column contamination

[seridi]

please, can anyone telle me how can I check if my column is contaminate or not.
My sample is dissolved in aqous phosphate buffer and some times wen I use 40/60 water -acetonitrile in my gradient the pressur increase.
how can i wasch my column i,I use in my LC-MS analysis C18 column
without column guard

[tom jupille]

Pressure changes during a gradient are normal, a result of the change in solvent viscosity. The pressure should return to its initial value when you return to the initial composition. You should see the same pressure change with or without injection of a sample.

In general, if the pressure returns to its initial value, and if your chromatography looks OK otherwise, I wouldn't worry about it. If pressure increases with every injection, then you might want to reverse-flush your column. Just turn it around and run in the reverse direction at your usual flow rate. Be sure to check with the column manufacturer before you do this. Not all columns can be reverse-flushed!

It's hard to be more specific without additional details on your column dimensions, flow, gradient conditions, sample volume, etc.

[josebenjamin]

Dear Seridi,

If the only indication you have of possible column contamination is the pressure changes, then it is possible you have none. Under the conditions you seem to be working the change in Methanol/water ratios will produce a normal pressure change.

However, it could also be that at some methanol/water proportion, the solubility of your phosphate salt is drastically decreased, and that you may even have salt precipitation. This quite obviously will increase the pressure, and may even plug the column. My experience is that mobile phases with more than 60% organic solvent can have this problem, particularly at salt concentrations higher than 0.02M.

Good luck

Josebenjamin

[tom jupille]

There is more information on buffer solubility in a paper published last year by Pete Carr:
http://www.lcgcmag.com/lcgc/article/art ... p?id=97119