NERY, A.L.P. ; LIEGEL, R. M. ; FERNANDEZ, C. Fluorescence and Chemiluminescence: Teaching Basic...

10/07/2015 Chemical Education Journal(CEJ), Vol.13, No.2/Registration No.1322

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Chemical Education Journal (CEJ), Vol. 13, No. 2 /Registration No. 1322 /Received October 3,2009.URL = http://chem.sci.utsunomiyau.ac.jp/cejrnlE.html

Fluorescence and Chemiluminescence:Teaching Basic Principles by Simple Demonstration

Experiments

Ana Luiza P. Nery1, Rodrigo M. Liegel2 and Carmen Fernandez3 [*] 1 Escola Vera Cruz; Rua Baumann, 73, Sao Paulo SP, 05318000 Brazil.

2 Colegio Santa Cruz; Av. Arruda Botelho, 255, Sao Paulo SP, 05466000 Brazil.3 Instituto de Quimica, Universidade de Sao Paulo, 26077 Sao Paulo SP, Brazil.

email: carmen iq.usp.br

Keywords: curriculum; demonstrations; excited states; luminescence; photochemistry; UVVisSpectroscopy

Contents

Abstract1. Introduction2. Materials and Equipment3. Description of the Experimentsa) Chlorophyll fluorescenceb) Brown eggshell fluorescencec) Riboflavin (B2 vitamin) fluorescenced) Tonic water fluorescencee) Optical Whitenersf) Chemiluminescence: Light sticks emissiong) Producing a light stick4. Final Remarks5. Safety6. Acknowledgements7. References

Abstract

The phenomenon of luminescence generated either by a chemical process (chemiluminescence) or byan external source of energy (fluorescence and phosphorescence) is an important analytical tool.Many biological substances emit characteristic fluorescence. Chlorophyll from leaves emits redfluorescence; protoporphyrin IX, found in the brown chicken eggshell and riboflavin (B2 vitamin)also exhibit characteristic fluorescence emission. Substances present in detergents (optical whiteners)are responsible for the fluorescence observed in white clothes from people dancing under black light.

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Light sticks, objects used by kids, divers and campers owe their chemical ground to thechemiluminescence. Although the relevance of these phenomena, most chemistry curricula omitdiscussion about their photophysical principles, even though include some treatment of lightabsorption and spectroscopy. In general, the photophysical principles are left to graduate studies. Weconsider that, due to the visual appeal of these phenomena, it is possible to discuss basicphotophysical principles in undergraduate level and, thus, we are suggesting simple demonstrationexperiments in this paper.

1. Introduction

The phenomenon of luminescence is frequently omitted from most undergraduate chemistryclasses, even though chemistry curriculum includes some treatment of light absorption andspectroscopy. On the other hand, due to its strong visual appeal, we consider that luminescentphenomenon could be used as a good strategy to teach basic photophysical principles even atundergraduate level. Moreover, luminescence is an important analytical tool, in particular forbiomedical applications since luminescent probes can be used as sensors for chemical environment(Leiner, 1991, Lakowicz, 2006). This paper describes some simple demonstration experimentsinvolving the phenomenon of luminescence generated either by a chemical process(chemiluminescence) or by an external source of energy (fluorescence and phosphorescence) in orderto promote a discussion about photophysical principles in classroom.

Light that is incident on a colored sample is partly absorbed by it. The first step of a chemicalor a physical process induced by light involves absorption of a quantum of light by a molecule,producing an electronically excited state. In this initial step the molecule absorbs a discrete amount oflight, which is enough to promote an electron to a higher energy level; the photon must contain aquantity of energy that exactly matches the energy of the electronic transition. The molecule is said togo from the ground to an excited state. Once in the excited state, the molecule has several availablepathways to release the absorbed energy. The result of the absorption may appear as heat or light.Following light absorption, different processes may occur: (i) nonradiative decay: the process ofvibrational relaxation, in which the excess energy is transferred to the surroundings as thermal motionof the environment (heat); (ii) fluorescence or phosphorescence in which a photon of the same orlower energy is emitted while the molecule returns to the ground state; (iii) chemical change, whenenergy results in changes in bonding structures, or a combination of these. The total energy change ofthe processes is always exactly equal to the energy of the photon that was absorbed.

