Turning on the Light: Lessons from Luminescence

Chemistry for Everyone

Turning on the Light: Lessons from Luminescence

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Patricia B. O’Hara* Department of Chemistry, Amherst College, Amherst, MA 01002; *[email protected] Carol Engelson West Springfield High School, 425 Piper Rd., West Springfield, MA 01089 Wayne St. Peter Hall High School, 975 N. Main St., West Hartford, CT 06117

How many of us have walked the shores at night and been mystified by the eerie green glow of marine organisms in the surf? Perhaps you paused by a meadow on a summer evening to watch the staccato flashes of light from fireflies seeking to attract their mates. What are the energy sources for these mysterious lights that seem to glow without the input of electricity or heat? Are the phenomena that cause these cold lights the same as those responsible for the light emitted from glow-in-the-dark stickers, or emergency light sticks? Are they related to incandescent light bulbs and fluorescent lights? The goals of this article and related laboratory exercises are to give secondary school teachers a context in which all of these phenomena, broadly grouped under the heading of luminescence, can be explored and explained with their students (1). Five laboratory exercises are provided in the Supplemental MaterialW so that students can perform their own examinations of some of the underlying principles of luminescence spectroscopy in a laboratory setting. Macroscopic Energy Fluorescent jellyfish, fireflies, stickers, light sticks, fluorescent and incandescent lights, fireworks, and even shattered wintergreen hard candies all emit light when atoms and molecules in an excited energy state return to a ground energy state. Most of us are familiar with the macroscopic world in which energy is divided into kinetic and potential energy. Kinetic energy is the energy possessed by an object because of its motion, which is why mobiles are called kinetic sculptures. Potential energy is the energy “stored” in a system and is often thought of as the potential that this system has to do work owing to its position. Water tumbling over Niagara Falls has potential energy owing to gravity and, when it falls, that potential energy is converted (eventually) into electrical energy used to light our homes and cities. In that same way an individual atom or molecule can possess potential energy that can be converted into light. Here we describe some of the major processes by which light is created in these systems, and how the wonder inspired by these processes can be used to motivate secondary school students to learn some basic ideas of science. Microscopic Energy In chemical terms, energy is stored in bonds, and whenever bonds are broken or formed, there is a potential for some energy to be released and for the system to do work. The potential energy stored in gasoline is converted into mechanical energy every time you drive your car. In the world of large www.JCE.DivCHED.org

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systems and many, many particles, energy seems to be continuous: it is theoretically possible for your car to go 20, 53.5, or 74.12434 mph and every speed in between. In the world of small, small systems, at the molecular level, things are a bit different. Unlike the macroscopic world, a molecule can only exist in certain discrete energy states, which are also referred to as quantum states. The lowest of these is called the ground state, followed by a first excited state and so on, but the energy is not continuous. Therefore, it is not possible for a molecule to contain a quantity of energy that is between the ground and the first excited state, or between the first and second excited states. In addition, we find it useful to subdivide the types of energy states a molecule can have. For the purposes of this discussion, we like to think separately about the energy of a single atom or molecule as electronic (where the electrons that are part of the atom change without being removed from the atom), vibrational (where atoms bonded to one another move relative to one another), and rotational (where the whole molecule tumbles in space). The electrons in atoms and molecules can move between different energy states by either absorbing energy (moving to a higher energy state) or emitting energy (moving to a lower energy state). The energy difference between any two quantum states is fixed, and therefore the energy required to move between them is also fixed. That energy is said to be quantized. Light Energy

Incandescence and Luminescence When an atom or molecule absorbs energy, it enters an excited state. To return to its ground (or resting) state, it must find a way to divest itself of the excess energy. Sometimes the energy needed to excite or relax an atom or molecule takes the form of electromagnetic energy, or light. When light is emitted by an atomic or molecular system, photons (quanta of electromagnetic radiation) carry off the extra energy of the system. The exact nature of how the system gets excited, and what excited energy level the system is in, will determine whether the process of light emission is classified as incandescence or luminescence. Incandescent processes are always caused by heating a material until it glows. Thus ordinary light bulbs derive the energy to excite the tungsten metal from the heat produced by electricity flowing through the tungsten filament. The remainder of this article deals with processes by which light is emitted without a simultaneous change in temperature, and is classified as luminescence or cold light. Luminescent processes include triboluminescence, fluorescence (resonant emission and redshifted emission),

