HPLC Columns For Highly Retained Compounds

[Rosi Lan]

Which column is recommended when separating neutral or other highly retained compounds?

[tom jupille]

That depends on what the "highly retained" compounds are. In the absence of any other details, a good starting point for most people is a good C18 column based on "type B" silica with a "full range" gradient from water (or buffer) to organic solvent.

[Rosi Lan]

That depends on what the "highly retained" compounds are. In the absence of any other details, a good starting point for most people is a good C18 column based on "type B" silica with a "full range" gradient from water (or buffer) to organic solvent.

Thanks, very helpful！：）

[Vlad Orlovsky]

You might want to consider low loading C18, or something with a shorter chain(C4, C8) or more polar stationary phase. This will shorten your method and save some ACN (or MeOH) you will use in the mobile phase.

[Kazimierz]

Dear Rosi,

"Highly Retained" I unerstand as Late Retention Peaks when You using Reverse Phase HPLC columns.

Plese try with HILIC columns f.e Silica Si column and 90% Acetonitrile as mobile eluent HPLC phase. In this way, the higly retained compounds will be eluated first, near the chromatogram front. The larger size column are better because easer packed by manufacter.

Serum clinical sample is depronized with acetonitrill or HCLO4 and next acetonitril.

For example - Ganciclovir is separated on C18 column but highly retained pek appear with rt=22 minute. However, in HILIC mode and Si column, the late pik going in front and ganciclovir posses nice clear chromatogram.

[H.Thomas]

I doubt that "Rosi" is still reading here, since she only wrote this one post and it is 3 months old, but for the sake of discussion:

We do not know, if she just wanted to get rid of the late eluters, or if these were her analytes.

I would second the advice to try a simple C18. One could change the strong eluent from MeOH or ACN to IPA/ACN. We use this eluent for HPLC of intact triglycerides - works well and the backpressure is comparable to MeOH.

HILIC will lead, as Kazimierz wrote, to the disappearance of the RP-late-eluters in the solvent front. I once had the task of developing a method, where very hydrophobic substances (glycolipid biosurfactants) had to be separated in HILIC mode. I ended up using ACN with 40% DCM and 10% water in ACN as eluents. I don't know if this can still be called HILIC, but it worked - even though there was no water in the weak eluent.