Raman Spectroscopy in the Undergraduate Curriculum Downloaded from pubs.acs.org by UNIV OF MICHIGAN ANN ARBOR on 11/20/18. For personal use only.
Chapter 2
Investigating the Similarities and Differences among UV/Vis, Infrared, Fluorescence, and Raman Spectroscopies through Discussion of Light–Matter Interactions Julia B. Wiester* Department of Chemistry, Saint Xavier University, 3700 West 103rd Street, Chicago, Illinois 60655, United States *E-mail:
[email protected].
Spectroscopy is an invaluable tool in chemistry and is introduced throughout the undergraduate curriculum. At the fundamental level, spectroscopy measures the interactions between electromagnetic radiation and matter. Usually, students practice measuring spectra to characterize molecules and analyze experimental results. However, too often, little emphasis is placed on the specific interactions of the electromagnetic radiation with matter, which leads to a lack of differentiation and understanding of applicability among spectroscopic techniques. Here, a discussion of scattering, absorption, and emission processes is used to assist in the understanding of different spectroscopic techniques such as infrared and UV/vis (absorption), fluorescence (emission), and Raman (scattering). The purpose is to provide instructors with different visual frameworks and exercises for the introduction of spectroscopy in the undergraduate classroom and laboratory. The intended outcome is for students to understand the experimental spectroscopic techniques at a deeper level, giving rise to improved critical thinking skills in regards to scientific processes.
© 2018 American Chemical Society
Introduction Imagine traveling on a cross-country road trip with a map or app that only allows you to see one-tenth of a mile at any given time. Although you may reach your destination, you may not actually know how you got there. In contrast, what if you were trying to travel from New York to Boston but only had a large map of the United States? Or perhaps you have a map of Chicago while navigating the downtown area, but half of the landmarks and street names are missing. These maps would not be helpful either. This example is a useful analogy for teaching: it demonstrates the importance of providing the appropriately sized map for the situation and the need for different levels of detail for the specific goal. In courses like physical chemistry and instrumental analysis, we know that mathematical analysis and the ability to solve specific and detailed chemistry problems is important in the classroom and lab, in particular when discussing spectroscopy. When teaching these topics, it is easy to get wrapped up in the details and mathematical procedures while skimming over how the quantum mechanical processes relate to the corresponding spectroscopic techniques. Too often details are the primary focus without framing the argument, or the big picture is presented but without details, making it hard to understand further. We need to continually provide context and the appropriate size “map” for the students. When discussing spectroscopy, we may not emphasize, how does light interact with the matter? This revelation is astonishing considering that spectroscopy measures this exact feature. Yet, the overarching concept of light–matter interactions are not the focus of undergraduate instrumental or physical chemistry textbooks or courses, leaving students to wonder, what is actually happening to the molecule? In order to better grasp spectroscopic methods, students must be able to differentiate between various forms of spectroscopy. When prompted to compare and contrast, students often simply define. Determining the similarities and differences between concepts requires a deeper level of understanding beyond definitions and is higher on various hierarchical learning models (1). Comparing and contrasting Raman, infrared (IR), and fluorescence requires an understanding of the different ways in which electromagnetic radiation interacts with matter. Additionally, we must focus on what is being measured when these techniques are employed in the laboratory to acquire information about molecules. In this chapter, electromagnetic radiation and the relationships between energy, wavelength, frequency, and wavenumber are first introduced. Specifically, the proportionality between variables is emphasized as well as units. Next, the different ways light interacts with matter (scattering and absorption) is discussed. This section then transitions into the different processes that occur with each interaction, relating them to energy levels. The measurement of electromagnetic radiation and how it relates to different processes is discussed. Finally, an activity comparing three different molecules using different spectroscopic methods is presented. Concept maps and visual tools are used throughout to assist students in processing the information. Concept maps are an effective tool to help show relationships between topics and an even better study tool for students to develop themselves (2). 14
Electromagnetic Radiation Before discussing how light interacts with matter, electromagnetic (EM) radiation—commonly referred to as light—must be introduced. The classical model depicts EM radiation as sinusoidal waves consisting of both electric and magnetic components. The radiation can be described using wave properties such as wavelength, frequency, velocity, and amplitude (3, 4). Indeed, experimental observations, such as diffraction due to wave interference, support the wave model of EM radiation. However, certain phenomena and observations could not be explained by the wave model. In the late 19th and early 20th centuries, physicists proposed that light could be viewed as particles or wavepackets, later called photons, which could explain phenomena such as blackbody radiation and the photoelectric effect, previously unexplained by the wave model. This led to the wave–particle duality of light and later the wave–particle duality of matter and nature in general (3, 4). As early as general chemistry, students are introduced to the wave–particle duality of nature for both matter and light. After these concepts are discussed in general chemistry, they are incorporated throughout the chemistry curriculum, but especially in quantum mechanics and instrumental analysis. Spectroscopy is best conceptualized and depicted with the photon model of light requiring quantized energy; however, light is typically described with wave properties such as wavelength and frequency. The energy of a photon is proportional to the frequency of radiation (3, 4). Due to the different ways light is measured and quantified, it is important to convert among the descriptors. Throughout introductory quantum mechanics, these conversions ideally become second nature for the students. Still, even with upper-level chemistry students, it is always worthwhile to spend time reviewing the various properties and equations used to describe EM radiation. Specifically, the relationships between properties should be emphasized. Electromagnetic radiation can be described by energy, frequency, wavenumber, and wavelength. Energy, frequency, and wavenumber are all proportional to one another, while wavelength is inversely proportional to those quantities. Wavenumber, the inverse of wavelength, is often the more difficult term for students to grasp. Wavenumber has the units of inverse length, commonly cm-1. It can be described as the number of waves in a certain distance whereas frequency is the number of waves in a given time (s-1). Equations 1, 2, and 3 demonstrate how energy (E) is related to frequency (ν), wavenumber (ν̃), and wavelength (λ). Planck’s constant is represented by h, and the speed of light is c.
