Fluoresence 1 Flashcards
What does fluorescence spec measure
The emmision of light
Are all chromophores fluorophores
No
Uses of fluorescent spec
Identifying bio molecules
Measuring concentrations
Looking at solvent exposure
Measuring size and shape of protiens
Measuring rigidity of environment
Measuring distances
Studying interactions between bio molecule
What do the dashed lines show on flourecnse spectrum
The absorbance
What is in the x axis of fluorescent spectrum
Wavelength (nm)
What is a jablonski diagram
A diagram with both absorption and emmision (fluorescence)
Explain the steps in the jablonski
Excitation (molecule absorbs and goes to highest s1 energy level)
Thermal decay (the energy is lost as heat, not light) goes to ground s1 level
Molecules goes down an electronic level in S0 and emits a photon in the process
More thermal decay to ground state
What is a stokes shift in terms of flourensces
The shift in wavelength (to a higher wavelength) from absorbance to emmision
What is the exception for emmision spectrum going to a longer wavelength then excitation soectrum
Atoms in the vapour phase because they don’t have that thermal decay step
Emmision can be from
And which takes a longer time
Phosphorense or flourescnse
Phosphorense
What is the wavelength range of typical stokes shifts
20-200nm
What do stokes shifts depend on
Vibrational levels in the excited and ground state
Which fluorophore has that largest stokes shift and what is its
Lowest
NADH 130
Benzene in phe (23)
What is the Franck condon principle
During absorption/excitation, the nuclei of the molecules don’t move
Only the electron cloud changes (only electrons move)
This is because excitation happens fast so there’s no time for the nuclei to move
What happens to the eiullibfium bond distance between atoms on the excited state
Why
In the excited state, the bond distance between them becomes longer
This is because the bonding between the atoms becomes weaker (since the electron has moved to an antibonding orbital)
What does it mean for frank condin when we say the nuclei don’t move
This means that during excitation the molecules go to a upper vibrational level of the excited electron state
They go straight up in energy and the actual nuclei don’t move, the electron cloud surrounding it vibrates
What does it mean for frank condin when we say the nuclei don’t move
This means that during excitation the molecules go to a upper vibrational level of the excited electron state
They go straight up in energy and the actual nuclei don’t move, the electron cloud surrounding it vibrates
Explain the steps of the Morse curve depiction of extubation and emission and why it’s like that
Step 1: molecule is excited and goes straight up to a excited electron level (higher level) and upper vibrational level (vibrates more)
The transition is straight up because the nuclei don’t have time to change position during the excitation
Step 2: thermal decay occurs where the flurorophore goes down vibrational levels (not electronic levels)
During this excitation decay, the interatomic distance is longer
Step 3:
The molecule goes down a electronic level and emits a photon quickly
The transition is straight down from its previous position in the upper electronic state (bc of Franck condon)
Step 4:
More thermal decay in the lower electronic level back to original interatomic distance
Explain the steps of the Morse curve depiction of extubation and emission and why it’s like that
Step 1: molecule is excited and goes straight up to a excited electron level (higher level) and upper vibrational level (vibrates more)
The transition is straight up because the nuclei don’t have time to change position during the excitation
Step 2: thermal decay occurs where the flurorophore goes down vibrational levels (not electronic levels)
During this excitation decay, the interatomic distance is longer
Step 3:
The molecule goes down a electronic level and emits a photon quickly
The transition is straight down from its previous position in the upper electronic state (bc of Franck condon)
Step 4:
More thermal decay in the lower electronic level back to original interatomic distance
Emmision spectra is always shifted to ______
Than excitation
Longer wavelengths
Because the emitted light is at a higher energy than the excitation light
What measures fluorescence
What’s soecial about it
Fluorimeter
Has two monochrometers
In a fluorimeter how do you adjust the monochromoter to get the excitation spectrum
What about the emmision spectrum
You have to scan the wavelength of first monochromator with the second one at a fixed wavelength
Fix a wavelength on the 1st and scan the wavelengths monochormator with the 2nd
lol
Lol
What is the inner filter effect
Due to the detector measuring light coming from the centre of the cuvette
before even reaching the centre of the cuvette, the light can be absorbed by the sample
Or the emitted light may get absorbed by the sample and not reach the detector
This means the excitation light from the lamp might not reach the centre of the cuvette
And emitted light might not reach the detector