Vol. 82 No. 1 January 2005

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Journal of Chemical Education

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phosphorescence, chemiluminescence, and bioluminescence (2, 3). The conditions under which each of these processes occurs are summarized in Figure 1.

emission of light with heating? yes

no

incandescence

luminescence from mechanical forces? yes

no

with excitation by light?

triboluminescence yes

no

immediately? yes

inanimate? yes

no

fluorescence

no

chemiluminescence

phosphorescence

bioluminescence

Figure 1. Possible luminescent fates for an atom or molecule after electronic excitation.

A

B

iii

S1 i

ii

ii

ν4 ν3 ν2 ν1

S1

iv

S0

S0

fluorescent emission in atoms

fluorescent emission in molecules

C v

S1

T1 vi

S0 phosphorescent emission from molecules

Figure 2. What can happen after absorption of light? (A) Up arrow (i) indicates excitation light. Down arrow represents emission of light from atoms that occurs when an electron moves from state S1 to S0 and is called direct or resonant fluorescence (ii). (B) Up arrows indicate excitation with photons of increasingly short wavelengths (higher energy). Emission of light in molecules can occur as simple resonant fluorescence (ii), or alternatively, some of the excited energy can be lost through internal conversion (iii) between vibronic levels ν1, ν2, ν3, ν4 in S1 and then emitted as fluorescent light which is red-shifted (iv). (C) Up arrow indicates excitation light. Emission in some molecules shows intersystem crossing (v) that results in phosphorescence (vi).

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Triboluminescence You can visualize the process of triboluminescence by breaking a wintergreen (methyl salicylate) Lifesavers candy either with a hammer, with a pliers, or in the mouth of a volunteer in a darkened room. While this effect can be seen by cracking virtually any hard candy confection, the presence of the methyl salicylate in the sugar crystal makes for a particularly bright emission. Triboluminescence occurs when emission of light is caused by a mechanical shock or strain in certain highly ordered crystalline systems. The effect was first observed by Sir Francis Bacon in the early 1600s from the abrasion of hard sugar with a knife (4). The reasons why some systems are more prone to triboluminescence than others remain mysterious and the ultimate cause of the effect is still debated. A recent patent application cites a process for impregnating gift wrap with a triboluminescent material (menthyl 9-anthracene carboxylate) so that unwrapping the gift would be accompanied with the emission of sparks of light (5). Fluorescence Fluorescent emission is the most straightforward of the various emission processes. When atoms and some molecules are excited to higher electronic energy states (either directly by absorption of light or indirectly through some other process) they return to the ground state by emission of a photon of exactly the same energy as that used to excite them. This type of fluorescence, called resonant fluorescent emission, is shown in Figure 2A. Light in fireworks, flame tests, and stars occurs as a result of this type of resonant emission. While it is also possible to observe resonant emission in molecules, one unique feature of molecules as shown in Figure 2B is redshifted fluorescent emission of light. This occurs when the photon that is emitted from the system has a wavelength that is longer than the wavelength of excitation (lower in energy). A redshift in the fluorescence, first characterized by Sir George Stokes in 1852, occurs when the molecule that is electronically excited undergoes a two-part relaxation. In the first phase, in a process known as internal conversion, the molecule undergoes rapid thermal relaxation (1 picosecond or less than 1 trillionth of a second) down to the ground vibrational state of the still excited electronic state as shown in Figure 2B. In the second phase, the molecule emits a photon within nanoseconds (one billionth of a second) of excitation to return it finally to the ground state. The photon emitted is always of lower energy (longer wavelength or redshift) than the excitation photon because of the energy lost in the excited state. Detailed experimental protocols that illustrate these aspects of luminescence spectroscopy are provided in the Supplemental Materials.W Both absorption (transmission) and emission of light by a sample can be measured with a very simple spectrometer that can be built using directions included in the Supplemental Material.W It is possible to interface this device with a personal computer (6). In experiment 1, Differentiating Between Light Sources, stu-