The significance of conversions is especially applicable to spectroscopy, because different conventions are used as well as different units for radiation. For UV/vis spectroscopy, the wavelength of light in nm is the common measurement. 15
This energy is enough to excite a molecule to a higher electronic state. IR radiation (typically measured with wavenumbers in cm-1) has about 10 to 100 times less energy than UV/vis radiation and therefore cannot excite a molecule to an electronic state, only a vibrational energy level. Rotational energy levels are closer together than vibrational energy levels and so require even lower energy photons for excitation; these photons are in the microwave range of radiation. From these methods, it is apparent that relating units of EM radiation as well as relative energies is an important skill in spectroscopy.
Exercise 1: Energy Concept Map and Energy Conversions Energy Concept Map To conceptualize the relationships, students are encouraged to sketch a concept map either during class or as a study tool. They are to include equations, relationships, and typical units. Figure 1 is an example of a concept map depicting the relationships among the variables in eqs 1–3. It is straightforward for the students to show how energy is related to each variable; however, relating frequency, wavenumber, and wavelength to each other requires an additional step. Many students can see these relationships and equations immediately from eqs 1–3, while others choose to algebraically solve for the desired term by equating eqs 1–3 to each other.
Figure 1. Example of a concept map showing different properties of electromagnetic radiation. 16
Energy Conversions Following the concept map activity, it is important to give students relevant examples to practice converting between terms. Different spectroscopies utilize different descriptions of energy. Students often move from one spectroscopic method to another without considering what is being measured or the magnitude of the energy used. For example, UV/vis spectroscopy typically records spectral intensity as a function of wavelength in nm whereas IR spectroscopy records spectral intensity as a function of wavenumber in cm-1. Considering the units of the spectra is one part of furthering this understanding of light–matter interactions. Students are asked to convert between units for different color laser pointers including an IR laser (5). They are asked to calculate the quantities listed in Table 1 for blue, green, red, and IR light. The underlined numbers are provided for the students, and the other quantities are for the students to calculate themselves. Wavenumbers always add an extra complication because they are usually less familiar to students. It is suggested to review this conversion in particular with students. An example is provided here: Convert the wavelength of 633 nm to wavenumbers in cm-1. Wavenumber and wavelength are inversely related (eq 4):
Students know that nano- is 10-9 and centi- is 10-2, but when the unit is in the denominator, like it is for wavenumbers, it adds an extra layer of difficulty. It is encouraged to use dimensional analysis to cancel units. Additionally, some students prefer to convert unit-to-unit directly, while others prefer to pass through the base unit of meters.
After completing these conversions, students are asked to compare the visible light quantities with the IR light and prompted with the following questions to relate the energies to spectroscopy methods they may already be familiar with: Which is higher energy, visible or IR light? (Visible light is higher energy than IR.) What does the magnitude of the energy say about the relative energy states that visible or IR spectroscopy measures? (Since visible light is greater energy, it can excite species to a higher energy state than IR light can.) UV/vis spectroscopy is used to measure electronic transitions and IR for vibrational transitions: compare the relative energies of electronic and infrared excited states. (The first excited electronic state, excited by UV or visible light, is higher energy than the first excited vibrational state, excited by IR light.) Summarize the relative energies and explain why UV/vis absorption spectroscopy is used to study electronic transitions and IR spectroscopy is used to examine vibrational frequencies of molecules. (UV/vis spectroscopy utilizes UV and visible light, which has enough energy to excite molecules to a higher electronic state. IR light has a lower energy, and therefore only enough to excite a molecule to a 17
higher vibrational energy level.) For students familiar with IR functional group vibrations, what is a potential material composing the IR laser if the range of the laser is from 1700 cm-1 to 2300 cm-1? (The IR laser is a carbon monoxide laser, and the vibrational frequency of CO is 2150 cm-1 (5, 6).)
