nmr practical considerations -...

©2013 Gregory R. Cook, NDSU

NMR Practical Considerations

Monday, January 28, 13

©2013 Gregory R. Cook, NDSU 2

Sample Preparation‣ Choose a high quality 5 mm NMR tube that is free of defects.

‣ Low quality tubes may be cause poor lineshape and difficulty in shimming

‣ Decide on sample amount

‣ 1H NMR - 1 to 20 mg. Higher concentration leads to line broadening and difficulty shimming

‣ 13C NMR - 100 to 300 mg for good S/N but less is ok. Low concentration samples will require more scans.

‣ Choose an appropriate solvent

‣ Need a solvent that will solubalize your analyte

‣ CDCl3 common - can be acidic.

‣ The more D, the easier it is to lock.

‣ D2O, acetonitrile-d3, acetone-d6, benzene-d6, DMSO-d6, THF-d6, CD2Cl2



Sample Preparation‣ Use the appropriate amount of deuterated solvent

‣ Sample should be completely dissolved. Particulatescan lead to poor lineshape.

‣ 0.7 mL (50 mm) in a 5 mm tube is best.

‣ Solvents may contain water. Tip - filter solventthrough a small plug of basic alumina or otherappropriate drying agent.

‣ Cap the tube and mount in spinner

‣ Sample does spin. Do not touch the edges ofthe spinner. Parafilm or tape on the tube canimbalance the sample and cause problems.

‣ NMR sample tube must be positioned tothe correct depth.



Lock‣ Lock the sample on the deuterium signal

‣ Locking is done to compensate for drifting magnetic field strength.

‣ Magnet drift is relatively small (a few Hz/hr) compared to the magnet strength but can cause broadening of signal over time.



Lock

‣ Z0 - DEUTERIUM CHANNEL

‣ Turn off lock and spin. Increase lockpower and lockgain. Adjust Z0 until you have a sine wave. Turn on lock and spin. It will not be steady because the power is on high. Lower power. Adjust lockphase to maximize signal.



Spin

‣ Why do we spin the sample?

‣ Spinning at ~20 Hz will average out the inhomogeneities in the sample on the X,Y plane.

‣ Spinning does NOT affect the Z axis very much.



Shimming

‣ Small adjustments to magnetic field

‣ field should be as uniform as possible

‣ shimming coils in all three dimensions to “shape” the field

‣ need to pay attention mostly to Z as most inhomogeneities are averaged out by spinning



Shimming



Recognizing shims that are off



Adjusting Shims

‣ Adjust Z1 until lock level is maximum

‣ Adjust Z2 until lock level is maximum

‣ Repeat

‣ This method of iteration is ok but not best

‣ if Z2 is way out of adjustment you may neverfind the optimal shims

‣ Adjust Z2 up or down until lock level drops dramatically

‣ Maximize lock with Z1. If max is higher than the starting point continue moving Z2 in the same direction and optimizing Z1.

‣ If max is lower, you moved Z2 the wrong direction.

‣ If you need to adjust Z3 you must re-optimize Z1 and Z2 as described.

‣ svs(‘filename’); rts(‘filename’) su



NMR Artifacts

‣ Good Spectra of Ethyl Benzene (expanded methyl triplet)

‣ symmetric lorenzian peak shapes, good shimming



NMR Artifacts

‣ Poor spectra of Ethyl Benzene (expanded methyl triplet)

‣ asymmetric non-lorenzian peak shapes



NMR Artifacts


‣ symmetric broadened non-lorenzian peak shapes



NMR Artifacts


‣ asymmetric broadened, non-lorenzian peak shapes with multiple maxima



NMR Artifacts

‣ Poor spectra of Ethyl Benzene

‣ Poor signal to noise



NMR Artifacts

‣ Center glitch - artifact in exact center of spectrum

‣ slight quadrature detector imbalance, usually disappears with more scans



NMR Artifacts

‣ Distorted spectra

‣ gain overload, turn on autogain (gain=’n’), sample may be too concentrated, dilute or reduce pulse width to very small number (1)



