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Imperial (Y2) - Structural Biology > Steven Matthews - NMR > Flashcards

Steven Matthews - NMR Flashcards

1
Q

What are the main applications/uses of NMR?

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2
Q

What is Nuclear Spin?

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3
Q

For a spin ½ nucleus, what happens when we place the nuclear magnetic field into external magnetic field?

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4
Q

What does the energy level between these two states depend on? Can we manipulate the number of nuclei in these two states?

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5
Q

Is NMR as sensitive as other techniques that use higher magnetic fields ?

A

NMR is not as sensitive as other techniques that use higher magnetic fields

Because we are dealing with very small energy differences between spin states.

Example - The energy differences are 10-5 smaller than that associated with infrared.

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6
Q

Examples of frequently used spin nuclei, spin state and their relative sensitivity?

A

Proton has largest γ as it has the smallest nucleus

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7
Q

In the past how were NMR spectra recorded?

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8
Q

What does the Larmor precession of nuclear spins refer to?

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9
Q

How can we represent the net magnetisation of spin 1/2 nuclei in the presence of strong external magnetic field?

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10
Q

What happens to the net magnetization when we apply a radiofrequency pulse?

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11
Q

Describe what happens to the spin states in the images when a radiofrequency pulse is applied at 90o?

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12
Q

What is the relationship between the length of a frequency pulse and the number of frequencies it contains?

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13
Q

Outline the basic principle behind NMR?

Hint - Pulse, free induction decay and Fourier transform

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14
Q

What are the different parameters on an NMR spectra?

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15
Q

What is meant by the term chemical shift?

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16
Q

What scale is used to measure the chemical shift?

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17
Q

When do we get chemical shift equivalence? When is the chemical shift for a proton the same?

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18
Q

What are the chemical shift ranges, what type of functional groups do we find to the left (downfield) and right (upfield) on the chemical shift spectrum?

A

Think of it as electron density

Left (downfield) - Low electron density around nucleus - Presence of electron withdrawing group

Right (upfield) - high electron density around nucleus

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19
Q

What the hell can we actually use chemical shifts for?

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20
Q

What do we use integrals for/relative height of the peak? What happens to the integrals when the NMR reflects a mixture of molecules?

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21
Q

In NMR, what does coupling refer to?

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22
Q

What is scalar coupling?

Example - Two spin system?

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23
Q

What are the different possible scalar coupling patterns? What can they be used for?

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24
Q

Typical scalar coupling values?

A

Coupling constant (J) - Hz

Indicate the degree of splitting that is found between these interactions - to what extent the peaks split

