Flourometry
Terms
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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?
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1. Excitation with UV lite
2. Reorientation
3. Emission of Visible lite - what 3 types of molecules flouresce the best?
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1. Molecules w/ conjugated double bonds.
2. Molecules w/ ring closures
3. Metal chelated molecules. - What is Quantum efficiency?
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the YIELD:
Ratio of #flouresc molecules to #excited molecules. - how many components are in a flourometer, and what are they?
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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?
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-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?
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1ary selects the absorption wavelength.
2ary selects the flourescent wavelength, has wide bandpass. - what are the limitations for the sample holder in flourometers?
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-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?
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1. Fluorometers
2. Spectrofluorometers
3. Specific use instruments. - whats the difference between a flourometer and spectrofluor.?
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-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?
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Flourometer has to chng filters,
Spectro scans over various wavelengths to select. - what is each instrument used in?
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Flourometer: clinical practice
Spectrofluorometer: research b/c it is more sophisticated. - what are 2 types of specfc use flourometers?
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1. Morphine analyzer
2. Calcium titrator - what is the 2 ADVANTAGES of flourometry?
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1. Sensitivity
2. Specificity - how sensitive is flourometry?
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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?
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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?
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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.
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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?
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Stopcock grease
Cleaning agents
Chemical impurities
Filter paper residues. - What are 2 clinical applications of flourescence?
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1. Meausring trace quantities of stuff: Drugs (monitor salicylate, heart therapy), Enzymes, Amino acids.
2. Immunoflouresc. w/ Ab tag