Focus
Nonlinear Raman Spectroscopy In the past decade the laser has come to be applied more and more to analytical chemistry, and it has been exceptionally important in the development of modern Raman spectroscopy. At the Conference on Lasers as Reactants and Probes in Chemistry, held at Howard University in May, Alan Eckbreth of United Technologies Research Center presented a paper on the use of nonlinear Raman spectroscopy in analysis. A paper by Mary Wirth of the University of Wisconsin—Madison, on lasers in analytical chemistry, also touched on some of the nonlinear Raman techniques. The introduction of the dye laser, which is tunable over a broad range, opened up a number of exciting new chemical applications for the nonlinear techniques. "In several instances," reported Eckbreth, "nonlinear Raman techniques are now supplanting longentrenched standard analytical approaches." In conventional (spontaneous) Raman spectroscopy, a laser is focused on a sample, and the scattered radiation is collected at right angles to the incident beam. Scattered radiation may originate from elastic collisions between laser photons and sample molecules (Rayleigh scattering), or from inelastic collisions in which the photons couple to molecular vibrations (Raman scattering), as shown in Figure 1. The Raman scattered radiation may either be red-shifted (Stokes) or blueshifted (anti-Stokes) from the original laser frequency. The frequency difference between these Raman-shifted photons and the incident laser photons corresponds to a vibrational and/ or rotational frequency of the molecule doing the scattering. Under normal conditions, more sample molecules exist in the ground vibrational state than in any of the excited vibrational states. Since an excited vibrational level must be populated for anti-Stokes Raman scattering to take 0003-2700/82/ A351-1021$01.00/0 © 1982 American Chemical Society
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place, the anti-Stokes lines are generally much weaker than the Stokes lines in the Raman spectrum. There are two serious problems with conventional Raman spectroscopy. First, there is the poor efficiency of the Raman effect. Even in favorable cases, only about 1 0 - 8 of the incident laser photons are converted to Raman signal. Second, the quantum yield for the generation of fluorescence can be as high as unity for certain molecular systems, and a fluorescence signal of even much lower magnitude will swamp the weak Raman signal. Here is where the nonlinear techniques are advantageous, both because nonlinear
Raman spectroscopy is often more sensitive than conventional Raman spectroscopy and because the nonlinear techniques can often discriminate effectively against fluorescence. Some terminology relating to Raman phenomena is defined in the box, Raman Terminology, and energy level diagrams for some of the nonlinear techniques are seen in Figure 2. The nonlinear techniques include stimulated Raman scattering (SRS), hyper Raman, stimulated Raman gain (SRG), inverse Raman scattering (1RS), coherent anti-Stokes Raman spectroscopy (CARS), and coherent Stokes Raman spectroscopy (CSRS).
ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982 · 1021 A
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Conventional (spontaneous) Raman scattering, explained Eckbreth, is a linear process in which a small frac tion of the incident photons exchanges quanta of vibrational/rotational ener gy with sample molecules and is inco herently scattered to "Stokes or antiStokes frequencies. This process is shown again in Figure 2a for reference. Stimulated Raman scattering (SRS, not to be confused with stimulated Raman gain, SRG), Figure 2b, is a nonlinear technique in which the spontaneous Raman radiation is am plified by factors as high as 10 13 (1). The amplified radiation emerges as a coherent beam coincident with the di rection of the incident laser radiation. The amplification process sets up in tense molecular vibrations in the sam ple at a frequency corresponding to the strongest Raman transition for the molecule. Although SRS has not found great utility for chemical analysis, it has found some application in extending the tuning ranges of existing lasers. For example (2), the output of a dye laser can be focused into a cell con taining hydrogen gas. Stimulated Raman scattering occurs in the cell, creating an intense beam of radiation shifted from the laser frequency by the vibrational frequency of hydrogen.
