IUPAC Recommended Chemical Shift Referencing

  1. To ensure correct referencing,

    2.  
  2. If the sample contains DSS, set the cursor on the DSS line in the 1H spectrum of the sample.  Type  rl(0p) to set the DSS line to 0 ppm.

    3.  
  3. If the sample does not contain DSS, use DOH line for referencing the 1H dimension, using the temperature vs. chemical shift of DOH chart.  Find the chemical shift value corresponding to the temperature, and type  rl(x.xxp)  where x.xx, is that chemical shift value for DOH line.

    4.  
  4. Note down the value of 'tof'.  Set the cursor at 0 ppm and type movetof.

    5.  
  5. Make sure that the value of 'dof2' is set correctly. Sometimes, 'dofb' is used for setting the center of the 13C channel instead of 'dof2' (while referencing pfg_C_cbcaconh or pfg_C_cbcanh spectra type dof2=dofb ).

    6.  
  6. Type  spcfrq . This command will display the following:

    7. RF Channel 1 (Obs) frequency is   499.7041891     { zero ppm for 1H channel, F0H }
    RF Channel 2 (Dec) frequency is     50.6405538     { center line for 15N channel, FCentN }
    RF Channel 3 (Dec2) frequency is  125.6577895    { center line for 13C channel, FCentC }
    RF Channel 4 (Dec3) frequency is  125.6727905    { center line for 13C' channel, FCentC' }
    Note: The last 2-3 digits of these numbers do change with time. This calculation procedure adjusts the referencing so that these changes do not appear in the ppm values of spectra acquired at different times.
     
  7. After noting down these numbers, change 'tof' back to the original value. Check referencing for the 1H dimension again as described in steps 2 or 3.

    8.  
  8. Referencing 15N channel (channel 2):


    9. F0N / F0H  =  0.101329118

    (Note: 15N referencing is relative to liquid NH3.)

      e.g. F0N  =  0.101329118 x 499.7041891  =  50.63458474


    { ( FCentN  -  F0N ) /  FCentN }  x  106  =  Chemical shift (ppm) at the center line of 15N channel



     
     
  9. Referencing 13C channel (channel 3):


    10. F0C / F0H  =  0.251449530


      e.g. F0C  =  0.251449530 x 499.7041891  =  125.6503835


    { ( FCentC  -  F0C ) /  FCentC }  x  106  =  Chemical shift (ppm) at the center line of 13C channel


  10. Referencing 13C' channel (channel 4): For HNCO, HN(CA)CO, and (HA)(CA)CO(CA)NH experiments.


    11. F0C' / F0H  =  0.251449530


      e.g. F0C'  =  0.251449530 x 499.7041891  =  125.6503835


    { ( FCentC'  -  F0C' ) /  FCentC' }  x  106  =  Chemical shift (ppm) at the center line of 13C' channel



     
  11. Literature References:
    1. Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J., Oldfield, E., Markley, J.L., and Sykes, B.D. ; "1H, 13C and 15N chemical shift referencing in biomolecular NMR"; J. Biomol. NMR, 6, 135-140 (1995).
    2. Wishart, D.S., and Nip, A.M. ; "Protein chemical shift analysis: a practical guide"; Biochem. Cell Biol., 76, 153-163 (1998).