peak identification in a nornicotine chromatogram

[RachelDavis]

Does anybody have experience with nicotine metabolites? Following extraction from animal tissue, we're trying to isolate nornicotine, but keep seeing a "double" peak, as illustrated below. Nornicotine (in this mobile phase) shows up around 9.5 minutes, the later of the two apparent peaks. Trying to figure out how to eliminate this contaminant, or at least identify it. Thanks! (For the record, using a Waters uBondapak C18 column, & ammonium acetate / methanol mobile phase.)

-Rachel

PS. I've already tried slowing the flow rate, but it wouldn't separate peaks within any reasonable amount of time. Also, smaller injection volume isn't an option, since we're near detection limit for other compounds in the sample.

[HPLCCONSULT]

I have separated the chiral enantiomers of nicotine related samples, but you appear to only need a basic method of achiral separation so this will not help...

Based on the info you provided (chromatogram helps as it is clear you have at least two species present) about your method it appears that you have reached the first stage of method development and just need to continue to try different things. Vary mobile phase composition (pH too) and column type until you have baseline separated peaks. This can take some time, but the result can be satisfying. Using RP you might want to start with screening gradient runs using ACN and MeOH as the organic phase to narrow down the best compositions. I have no idea what your experience level is, but I would also do a literature search to see if there are any "stock" methods out there worth using as well.

*Provide us with more information about your experience level and what you have already tried and the forum members may be able to provide more help.

[RachelDavis]

Thanks for the comments so far. I'm actually relatively new at this, pioneering HPLC methods in a lab otherwise focused upon molecular biology. The current mobile phase & extraction method were borrowed from others who have published similar work.

[HW Mueller]

Presumably you are using UV detecion. If so, take a spectrum of different parts of the peaks to check whether this might be due to a sample solvent, mobile phase incompatibility, etc. Careful with interpreting UV specs! In this connection it would be helpful if you told us what the sample solvent/matrix is.

[Bryan Evans]

These are difficult compounds - you'll need ion-pairing and gradient elution to have success separating this compound on ODS.

Below is nicotine & metabolites on Unison UK-Amino & Cadenza CD-C18:
http://www.imtaktusa.com/site_media/fil ... TI379E.pdf

(I'm not sure if those are 2 different species or just poor peak shape of 1 species)

[Uwe Neue]

You need to give a bit more information: what is the mobile phase composition, what is the pH, what is the sample solvent? I suspect that the problem is somewhere burried in this info.

[grzesiek]

I don't like the picture at all
Tried MS-ing it? You know what type of compound is the smaller one?
You sure this is another compound? Replaced mobile and stationary phases?

[RachelDavis]

You need to give a bit more information: what is the mobile phase composition, what is the pH, what is the sample solvent?

The mobile phase is 70:30 0.1 M ammonium acetate : methanol, with 0.4% triethylamine, at pH 4.4.

The sample solvent is 100% methanol.

I haven't used gradient elution thus far -- what would you recommend as the start- and end-points of the gradient for troubleshooting?

Sadly, no MS capabilities in our facility.

[skunked_once]

(I'm not sure if those are 2 different species or just poor peak shape of 1 species)

from Bryan Evans

The mobile phase is 70:30 0.1 M ammonium acetate : methanol, with 0.4% triethylamine, at pH 4.4.

The sample solvent is 100% methanol.

It's bad practice to use a sample solvent that is stronger than your mobile phase. This could result in unusual peak shapes. Try dissolving your sample in the mobile phase and rerun the samples.

[grzesiek]

maybe you could provide entire chromatogram to us