Chemistry for Everyone

Turning on the Light: Lessons from Luminescence W

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 systems and many, many particles, energy seems to be con-
been mystified by the eerie green glow of marine organisms tinuous: it is theoretically possible for your car to go 20, 53.5,
in the surf? Perhaps you paused by a meadow on a summer or 74.12434 mph and every speed in between. In the world
evening to watch the staccato flashes of light from fireflies of small, small systems, at the molecular level, things are a
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seeking to attract their mates. What are the energy sources bit different. Unlike the macroscopic world, a molecule can
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for these mysterious lights that seem to glow without the in- only exist in certain discrete energy states, which are also re-
put of electricity or heat? Are the phenomena that cause these ferred to as quantum states. The lowest of these is called the
cold lights the same as those responsible for the light emit- ground state, followed by a first excited state and so on, but
ted from glow-in-the-dark stickers, or emergency light sticks? the energy is not continuous. Therefore, it is not possible for
Are they related to incandescent light bulbs and fluorescent a molecule to contain a quantity of energy that is between
lights? The goals of this article and related laboratory exer- the ground and the first excited state, or between the first
cises are to give secondary school teachers a context in which and second excited states. In addition, we find it useful to
all of these phenomena, broadly grouped under the heading subdivide the types of energy states a molecule can have. For
of luminescence, can be explored and explained with their the purposes of this discussion, we like to think separately
students (1). Five laboratory exercises are provided in the about the energy of a single atom or molecule as electronic
Supplemental MaterialW so that students can perform their (where the electrons that are part of the atom change with-
own examinations of some of the underlying principles of out being removed from the atom), vibrational (where at-
luminescence spectroscopy in a laboratory setting. oms bonded to one another move relative to one another),
and rotational (where the whole molecule tumbles in space).
Macroscopic Energy The electrons in atoms and molecules can move between dif-
ferent energy states by either absorbing energy (moving to a
Fluorescent jellyfish, fireflies, stickers, light sticks, fluo- higher energy state) or emitting energy (moving to a lower
rescent and incandescent lights, fireworks, and even shattered energy state). The energy difference between any two quan-
wintergreen hard candies all emit light when atoms and tum states is fixed, and therefore the energy required to move
molecules in an excited energy state return to a ground en- between them is also fixed. That energy is said to be quan-
ergy state. Most of us are familiar with the macroscopic world tized.
in which energy is divided into kinetic and potential energy.
Kinetic energy is the energy possessed by an object because Light Energy
of its motion, which is why mobiles are called kinetic sculp-
tures. Potential energy is the energy “stored” in a system and Incandescence and Luminescence
is often thought of as the potential that this system has to do When an atom or molecule absorbs energy, it enters an
work owing to its position. Water tumbling over Niagara Falls excited state. To return to its ground (or resting) state, it must
has potential energy owing to gravity and, when it falls, that find a way to divest itself of the excess energy. Sometimes
potential energy is converted (eventually) into electrical en- the energy needed to excite or relax an atom or molecule takes
ergy used to light our homes and cities. In that same way an the form of electromagnetic energy, or light. When light is
individual atom or molecule can possess potential energy that emitted by an atomic or molecular system, photons (quanta
can be converted into light. Here we describe some of the of electromagnetic radiation) carry off the extra energy of the
major processes by which light is created in these systems, system. The exact nature of how the system gets excited, and
and how the wonder inspired by these processes can be used what excited energy level the system is in, will determine
to motivate secondary school students to learn some basic whether the process of light emission is classified as incan-
ideas of science. descence or luminescence. Incandescent processes are always
caused by heating a material until it glows. Thus ordinary
Microscopic Energy light bulbs derive the energy to excite the tungsten metal from
the heat produced by electricity flowing through the tung-
In chemical terms, energy is stored in bonds, and when- sten filament. The remainder of this article deals with pro-
ever bonds are broken or formed, there is a potential for some cesses by which light is emitted without a simultaneous
energy to be released and for the system to do work. The change in temperature, and is classified as luminescence or
potential energy stored in gasoline is converted into mechani- cold light. Luminescent processes include triboluminescence,
cal energy every time you drive your car. In the world of large fluorescence (resonant emission and redshifted emission),

www.JCE.DivCHED.org • Vol. 82 No. 1 January 2005 • Journal of Chemical Education 49

