2CALIS-1 and 2CALIS-2 are pulse sequences for the accurate calibration of the RF field strength and have twice the sensitivity of the earlier CALIS sequences.. 2CALIS-2 is intended for calibration of e.g., 13C or 15N pulses on natural abundance samples whilst 2CALIS-1 is recommended primarily for enriched samples. Both experiments can be performed without prior knowledge or guess of the RF field strength and no delays in the pulse sequences are critically dependent on coupling constants.

Which CALIS sequence to use?

The choice of whether to use CALIS or 2CALIS is relatively simple. In the case of small molecules where the nucleus to be calibrated is relatively abundant e.g. 13C. The sequence 2CALIS-2 is the preferred choice since it doesn't require decoupling and any residual signal from non 13C attached protons is unlikely to interfer with the calibration. For fully enriched samples the sequences 2CALIS-1 and 2CALIS-2 will always be preferred since supression of unwanted signals from I spins not connected to S spins is not an issue. The choice between 2CALIS-2 and CALIS-1 for more complex molecules is not as simple. If the spectrum is crowed and/or the S spin is of very low abundance the use of CALIS-1 is likely to be preferred as it has the same sensitivity and offers complete supression of signals not connected to S spins. However, in the case of lower field instruments or with molecules which have larger linewidths it may be possible to make use of 2CALIS-1 as the cancelation of the I spin signals not connected to an S spins is automatically in these cases superior. In other words provided one is aware of the caveats associated with difference spectroscopy 2CALIS-1 may be an appropriate choice. Indeed in the case of proteins, it offers a much simpler calibration.


2CALIS-2 pulse sequence 2CALIS-2 pulse sequence 2CALIS-2 pulse sequence

For theta= pi/2 the signal goes through a zero crossing. The same phasing is be applied to the spectra recorded with the different vales of theta. The delay tau = 2(1JIS)-1. The delay delta = the lenght of the gradient G0 plus either 2 or 0 theta depending on whether the pulse to be calibrated is equal to 1 or 3 theta respectively. The open and filled bars represent pi or pi/2 pulses respectively. The shaped pulses are selctive pi/2 applied at the frequency of water. It should be noted that in the majority of cases only the last pair of shaped pulses is required for adequate water supression.

The gradients are set so that the gradient G0 is significantly larger than the others. The gradients G1 & G2 are simply small gradients to eliminate the effects of incomplete inversion by the pi pulse and should be small and so so as not to accidently refocus undesired pathways.

The recommended phase cycle is an even number of steps out of:

  • phi = 2y, -2y
  • Calibration pulse = theta, 3theta, 3theta, theta
  • Receiver phase = x, -x.


example 2CALIS-2 calibration spectra Example calibration of the 13C RF field strength using the 2CALIS-2 sequence on mannose and shows both the alpha and beta anomeric signals. example 2CALIS-1 calibration spectra Example calibration of the 13C RF field strength using the 2CALIS-1 sequence on the small protein Cytochrome c and shows the complete aliphatic region.


  1. Andrew J. Benie, Ole W. Sørensen 2CALIS doubling the sensitivity of CALIS for the calibration of the rf field strength for indirectly observed nuclei J. Magn. Reson. In press

  2. Andrew J. Benie, Ole W. Sørensen New methods for calibration of the RF field strength for indirectly observed nuclei J. Magn. Reson. 180 316-319 2006

Code and procedures for Varian and Bruker.

Document: 2CALIS (index.shtml)
Last modified: 2006-09-14