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Absorption of one wavelngth, emission of a longer wavelngth with lower energy.
what 3 steps occur in flourescence?
1. Excitation with UV lite
2. Reorientation
3. Emission of Visible lite
what 3 types of molecules flouresce the best?
1. Molecules w/ conjugated double bonds.
2. Molecules w/ ring closures
3. Metal chelated molecules.
What is Quantum efficiency?
the YIELD:
Ratio of #flouresc molecules to #excited molecules.
how many components are in a flourometer, and what are they?
1. Stable power supply
2. Light source
3. Primary filter
4. Sample holder
5. Secondary filter
6. Detector
what does power get supplied TO?
-Uv light source
What type of light source is in the flourometer?
-What type of bulbs can be used?
-UV light emitting at 200-800 nanometers.

-Hydrogen, Deuterium, Xenon/Mercury Arc
How many filters?
What are they?
-Primary filter
-Secondary filter
-Both are Wratten (gelatinous)
What does each filter do?
1ary selects the absorption wavelength.

2ary selects the flourescent wavelength, has wide bandpass.
what are the limitations for the sample holder in flourometers?
-cuvettes made of
-GLASS if excit wvlngth is over 320 nanometers
-QUARTZ if less.
or just use POLYSTYRENE
what is the detector in a flourometer?
PMT, which feeds the meter readout with info.
what angle is between the two filters?
90 degrees to prevent overlap of flourescent and excitation wavelengts.
what 3 types of instruments use flourometry in them?
1. Fluorometers
2. Spectrofluorometers
3. Specific use instruments.
whats the difference between a flourometer and spectrofluor.?
-fluormtr uses a FILTER, and Mercury arc noncontin. lamp.

-Spectrofl. uses a GRATING, and Xenon arc continuous output lamp.

-Flour has to chng filters, spectro scans over various wavelengths to select.
how do they operate differently?
Flourometer has to chng filters,

Spectro scans over various wavelengths to select.
what is each instrument used in?
Flourometer: clinical practice

Spectrofluorometer: research b/c it is more sophisticated.
what are 2 types of specfc use flourometers?
1. Morphine analyzer
2. Calcium titrator
what is the 2 ADVANTAGES of flourometry?
1. Sensitivity
2. Specificity
how sensitive is flourometry?
1000x more than absorption photometry!

Detection of 0.1 ppBILLION
Absorpn detects 0.1 ppMILLION
how many problems are associated w/ flourometry?
What are they?
A LOT! - 6:
1. Limited choice of standard.
2. Quenching
3. Photodecomposition
4. pH effect
5. Temp effect
6. Other contamination
why is the reference compound or standard limited?
because the analyte being measured may not be available - morphine for e.g.
What is quenching?
Returning to ground state by a mechanism other than emission, results are decreased
What 4 things cause quenching?
1. Molecular dissociation
2. Interaction w/ other stuff
3. Solution contaminants
4. Sample concentration
why would molecules dissociate?
if the excitation wavelength has more energy than the bond strength of samples
what solution contaminants cause quenching?
oxygen, nitrogen compounds. NaCL so can't use saline diluent.
how does sample concentration affect flourescence?
If the sample is too concentrated, all the light is absorbed by molecules at one side of cuvette; must dilute to correct.
What is "Photodecomposition"?

How does it affect results?
How is it corrected?
the decomposition of sample due to intense UV lite.

Results are decreased. Lower intensity or complex w/ substance to alter absorption peak.
How does pH affect flouresc.?

How does it affect results?
How is it corrected?
By modifying the resonance structure of a molecule.
Results will vary.
Control the pH to correct.
How does Temp effect flouresc?
How does it affect results?
How is it corrected?
by promoting vibration and collision, thus energy loss.
Results: decreased
Correct: maintain T or cool soln
What can contaminate sample?
Stopcock grease
Cleaning agents
Chemical impurities
Filter paper residues.
What are 2 clinical applications of flourescence?
1. Meausring trace quantities of stuff: Drugs (monitor salicylate, heart therapy), Enzymes, Amino acids.
2. Immunoflouresc. w/ Ab tag

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