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Hyper Raman scattering is shown in Figure 2c. Eckbreth explained that this phenomenon may be thought of as Raman scattering resulting from two incident laser photons. Unlike the other nonlinear phenomena he de scribed, which are third-order pro cesses, hyper Raman is a second-order process, arising from the quadratic electric field term for the laser, known as hyperpolarizability. "The process is extremely weak and thus experimen tally difficult," said Eckbreth. "Al though more spectra are being re ported, it is unlikely ever to be com petitive with the coherent third-order processes." Stimulated Raman Gain/Loss
Figure 2. Quantum diagrams of the nonlinear Raman processes, with spontaneous Raman included as a reference. Levels shown are virtual states ( ) and ground and excited vibrational levels ( ) in the ground electronic state of the molecule. Ground vibrational levels are marked " G . " For each Raman process shown, arrows entering at left are incident photons, up arrows represent photon absorption, down arrows represent photon generation, and arrows at right are out put photons. Note conservation in the number of photons created and lost in each process (each photon is represented as an arrow), v, = laser frequency, vs = Stokes frequency, va = anti-Stokes frequency, vv= vibrational frequency. Figure courtesy of Alan Eckbreth 1 0 2 2 A · ANALYTICAL CHEMISTRY, V O L . 5 4 , NO. 9, AUGUST 1982
Stimulated Raman gain (SRG) and inverse Raman scattering (1RS) are two recently developed nonlinear techniques of analytical interest. Since they are closely related, Eckbreth rec ommended that a more general no menclature would be appropriate— stimulated Raman gainAoss. But since the loss technique was disclosed first and named inverse Raman scattering,
Focus this term persists in common usage. Essentially, the techniques are two sides of the same coin, one involving stimulated gain at a Stokes-shifted frequency, the other involving stimu lated loss at an anti-Stokes-shifted frequency. SRG (Figure 2d), explained Eckbreth, may be viewed as an in duced emission process at the Stokes frequency {vs). This gain is realized by directing two lasers into the sample, a pump laser (I>J) and a tunable probe laser. The intensity of either of these beams is monitored as the probe laser is scanned. When the frequency of the probe laser is scanned through vs, a frequency Stokes-shifted from the pump, the molecule will vibrate at the frequency vv, where νυ = νι — vs. As a result of this vibration being driven, vs gains in intensity and vt decreases in intensity. In 1RS (Figure 2e), on the other hand, the pump frequency at c; expe riences gain and the anti-Stokes-shift
ed probe laser frequency loses intensi ty as the probe is scanned through a vibrational resonance. Stimulated loss occurs at the anti-Stokes frequency. The term inverse Raman refers to the fact that, at resonance, the probe ra diation is attenuated. In spontaneous Raman spectroscopy, of course, radia tion at Raman-active frequencies would be generated in the course of the experiment. Both SRG and 1RS are coherent processes. In a Raman experiment, the scattering arises from an oscillating polarization created by the interaction of the incident laser and sample mole cules. In a coherent process, a phase relationship exists between the oscil lating polarization in different parts of the sample (2). Since this polarization is driven by a coherent incident laser, the Raman output in such a process is also coherent and highly directional. This coherent Raman output beam can then be easily separated from in-
Raman Terminology In the past few years, a series of new Raman techniques has found broad ana lytical application. The terminology devised to describe these techniques, e.g., nonlin ear, resonance enhancement, surface en hancement, and coherence, may at times seem inscrutable. The information below may help clarify the situation. Raman scattering arises from an oscil lating polarization induced in the sample matrix by the incident laser beam. Con ventional Raman scattering is a linear process, originating from induced sample polarization dependent on the first power of the electric field strength of the incident beam. Other Raman techniques that depend on polarization induced by second and higher order electric field strength terms are known as nonlinear techniques. These in clude stimulated Raman scattering (SRS), hyper Raman, coherent anti-Stokes Raman spectroscopy (CARS), coherent Stokes Raman spectroscopy (CSRS, pronounced "scissors"), stimulated Raman gain (SRG), inverse Raman scattering (1RS), photoacoustic Raman spectroscopy (PARS), and Raman-induced Kerr effect spectros copy (RIKES). The topic of resonance enhancement was discussed in detail in a 1979 REPORT in this JOURNAL (Morris, Michael D.; Wallan, David J. Anal. Chem. 1979,51,182-92 A). Resonance enhancement occurs when the frequency of the incident beam corresponds to the energy of an electronic transition of
