IR Spec Flashcards
What is wave number
1/wavelength
IR SPECTRAL REGIONS SLIDE 3
Write in notes
What are the three types of motion
Translation,
rotation (atoms of diff masses and bond between them causes thing to spin 180 on verticals axis
vibration (moving in and out by compression or stretching)
What is # of vibration modes for non linear molecule
Linear
Also called normal modes
On sheet slide 4
What has to happens for a transition in energy to be infrared?
Needs to be a change in the dipole moment involved
Ex. N2 give no dipole so not IR
C=O has dipole so is IR
What are the two models of vibration and what do they mean
Harmonic osciloscillator (compression and stretching in the bond makes increase energy)
Anharmonic oscillator: compression make energy increase but stretching makes the bond break at some point so energy doesn’t increase as much)
Graph of PE and interatomic distance
Wave number for vibrations formula
Reduced mass formula
On sheet slide 6
What diff in ir spec than uv
Has IR light source
IR Optics and detector
Scans wave numbers
What is FTIR
What is a Fourier transformation
The machine irradiate the sample with all wave numbers at the same time
then arrange all the signals that the sample gave out that corresponded to those wavenumbers
and get a spectrum with the full range of the samples absorbance at diff wavenumbers
Math procedure to analyze the data and get absorbance vs wave number
How does ftir spec machine work
The beam splitter sends light in two directions
Light goes to a moving and fixed mirror
Moving mirror affects the light and gives a Doppler effect
An interferogram that shows signal intensity vs the mirror position is made from this.
Then math happens to give spectrum of abs vs wavenumber
Is an interferogram a spectrum
No
Need to do math processing to it to get absorbances vs wavenumbers ir spectrum
What are the windows of IR sample holders/cells made of
What it’s important to note for biological sample
Nacl, Kbr, LiF, Caf2 because they’re transparent to IR radiation (so light goes directly through them)
For biological samples you use CaF2 (doesn’t dissolve) and not Kbr since Kbr dissolves in water am can go into the sample
Why did the experiment on slide 10 do two runs with both h20 and deuterated water for IR experiment
It’s to show that the two solvents may overlap in absorbance at the same wavenumbers as the protein of interest
so they did both measurements to see if a peak in one solvent can’t be seen in the other solvent
Slide 11 IR absorbance values
Okay
How do you overcome the problem of H2O absorbance
Use a short path length and high sample concentrstion (to get enough signal from sample in small length)
Use D2O (deuterated water) since it’s transparent at 1300-2100 and 2800-4000
Use FTIR which lets us subtract high values for the blank
What are the advantages of FTIR
because FTIR source is a laser, the precise wave numbers let’s us subtract the large absoebance from the solvent (via the blank)
High signal to noise ratio (since it amplifies the signal every scan it does and cancels the noise signals during each scan)
High resolution (narrower) bands because of the math stuff during or after the Fourier transformation
What is Fourier deconvolution
What are the ways to do it
The math processing to get narrower bands
Can do by multiplying the signal intensity of the interferogram by an exponential function (this exaggerates the dip in the peaks and gives better resolution)
Or second derivatives
How do you get narrower bands on an IR spectrum
What’s the downside to this
Use the second derivative spectra
Th baseline of the spectra might be distorted and the noise could be amplified
What are the biochemical applications of IR spec
Get info about:
Specific bonds or groups in a molecule or ligand (ex. carbon monoxide binding to heme)
Can detect H/D (hydrogen deuterium) exhange to analyze the exposure of certain groups to the solvent (ex. peptide linkages, SH)
Can find The secondary structure of proteins (like composition of hemogloibin)
Comp of hemoglobin slode 15
In notes
What is amide 1 two and 3
1: 1650, the c=o stretch, depends on protien conf, used to find % of secondary structure
- 1550, the stretch and wagging of C N H, sensitive to exchange N-H with N-D, used to monitor exposure of the backbone
- 1300, stretch of CN bond
If the bond order (double triple bonds ) increases what happens
The bind force constant increases, meaning wavenumber increases
Slide 16 carbon oxygen stretching numbers
In notes
Slide 17 Amide 1 wavenumber and corresponding secondary structure
In notes
Slide 18 Example of amide 1
Top is diff spectrum
Sencond is enhanced diff spectrum
Upside down is second derivative
Shows that hemoglobin is at higher wavenumber than concanavlin
What’s the difference in hydrogen and deuterium
Deut heavier, changes ir spec wavenumber if a d2o exchange happens
What is isotopic labelling and why do we use it
Change c12 to c13 or N14 to N15
because we want to get a protein whos IR wavenumber is separate from the wavenumber of other proteins
Ex. If we have two proteins that are bound together, you can make the heavy version of one of them, let them both bind and measure both spectra to see the difference in the heavy vs the normal
What can d2o exchange of the h on nitrogen to D help with
What can isotopic labelling help with
Monitoring exposure
Monitor the binding of one protein (if two proteins are bound the h is less likely to exhange with d2O)
Assigning bands on specific groups (like making the CO2 in asp 13CO2 to give it a specific band)
When you do isotopic labelling what does this do to wavenumber
Mass increases, so mew (reduced mass) increases, meaning wavenumber decreases
What can the amide II band be used for
To follow the H to D exchange overtime to monitor “exposed” amides (peptide backbone)
Why does amide II with exhange show more effect than amide I
Because the amide 2 includes the N wobble and stretch, so the h switching with D is shown
Amide 1 only looks at the c=O stretch so not showing the nh turning to nD as much
Review questions slide 23-25
In notes
