1. 1 NMR Practical Aspects (I) Processes involving in a NMR study: 1. Sample Preparation/Considerations 2. Setting up Spectrometer 3. Setting up acquisition parameters and carrying out experiment 4. Processing raw NMR fid data into NMR spectrum 5. Plotting of NMR Spectrum (1D with integrals of peak intensities) 6. Analysis of NMR data References: Basics of NMR: http://www.cis.rit.edu/htbooks/nmr Practical: http://arrhenius.rider.edu/nmr/NMR_tutor/pages/nmr_tutor_home.html

2. 2 Safety Precautions: Very Strong Magn etic Field! 10G 5G No Entry for Person with Heart Pace Maker Keep and Secure Ferromagnetic Objects away from the magnet Aluminum or Stainless Steel Ladder

3. 3 Sample Considerations: Molecular weight (MW): Larger molecules => tumbling slower (  c  ) => relax faster (i.e. shorter T 2 ) => broader line-widths => lower sensitivities MW 

4. 4 Concentration: relatively high conc. required because of low sensitivity. At least ~0.1 mM for small organics; 1- 5mM for protein or large macromolecules. For a given S/N, half conc. takes four times longer expt. time. Solubility (  => S/N  ), aggregation or polymerization (S/N  ). Stability – tens of hours in the spectrometer may be required. E.g. oxidation, microbial contamination, hydrolytic breakdown. Temperatures for data collection: T  => S/N  need to consider the temperature dependence of stability Buffer/solvent selection: affect solubility, stability, position of spectral absorption lines can be solvent dependent, viscosity (  => S/N  ) Sample size: standard 5 mm tube need ~0.5ml sample volumn other tubes to enhance sensitivity e.g. 10 mm tube, Shigemi tube Sample Considerations:

5. 5 Setting up Spectrometer procedures: 1. Insert Sample and set Temperature 2. Locking : Manual or Auto-locking 3. Tuning the probe: Manual or Auto-tuning 4. Shimming: Manual, Gradient shimming or Auto-shimming

6. 6 Even in the best spectrometers the field strength varies to some extent over time The position of the deuterium peak is monitored To counteract the field drift a lock field is applied to maintain a constant deuterium resonance position Locking the deuterium signal from solvent to maintain high magnetic field stability and high line resolution Setting up Spectrometer: Locking Deuteriated solvent is usually used to provide the Deuterium Lock signal e.g. CDCl3, D2O, CD3OD

7. 7 Setting up Spectrometer: Tuning the Probe Variations in the polarity and dielectric constant of the solvent will affect the probe tuning. Tune each coil to be resonant at the Larmor frequency for the corresponding nucleus: e.g. 1 H, 13 C, 15 N Two capacitors (tune and match) are adjusted to achieve maximum power transfer into and out of the probe Minimize reflected power to get best match Align the minimum to expected freq to get best tune Expected freq of the selected nucleus Tuning with ‘wobb’ curve

8. 8 The process of making the magnetic field surrounding the sample as homogenous as possible A series of shim coils correct minor inhomogeneities in the static magnetic field Good Shims => Sharp lines! All the nuclei in the sample “feel” the same magnetic field Poor Shims => Broad lines and poor line-shapes! Variable magnetic field across the sample – nuclei in different regions of the sample will resonate at slightly different frequencies Setting up Spectrometer: Shimming

9. 9 Shimming is judged at 50%, 0.55% and 0.11% of peak height in Hz

10. 10 Spinning Sidebands observed at multiples of spinning rate What is the spinning rate here? 24 Hz No spinning for biomolecule samples because natural linewidth is large

11. 11 Setting up acquisition parameters For each dimension: Spectral Frequency (sf) : Center of all NMR peaks or H 2 O peak position for aqueous sample Spectral Width (sw) in Hz : Covering all NMR peaks Number of data points (np) for direct detection dimension or number of increments for indirect dimensions: Depends of digital resolution required or experimental time available. Calibrate the 90 degree pulse width (pw90 in us) and set power (in dB) for the pulses. Global parameters: Recycling delay (d1) : >= 5T 1 Number of scans per fid (nt) : signal averaging to get better signal to noise (S/N) Receiver Gain (rg) : maximize DAC usage

12. 12 tof is the transmitter offset frequency from a base frequency e.g. 600.13 MHz Presaturation Experiment on protein Sample in H 2 O [H 2 O]=55 M >> [protein]=1mM > DAC resolution (e.g. 65535) => can’t see weak protein signals sw sf sf+sw/2sf-sw/2

13. 13 Some useful terminologies and relationships: Sampling rate: sr = 1/(dt) in Hz; dt=dwell time Optimal sampling rate is based on Nyquist criterion => sw = sr / 2 i.e. highest observable frequency = sample rate / 2 or dt = 1/(2*sw) in second Acquisition time: aq = dt * np These two frequencies are 10 and 110 Hz sampled at Dt=0.01 seconds. The FT would have a SW of 100 Hz, and the two peaks are indistinguishable. This is called aliasing and can be a big experimental problem if you are not careful. How to detect aliasing peaks? Alter sw

14. 14 Digital Resolution: dr = sw / np in Hz should be < natural line width Improve dr either by  np or  sw line width (lw)= 1/(  T 2 ) Larger for bigger molecules lw < 1 Hz for most organics 10-30 Hz for proteins

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17. 17 Relationship between pulse width, power and Bandwidth Bandwidth (bw) = 1/(4 * pw) should be > spectral width Power  6dB => pw 90  by a factor of 2 Therefore can increase power until pw 90 gives large enough bw FT

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19. 19 Therefore the actual number of increments for indirect dimensions is usually a compromise between digital resolution required and experimental time available. 3D data will requires increments on ni and ni2 dimensions i.e. ni * ni2 FID’s Total time is in the order of 10 to 100 hours

20. 20 Optimal Recycling Delay: For full relaxation and restoration of equilibrium Magnetization Recycling delay (d1) >= 5T 1 But T 1 can be very long for some nucleus e.g. 13 C => very long acquisition time for full relaxation And signal averaging gives S/N  Therefore one can get best use of experimental time (T exp ) to get best S/N by using the Ernst angle (  e ) for excitation in 1D experiment: cos  e = exp(-T exp / T 1 ) t1t1 t1t1 ee