NMR Artifacts

‣ Rolling baseline

‣ improperly set zero- or first-order phase parameters, very broad background peaks from solids can cause this, zero lp and rp and phase



NMR Artifacts

‣ Aliased or Folded Peaks

‣ resonances outside the spectral windowsetsw(upper limit, lower limit) or sw=sw*2



Spectral Width



NMR Artifacts

‣ Slanted Baseline

‣ first point distortion of the FID, can be caused by peaks near edge of spectrum, increase spectral width



NMR Artifacts

‣ Spinning Sidebands

‣ adjust first-order non-spinning ships - X and Y then X and ZX then Y and ZY, then X and Y again. Recheck Z1 and Z2. If it doesn’t work, increase spin to 30 Hz.



NMR Artifacts

‣ Poor Digital Resolution

‣ acquisition time is too short or Fourier number is too smallat=4 or fn=4*np (zero filling)



Zero Filling

‣ Add zero points to the end of the FID

‣ This helps to “digitally” resolve broad peaks that don’t have enough points. It would be best to redo the spectra with more points.



NMR Artifacts

‣ ‘sinc wiggles’

‣ acquisition time is too short and FID is clipped, usually with large peaks, increase at



VNMR Parameters

‣ Standard proton NMR pulse sequence

‣ remember the s2pul stander 2 pulse sequence



VNMR Parameters

‣ Proton NMR Parameter Window



VNMR parameters

‣ Spectral Width sw in Hz - total width of the spectrumsetsw(upper limit, lower limit) e.g. setsw(12.0p, -1.0p)movesw sets the spectral width based on position of cursors

‣ You can set sw directly but need to define the transmitter offset (the midpoint of the spectral region)sw=10.0p tof=5.0pmovetof moves the offset to the current cursor position

‣ Aquisition Time atat = np /(2xsw). 1/at is the best spectral resolution obtainable (assuming perfect shimming), e.g., with a 2 second acquisition time you will not be able to resolve peaks less than 0.5 Hz apart.

‣ Acquired Complex Points np - number of data points aquired. Increase this to increase the resolution.

‣ Recycle Delay d1- relaxation delay - 1.5 sec is usually good for most spectra, increase for samples that have long T1



VNMR parameters

‣ Number of Transients nt - the number of scans. Increase for more scans. Set to a high number (thousands) for long carbon scans. Can stop it at any point.

‣ Blocksize bs - the number of scans obtained before the data is saved to the disk. You can do a wft to see the spectra as it is acquired after every block of data is saved. When you have good S/N you can stop it.

‣ Steady State ss - steady state scans. Dummy scans done at the beginning of the experiment - has all the pulses and delays of the experiment but does not acquire the FID. Used in cases where equilibrium will change after initial scans. Mostly used in 2D experiments.

‣ Nucleus tn - transmitter nucleus being observed

‣ Observe Pulse pw - pulse width. For maximum signal should be 90-degree pulse. Depends on power.

‣ Power tpwr - transmitter power. A lower power results in a weaker pulse thus you need a longer time for a 90-degree pw.



VNMR other useful commands

‣ aa - abort acquisitionaph - autophaseaxis - scale units; axis=’h’, axis=’p’df - display FIDdpf - display peak frequenciesdpir - display integral regionsdps - dispaly pulse sequenceds - display spectrumdscale - display scaledssa - display stacked spectradssh - display stacked spectra horizontallyf - full spectrumga - acquire and processnl - nearest linepir - plot integral regionspl - plot spectrumpll - plot line list

‣ plot - plot everythingppa - plot partial parametersppf - plot peak freqenciespscale - plot scalera - resume acquisition (stopped)rl - reference linesa - stop acquisitionsu - setup hardware parameterssvf(‘filename’) - save FIDwp - width of chart in ppmvs - vertical scalewft - weighted fourier transform



Determining Ratios by NMR Integration

‣ The integration of NMR peaks is usually straightforward if the ratio is <20:1 (95:5).

