Chemical Shift Referencing
1H Referencing
For aqueous samples the recommended standard is DSS (2,2-dimethyl-2-silapentane-5-sufonic acid). Prepare a 10 mM solution of DSS in the same buffer as your sample and acquire a 1D 1H spectrum with solvent presaturation. Make sure that td and si are both at least 16k in order to obtain adequate digital resolution.
Calibrate the DSS spectrum manually... Zoom in on the up-field DSS peak.
Click on the calibrate button in the left menu bar,
move the cursor to the center of the DSS
peak and press the middle mouse button. Enter in the dialog box the chemical
shift as exactly 0.0 ppm.
Record the value of the parameter SR (found in the edp menu). All subsequent 1H spectra acquired with the same buffer solution and temperature can be referenced by setting SR to this value.
X-nucleus Referencing
X nuclei may be indirectly referenced to the DSS 1H spectrum by calculating the corresponding SR parameter using the equation
SR(X) = [BF(H) + SR(H)]*gX/gH - BF(X)
where BF(H) = 499,865,000,
and gX/gH and BF(X) are:
| gX/gH | ||||
|---|---|---|---|---|
| X-Nucleus | DSS | TMS | TSP | BF(X) |
| 15N | 0.10132905 | 0.10132914 | 0.10132900 | 50,650,896 |
| 13C | 0.25144952 | 0.25145002 | 0.25144954 | 125,691,104 |
For an inverse experiment such as an HSQC the calculated value can be entered in the edp menu, or from the command line by the command
1 SR VALUE
where VALUE is in Hz.