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Complex NMR experiments: 2D, selective, etc.

Contents

• Introduction to 2D NMR experiment: gradient selected COSY and DQF COSY

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• 2D NOESY

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• 2D 1H-13C HSQC , multiplicity edited

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• 2D 1H-13C HMBC

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• DOSY

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• Organic Structure Elucidation (separate document)

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Introduction to 2D NMR experiment: gradient selected COSY and DQF COSY

Basic 2D NMR experiment setup. Gradient selected and DQF COSY.

Prerequisite: You MUST have mastered performing 1D 1H NMR on manual Bruker instruments (NOT with ICON-NMR) before you may set up a 2D experiment.

The tutorial covers a basic gradient-selected COSY (more sensitive) and a double-quantum filtered COSY (less sensitive but it removes all singlets and gives a cleaner 2D plane).

https://youtu.be/djtE96oh6Ak

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2D NOESY

Prerequisite: You MUST practice the COSY before you can set up NOESY.

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Setup of the experiment

To set up two-dimensional NOESY, please, read and follow step-by-step guidelines from the Topspin Guide Book: Advanced NMR experiments:   2D_NOESY.pdf. This book is available in your Topspin installation in Manuals section.

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Optimization of parameters

• Mixing time, D8
  (optimally, should be around average T1 of a molecule):
  
  
  • 2-3 sec for Mw < 250 Da
    
    
  • 1 sec for 250 < Mw < 400 Da
    
    
  • 0.5 sec for Mw >400 Da

  NOTE: In practice, one will perform several NOESY experiments with different D8. For example, for a larger molecule (> 400 Da), I would set up experiments with D8 set to 0.1 sec, 0.25 sec, 0.5 sec, 0.75 sec, and 1 sec thus probing a range of mixing times around the optimal value of 0.5. Protons that are very close in space will develop NOE much sooner than more distant protons. As D8 increases, long-distance NOEs add to the picture while the cross-peaks between the closer-spaced protons remain too. The maximum mixing time is expected to show cross peaks for all protons in the molecule that are within 5 Angstroms. 
  
  KEEP IN MIND: the longer the D8, the lower the overall amount of signal (sensitivity) in the NOESY spectrume. You may need a larger NS to observe NOE cross peaks at long D8 values!

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• Recycle delay, D1
  

Mw, Da	If Mw is unknown, use # protons **)	D1, seconds
< 275 Da	< 30 protons	2 sec
275-350 Da	30-40 protons	1.5 sec
> 350 Da	> 40 protons	1 sec

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• Direct dimension points, TD F2
• 2048

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• Increments in the indirect dimension, TD F1
  • 128 for a quick survey of a signal
  • 256 or 512 for higher resolution (if 128 appeared not adequate)

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• Number of scans, NS
  • 4  - only for quick survey of the overall signal
  • 16  (required by the phase cycle)

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Processing

Topspin ProcPars Tab

• SI F1 = 1024
• SI F2 = 2048
• SR F1 and SR F2 = 0 Hz

Phasing

Phase 2D to make diagonal peaks positive.

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Interpretation

Below are quick notes on properties of NOE cross peaks. For comprehensive discussion, see
Burns and Reynolds, "MInimizing risk of deducing woring natural product structures from NMR data", Magn Reson Chem, 2021, 59: 500-533

and

High-Resolution NMR Techniques in Organic Chemistry, 3rd Edition, by Timothy D.W. Claridge. Elsevier Science (May 27, 2016), ISBN-10 ‏ : ‎ 0080999867, ISBN-13 ‏ : ‎ 978-0080999869

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Small to medium sized molecules, approx. < 600 Da, in a low-field spectrometer (400-500 MHz)

• NOE cross peaks appear with an opposite sign relatively to diagonal peaks
  
  
• EXSY, exchange peaks appear with the same sign as diagonal peaks.
  • Exchange with water is observed as exchange cross-peaks at water frequency
  • A problem: exchange peaks may overlap with NOE cross-peaks cancelling each other.
    
    
• COSY artifacts: split (dispersive) cross-peaks with positive and negative components. 
  • Helpful trick: Using maximum mixing time maximizes NOE but not the COSY, therefore the COSY artifacts may be identified. 
    
    
• Pulse program artifacts: peak patterns looking as a mirror image of a diagonal, or pairs of peaks running parallel to the diagonal. They are unrelated to the NOE effect, - to be ignored.
  

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Larger molecules, > 600 Da, in a low-field spectrometer (400-500 MHz)

• NOE effect vanishes beyond 600 Da at low field magnets and changes sign to positive as molecular weight increases further (1-2 kDa)
  
  
• NOE cross peaks in a higher molecular weight region will appear with  the same sign as diagonal peaks
  
  
• EXSY, exchange peaks also appear with a positive sign: you cannot distinguish NOE cross-peak from exchange cross-peaks in this molecular weight range!
  
