detergent and lipids in FPLC

2 posts / 0 new
Last post
Aggi
Aggi's picture
detergent and lipids in FPLC

Hi there,

I'm currently trying to run peptide samples containing detergent (1%) and some hydrophobic substance in a GFC system. I want to see the separation of peaks and difference in retention times of different combination of samples. Probably will use PBS or saline as mobile phase.

I'd like to know whether having a detergent in the sample will affect the run?
is having the hydrophobic substance will affect the quality of column (it will stick to it?). i'm using superdex.

The hydrophobic substance was only soluble in a certain buffer with detergent added to it. so if i inject it in a system where only PBS or saline is present, will it then precipitate out of solution? if so, is there any suggestion as to how to do it? if using detergent in the mobile phase, the back pressure might be too high.

Thank you.

-Anggia-

Dr. Analytical
Dr. Analytical's picture
In gel filtration

In gel filtration chromatography (GFC), you must dissolve your sample in a "good" solvent. But your sample must also be soluble in the mobile phase. The best method is to dissolved your sample in the actual mobile phase that you are using. You are correct that the sample may precipitate in a different solvent. To check this, dilute some of the sample with mobile phase and watch for the solution to become cloudy. It may take a few minutes. However, usually your sample solution is not very concentrated, and at dilute concentrations it will be soluble even in mobile phase. But there is no problem with using a detergent in the mobile phase. Visit the website for the column manufacturer, and look for GFC applications of proteins. There are many other ways to do this separation.

Also, I hope you realize that GFC is not a high resolution technique. Very small changes in molecular weight will not be noticed. You must choose your column carefully so that it can separate molecules in the size range where you want to see differences.

Good luck!