Flourometry

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what is FLOURESCENCE?: 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?: 6:
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
-Photodetector.
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?: Two.
-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?: Halides
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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