Black light source: UV-A light (315 - 400 nm)
Mortar and pestle
Light sticks (different colors)
Ethyl acetate
Hydrochloric acid 0.1 mol L^-1
3-5 spinach leaves
Brown eggs
Multivitamin tablet
Tonic water
Detergent

3. Description of the Experiments

For the following experiments all background light must be turned off and the room must be as dark as possible. The UV source must be kept in a box with a frontal slit to avoid students to look directly to the light, which may cause retina damages. It is important to compare the observed colors under visible and UV excitation, mainly in the case of chlorophyll, since its solution is green under visible 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 (Nelson and Cox, 2000). Chloroplasts of higher plants always contain chlorophyll a and b. The structure of the chlorophyll molecules consists of several conjugated nitrogen-containing rings surrounding a magnesium ion by coordinate covalent bonds (Figure 2). This structure resembles the protophorphyrin of the heme group of hemoglobin; one striking difference between chlorophyll and the heme group of hemoglobin (besides other structural differences) is that in chlorophyll a Mg²⁺ ion occupies the central position, instead of the Fe²⁺ ion that is present in hemoglobin.

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

Although both forms of chlorophyll are green, their absorption spectra are sufficiently different to allow them to complement each other�fs range of light absorption in the visible region. The green color is due to absorbance range, which maxima are at 412 nm (blue region) and 666 nm (red region) 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 by chlorophyll (see Figure 3) results in a green solution (solution reflects green color). In the absence of visible 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 reabsorbed by the solution. The drastic change of color in the case of chlorophyll is an experiment that has a beautiful visual appeal, motivating students to pay attention to phenomena related to light absorption and 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. If the 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, no fluorescence can be observed, since absorption and emission spectra overlap and fluorescence reabsorption may occur.

b) Brown eggshell fluorescence

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

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 fluorescence would 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 are carbon 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 one can observe a peculiar spectacle of the gradual increase of the fluorescence emission from the solution. The previous very week fluorescence of the intact eggshell disseminates through the solution as an intense purplish emission (Brandl, 1998).

Procedure: Wash a brown eggshell and irradiate it with black light. Put the eggshell in a beaker, add about 50 mL of ethyl acetate and irradiate again. Add some drops of HCl 0.1 mol L^-1 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 as milk and eggs and also on multivitamin tablets. Under UV-A excitation, its fluorescence is observed by naked eyes as an intense green-yellow 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 and add 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 active ingredient 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 its bitter flavor. Quinine is found in the root, bark and branches of cinchonas trees, native to South America and has been used as a treatment for malaria for hundreds of years. It does not cure the disease, but does help to suppress parasitic activity and thus reduce life-threatening fever ( Krettli et al., 2001). It is a strongly fluorescent compound in dilute acid solution emitting at ca. 450 nm and is used as an important fluorescence standard in chemistry (Pandey et al., 1999). Quinine solutions are colorless, which indicates that it has no meaningful absorption in the visible range and that excitation must necessary occur at UV region of the electromagnetic spectrum, within its absorption bands at 250 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 molecule in its excited electronic state can be deactivated by a collision with a chloride ion before it has a chance to return to its ground state by the emission of a photon. The chloride ion acts as a fluorescence quencher, reducing the fluorescence quantum yields. The fluorescence quenching of quinine can be related to the halide concentration, and can be used for the sensing of chloride ions for analytical 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 jars and compare both samples. Discuss the observed phenomena: fluorescence and quenching on basis on the Jablonski diagram.

e) Optical Whiteners

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

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

f) Chemiluminescence: Light sticks emission

Many chemical reactions produce both light and heat. In these cases, the energy source that causes electrons to move from the ground to the excited state comes from the chemical reaction. A burning candle is such a reaction. When a candle is lit, its flame both glows and becomes hot. It is much less common for a chemical reaction to produce light without heat. Such reactions, where energy is given off as visible light, are called chemiluminescent reactions. Chemiluminescence is defined as the production of light energy from a chemical reaction without the use of heat, for this reason it is also known as "cold light". When light emission derives from living organisms the phenomena is called bioluminescence. Chemiluminescence is the chemical ground of light sticks, these objects that, besides wonder kids, are used by trick-or-treaters, divers, campers, and for decoration and fun. A CYALUME^R (trademark of American Cyanamid Company) light stick is a plastic tube divided into two compartments. Although there is more than one recipe for a light stick, a common commercial one uses a solution of hydrogen peroxide kept in one of the compartments and a solution of a phenyl oxalate ester, for example bis(2,4-dinitrophenyl)oxalate (DNPO) or bis-(2,4,6-trichlorophenyl)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 mix with those in the plastic tube, allowing the chemical reaction to take place. The basic premise is that the reaction between the two chemicals releases enough energy to excite the electrons in the fluorescent 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 of the reaction (Table 1).

Table 1: Fluorescence emission of selected dyes.

Mechanistic studies indicate the occurrence of a Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism (Schuster, 1979), with the formation of a 1,2-dioxetanedione as intermediate, which transfers energy to the dye molecule. The dye molecule acts as a catalyst of the process. Light emission is observed when the excited molecule of the dye (Fl*) returns to the ground state (Stevani and Baader, 2000). This mechanism suggests that if the dye molecule (Fl) can be excited 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 of different colors show very similar chemiluminescence and laser-induced fluorescence spectra.

Procedure: Irradiate different color light stick. Observe and discuss the observed emission. Break some of them. Discuss different kinds of excitation source: light and chemical reaction. Irradiate a light 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 colors by using different dyes, for example chlorophyll solution, allowing comparison of two different processes: photophysical (fluorescence) and chemiluminescence. In a beaker containing about 100 mL of ethyl acetate, add 10 mL of 2.7 mol L^-1 H₂O₂ aqueous solution and about 50 mg of a fluorescent dye. 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 spectroscopy may help students to understand some everyday phenomena. The discussion of experiments like those suggested in this paper may be useful for the process of giving meaning to the surrounding world through chemical knowledge. The examples here discussed are a very effective pedagogical tool for the demonstration of two chemical phenomena involving light: fluorescence and chemiluminescence. Such phenomena have been widely used as analytical tools, mainly in biomedical analysis, due to their high sensibility.

5. Safety

Ultraviolet light is potentially damaging to exposed skin and especially eyes. Do not stare at the black-light source. Ethyl acetate is flammable. Hydrochloric acid is corrosive, can cause severe burns to the skin in its concentrated form, and should be handled with care. There are no significant hazards associated the fluorescent compounds.

6. Acknowledgements

The authors acknowledge financial support from FAPESP, CAPES, CNPq, Dow Chemical Company and Pro-Reitoria de Pesquisa (Universidade de Sao Paulo).

7. References

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