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Chapter 1
Introduction to Raman Spectroscopy in the Undergraduate Curriculum Christian S. Hamann and Matthew D. Sonntag* Department of Chemistry and Biochemistry, Albright College, 1621 N. 13th Street, Reading, Pennsylvania 19604, United States *E-mail:
[email protected].
Raman spectroscopy is a powerful tool that is utilized in many disciplines and is becoming more used in the undergraduate curriculum. In this chapter we provide a brief introduction to the history of Raman spectroscopy, provide a summary of several Raman experiments available with basic Raman instrumentation, list several of the challenges associated with introducing Raman spectroscopy into the undergraduate curriculum, and summarize the resources available within this volume for faculty who wish to implement the ideas and experiments presented herein.
Introduction The ability to observe inelastically scattered light from molecules was proposed in 1923 by Smekal (1) and first observed by Krishnan and Raman in 1928 (2) and the Nobel prize was awarded for its observation in 1930 (3). For the next several decades the Raman literature was dominated by structural and physical applications with little impact in education or chemical analysis. This was primarily due to low signal intensity, fluorescence interference, and the cost of instrumentation. The technology associated with these early Raman experiments used double grating monochromators, single channel detectors with long focal lengths (~1 meter) and broad bandwidth sources (4). These components involved large costs and maintenance to achieve reliable results. In the 1980s and 90s several technical advances made Raman spectroscopy more competitive with other analytical techniques and allowed for its incorporation into the educational curriculum and more routine chemical analysis (4). These © 2018 American Chemical Society Sonntag; Raman Spectroscopy in the Undergraduate Curriculum ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
advances include the invention of the holographic notch filter, air-cooled lasers (such as diodes), multi-channel detectors (such as charge-coupled devices), and personal computers. While infrared spectroscopy is generally the more common vibrational technique in undergraduate education, Raman spectroscopy provides complementary vibrational information. While both techniques are nondestructive and can be used for samples in the solid, liquid or gas phase, Raman requires little in the way of sample preparation. An additional advantage of Raman spectroscopy compared to IR is the ability to perform analysis through a container or via standoff detection (5, 6). This allows for safe analysis of controlled substances or other materials deemed to be safety hazards due to toxicity. Perhaps one of the most beneficial advantages of Raman spectroscopy, particularly for biological analysis, is its insensitivity to water. Water is a very weak Raman scatterer, typically much weaker than other materials, and the spectrum of water is simple, with only a few peaks leading to minimal interference with the analyte of interest. Thus, studies of analytes in aqueous solutions is straightforward. In most cases the Raman signal from the solute will be much more intense than the water, even when the water is present in great excess. These advantages, along with several technical advances discussed above, have led to widespread implementation of Raman spectroscopy as a research tool in diverse fields. This necessitates training our students in the technique as it has become an extensively utilized analytical tool across many disciplines. For example, Raman spectroscopy has been heavily implemented in industry fields such as pharmaceuticals and synthesis as a method of process control and development as well as multi-component analysis (7, 8). In the areas of art conservation and archeology, Raman spectroscopy has emerged as a valuable analytical tool based on its ability to perform non-destructive analysis, mapping capabilities, and portable instrumentation (9). Additionally, a variant, surface-enhanced Raman spectroscopy (SERS), has been utilized due to its ability to perform extractionless, non-hydrolysis analysis directly on individual art fibers. This analysis can be performed with microgram samples and also quenches any fluorescence that may be present in the sample (10). Raman spectroscopy has been used in the context of forensic science for a wide variety of analysis including gunshot residue, inks and dyes, explosives, and serology among many others (11–13). The medical community has also begun using Raman spectroscopy as a diagnostic and sensing platform. It has been utilized to detect a variety of cancers, evaluate blood cultures for pathogens, study macular degeneration, and as a surgical assistant to assess tissue health (14). Raman spectroscopy has also been used to measure glucose levels for diabetes research (15). With so many applications, the impetus to teach this technique to undergraduate students is clear. However, the challenge in introducing Raman spectroscopy for many primarily undergraduate institutions is the purchase cost of these instruments, particularly instruments capable of multiwavelength excitation. The acquisition of such instruments was supported by many years by the National Science Foundation through a variety of grant programs. These included the Instrumentation and Laboratory Improvement (ILI), the Course Curriculum 2 Sonntag; Raman Spectroscopy in the Undergraduate Curriculum ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