The photophysical processes occurring from absorption to emission are often shown in a socalled Jablonski diagram, which is a simplified representation of the relative positions of electronicand vibrational energy levels of a molecule (Figure 1). Of course all possible energy routes cannot beencompassed in a single figure, and different forms of the diagram can be found in different contexts.



Figure 1. Simplified Jablonski diagram. The sequence of events leading to fluorescence andphosphorescence are shown. S0 is the ground state, S1 and S2 are excited singlet states; T1 is anexcited triplet state. 0, 1, 2 represent vibrational levels. Straight lines represent transitions involvingphotons, dotted lines represent vibrational or thermal transitions.

Luminescence is defined as light emission in the visible range (400 to 700 nm) of theelectromagnetic spectrum as a result of an electronic transition. Lightinduced luminescence can be oftwo types: fluorescence or phosphorescence. Radiative transitions involve light emissions. Radiationis called fluorescence if spin multiplicities both in ground and excited state are the same.

Absorption of a photon (hA in figure 1) populates the vibrational levels of S1 (or S2). Thisprocess is very fast and happens within 1015 s. In the next 1012 s the molecule relaxes to the lowestvibrational level of S1, a process called internal conversion. Since emission typically occurs after 109

s the molecule is fully relaxed at the time of emission, hence, as a rule, emission occurs from thelowest vibrational level of S1 (KashaLs rule) and the fluorescence spectrum is generally independentof the excitation wavelength. As a consequence, light emission accompanying decay of excitedmolecules is always at longer wavelength (lower energy) than that of the absorbed light. Afteremission (hF in Figure 1) the molecule returns to the ground state, possibly after vibrationalrelaxation. This completes the simplest case of fluorescence: excitation, internal conversion, emissionand relaxation. On the other hand, phosphorescence involves excited and ground states with differentspin multiplicities. In this case, after excitation to a vibrational level of S2, for example, anintersystem crossing (ISC) to an electronic triplet excited state occurs. As a result, in contrast tofluorescence, in the phosphorescence phenomena light is emitted over a longer period of time sincethe excited molecule undergoes a transition to a different excited state; a metastable state. Metastablemeans that the electronic state is not completely stable, but it is also not particularly unstable either.Thus, not all electrons in the metastable state will return to the ground state at once. The radiativedecay (hP in Figure 1) from the triplet state is a slow process:the return to the singlet ground state isspinforbidden. As the electrons of a phosphorescent molecule return to the ground state the moleculecontinues to emit light. Perhaps an easy way to identify fluorescence or phosphorescence is if you cansee the emission after the illuminating source is removed, it is phosphorescence. If the emission stopswhen the illuminating source is removed, it is fluorescence. Phosphorescent objects should be familiarto almost everybody: stickon stars, frisbees, cereal box goodies, watch faces, posters and so on.

Many ordinary materials such as green vegetables, tonic water, detergents, and brown



eggshells, contain substances that may emit fluorescence under determined conditions. This paperdescribes experiments for teaching photochemistry in an undergraduate chemistry class, with the aimto discuss some photophysical processes, relating the observed properties of fluorescent compounds toenergy levels diagrams and to absorption and emission spectra. Another important fact to be pointedout refers to the use chemical energy, besides electromagnetic energy, as excitation source.

2. Materials and Equipment

Black light source: UVA light (315 400 nm)Mortar and pestleLight sticks (different colors)Ethyl acetateHydrochloric acid 0.1 mol L135 spinach leavesBrown eggsMultivitamin tabletTonic waterDetergent

3. Description of the Experiments

For the following experiments all background light must be turned off and the room must be asdark as possible. The UV source must be kept in a box with a frontal slit to avoid students to lookdirectly to the light, which may cause retina damages. It is important to compare the observed colorsunder visible and UV excitation, mainly in the case of chlorophyll, since its solution is green undervisible light and emits in the red range of the visible spectra under UV excitation.

a) Chlorophyll fluorescence

Chlorophyll, a natural pigment found in green plants is an example of a fluorescent compound.It is the most important light absorbing pigment in thylakoid membranes in the chloroplasts (Nelsonand Cox, 2000). Chloroplasts of higher plants always contain chlorophyll a and b. The structure of thechlorophyll molecules consists of several conjugated nitrogencontaining rings surrounding amagnesium ion by coordinate covalent bonds (Figure 2). This structure resembles the protophorphyrinof the heme group of hemoglobin; one striking difference between chlorophyll and the heme group ofhemoglobin (besides other structural differences) is that in chlorophyll a Mg2+ ion occupies thecentral position, instead of the Fe2+ ion that is present in hemoglobin.

chlorophyll a chlorophyll b



Figure 2 . Structural formulas of chlorophyll a and chlorophyll b.

