JCE Classroom Activity: #91

Fluorescent Fun: Using a Homemade Fluorometer WM. Farooq WahabDepartment of Chemistry, University of Karachi, Karachi-75270-Pakistan; farooq.w@gmail.comIn this Activity, students investigate the fluorescence of highlighter marker ink and the principles employed in studyingfluorescent molecules using a homemade fluorometer and different colored filters.

BackgroundSome compounds glow when they are excited by light. The emission of light ceases when the exciting radiation isstopped. This phenomenon is called fluorescence. Fluorescence is different from phosphorescence (1, 2), which is theafterglow observed after the exciting radiation is stopped, e.g. in glow-in-the-dark toys. Both processes, however, re-quire light as a means of exciting the molecules. Fluorescence is a common phenomenon observed in tonic watercontaining quinine, fluorescent tubelights, and highlighter marker inks. Recently, it was discovered that flowers namedfour o’clocks fluoresce in daylight (3). Analyses based on fluorescence are becoming extremely important in detectingair and water pollutants because many organic pollutants emit light upon excitation by ultraviolet light. Fluorometersemploy intense light to excite the analyte molecules and monitor the intensity of emitted light. The intensity of theemitted light is usually proportional to the concentration of analyte present.

Integrating the Activity into Your CurriculumFluorometry has a variety of applications that range from detecting ultra-trace levels ofdrugs, to measuring carcinogens in cigarette smoke (aromatic hydrocarbons), to monitor-ing algal growth in water. This Activity helps students understand how certain moleculesinteract with light, and that fluorescence is one of the ways these molecules can lose theirexcess energy after they absorb light energy.

About the ActivityInk can be extracted into water from a fluorescent yellow highlighter marker. The inkfluoresces brilliant green in daylight. Different brands of yellow highlighters yielded ahighly fluorescent ink with a wavelength of maximum absorption at 455 nm. A brightlight source such as a 6-V flashlight or a bright white LED (4) serves as the light source.Colored cellophane sheets (gift wraps), colored squares printed on an inkjet printer transparency, or color filter paddles (4)serve as wavelength filters. The instructor may wish to record the transmission spectrum of the sheets on a scanningspectrophotometer; this will give an idea of what wavelengths are able to pass through the sheets. Related concepts andtransmission spectra of colored cellophane sheets are in an accompanying article (5). The blue component of the whitelight, allowed to pass through the blue filter, causes maximum fluorescence in the ink whereas with other colors thefluorescence is considerably less. Photos, a “More Things to Try” section that uses a manual spectrophotometer orcolorimeter, and transmission spectra of colored inkjet printer transparencies are in this issue of JCE Online.W

Answers to Questions1. If viewed at a 180o angle, we would see the transmitted light of the flashlight as well as fluorescence from the ink.

Since we are interested in seeing the light emitted by the molecules (not the light transmitted through the solution),we look at an angle where the chances of bending or scattering of the incident light are minimum, at right angles.

2. The blue color filter, which results in maximum fluorescence, corresponds to the wavelength that the fluorescentink absorbs the most. The emitted wavelengths appear green. Blue color corresponds to shorter wavelengths (highenergy) and the green color to longer wavelengths (lower energy). Note: This wavelength relationship is known asthe Stokes shift. It would be helpful to provide students with a chart relating colors to wavelength if you are able to.

3. Part of the incident light that causes fluorescence is absorbed by the first solution; the remaining transmitted lightdoes not excite fluorescence as strongly in the second solution.

4. There is a direct relation between light source intensity and the fluorescent intensity. The solution glow with theflashlight is not as bright as that caused by sunlight. Note: Real fluorometers use an extremely bright light sourcesuch as a xenon arc lamp.

References and Additional Related Activities (URLs accessed May 2007)1. O’Hara, Patricia B.; Engelson, Carol; St. Peter, Wayne. Turning on the Light: Lessons from Luminescence. J. Chem.

Educ. 2005, 82, 49–52.2. An introduction to fluorescence spectroscopy. http://homepages.wmich.edu/~rsung/files/IntroFluor.pdf3. Botany: floral fluorescence effect. http://www.nature.com/nature/journal/v437/n7057/fig_tab/437334a_F2.html4. White photon microlight (#LED-WHT) and Color filter paddles (#FIL-100) are available from Educational Inno-

vations, http://www.teachersource.com/catalog/index.html, 888/912–7474.5. Wahab, M. Farooq. Fluorescence Spectroscopy in a Shoebox. J. Chem. Educ. 2007, 84, 1308–1312.

Instructor Informationphoto by M

. Farooq Wahab

Fluorescent ink extractedinto water from a highlightermarker illuminated from thetop by a white LED (notshown).

