as low as 3-5 cm-1 can be efficiently detected.

In earlier times people primarily used single-point detectors such as photon-counting Photomultiplier Tubes (PMT). However, a single Raman spectrum obtained with a PMT detector in wavenumber scanning mode was taking substantial period of time, slowing down any research or industrial activity based on Raman analytical technique. Nowadays, more and more often researchers use multi-channel detectors like Photodiode Arrays (PDA) or, more commonly, a Charge-Coupled Devices (CCD) to detect the Raman scattered light. Sensitivity and performance of modern CCD detectors are rapidly improving. In many cases CCD is becoming the detector of choice for Raman spectroscopy.

The intensity of Raman scattering is proportional to the fourth power of the frequency of scattered light. Thus, FT Raman instruments, with their near-infrared lasers, require higher power levels than instruments employing visible lasers. In addition, the near-infrared detectors used with FT Raman spectrometers are less sensitive than the CCD array detectors which are used with excitation instruments designed to operate solely within the confines of the visible portion of the electromagnetic spectrum of light.    

Applications in Forensic Science

Until fairly recently, the applications of Raman Spectroscopy to forensic science studies have been relatively sparse. The main reasons for this were the high backgrounds encountered in the Raman spectra of most samples due to either impurities or inherent fluorescence, the high cost of instrumentation, the difficult adjustments of sample position, the alignment of special designed optical apparatus, and the length of the experimental procedures. These, along with the more limited collections of library data, cause Raman spectroscopy to be a less attractive technique than the widely used Fourier Transform Infrared Spectroscopy (FTIR). 

More recent developments have, however, made it possible to reduce some of these limitations and to add this method to the preferred FTIR spectrometers. Continual improvements in detector, microscope, and laser design have now opened Raman spectroscopy to a much wider audience and made the technique more applicable to a routine analysis (8 – 12).

The advantages of Raman spectroscopy over IR absorption spectroscopy in the analysis of forensically significant materials have been clearly outlined. These advantages have been utilized in the rapid analysis of amphetamine-type narcotics in both tablet form and aqueous solution. The high spatial resolution (~ 1 micron) inherent in microscope-based Raman systems allows for the analysis of micron-sized particles, such as explosives and propellant residues. Trace analysis (< ppm) of narcotics and explosives has also been demonstrated by using techniques such as Surface Enhanced Raman Spectroscopy (SERS) and Surface Enhanced Resonance Raman Spectroscopy (SERRS). Furthermore, the development of fiber optic Raman probes has allowed the implementation of portable devices for in situ examination of narcotic and explosive materials.

Moreover, because of the differences between the two methods and the wide range of Raman scattering cross sections which occur for various types of compounds, Raman data for complex mixtures may serve to help identify some components not detected by IR spectroscopic methods – particularly in complex substances that include both inorganic and organic compounds having a wide range of molar concentrations.

In general, the symmetric vibrations of a molecule, which are often IR inactive or produce only weak IR absorptions, will give rise to prominent Raman peaks. Some inorganic compounds are strong Raman scatterers because of the large changes in polarizabilities which occur for their vibrations. For example, the IR absorptions of inorganic pigments are generally broad, whereas Raman peaks of inorganic compounds are usually narrow. Some organic pigments may have relatively large Raman scattering cross sections because they contain aromatic groups or highly conjugated groups having a large degree of molecular symmetry. 

In one early study, Raman spectroscopy was not only capable of distinguishing between the types of polymers used in the manufacture of broken plastic automobile lenses, but was also found sensitive to the thermal history of the item during its production. The detection of these internal stress conditions and their characterization by FT-Raman led to the differentiation of manufacturers of the same products.

In addition, the FT-Raman technique is useful in forensic document examination of paper because of its ability to identify mineral components and some of the minor components which are associated with the origin of the cellulose and technology of pulp and paper production.

Finally, reading the widely varying composition of street-seized drug samples, the vast range of possible diluents and impurities that may be present poses several problems in the area of illicit drug analysis. The primary difficulty lies in the presence of fluorescent materials that can obscure the Raman signal, making accurate measurements difficult or impossible. The practical solution is to use excitation wavelengths either in the UV or near-IR regions of the spectrum. Multiple diluents further exacerbate the problem unless the Raman signal from the diluent is weak with respect to the signal from the narcotic.

Despite these issues, Raman spectroscopy is finding a wider niche in forensic applications, given that it provides information that is complimentary to absorption IR spectroscopy and is generally non-destructive.

*Note: Raman scattering can occur with a change in vibrational, rotational or electronic energy states of a molecule. Chemists and forensic scientists, however, are concerned primarily with the vibrational Raman Effect. We have therefore used the term Raman Effect to refer strictly to the effects of atomic / molecular vibrations.

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References / Reading

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1) Raman, C.V., Krishnan, K.S.

“A Change of Wavelength in Light Scattering”

Nature (London), Vol. 21, p. 501, p. 619 (1928)

2) Colthrup, N.B. and Daly, L.H.

Introduction to Raman and Infrared Spectroscopy

3^rd Edn., Academic Press, San Diego, CA (1990)

3) Turrell, G. and Corset, J. (Eds.)
Raman Microscopy: Developments and Applications
Academic Press, London (1996)

4) Smith, E. and Dent, G.
Modern Raman Spectroscopy: A Practical Approach
John Wiley & Sons, NY, NY (2005)

6)  Smith, E., Dent, G.

Modern Raman Spectroscopy: A Practical Approach

Wiley & Sons, New York, NY (2005)

7)  Mulvaney, S.P., Keating, C.D. 

“Raman Spectroscopy”

Analytical Chem. Vol. 72, p. 145 (2000)

8)  Hendra, P.J. et al.

“Fourier Transform Raman Spectroscopy of Illicit Drugs”

J. Raman Spec., Vol. 20, p. 745 (1989)

9) Kuptsov, A.H.

“Applications of FT Raman Spectroscopy in Forensic Science” 

J. For. Sci. Vol. 39, p. 305 (1994)

10)  Miller, J., Bartick, E.

“Forensic Analysis of a Single Fiber”

Applied Spectroscopy, Vol. 55, p. 1729 (2001)

11)  Suzuki, E.M. and Carrabba, M.

"In Situ Identification and Analysis of Automotive Paint Pigments Using Line Segment Excitation Raman Spectroscopy I. Inorganic Topcoat Pigments"

J. For. Sci., Vol. 46, p.1053 (2001)

12) Ryder, A.G.

“Classification of Narcotics in Solid Mixtures”

J. For. Sci., Vol. 47, p. 275 (2002)

13) Massonnet,G.

“Evaluation of Raman Spectroscopy for the Analysis of Colored Fibers ”

J. For. Sci., Vol. 50  (2005)