NMR

Question 1

Background

Answer 1

Nucleus positive charged - treated as if it’s spinning
Nucleus takes up one of a fixed number of orientations
Different isotopes of same element have different orientation
1H, 13C main isotopes
Orientation = energy level
Nucleus absorbs energy = jumps to higher level
Nucleus emits energy = jumps to lower level
Detected emitted energy as electromagnetic radiation - forms NMR spectrum

Question 2

Fourier transform NMR

Answer 2

Samples placed in magnetic field pulse of electromagnetic radiation covering a range of frequencies for a specific emitted by emitter coil
Nucleus absorbs and re emits radiation
Emitted radiation detected, frequencies converted to spectrum by a mathematical process - Fourier transformation
Spectra can be added together to reduce baseline noise and detect weaker peaks

Question 3

NMR spectrum

Answer 3

Sample solution placed in glass NMR tube, spinner added around tube, tube added to spectrometer from top

There a flow of air coming up from central bore of spectrometer, NMR sample gently lowered into position by reducing upwards flow of air, tube held in position by poles of the magnet by upwards flow of air

Air flow spins sample to improve spectra, sample tube ejected by increasing upwards flow of air

Emitter and receiver coils contained in a probe, different probes used for different applications, NMR tube sits on top of probe

Question 4

NMR magnet

Answer 4

Superconducting magnet - has to be cooled to liquid helium temperatures
Magnet contained in liquid helium reservoir

Question 5

NMR of solids - MAS

Answer 5

Solids give spectra with broad peaks, various techniques to narrow peaks

MAS - solid sample packed in rotor - held at angle - 54.74 to applied magnetic field - sample spun by flow of air - smaller rotor spins faster

Question 6

D20 shake

Answer 6

If presence of OH, NH in organic compound is suspected - spectrum run - D20 added - protons replaced with D - peaks due to OH, NH disappear - avoids peaks from organic solvents

Question 7

Multipulse techniques

Answer 7

Multiple pulse techniques - sequence of pulses emitted by spectrometer at NMR frequency of nucleus being studied - pulses last for specific time lengths - given as angles - larger angles = longer pulse

Pulses separated by time intervals - frequencies emitted by nucleus observed

Question 8

Coupling and decoupling

Answer 8

Coupling - magnetic interaction that occurs between magnetic nuclei - can occur between 1H and 13C along a carbon chain

Decoupling - nucleus of interest 13C or 15C irradiated - decoupling removed interaction between 1H nucleus and adjacent nucleus it’s coupling to

Question 9

INEPT

Answer 9

Insensitive nuclei enhanced polarisation transfer

Multipulse technique - increases sensitivity of nuclei that gives weak peaks

13C peaks increased 4X
15N peaks increased 10X

Question 10

DEPT

Answer 10

Distortionless enhancement by polarisation transfer

Also improves sensitivity of weak peaks - improvement effected by number of protons on the insensitive nucleus - can be used in 13C to determine number of protons attached to each carbon

Question 11

Correlation spectroscopy

Answer 11

Used to identify which nucleus are close to each other - can be close through bonds or space - shown as contour plots

COSY, HSQC, HMBC

Question 12

COSY

Answer 12

Identifies protons on adjacent carbon atoms - couple to each other - sample irradiate with a sequence of pulses at the 1H frequency - protons that couple identified as cross peaks

Cross peaks between Ha - Hb, Hb - Hc

Cross peaks by couple protons appear asymmetrically on either side of diagonal

Question 13

HSQC

Answer 13

Heteronuclear correlation - identifies carbon atoms that are directly attached to each other

Cross peaks between Ha - Ca
No cross peaks between each proton and other carbons

Question 14

Protein structure determination - XRD

Answer 14

Single x ray diffraction - beam of monochromatic x ray directed at crystal of protein - x ray beam diffracted - passes through crystal - emerges at specific angles relative to undiffracted beam - Positions and intensities of beam recorded

Performed using four circle diffractometer - crystal mounted on goniometer - directly over centre of turntable - crystal can be rotated around 3 axes - detector roared into position around crystal to detect each diffracted beam

Question 15

NMR VS XRD

Answer 15

XRD determines long range order - atoms in same relative position in a molecule - in an ordered arrangement of molecules - in a crystal structure
NMR determines short range order - what chemical environment is - what the atoms are - within a short distance of each other

XRD - protein must be in crystalline form
NMR - can be in solution or solid phase

Question 16

NMR for protein structure determination

Answer 16

Proteins to be analysed labelled with 15N and 13C isotopes - experiments carried out to obtain NMR spectra

Chemical shifts of each leak determined - assigned to particular atoms - data and cross peaks used to calculate structure

3 main techniques to determine structure
Triple resonance - experiments to assign chemical shifts to specific atoms - identify which atoms are related to each other in protein chain
Chemical shift measurements to predict torsion angles - determines which section of protein forms alpha helix, beta pleated sheet
Nuclear overhauser effect spectroscopy - NOESY - identifies which atoms are physically close in space