 Chemistry for Everyone

emission of light phosphorescence, chemiluminescence, and bioluminescence

(2, 3). The conditions under which each of these processes
with heating? occurs are summarized in Figure 1.
yes no Triboluminescence
You can visualize the process of triboluminescence by
incandescence luminescence
breaking a wintergreen (methyl salicylate) Lifesavers candy
from mechanical forces?
either with a hammer, with a pliers, or in the mouth of a
volunteer in a darkened room. While this effect can be seen
yes no
by cracking virtually any hard candy confection, the pres-
ence of the methyl salicylate in the sugar crystal makes for a
triboluminescence with excitation by light? particularly bright emission. Triboluminescence occurs when
emission of light is caused by a mechanical shock or strain
yes no
in certain highly ordered crystalline systems. The effect was
first observed by Sir Francis Bacon in the early 1600s from
immediately? inanimate?
the abrasion of hard sugar with a knife (4). The reasons why
yes no yes no some systems are more prone to triboluminescence than oth-
ers remain mysterious and the ultimate cause of the effect is
fluorescence chemiluminescence still debated. A recent patent application cites a process for
impregnating gift wrap with a triboluminescent material
phosphorescence
(menthyl 9-anthracene carboxylate) so that unwrapping the
bioluminescence
gift would be accompanied with the emission of sparks of
light (5).
Figure 1. Possible luminescent fates for an atom or molecule after
electronic excitation. 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
ν4 by absorption of light or indirectly through some other pro-
A B ν3 cess) they return to the ground state by emission of a pho-
iii ν2
S1 ν1 S1 ton of exactly the same energy as that used to excite them.
This type of fluorescence, called resonant fluorescent emis-
i ii ii iv
sion, is shown in Figure 2A.
Light in fireworks, flame tests, and stars occurs as a re-
sult of this type of resonant emission. While it is also pos-
S0 S0
sible to observe resonant emission in molecules, one unique
fluorescent emission fluorescent emission feature of molecules as shown in Figure 2B is redshifted fluo-
in atoms in molecules rescent emission of light. This occurs when the photon that
is emitted from the system has a wavelength that is longer
C than the wavelength of excitation (lower in energy). A red-
v shift in the fluorescence, first characterized by Sir George
S1 T1 Stokes in 1852, occurs when the molecule that is electroni-
cally excited undergoes a two-part relaxation. In the first
phase, in a process known as internal conversion, the mol-
vi
ecule undergoes rapid thermal relaxation (1 picosecond or
less than 1 trillionth of a second) down to the ground vibra-
S0
tional state of the still excited electronic state as shown in
phosphorescent Figure 2B. In the second phase, the molecule emits a pho-
emission from molecules ton within nanoseconds (one billionth of a second) of exci-
tation to return it finally to the ground state. The photon
Figure 2. What can happen after absorption of light? (A) Up ar- emitted is always of lower energy (longer wavelength or red-
row (i) indicates excitation light. Down arrow represents emission shift) than the excitation photon because of the energy lost
of light from atoms that occurs when an electron moves from state in the excited state.
S1 to S0 and is called direct or resonant fluorescence (ii). (B) Up Detailed experimental protocols that illustrate these as-
arrows indicate excitation with photons of increasingly short wave- pects of luminescence spectroscopy are provided in the
lengths (higher energy). Emission of light in molecules can occur
Supplemental Materials.W Both absorption (transmission) and
as simple resonant fluorescence (ii), or alternatively, some of the
excited energy can be lost through internal conversion (iii) between
emission of light by a sample can be measured with a very
vibronic levels ν1, ν2, ν3, ν4 in S1 and then emitted as fluorescent simple spectrometer that can be built using directions in-
light which is red-shifted (iv). (C) Up arrow indicates excitation light. cluded in the Supplemental Material.W It is possible to in-
Emission in some molecules shows intersystem crossing (v) that re- terface this device with a personal computer (6). In
sults in phosphorescence (vi). experiment 1, Differentiating Between Light Sources, stu-

