Fluoresence

Question 1

What is the difference between Fluoresence and absorption?

Answer 1

• 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.

Question 2

What is fluoresence?

Answer 2

• 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.

Question 3

instruments required for fluorimitery

Answer 3

1. spectrofluorimeter
2. High energy light source - xenon arc lamp
3. 2x monochromators - excitation & emission
4. Detector - allighned 90 degrees to lamp to minimise high energy light reaching detector that has not passed through the sample
5. PMT - used as detector/amplifier

Question 4

Describe the mechanism of Fluoresence, include in your answer the singlet and triplet states.

Answer 4

1. 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 (↑↓).
2. 1x Electron moves to excited state (S₁) retaining paired spin.
3. Vibrational relaxation/internal conversion occurs resulting in the excited molecule to drop to a lower energy level.
4. 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
5. 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)

Question 5

Why is a high power light source used in fluorescence but not in absorption?

Answer 5

• 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.

Question 6

What are the differences in instrumentation in absorption and fluorescence?

Answer 7

See image.

Question 8

What are the factors affecting fluorescence.

Answer 8

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

Question 9

What is Quenching? Name the two types.

Answer 9

• A reduction in the light emitted during fluorescence
• Self/Concentration quenching
• Chemical quenching

Question 10

Chemical quenching

Answer 10

• 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

Question 10

Self quenching

Answer 10

1. Seen at high concentrations (>0.005%)
2. Significant amount of incidence light is absorbed before reaching central part of cuvette
3. emitted light is concentrated at the face of the cell rather than the body of the solution
4. can cause calibration curve
5. may be solved by further dilution of the sample.