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25
What is one key difference that arises when performing NMR in an organic solvent vs. aq. solution?
Main difference - Exchangeable -OHs & -NH3 will only appear on the spectrum under specific conditions 1. **When NMR is performed in H2O** --\> OH shows no resonances due to rapid exchange with the high concentration of H2O (chemical shift averaged to H2O) unless exchange is slowed through internal H-bonding Basically Protons attached to OH exposed to H2O will rapidly exchange - NMR can pick up on this - will be averaged out to H2O chemical shift Likewise, NHs are similar to OH but are exchange at a slower rate at physiological pH - more readily observed 2. **When NMR is performed in Organic solvents -** no free exchange of protons with these groups - Hence, we will get a peak for OHs and NHs (just like any other proton environments) + will contribute to the coupling (splitting) of peaks! But! If we were to add D2O to the organic solvent solution - we lose that proton signal plus any coupling it may have
26
Are NMR spectra normally recorded in H2O or organic solvents?
Normally NMR spectra are recorded in Water
27
What do these two graphs show you?
Remember... T1 - Refers to the return towards the net bulk magnetization - equilibrium T2 - Dispersal of spins --\> entropic increase\* - move away from spin coherence - increase in disorder Answer from the overlord is pending! - Answered
28
Why does the spin-spin relaxation change depending on the size of the molecule?
29
What does dipolar coupling refer to?
30
How is the Nuclear Nuclear Overhauser effect (NOE) used to indirectly measure dipolar coupling?
31
Why would we use Carbon-13 NMR?
32
Three type of Carbon-13 (13C) NMR Spectra we should be aware of?
33
Are the NMR spectra of peptides and proteins very complex
34
If an NMR spectra is very complex, how can we make it simpler to analyse?
35
How do we encode a second frequency dimension into our 2D NMR?
36
How would a 2D 1H-1H Correlation Spectroscopy (COSY) look like?
1. Peaks that lie on the diagonal line correspond to the peaks one would see on a 1D NMR 2. Cross peaks - They identify proton pairs that are in close proximity COSY - Number of bonds away (Max 3-4) NOESY - Less than 5Å away
37
What do you mean by 2D NMR spectra can be used to resolve overlaps?
38
Draw a 2D COSY spectrum for the following molecule
39
Does each amino acid have its own COSY spectrum?
40
When drawing any sort of NMR spectra, whether that 1D, 2D etc., what should you include?
Apart from drawing the spectra out YOU MUST include a written description to explain why you drew the spectra they way you did? e. g. 1. What did you give that group that chemical shift - shielding? 2. What did you assign does scalar coupling patterns - peak splitting? 3. Why did you draw does cross-peaks on the 2D spectra?
41
When would we observe a cross-peak between two environments on a NOESY spectra?
42
Apart from COSY and NOESY, what other 2D spectra are there which we can use?
43
Outline what a 13C-1H HMQC & 13C-1H HMBC are...
44
Can you obtain integrals from a 2D NMR spectra?
2D spectra can be used to obtain integrals for example to quantify the number of carbons in HMQC and HMBC but.. It is more accurate to run a 1D spectra.
45
On a 2D spectra are the frequencies on the X or Y axis recorded directly?
Remember we for a 2D spectra we encode one frequency into another
46
Why do elementary (Protons, neutron, electron) particles have magnetic moment?
They possess charge + spin (angular momentum) Hence, movement of a charged creates a magnetic field --\> a magnetic moment is created Thus, depending on the nuclear make of a nucleus we can get a magnetic moment
47
Why does the proton have greatest magnetic moment?
Higher charged density results in greater magnetic moment A proton is the smallest/highest charged density --\> resulting in the greatest magnetic moment
48
How do spin 1/2 particles, behave in an external magnetic field?
With spin ½ we have two orientations which means that they can align **parallel** or **anti-parallel** to th external magnetic field Since it can’t align perfectly it processes around the external magnetic field
49
How many different states does a particle with a spin quantum number equal to 1 have (I=1)?
Number of orientations - determined using the following equation 2I + 1 Where 'I' is the spin quantum number Spin 1 has three orientations which are... -1, 0 and +1
50
What would happen if we were to increase the strength of the external magentic field?
Increase external magnetic field --\> the energy difference between spin states increases Hence, we use have to use a higher energy pulse to match this energy difference
51
When running NMR, do you need highly concentrated solutions of your protein?
Yes, it is preferable to have highly concentrated solutions of your protein. e.g. ~1mM or 15mg ml-1 for a 15 kDa protein
52
What are the chemical shifts (rough estimate) that correspond to -CH3 compared to a aromatic group?
1. -CH3 proton --\> typically exhibits a chemical shift of 1ppm 2. Aromatics proton --\> typically exhibit a chemical shift of 7ppm Note - Generally speaking the chemical shifts of protons range from 0 to 9 ppm!
53
What size protein is NMR limited to?
Generally limited to proteins that are less than 50kDA - but the resolving power is certain to increase in the future
54
What are the different scalar coupling patterns? How can we figure out the intensity ratio for each pattern?
55
What is the N+1 rule - Peak splitting?
56
How do you use the N+1 rule, when there are two hydrogen environments surrounding that are not equivalent, surrounding a central hyrdogen environment?
Apply the N+1 rule to each surrounding hydrogen environment and multiply them together - to get the splitting pattern Notice that in this case we only have 6 peaks as some peaks overlap But note.... You can either have 4 x 3 - Quartet of triplets or... 3 x 4 - Triplet of quartets
57
How can you draw the following NMR spectra... 4 x 3 - Quartet of triplets or... 3 x 4 - Triplet of quartets
58
Why do we get the intensity ratios that follow the pascals triangle?
59
Explain why the following molecule would produce a doublet with a 1:1 ratio on the NMR spectra?
60
Why would the following molecule give rise to a triplet with an intensity ratio of 1:2:1?
61
Why would the following molecule give rise to a triplet with an intensity ratio of 1:3:3:1?
62
How is intensity represented on 2D NMR spectra?
Intensity is depicted as countour lines - just like on a map that shows height
63
How is 2D NMR data collected experimentally?
64
Outline how to analyse HMQC/HSQC.
65
Outline how to analyse HMBC?
66
Why do we get signals on HMBC NMR spectra that don't correspond to a H1 peak?
Known as satellite signals that lay symmetrically either side of a proton signal. These signals are not a legitimate HMBC signal but rather HMQC signals bleading through on to our HMBC spectra - Show 1J Bonding
67
On a HMBC, is there any correlation between 2J and 3J coupling and the signal strenght?
In Aromatic/conjugated systems 3J signal will almost always be stronger than 2J In Aliphatic systems 2J tend to be stronger - but is more subjective

Imperial (Y2) - Structural Biology (6 decks)

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