Light and Molecules, Secondary Level Use of Luminol in Forensic Analysis

Summer 2006 Light and Molecules, Secondary Level Use of Luminol in Forensic Analysis . OBJECTIVE: To detect trace amounts of an oxidant such as hemoglobin using chemiluminescence. BACKGROUND: Chemiluminescence is the emission of light as a result of a chemical reaction. Ancient documents almost two thousand years old record Pliny the Elder’s observation that a walking stick rubbed with the luminescent jellyfish, pulmo marinus, would “light the way like a torch”. The term chemiluminescence was first used in 1888 by Eilhardt Weidemann in his studies of reactions that produced light without heat or “cold light”. We have come to learn that this light is derived from unstable high energy intermediates in some chemical reactions. A + B → high energy intermediate → products + light In this experiment, you will use a basic solution of luminol, 3-aminophthalhydrazide, to react with an oxidant (bleach) to generate the cold blue light characteristic of this reaction. The molecular structure of luminol and proposed reaction are shown below. Luminol 3-aminophthalate (3-APA) The cold blue emission of luminol is best viewed in a darkened room. Chemiluminescence persists for seconds to minutes depending on the concentration of oxidant, and several other factors. FORENSICS: Crime scene investigators, police detective specialists who have advanced training in forensic science, consider luminol to be an important tool in their investigation of crime scenes. Blood contains an oxidant, hemoglobin, powerful enough to oxidize luminol for extended periods of time after a crime has been committed and the reaction is sensitive enough to detect the presence of blood even after the article contaminated with blood has been washed. A number of prime time television shows have made use of this chemiluminescent reaction as part of their crime scene investigations. A quick search of newspapers like the New York Times reveals 38 references to the use of luminol in the last two years. It is important to emphasize here that a positive luminol result is suggestive, but not proof that blood is present on the sample. Should detectives wish to make a conviction, further evidence is necessary, since there are many other molecules besides blood (such as bleach, or any other oxidizing material) that can react with luminol. It is also a destructive assay. Blood assayed by luminol cannot also be used for DNA analysis, so research is active in the forensics field to develop an indicator for blood that is as sensitive as luminol but more specific and non-destructive. 1 Summer 2006 THE SCENE: A valuable balance is missing from your chemistry laboratory. There is a broken beaker by the sink and a few drops of blood. Several other pieces of laboratory equipment are scattered about on the floor. OBJECTIVE: It is your job to use the chemiluminescence of luminol to discover which pieces of laboratory equipment may have been exposed to “blood”. (Note: no real blood has been used in this experiment.) MATERIALS: luminol (3-aminophthalhydrazide), sodium carbonate, sodium perborate (catalyst), a spray bottle for the luminol solution. Obtain from your instructor an assortment of laboratory apparatus that might potentially have been exposed to blood (i.e. spatula, pipette, stirring rod, thermometer). Caution: gloves and goggles must be worn for safety at all times when handling these solutions. PROCEDURE: 1. Prepare a basic solution of luminol by dissolving 5.0 g of sodium carbonate in 100 mL of distilled water. Add 0.1 g of luminol reagent, and stir or swirl to dissolve. Transfer contents to a spray bottle. 2. Sodium perborate is not indefinitely stable. Swirl 0.7 g of sodium perborate catalyst to dissolve in the the 100 mL of luminol solution in your spray bottle just before analysis. 3. Darken the room and spray the luminol solution onto the potentially contaminated pieces of laboratory equipment to discover which produce the cold blue emission of luminol. You may need a few sprays to see the luminescence but you will most certainly have material left over. Record your observations below: Reaction with spatula Reaction with pipette Reaction with stir rod. Reaction with thermometer ANALYSIS: 1. Which pieces of equipment are contaminated? 2. Review the relationships of color to wavelength. What is the approximate wavelength of light emitted by the luminol reaction? 3. Using the relationships below, calculate the change in energy (per photon and per mole) as luminol intermediates return to the ground state by emitting a photon. E = h  per photon E = energy h (Planck’s constant) = 6.626 x 10-34 J.s N (Avogadros number) = 6.02 x 1023 mole-1 c=   c (speed of light) = 2.998 x 108 m/s  = wavelength  = frequency 2 Summer 2006 ANSWER SHEET For example: Record your observations below: Reaction with spatula Reaction with pipette Light blue emission lasts several sec (not indefinitely) No reaction after repeated spraying Reaction with stir rod. No reaction after repeated spraying Reaction with thermometer No reaction after repeated spraying ANALYSIS: 1. Which pieces of equipment are contaminated? On the basis of these results, it appears as though the spatula was the only piece of equipment to be exposed to “blood” 2. Review the relationships of color to wavelength. What is the approximate wavelength of light emitted by the luminol reaction? Blue light is approximately 400 nm = 4.00 x 10-7m 3. Using the relationships below, calculate the change in energy (per photon and per mole) as luminol intermediates return to the ground state by emitting a photon. E = h  per photon c=   E = energy c (speed of light) = 2.998 x 108 m/s H (Planck’s constant) = 6.626 x 10-34 J.s  = wavelength 23 -1 N(avogadros number) = 6.02 x 10 mole  = frequency Per photon: E = (6.626 x 10-34 Js)(2.998 x 108 m/s) / (4.00 x 10-7m) Per mole: E= 4.97 x 10-19 J x 6.02 x 1023 mole-1 = 2.99 x 105 J/mole = 4.97 x 10-19 J = 299 kJ 3 Summer 2006 TEACHER NOTES BACKGROUND: 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 chemiluminescence, a process in which an inanimate system is excited by the energy of a chemical reaction (1). In these devices, two reactants are separated by a chamber. 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 (2,3). Luminol has gained attention from the print media and television, where it is now in widespread use at many crime scene investigations. Here, students can play an active role in preparing a luminol solution and using it to detect the presence of simulated “blood” (a diluted bleach solution) via chemiluminescence on pieces of equipment left behind at a crime scene. 1. Carlson, R.; Lewis, S.W.; and Lim, K.F. Seeing the Light: Using Chemiluminescence to Demonstrate Chemical Fundamentals. Aust. J. Chem. Ed. 2000 14, 51-53. 2. Schreiner, R., Testen, M.E., Shakhashiri, B. Z., Dirreen, G. E. and Williams, L. G. Chemical Demonstrations: A Handbook for Teachers of Chemistry, Volume 2 (The University of Wisconsin Press) 1983. 3. Lister, T., Classic Chemical Demonstrations (Royal Society of Chemistry, London) 1996. NOTES ON MATERIALS 1. Chemicals can be purchased online: a. sodium perborate CAS # 7632-04-4 b. sodium carbonate CAS # 5968-11-6 (Arm & Hammer washing soda will work just as well) c. luminol CAS # 521-31-3 2. Simulated blood can be prepared from household bleach (sodium hypochlorite) – diluted 10 ml per 100 ml of water to which a drop of red food coloring is added. Transfer the contents to a spray bottle. Before class, spray one or more of the assembled pieces of laboratory equipment with the bleach solution, our simulated blood. Once dry, it is nearly invisible. 3. Both the bleach solution above and the luminol solution will keep at room temperature for several months. 4