Chromatography Forum

I had just start to work with UPLC and I´m having big issues with pressure. I´m doing amino acids PITC derivatives separation on an Hypersil Gold C-18 column, 50 x 2,1 mm, 1,9 microns. The original analysis method was developed for HPLC and I´ve adapt it to UPLC with very good results in separation but I´m having problems with pressure although I filter the buffer every day, and I prepare it freshly every two days, I also filter samples and I use an precolumn filter. My buffer is 140 mM (NaCOOH) and I wonder if may be is too concentrated for UPLC, could anyone give me any advice?

That's not very much to go on... What exactly is your pressure problem? (consistently high? building up? how high?) What's in your samples apart from amino acids? (solvents?) Do you use a gradient? Flowrate?

What is the other component apart from the buffer - acetonitrile?
What is the organic versus inorganic ratio?

Best Regards

And what pore size filter?

I´m using a gradient from 96:4 Buffer:Acetinitrile to 50:30, at 0,7 ml/min and oven at 36ºC. The pressure now, at gradient initial conditions is around 7500 psi. I installed this column yesterday and the pressure just after conditioning, was around 5400 psi and this morning it was reaching 6200 psi. My samples these days are only standards, they are on buffer: Acetonitrile 95:5.

Thank you in advance for your help.

That pressure doesn't seem very high to me, especially at such high a flow rate.

The reason I asked about the pore size on the filter is that UHPLC columns typically have 0.2 micron inlet screens. If you are filtering through, say, 0.35 micron filters, you may indeed be letting particles through that will clog the column inlet. If the pressure drops back when you replace the precolumn filter, that's your problem.

Thank you Tom but I´m using a 0,2 microns filter ...

hi

I assume the initial pressure keeps increasing over time. Try running blank injections and see if the pressure increases, at least then you can say if the issue is the std / samples or mobile phase.

Are you premixing your mobile phases or are you using pure buffer and pure acetonitrile?
If your're not premixing yet, I'd strongly suggest to give it a try. Upon external mixing, you might see some cloudiness in your eluents which gives you the proof that the buffer salt is precipitating.
Your problem might also arise due to different mixing characteristics and significantly lower dwell volume of the UHPLC as compared to the traditional HPLC. Premixing might help...