the molecule. The enhanced Raman signals •have intensities that are 102-106 times greater than normal Raman intensities. Thus, lower limits of detection are a char acteristic of the resonance Raman tech niques. Both linear and nonlinear Raman techniques can benefit from resonance signal enhancement, though operating in the resonance region may not be feasible for all samples under study. Another form of enhancement has been observed in the spectra of a variety of ad sorbed species (such as pyridine and cya nide ions, CO, and benzoic acid) on several metal surfaces (such as Cu, Ag, and Au). Surface-enhanced Raman scattering (SERS), the technique based on this phe nomenon, is currently attracting a great deal of interest among those concerned with surface adsorption. In the analytical chemistry community there is particular interest in SERS among the electrochemists, who are interested in a better under standing of events at electrode surfaces. Coherent Raman spectroscopy is a term that refers to a series of closely related nonlinear Raman techniques in which the scattered Raman radiation emerges from the sample as a coherent beam—coherent meaning that the photons are all in phase with one another. The coherent techniques include SRS, CARS, CSRS, SRG, and 1RS. Although most of the nonlinear Raman techniques are also coherent techniques, there is one incoherent nonlinear Raman process, hyper Raman.
1024 A · ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982
coherent emissions, such as fluores cence. As mentioned above, fluorescence radiation has long been a problem in conventional Raman spectroscopy. Extensive sample purification, addi tion of quenching agents, and time resolution of Raman signals from the much slower fluorescence emission have all been employed in an attempt to ameliorate the problem, but none of these approaches can match the excel lent fluorescence rejection of the co herent Raman processes, including SRG, 1RS, and CARS. This is particu larly important for the analysis of compounds of biochemical or clinical interest, many of which have high flu orescence quantum yields. "In such systems," explained Eckbreth, "spon taneous Raman scattering yields lowquality spectra at best, whereas in verse Raman provides spectra of fairly high quality." 1RS is preferable to SRG for fluorescing samples, since the signal beam in 1RS (va) is upshifted away from intense fluorescence inter ferences. CARS/CSRS Of the various nonlinear Raman techniques, CARS has received the most attention for its applicability to chemical analysis. In CARS (Figure 2f), two laser sources, a pump (vi) and a Stokes-shifted probe (vs) are mixed and generate a coherent beam at a third frequency, v$ = 2vi — vs. Re membering (see Figure 2a) that vs = vi — v0, where va is a vibrational reso nance of the molecule, we can see that (2vi - vs) = 2vl - (PI - νυ) = vi+ νυ = va. Therefore, the frequency (va) generated in the CARS process by the mixing of the pump frequency and the Stokes-shifted probe frequency is equal to the anti-Stokes frequency for that vibrational transition. Coherent Stokes Raman spectroscopy (CSRS, pronounced "scissors") is a similar process, but here Stokes radiation is generated by the mixing of a pump frequency and an antiStokes-shifted probe frequency (Figure 2g). In a CARS experiment, a varying third frequency (v3 = 2vi — vs) gener ated by the mixing of the pump and probe frequencies appears continuous ly as the probe frequency is scanned (the probe is commonly a tunable dye laser). The power generated at this third frequency is generally weak, but is greatly enhanced when the pump and probe frequencies differ by the energy of a vibrational transition (vi — vs = νυ), and only at this point will v3 equal va.