Although both forms of chlorophyll are green, their absorption spectra are sufficientlydifferent to allow them to complement each otherfs range of light absorption in the visible region. Thegreen color is due to absorbance range, which maxima are at 412 nm (blue region) and 666 nm (redregion) for chlorophyll a (Figure 3).

Figure 3 . Absorption spectrum of a chlorophyll a solution.

Comparing the color of chlorophyll solution under visible light and UV irradiation is an



excellent opportunity to discuss light absorption and emission. Blue and red light absorption bychlorophyll (see Figure 3) results in a green solution (solution reflects green color). In the absence ofvisible light, under enough energy to promote an electronic transition red fluorescence is observed.Chlorophyll fluorescence occurs in the red region of visible light, between 675 and 685 nm.Fluorescence is not observed when the solution is concentrated, since the red emission is reabsorbedby the solution. The drastic change of color in the case of chlorophyll is an experiment that has abeautiful visual appeal, motivating students to pay attention to phenomena related to light absorptionand emission.

Why does not chlorophyll fluoresce under visible light? As a matter of fact, it does. However,the higher intensity of the emitted green light does not allow the red fluorescence to be observed. Ifthe solution were irradiated in a dark room with blue light, fluorescence emission would be observed.

Procedure: Grind fresh spinach leaves with a mortar and a pestle in about 50 mL of ethyl acetate.Transfer the green solution to a beaker. Irradiate it with the black light. If no fluorescence is observed,dilute the solution with more ethyl acetate and irradiate again. If the solution is too concentrated, nofluorescence can be observed, since absorption and emission spectra overlap and fluorescencereabsorption may occur.

b) Brown eggshell fluorescence

Protoporphyrin IX, the intermediate in the biosynthesis of heme group of hemoglobin, shows avery stark fluorescence under UV light excitation. In contrast to molecules with the heme group, inprotophorphyrin IX structure there is no central metallic atom (Figure 4).

Protoporphyrin IX

Heme group

Figure 4 . Structural formulas of protoporphyrin IX and heme group.

Protophorphyrin IX is found in some bones and mollusk shells. It is responsible for the brown



color of chicken eggshells. If an eggshell were irradiated with black light, a very weak fluorescencewould be observed. However, after treatment of the eggshell with hydrochloric acid in ethyl acetate,one can immediately observe the formation of bubbles on the surface of the egg. The bubbles arecarbon dioxide resulting from the reaction of the CaCO3 in the eggshell with hydrochloric acid:

CaCO3(s) + 2 HCl (aq) CaCl2 (aq) + CO2(g) + H2O (l)

As the eggshell reacts, protoporphyrin IX is liberated to the solution, and under UV light onecan observe a peculiar spectacle of the gradual increase of the fluorescence emission from thesolution. The previous very week fluorescence of the intact eggshell disseminates through the solutionas an intense purplish emission (Brandl, 1998).

Procedure: Wash a brown eggshell and irradiate it with black light. Put the eggshell in a beaker, addabout 50 mL of ethyl acetate and irradiate again. Add some drops of HCl 0.1 mol L1 to the solution,irradiate again and observe fluorescence during gas liberation.

c) Riboflavin (B2 vitamin) fluorescence

Riboflavin (Figure 5), or vitamin B2, is a fluorescent compound found in many foods, such asmilk and eggs and also on multivitamin tablets. Under UVA excitation, its fluorescence is observedby naked eyes as an intense greenyellow emission (Chatellier and White, 1988).

Figure 5 . Structural formula of riboflavin.

Procedure: Grind a multivitamin tablet with a mortar and a pestle. Transfer the solid to a beaker andadd about 50 mL of water. Irradiate it with black light.

d) Tonic water fluorescence

May be one of the most known fluorescent ordinary products is tonic water, whose activeingredient is quininium cation (Figure 6).



Figure 6 . Structural formula of quininium cation.

