Sometimes Peak Defects Are Not the Sign of a Problem

Last month, I demonstrated the feasibility of a 250 µL injection in a split/splitless injection port, using the EPA Method 521 (nitrosamines in drinking water) list. As you can see in the chromatogram below, seven of the eight peaks are nice and symetric. Peak number 2, however, shows exaggerated fronting.

250µL injection 521 scan mode 0.1µg-mL

N-nitrosomethylethylamine is the only compound on this list that exists as a set of enantiomers. I suspected that the front defect on peak 2 was due to the partial resolution of a minor isomer. A suspicion which I recently confirmed while working on a rapid 8270 screening run using a short, narrow bore column (results shown below).

n-nitrosomethylethylamine entiomer chromatography

I was able to achieve this partial separation by taking advantage of the higher efficiency of a narrow bore, thin film column and minimizing my injection band by combining split injection with a narrow inlet liner. You may have noticed that the n-nitrosomethylethylamine peak in the first chromatogram is approximately 20 seconds wide while the partially resolved peaks in the second (8270) chromatogram are about 1.2 seconds wide each (or 2.4 seconds wide total). In order to properly define peaks which are this narrow, you need a fast acquisition speed. Not being able to resolve the enantiomers is not an issue, but if you look at the aniline – bis(2-chloroethyl)ether separation, you’ll see that a slow acquisition rate could easily result in the loss of resolution for this isobaric pair. I used a 7890-5975C for this work, but a 5973 with the fast electronics package should be sufficient.

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5 Responses to “Sometimes Peak Defects Are Not the Sign of a Problem”

  1. John C. says:

    Unless I’m mistaken or missing something, a chiral stationary phase is not used for that method, so you’re not separating enantiomers. That would be quite a feat. You may be observing tautomerization, or a minor isomer, but you’re not separating enantiomers on a non-chiral stationary phase.

  2. John C. says:

    And looking at this again, and this time at the structures, N-nitrosomethylethylamine doesn’t have a chiral center, so even if you were using a chiral stationary phase, there are no enantiomers to separate.

  3. It has a chiral center…you have to consider the unbonded pair. A few of us in the applications group have been trying to determine what is going on here. It was pointed out that N-nitrosomethylethylamine should undergo nitrogen inversion, but we thought it was weird that one conformation appeared to be favored 2:1 (we would expect a racemic mixture). I agree that tautomers at equilibrium or a minor isomer would make more sense, but none of us can think of likely structures or compounds. The nominal mass spectra for the two peaks are identical. As for the chiral selectivity of the 5ms, I agree, it would be surprising if this is really what is going on, but I don’t know enough about the polymer or the way it is made to confirm that it is not at least somewhat selective for optical activity.

  4. Jonathan Smuts says:

    Nitrogen can indeed be chiral but only when there is a barrier of rotation. The most famous example of this is Troger’s base where the two nitrogen centers are evidently fixed and thus cannot invert. The N-nitrosomethylethylamine above has no barrier to rotation and can freely invert; it certainly has less barrier to rotation than compounds 5 and 6 and there you do not see any peak broadening. The lack of a chiral stationary phase, already mentioned, also supports this. Something else must be going on here.

  5. Yuki says:

    Chris is right here. Some got confused because one of the N-nitrosomethylethylamine s Chris depicted in Lewis structure should have the N=O part of the -N-N=O bond pointing the other direction from its counterpart(one on the right picture pointing up, so the one on the left should also be pointing up because the one on the left is inverted as a whole in the picture). Also in terms of nomenclature, this is not an enantiomer or tautomer, this is called geometrical isomer because it is at a double bound.

    Jonathan, it has a barrier of rotation because of a double bond character of -N-N part. Resonance structure can be drawn easily.

    As for non-chrial stationary phase showing 2 peaks possibly geometrical isomers, another well known example is 2 peaks seen if you add methoxime and TMS to carbohydrates, because methoxime also has the same geometrical isomers as N-nitrosomethylethylamine.

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