3 - NMR and Nascent Chain Folding Flashcards
What is nuclear spin?
All nuclei spin about their axis, creating a localised magnetic field. Usually the direction of the N-S axis of this field is random, and they all have equal energy.
What effect does an external magnetic field have on the nuclear spins of the atoms?
when a strong external magnetic field is applied the magnetic axis sort into two states; they orient themselves either with the direction of the magnetic field or against it.
When an external field is applied, how populated are the different spin states?
Aligning the axis with the external field is a lower energy state then aligning against it, though only slightly; at room temperature both are populated (according to the Boltzmann distribution) with only a slight bias towards alignment. However this bias is enough to give the sample a net magnetic moment.
What is the Larmor frequency?
When in an external magnetic field, the N-S axis of the nuclear magnetic field precesses around the fixed direction of B0. The frequency of this precession is called the Larmor Frequency, and is determined by the gyromagnetic ratio; a constant for each different nucleus.
What does the precession of the nuclei allow for?
The precession allows the nuclei to resonate with electromagnetic radiation when the frequency of the wave is equal to the Larmor frequency. This puts it in the radio frequency range; 10-1000 MHz. The machine being used must be tuned to the frequency of the nuclei in question.
What does the frequency of an NMR machine describe?
The higher the maximum frequency of the NMR machine the higher the sensitivity and resolution of the resulting data.
Most NMR machines operate at 500-700MHz, and are capable of looking at multiple nuclei of a biological sample. The 800MHz and the 950MHz machines are capable of being used for high-throughput metabolomics studies.
How can nuclear spin be thought of quantum mechanically?
Nuclear spin is a quantum mechanical property of the quarks within the nucleus, hence why the total spin number of the nucleus is dependent on the baryon combination that constructs it. The total spin of the element/isotope (I) can be any number that is a multiple of ½ - 0, ½, 1, 1½, 2, 2½, etc.
What nuclei are NMR active?
Only nuclei with odd mass numbers are NMR active, with the exception of 2H and 15N, but the most common nuclei in NMR are 1H and 13C which are both I = ½ (‘spin-half’ nuclei).
The total number of spin states = 2I + 1, so these have 2 spin states; up and down.
How is the sample loaded into an NMR machine?
When the sample is within the magnetic field produced by the superconducting (and super-cooled – 4.2K) magnets it is spun to ensure field homogeneity.
What happens when a sample is probed by radio waves?
The radio transmitter and receiver are tuned to the Larmor frequency in question and the transmitter saturates the sample, flipping the spin states of the nuclei to the higher energy level.
What is measured in NMR spectroscopy?
The change in the effective energy gap between the spin states, and hence the radio frequency at which they absorb and emit. This will be different for different nuclei due to their electronic environment.
What is electronic shielding?
Electrons themselves also spin, usually in a way that opposes the external magnetic field. This reduces the effective B0 upon the nucleus. Hence the larger the decrease in the energy separation the more electrons are in the area of that nucleus.
How is electronic shielding of the nuclei measured?
This must be measured relative to a known structure, often TMS, which is included with the sample.
How can measurements of shielding build up a picture of the molecule?
Since the strength of the signal is proportional to the population of nuclei in the same electronic environment, a picture can be built up of the molecule being imaged.
The variation in RF over time can be converted into a spectrum of populated frequencies through a Fourier transform.
What else can effect the difference in energy between the spin states?
The difference in spin energy levels between a nucleus’s spin state is also dependent on the spin state of the nuclei around it. This is called spin-coupling; when an adjacent nucleus is in the spin up state the nucleus in question will have a slightly different energy of absorption then when the adjacent nucleus is in ‘spin down’.
What does spin coupling do to the spectrum?
This splits the peak, and allows identification of the atoms adjacent to each nucleus. This allows us to use the scalar and dipolar couplings to match the set of signals with a molecular structure. This is called Resonance Assignment.
What is scalar and dipolar coupling?
Scalar coupling is though bonds, position of nuclei relative to one another constant-ish.
Dipolar coupling though space - not connected by bonds.
What is the nuclear overhauser effect.
Coupling through space as opposed to through adjacent bonds is a phenomenon known as the Nuclear Overhauser Effect (NOE). This can be used to analyse relative positions of atoms up to 5Å apart.
What does the 1D spectrum of a protein look like?
Being huge molecules, proteins produce hundreds of thousands of signals and resonance assignment becomes very difficult. Very little structural information can be derived from 1D NMR.
To get around this issue, 2D NMR can be used.