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 Chemistry for Everyone

dents are introduced to the interaction of light and matter. minescence, a process in which an inanimate system is ex-
In experiment 2, Relative Quantum Yield, common dyes are cited by the energy of a chemical reaction (8). In these de-
used to introduce students to quantitative transmission and vices, two reactants are separated by a membrane. The
fluorescence measurement in the computation of a quantum reaction is initiated by breaking the membrane between the
yield using this instrument. chambers, thereby mixing the reactants. The reaction
Many applications of fluorescence exist in the real world. progresses with the spontaneous emission of red, blue, vio-
Vanishingly small quantities of protein or nucleic acid can be let, or green light and over time when the reaction is com-
detected in medical diagnostic by complexation with plete, the mixture no longer emits light. Chemiluminescence
fluorescent dye molecules or naturally fluorescent proteins. derived from the oxidation of luminol has been a staple of
Some detergents that claim to be “whiter than white” not only chemical demonstrations for introductory students in chem-
reflect incident light but also cause white light to be emitted istry (9, 10). Luminol has gained attention from the print
and therefore appear whiter than the original garment. The media and television, where it is now in widespread use at
detergent contains colorless dye molecules that absorb invisible many crime scene investigations. Our procedure to use
ultraviolet light and then fluoresce white light when they luminol to detect the presence of simulated blood (a dilute
absorb sunlight. Chlorophyll, one of the principal biological solution of bleach) via chemiluminescence is described in
molecules responsible for capturing the energy of sunlight by experiment 5, Use Of Luminol In Forensic Analysis.
green plants, is a fluorescent molecule. It is easily extracted
from plants, and its bright red fluorescence can be observed Bioluminescence
in a darkened room by shining an ultraviolet lamp on a A related phenomenon is the emission of light from bio-
solution of chlorophyll and water. Instructions for this simple logical organisms such as fireflies and jellyfish, a process called
experiment are given in the experiment 3, Chlorophyll bioluminescence. We know that the function of this light in
Fluorescence. fireflies, derived from the oxidation of luciferase, is for the
female to attract a mate. In the Pacific jellyfish, Aequoria
Phosphorescence victoria, blue light is first emitted via bioluminescence
Many students will be familiar with glow-in-the-dark through the oxidation of the protein, aequorin, and then these
stickers or stars, in which an initial “charging” of the system photons are absorbed by green fluorescent protein (GFP),
with a light bulb can cause material to glow for hours. What which subsequently fluoresces green. It is unclear in the jel-
you are observing is phosphorescence, a process very similar lyfish whether the emission of light is intended to attract a
to fluorescence but long lived (7). When light shines on a mate or food. Molecular geneticists have devised a clever
phosphorescent material, there is a slight delay in the emis- means to use the GFP fluorescence to tell whether or not
sion of light from the sample. This delay in which the sys- they have been successful in inserting a new gene into a host
tem loses energy and returns to the ground state might be as organism and inducing that organism to express (make) the
long as minutes to hours. Rocks containing the element phos- protein coded for by the gene under investigation. They do
phorus are naturally phosphorescent, hence the name of this this by attaching the gene for GFP onto the end of the gene
process. At the molecular level, what has happened is the same under investigation. If, after inserting the new gene into the
kind of excited electronic state that was generated in fluores- host cell and inducing protein expression, the host cells glow
cence, however in phosphorescence, the species undergoes a green, then you can be sure that the experiment has succeeded
further transformation before it returns to the ground state. (11) and you have made a transgenic organism. For teach-
A ground state for an atom or molecule can have either one ers, two excellent resources that can be used to introduce stu-
or two electrons in each level, but if two electrons are in the dents of biology or biochemistry to these processes are
same energy level, they must have opposite magnetic spins; provided through the Educational Division of Bio-Rad. One
we say that their spins are paired. In phosphorescence, as educational resource kit, pGLO Bacterial Transformation Kit,
shown in Figure 2C, the excited electron changes its mag- provides necessary chemicals and equipment for a class of 24
netic spin by a process known as intersystem crossing. The students to transform harmless bacteria with the gene for GFP.
new state for the system in which the remaining ground-state With the GFP-producing colonies in hand, it is then pos-
electron and excited-state electron now have the same mag- sible, with the Green Fluorescent Protein Chromatography
netic spin is called a triplet state. Once this happens, the Kit, for a similarly sized class to perform the isolation and
molecule is still excited, and it is very difficult to get back purification of the GFP with almost all of the supplies and
down to the ground state because to do so would require an chemicals provided in the kit. These kits have a high success
additional spin flip and a change in electronic energy level. rate for our students and complementary curricular support
Very difficult to a molecule means it might take the mol- materials are provided from Bio-Rad.
ecule as much as a millisecond (one thousandth of a second)
to do this. Experiment 4, Understanding The Differences Conclusion
Between Fluorescence And Phosphorescence, enables students
to get hands-on experience with these two related processes. Light emission, in all of its many forms and manifesta-
tions, has the capacity to excite the imagination of students.
Chemiluminescence Some of the processes discussed here are part of the techno-
Often light is emitted from a system without the input logical revolution that is taking place all around us. DNA
of electrical, mechanical, or electromagnetic energy. Here, the microarrays have the capacity to use fluorescence to display
energy to excite the system is derived from a chemical reac- thousands to tens of thousands of genes, proteins, or other
tion. Glow sticks, bracelets, and necklaces exhibit chemilu- markers on a tiny microchip. The presence or absence of a