and Laboratory Improvement (CCLI), and the Transforming Undergraduate Education in Science, Technology, Engineering, and Mathematics (TUES) programs. These opportunities allowed many institutions to acquire Raman instrumentation. Unfortunately, the only current general program available is the highly competitive Major Research Instrumentation (MRI) program, which has a focus on research rather than curricular needs. Furthermore, many Raman instruments do not meet the cost restrictions of the MRI program necessitating purchase directly through departmental or college budgets at a time when budgets are often tight. Although recent technological advances have drastically reduced their cost, instruments capable of multiwavelength excitation remain expensive. However, single wavelength research grade instruments now have prices that are comparable to Fourier transform infrared instruments. A variety of companies (including Horiba, Renishaw, B&W Tek, Ocean Optics, among many others) have entered the market for Raman instrumentation further contributing to the affordability of these instruments. This has led to the release of portable and handheld instruments that are also capable of sensitive measurements at cost-effective prices (16, 17). With the expansion of the utility of Raman spectroscopy, multiple organizations realized the value of the technique as integral to the knowledge of a well-trained chemist, most notably, the American Chemical Society (ACS). The mission of the ACS Committee on Professional Training (CPT) (18) states that it “promotes excellence in postsecondary education and provides leadership to the ACS in the professional training of chemists (19).” Its membership includes representatives from a broad range of chemistry subdisciplines and from a broad range of institutions (colleges, universities, industry). On a periodic basis the CPT publishes a bulletin titled ACS Guidelines for Bachelor’s Degree Programs that provides details about how institutions can meet ACS certification. The most recent issue of the Guidelines was published in 2015 (20). The portion of the guidelines applicable to the goals of this ACS Symposium Series is found in Chapter 4 (Infrastructure), Section 2 (Instrumentation): Characterization and analysis of chemical systems require an appropriate suite of modern, high quality, and properly maintained instrumentation and specialized laboratory equipment that are utilized in undergraduate instruction and research (20). The only instrument specifically called for is a nuclear magnetic resonance (NMR) spectrometer, one that is actually used by undergraduate students (20). In this same section, five other categories of instruments are provided and approved programs must own and maintain instruments from four of the five categories (20). These categories include optical molecular spectroscopy (of which Raman spectroscopy is an option); optical atomic spectroscopy; chromatography and separations; and electrochemistry (20). Selections may be driven by what instruments are available and what expertise individual faculty members bring to their respective institutions. Many, to some extent, rely on the inspiration provided by publications such as the Journal of Chemical Education and the ACS Symposium Series for experiments that we tailor to our own curricular 3 Sonntag; Raman Spectroscopy in the Undergraduate Curriculum ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
needs. Central to the justification for this Volume is the observation that Raman spectroscopy is arguably the least common of the optical molecular spectroscopy techniques called for in the ACS Guidelines. Yet its profile is increasing, its applications are broad, its sample capacity almost limitless. And the education community is taking notice. This Volume of the Series focuses on the intentional incorporation of Raman spectroscopy into the undergraduate curriculum as a tool for engaging students in the study of molecular structure and function. Recent advances in instrumentation (highlighted earlier in this introductory chapter) have made hands-on Raman spectroscopy a reality for an ever-increasing number of undergraduate students. While Raman spectroscopy has much distance to cover before being thought of in the same way as such workhorse techniques as Fourier transform infrared, fluorescence, ultraviolet-visible, or NMR spectroscopy, this technique is gaining significant inroads. The existence of this Volume is a testament to that progress. Publication rates strongly indicate that advances in the availability of Raman spectroscopy instruments are having a direct impact on undergraduate chemistry education. Searching the titles, abstracts, and “anywhere” in the Journal of Chemical Education using each spectroscopy as a keyword results in the data shown in Table 1.
Table 1. Tabulation of articles appearing in the Journal of Chemical Education containing different types of spectroscopy Spectroscopy
Title
Abstract
Anywhere
infrared (Fourier transform)
207 (11)
370 (43)
2966 (304)
fluorescence
160
337
1967
Raman
66
104
951
ultraviolet-visible
6
19
871
NMR
472
1015
3699
Looking at abstracts alone, NMR spectroscopy surpasses Raman spectroscopy by a factor of 10 to 1 (1015 abstracts versus 104 abstracts). While this might be considered cherry-picking the data, this result does not come as much of a surprise. Further consideration of these “back of the envelope” numbers given to the actual content of each paper tells a more nuanced story. Figure 1 contains a summary of the publication history of the Journal of Chemical Education. The upward trend in publications related to implementing Raman spectroscopy experiments is clear! Note that Sir Raman won the Nobel Prize in Physics in 1930 so the first bracket is 1930-1939 (the Journal began publication in 1924). Also note that the 2010-present bracket shows the highest number of articles in which students actually use the instrument and this decade is not yet over. Another trend that is apparent: applied computational chemistry is not only increasing its presence in the pedagogy literature, it is being used more frequently as a complement to Raman spectroscopy. Although this introductory 4 Sonntag; Raman Spectroscopy in the Undergraduate Curriculum ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
chapter is not specifically focused on literature-mining (a study unto itself), these attention-capturing numbers underscore the convergence of the value of Raman spectroscopy in research and application, the availability of instrumentation, and implementation by the community of undergraduate educators.
Figure 1. Number of publications per decade in the Journal of Chemical Education. Raman spectroscopy continues to innovate as new variations of Raman spectroscopy continue to be developed in order to push the sensitivity, spatial resolution, and temporal resolution of the technique. It has continued to be applied to study a wide variety of problems in diverse fields. Some of these developments will be discussed in the chapters herein. As part of this introductory chapter we provide an overview of many of the techniques and experiments that can be used by both the novice and the advanced user to incorporate Raman spectroscopy into all levels of the curriculum.
