Fluoresence spectroscopy

Question 1

Why only some molecules fluoes?

Answer 1

It depends on the “overlapping” of the types of energy levels. If the gap is large, fluoresence will be expelled, if its too short, only heat will be given out.

Question 2

Emission

Answer 2

The expelled spectrum is of longer wavelength than the incident spectrum, this is because as the electrons are absorbed, they transition to the lowest energy state of that level and relax, this is followed by transition to the ground state (fluorescence)

Question 3

The fluorimeter

Answer 3

Light >
Excitation monochromator >
Sample >
Emission monochromator >
Detector >
Display >

It provides greater sensitivity than absorbtion spec.
This is because the emission wavelength is different to the excitation, scattered light can be eliminated. Also, emitted light against a dark background than changed in light intensity against a bright background.

It has 2 monochromator, hence you can measure 2 types of spectra.

Question 4

Uses of fluorescence

Answer 4

measuring concentrations: fluorescence intensity is proportional to the concentration of the fluorophore at low concentrations.

Question 5

Fluoescence change with solvent polarity

Answer 5

The spectrum will increase intensity with reduced polarity.. This is because the excited state is more polar than ground state, hence it will interact with polar solvents, reducing the fluorescence. This is called “red shift”. “blue shift” also occurs when the flurophore does not interact with the solvent.

Question 6

intrinsic and extrinsinc fluorophore

Answer 6

Typtophan fluorescence and chlorophyll (intrinsic)
The emission spec will deduce how many tryptophan is present, by conformation change of the cell, inducing the tryptophan to move towards the cell membrane, this interacts and provides less fluor.

A second technique is using a quencher.Its a substance that reduced the quantum yield. The quencher can access the tryptophan residue which physically “collides”.
this is called collisional quenching.

Question 7

Resonance energy transfer

Answer 7

Excitation energy can be transferred from an excited molecule to an acceptor molecule who absorbtion bands overlap the emission bands of the donor.

Question 8

Fluorescence polarisation

Answer 8

To measure mobility of molecules.
If molecules stay on the same orientation, the expelled light will remain the same plane polarised light, if the molecules move, the polarisation is decreased.

The higher value of P or r, the less the molecules are moving.

This can deduce the viscosity, size.