Quinine is added to the beverage as the hydrochloric or sulfuric acid derivative, giving itsbitter flavor. Quinine is found in the root, bark and branches of cinchonas trees, native to SouthAmerica and has been used as a treatment for malaria for hundreds of years. It does not cure thedisease, but does help to suppress parasitic activity and thus reduce lifethreatening fever ( Krettli etal., 2001). It is a strongly fluorescent compound in dilute acid solution emitting at ca. 450 nm and isused as an important fluorescence standard in chemistry (Pandey et al., 1999). Quinine solutions arecolorless, which indicates that it has no meaningful absorption in the visible range and that excitationmust necessary occur at UV region of the electromagnetic spectrum, within its absorption bands at250 and 350 nm.

When NaCl is added to the solution, the fluorescence emission is reduced. This phenomenon,called fluorescence quenching, can be explained based on the Jablonski diagram. A quinine moleculein its excited electronic state can be deactivated by a collision with a chloride ion before it has achance to return to its ground state by the emission of a photon. The chloride ion acts as afluorescence quencher, reducing the fluorescence quantum yields. The fluorescence quenching ofquinine can be related to the halide concentration, and can be used for the sensing of chloride ions foranalytical and clinical purposes (Martin and Narayanaswamy, 1997).

Procedure: Pour tonic water into two jars and turn on black light. Add some NaCl to one of the jarsand compare both samples. Discuss the observed phenomena: fluorescence and quenching on basis onthe Jablonski diagram.

e) Optical Whiteners

Another simple fluorescent experiment to be demonstrated is shining black light onto whiteclothes. The observed fluorescence is due to the presence of optical whiteners in detergents; a class oforganic compounds that have very specific fluorescent properties. They have excitation maxima in thenear UV (generally 340 400 nm) and emit in the 430 460 nm wavelength range, generally peakingaround 440 450 nm.



Procedure: Put some detergent crystals under the black light and observe the fluorescence. Add thedetergent into water and irradiate again.

f) Chemiluminescence: Light sticks emission

Many chemical reactions produce both light and heat. In these cases, the energy source thatcauses electrons to move from the ground to the excited state comes from the chemical reaction. Aburning candle is such a reaction. When a candle is lit, its flame both glows and becomes hot. It ismuch less common for a chemical reaction to produce light without heat. Such reactions, whereenergy is given off as visible light, are called chemiluminescent reactions. Chemiluminescence isdefined as the production of light energy from a chemical reaction without the use of heat, for thisreason it is also known as "cold light". When light emission derives from living organisms thephenomena is called bioluminescence. Chemiluminescence is the chemical ground of light sticks,these objects that, besides wonder kids, are used by trickortreaters, divers, campers, and fordecoration and fun. A CYALUMER (trademark of American Cyanamid Company) light stick is aplastic tube divided into two compartments. Although there is more than one recipe for a light stick, acommon commercial one uses a solution of hydrogen peroxide kept in one of the compartments and asolution of a phenyl oxalate ester, for example bis(2,4dinitrophenyl)oxalate (DNPO) or bis(2,4,6trichlorophenyl)oxalate (TCPO), together with a fluorescent dye (Fl) (Shakhashiri et al., 1981).

When a light stick is bent, the glass vial breaks, and the chemicals that are inside the glass mixwith those in the plastic tube, allowing the chemical reaction to take place. The basic premise is thatthe reaction between the two chemicals releases enough energy to excite the electrons in thefluorescent dye (Fl). Scheme 1 shows chemiluminescent decomposition of TCPO (Mohan and Turro,974):

Scheme 1 :

The emission of the fluorescent dye (Fl) is what determines the observed color as product ofthe reaction (Table 1).



Table 1: Fluorescence emission of selected dyes.

dye color9,10diphenylanthracene blue

9,10bis(phenylethynilanthracene) greenRubrene yellow

Rhodamine 6G orangeRhodamine B red

Mechanistic studies indicate the occurrence of a Chemically Initiated Electron ExchangeLuminescence (CIEEL) mechanism (Schuster, 1979), with the formation of a 1,2dioxetanedione asintermediate, which transfers energy to the dye molecule. The dye molecule acts as a catalyst of theprocess. Light emission is observed when the excited molecule of the dye (Fl*) returns to the groundstate (Stevani and Baader, 2000). This mechanism suggests that if the dye molecule (Fl) can beexcited by some other energy source (UV light for example), the same light emission should result,even after reagents were totally given up. Salter et al. (1999) have observed that light sticks ofdifferent colors show very similar chemiluminescence and laserinduced fluorescence spectra.