  
• COSY artifacts: split (dispersive) cross-peaks with positive and negative components. 
  • Helpful trick: Using maximum mixing time maximizes NOE but not the COSY, therefore the COSY artifacts may be identified. 
    
    
• Pulse program artifacts: peaks looking as a mirror image of a diagonal, or pairs of peaks running along the diagonal. They are unrelated to the NOE effect, - to be ignored.
  

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NOTE: The 600 Da boundary for NOE is approximate. The change of sign of NOE is controlled not by molecular weight or shape but by the rotational diffusion coefficient of the molecule in the current solvent as it compares to the spectrometer field strength. Greater sovent viscosity, lower temperature, and stronger magentic field all shift this boundary to smaller molecular weights. Example: 650 Da molecule exhibits positive NOE in DMSO at 800 MHz that is the "zero NOE" boundary shifted to lower Mw at 800 MHz in DMSO. In practice, it is advisable to record both NOESY and ROESY and compare results for your sample/solvent/temperature/magnet combination.

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2D 1H-13C HSQC (multiplicity edited)

Prerequisite: You MUST have practiced the COSY before you can set up HSQC.

The multiplicity edited experiment is used in all assignment work. The resulting 2D plan contains CH and CH3 peaks in the same sense (usually, phased to positive) and CH2 peaks appearing negative.

To set up HSQC on a Bruker instrument

1. Use a parameter set CMCse_HSQC

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2. Parameters to adjust:
   
   
   1. Proton parameters: O2 and SW2 - same as in COSY
      
      
   2. Carbon parameters: O1 and SW1 (center frequency and spectral width in indirect dimension).
      
      
   3. NS mus be an integral number of phase cycles (check PulseProg tab for phase cycle used in the program). If sensitivity in your experiment is not enough you will increase NS in the phase cycle steps.
      
      
   4. Use TD F1 = 128 for your initial experiment (gives 64 increments in indirect dimension). If you will need more resolution you will reacquire later.

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3. Parameters to NOT touch: TD F2, AQ, and D1. 
   
   This experiment uses heteronuclear decoupling, which may damage the probe. These parameters are set to safe values by default.

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4. Issue 'pulsecal' to calibrate a proton pulse

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5. Issue 'rga' to adjust Gain

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6. Issue 'expt' to see experimental time

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Approximate acquisition time for HSQC

• 400/500 MHz
  • 100 mM sample needs one hour
  • 50 mM sample needs about four hours
  • 20 mM sample needs an overnight run (some 16-18 hours)

• 800 MHz with a cryoprobe
  • 20 mM sample  - 2-4 hours
  • 10 mM sample  - 8-16 hours
  • 5 mM sample - 24 hours or more

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2D 1H-13C HMBC

Prerequisite: You should have acquired 1D carbon and 2D HSQC on your sample prior to setting up the HMBC.

To set up HMBC on a Bruker instrument

1. Use a parameter set CMCse_HMBC

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2. Parameters to adjust:
   
   
   1. Proton parameters: O2 and SW2 - same as in COSY or HSQC

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   2. Carbon parameters: O1 and SW1 - same as in 1D carbon.
      
      IMPORTANT: SW1 must be larger than that of HSQC because HSQC only detects protonated carbons while HMBC is capable of detecting all carbons.

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   3. NS must be an integral number of phase cycles (check PulseProg tab for phase cycle used in the program). If sensitivity in your experiment is not enough you will increase NS in the phase cycle steps.
      
      IMPORTANT: HMBC is less sensitive than HSQC. You should double the number of scans relatively to HSQC on the same sample.

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   4. Use TD F1 = 128 for your initial experiment (gives 64 increments in indirect dimension). This will be coarse but reasonably fast. If you will need more resolution you will reacquire later.

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   5. Set AQ according to your molecular weight:
      
      
      • AQ=0.3 sec for Mw <= 400 Da
        
        
      • AQ=0.4 sec for Mw > 400 Da

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   6. D1 must be set considering the total recycle delay: DR = D1 + AQ
      

Mw, Da	If Mw is unknown, use # protons **)	Recycle delay, DR, seconds
< 275 Da	< 30 protons	2 sec
275-350 Da	30-40 protons	1.5 sec
> 350 Da	> 40 protons	1 sec

For example, for the compound of 500 Da, I will use AQ=0.4 sec. The DR must be 1 sec, therefore, D1 = DR - AQ = 1 - 0.4 = 0.6 sec.

