Chromatography Forum

I am using a Zorbax C8 250X4.6mm, 5µ column. My flow rate is 1 ml/min. Injection volume is 50µl. Mobile phase-Buffer Phosphate 0.05M/Acetonitrile.
All the peaks have a very good shape on a new column, after some period of usage, ALL the peaks start to be distorted (they have tailing, the height is considerably reduced and further peaks distortion and splitting can occur). This phenomenon repeated again and again as I change the column (4 columns are not good now). After I used the guard with the new (5-th) column, the phenomenon occur even faster! The phenomenon is irreversible-I washed the column with Acidic Water, Acetonitrile, Acidic Methanol-without any success.
What is the reason? It seems to be physical problem (symptom is show up on all the peaks in the chromatogram)… But may be it is chemical problem -I suspect about xanthan gum that inside the sample injected (we try all the filters, but xanthan gum pass all of them), may be it remains on the stationary phase of the column. Please help! I am despaired and don't know what to do…

As you imply it is something that is irreversibly bind to your column. If this is the case a C18 clean up should do the job as you will be able to elute your analytes but not the xanthan gum or whatever the compound to blame...

If xanthan gum is the problem, you can try flushing your column with warm (60[sup]o[/sup]C) water:methanol (95:5). Don't use a column heater, but heat the water to 60[sup]o[/sup]C on a hot plate. Backflushing will be more effective, but check with your column manufacturer before you do this since it could damage some columns. To avoid the problem in the future, you can use a C18 solid phase extraction cleanup prior to injection.

I assume you know that you need to flush your column with your mobile phase without the phosphate buffer after running samples. If this is not done, phosphate salts can precipitate in the column and cause physical damage.

If we point a finger at the xanthan, are your sample solutions noticeably thickened by having this component in there? Literature suggests that the effect on solution viscosity is one of its most widely exploited properties. Additionally, it is stated that a lack of significant change in viscosity with temperature is somewhat unique - trying to clean your column of xanthan with heated solutions would presumably offer no additional benefit. The fact it is soluble in hot and cold aqueous solution makes one wonder if deposition would even be a concern.

To summarise the thickness issue.. perhaps peak shape is affected by inefficient mixing when injected into the mobile phase. Do the problems remain when using a diluted sample or a low injection volume?

I work with formulations containing a pectin and that poses a problem for reverse phase with acidic/organic mobile phase.
Running the column backwards with 100% water should do the trick (assuming it's ok for your particlular column).

You should modify your method to have a wash step at the beginning to pass the xanthan gum. Start off with < 5% organic and a neutral buffer then transition to acidic buffer and an organic gradient.