Fluoresence 1

Question 1

What does fluorescence spec measure

Answer 1

The emmision of light

Question 2

Are all chromophores fluorophores

Answer 2

No

Question 3

Uses of fluorescent spec

Answer 3

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

Question 4

What do the dashed lines show on flourecnse spectrum

Answer 4

The absorbance

Question 5

What is in the x axis of fluorescent spectrum

Answer 5

Wavelength (nm)

Question 6

What is a jablonski diagram

Answer 6

A diagram with both absorption and emmision (fluorescence)

Question 7

Explain the steps in the jablonski

Answer 7

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

Question 8

What is a stokes shift in terms of flourensces

Answer 8

The shift in wavelength (to a higher wavelength) from absorbance to emmision

Question 9

What is the exception for emmision spectrum going to a longer wavelength then excitation soectrum

Answer 9

Atoms in the vapour phase because they don’t have that thermal decay step

Question 10

Emmision can be from

And which takes a longer time

Answer 10

Phosphorense or flourescnse

Phosphorense

Question 11

What is the wavelength range of typical stokes shifts

Answer 11

20-200nm

Question 12

What do stokes shifts depend on

Answer 12

Vibrational levels in the excited and ground state

Question 13

Which fluorophore has that largest stokes shift and what is its

Lowest

Answer 13

NADH 130

Benzene in phe (23)

Question 14

What is the Franck condon principle

Answer 14

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

Question 15

What happens to the eiullibfium bond distance between atoms on the excited state

Why

Answer 15

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)

Question 16

What does it mean for frank condin when we say the nuclei don’t move

Answer 16

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

Question 17

What does it mean for frank condin when we say the nuclei don’t move

Answer 17

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

Question 18

Explain the steps of the Morse curve depiction of extubation and emission and why it’s like that

Answer 18

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

Question 19

Explain the steps of the Morse curve depiction of extubation and emission and why it’s like that

Answer 19

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

Question 20

Emmision spectra is always shifted to ______
Than excitation

Answer 20

Longer wavelengths

Because the emitted light is at a higher energy than the excitation light

Question 21

What measures fluorescence

What’s soecial about it

Answer 21

Fluorimeter

Has two monochrometers

Question 22

In a fluorimeter how do you adjust the monochromoter to get the excitation spectrum

What about the emmision spectrum

Answer 22

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

Question 23

lol

Answer 23

Lol

Question 24

What is the inner filter effect

Answer 24

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

Question 25

How do you fix the inner filter effect

Answer 25

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

Question 26

What is the time difference between absorbance and excited state

What does this mean

Answer 26

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

Question 27

Do all excited fluorophore emit a photon?

What other things can happen

Answer 27

No

Many other things can happen like

Intersystem crossing (phosphorescence)

Energy transfer to an acceptor

Thermal inactivation (loss as heat)

Collision with other molecule

Question 28

Why is the fluoresces measured at 90 degrees to the cuvette instead of straight across the cuvette?

Answer 28

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)

Question 29

To get an emmision spectrum what do we do to the first monochromator?

Answer 29

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

Question 30

When collecting emoted light are we capturing most of it?

Answer 30

No most of it is coming off the sample and only some goes to the second monochromator

Question 31

What is a band pass

Answer 31

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

Question 32

Other than the first monochormator with is being light to the sample where else can a band pass be set

Answer 32

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

Question 33

Who sets the band pass

Answer 33

The person using the machine (operator)

Question 34

What is the typical range of a band pass

Answer 34

1-10nm

Question 35

In both the emmision and excitation band passes, what does it mean if you have a higher band pass

Answer 35

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

Question 36

What is the trade off of setting a band pass

Answer 36

The S/N ratio is higher, but the resolution is lower

Question 37

If product of the two band pass values are the same what does this mean

Answer 37

They give the same intensity

Question 38

What is quantum yield ?

Answer 38

It measures how many of the excited fluorophores actually emit light

Question 39

Why is tryptophan so good for fluoresce experiments

Answer 39

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

Question 40

What is the order of quantum yield for chromophores

What has the lowest

Answer 40

Trp>tyr>phe

DNA and rna bases

Question 41

If something has a quantum yeild of 0.2 what does this mean

Answer 41

Out of every five molecules of the thing 20% of those will emit light

So one will emit light

Question 42

What is an efficient way of measuring quantum yeild

What do we have to make sure and why

Answer 42

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

Question 43

What does the intensity of the fluorescence (If) depend on

What assumption are we making when doing this

Answer 43

Depends on the intensity of the light absorbed (Iabs)

only when there is no inner filter effect

Question 44

Slide 20 and 21

Answer 44

Derivation write in sheet

Question 45

In contrast to absorbance, the fluoresce red and blue shifts correspond to what

Answer 45

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

Question 46

Why does a red shift happen when a more polar solvents is there for fluoresces

Answer 46

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

Question 47

Why does a red shift happen when a more polar solvents is there for fluoresces

Answer 47

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

Question 48

If fluorescence more or less sensitive to solvent than absorbace

Why is the dipole moment effect stronger in fluoresce than absorbance

Answer 48

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