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Bchem 471 > Fluoresence 1 > Flashcards

Fluoresence 1 Flashcards

1
Q

What does fluorescence spec measure

A

The emmision of light

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2
Q

Are all chromophores fluorophores

A

No

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3
Q

Uses of fluorescent spec

A

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

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4
Q

What do the dashed lines show on flourecnse spectrum

A

The absorbance

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5
Q

What is in the x axis of fluorescent spectrum

A

Wavelength (nm)

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6
Q

What is a jablonski diagram

A

A diagram with both absorption and emmision (fluorescence)

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7
Q

Explain the steps in the jablonski

A

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

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8
Q

What is a stokes shift in terms of flourensces

A

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

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9
Q

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

A

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

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10
Q

Emmision can be from

And which takes a longer time

A

Phosphorense or flourescnse

Phosphorense

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11
Q

What is the wavelength range of typical stokes shifts

A

20-200nm

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12
Q

What do stokes shifts depend on

A

Vibrational levels in the excited and ground state

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13
Q

Which fluorophore has that largest stokes shift and what is its

Lowest

A

NADH 130

Benzene in phe (23)

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14
Q

What is the Franck condon principle

A

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

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15
Q

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

Why

A

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)

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16
Q

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

A

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

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17
Q

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

A

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

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18
Q

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

A

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

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19
Q

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

A

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

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20
Q

Emmision spectra is always shifted to ______
Than excitation

A

Longer wavelengths

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

21
Q

What measures fluorescence

What’s soecial about it

A

Fluorimeter

Has two monochrometers

22
Q

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

What about the emmision spectrum

A

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

23
Q

lol

A

Lol

24
Q

What is the inner filter effect

A

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

25
Q

How do you fix the inner filter effect

A

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

26
Q

What is the time difference between absorbance and excited state

What does this mean

A

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

27
Q

Do all excited fluorophore emit a photon?

What other things can happen

A

No

Many other things can happen like

Intersystem crossing (phosphorescence)

Energy transfer to an acceptor

Thermal inactivation (loss as heat)

Collision with other molecule

28
Q

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

A

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)

29
Q

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

A

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

30
Q

When collecting emoted light are we capturing most of it?

A

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

31
Q

What is a band pass

A

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

32
Q

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

A

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

33
Q

Who sets the band pass

A

The person using the machine (operator)

34
Q

What is the typical range of a band pass

A

1-10nm

35
Q

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

A

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

36
Q

What is the trade off of setting a band pass

A

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

37
Q

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

A

They give the same intensity

38
Q

What is quantum yield ?

A

It measures how many of the excited fluorophores actually emit light

39
Q

Why is tryptophan so good for fluoresce experiments

A

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

40
Q

What is the order of quantum yield for chromophores

What has the lowest

A

Trp>tyr>phe

DNA and rna bases

41
Q

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

A

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

So one will emit light

42
Q

What is an efficient way of measuring quantum yeild

What do we have to make sure and why

A

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

43
Q

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

What assumption are we making when doing this

A

Depends on the intensity of the light absorbed (Iabs)

only when there is no inner filter effect

44
Q

Slide 20 and 21

A

Derivation write in sheet

45
Q

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

A

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

46
Q

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

A

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

47
Q

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

A

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

48
Q

If fluorescence more or less sensitive to solvent than absorbace

Why is the dipole moment effect stronger in fluoresce than absorbance

A

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

Bchem 471 (24 decks)

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