Fluorescence SpectroscopyFluorescence SpectroscopySheds Sheds Light Light on the Who-Dunnit! on the Who-Dunnit!

What are the Features of Light?What are the Features of Light?

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Gamma Rays X Rays UV IR Microwave TV Radio

nm cm meters

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UVB UVA Near IR

VISIBLE

• R - Red

• O - Orange

• Y - Yellow

• G - Green

• B - Blue

• I - Indigo

• V - Violet

The Electromagnetic SpectrumFeatures of LightLuminescence Spectroscopy

High Energy Low 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 = 10-9 sec or 0.000000001 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?Why Do Molecules Glow, or Fluoresce?

The Experiment: How Did Our Friend Die?The Experiment: How Did Our Friend Die?

Visible, White Light Dispersed:

The Technique: The LS55 Fluorescence SpectrometerThe 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 …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

Emission Spectrum of AntifreezeExcitation Wavelength = 395 nm

Spectrum Name: antifreeze2.sp Description: Antifreeze Spectrum

Date Created: Wed Jan 09 11:44:01 2002

Data Interval: 0.50 nmInstrument Model: Arithmetic

Scan Speed: 960 nm/minEmission Slit Width: 4.00 nmExcitation Slit Width: 3.00 nm

Time: 10:58:17 AMDate: 1/17/2002

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Excitation Wavelength = 395 nmEmission Spectrum of Bourbon

Spectrum Name: BOURBON.SP Description: Bourbon Sample

Date Created: Wed Jan 09 10:59:44 2002

Data Interval: 0.50 nmInstrument Model: LS55

Scan Speed: 960 nm/minEmission Slit Width: 15.00 nmExcitation Slit Width: 8.00 nm

Time: 10:58:17 AMDate: 1/17/2002

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Excitation Wavelength = 395 nm

Antifreeze Emission

Bourbon Emission Emission Spectrum of Victim's Bourbon Drink

Spectrum Name: VICDRINK.SP Description: Victim's Bourbon Drink

Date Created: Wed Jan 09 11:01:51 2002

Data Interval: 0.50 nmInstrument Model: LS55

Scan Speed: 960 nm/minEmission Slit Width: 15.00 nmExcitation Slit Width: 8.00 nm

Time: 10:58:17 AMDate: 1/17/2002

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1. Photoluminescencea. Fluorescence (10-9 sec)b. Phosphorescence (10-6 sec)

2. Chemiluminescence3. Bioluminescence4. Electroluminescence5. Radioluminescence