Table 1. Comparison of Laser Colors for Unit Conversion Practice Color
Blue
Green
Red
IR
Wavelength (nm)
405
532
633
5000
Wavelength (m)
4.05 x 10-7
5.32 x 10-7
6.33 x 10-7
5.00 x 10-6
Energy (J)
4.90 x 10-19
3.73 x 10-19
3.14 x 10-19
3.97 x 10-20
Energy (eV)
3.06
2.33
1.96
0.248
Frequency (s-1)
7.40 x 1014
5.64 x 1014
4.74 x 1014
6.01 x 1013
Wavenumber (cm-1)
24,691
18,797
15,798
2000
Light Interacting with Matter After a discussion of electromagnetic radiation, it is time to transition to the specific interactions of radiation with matter. Matter is composed of a collection of electric charges (protons and electrons as well as neutrally charged particles, neutrons). It is important to consider what is happening on a molecular and electronic level in order to fully understand the accompanying spectroscopies. Specifically, scattering and absorption will be discussed. Although scattering and absorption are not two mutually independent processes (7), for this discussion they will be treated as separate. Scattering The fundamental basis for scattering is based on the heterogeneity of a system, whether it is heterogeneity on an electronic, molecular, or bulk scale (7). When EM radiation is incident on matter, the electric charges are accelerated as they oscillate with the electric field and radiate EM energy, called scattering. In other words, scattering is a result of accelerated electric charges radiating light at the same energy as illumination (7–9). For a detailed analysis of scattering, instructors are recommended to review reference (7). The size of the matter and spacing of particles must be considered when discussing scattering. An analogy often used is the slab–particle analogy, where the slab describes matter with a relatively flat surface and large size. Commonly observed slab–light behavior include reflection from a smooth surface, diffraction through a prism, or transmission through a glass window. These are in essence scattering processes as well. The scattered radiation on a bulk scale interferes with the other scattered radiation so that the net result is reflection and transmission (7). Transmitted light appears to be unaltered in terms of frequency or wavelength in 18
slabs or bulk matter from the destructive interference (4). Although the frequency of transmitted light is unchanged, the speed is slowed while traveling through media, due to the retention and reemission of radiation. The ratio of the speed of EM radiation in matter compared to vacuum is the refractive index. The refractive index and incident angle contribute to the amount of light reflected, refracted, and transmitted (4). For a single particle, there is not enough destructive interference to “cancel” out the waves, so the scattering is more apparent as light is radiated in all directions from the original path (4, 7). The scattering is considered elastic when the radiated light (secondary radiation) has the same wavelength (and therefore frequency) as the incident light (4). Elastic scattering by species significantly smaller than the wavelength of radiation, such as molecules, is called Rayleigh scattering. The scattering intensity is proportional to the fourth power of frequency, so higher frequency light is scattered more (4). Indeed, this is the underlying mechanism for the blue sky (4, 7, 9). Mie scattering describes scattering from larger particles that are close to the size of the wavelength of visible light such as nanoparticles and colloids. In contrast to elastic scattering, Raman scattering describes radiated light that has quantized energy differences compared to the incident light. The energy differences between incident and scattered light correspond to rotational or vibrational energy levels within a molecule (4). For a molecule to undergo Raman scattering, it must undergo a change in polarizability during the course of vibration, and therefore molecular symmetry affects whether a vibration is Raman active. Polarizability is a measure of the deformability of a bond, or how easily the electron clouds of a molecule can be distorted (4, 8, 9). The induced polarization, P, is described by eq 5 where α is the polarizability and E is the incident electric field.
Though not the focus of this chapter, instructors are directed to reference (9) for an in-depth discussion of polarizability and symmetry in relation to Raman scattering. Figure 2a is a general depiction of scattering in which the electrons oscillate (due to the incident EM field), represented by the lighter sphere, and energy is radiated in all directions at the same energy as incident light. Transmission occurs when scattered light destructively interferes with itself so that the net result is light appearing to be unaltered, as portrayed in Figure 2b. Scattered radiation with the addition of Raman scattered light, represented by dashed lines, has a different energy compared to the incident light and is visualized in Figure 2c. In each scattering example, light is radiated in all directions by the electric charges that were displaced (accelerated), and this radiation can be considered secondary radiation (7).