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dents are introduced to the interaction of light and matter. In experiment 2, Relative Quantum Yield, common dyes are used to introduce students to quantitative transmission and fluorescence measurement in the computation of a quantum yield using this instrument. Many applications of fluorescence exist in the real world. Vanishingly small quantities of protein or nucleic acid can be detected in medical diagnostic by complexation with fluorescent dye molecules or naturally fluorescent proteins. Some detergents that claim to be “whiter than white” not only reflect incident light but also cause white light to be emitted and therefore appear whiter than the original garment. The detergent contains colorless dye molecules that absorb invisible ultraviolet light and then fluoresce white light when they absorb sunlight. Chlorophyll, one of the principal biological molecules responsible for capturing the energy of sunlight by green plants, is a fluorescent molecule. It is easily extracted from plants, and its bright red fluorescence can be observed in a darkened room by shining an ultraviolet lamp on a solution of chlorophyll and water. Instructions for this simple experiment are given in the experiment 3, Chlorophyll Fluorescence.

Phosphorescence Many students will be familiar with glow-in-the-dark stickers or stars, in which an initial “charging” of the system with a light bulb can cause material to glow for hours. What you are observing is phosphorescence, a process very similar to fluorescence but long lived (7). When light shines on a phosphorescent material, there is a slight delay in the emission of light from the sample. This delay in which the system loses energy and returns to the ground state might be as long as minutes to hours. Rocks containing the element phosphorus are naturally phosphorescent, hence the name of this process. At the molecular level, what has happened is the same kind of excited electronic state that was generated in fluorescence, however in phosphorescence, the species undergoes a further transformation before it returns to the ground state. A ground state for an atom or molecule can have either one or two electrons in each level, but if two electrons are in the same energy level, they must have opposite magnetic spins; we say that their spins are paired. In phosphorescence, as shown in Figure 2C, the excited electron changes its magnetic spin by a process known as intersystem crossing. The new state for the system in which the remaining ground-state electron and excited-state electron now have the same magnetic spin is called a triplet state. Once this happens, the molecule is still excited, and it is very difficult to get back down to the ground state because to do so would require an additional spin flip and a change in electronic energy level. Very difficult to a molecule means it might take the molecule as much as a millisecond (one thousandth of a second) to do this. Experiment 4, Understanding The Differences Between Fluorescence And Phosphorescence, enables students to get hands-on experience with these two related processes. Chemiluminescence Often light is emitted from a system without the input of electrical, mechanical, or electromagnetic energy. Here, the energy to excite the system is derived from a chemical reaction. Glow sticks, bracelets, and necklaces exhibit chemiluwww.JCE.DivCHED.org

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minescence, a process in which an inanimate system is excited by the energy of a chemical reaction (8). In these devices, two reactants are separated by a membrane. The reaction is initiated by breaking the membrane between the chambers, thereby mixing the reactants. The reaction progresses with the spontaneous emission of red, blue, violet, or green light and over time when the reaction is complete, the mixture no longer emits light. Chemiluminescence derived from the oxidation of luminol has been a staple of chemical demonstrations for introductory students in chemistry (9, 10). Luminol has gained attention from the print media and television, where it is now in widespread use at many crime scene investigations. Our procedure to use luminol to detect the presence of simulated blood (a dilute solution of bleach) via chemiluminescence is described in experiment 5, Use Of Luminol In Forensic Analysis.

