We are using a C-18 column (Nucleosil 100, 5 u, 150mm x 4.6 mm, PEEK hardware) to seperate and identify Tc-99 using a Flow Through LS detector. We are running an Isocratic gradient of 1.8mmolar TBA (Tetrabutylammonium Acetate) and ACN in a 70/30 mixture at 1 ml/min. For some reason, the pressure continues to rise as the column is used. I have a prefilter and precolumn in place before the column, and flush every night with a 50/50 ACN/Water mixture. I have even tried backflushing the column with no reduction in pressure. The columns start at 1400 psi, but by the end of the day are at 3200-3500. By the next day, the pressure is over 5000 psi. I have tried a second column, and even a Supelco C-18 column with the same results.
Any help would be greatly appreciated!

Try changing some of your PEEK tubing lines (or back flush them), maybe you got some particulate matter stuck in the head of one of the lines. This might explain why you don't see an improvement with a column change.

Sounds like one of our problems that prompted me to start the chain "Frits vs. sieves.." of june 26. (Seems to me that something in that direction was discussed even earlier).
If you use a light scattering ? detector your Tc must be attached to a large molecule, a protein?
Does your column start out at 5000 psi on the next day, or does it rise to that within a few minutes after starting flow? Is your flow resistance lower in the backward direction?
What solution is in your column over night?

We have tried changing the PEEK tubing, even bypassing the Injector, but the pressure still stays high (although it was an excellent suggestion to try).
In terms of the Frits vs Sieves, (by HW Mueller), I have read with interest the chain you started earlier, but was not quite clear on which manufactores use a sieve instead of a frit (which I gather is better at not clogging).
The detector we are using is not a light scattering, but Liquid Scintillation. It doesn't need to attach, just produce a photon which causes the formation of light, which is measured.
The column usually starts high the next morning right off the bat, although it does take a few minutes to reach the max pressure. The resistance is just about the same forwards and backwards. The column is normally left in a 50/50 ACN/Water mixture at night.

What else is in your sample besides Tc-99?

Right, anonymous (June 29). . . , are there macromolecules involved?