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Figure 2. Visual depiction of different scattering (a–c) and absorption (d–e) processes when EM radiation (dark black arrows) is incident on a particle. In each of the scattering processes, EM radiation is incident on the particle, inducing electron oscillation (lighter sphere), and energy is radiated out at the same energy (lighter gray arrows) in all directions. When light is transmitted (b), scattered light destructively interferes with itself, so that the light appears unaltered (dark black arrows). In the case of Raman scattering (c), light of different energy is also scattered (dashed lines). Absorption (d) is depicted by fewer arrows passing through the particle, as some of the radiation is converted to internal energy of the species. Fluorescence (e) is similar because radiation is absorbed, but then radiation of a lower energy is emitted (shaded thick arrow). Absorption Depending on different properties of the material such as size, shape, refractive index, dielectric, surroundings, orientation, and others, the particle may transform incident EM radiation into other forms of energy like heat; this is called absorption (7, 8). Another way of thinking about absorption is that the radiation is attenuated or dampened upon interaction with the species (Figure 2d). Since energy is conserved, the radiation is converted to internal energy of the species, giving rise to excited translational, rotational, vibrational, or electronic energy states of the species, represented by a slightly darkened sphere in Figure 2d. For a substance to absorb radiation, the energy of the excitation photon needs to match the energy difference between the ground and excited state. Microwave radiation has wavelengths of 1 mm–1 m and is lower energy than infrared and visible radiation. However, microwave radiation has enough energy to excite molecules to an excited rotational energy state. Rotational absorption spectroscopy is useful for determining parameters such as bond lengths because the rotational frequency depends on the moment of inertia of the molecule (3). Infrared radiation has higher energy (and therefore shorter wavelengths) than microwave radiation and can excite molecules to higher energy states, such as vibrational energy states. When molecules are excited to a higher vibrational state, the amplitude of molecular vibration is increased. As the energy difference between electronic energy states is 10 to 100 times larger than vibrational energy levels, we can expect the light that excites these transitions to be 10 to 100 times 20
higher in energy. Indeed, UV/vis light is two to three orders of magnitude greater in energy than IR light and can excite electronic transitions (4). There are many possible transitions for each electronic absorption because each electronic state contains several vibrational states and each vibrational state contains multiple rotational levels. For example, ultraviolet and visible radiation can energize a molecule to any vibrational level of an excited electronic level. Once light is absorbed by a species, there are several different pathways for the species to return to its ground state, also called relaxation. Nonradiative relaxation or decay describes the return of the particle to a lower energy state without the emission of a photon. Instead, the energy is lost typically by collision with other molecules where the energy is converted to kinetic energy, leading to a small temperature increase. This is the common pathway for chromophores, molecules that absorb visible light to an excited electronic state. It is also the typical pathway for molecules that absorb microwave or infrared radiation: molecules return to the ground state via nonradiative decay (4). In contrast, radiative decay describes the process in which a molecule (which had previously absorbed energy) emits a photon in the process of returning to the ground electronic state. This is most often in the UV/vis range and at a lower energy than was incident (4). In other words, radiation is emitted during the relaxation process (10). Phosphorescence and fluorescence are two types of radiative decay and are considered emission processes. Phosphorescence is a slower process in which intersystem crossing occurs to a state of higher spin multiplicity. Since this transition is forbidden, it occurs on a slower time scale, greater than 10-5 s. Fluorescence is a relatively faster process that does not include a change of electron spin and occurs faster than 10-5 s (4). Figure 2e depicts the fluorescence process with a shaded arrow. For the examples and activities described here, fluorescence will be the only type of radiative decay discussed.
Energy Level Diagrams Schematically depicting the energy transitions for the processes described in the previous sections is a natural transition between describing the light–matter interactions and spectroscopic measurements. Scattering Energy Transitions The most common way to describe energy transitions for scattering processes involve the ever-elusive “virtual” state. The virtual state is not an energy eigenstate and therefore does not correspond to a specific discrete energy level (4, 9). As described previously, scattering is the result of the interaction of EM radiation with the electric charges of matter in which the electric charges oscillate, forming an induced dipole, which then radiates (scattered) light. This induced dipole can be described as a virtual state, or an intermediate, before the (accelerated) electric charges emit the radiation out into the surroundings. In contrast to many processes described in quantum mechanics and spectroscopy, 21
scattering is not directly quantized, as the molecule can be excited to many different “virtual” states (4). The majority of the light is radiated out at the same energy as the incoming light, and this process is considered an elastic process. Elastic scattering is depicted in Figure 3 on the left side of the diagram.
Figure 3. Energy level diagram comparing the different light–matter interactions. In this schematic, the length of the straight arrows are proportional to energy of the photon involved in the process. Raman scattering occurs when the radiation is reemitted at a different energy than was incident. Since energy is conserved, the difference in energy must be accounted for. As a result, the molecule ends in a different vibrational state than it started in. In Raman scattering, it is the difference between incident and scattered light that is quantized. The difference in radiation energies correspond to the difference of vibrational energy states. When a molecule ends in a vibrational energy level higher than it started, the scattering is referred to as Stokes scattering. Anti-Stokes scattering describes the scattered light that is higher in energy than the incident light, and as a result the molecule ends in a lower vibrational state than it started. Raman scattering (Stokes and anti-Stokes) is depicted in Figure 3 by dashed arrows, which indicate an energy different than was incident. If the energy of incident light happens to match the energy difference between the ground and excited electronic states, the virtual state correlates with an electronic excited state and amplified Raman scattering occurs. This phenomenon is known as resonance Raman scattering (4). Absorption Energy Transitions The absorption process is easier to visualize on an energy level diagram because the EM radiation excites a quantized energy state of the molecule. As seen in Figure 3, when a molecule absorbs light, it moves “directly” to the higher energy state. For UV/vis radiation, it is enough energy for the molecule to be excited to a higher electronic state. For IR radiation, the energy excites the molecule to a higher vibrational state. The molecule can nonradiatively decay back to lower energy levels (as represented by the curved arrows in Figure 3). Alternatively, the molecule can release energy in the form of a photon to return to 22
lower energy levels. This form of radiative decay is fluorescence when there is no change in electron spin states. The specific properties of molecules that cause it to fluoresce and the fluorescence intensity are discussed thoroughly in various textbooks (3, 4, 10). Figure 3 depicts the radiative decay process of fluorescence by shaded thick arrows.
