Ugly peak shape of amine compound

[Anthony_Ng]

Dear all,

Let me state our current situation first:
We have only 1 C18 column in the LC-MS instrument. The only mobile phase we use is MeOH and H2O.

Usually C18 give us a good peak shape for simple/small molecules. However, some colleages give us compounds with mass > 1550 and with 2-3 amine groups in it. Also some guanine-like groups are attached.

This cpd give us a terrible peak shape at about RT 12min and I start to add 0.1% formic acid in H2O mobile phase. Hence, the peak is sharp and shift to injection peak region.

I am not sure if I am correct: By adding formic acid, we made the amine group protonated, hence the cpd become ionic and then no interaction with C18 column.

So, should I start to consider buying HILIC to run those amine protonated samples? Can I use MeOH in HILIC? And any other choice apart from HILIC?

Thanks in advance!

[Bryan Evans]

Keep at it with reversed-phase. Use this for mobile phase A:

20mM acetic acid / 20mM ammonium acetate = 50/50

(or some variation of this)

You have plenty of retention on ODS. You need to optimze the method
for peak shape, not retention (meaning you do not need normal phase).

[Vlad Orlovsky]

Your problem is residual silanols. Try to use lower pH mobile phase, may be you will be able to suppress it, but with three amino groups it might be hard. I don't expect your compound to work well on HILIC because it is very hydrophobic. Polarity of amines on compound with 1500 molecular weight might not be enough to provide good retention. You can try mixed-mode reversed-phase columns in reversed-phase cation-exclusion mode. In this mode both your compound and stationary phase are charged positively and basic groups on the surface of silica shield your compound from silanol interactions:

http://www.sielc.com/application_151.html
http://www.sielc.com/compound_110.html
http://www.sielc.com/application_065.html

These are different but applications show improved peak shape for basic hydrophobic analytes.

[Bryan Evans]

Also see if you can buffer your sample diluent.