HILIC

[XarixTheAlchemist]

In our lab we have a silica column, can i use it in HILIC mode separation?
Thank you for the answers!

[carls]

Bare silica is good for HILIC. If the silica column is currently in normal phase mobile phase be sure to flush thoroughly with IPA before going to ACN.

[XarixTheAlchemist]

and i think so, but when I run seven compound (benzimidazoles and their metabolites) there has one single peak before first minute and the column was not use for normal phase
Thank you if you help me

[tom jupille]

HILIC has just about as much variability as does reversed-phase, so without knowing details about your conditions, it's impossible to make any specific suggestions.

So:
isocratic or gradient?
if isocratic, what aqueous/organic ratio?
if gradient, what initial/final conditions and gradient time?
what buffer and pH?
column dimensions? flow rate?
what silica (exactly: vendor, name, particle size)

[carls]

All bare silica columns are shipped with normal phase eluent unless it is specifically a HILIC column. Not flushing the hexane out will cause all sorts of weird behavior. Unless you flushed the column with IPA all of the hexane has not been removed.

[XarixTheAlchemist]

Thank you for the advice about OPA I will try.
The column is Poroshell Sil(50x??mm, 2,7um), and the gradien is from 98%AcN 0,1%formic acid(A) to 90% for 3,5 min
and 100mM Ammonium formate(B)
Thank you again for the answers
Best regards

[tom jupille]

You're changing waaaay too many parameters at one time.

Some suggestions:
1. HILIC depends on forming a water-rich layer at the stationary phase surface. With only 2% aqueous in your initial mobile phase, you may not have enough water in there to form a stable layer.
2. It takes time for that layer to form. Equilibration times for HILIC tend to be somewhat longer than for RP (but not as bad as Ion Pair!).
3. Until you understand what's going on, it would be best to use the same buffer and concentration in both the A and B components of your mobile phase.

[carls]

As Tom has suggested start with 90% ACN isocratic and use the same buffer and concentration in both A and B, i.e. 10mM ammonium acetate/formate pH 5/3.5 . Flush any buffer out of your column with 50/50 ACN/water before flushing with isopropanol. After flushing with IPA equlibrate the column for 30minutes with 90% ACN mobile phase (minimum of 25 column volumes). Run your analytes isocratically with 90% ACN. If they still elute unretained you could try 95% ACN, however, if 95% ACN does not give a retention factor of at least 2 then you are probably better off with reversed phase. Prior to running at 95% the column will need to be equilibrated at 95% for at least 20 column volumes.