Chromatography Forum

Hi,

I have run MS mostly for identification before, but today I have used it for quantitation for the first time.

Sample was a peptide and I spiked the sample with a deuterated version of the same molecule (peptide Mw is about 1000, the deuterated 8 units more)

When I extract the two masses I can see that the apex of the M and M+8 peaks are about 5 seconds apart (it is the same injection). The M+8 elutes first. Is this common to see this phenomenon?

Yes there often are small RT differences between natural and deuterated standards. Five seconds is quite a bit but if you used an isocratic separation or shallow gradient, it would not surprise me. 13C standards usually elute much closer to the natural molecules.

I cannot recall why (I had looked this up a few years back - fascinating history of science in this topic), but it is common to see an earlier elution of the labeled standard.

Sometimes it can be an advantage to have them separated. I once did some Melamine samples and found that if the Melamine and the deuterated analog eluted together it cause an enhancement of the recovery of both. When a sample was analyzed and there was no Melamine present, the internal standard recovery came out low compared to the recovery in the standards. When I adjusted the gradient to allow separation of the two peaks, then the internal standard had the same recovery in both standards and blank samples.

In GCMS if you are using 1,4-Dichlorobenzene-d4 as an internal standard for 1,4-Dichlorobenzene you will have problems because the masses used to quantify the non deuterated are also present in the deuterated at a small amount, therefore separation is also desirable.

I use a shallow gradient, and the retention time of the peak is about 21 minutes (so 5 seconds is not a huge difference).

Glad to hear that it is a known phenomenon. I was starting to wonder if I had the right compound in the sample...

just to add: 13C is better than 2H because the fractional change in mass is very small (13/12 instead of 2/1) which means that 13C behaves more closely to 12C than 2H does to H. You can imgaine that the energy in a vibrating C-H bond is quite different to a C-D bond because the weight on the end of the spring is twice the size.

Ouch, James_Ball, yes, I can see that 4-times-deuterated dichloro-anything would be a catastrophe, because the +4 peak of a dichloro compound is going to be quite large (about 1/9th of the base peak, ignoring all other elements). I bet you dream of having a fully-labelled 13C version of it as your internal standard.