To my understanding your column is a plain bare silica and these do offer limited selectivity in HILIC for structurally similar compounds. It might help to alter the type and concentration of the salt in your mobile phase (you did not mention what you use), but I would rather go for a column with a bonded hydrophilic stationary phase, such as zwitterionic or amide, which are know to provide more retention and selectivity under HILIC conditions.

Thank you for your response. Yes, the column we initially ordered was based on an amide phase, but we unfortunately received a bare silica column instead..

Regarding the mobile phase, I initially used 10 mM ammonium acetate. Subsequently, I switched to a methanol/water mixture in equal ratio. However, I am encountering difficulties in detecting glycerol under these conditions. On the other hand, I am able to observe a clear separation of serinol and 3-amino-1,2-propanediol.

Could you provide any insights or recommendations on how to improve the detection of glycerol? Any additional suggestions for optimizing the mobile phase or column conditions would be greatly appreciated.

Thank you for your assistance.

Best regards,

So it seems you have two issues, both detection and separation? Maybe the free online booklet "A Practical Guide to HILIC" (available on ResearchGate, although it quite some years since we wrote it) could be a source of useful tips for you? For the separation I recommend you stick with acetonitrile since methanol tend to give too much hydrogen bonding thus competing with the HILIC retention mechanisms and weakening the immobilized water layer that contributes to retention. You might need 70-90% acetonitrile for sufficient retention (more ACN = higher retention). Salt levels often need to be higher in HILIC compared to reversed phase due to the electron rich nature of separated compounds and the stationary phases. For detection; can you find glycerol in UV or RI without the column? In UV you might need lower wavelengths, but in RI there is often not much to do other than having a sensitive detector and increasing the concentration of the analyte. Also keep the temperature stable to avoid drift in RI.