hello everybody!
I plan to establish several gradient methods on normal phase (plain silica) and am doing right now first tests on reproducibility (10 injections in a row and day-to-day. Eluents are heptane and ethylacetate, 100% hep to 50:50 in 15 min. equilibrating is 6 min. The peak (abietine acid) shifts more than 30 sec within 10 injections and more than a minuit from day to day.
0,05% acetonitrile is added to the Heptane, but watercontent in ethylacetate is not controlled till now. Is it needed at all? Is acetonitrile sufficiant as desactivator?
thanks

Hello Anon,
Your normal phase system is very sensitive for H2O in any part of solvents or samples.
You shouldt use dry solvents...
BTW this is the reason why the RPLC sytems are more common than NPLC

There are several ways to get around the water content problem on silica. You cna work with dry solvents, but then you may need to equilibrate your column forever, and then there may still be differences between dry on one day and dry next month.

In the old times, we used a different approach to get good reproducibility. We used solvents that had a controlled water content. The simplest way to do this is to use a solvent mixture that is half saturated with water. The way this works is as follows. You make your mobile phase, then divide it into two equal portions. To one of them you add just enough water that it won't completely dissolve. You stir this half for a while, until it is saturated with water. Now you decant the water, and add the water saturated half to the dry half that you had put aside. Now you have a solvent that is half-saturated with water. And you have a more reproducible chromatography.

I have a question, based purely on curiosity, having never done old fashioned normal phase, but only HILIC: Why not use saturated rather than half-saturated? I would think that the half-saturated would still be able to absorb water from the air, while saturated would not.

Are Cyano, Amino and diol columns amendable for gradient elution when operated in classical Normal Phase chromatography? What about their equilibration time if we run gradient from 5% ISP in hexane to 10% IPA in hexane?

TIA.

MG:
When you work with water saturated solvents, you accumulate water in the pores. Ultimately, the pores will be full of water and you are doing partition chromatography. This is the kind of stuff that people did before bonded phases came along.
TIA:
Bonded phases are supposedly less sensitive to the water accumulation problem. And yes, gradient elution can be done in normal phase chromatography, just as in RP.