Module 3b: Luminiscence Flashcards
Define luminiscence
any emission of light when an electron returns from an excited state (by any other energy other than heat) to ground state
There are four types of luminiscence which are
- fluorescence
- phosphorescence
- chemiluminiscemce
- electrochemiluminiscence
Luminiscence should be observed at what direction and why?
Perpendicular to the incident light to avoid detection of scattered radiation
In quantitative analysis, the intensity of luminiscence is proportional to
concentration of species (C)
incident radiant power (P0)
constant depending on emitting molecule (k)
An atom or molecule that can fluoresce
fluorophore
Makes Fluorescence measurements 100 to 1000 times more sensitive than absorbance measurements
- intense light source
- digital signal filtering
- sensitive emission photometers
Principle of Fluorometry
Molecule that has greater energy that the environment fluoresces where returning to ground state.
Test based on the principle of fluorescence
Fluorometry
Components of fluorometers
- light source (excitation source)
- excitation or primary monochromator (larger than absirbance spectrophotometers)
- sample cell
- emission or secondary monochromator
- detector
Difference between fluorometer and spectrofluormeter
Fluorometer - uses interference or glass filters
Spectrofluorometer - prism or grating monochromator
Application of Fluorometry
- fluorescent tags or labels
- hematofluorometry of zinc protoporphyrin in whole blood
- flow cytometry
Concentration effect that refers to the loss of excitation intensity across the cuvet path length as the fluorophor absorbs the excitation light
Inner filter effect
Concentration effect where a macromolecule, like an antibody, is labeled heavily with a fluorophor, and radiationless energy transfer occurs.
concentration quenching
The difference between excitation light and emitted fluorescence in fluorometry
Stoke’s Shift
When the excitation and emission spectra overlap in fluorescence.
Light Scattering
Limitation of fluorometer in which a serum or urine sample contain many compounds that fluoresce which contributes to unwanted background fluorescence.
sample matrix effects
The most serious contributors to sample matrix effect in fluorometry
proteins and bilirubin
The temperature in fluorometry must be regulated within
+/- 0.1 °C
Fluorescence intensity ________ with increasing temperature by approximately
decreases
1-5 percent per degree Celsius
Limitation of fluorrometry where weakly fluorescing or dilute solution are affected by intense light sources
photodecomposition
Six limitation of Fluorometry
- concentration effects
- Light scattering
- solvent and cuvette effects
- sample matrix effects
- temperature effects
- photodecomposition
Test that is similar to flurometry but is distinguished in that it continues to occur even
after the radiation causing it has ceased.
Phosphorescence
Difference between the emitted light in Fluoremetry amd Phossphorescence
There is a larger shift in wavelength in phosphorescence
Types of luminescence in which the excitation event is caused by a chemical or electrochemical reaction, not by photolumination
Chemiluminiscemce and bioluminiscence
Principle of chemiluminiscence
Compounds react with an oxidizing agent, with a catalyst,
It shifts into excited state.
Light is emitted as the product returns to ground state.
Common substrates in chemiluminiscence
luminol
isoluminol
acridinium estees
luciferin
Common oxidants in chemiluminiscence
hydrogen peroxide
hypochlorite
oxygen
Commonly used catalysts in Chemiluminiscence
enzymes (alkaline phospahatase, horseradish peroxidase, microperoxidase) metal ions (copper, ferric phthalocyanine complex)
substrate, oxidizer, and catalyst used in forensics as diagnostic tool for the detection of blood and other biological fluids.
luminol, hydrogen peroxide, peroxidase
A special form of chemiluminescence occurring within biological systems.
bioluminiscence
Biological substrate, oxidizer and catalyst in fireflies
Luciferin, oxygen, luciferase
another example of biological caatalysts in bioluminiscemce
aequorin
Type of luminescence where the reactive species that produce the chemiluminescent reaction are electrochemically generated.
Electrochemiluminiscence
Advantages of Electrochemiluminiscence
reagent stability
simple reagent prepartion
enhanced sensitivity
A physical phenomenon resulting from the interaction of light with particles in solution.
Light scattering
Difference between fluorometry and light scattering techniques
scattered light has smiliar frequency as incident light
Best application of Light scattering techniques
Immunoassay of specific serum proteins and haptens
Two types of light scattering techniques
turbidimetry
nepehlometry
Measures the decrease in intensity of the incident light at 180 degrees from the incident beam
turbidimetry
It is the detection of light energy that js scattered or reflected toward a detector that is not in the direct path of the transmitted light, usually measured at a right angle.
nepehelometry.
What is measured in turbidimetry?
blocked light
what is measured in nephelometry?
scattered light
Nephelometry that is commonly used in measuring macromolecules with size larger than the wavelength of incident light.
Forward scattering nephelometer
