Absorption Spec Flashcards
What is a chromophore
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
What are UV visible chromophores
Give examples of
Aromatic of conjugated systems that loose their energy difference into the uv range
Ex benzene, indole, phenol, amide, aromatic bases of nucleic acids
What are infrared chromophores
They have vibrations that involve the stretching or bending of bonds
What are examples of chromophores in both uv and IR
Benzene (UV)
Amide 1 vibration (IR)
Slide 3 max absorbances
Write down on sheet
How to find amount of amides in a protien
AA- 1-asn-gln
Why is the extinction coeffienct on the y axis of the absorption graph at log scale
To see the high extinction coefficients at low wavelengths
What is speacial about the number of absorption peaks for a single chromophore
Many diff absorption peaks at diff wavelength
What is an example of why we need buffer when measuring absorbance
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
Does the uv spectrum of trp change at high ph
No
Under diff ph conditions, why would amides in the peptide back bone absorb diff
Because the polypeptide shape is diff at diff ph
Can form helix, random coil, or beta strands
For lysine at What pH is each formed
helix,
random coil,
beta strands
helix, pH 10.8 low temp (25 deg)
random coil, 6 (25 deg)
beta strands 10.8, high temp (52 deg)
Why would lysine be random coil at ph 6?
It’s pka is 10.8 so at ph 6 it’s protonated
Carrying a charge make it change conformation
The spectrum of a molecule is ____
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
Where do nucleic acids absorb
Higher energy transitions
Why do we not measure spectra below 190nm
260nm
Idk
Because O2 absorbs there and we don’t want that measurement
What contributes to the width of an absorbance peak
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
Some spectra have a
Vibronic fine structure
If small bumps on peak it’s a
If peak near beginning
If around 277
Benzene chromophore so phenylalanine
Amide (196)
Tyr (phenol) or trp (indole)
How is the area of a peak measure
For triangular peak: w(1/2) x Emax
For Gaussian : 1.06 x w(1/2) x Emax
What is oscillator strength (f) of a transition
What is it proportional to
The probability that a photon will be absorbed
Proportional to the area under the absorbance curve
What is the occislator strength for one transition in one chromophore
0 to 1
What is oclsccilator strength proportional to
The area under the peak
Oscillator strength equation slide 12
Ok
What is the oscillator strength of strong transitions, what about weak
What does of mean if f > 1
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
Slide 13 sample calc for f
Why didn’t we half Width of peak
The spectrum of a chromophore has a dependence on what
The change in energy depends on what
Its surroundings
The substituents on the chromophore and on the solvent
Benzene toluene and phe in uv and water wavelengths
Slide 14 write on sheet
What causes red and blue shifts
What is a blue shift
What is a red shift
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
What solvents give
Blue shift
Red shift
Blue shift : polar solvents like h2O (or aqueous buffer)
Red shift: less polar solvent like 20% glycerol of DMSO (dissolves insoluble molecules) + h2O
Delta E is greater for what type solvent
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
Why is there a red shift of trp and tyr when the buffer is made less polar
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
What could be considered the solvent of a chromophore
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
What does protein denaturation do to the red and blue shift of a protein
Denaturation moves the buried chromophore to a more polar solvent (the water surrounding the protein)
which gives a small blue shift
What is meant by coupling between chromophores
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
What is the result of coupling
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
What is an example of coupling
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
Review q slode 19
Ok
