Chromatography Forum

Hi guys,

We have a new fairly quick method for Vitamin A anlysis. We quantify retinoic acid, retinol, retinal, retinyl acetate, and nwo retunyl palmitate in 10 min via HPLC-UV. Here's the problem: retinyl palmitate shows 0.2-1% carry-over. None of the other species have any detectable carry-over or only very low acceptable carry-over.

The gradient is held at 95% B for another 1.5 min (3 column volumes) after the palmitate has eluted and the syringe is washed 3x with IPA, and the valve is washed 1x with water and 3x with IPA. Increasing time at high B and adding a wash did not attenuate the problem at all, which is strange.

Where do you think the carry-over comes from? It obviously has to do with the hydrophobicity of the compound. I am thinking about adding 10% or so octane to my organic IPA wash. I would like to avoid letting the method run for much longer at high B after elution of the palmitate since time = $$$.

Let me know if you have experienced anything similar and how you overcame the problem (or didn't).

Mobile phase A: ACN/water mix. B: 90:10 IPA/ACN.


Many thanks as always,

Arne

Hey there - Hydrophobic species can and do stick to RP columns and decreasing the polarity of your column wash a bit can't hurt, but I can see this kind of thing happening in a situation where your needle wash solvent is not strong enough. I might try a stronger solvent there along with a longer wash time, if that's an option, before fiddling further with the mobile phase composition.

Good luck and let us know how things turn out!

CJ

to avoid this problem , i think you have to make a delay time ( washing with mobile phase ) after every injection at least 10 min.

ph/Amr Tarek
Instrumental Unit Head at Otsuka pharmaceuticals