Oily Sample Analysis by NP-LC

[DB2]

I have been attempting to create a Normal-Phase LC method for the analysis of degradation products of a small polar molecule of approximately 300 daltons. The analyte molecule is basic and is dissolved within an oily matrix. As such, the sample is being dissolved in chloroform before being injected. The analyte molecule and degradation products absorb at 290nm.
I have attempted the analysis with several different columns as well as normal phase solvent systems of differing relative polarity. Thus far, I have achieved the best results using a stable bond Cyano column. Elution is achieved by gradient from 95:5 to 50:50 hexane, and ethanol respectively. I am achieving good retention of my main band however it exhibits broad tailing. I am not able to detect major and minor degradation products that are present. I initially tried adding 0.1% TFA to the strong solvent in the hopes of sharpening the peaks but this had no effect. Thus far, I have assumed that the main band which displays extreme tailing is masking the degradants.
Is it possible that adding 0.1% TEA or isopropylamine might help? Anyone have any experience with the analysis of oily samples on a NP-LC system? Many thanks for any/all info.

[Bryan Evans]

Interesting problem.

Imtakt has an LC-ELSD application for nicotine derivatives and metabolites.

Normal Phase: Unison UK-Amino
Reversed Phase: Cadenza CD-C18

http://www.imtakt.com/TecInfo/TI379E.pdf

I'm not sure how stable a CN phase will be under these condtions (0.5% TFA). But it's no problem for Unison UK-Amino.

[Uwe Neue]

In normal phase, the way to reduce tailing is to add a competing compound of the same type as the analyte (thus the addition of TFA is not the right idea). For an analyte with amine functions, I would add triethyl amine or something similar to the mobile phase. Don't worry, this is not an aqueous medium, so you won't dissolve the silica. Otherwise, I would start with silica, and go to bonded phases only, if I do not get the selectivity that I want.

[Uwe Neue]

Hot off the press... Published on-line today in the Journal of Chromatography...

Optimization of normal-phase chromatographic separation of compounds with primary, secondary and tertiary amino groups
Authors: M. Kagan, M. Chlenov, S. Melnikov, A. Greenfield, J. Gross, R.C. Bernotas

…Several classes of amines, including benzylamines, anilines, ephedrines, tryptamines, and azatryptamines were chromatographed using mixtures of hexane and ethoxynonafluorobutane with methylene chloride and methanol. Peak tailing, diminished selectivity and low plate count were minimized by the addition of volatile amines to the mobile phase. The optimal additive was n-propylamine at 0.1% concentration…

[DB2]

many thanks for the advice....I am going to give n-propylamine a shot......stay tuned....