Exercise 2: Compare and Contrast the Different Processes Using the energy level diagram (Figure 3), students are asked to compare and contrast the different processes: elastic scattering, Raman scattering, UV/vis absorption, IR absorption, and fluorescence emission. Specific prompts are as follows: Group the processes by scattering, absorption, and radiative decay. (IR and UV/vis are absorption processes, but differ by the excited energy state, IR to an excited vibrational state of the ground electronic level and UV/vis to an excited electronic state. Fluorescence is an emission process that follows absorption to an excited electronic state. Raman is a scattering process, in which the final vibrational state differs from the initial.) How do the initial and final states compare for the processes? (For absorption processes like IR and UV/vis, the final state is a higher energy than the initial state. For fluorescence, the final state after emission is lower in energy than the excited state. Raman Stokes scattering is similar to IR absorption in that the final state is a higher energy than the initial state.) Here they will see a similarity between Stokes Raman scattering and IR absorption in that the “final” states are both an excited vibrational state. However, by examining the processes visually with the energy level diagram, students observe that the path in which the molecule ends in the vibrational state is different. How are fluorescence and Raman similar? How are they different? (Both Raman and fluorescence involve the radiation of a photon to move to a lower energy level. They are different because fluorescence involves absorption to a quantized energy level and Raman is a scattering process in which excitation is not a quantized energy state. Additionally, the time scales are different.) This can lead back to the discussion on scattering vs absorption and also the timescale of such processes. For example, Raman scattering is on the timescale of less than 10-14 s while fluorescence is typically around 10-6 to 10-10 s (4). Finally, the proportionality of arrow length to magnitude of energy is discussed. How does arrow length compare to energy absorbed, scattered, or emitted? (The longer the arrow, the larger the energy involved in each process.) Compare the energy of photons involved in the processes by comparing the arrow length in the diagram, specifically UV/vis, IR, fluorescence, and Raman. (The arrows for UV/vis absorption are much longer than those for IR absorption, therefore higher energy photons are required for excitation to an excited electronic state compared to a vibrational energy level. The arrows for fluorescence emission are shorter than the arrows for UV/vis absorption, so the emitted fluorescence is lower energy than the UV/vis absorbed energy. Raman Stokes scattering has a shorter arrow than the incident light, so Stokes scattering is lower energy and frequency.) In a different part of this exercise, students are asked to compare each of the processes discussed previously with a person walking or jumping up a staircase. 23
Each step represents a vibrational energy level whereas each story or floor landing represents an electronic energy level, as depicted in Figure 4. This analogy allows students to see that although molecules may end in the same vibrational energy level in IR absorption and Raman spectroscopy, the manner in which it got there differed. An example prompt is: Describe how a person would move or jump on a staircase in order to represent IR absorption and Raman scattering. (In IR absorption spectroscopy, the person would simply take a single step up to a higher vibrational level (step). For Raman, the person may jump up a few stairs—not land—but come down to end on the first step.) This is also a fun way to get students up and moving during a long lecture class.
Figure 4. The energy–state staircase analogy.
Measuring the Light–Matter Interactions: Spectroscopy Finally, with an understanding of what is happening on a molecular or particle level when light interacts with matter and how this affects the energy states of the system, spectroscopy can be discussed. Spectroscopy is the measurement of light–matter interactions. Although the spectra for the different processes may appear similar at first, we have seen how different the processes actually are and will apply those differences to understand what is being measured for each spectroscopic technique. Raman Spectroscopy Raman spectroscopy usually measures the Stokes scattering of a substance, in which light is scattered at a lower energy than the incident light. The photons that are collected by the detector are from the Raman scattered light, which is a lower energy—and therefore frequency and wavenumber—than the excitation radiation, usually a laser. The difference between the incident and scattered light is quantized and corresponds to the vibrational energy levels of the molecule. This difference is known as the Raman shift. Typically, it is the signal intensity plotted as a function of the Raman shift that is displayed in Raman spectroscopy. 24
The shift represents the excited vibrational state of the molecule and therefore the vibrational mode of the molecule. The Raman shift is displayed in wavenumbers, cm-1. Since the excited state is a virtual state and not quantized, the energy of the excitation light does not have to correspond to the difference in energy levels as it does in absorption spectroscopy. Therefore, many different excitation frequencies can be used for Raman. Indeed, the benefits and properties of certain excitation frequencies for various applications and species have been discussed (11). Absorption Spectroscopy Absorption spectroscopy measures the ratio between incident radiation and transmitted radiation in order to determine the energy that is absorbed by the sample.