Bioluminescence A related phenomenon is the emission of light from biological organisms such as fireflies and jellyfish, a process called bioluminescence. We know that the function of this light in fireflies, derived from the oxidation of luciferase, is for the female to attract a mate. In the Pacific jellyfish, Aequoria victoria, blue light is first emitted via bioluminescence through the oxidation of the protein, aequorin, and then these photons are absorbed by green fluorescent protein (GFP), which subsequently fluoresces green. It is unclear in the jellyfish whether the emission of light is intended to attract a mate or food. Molecular geneticists have devised a clever means to use the GFP fluorescence to tell whether or not they have been successful in inserting a new gene into a host organism and inducing that organism to express (make) the protein coded for by the gene under investigation. They do this by attaching the gene for GFP onto the end of the gene under investigation. If, after inserting the new gene into the host cell and inducing protein expression, the host cells glow green, then you can be sure that the experiment has succeeded (11) and you have made a transgenic organism. For teachers, two excellent resources that can be used to introduce students of biology or biochemistry to these processes are provided through the Educational Division of Bio-Rad. One educational resource kit, pGLO Bacterial Transformation Kit, provides necessary chemicals and equipment for a class of 24 students to transform harmless bacteria with the gene for GFP. With the GFP-producing colonies in hand, it is then possible, with the Green Fluorescent Protein Chromatography Kit, for a similarly sized class to perform the isolation and purification of the GFP with almost all of the supplies and chemicals provided in the kit. These kits have a high success rate for our students and complementary curricular support materials are provided from Bio-Rad. Conclusion Light emission, in all of its many forms and manifestations, has the capacity to excite the imagination of students. Some of the processes discussed here are part of the technological revolution that is taking place all around us. DNA microarrays have the capacity to use fluorescence to display thousands to tens of thousands of genes, proteins, or other markers on a tiny microchip. The presence or absence of a


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particular gene, piece of DNA or RNA, or protein is most often detected by the fluorescence of a complementary molecule that binds to the molecules of interest. Prolume Ltd., a company whose primary focus is cancer research, is exploring the use of the harmless GFP to create glow-in-the-dark drinks and food, including icing for birthday cakes. With a future that has the potential to include glowing birthday cakes, sparking gift wrap, personalized fluorescent DNA identification cards, and transgenic green animals and plants, we would be wise to educate both ourselves and our students in the background processes that make this all possible. W

Supplemental Material

Instructions for the students and notes for the instructor for the five experiments are available in this issue of JCE Online. Literature Cited 1. Harvey, E. N. A History of Luminescence; American Philosophical Society Press: Philadelphia, PA, 1957.

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2. Principles of Fluorescence Spectroscopy, 2nd ed.; Lakowitz, J. R., Ed.; Plenum Publishing Corp.: New York, 1999. 3. Weidemann, E. Ann. Phys. Chem. 1888, 34, 446. 4. Bacon, F. Advancement and Proficience of Learning; Oxford University Press: Oxford, United Kingdom, 1605. 5. Geddes, N. J.; Sage, I. C.; Rozelaar, C. F.; Mason, I. R.; Grant, H. Manufacture of Triboluminescent Materials in Paper Products for Wrapping or Gift Papers (Qinetiq Limited, United Kingdom) PCT Int. Appl., 2002, WO 2002062914. 6. Riley, S. A.; Nishimura, A. M. J. Chem. Educ. 1997, 74, 1243. 7. Lisensky, G. C.; Patel, M. N.; Reich, M. L. J. Chem. Educ. 1996, 73, 1048. 8. Carlson, R.; Lewis, S. W.; Lim, K. F. Aust. J. Chem. Ed. 2000, 14, 51. 9. Schreiner, R.; Testen, M. E.; Shakhashiri, B. Z.; Dirreen, G. E.; Williams, L. G. Chemical Demonstrations: A Handbook for Teachers of Chemistry; The University of Wisconsin Press: Madison, WI, 1983; Vol. 1. 10. Lister, T. Classic Chemical Demonstrations; Royal Society of Chemistry: London, 1996. 11. Chan, A. W. S.; Chong, K. Y.; Martinovich, C.; Simerly, C.; Schatten, G. Science 2001, 291, 309.


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Turning on the Light: Lessons from Luminescence

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