Secondary Referencing with Unified
Chemical Shift Scale
updated Aug 2015
For referencing chemical shift of any nucleus, including 1H, 19F and
X-nuclei, we follow one of several methods recommended by IUPAC whenever possible (proposed in 2001 and updated
IUPAC recommends the use of a "Unified Chemical Shift Scale" and that ALL
chemical shifts be referenced against the 1H signal from very dilute
TMS (tetramethylsilane, ~1% or less) in chloroform,
either directly or indirectly. The TMS reference is called the "primary reference".
You can imagine that all nuclei share the same frequency scale ranging from 0
MHz to 500 MHz (on a 500Mhz spectrometer). In principle, only ONE referencing
point is needed for all nuclei. This is the foundation of "unified chemical shift scale" concept.
This unified scale is simply the ratio (Xi) of the resonance frequency of any nucleus relative to the 1H frequency of
TMS, and was supposed to replace the traditional chemical shifts (in ppm) all together. But, this reporting method
has not seen widespread use. For historic reasons, each nucleus and research
field used their own 0ppm reference compound for a particular nucleus (imagine
discrete zero ppm points in different regions of the frequency scale).
Traditional, individual nucleus 0ppm reference is called
"secondary reference" following the introduction of the unified scale (see below).
Most chemical shift referencing is now done under the umbrella of the "unified chemical shift" scale
but using the ppm referencing against traditional reference compounds.
To continue to use such historic referencing compounds and also to comply with other
IUPAC recommendations, IUPAC summarized tables of the frequency ratios of each nucleus
in its historic reference compound (i.e P31 in H3PO4, see Tables 1 and 2) over that of TMS 1H
signal. This ratio (Xi), measured precisely and field independent, allows indirect establishment of the zero ppm
point of traditional (secondary) reference compound once the resonance frequency of the
TMS 1H signal (primary) is known. The chemical shifts of all other reference compounds can be calculated based
on the calculated TMS 1H frequency using the pre-measured Xi value. In fact,
based on a similar principle, if the frequency of the deuterium lock signal (2H) (or any nucleus in general)
is known, the ppm value of any other nucleus can also be calculated using the Xi values.
Referencing using the pre-determined Xi ratios along with
any resonance with a known chemical shift value in ppm dramatically simplifies
chemical shift referencing with traditional reference compounds. This "secondary referencing" method is not super-precise in the traditional sense but it is sufficient for
most work conducted here, unless a strict comparison of shift values across multiple samples
is needed. In that case, extreme care has to be taken regardless what reference method is used.
Description of Referencing Method in Publication
If a direct, internal or external reference compound is used, this should be stated.
In most other cases, we use the "secondary referencing" method above. The description needs to be
clear about how the referencing is done (modify as needed):
- If residual solvent signal (such as 1H of CDCL3 at ~7.24ppm) is used to reference the spectrum, the description should be
something like this:
"Residual 1H resonance from deuterated solvent is used to reference
the 1H spectrum with the methyl resonance of TMS at 0.0ppm, according to IUPAC recommended method and the manufacturer's protocols (add references above if necessary)."
- If the 13C solvent signal is used to reference the 13C spectrum, the following describes the method:
"The 13C spectrum is referenced through the solvent 13C resonance (or with the solvent 13C resonance set to XXXppm) according to IUPAC recommended secondary
referencing method and the manufacturer's protocols (add references above if necessary)."
- If a spectrum from any nucleus is referenced via the "By Solvent" method, the following describes the method:
"The spectrum is referenced through the solvent lock (2H) signal
according to IUPAC recommended secondary referencing method and the manufacturer's protocols (add references above if necessary)."
For most applications:
If a more accurate chemical shift reference is required:
- First choice: if a residual solvent signal in the spectrum can be located and has a known
ppm value in the solvent table, use this signal.
- Second choice: if no residual solvent signal is available or can be reliably identified, use
the lock signal (2H or D) to reference the spectrum of any nucleus (enter setref or click the By Solvent
button under Process->Default).
- First choice: add TMS to your sample, measure the 1H spectrum and reference it by
the 0.0ppm TMS peak. For referencing any X-nucleus spectrum from this sample, see below on how to use the mref command
to transfer the reference of 1H spectrum to the X-nucleus spectrum.
- Other choices:
Two other traditional methods of referencing is (1) use a
a separate tube containing the reference compound (external referencing) or (2)
using a compound sealed in a capillary tube inside the same NMR tube. An external
referencing with samples in separate tubes does not give better reference
accuracy, is tricky to apply properly due to field correction factors, and
should be limited. For more details and pros and cons of each referencing
method, see the IUPAC documents linked above. Remember that it is not recommended to lock or shim
your sample after a separate reference sample is used to collect the reference
Note TMS has a melting point of -99C and a low boiling point of 27C. Collect a
1H spectrum at ~25C and reference your spectrum against the TMS 1H signal. Outside
this temperature range, you could still use the reference done within the above
temperature range, by using ppm values of peaks from your compounds.
Alternatively, but less precise, try to use the lock signal from the solvent to
roughly reference your spectrum (with the command setref, see
below), or choose to have other internal or external reference standards.
If super accurate chemical shift values are desired, you must consider and
correct a number of factors that affect chemical shift referencing for each
method, such as temperature, solvent, bulk magnetic susceptibility, sample
geometry, etc. The truth is that such occasion arises only rarely and no
matter what you do, referencing contains uncertainty once the sample condition
deviates from well-known conditions.
More Details on Chemical Shift Referencing Procedures
Use Residual Solvent 1H or Solvent 13C Signal (rough referencing)
This should work sufficiently well for most situations unless a very accurate referencing is needed.
Simply locate the residual 1H or solvent 13C signal and set the chemical shifts to
the known values.
1H Referencing with Internal TMS (<1%)
- Ideally, with temperature at ~25C (doesn't have to be accurate at all),
measure a simple 1H spectrum and locate the TMS peak on the right side of
the spectrum. Reference the TMS peak to 0.0ppm (put cursor over peak and
type: nl rl(0.0p) )
For All Nuclei (including 1H, 19F, and X-nucleus)
- Type setref or click Process->Default->By Solvent under any experiment to use the lock signal
to reference your spectrum of any nucleus.
In some situations, you may obtain more accurate X-nucleus referencing by using a carefully referenced 1H spectrum (using an internal TMS signal
for example) to indirectly reference all other nuclei. However, if the 1H referencing is rough (such as from solvent residual
signal or lock signal), you might as well just use the referencing by the lock (setref) method for
the other nucleus.
- move reference parameters of source experiment to target experiment
- Only applies to direct dimension of source and target experiments
- Detection nucleus in both experiments can be any nucleus
- Source and target experiments must be done at same condition with same
- Example: mref(1,2) reference the
spectrum of exp2 (i.e. 13C) using the 1H reference of exp1
- Use IUPAC Xi values
- use reference of direct dimension to indirect dimensions in 2D or 3D.