Practically speaking, diverse methods may be used to reference chemical shifts in an NMR experiment, which can be subdivided into indirect and direct referencing methods. Indirect referencing uses a channel other than the one of interest to adjust chemical shift scale correctly, i.e. the solvent signal in the deuterium (lock) channel can be used to reference the a H NMR spectrum. Both indirect and direct referencing can be done as three different procedures:
# Internal referencing, where the reference compound is added directly to the system under study." In this common practice, users adjust residual solvent signals of H or C NMR spectra with calibrated spectral tables. If substances other than the solvent itself are used for internal referencing, the sample has to be combined with the reference compound, which may affect the chemical shifts.
# External referencing, involving sample and reference contained separately in coaxial cylindrical tubes." With this procedure, the reference signal is still visible in the spectrum of interest, although the reference and the sample are physically separated by a glass wall. Magnetic susceptibility differences between the sample and the reference phase need to be corrected theoretically, which lowers the practicality of this procedure.
# Substitution method: The use of separate cylindrical tubes for the sample and the reference compound, with (in principle) spectra recorded individually for each." Similar to external referencing, this method allows referencing without sample contamination. If field/frequency locking via the H signal of the deuterated solvent is used and the solvents of reference and analyte are the same, the use of this methods is straightforward. Problems may arise if different solvents are used for the reference compound and the sample as (just like for external referencing) magnetic susceptibility differences need to be corrected theoretically. If this method is used without field/frequency locking, shimming procedures between the sample and the reference need to be avoided as they change the applied magnetic field (and thereby influence the chemical shift).
Modern NMR spectrometers commonly make use of the absolute scale, which defines the H signal of TMS as 0 ppm in proton NMR and the center frequencies of all other nuclei as percentage of the TMS resonance frequency:
The use of the deuterium (lock) channel, so the H signal of the deuterated solvent, and the Ξ value of the absolute scale is a form of internal referencing and is particularly useful in heteronuclear NMR spectroscopy as local reference compounds may not be always be available or easily used (i.e. liquid NH for N NMR spectroscopy). This system, however, relies on accurately determined H NMR chemical shifts enlisted in the spectrometer software and correctly determined Ξ values by IUPAC. A recent study for F NMR spectroscopy revealed that the use of the absolute scale and lock-based internal referencing led to errors in chemical shifts. These may be negated by inclusion of calibrated reference compounds.