Equation 6 describes the relationship between transmittance (T), absorbance (A), and the power of the radiation before (P0) and after (P) passing through the sample. Transmittance is the ratio of these radiant powers (4). A large absorption for a given wavelength of light means that only a small amount of radiation was transmitted to the detector. Typically a large range of energies of EM radiation is irradiated onto the sample so that the absorption (or transmittance) is displayed as a function of wavelength or wavenumber. UV/vis absorption spectroscopy typically measures absorption as a function of wavelength in nm, while IR spectroscopy typically measures transmittance as a function of wavenumber in cm-1. Fluorescence Spectroscopy Fluorescence spectroscopy measures the photons that are emitted from an excited sample. EM radiation corresponding to the electronic absorption of the molecule excites the molecule to a higher electronic energy state, where it radiatively decays back to the ground electronic energy state, emitting a photon in the process. The intensity of emitted light is displayed as a function of wavelength (nm). The emission spectrum occurs at lower energies—higher wavelengths—than the excitation radiation. This can be visualized in Figure 3 as the molecules are excited to a higher vibrational level of the excited electronic state then nonradiatively decay to the ground vibrational state of the excited electronic state. The lifetime of these excited vibrational states is about 10-15 s compared to the longer lifetime of the excited electronic state (10-8 s) (4).
Exercise 3: Summary of Spectroscopic Measurements Here students are asked to summarize the measurements in each type of spectroscopy discussed. Specifically, what is the detector collecting? (Photons.) Considering absorption and fluorescence, how does the number of photons collected translate to the spectrum appearance and the interaction of the photons with the sample? (For absorption, if fewer photons are collected by the detector 25
at a given wavelength, then more photons are absorbed and fewer are transmitted. The spectrum would display this as a minimum in transmission mode and a maximum in absorption mode. In fluorescence, because it is the emitted photons that are measured, the more photons emitted or fluoresced, the more intense or taller the spectral peak.) In Raman spectroscopy, the position of the peaks tend to cause the difficulty. The following examples will help elucidate the relationship between the Raman peak and process that is occurring. If a Raman peak at 992 cm-1 corresponds to a ring-stretching mode of benzene (12), sketch a sample spectrum with the peak. (Students will sketch a spectrum of the Raman shift and place a peak at 992 cm-1.) What wavenumber of photon was collected by the detector? (Light of wavenumber of 992 cm-1 was not what was collected by the detector, which is the most common answer by undergraduate students.) Typically further prompts are required: Convert the laser light wavelength that went into the benzene sample to wavenumbers. (633 nm =15,798 cm-1.) Students then see the stark difference between the incident light wavenumber and the observed peak, leading them to consider another step. Using Figure 3, determine the wavenumber of the Rayleigh scattered light. (Rayleigh scattering is the same energy as incident, therefore 15,798 cm-1.) Reminding students that the displayed peak (992 cm-1) is the Raman shift and the difference between incident and scattered light, calculate the wavenumber of the Raman scattered light. (15,798 cm-1-992 cm-1 = 14,806 cm-1. Therefore, the detector collected photons with wavenumber 14,806 cm-1.) It is also a good opportunity to emphasize and review wavenumber definition: it is the number of waves in a given distance. Incident light had 15,798 waves per centimeter while Raman scattered light was only 14,806 waves per centimeter, 992 fewer waves per centimeter than the incident light. Since energy is proportional to wavenumber, the light that was Raman scattered is lower energy than the incident light, in particular an energy of 1.96 x 10-20 J (0.123 eV) lower.
