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
How do you fix the inner filter effect
Dilute the sample (high concentration means high inner filter effect)
when you do this the optical density is lower (because concentration is lower) and the observed fluoresce intensity is similar to the corrected
Also use the correction equation
What is the time difference between absorbance and excited state
What does this mean
Absorbances is 10^-15 second (femtosecond)
And excited state is 10^-9 (nanosecond)
Excited state is 1 million fold slower
This means that during the excited state, many things can happen which lead to a loss of energy
Do all excited fluorophore emit a photon?
What other things can happen
No
Many other things can happen like
Intersystem crossing (phosphorescence)
Energy transfer to an acceptor
Thermal inactivation (loss as heat)
Collision with other molecule
Why is the fluoresces measured at 90 degrees to the cuvette instead of straight across the cuvette?
Because we need to filter out the samples transmitted light
If we just measured across from the cuvette we’d get the transmission of light (wrong signal)
To get an emmision spectrum what do we do to the first monochromator?
We have to set the first one at a fixed wavelength
This fixed wavelength is the max wavelength of the absorbance of our sample so we get the best signal
Then we can scan which wavength of light the sample emits by checking the second monochromator
When collecting emoted light are we capturing most of it?
No most of it is coming off the sample and only some goes to the second monochromator
What is a band pass
When we set a certain Wavelength of light to go through the first monochormator
We set a band pass (ex. 280 nm with a band pass of 10nm)
This means the light going through the first monochromator is 270-290nm
Other than the first monochormator with is being light to the sample where else can a band pass be set
At the second monochormator
We can make it so that the light being emitted has a wavelength of +/- some value
This is the emmision band pass
Who sets the band pass
The person using the machine (operator)
What is the typical range of a band pass
1-10nm
In both the emmision and excitation band passes, what does it mean if you have a higher band pass
For excitation, more light is reaching the sample so you get a higher excitation signal
For emmision more emitted light is reaching the detector so you get more emmision signal
But for both the resolution of peaks is lower
What is the trade off of setting a band pass
The S/N ratio is higher, but the resolution is lower
If product of the two band pass values are the same what does this mean
They give the same intensity
What is quantum yield ?
It measures how many of the excited fluorophores actually emit light
Why is tryptophan so good for fluoresce experiments
Because it has a high quantum yeild meaning it has a high chance of emitting
So we would mutate in a trp for easy measurements
What is the order of quantum yield for chromophores
What has the lowest
Trp>tyr>phe
DNA and rna bases
If something has a quantum yeild of 0.2 what does this mean
Out of every five molecules of the thing 20% of those will emit light
So one will emit light
What is an efficient way of measuring quantum yeild
What do we have to make sure and why
We can use a standard (something that definitely fluoresces) like fluorescein to find the Z value
We know the quantum yeild of that sample
Then find the ratio of the quantum yeild of your sample vs the standard sample
Make sure that the standard has the same wavelength of excitation and emission because the instrument might not have the same sensitivity over a large range of wavelengths
What does the intensity of the fluorescence (If) depend on
What assumption are we making when doing this
Depends on the intensity of the light absorbed (Iabs)
only when there is no inner filter effect
Slide 20 and 21
Derivation write in sheet
In contrast to absorbance, the fluoresce red and blue shifts correspond to what
Red is more polar solvents (but still lower energy, only diff is the solvent interacts with the excited state)
Blue is less polar solvent
In absorabce it the opposite
Why does a red shift happen when a more polar solvents is there for fluoresces
The polarizabitilty effect: the excited state interacts more with polar solvents
Dipole moment effect: the excited state interacts with polar solvent more than ground state
This is because there’s more time during the excited state for the solvent dipoles to reorient so they interact more favourably with the excited state
Why does a red shift happen when a more polar solvents is there for fluoresces
The polarizabitilty effect: the excited state interacts more with polar solvents
Dipole moment effect: the excited state interacts with polar solvent more than ground state
This is because there’s more time during the excited state for the solvent dipoles to reorient so they interact more favourably with the excited state
If fluorescence more or less sensitive to solvent than absorbace
Why is the dipole moment effect stronger in fluoresce than absorbance
More
Because absorabce happens to fast for the dipoles to reorient , the excited state is longer and these more time for the effect to happen