Procedure: Irradiate different color light stick. Observe and discuss the observed emission. Breaksome of them. Discuss different kinds of excitation source: light and chemical reaction. Irradiate alight stick whose chemicals have already been given up.

g) Producing a light stick

Light stick reaction may be produced in class. In this case it is possible to produce other colorsby using different dyes, for example chlorophyll solution, allowing comparison of two differentprocesses: photophysical (fluorescence) and chemiluminescence. In a beaker containing about 100 mLof ethyl acetate, add 10 mL of 2.7 mol L1 H2O2 aqueous solution and about 50 mg of a fluorescentdye. Add ca. 5 mg of TCPO or DNPO, turn off the lights and observe.

4. Final Remarks

Basic notions of quantum chemistry, quantization of molecular energy levels and spectroscopymay help students to understand some everyday phenomena. The discussion of experiments like thosesuggested in this paper may be useful for the process of giving meaning to the surrounding worldthrough chemical knowledge. The examples here discussed are a very effective pedagogical tool forthe demonstration of two chemical phenomena involving light: fluorescence and chemiluminescence.Such phenomena have been widely used as analytical tools, mainly in biomedical analysis, due totheir high sensibility.

5. Safety



Ultraviolet light is potentially damaging to exposed skin and especially eyes. Do not stare atthe blacklight source. Ethyl acetate is flammable. Hydrochloric acid is corrosive, can cause severeburns to the skin in its concentrated form, and should be handled with care. There are no significanthazards associated the fluorescent compounds.

6. Acknowledgements

The authors acknowledge financial support from FAPESP, CAPES, CNPq, Dow ChemicalCompany and ProReitoria de Pesquisa (Universidade de Sao Paulo).

7. References

Brandl , H. (1998). Trickkiste Chemie, Munchen: Bayerischer Schulbuch Verlag.

Chatellier , D. S.; White, H. B. (1988). " What color is egg white? A biochemical demonstration of theformation of a vitaminprotein complex using fluorescence quenching". J. Chem. Edu . 65, 814815.

Krettli , A. U.; AndradeNeto, V. F.; Brandao, M. G. L.; Ferrari, W. M. S. (2001). Mem. Inst.Oswaldo Cruz 96 , 10331042.

Lakowicz , J. R. (2006). Principles of Fluorescence Spectroscopy, Berlin: Springer, Third Edition.

Leiner , M. J. P. (1991). "Luminescence chemical sensors for biomedical applications: scope andlimitations". Anal. Chim. Acta 255, 209222.

Martin , A.; Narayanaswamy, R. (1997). " Studies on quenching of fluorescence of reagents inaqueous solution leading to an optical chlorideion sensor". Sensor Actuat. B 39, 330333.

Mohan , A. G.; Turro, N. J. (1974). "A facile and effective chemiluminescence demonstrationexperiment". J. Chem. Edu. 51,528529.

Nelson, N. L.; Cox, M. M. (2000). Lehninger Principles of Biochemistry.New York: Worth Publisher.

Pandey , S.; Borders, T. L.; Hernandez, C. E.; Roy, L. E.; Reddy, G. D.; Martinez, G. L.; Jackson, A.;Brown, G.; Acree, W. E., Jr. (1999). " Comparison of Analytical Methods: Direct Emission versusFirstDerivative Fluorometric Methods for Quinine Determination in Tonic Waters." J. Chem. Edu .76, 8587.

Salter , C.; Range, K.; Salter, G. (1999). " LaserInduced Fluorescence of Lightsticks". J. Chem. Edu .76, 8485.

Schuster , G. B. (1979). "Chemiluminescence of organic peroxides. Conversion of groundstatereactants to excitedstate products by the chemically initiated electronexchange luminescencemechanism". Acc. Chem. Res. 12, 366373.

Shakhashiri , B. Z.; Williams, L. G.; Dirren, G. E.; Francis, A. (1981). ""CoolLight"Chemiluminescence". J. Chem. Edu. 58, 7072.

Stevani , C. V.; Silva, S. M.; Baader, W. J. (2000). "Studies on the Mechanism of the Excitation Stepin Peroxyoxalate Chemiluminescence". Eur. J. Org. Chem. 24, 40374046.



[*] corresponding author

Tel: +55 +11 30913865Fax: +55 +11 38155579

carmen iq.usp.br

NERY, A.L.P. ; LIEGEL, R. M. ; FERNANDEZ, C. Fluorescence and Chemiluminescence: Teaching Basic...

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