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JCE Classroom Activities are edited by Erica K. Jacobsen and Julie Cunningham

JCE Classroom Activity: #91 Student Activity

Fluorescent Fun: Using a Homemade FluorometerHave you ever thought about how a fluorescent highlighter marker actually highlights the text? Why does the inkappear to be brilliant and glowing on the paper? Why does the glow disappear if you move the paper into the dark?When illuminated, the ink absorbs light and rapidly re-emits it, giving the text a glowing look. Thus, to see fluorescence,we need a light source to excite the electrons in the molecules to an excited electronic state. Once excited, the electronsin the molecules return to ground state and lose their surplus energy as light. You may ask why other solutions do notglow in light. This is because relatively few molecules re-emit light after light absorption; the rest lose the energy bycolliding with other molecules, which raises the temperature. You can construct a homemade fluorometer to investigatethe properties of fluorescent ink.

Try ThisYou will need: a fluorescent yellow highlighter marker; opaque cardboard box with lid such as a shoebox(27 � 17 � 9 cm or similar); 400-mL beaker; a clear, colorless container shorter than the height ofthe box, such as a 9-oz. plastic or glass beverage cup; distilled water; transparent cellophane sheets ofvarious colors (blue, green, yellow, and red; deep colors recommended for better observation); ruler;boxcutter; black tape; scissors; stapler; bright light source such as a flashlight or a white LED; two250-mL glass containers; small glass container or cuvette; dark room; coffee filter.

__1. Construct a fluorometer (see figure). Using a boxcutter, cut slits S1 (0.5 cm high, 2 cm wide) and S2 (1 cm high,2 cm wide) on adjacent sides of the box, both 3.5 cm from the bottom of the box. The edge of S1 is 6 cm from theshared edge AB (see figure); the edge of S2 is 3 cm from AB. S1 is the entrance for exciting light and S2 is forobserving the fluorescence emission. Cut circular hole C in the shoebox lid, about 5 cm wider than the diameterof your clear, colorless glass container. Cut hole C 1 cm from the side in which S1 is made and 2 cm from the S2side. Make a cardboard cover for the hole to prevent stray light from entering. Place the box lid on the shoebox andseal shut with black tape. Seal any additional cracks or seams in the box with black tape.

__2. Prepare a color (wavelength) filter. Cut two or three 4-cm squares from a sheet of colored cellophane. Stack thesquares and staple the stack together near the edges of the squares. Repeat using other colors of cellophane.

__3. Fill a 400-mL beaker with distilled water. Dip the tip of a fluorescent yellow highlighter marker in the watermultiple times until the solution becomes slightly fluorescent green in room light. Filter any particles from thesolution using a coffee filter.

__4. Prepare a table with four columns: solution (water or ink), color of exciting light (i.e. filter color), intensity offluorescence (high, low, not observable), and color of fluorescence.

__5. To a clear, colorless glass or plastic container that is shorter than the height of the box, add distilled water until theliquid is above the height of slits S1 and S2. Insert the container into hole C. Place the cardboard cover over holeC. Darken the room. Turn on a flashlight or white LED in front of slit S1 and view from slit S2. Record yourobservations in the table you made in step 4. You may see a small amount of white light due to scattering andrefraction of light by the container.

__6. Repeat step 5, but replace the distilled water with highlighter ink solution from step 3. Are the results different?Turn off the flashlight. How does the solution appear? Now, place a filter from step 2 in front of the excitation slit(S1). Turn on the flashlight in front of the filter. Observe the emitted light at the emission slit. Record yourobservations. Repeat for the other color filters. Which filter color results in maximum fluorescence?

__7. Outside of the shoebox fluorometer, fill two 250-mL glass containers with the highlighter ink solution fromstep 3. Allow a flashlight beam to pass through one container and into the other. View the solutions from the top.Compare the appearance of the two solutions.

__8. Place 10 mL of ink extract in a small glass container. Add enough distilled water to make it appear colorless.Darken the room. Shine the flashlight onto the solution, observe, then turn off the flashlight. Bring the dilutedink solution into full sunlight. Record your observations. (It may help to focus sunlight onto the solution using amagnifying glass.)

Questions1. Why should the excitation slit (S1) be at a 90o angle to the emission slit (S2)? What is the difficulty with using a 180o

angle?2. What is the relationship between the energy of the excitation wavelengths compared to the emitted wavelengths?3. In step 7, how does the appearance of the two solutions differ? What could be the reason for this?4. What is the relationship between the intensity of exciting light and the intensity of fluorescence based on step 8?

Information from the World Wide Web (accessed May 2007)Fluorescence microscopy: basic concepts in fluorescence. http://micro.magnet.fsu.edu/primer/techniques/fluorescence/

fluorescenceintro.html

1312B Journal of Chemical Education • Vol. 84 No. 8 August 2007 • www.JCE.DivCHED.org

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