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 Chemistry for Everyone

particular gene, piece of DNA or RNA, or protein is most 2. Principles of Fluorescence Spectroscopy, 2nd ed.; Lakowitz, J. R.,
often detected by the fluorescence of a complementary mol- Ed.; Plenum Publishing Corp.: New York, 1999.
ecule that binds to the molecules of interest. Prolume Ltd., a 3. Weidemann, E. Ann. Phys. Chem. 1888, 34, 446.
company whose primary focus is cancer research, is explor- 4. Bacon, F. Advancement and Proficience of Learning; Oxford
ing the use of the harmless GFP to create glow-in-the-dark University Press: Oxford, United Kingdom, 1605.
drinks and food, including icing for birthday cakes. With a 5. Geddes, N. J.; Sage, I. C.; Rozelaar, C. F.; Mason, I. R.; Grant,
future that has the potential to include glowing birthday H. Manufacture of Triboluminescent Materials in Paper Prod-
cakes, sparking gift wrap, personalized fluorescent DNA iden- ucts for Wrapping or Gift Papers (Qinetiq Limited, United
tification cards, and transgenic green animals and plants, we Kingdom) PCT Int. Appl., 2002, WO 2002062914.
would be wise to educate both ourselves and our students in 6. Riley, S. A.; Nishimura, A. M. J. Chem. Educ. 1997, 74, 1243.
the background processes that make this all possible. 7. Lisensky, G. C.; Patel, M. N.; Reich, M. L. J. Chem. Educ.
1996, 73, 1048.
W
Supplemental Material 8. Carlson, R.; Lewis, S. W.; Lim, K. F. Aust. J. Chem. Ed. 2000,
14, 51.
Instructions for the students and notes for the instruc- 9. Schreiner, R.; Testen, M. E.; Shakhashiri, B. Z.; Dirreen, G.
tor for the five experiments are available in this issue of JCE E.; Williams, L. G. Chemical Demonstrations: A Handbook for
Online. Teachers of Chemistry; The University of Wisconsin Press:
Madison, WI, 1983; Vol. 1.
Literature Cited 10. Lister, T. Classic Chemical Demonstrations; Royal Society of
Chemistry: London, 1996.
1. Harvey, E. N. A History of Luminescence; American Philosophi- 11. Chan, A. W. S.; Chong, K. Y.; Martinovich, C.; Simerly, C.;
cal Society Press: Philadelphia, PA, 1957. Schatten, G. Science 2001, 291, 309.

52 Journal of Chemical Education • Vol. 82 No. 1 January 2005 • www.JCE.DivCHED.org