Exercise 4: Prediction and Comparison of Spectra Finally, it is time to put all the processes, measurements, and techniques into practice. As discussed in the introduction, comparing and contrasting concepts is a significant step in the learning hierarchy. This exercise encourages students to compare and contrast the processes of Raman scattering, IR absorption, fluorescence, and UV/vis absorption as well as the corresponding spectroscopic techniques by predicting spectral results. The predictions can be followed by a chemical literature search to qualify predictions or can be tested experimentally in a physical chemistry or instrumental analysis lab. Most published experiments in chemical education focus on two or three techniques for a given molecule (13–17). For example, Clarke and Oprysa (18) described an experiment using a fluorometer to measure Raman and Rayleigh scattering of water simultaneously with the fluorescence of either quinine or fluorescein. Additionally, they compared the Raman shift to the IR spectrum of water. This experiment is a good exercise to examine the use of a fluorometer for scattering measurements, especially if a Raman spectrometer isn’t available. However, comparison of all four techniques 26
with the same molecules provides an interesting learning opportunity. With the availability of a Raman spectrometer, Clarke and Oprysa’s experiment can be expanded to compare Raman, IR, fluorescence, and UV/vis of three different dyes. However, it can be adapted as an in-class activity to search the chemical literature as well. The purpose of this exercise is to (1) predict and/or describe spectra (fluorescent, UV/vis, Raman, IR) qualitatively for three different fluorophores, (2) explain the transitions that are occurring for each process, and (3) find literature values for spectra, and/or measure spectra experimentally if possible. Three fluorophores are used in this activity: quinine monohydrochloride dihydrate, fluorescein, and rhodamine B. These fluorophores were chosen based on their availability in undergraduate labs and the range of emission spectra. Table 2 describes the information for each dye. Students should make these observations either via experiment or literature search and complete the table.
Table 2. Observations and Concentrations of Three Different Fluorophores Fluorophore
Solid Color
Aqueous solution color (white light)
Aqueous solution color (UV light)
Concentration (M)
Quinine
White
Colorless
Blue
3 x 10-3
Fluorescein
Orange
Yellow green
Yellow green
3 x 10-4
Rhodamine B
Dark green
Magenta
Magenta orange
3 x 10-4
For those performing lab experiments, students are instructed to make 100 mL stock solutions from the solid dyes at the concentrations specified in Table 2. They are encouraged to note the colors of the solid and aqueous solutions. Based on the colors of the solid dyes and aqueous solutions, students are asked to predict the absorption peak wavelength and the emission peak wavelength. For absorption, they can use a color wheel to predict absorption peaks. Substances appear the color that is complementary to the color absorbed. For fluorescence emission, the color emitted is the color observed.
UV/Vis Absorption Spectroscopy The first step is to acquire UV/vis spectra for each dye. The stock solutions prepared according to Table 2 should be diluted about 10-fold. Students are then asked to compare the measured λmax, the wavelength of maximum absorbance, with their predicted results. Figure 5 displays normalized absorption spectra for each of the dyes at a 10-fold dilution.
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Figure 5. UV/vis absorbance spectra for aqueous solutions of quinine (λmax 331 nm), fluorescein (λmax 477 nm), and rhodamine B (λmax 553 nm). Fluorescence Spectroscopy Before measuring the fluorescence emission, students are asked to predict the value based on the absorption spectrum and the energy level diagram in Figure 3. Since the arrows in Figure 3 for fluorescence are shorter than UV/vis absorption (due to vibrational relaxation), fluorescence should occur at lower energies, or longer wavelengths, than the absorption maxima. Therefore, quinine is expected to emit light with wavelengths higher than 331 nm (potentially in the visible range), fluorescein should emit light with wavelengths higher than 477 nm, and rhodamine B should emit light with wavelengths higher than 553 nm. To examine the fluorescence emission of the three dyes, different excitation lights were used for each dye based on its absorption spectrum, as well as 633 nm light. The 633 nm light was chosen because it is a common laser used for Raman spectroscopy, so exciting at this wavelength provides the potential to compare techniques directly. Sample spectra for the dyes are shown in Figure 6. Students observe that the emission λmax did not change for different excitation wavelengths; however, the intensity did, indicating that the excitation light was not optimized to the absorption band of the fluorophore for each spectrum. Students are asked, is the emission wavelength higher or lower than the absorption wavelength? Explain. (Fluorescence emission is at lower energy than excitation energy due to vibrational relaxation in the excited electronic state. Lower energy means a longer wavelength.) Why doesn’t the λmax change even with different excitation wavelengths? (Because the emission occurs from the ground vibrational state of the excited energy level down to the ground state. Even with higher excitation energies, the molecule undergoes nonradiative decay through the vibrational states of the excited energy level to the ground vibrational state.) Are there certain excitation wavelengths that don’t lead to fluorescence? Can a signal still be detected? Explain. (We would not expect to observe fluorescence when the molecule is excited by energies lower than required to excite to the electronic energy level. There would not be enough energy to move the molecule to the excited electronic state. However, scattering can still be observed, either Rayleigh or Raman, since the molecule would only be excited to a “virtual state,” and this would be observed at a wavelength different than fluorescence (18).) 28
How do your measured spectra for UV/vis and fluorescence compare to literature values? Explain any discrepancies. (Most literature values should be close, unless different solvents are used. This could lead to a discussion on the effect of solvents in fluorescence.)
Figure 6. Normalized fluorescence spectra of quinine (λmax 392 nm), fluorescein (λmax 527 nm), and rhodamine B (λmax 609 nm).
