Fluoresence Flashcards
What is the difference between Fluoresence and absorption?
- In absorption, a sample absorbs light energy, causing its electrons to jump from a lower energy level to a hugher energy level however in Fluoresence, the sample that absorbs light then emits it as the elctrons return from a higher energy state to a lower energy state.
What is fluoresence?
- Sample absorbs light in the UV spectrum and emits light in VIS-L spectrum
- Light emitted is always of a longer wavelength than light being absorbed - Stokes Law
- Some energy is lost as heat.
instruments required for fluorimitery
- spectrofluorimeter
- High energy light source - xenon arc lamp
- 2x monochromators - excitation & emission
- Detector - allighned 90 degrees to lamp to minimise high energy light reaching detector that has not passed through the sample
- PMT - used as detector/amplifier
Describe the mechanism of Fluoresence, include in your answer the singlet and triplet states.
- Sample absorbs light causing one of the electrons to to be excited from ground state (S₀) to higher energy level - both electrons have a paired spin (↑↓).
- 1x Electron moves to excited state (S₁) retaining paired spin.
- Vibrational relaxation/internal conversion occurs resulting in the excited molecule to drop to a lower energy level.
- The electron then relaxes back to ground state, emitting light in the process (FLuoresence).
OR
Intersystem crossing occurs where the excited molecule moves into a triplet state (T₁), the electrons spin flips so it is now parallell with the electron in ground state - Transition from T₁ to ground state is slower (1-2s), causing delayed emission (Phosphorescence) - this process is spin forbidden (much slower process and less likely to occur)
Why is a high power light source used in fluorescence but not in absorption?
- Fluorescence is usually weak.
- the sample is a dilute sample so more light energy is required to absorb the little concentration in the sample to produce a detectable signal.
- However in Absorption measures the change in light intensity from insidence to transmitted, so even a low power light source can detect a noticable change.
What are the differences in instrumentation in absorption and fluorescence?
See image.
What are the factors affecting fluorescence.
Equation (IF = 2.303 ØF I0 εcL ) shows the following factors affect fluorescence:
* Source intensity
* Fluorescence efficiency (quantum yield)
* Concentration
* Pathlength
In practice, the following factors are also taken into consideration:
* Quenching
* Self absorption
* Avoidance of matrix effects
What is Quenching? Name the two types.
- A reduction in the light emitted during fluorescence
- Self/Concentration quenching
- Chemical quenching
Chemical quenching
- involves the removal of the energy from an excited molecule by another molecule usually as the result of a collision.
- This can be important in an analysis since the fluorescence of the analyte might be quenched by the molecules of some compound present in the sample - matrix effect.
- There are two possible quenching mechanisms:
A* + Q => A + Q* collision A* + Q => AQ* => AQ complex formation
A = fluorescent analyte molecule
Q = quenching species
Self quenching
- Seen at high concentrations (>0.005%)
- Significant amount of incidence light is absorbed before reaching central part of cuvette
- emitted light is concentrated at the face of the cell rather than the body of the solution
- may be solved by further dilution of the sample.
Advantages of Fluorescence
- Flourimetry is more specific than UV spec
1. Use of two monochromators
2. Not all molecules with a chromophore fluoresce - Has a very low detection limit making it more specific for analysis of small amounts of potent drugs
- Easier to measure a small signal (fluorescence intensity) rather than two large signals (absorbance)
- Flourescence intensity may be increased by increasing Io. Unlike in absorbance
Factors affecting Fluorescence?
- Source intensity
- Fluorescence intensity
- Concentration
- Path length
In practice:
Quenching
Self absorption
Avoidance of matrix effects