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Multiple-bond J(CH) setting CNST13



• common: CNST13 = 8 Hz - will be sensitive for J couplings of 3-4 Hz but will miss smaller couplings;
  
  
• optional:  CNST13 = 4 Hz - will detect more peaks for small couplings but may miss stronger ones.
  NOTE: Remember that the experiment becomes less sensitive with this setting: you should double the NS.
  
  

Issue getprosol to set probe parameters

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Issue pulsecal to calibrate a proton pulse

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Issue rga to adjust Gain

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Issue expt to see experimental time

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Processing HMBC in Topspin

• Set in ProcPars:
  
  
• SI F1 = 2048
  
  
• SI F2 = TD F2

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• Enter on a command line:
  
  
• proc2d y
  
  
• xf2m

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Processing HMBC in MNova

• Use default settings

 

 

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DOSY

• DOSY on Bruker 400 and 500

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• DOSY on Varian 600

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DOSY on Bruker 400 and 500

Bruker guidelines on DOSY expts (PDF)

NOTES: D of residual H2O in pure D2O at 298K is 1.902e-9 m2/s (in Claridge, p317 in 2nd and p397 in 3rd) => log D = -8.72

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  DOSY measurement

1. Have VT off for enough time - 15 min
   
   
2. Create a proton 1D
   
   
3. Verify that D1+AQ >= 3 T1
   
   
4. Run a proton 1D
   
   
5. Create DOSY experiment:
   
   
   1. For Proton DOSY: 
      1. Create new experiment with "DOSY" parameter set
      2. run pulsecal
         
         
   2. For other nucleus (31P, 13C, 113Cd, etc.): Open the Setup_DOSY template for this nucleus. Create new experiment with Start:Create Dataset:Use current parameters. If you want to use a nucleus that have never been used for DOSY, we have to create an experiment for this nucleus before you can proceed.
      
      
   
   
6. Verify that
   1. Verify that D1+AQ >= 3 T1
      
   2. p30 < 3ms
   3. p30/(D1+AQ) < 0.05
      
      
7. Run test of first and last spectral intensities: issue xau dosy 2 95 2 l y y 
   NOTE: symbol "l" is a lowercase "l" as in "lower".
   
   
8. Extract first and second fids and overlay - they must have intensities 10:1 or 20:1
   Considerations (from Dosy an Diffusion by NMR, p.9): The smallest signal to be detected (i.e. at highest gradient strength) has to be above the noise. If the signal intensity is already totally gone, reduce the gradient strength (gpz6). If the signal is still to big, you have to increase either the diffusion time ∆ (d20) or the gradient length δ (p30 - no more than to 3 ms!!!). Increasing δ is favorable, because it results in a bigger effect. δ2 is determining the signal attenuation, while ∆ is only affecting the exponential decay function linearly (see chapter 1). If you change ∆, you have to take the relaxation into account (T1 relaxation for all STE type sequences).
   
   
9. After you made adjustments, check if
   1. p30 < 3ms
   2. p30/(d1+aq) < 0.05
      
      
      
10. Run full experiment:
    
    1. set NS to phase cycle, or 1/2 of it. DS to 8
    2. issue xau dosy 2 95 N l y y where N is number of gradient steps (10-15)

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DOSY Processing

1. Phase the first fid:
   1. issue rser 1
   2. process
   3. phase
   4. press [nD] button to transfer phase values back to DOSY
   5. close Temp window
      
      
2. Edit ProcPars: ABS1 and ABS2 limits to 1000,1000 and -1000, -1000
   
   
3. Set SI for F1 to 2N 
   (that is to TWICE the number of gradient steps; ignore the fact it is adjusted automatically to power of 2)
   
   
4. xf2; abs2
   
   
5. setdiffparm (this command needs to be issued only once in the experiment)
   
   
6. Issue eddosy
   Set:
   1. PC to minimum S/N peaks you would like to include
   2. F1mode to Peaks
   3. Imode to Intensity)
      
      
7. dosy2d setup
   (issue this command if you want to automatically setup D max and min limits)
   
   
8. Edit DISPmin and max limits to show necessary diffusion coefficient range.
   
   
9. issue dosy2d  to process to DOSY representation
   
   
10. Go to Spectrum, check if you have enough contour levels (Right click, Edit contour levels, set Level increment to 1.4, Number of levels to 64, click Fill and Apply)
    
    
11. NOTE If you nee to rerun DOSY with new range of D or other parameters, issue:
    1. xf2; abs2
    2. make necessary adjustments
    3. dosy2d

 

 

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DOSY on Varian 600

1H Varian protocol (PDF)

31P Varian protocol (PDF)

 

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