IR Spectroscopy Next, students are instructed to obtain IR spectra for each of the powdered samples. This allowed us to avoid the interference from water. Figure 7 shows the IR spectra for quinine, fluorescein, and rhodamine B. Certainly, a valuable exercise would be to identify the various functional groups in each dye and their corresponding vibrational frequencies. This is done widely in organic chemistry and instrumental analysis, and peak identification can be found through a literature search. However, the objective here is to observe the spectra qualitatively and compare the measurements of different techniques. For example, in the fluorescein IR spectrum (Figure 7 middle), the 1327 cm-1 stretch can be assigned to the xanthene ring C-C stretch (19). Students are asked to interpret the meaning in terms of the molecule–IR radiation interaction: What is transmitted and collected by the detector? What is absorbed? What happens when the light is absorbed? (The xanthene ring C-C stretch vibrates at a frequency corresponding to 1327 cm-1, and as result absorbs radiation with 1327 cm-1. Therefore, transmission for this wavenumber of light is decreased, as seen from the inverted peak on the IR spectrum.)
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Figure 7. Infrared spectra of quinine (top), fluorescein (middle), and rhodamine B (bottom). Raman Spectroscopy The last spectroscopic method in this exercise is Raman spectroscopy. Raman spectra may be acquired in the lab, or reference spectra can be used, since many undergraduate laboratories do not have access to Raman spectrometers. Here, the Raman shift of each of the peaks was compared to the IR spectral peaks through a literature analysis. Raman spectrometers typically automatically calculate and display the Raman shift so it correlates with an IR vibrational spectrum. Although 30
this is useful for analytical purposes, it makes it significantly more difficult for a student to conceptually understand the difference between IR absorption and Raman scattering. For this part of the exercise, students search the chemical literature for Raman spectra of quinine (20), fluorescein (19), and rhodamine B (21, 22). This activity will acclimate students to searching and reading the chemical literature to help explain experimental observations. For example, the xanthene C-C stretch of fluorescein shows a Raman shift of ~1327 cm-1 (19). Students should see that the same stretches have similar displayed wavenumbers in each spectrum. However, once again it is important to emphasize the difference in the processes. The calculation in Exercise 3 can be repeated here. With an excitation wavelength of 633 nm (15,798 cm-1), what is the wavenumber of scattered light for the xanthene C-C stretch of fluorescein? (15,798 cm-1 – 1327 cm-1 = 14,471 cm-1. Excitation light was 15,798 cm-,1 and 14,471 cm-1 was Raman Stokes scattered, so 1327 cm-1 is the shift or difference. This is where the peak appears.) What light is collected by the detector? (14,471 cm-1, the scattered light.) Once students find sample reference Raman spectra and determine what is scattered, they are asked to mark the excitation peak and Rayleigh scattering peak on the spectrum. Again, students must consider the process that is occurring for Raman scattering and what is being measured and displayed for the spectrum. Students usually correctly determine that these peaks should be at the same position, but where are they in relation to the spectral peaks displayed? This allows for several learning opportunities because of misconceptions about the displayed spectrum. For one, students may begin by converting the excitation laser light (633 nm wavelength for example) to wavenumbers. When they note that this wavenumber (15,798 cm-1) is not on the range of the display, they place it far off the page past the highest wavenumber of ~3000 cm-1. Understandably, without a full understanding or proper instruction, the wavenumber axis is mistaken for scattering frequency instead of Raman shift. This feature must be emphasized for the student: that it is the difference between excitation and Raman scattered light that is displayed. Compared to the energy level display of Figure 3, students can see that the excitation frequency is equal to the Rayleigh peak frequency and therefore corresponds to a 0 shift. In other words, 633 nm light (~15,798 cm-1) was incident on the sample, and 633 nm light (~15,798 cm-1) was Rayleigh scattered. The Rayleigh and excitation peak can be sketched in at the 0 marking on the spectrum. Following the UV/vis, fluorescence, IR, and Raman exercises, a small group discussion can be performed to summarize all findings, and the different spectra can be compared and contrasted all together. This is a good opportunity for students to see how different experimental methods provide different information about molecules. Finally, there is ample opportunity to discuss selection rules for IR and Raman. Although not the focus of this chapter, the difference in light–matter interactions between IR absorption and Raman scattering means there are different selection rules. Raman scattering occurs if there is a change in polarizability during a vibration while IR absorption occurs if there is a change in the dipole moment. The molecules used in this exercise are likely too complex 31
to investigate the nature of these selection rules thoroughly, but other simpler molecules could also be used and added to this exercise as well.
Summary Here, the basic principles of light–matter interactions were discussed as an introduction to different forms of spectroscopies. The objective was to provide students with an overview of the different processes, while still considering what is occurring on a molecular level. To transition to spectroscopy, energy level diagrams were explored so that students could visualize what could be measured in an experiment. Finally, spectroscopy was discussed with examples that compared the different methods using the same compounds.
Acknowledgments J. Wiester thanks Mark Westerhoff and Bindhu Alappat for useful discussions and assistance in editing and revisions. Additionally, J. Wiester thanks Mark Westerhoff for instrumentation assistance and help.
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