Absorption Spec Flashcards

1
Q

What is a chromophore

A

A group of bonded atoms (that are part of a molecule) and act as a unit for excitations to that they have a characteristic spectrum

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2
Q

What are UV visible chromophores

Give examples of

A

Aromatic of conjugated systems that loose their energy difference into the uv range

Ex benzene, indole, phenol, amide, aromatic bases of nucleic acids

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3
Q

What are infrared chromophores

A

They have vibrations that involve the stretching or bending of bonds

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4
Q

What are examples of chromophores in both uv and IR

A

Benzene (UV)

Amide 1 vibration (IR)

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5
Q

Slide 3 max absorbances

A

Write down on sheet

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6
Q

How to find amount of amides in a protien

A

AA- 1-asn-gln

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7
Q

Why is the extinction coeffienct on the y axis of the absorption graph at log scale

A

To see the high extinction coefficients at low wavelengths

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8
Q

What is speacial about the number of absorption peaks for a single chromophore

A

Many diff absorption peaks at diff wavelength

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9
Q

What is an example of why we need buffer when measuring absorbance

A

At ph 12 tyrosine gets deprotonated and turns to tyrosinate

This changes it’s absorbance from the ph 7 tyrosine, which is why we need a buffer to keep a certain ph

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10
Q

Does the uv spectrum of trp change at high ph

A

No

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11
Q

Under diff ph conditions, why would amides in the peptide back bone absorb diff

A

Because the polypeptide shape is diff at diff ph

Can form helix, random coil, or beta strands

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12
Q

For lysine at What pH is each formed
helix,
random coil,
beta strands

A

helix, pH 10.8 low temp (25 deg)

random coil, 6 (25 deg)

beta strands 10.8, high temp (52 deg)

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13
Q

Why would lysine be random coil at ph 6?

A

It’s pka is 10.8 so at ph 6 it’s protonated

Carrying a charge make it change conformation

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14
Q

The spectrum of a molecule is ____

A

The sum of the spectra for all chromophores present

Like in a graph it’s show a lot of peaks because that’s all the chormophores

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15
Q

Where do nucleic acids absorb

Higher energy transitions

Why do we not measure spectra below 190nm

A

260nm

Idk

Because O2 absorbs there and we don’t want that measurement

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16
Q

What contributes to the width of an absorbance peak

A

When a chromophores undergoes excited it arrives a various vibrational levels

And the same chromophores have slightly different solvent interactions so that their ground state energy is higher or lower

Due to this, their change in energy when reaching the excited state changes and the summing of many different absorbance lines makes the broad peak

17
Q

Some spectra have a

A

Vibronic fine structure

18
Q

If small bumps on peak it’s a

If peak near beginning

If around 277

A

Benzene chromophore so phenylalanine

Amide (196)

Tyr (phenol) or trp (indole)

19
Q

How is the area of a peak measure

A

For triangular peak: w(1/2) x Emax

For Gaussian : 1.06 x w(1/2) x Emax

20
Q

What is oscillator strength (f) of a transition

What is it proportional to

A

The probability that a photon will be absorbed

Proportional to the area under the absorbance curve

21
Q

What is the occislator strength for one transition in one chromophore

A

0 to 1

22
Q

What is oclsccilator strength proportional to

A

The area under the peak

23
Q

Oscillator strength equation slide 12

A

Ok

24
Q

What is the oscillator strength of strong transitions, what about weak

What does of mean if f > 1

A

Strong : >0.1

Weak: <0.01

Means there’s more than one chromophore in the molecule

Or there is more than one transition in a single chromophore

25
Q

Slide 13 sample calc for f

A

Why didn’t we half Width of peak

26
Q

The spectrum of a chromophore has a dependence on what

The change in energy depends on what

A

Its surroundings

The substituents on the chromophore and on the solvent

27
Q

Benzene toluene and phe in uv and water wavelengths

A

Slide 14 write on sheet

28
Q

What causes red and blue shifts

What is a blue shift

What is a red shift

A

the small diff in the max wavelength of a chormophore due to the diff solvents it’s in

Going to a shorter wavelength (more separation between levels), higher energy, molecule is in more polar solvent

Going to longer wavelength (less separation between energy levels) , lower energy, thing in is less polar solvent

29
Q

What solvents give

Blue shift

Red shift

A

Blue shift : polar solvents like h2O (or aqueous buffer)

Red shift: less polar solvent like 20% glycerol of DMSO (dissolves insoluble molecules) + h2O

30
Q

Delta E is greater for what type solvent

A

More polar solvents

Because the polar solvent interacts better with the ground state than with the excited state

This makes the change in energy higher , and higher energy means shorter wavelength, means blue shift

31
Q

Why is there a red shift of trp and tyr when the buffer is made less polar

A

Intially the ground state of indole and phenol interact better with the polar water (making blue shift)

But once the buffer is made less polar by the addition of dmso, there becomes a small red shift

Smaller change in energy, longer wavelength , red shift

32
Q

What could be considered the solvent of a chromophore

A

The surrounding amino acids of that chromophore

so the solvent for the trp indole would be the other amino acids that the trp is in

And just the solution it’s in

33
Q

What does protein denaturation do to the red and blue shift of a protein

A

Denaturation moves the buried chromophore to a more polar solvent (the water surrounding the protein)

which gives a small blue shift

34
Q

What is meant by coupling between chromophores

A

When similar chromophores are close together and have the correct orientation they interact

This way they act as a UNIT for excitation

This gives two absorbance transitions at slightly diff energies because they are still two diff chromophores

35
Q

What is the result of coupling

A

The max extinction coefficient (Emax) goes down

Originally, the peaks would sum and E max would be higher

But now when interacting they have diff energies so diff wavelengths and when summing they slightly cancel each other

36
Q

What is an example of coupling

A

There’s dsDNA which is stacked and heating turns it into ssDNA which is unstacked

The unstacked ssDNA has chromophores that are not coupled (since they’re not stacked) so the absorbance is higher than the dsDNA

37
Q

Review q slode 19

A

Ok