Focus
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The basic idea in CARS/CSRS is to monitor this enhanced third frequen cy for the information it provides about the chemical system under study. Of the two techniques, CARS is by far the most popular for analytical applications, since va in CARS is in the anti-Stokes region, where fluores cence is not a problem. CARS offers the analyst a number of advantages over spontaneous Raman scattering (3,4): • CARS is a more efficient process by orders of magnitude than spontaneous Raman. Spontaneous Raman yields only one scattered photon in ~ 1 0 8 in cident photons, while CARS may yield one scattered photon in 10 2 -10 3 inci dent photons. • The radiation produced in sponta neous Raman is scattered into 4π steradians, and only a small amount of this radiation can be collected. In CARS, on the other hand, the output is coherent and laserlike. This signal can be separated from the incident ra diation and collected with an efficien cy of approximately 90%. • As mentioned above, the CARS sig nal is generated in the anti-Stokes re gion, where it is removed in frequency from fluorescence emission, whereas most of the intensity in spontaneous Raman appears at Stokes-shifted frequencies (the anti-Stokes lines are relatively weak). • In spontaneous Raman, a wide range of frequencies is generated, ne cessitating the use of a monochromator. Resolution in spontaneous Raman is then limited by the performance of this monochromator. In CARS only one frequency (va) is monitored. No monochromator is needed for detec tion, and the spectral resolution in CARS depends only on the spectral width of the exciting laser. Thus, high resolution in conventional Raman spectroscopy is ~0.1—0.01 cm - 1 , with routine scanning at 5 c m - 1 . In CARS, routine spectra are recorded at ~0.4 c m - 1 , with high resolution at 10 -3 — 10~ 4 cm" 1 (4). A serious drawback of CARS is the presence of a nonresonant background signal from the matrix that can ob scure the resonant CARS signal at low sample concentrations. In convention al CARS, this background signal has limited the sensitivity to ~ 1 % in solu tion. Two methods, polarization suppres sion and resonance enhancement, have been widely used to eliminate or mitigate the nonresonant background signals in CARS. Polarization sup pression involves appropriate adjust ment of the pump, probe, and CARS
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1026 A · ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982
detection polarization orientations. Eckbreth predicts that "polarization CARS approaches will improve detec tion sensitivities by perhaps two to three orders of magnitude" (5). Detec tion limits can also be improved con siderably by utilizing the electronic resonances of the molecule (reso nance-enhanced CARS), since greatly enhanced intensity is obtained when the pump frequency (i>/) approaches the frequency of an allowed electronic dipole transition (ve). With reso nance-enhanced CARS, detection lim its as low as about 10~ 7 M have been reported. Relative to other analytical techniques, however, this is still a fair ly high number, and CARS is thus not widely applicable for trace analysis. CARS has proven useful in a num ber of analytical applications. Eck breth spoke in detail at the conference on the use of CARS in the diagnostic probing of hostile combustion envi ronments, including flames and plas mas. Recently, attention has also been directed to the other side of the tem perature spectrum, high-resolution CARS studies of cold supersonic jets (6). Due to its coherence and upward frequency shift, CARS discriminates very effectively against fluorescence, which is incoherent. Thus, CARS is attractive for the analysis of com pounds with intrinsically high fluores cence quantum yields, including many of the biological compounds under in tense study today. CARS finds other applications in high-resolution Raman spectroscopy and as a tool for photo chemical analysis and chemical kinet ics measurements, especially on the nanosecond and picosecond time scale. Recent CARS work has included the development of liquid chromato graphic CARS detection (LC/CARS) for the identification of environmental pollutants in water (3). Stuart A. Borman
References (1) Barrett, J. J. In "Chemical Applica tions of Nonlinear Raman Spectrosco py"; Harvey, Albert B., Ed.; Academic Press: New York, 1981; Chapter 3. (2) Wright, John C. In "Lasers in Chemi cal Analysis"; Hieftje, Gary M. et al., Eds.; Humana Press: Clifton, N.J., 1981; Chapter 4. (3) Carreira, L. A. et al. In "Chemical Ap plications of Nonlinear Raman Spectros copy"; Harvey, Albert B., Ed.; Academic Press: New York, 1981; Chapter 8. (4) Harvey, A. B. Anal. Chem. 1978,50, 905-12 A. (5) Eckbreth, Alan C; Schreiber, Paul W. In "Chemical Applications of Nonlinear Raman Spectroscopy"; Harvey, Albert B., Ed.; Academic Press: New York, 1981; Chapter 2. (6) Levy, Donald H. Science 1981,214, 263-69.
