In the Classroom
Tested Demonstrations
Luminosity, My Dear Watson, Luminosity!—Or, Are Those Bloodstains? submitted by:
checked by:
Barbara A. Burke,* Kamran Golestaneh, and Helene Samson Chemistry Department, California State Polytechnic University, Pomona, Pomona, CA 91768 Galen P. Mell
Division of Biological Sciences, University of Montana, Missoula, MT 59812-1002
Luminol demonstrations are well known and have been described in great detail (1–5). The procedures given here are based on those that have been used by forensic scientists to detect the presence of latent bloodstains on a variety of porous substrates (6–9). Many of these demonstrations use large volumes of solutions in order to show off the spectacular effects of luminol reactions. The demonstration described here was developed as part of an ongoing effort in the chemistry department to minimize the amount of hazardous waste generated.
with water in a test tube and then applied to the fabric or paper and allowed to dry. (Dilutions up to about 1:150 can be detected.) Use capillary melting-point tubes to apply diluted blood to the fabric or paper. The bloodstained fabric or paper can be stored in plastic baggies for several weeks.
Demonstration
Solution A Dilute 5 mL of 3% hydrogen peroxide, H2O2, to 100 mL. Store in a tightly sealed polyethylene bottle in a refrigerator. This will keep for several weeks.
In a darkened room, a solution containing dilute hydrogen peroxide (solution A) is mixed with a solution of luminol in a carbonate buffer (solution B) as it is sprayed on a bloodstained piece of cloth or paper to produce the characteristic bluish glow of luminol. Only the portion impregnated with the blood will luminesce. Horse and sheep blood, rather than human, are used for this demonstration. Transition metal ion solutions of Cu2+, Fe3+, and Co2+ can also be used because they will promote luminescence when in contact with a mixture of solutions A and B. A two-reservoir spray bottle was constructed and used in this demonstration (Figs. 1, 2) Rather than constructing a bottle, one may use a plastic T-connector and some flexible plastic tubing to modify a household spray bottle in order to draw solutions A and B simultaneously from two Erlenmeyer flasks (Fig. 3). Alternatively, solutions A and B can be mixed a few minutes before the demonstration and placed in a spray bottle. The two solutions, once mixed, can be stored for only a week or so. Another variation is to spray the bloodstain sequentially with solution A and solution B in separate spray containers. Preparations Prior to Demonstration
Bloodstains Use either fabric or paper. In either case, test to be sure there are no brighteners present by spraying a small sample with the mixture of solutions A and B. (If the fabric has brighteners, wash it 2 or 3 times with water only to remove the brighteners.) Horse or sheep blood that has been stabilized with citrate to prevent clotting is used as is or diluted
Transition Metal Ion Solutions Prepare 0.1 M solutions of the chloride or sulfate salts of the metal ions Cu2+, Co 2+, Fe3+. Concentrations of 0.1– 0.001 M Cu2+, 0.1–0.0001 M Co2+, and 0.1–0.01 M Fe3+ can be detected. Apply to the fabric or paper as above.
Solution B Dissolve 0.4 g of sodium carbonate, Na2CO3; 0.02 g of luminol (3-aminophthalhydrazide), C8H7O2N3; 2.4 g of sodium bicarbonate, NaHCO 3; and 0.05 g of ammonium carbonate monohydrate, (NH4)2CO3⭈H2O, in that order, in 100 mL of solution. This will keep for several weeks when refrigerated. Two-Reservoir Spray Bottle Two different types have been used. Both are relatively easy to make. See Figures 1 and 2. To make the bottle in Figure 1, use a razor blade or sharp knife to split the plastic bottle, just below the neck, in half. Insert a stiff piece of plastic and glue (epoxy works well) it in place. The bottle in Figure 2 was constructed by cutting two holes (the size of the caps of the smaller bottles) in the bottom of the spray bottle. Holes for the sprayer tubing were made in the caps of the smaller bottles and these caps were then glued into the precut holes on the bottom of the spray bottle. In this way bottles containing solution A and solution B could be screwed into the caps just before the demonstration. These systems made it more convenient to store the solutions when not in use. Alternatively, a plastic T-connector and some flexible plastic tubing can be used to modify a household spray bottle in order to draw Solution A and Solution B simultaneously from two Erlenmeyer flasks. These may be taped together for ease of use (Fig. 3).
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JChemEd.chem.wisc.edu • Vol. 76 No. 1 January 1999 • Journal of Chemical Education
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In the Classroom
Safety Precautions Normal safety precautions are needed for the preparation and handling of the solutions used in this demonstration. Follow the guidelines for handling and waste disposal on the manufacturer’s Materials Safety Data Sheet (MSDS) for each chemical used. CAUTION: Spray reagents should be used in a darkened room with adequate ventilation. If used up close, a mask and gloves should be worn. Discussion
Figure 1. Construction of two-reservoir plastic spray bottle. Two chambers are created by splitting lower portion of bottle and inserting plastic divider (see text).
This demonstration can be quite spectacular when a large picture made of diluted blood is sprayed in a darkened room. The sensitivity of the luminol test can be easily shown by spraying a series of pieces of bloodstained fabric or paper containing a wide range of blood dilutions. The luminol test is a presumptive or screening test in forensic science. Many chemicals give false-positive results. Therefore, at a crime scene, a negative test means that the stain is definitely not blood, but a positive test means only that it might be blood. Samples of that stain would then need to be tested further to determine whether it is blood. Some examples of chemicals that will give false-positive results for the luminol reaction to detect the presence of blood can be shown using fabric or paper stained with transition metal salts. This part of the demonstration is instructive to students as a reminder of the importance of confirmatory tests when dealing with the identification of unknowns. Literature Cited
Figure 2. Alternative construction of two-reservoir spray bottle (see text).
1. Shakhashiri, B. Z. Chemical Demonstrations, A Handbook for Teachers of Chemistry, Vol. 1; The University of Wisconsin Press: Madison, WI, 1983; pp 125–204. 2. Gilbert, G.; Alyea, H. N.; Dutton, F. B.; Dreisbach, D. Tested Demonstrations in Chemistry and Selected Demonstrations from the Journal of Chemical Education, Vol. I; Department of Chemistry, Dennison University: Granville, OH, 1994; pp. H41–H55. 3. Bindel, Thomas H. J. Chem. Educ. 1996, 73, 356. 4. Schechinger, L.; Waldman, A. S. J. Chem. Educ. 1995, 72, 243. 5. Alyea, H. N.; Dutton, F. B. Tested Demonstrations in Chemistry, 6th ed.; Division of Chemical Education of the American Chemical Society: Easton, Pennsylvania, 1965; p 186. 6. Forensic Science Handbook; Saferstein, R., Ed.; Prentice-Hall: Englewood Cliffs, NJ, 1982; pp 272–276. 7. Cunliffe, F.; Piazza, P. B. Criminalistics and Scientific Investigation; Prentice-Hall: Englewood Cliffs, NJ, 1980; pp 181– 183. 8. Crime Investigation, 2nd ed.; Kirk, P. L.; Thorton, J. I., Eds.; Wiley: New York, 1974; pp 184–189. 9. Kind, S.; Overman, M. Science against Crime; Nature and Science Library, Doubleday: Garden City, NY, 1972; p 43.
Figure 3. Use of spray nozzle from household spray bottle to simultaneously draw solutions from two reservoirs.
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Journal of Chemical Education • Vol. 76 No. 1 January 1999 • JChemEd.chem.wisc.edu
