Chromatography Forum

Hi All,
I have a problem separating steroids, and I'm wondering if anyone would have any advice to offer?

I am trying to separate three steroid peaks.
Steroid 1 and Steroid 2 are closely related. Between them lies a small peak, which may be a possible intermediate between the two, as it is a related peak. The problem is, while the intermediate is sufficiently resolved from Steroid 2, it elutes on the tail of Steroid 1, and is too close for integration purposes.
I am currently using a C18 column, 5um particle size, 4.6mm X 250mm dimensions. My mobile phase is 60/40 MeOH/H2O. Flow is 1.0ml/min
Thanks in advance for your help.
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First, try acetonitrile instead of methanol. About 45% acetonitrile will give you about the same retention. If the intermediate peak is now closer to peak 2, and insufficiently resolved, try a mix in between methanol and acetonitrile.

If there is no hope with acetonitrile, try THF, about 40%. Otherwise the same game as above.

If you still do not get it resolved, use a different column. We can give advise on the best options, if you tell us more about your steroids - functional groups, differences between the three etc.

As noted bu Uwe, try using acetonitrile in mobile phase first, and ensure your sample is dissolved in the mobile phase. Not sure what your detection is, but some simple steroids don't have any strong chromophores, which can limit use of some other solvents.

If that doesn't work, I've found just adding 1 - 10 mM of ammonium acetate to the water can improve the peak shape and separation of some steroid derivatives on C18 reverse phase systems, but if using UV, please be aware that such additions can also affect UV spectrum ( for example the relatively narrow Trilostane UV spectrum peak moves from 252 to 282nm).

Further to Bruce's suggested use of NH4OAc to improve peak shape/resolution, you could instead try 0.1 % H3PO4 if you need wavelength detection @ 210 nm.

Since we all learn from these problems, PLEASE post results (negative as well as positive).

Good Luck,

Motoball