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Bchem 471 > IR Spec > Flashcards

IR Spec Flashcards

1
Q

What is wave number

A

1/wavelength

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2
Q

IR SPECTRAL REGIONS SLIDE 3

A

Write in notes

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3
Q

What are the three types of motion

A

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)

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4
Q

What is # of vibration modes for non linear molecule

Linear

Also called normal modes

A

On sheet slide 4

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5
Q

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

A

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

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6
Q

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

A

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

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7
Q

Wave number for vibrations formula

Reduced mass formula

A

On sheet slide 6

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8
Q

What diff in ir spec than uv

A

Has IR light source

IR Optics and detector

Scans wave numbers

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9
Q

What is FTIR

What is a Fourier transformation

A

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

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10
Q

How does ftir spec machine work

A

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

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11
Q

Is an interferogram a spectrum

A

No

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

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12
Q

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

What it’s important to note for biological sample

A

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

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13
Q

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

A

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

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14
Q

Slide 11 IR absorbance values

A

Okay

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15
Q

How do you overcome the problem of H2O absorbance

A

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

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16
Q

What are the advantages of FTIR

A

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

17
Q

What is Fourier deconvolution

What are the ways to do it

A

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

18
Q

How do you get narrower bands on an IR spectrum

What’s the downside to this

A

Use the second derivative spectra

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

19
Q

What are the biochemical applications of IR spec

A

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)

20
Q

Comp of hemoglobin slode 15

A

In notes

21
Q

What is amide 1 two and 3

A

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

  1. 1550, the stretch and wagging of C N H, sensitive to exchange N-H with N-D, used to monitor exposure of the backbone
  2. 1300, stretch of CN bond
22
Q

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

A

The bind force constant increases, meaning wavenumber increases

23
Q

Slide 16 carbon oxygen stretching numbers

A

In notes

24
Q

Slide 17 Amide 1 wavenumber and corresponding secondary structure

A

In notes

25
Q

Slide 18 Example of amide 1

A

Top is diff spectrum

Sencond is enhanced diff spectrum

Upside down is second derivative

Shows that hemoglobin is at higher wavenumber than concanavlin

26
Q

What’s the difference in hydrogen and deuterium

A

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

27
Q

What is isotopic labelling and why do we use it

A

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

28
Q

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

What can isotopic labelling help with

A

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)

29
Q

When you do isotopic labelling what does this do to wavenumber

A

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

30
Q

What can the amide II band be used for

A

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

31
Q

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

A

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

32
Q

Review questions slide 23-25

A

In notes

Bchem 471 (24 decks)

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