Absorption Spec

Question 1

What is a chromophore

Answer 1

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

Question 2

What are UV visible chromophores

Give examples of

Answer 2

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

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

Question 3

What are infrared chromophores

Answer 3

They have vibrations that involve the stretching or bending of bonds

Question 4

What are examples of chromophores in both uv and IR

Answer 4

Benzene (UV)

Amide 1 vibration (IR)

Question 5

Slide 3 max absorbances

Answer 5

Write down on sheet

Question 6

How to find amount of amides in a protien

Answer 6

AA- 1-asn-gln

Question 7

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

Answer 7

To see the high extinction coefficients at low wavelengths

Question 8

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

Answer 8

Many diff absorption peaks at diff wavelength

Question 9

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

Answer 9

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

Question 10

Does the uv spectrum of trp change at high ph

Answer 10

No

Question 11

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

Answer 11

Because the polypeptide shape is diff at diff ph

Can form helix, random coil, or beta strands

Question 12

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

Answer 12

helix, pH 10.8 low temp (25 deg)

random coil, 6 (25 deg)

beta strands 10.8, high temp (52 deg)

Question 13

Why would lysine be random coil at ph 6?

Answer 13

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

Carrying a charge make it change conformation

Question 14

The spectrum of a molecule is ____

Answer 14

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

Question 15

Where do nucleic acids absorb

Higher energy transitions

Why do we not measure spectra below 190nm

Answer 15

260nm

Idk

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

Question 16

What contributes to the width of an absorbance peak

Answer 16

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

Question 17

Some spectra have a

Answer 17

Vibronic fine structure

Question 18

If small bumps on peak it’s a

If peak near beginning

If around 277

Answer 18

Benzene chromophore so phenylalanine

Amide (196)

Tyr (phenol) or trp (indole)

Question 19

How is the area of a peak measure

Answer 19

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

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

Question 20

What is oscillator strength (f) of a transition

What is it proportional to

Answer 20

The probability that a photon will be absorbed

Proportional to the area under the absorbance curve

Question 21

What is the occislator strength for one transition in one chromophore

Answer 21

0 to 1

Question 22

What is oclsccilator strength proportional to

Answer 22

The area under the peak

Question 23

Oscillator strength equation slide 12

Answer 23

Ok

Question 24

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

What does of mean if f > 1

Answer 24

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

Question 25

Slide 13 sample calc for f

Answer 25

Why didn’t we half Width of peak

Question 26

The spectrum of a chromophore has a dependence on what The change in energy depends on what

Answer 26

Its surroundings The substituents on the chromophore and on the solvent

Question 27

Benzene toluene and phe in uv and water wavelengths

Answer 27

Slide 14 write on sheet

Question 28

What causes red and blue shifts What is a blue shift What is a red shift

Answer 28

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

Question 29

What solvents give Blue shift Red shift

Answer 29

Blue shift : polar solvents like h2O (or aqueous buffer) Red shift: less polar solvent like 20% glycerol of DMSO (dissolves insoluble molecules) + h2O

Question 30

Delta E is greater for what type solvent

Answer 30

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

Question 31

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

Answer 31

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

Question 32

What could be considered the solvent of a chromophore

Answer 32

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

Question 33

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

Answer 33

Denaturation moves the buried chromophore to a more polar solvent (the water surrounding the protein) which gives a small blue shift

Question 34

What is meant by coupling between chromophores

Answer 34

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

Question 35

What is the result of coupling

Answer 35

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

Question 36

What is an example of coupling

Answer 36

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

Question 37

Review q slode 19

Answer 37

Ok