The Basics of Raman Spectroscopy Raman spectroscopy is a form of inelastic light scattering where a photon excites a sample. When a photon interacts with a sample it can scatter one of two ways, either elastically or inelastically. Elastic, or Rayleigh, scattering does not change the frequency of the incoming photon and no information on the vibrational structure of the molecule is generated. The photon can inelastically scatter off the molecule either imparting or losing energy to the molecule itself. During this inelastic process the total energy of the system must remain constant, so the photon shifts to a different energy or frequency. This energy difference is equal to the energy difference between the initial and final vibrational state of the molecule. If the final state is higher in energy than the initial state, the scattered photon is shifted to a lower frequency and is deemed a Stokes shift. If the final state is lower 5 Sonntag; Raman Spectroscopy in the Undergraduate Curriculum ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
in energy the photon is shifted to a higher frequency and is termed an anti-Stokes shift (see Figure 2 for details).
Figure 2. Energy level diagram showing the states involved with Raman spectroscopy and the different types of scattering. The process involved in infrared spectroscopy is shown as a comparison. (see color insert) For the Raman effect to express itself for a molecule, that molecule must incur a change in its electric dipole polarizability during the vibrational motion. The intensity of the Raman scattering is proportional to the magnitude of the polarizability change. In a basic Raman experiment, monochromatic light illuminates and interacts with the molecules of a sample. The light is scattered by a molecule and the inelastically scattered light is used to construct the Raman spectrum, plotted as intensity versus frequency shift. This inelastic light constitutes a small fraction (~1 x 10-7) of the total light yet provides useful chemical and structural information. The Rayleigh scattering (elastically scattered light) is roughly 1000 times more intense than the inelastically scattered light and must be filtered out before reaching the detector. Basic Raman instrumentation can be either dispersive or non-dispersive. While both utilize lasers, non-dispersive instruments resemble Fourier transform infrared instruments in that they use an interferometer to obtain the Raman spectrum. In dispersive instruments, gratings are used to separate the light which is then detected on a multi-channel detector such as a charge-coupled device. Filters are used to remove the intense Rayleigh-scattered photons leaving only the inelastic scattered light to be collected. Spectra are quantitative, and the resulting data may be used to determine the number and/or ratio of molecules present in a sample (21). For liquid or solid non-fluorescent samples, basic Raman spectroscopy should be sufficient to obtain high quality spectra for analysis (22, 23). For dilute solutions, fluorescent species, or complex mixtures it may be necessary to utilize more advanced techniques. 6 Sonntag; Raman Spectroscopy in the Undergraduate Curriculum ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Enhanced Raman Spectroscopy While basic Raman spectroscopy has increased in its sensitivity due to advances in instrumentation, two main challenges remain. First, Raman cross sections are roughly 1010 smaller than the corresponding processes in infrared or fluorescence spectroscopy (24). Second, the visible light used for Raman excitation can also induce fluorescence that can easily overwhelm the Raman signal. Several different extensions of Raman spectroscopy have been developed to address these deficiencies including resonance Raman spectroscopy (25), surface- and tip-enhanced Raman spectroscopy (26–29), coherent anti-Stokes Raman spectroscopy (30, 31) and stimulated Raman spectroscopy (32), among many others. Although their mechanisms differ, they all aim to increase the sensitivity of the technique beyond the basic Raman experiment. Among these variants, surface-enhanced Raman spectroscopy (SERS) has emerged as a technique well-suited to undergraduate education due to its ease of implementation in both the classroom and laboratory setting. Several chapters within this volume detail the use of SERS in an undergraduate setting and as such, we will give a brief explanation of the technique and its benefits. For a more detailed description of SERS please see the chapters herein as well as several review articles (26, 27). SERS is a technique which exploits the electromagnetic properties of certain metals to increase the signal intensity by a factor of up to 1010 (33, 34). This is an upper limit for the possible enhancements, for the purposes of undergraduate work, the enhancement factors that are most likely to be obtained are on the order of 104 – 106. In addition to the large signal enhancement, SERS has the added benefit of quenching fluorescence due to the metal nanostructured surface. The instrumentation required for a SERS experiment is the same as basic Raman spectroscopy; however, SERS measurements require that the sample be placed on colloids or some other nanostructured metallic surface. The traditional metals used are gold, silver and copper although other metals such as aluminum can also be used (35). This enhancement is based on exciting the localized surfaced plasmon resonance (LSPR) of the surface thereby increasing the local electromagnetic (EM) field. As the local field strength increases, the total signal intensity increases as the size of the electric field to the fourth power (often shown as E4) (27). This mechanism predicts that the maximum enhancement is achieved when the LSPR frequency is coincident with the excitation frequency (36). Use of this technique has allowed a wide range of applications, all of which have been incorporated into undergraduate curricula including materials science, catalysis, art, pharmaceuticals, and can be used to study processes at the single molecule level (37–44). Practically speaking there are some limitations involved in the implementation of SERS; principally that it requires the use of a metallic nanostructured substrate. Only certain metals (primarily noble metals) possess a LSPR in the visible regime that would be suitable for SERS experiments. This limits the types of surfaces that can be utilized in a given situation. Since SERS is a surface science technique, the analyte of interest must bind or remain close (
