- For choosing the internal standard, TMS or dioxane are the commonly used chemical shift references, but they are not the good internal standards because of their high volatility, and it’s difficult to make highly accurate concentrations of the standard solutions. TMSP is a good water-soluble chemical shift reference, but it will be absorbed to the NMR tube glass, so it’s not a stable internal standard. Sigma-Aldrich supplies nice internal standard compounds, see their brochure here.
- Since the spin-lattice relaxation time T1 for every H1 nuclear in the molecules is dependent on the concentration, the mixture composition, and the solvent you use, proton T1 measurements should be done first for the target NMR sample, (refer to our note 4). And the longest resulting T1 in your sample should be used to set recycle delay D1, which should be set to ~ 7 to 10 times of T1 to make sure fully relax back. (e.g. for a small molecule with MW of several hundred Da, if T1 is about 2 sec, D1 should be set to 15 to 20 sec, etc.)
- Use zg pulse sequence but not zg30 to improve S/N ratio and peak intensity. Use “pulsecal” macro to get calibrated proton 90° pulse P1.
- Set proper NS value and make S/N ratio higher than 1000, use “sino” macro and PROCPARS tab to set proper signal region and noise region, choose the smallest peak to check this.
- Set proper acquisition time AT (> 3*T2, in general, 1 to 5 sec.) to get enough S/N ratio. Receiver gain “rg” value won’t make ADC overflow!
- No spinning sample. The potential spin side-band could make spectra complicated.
- Set digital resolution (DR) < 0.4 Hz/pt. SW >= 20 ppm; try to set O1p around the center of the spectra and don’t overlap with any peak.
- When doing integration, don’t include the C13 satellites peaks.
Dwell time (DW) = 1/(2*SW);
AT = DW * TD = TD/(2*SW)
DR = SW/(0.5*TD)
Hongwei edited on 08/13/2025