ion pair for atypical samples

[david s]

hi Alex

The approach went like this , an existing method utilising THF and Water did not separate to baseline two synthetic analogues of cortisone. A quick bit of analysis using THF,Acetonitrile and methanol at various concentrations did not brink about any real meaningful improvment to the critical pair without seriously affecting or generating any other peaks in the chromatography. examination of the molecularity shows the only difference in the structure ( as confirmed by NMR and Mass sec) of the two molecules is the appearance of an additional double bond in ring A - This lead me to think that my best option is to try and exploit this difference in structure. It follows that 2 double bonds on ring A in a conjugate system must have a higher electron density at ring A and therefore experimenting with ion pair may bring about the separation, not through a truly ion pair chromatography but applying the principles of change affiliation the theory also being that molecule A which has the extra double bond ( and hence the stronger affilitiation due to localised electron density) should retain on the column for longer and come off molecule B. In practice a good separation is observed however molecule A does come off first. This is the bit that is confusing.

Now when you run on a new column, without the ion pair, the separation is not so good- introduction of the ion pair on the same column does improve the chromatography. and so the system is shown to be reliable and reproducible but i do not understand ( and hence my problem) the mechanics as to what is going on

hopefully my logic makes sense

[HW Mueller]

Well, I have not seen anybody who would dare to predict what the effects would be on adding ion pairing agent in HPLC of neutral compounds using a C-18 column.
So stated more bluntley than above, the effect you see probably has nothing to do with the IP agent (that means you are lucky), something else changed.

[AdrianF]

Cortisol is not ionized under such HPLC conditions, if this holds true for your derivatives I don´t see how you can call this ion pair chromatography. My guess would be that you just changed the surface properties of your column with the ion pair agent.

I totally agree with this comment. The diethylamine is only interacting with the column;not with the steroids which are not charged. If this is the case you would expect the more polar compound to elute first.

If you have the separation why worry about the mechanism.

No Tswett.

[david s]

HW, AdrianF thank you very much for your comments. in some ways it is a bit reassuring that this may well remain an enigma at least i am not missing something obvious. To answer your question Adrian, i suppose for completeness i would like to know the mechanism. it is true i do not need to know it but i would like to know it and this has been the point from the start of this string.

I dont know if what i have done is daring, what i would say is if you have a hunch or theory, it can be well worth checking it out and this is exactly what i have done. If there are holes or critisisms in my theory, i would like to hear them. they can only assist in making me a better chemist. i also accept that my theory does not explain the observation and so must be wrong. i suppose the main thing is that i got the improvment in separation of the critical pair.

If i do ever decipher / work out what is going on i will post something onto this string

many thanks for everyones help