Oswald, C. L.; Peterson, J. A.; Lam, H. W. Org. Lett. 2009, 11, 4504Monday, January 28, 13



‣ Have good clean NMR with good S/N

‣ Integrate separated peaks of the same type is best

‣ Can be done for structural isomers, diastereomers or mixtures of unrelated compounds

‣ “Pure by NMR” generally meant to be ~95% pure or better.

‣ This is generally accepted error limit that can be distinguished from the noise.

‣ Can we do better?



Quantitative NMR

‣ Consider 13C Satellites1.1% of the signal - a ratio of 178.5:1 for each satellite peak!

Claridge, T. D. W.; Davies, S. G.; Polywka, M. E. C.; Roberts, P. M.; Russe, A. J.; Savory, E. D.; Smith, A. D. Org. Lett. 2008, 10, 5433



Quantitative NMR

‣ 13C Satellites can be used as an internal standard for diastereomer mixtures up to 1000:1 (99.8% de)

‣ BUT you must run the NMR experiment under quantitative conditions

‣ use delays of 5*T1 (about 25s); this ensures that >99% of nuclei have relaxed fully before the next pulse

‣ other processing procedures should be followed as described in the paper - best to find a large single peak

‣ Collect the spectrum and integrate the minor isomer relative to the 13C-1H satellite of the major isomer (satellite integration = 1)

‣ When the height of the 13C-1H satellite of the major isomer is greater than the 12C-1H resonance of the minor isomer, the ratio has to be >180:1 (>98.9% de)




Quantitative NMR




Quantitative NMR


1.0000 13C

18.3561minor

178.5 12C

1 13C

= 9.7243 : 1

% de = 8.7243

10.7243= 81.35% de



Quantitative NMR




No-D NMR



No-D NMR

‣ One CAN run proton NMR in unlocked mode

‣ Concentration of most neat solvents is ca. 10 M

‣ Most reactions are run 0.1 - 10 M

‣ Most commercial reagent solutions are 0.5 - 2.5 M

‣ Thus, analyte to solvent ratios range from 100:1 to 10:1 typically



No-D NMR



No-D NMR

A known quantity of p-hydroxybenzoic acid (typically 50-70 mg) was added to a solution

(1.00 mL) of aqueous ethanol, and 40% aqueous NaOH (∼100 μL) was added to deprotonate and solubilize the standard acid. No-D 1H NMR data were collected

(4-8 transients) with an acquisition time and a pulse delay of 20 s each to ensure

complete relaxation and quantitative proton integration. Pulse widths (transmitter

power) were reduced to remove baseline spectral artifacts and

increase integration accuracy. Using these protocols the Ho vs Hm resonances

integrated reliably to 1.00 ± 0.02.



No-D NMR

‣ Shimming a No-D sample

‣ Use a reference tube of the same volume of deuterated solvent, lock and shim

‣ Load a previous optimized set of shim settings for the solvent being used

‣ Use a sealed capillary insert containing a deuterated sample of the same solvent in the NMR tube - a mp tube works well.

‣ Gradient shimming

‣ Shim using the FID

‣ Shim using the spectrum



No-D NMR

‣ Setting up the experiment

‣ Use a typical volume ~0.7 mL and concentration 0.1-1.0 M

‣ Setup spectrometer as you would for a normal deuterated NMR experiment

‣ Turn off Lock but spin the sample

‣ Acquire a single scan and phase the initial un-shimmed spectrum

‣ Select, expand, and note a “reporter resonance” of known peak shape (e.g. a solvent peak)

‣ Run FID/Spectrum macro gf and enter the interactive acq display proccess to observe real-time FID or spectrum



No-D NMR

‣ Shimming using FID

‣ Select FID buton and increase gain until FID level is 500 to 1000 to make it easier to observe small changes in level

‣ adjust shims, allow FID to stabilize until you have a maximum

‣ Shimming using Spectrum

‣ Shim by monitoring the increase in reporter peak intensity (numerical or graphical) and look for good narrow line shape

‣ In general

‣ Shimming by FID or spectrum is slower than by deuterium lock



No-D NMR


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