UPLC Acquity BEH columns

[patriciodp]

Hi! I´m trying to develop an analysis method to quantify vitamin A. The problem I have is that I´m using an Acquity BEH C18, 1,7um 50 x 2,0 mm and I observe that the pressure increases continously with subsequent injections until it passes the 15000 psi. It also happens when I pass 100% MetOH through a new column. I hope if someone has an explanation for this. Or if someone have had problems with this kind of columns.
Regards

[tom jupille]

Where does the pressure start?
How much time (and/or how many samples) does it take for the pressure to climb to 15k psi?
Does the pressure come back down?
Have you tried backflushing the column (and if so, what was the result).

UHPLC columns generally are much less tolerant of particulates than conventional columns. "Good column hygeine" for these columns includes filtration of *everything* (samples and mobile phases) through 0.2-micron filters.

[rwang]

Do you use a pre-column filter (Phenomenex KrudKatcher Ultra or Waters Vanguard Pre-column)? If not I strongly recommend using one. Better to trash a filter than a column.

Sounds like somewhere in your fluid line was clogged - UHPLCs use very thin capillaries that are much more prone to blockage than ordinary HPLC tubings.

BTW, what is your method for your samples? Just making sure there's no precipitation of buffers happening inside the column.

UHPLCs require a much higher standard level of "chromatography hygiene" and you are expected to change your aqueous buffer everyday.

Hope that helps.

[patriciodp]

In the lab, we filter every mobile phase through 0.22 um filters. The samples we inject are also filtered through 0.22 um. We also use pre-columns filters. The problem we have is that we take a new column and before we make the first injection we stabilize with 100% Methanol(that is the mobile phase) and the pressure starts at aproximately 2500 psi at 0,5 ml/min. About an hour later the pressure is at 6500 psi!!!! And we didn´t make any injections to the column that is new, without any usage. We´ve seen that problem with two of this columns.
It´s very strange.

[Klaus I.]

What happens when you don’t filter your solvent? For UHPLC-Applications, I have never observed disadvantages when I have used common LC-grade solvents. This filtering causes often trouble for UHPLC.

[lperelman]

Just a warning: VA sticks to many filter modalities. You will want to test filters using UV before you move to HPLC. I work with many retinoids, and they are quite difficult to handle. I use the exact same column, and we have had no problems with column pressure. Can you provide method details? Diluent? Mobile Phases? etc...

[Klaus I.]

The problem we have is that we take a new column and before we make the first injection we stabilize with 100% Methanol(that is the mobile phase) and the pressure starts at aproximately 2500 psi at 0,5 ml/min. About an hour later the pressure is at 6500 psi!!!! And we didn´t make any injections to the column that is new, without any usage. We´ve seen that problem with two of this columns.
It´s very strange.

This sounds different compared to you first post in this thread. Maybe Waters columns are shipped with 100 % acetonitrile or in mixtures with water up to ~50%. Methanol have a much higher viscosity then acetonitrile. How looks like the pressure curve during this hour of equillibration?

I have not much experience with 100 % methanol, but with a 5 cm UPLC-column the backpressure of 450 bar is maybe not to high for a flow rate of 0.5 mL/min. The backpressure of 100 bar at the beginning is maybe a indication (without knowing your exact system-setup), that the column is stored with 100 % acetonitrile when it was shipped.

You can also examine the conditions of the QC-testrun from the vendor, the backpressure is for Waters UPLC-colums also noted there (Be careful when you read-out the data from the column-chips, there are sometimes problems und you can read only the date from the column installed before).

And again the question: Why do you filter your solvents, especially when you only run the analysis with 100 % MeOH? What solvent-grade have your MeOH?

[patriciodp]

Thanks for all the answers. The methanol I use is HPLC grade. I know that hplc grade solvents are filtered through 0,22um before being bottled. But I can`t be sure that there aren`t any particulates, so I filter the methanol through 0,22um. The pressure rising in a new column being a cause of replacing the Acetronitrile of the columm by methanol is a good explanation. I think those columns are shipped with Acetonitrile. I´ll continue with my assays, and if I have many problems, we have an alternative HPLC method that works very good. Thanks to all who responded!!!!!

[Mattias]

I often find that the capillary after the column becomes clogged (Acquity with BEH-columns). You can see how much the pressure decreases if you disconnect the column outlet when the pump is running.

We have replaced the capillaries with less narrow ones, and the problem is gone.

[lperelman]

I think Klaus is onto something here. I have methods that use the UPLC BEH columns that do not respond well to rapid increases in flow rate OR changes in mobile phase. When we slowly increase the flow rate or new mobile phase, then we get good response. Go slower as you equilibrate your column. In fact, the care and use manual for the column says you should start at 0.1 mL/min and increase slowly until you achieve your final flow rate.

[Alexandre]

We never filter MS Grade solvents, 6 years already, just find a good brand. If you do, buy then HPLC grade they cost ~30-50% less. 2 mm ID UPLC columns never gave us problems. You need good work instruction to all your staff how to protect glassware from dust, special care for washing, never use UPLC solvent beakers and flasks for anything else etc.

However, check your filters, some are not compatible with strong organics you may block column just with dissolved plastic.