Fluorescence Spectroscopy Sheds Light on the Who-Dunnit! What are the Features of Light? Gamma RaysX RaysUVIRMicrowaveTVRadio nmcm meters UVBUVANear IR VISIBLE R - Red O - Orange Y - Yellow G - Green B - Blue I - Indigo V - Violet The Electromagnetic Spectrum Features of Light Luminescence Spectroscopy High EnergyLow Energy 1.The sample is “hit” by a wavelength of light; i.e. BLUE (~450 nm) 2.The sample absorbs, or uses up, a small amount of this initial energy, and wants to spit-out the remaining as Fluorescence 3.What remains is LESS energy; so what is emitted as fluorescence is of LONGER wavelength; i.e. GREEN (~600 nm) 4.Fluorescence = sec or sec Wavelength ( ) = the length of 1 Cycle; a or b Frequency (f) = number of oscillations per second Wave B has the Shorter Wavelength & Higher Frequency (and is the Stronger Wave) Types of Luminescence: Fluorescence and Bioluminescence Why Do Molecules Glow, or Fluoresce? The Experiment: How Did Our Friend Die? Visible, White Light Dispersed: The Technique: The LS55 Fluorescence Spectrometer Schematic of the LS55 Fluorescence Spectrometer 1.Most bourbons are Aged Inside of Charred, burned Oak Barrels 2.Burned Oak contains PolyAromatic Hydrocarbons (PAH’s). These PAH’s are transferred to the bourbon during Aging. 3.The Color and Flavor of Bourbon are derived from this Burned Oak Aging. 4.Trace amounts of these PAH’s are dissolved in the bourbon; these PAH’s fluoresce in the Visible Region at 480 nm. 1.Antifreeze contains approximately 95% of Ethylene Glycol, which is the compound that is responsible for the cooling properties. 2.Antifreeze also contains Fluorescein, which is the Aromatic Compound that provides the well-known Bright Green Color at 520 nm. Fluorescein is added so that a technician can easily diagnose a leak within the engine’s cooling system. 3.If ingested by Humans, Ethylene Glycol, while not immediately toxic, is metabolized within the body to Glycoaldehyde, and later metabolized to Glycolic Acid, Glyoxylic acid, and Oxalic Acid. 4.These three toxic metabolites cause Central Nervous System (CNS) Depression (i.e. drowsiness, and respiratory failure), followed by Cardiopulmonary Disorder, and later Renal Damage. 5.The Lethal Dose of Ethylene Glycol, for an adult human, is 100 milliliters. We know that the Deceased Had a Thermos in his Possession prior to his demise. We also know that the Victim Loved his Bourbon … Is it Possible that Someone Spiked Our Friend’s Beverage with a Lethal Dose of Poisonous Antifreeze? If this were true, then the drink would exhibit the typical green fluorescence, most commonly known as Antifreeze Green, with an emission at 520 nm. Further, bourbon would be identified with fluorescence at 470 nm in the emission spectrum. Fluorescence of Bourbon Fluorescence of Antifreeze Let Take a Look at the Contents of the Thermos, and See if it Fluoresces … Our Conclusions … Using the LS55 Spectrometer, we are able to identify the drink in the thermos as bourbon. We are also able to identify antifreeze in the victim’s drink, and have determined the amount (%) of antifreeze present in the bourbon. The results below indicate that the victim’s drink contained almost 30% antifreeze. Assuming that our victim had at least three drinks, collectively this would contain more than the 100 mL Lethal Dose of Ethylene Glycol. Victim’s Beverage 1.Photoluminescence a.Fluorescence (10 -9 sec) b.Phosphorescence (10 -6 sec) 2.Chemiluminescence 3.Bioluminescence 4.Electroluminescence 5.Radioluminescence