I expect compound X to be present in most samples for my current study, but Profiler shows a maximum concentration of zero. What’s going on?
8 February 2016 Comments Off on I expect compound X to be present in most samples for my current study, but Profiler shows a maximum concentration of zero. What’s going on? FAQ - Profiling Spectra
The simplest possibility to consider is that the compound in question is just not present. Having excluded that, for example by supplementary analysis confirming that the compound is indeed present, there are some other considerations.
If you are running reconstituted samples in pure D2O, Profiler may erroneously assign some compounds a maximum concentration of zero. In pure D2O, exchangeable protons are completely replaced by deuterium, effectively removing the signal for those protons from the NMR spectrum by reducing its intensity to near zero. When calculating maximum concentrations, Profiler only considers values that do not allow any cluster of a compound to exceed the measured intensity of the spectrum. If an expected cluster is effectively zero intensity, Profiler will predict a maximum concentration of zero for that compound.
Two other factors can also influence Profiler’s calculation of maximum concentration. Incorrect CSI settings can reference the spectrum to the wrong chemical shift or result in Profiler incorrectly interpreting intensity ratios with which it calculates all compound concentrations. Also, incorrectly setting the spectrum pH can result in clusters or transform windows starting in the wrong positions, meaning that Profiler will not use the correct spectrum locations in calculating maximum concentrations.