Two Phosopholipds, same retention time...

[glocke12]

Hi

I have two phospholipids that are very similar in MW (one is ~1350, the other is ~1495) that I am trying to separate via UPLC. I have tried several different things so far in terms of gradient and buffer, but they both have the same exact RT no matter what I try.

Any suggestions?

[Kristof]

Hi,
Could you tell us more about those phospholipids? Is the only difference a molecular weight? And it would be because difference in length of lipid chain I suppose?

Some time ago I saw some HPLC separations of closely-related lipophilic molecules with even smaller (or none) difference in MW using C30 column - have you tried that?

[glocke12]

Hi,
Could you tell us more about those phospholipids? Is the only difference a molecular weight? And it would be because difference in length of lipid chain I suppose?

Some time ago I saw some HPLC separations of closely-related lipophilic molecules with even smaller (or none) difference in MW using C30 column - have you tried that?

Hi

Lipid 1: Phospatidylinositol3,4,5-triphosphate MW= 1795
Lipid 2: Phospatidylinositol4,5-bisphosphate MW= 1513

The column I am using now is: Phenomenex Kinetex C8 2.1x100mm 2.6um, I have not yet tried a C30 column.

[Kristof]

Other idea - they have different charge so perhaps you could try ion exchange? With organic solvent in mobile phase to decrease interactions of lipid chains?

[carls]

What pH and buffer are you using? I would guess pH 7-8, would give you the best chance for selectivity. Also, what organic modifiers have you tried?

[unmgvar]

several things that can help
use a polar embedded column like the HP c-18 from sepax
http://www.sepax-tech.com/application_notes/OD1001.pdf

or you could try to reduce the temp, in the oven,
10 or even 5 degrees make better for separation, i don't think the column will be capable of handling the pressure on UHPLC

you could also try a cholester from nacalai
the C-30 also works best at lower temp, of 20 or less

you could also try a HILIC column i know it works also for the nucleotide mixture