Chromatography Forum

I am developing a method on a waters spherisorb C18 column. The mobile phase if 50% 0.02M PhosBuffer with 0.1%TEA ,10% ACN, 40% MeOH. The buffer is pH to 8.8prior to mixing with the organic. I assumed that when mixing the pH 8.8 buffer with the organic it should decrease the pH. Ph of the final solution however is 9.4 is this an apparent pH?

My concern is column stability the manufacturer recommends ph 2-8 and states phosphate buffers and high temp will decrease column life. Im runnign at 40C. Will this cause column bleeding/deterioration rapidly? Anyone know?

From what I've read, phosphate is harsh at high pH. High temperature at high pH is even worse. If you need to run under these conditions, you should buy a coulmn that is stable at high pH.

I am developing a method on a waters spherisorb C18 column. The mobile phase if 50% 0.02M PhosBuffer with 0.1%TEA ,10% ACN, 40% MeOH. The buffer is pH to 8.8prior to mixing with the organic. I assumed that when mixing the pH 8.8 buffer with the organic it should decrease the pH. Ph of the final solution however is 9.4 is this an apparent pH?

My concern is column stability the manufacturer recommends ph 2-8 and states phosphate buffers and high temp will decrease column life. Im runnign at 40C. Will this cause column bleeding/deterioration rapidly? Anyone know?

Nowadays many polymer based column are available in market which can be used at extreme pH, but try to avoid using phosphate buffer with 40% of methanol and at 40°C, it will form ppt and can clog your column and your system

Ideally develop a method within the pH range of the column. There's plenty of high pH stable columns (google them, I don't want to suggest some and get accused of manufacturer bias! )

If you must have sperisorb silica and must have a pH outside it's stable range then you should ensure you rinse high pH mobile phase out of the column after each usage with whatever storage solvent is recommended by the manufacturer (probably MeCN) to ensure it doesn't sit and disolve for extended periods.

Edit: Of course don't wash with neat organic if using a buffer which will crash out in your lines in neat organic. Wash with 1:1 organic:Aqueous or with water then organic.

thanks for all the advice. one correction it is water symmetry column. I am evaluationg lower pH now(7.5) it it looks like the same chromatography...not sure why the method was at pH 8.8 to begin with???

Your buffer is based on this equilibrium:
HPO4(2-) + H2O == H2PO4(-) + OH(-)
When you add the organic solvent, it lowers the dipole moment and dielectric constant. The doubly-charged HPO4(2-) is less favored in solution now, so the equilibrium shifts to the right.

In general, anionic buffers (not only phosphate) shift to the alkaline when you add organic solvent, while amine buffers shift to the acid.

Measuring pH in water/organic mixtures is legitimate, but the familiar 0-14 scale gets a bit bent in the process.

The lifetime of silica columns at a pH above 8 is limited, especially at elevated temperature. However, if it is limited, it does not mean that the column will die immediately.

At 40 degrees and pH 8.8, my guess would be that the column will last for a week or 2. This may not be good enough for you.
If you get the same chromatography at pH 7.5, stick with that. If on top of that you can get the same results at 30 degrees, go to 30 degrees, and your column lifetime will be within the range that one usually expects.

If you can do the same separation on an XBridge column, you can stick with the original conditions.