NMR Flashcards
What is nuclear spin?
• Elementary particles possess an intrinsic angular momentum (I) known as spin
• Spin is quantised ( has discreet values) and comes in multiples of ½ such that there are 2I+1 values between -I and +I (I is spin quantum number)
• E.g. if I=1/2 there are 2 states
• Protons, unpaired electrons and neutrons possess spin and I=1/2
• Nuclei comprising even protons and even neutrons have 0 spin angular momentum
• Nuclei with odd protons and odd neurons have integral spin quantum numbers
• The rest have ½ integral spin
How does the nucleus create a magnetic field
charged nucleus rotating with angular frequency creates a magnetic field, B
• Magnet field is associated with the atomic level
• Proton behaves as a magnet
• C12has 0 spin as protons and neutrons are even
• Out of a magnetic field the 2 states have the same energy
• You can probe a molecule by bringing magnetic fields close together
• If you put a particle in a large magnetic field it creates an energy gap between the 2 states
• The particles can align parallel (lower energy) or anti parallel (higher energy)
• Strength of the applied magnetic field determines the energy gap of a given nuclei
• Higher magnetic fields give higher sensitivity (energy gap gives sensitivity)
• Energy gap of 10^-5 is infrared —> not very high frequency —> good as x-ray and UV damage biological molecules
• Most small nuclei have ½ spin
• Bigger nuclei spin slower
• Magnetogyric ratio correlated to size of nucleus
Frequently used nuclei with spin
• The proton is the most sensitive nucleus
• 13C/15N are low abundance therefore low sensitivity
• The deuteron (2H) is NMR active
How is an NMR spectrum recorded
• Radiofrquency can be swept through all resonances and the absorption is measured
What lies Larmor precession of nuclear spins
• Magnetic moment associated with a spinning spherical charge will process in an external magnetic field (B0)
• Known as the larmor precession
• Absorption occurs when the radiofrequency matches the larmor frequency
• Axis is at a slightly offset angle so not actually parallel to the magnetic field
• External magnetic field applies residual force so the nuclei precess along the magnetic field
• Can probe this frequency
• Correlated to the frequency for absorption
• Get resonance with radio frequency
• Large numbers of spin 1/2 nuclei at equilibrium in a strong external magnetic field B0
• Parallel spin is lower energy so more populated
• Antiparallel is less populated at equilibrium
• Lots of spins are at larmor precession at appropriate frequency
• Results in a net parallel magnetic moment (M0) aligned with magnetic field
Effect of a radio frequency pulse
• Apply at 90 deg to z axis, along y axis so exerts force on magnetisation
• Radiofrequency pulse very short
• Micro seconds in length
• Net magnetisation shifts away from the z axis and towards the y axis
• This is due to spin flips between the +1/2 and -1/2 states until the spin precessions about the z-axis becomes coherent (bunched up and non-random)
• Generates a significant y component to the net magnetisation M
• At equilibrium they are evenly distributed around the cone
• Switch off the pulse then the nuclei precess about z and return to equilibrium
• Magnetisation spirals back to z
• Then you can pulse again
• We detect coherence as it gives a large magnetic component
Why use a short radio frequency pulse
• A short radiofrequency pulse contains many frequencies in a broad band and thus can excite resonances of all nuclear spins in a sample at the same time
• Shorter pulses contain more frequency info
• Can only construct a truncated wave by adding different sin waves together
• Need to add a range of frequencies to get constructive/destructive interference
• Fourier pairs are 2 functions: frequency domain and time domain
• Decaying radiofrequncy signal generated in the receiver coil is called the free induction decay (FID)
• Related to the traditional frequency spectrum through a Fourier transform
• Fourier transform flips frequency<—> time
NMR parameters
• Chemical shift- frequency axis
• Integral- area under peak
• Scalar coupling (J) – separation between adjacent doublets/ triplets
• Relaxation times (T1 and T2) – time taken to get back to x from y – manifests in line width
• Dipolar coupling (D) – doesn’t manifest in spectrum
• Nuclear Overhauser effect (NOE) – can’t see in spectrum
Chemical shift
• Most important parameter
• X axis
• If multiple protons in a sample e.g. ethanol, protons don’t all resonate at the same frequency
• Due to the e- cloud around the nucleus
• Electrons associated with atoms circulate about the direction of an applied magnetic field
• Causes a small, shielding local magnetic field at the nucleus
• Reduces B0 slightly
• Binduced is dependent on B0 and direction depends on where you are relative to the nucleus
• Inside the nucleus it is opposite to B0
• Add or subtract the vectors
• Electronegative groups withdraw electrons away from the nucleus, reducing the shielding effect
• Nucleus experiences larger magnetic field so goes to higher frequency
• Called deshielding
Chemical shift of aromatics
• Field from pi e- can oppose or reinforce the applied field (e.g. aromatics)
• Delocalised pi e- in an aromatic ring circulate in response to an applied magnetic field
• This ring current generates a local magnetic field which opposes the applied magnetic field
• Protons at the edge of the ring feel a larger magnetic field as they are deshielded
• Aromatic ring protons will therefore resonate at higher frequency and exhibit a downfield shift
• Aromatic groups behave like electronegative groups
How is chemical shift calculated
• Chemical shift expresses frequency as a fraction of lab magnetic field so you can compare different spectrometers recorded at different frequencies
• TMS (tetramethylsilane) is the reference
• TMS has 4 methyl groups that are all equivalent
• Very shifted right due to silicone
= shift downfield from TMS (in Hz) / spectrometer frequency (in MHz)
Chemical shift equivalence
• If rotation, reflection or inversion can swap the protons they are equivalent
• Aromatic ring can also spin
• Nuclei that are interchangeable by a symmetry operation or rapid exchange on the NMR timescale have the same chemical shift
• Conformational exchange gives an average value so you are presented with a single species
• Exchangeable protons can swap, if this is fast enough we get a single species
Chemical shift ranges
• Less shielded – high frequency
• More shielded – low frequency
• Chemical shift gives information regarding types of chemical groups
Using integrals
• The area under the peak gives us the relative numbers of nuclei
• Not always true – NOE
Scalar coupling (J) of 2 spin systems (AX)
• Adjacent, non-equivalent spin ½ nuclei will experience a spin-spin interaction
• This coupling is communicated through electrons (spin ½) in bonds (and up to 4 bonds)
• The frequency of HA is different dependent on the alignment of HX
• 2 possible states of A so X forms a doublet
• HX and Ha can’t be in same position/ have the same properties
• 2 different energy states manifest as 2 magnetically different environments
• Direct through bond interaction between 2 neighbouring spins
• For triplet there are 2 protons adjacent , 3 possible states: 2 parallel, 2 antiparallel, 1 of each
Scalar coupling to exchangeable OH/NHs
• Samples in organic solvents show splitting to OH but a small amount of D2O will remove splitting
• Any exchangeable group is replaced by D so peak disappears as D is instead of H
• If you dissolve in 100% D2O you see NO exchangeable groups, no amides or hydroxyls
• Note: biological samples in H2O show no OH resonances due to rapid exchange with the high conc of H2O unless exchange is slowed through internal H bonding
• NHs are more readily observed unless D2O is used
• Amides can be seen as exchange is slow on NMR timescale
• Can ignore OH groups
