IR Spec

Question 1

What is wave number

Answer 1

1/wavelength

Question 2

IR SPECTRAL REGIONS SLIDE 3

Answer 2

Write in notes

Question 3

What are the three types of motion

Answer 3

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)

Question 4

What is # of vibration modes for non linear molecule

Linear

Also called normal modes

Answer 4

On sheet slide 4

Question 5

What has to happens for a transition in energy to be infrared?

Answer 5

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

Question 6

What are the two models of vibration and what do they mean

Answer 6

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

Question 7

Wave number for vibrations formula

Reduced mass formula

Answer 7

On sheet slide 6

Question 8

What diff in ir spec than uv

Answer 8

Has IR light source

IR Optics and detector

Scans wave numbers

Question 9

What is FTIR

What is a Fourier transformation

Answer 9

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

Question 10

How does ftir spec machine work

Answer 10

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

Question 11

Is an interferogram a spectrum

Answer 11

No

Need to do math processing to it to get absorbances vs wavenumbers ir spectrum

Question 12

What are the windows of IR sample holders/cells made of

What it’s important to note for biological sample

Answer 12

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

Question 13

Why did the experiment on slide 10 do two runs with both h20 and deuterated water for IR experiment

Answer 13

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

Question 14

Slide 11 IR absorbance values

Answer 14

Okay

Question 15

How do you overcome the problem of H2O absorbance

Answer 15

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

Question 16

What are the advantages of FTIR

Answer 16

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

Question 17

What is Fourier deconvolution

What are the ways to do it

Answer 17

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

Question 18

How do you get narrower bands on an IR spectrum

What’s the downside to this

Answer 18

Use the second derivative spectra

Th baseline of the spectra might be distorted and the noise could be amplified

Question 19

What are the biochemical applications of IR spec

Answer 19

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)

Question 20

Comp of hemoglobin slode 15

Answer 20

In notes

Question 21

What is amide 1 two and 3

Answer 21

1: 1650, the c=o stretch, depends on protien conf, used to find % of secondary structure

2. 1550, the stretch and wagging of C N H, sensitive to exchange N-H with N-D, used to monitor exposure of the backbone
3. 1300, stretch of CN bond

Question 22

If the bond order (double triple bonds ) increases what happens

Answer 22

The bind force constant increases, meaning wavenumber increases

Question 23

Slide 16 carbon oxygen stretching numbers

Answer 23

In notes

Question 24

Slide 17 Amide 1 wavenumber and corresponding secondary structure

Answer 24

In notes

Question 25

Slide 18 Example of amide 1

Answer 25

Top is diff spectrum

Sencond is enhanced diff spectrum

Upside down is second derivative

Shows that hemoglobin is at higher wavenumber than concanavlin

Question 26

What’s the difference in hydrogen and deuterium

Answer 26

Deut heavier, changes ir spec wavenumber if a d2o exchange happens

Question 27

What is isotopic labelling and why do we use it

Answer 27

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

Question 28

What can d2o exchange of the h on nitrogen to D help with

What can isotopic labelling help with

Answer 28

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)

Question 29

When you do isotopic labelling what does this do to wavenumber

Answer 29

Mass increases, so mew (reduced mass) increases, meaning wavenumber decreases

Question 30

What can the amide II band be used for

Answer 30

To follow the H to D exchange overtime to monitor “exposed” amides (peptide backbone)

Question 31

Why does amide II with exhange show more effect than amide I

Answer 31

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

Question 32

Review questions slide 23-25

Answer 32

In notes