NMR Practical Considerations Gregory R. Cook, NDSU Monday, January 28, 13

Transcription

1 NMR Practical Considerations

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 1 H NMR - 1 to 20 mg. Higher concentration leads to line broadening and difficulty shimming 13 C NMR 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 2

3 Sample Preparation Use the appropriate amount of deuterated solvent Sample should be completely dissolved. Particulates can lead to poor lineshape. 0.7 ml (50 mm) in a 5 mm tube is best. Solvents may contain water. Tip - filter solvent through a small plug of basic alumina or other appropriate drying agent. Cap the tube and mount in spinner Sample does spin. Do not touch the edges of the spinner. Parafilm or tape on the tube can imbalance the sample and cause problems. NMR sample tube must be positioned to the correct depth. 3

4 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. 4

5 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. 5

6 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. 6

7 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 7

8 Shimming 8

9 Recognizing shims that are off 9

10 Recognizing shims that are off 10

11 Recognizing shims that are off 11

12 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 never find 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 12

13 NMR Artifacts Good Spectra of Ethyl Benzene (expanded methyl triplet) symmetric lorenzian peak shapes, good shimming 13

14 NMR Artifacts Poor spectra of Ethyl Benzene (expanded methyl triplet) asymmetric non-lorenzian peak shapes 14

15 NMR Artifacts Poor spectra of Ethyl Benzene (expanded methyl triplet) symmetric broadened non-lorenzian peak shapes 15

16 NMR Artifacts Poor spectra of Ethyl Benzene (expanded methyl triplet) asymmetric broadened, non-lorenzian peak shapes with multiple maxima 16

17 NMR Artifacts Poor spectra of Ethyl Benzene Poor signal to noise 17

18 NMR Artifacts Center glitch - artifact in exact center of spectrum slight quadrature detector imbalance, usually disappears with more scans 18

19 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) 19

20 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 20

21 NMR Artifacts Aliased or Folded Peaks resonances outside the spectral window setsw(upper limit, lower limit) or sw=sw*2 21

22 Spectral Width 22

23 NMR Artifacts Slanted Baseline first point distortion of the FID, can be caused by peaks near edge of spectrum, increase spectral width 23

24 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. 24

25 NMR Artifacts Poor Digital Resolution acquisition time is too short or Fourier number is too small at=4 or fn=4*np (zero filling) 25

26 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. 26

27 NMR Artifacts sinc wiggles acquisition time is too short and FID is clipped, usually with large peaks, increase at 27

28 VNMR Parameters Standard proton NMR pulse sequence remember the s2pul stander 2 pulse sequence 28

29 VNMR Parameters Proton NMR Parameter Window 29

30 VNMR parameters Spectral Width sw in Hz - total width of the spectrum setsw(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=5.0p movetof moves the offset to the current cursor position Aquisition Time at at = 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 sec is usually good for most spectra, increase for samples that have long T1 30

31 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. 31

32 VNMR other useful commands aa - abort acquisition aph - autophase axis - scale units; axis= h, axis= p df - display FID dpf - display peak frequencies dpir - display integral regions dps - dispaly pulse sequence ds - display spectrum dscale - display scale dssa - display stacked spectra dssh - display stacked spectra horizontally f - full spectrum ga - acquire and process nl - nearest line pir - plot integral regions pl - plot spectrum pll - plot line list plot - plot everything ppa - plot partial parameters ppf - plot peak freqencies pscale - plot scale ra - resume acquisition (stopped) rl - reference line sa - stop acquisition su - setup hardware parameters svf( filename ) - save FID wp - width of chart in ppm vs - vertical scale wft - weighted fourier transform 32

33 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,

34 Determining Ratios by NMR Integration 34

35 Determining Ratios by NMR Integration 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? 35

36 Quantitative NMR Consider 13 C Satellites 1.1% 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,

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

38 Quantitative NMR 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,

39 Quantitative NMR 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,

40 Quantitative NMR C minor C 1 13 C = : 1 % de = = 81.35% de 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,

41 Quantitative NMR 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,

42 No-D NMR 42

43 No-D NMR One CAN run proton NMR in unlocked mode Concentration of most neat solvents is ca. 10 M Most reactions are run M Most commercial reagent solutions are M Thus, analyte to solvent ratios range from 100:1 to 10:1 typically 43

44 No-D NMR 44

45 No-D NMR 45

46 No-D NMR 46

47 No-D NMR 47

48 No-D NMR 48

49 No-D NMR 49

50 No-D NMR 50

51 No-D NMR 51

52 No-D NMR 52

53 No-D NMR A known quantity of p-hydroxybenzoic acid (typically 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 ±

54 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 54

55 No-D NMR Setting up the experiment Use a typical volume ~0.7 ml and concentration 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 55

56 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 56

57 No-D NMR 57