Chromatography Forum

Transferring a method from HPLC to UPLC we now have encountered problems with column overpressures that were not seen on our old system. Our extract is a chloroform dip of plant leaf material which is directly diluted into ethanol prior to injection. Mobile Phase is ACN/H2O with formic acid for MS detection. Numerous samples will run at stable pressure then suddenly an increase occurs and off it goes up and up and up. Often this happens after a blank sample containing only ethanol is injected. Cannot seem to be able to recover column once this has happened with either back flushing or purging with high organic. We filter all solvents, change at least every two days, have 5% organic in our Aqueous phase and use the recommeded inline filter system but I think the problem is more related to the samples rather than bacterial build up as we don't see a pressure increase when the system is running through solvents alone - only after injection.

If surface waxes or other contaminants are been deposited on the column is there any way we could flush the column at the end of a run to get rid of these things enabling us to get a longer lifetime out of our columns? Sometime we are only getting a couple of hundred injections out of a column which for UPLC is only a few hours worth of runs.

It could be that the column frits are becoming blocked. The UPLC columns have quite a wide particle size distribution, and so need frits with a small mesh, therefore they are more likely to block than columns with larger particles which have larger frits.

I posted yesterday re the "Fused Core" columns which as they have a larger particle size and also a narrow particle size distribution, they have bigger frits less prone to blocking. Ypu may want to try one of these on your UPLC system. Link to post below

http://www.sepsci.com/chromforum/viewtopic.php?t=5492

Thanks for the tip developer1974. I am not familiar with the Agilent system that you have. Is this a fast or conventional HPLC? I have taken a look at the Sigma website and the columns look impressive. Do you think that the larger particle size would stop these system overpressures we are experiencing? I haven't much experience with choosing column types.

Try to filtrate your samples before you inject them. We had to do that for a couple of methods when we took them to the UPLC system.

We thought of filtration but it reallt would not be possible as this is going to be a very high throughput method and we are using the Waters 96 well plates to inject from. We have however considered centrifugung samples which may be a more suitable option but ideally we would like not to add any extra steps to our extraction procudure if possible as time is really a factor. This is why I was very interested in the possibility of using an alternative column or flushing the column somehow at the end of a sequence - a high ACN step has not helped so was looking for something that would flush out any waxes etc.. that may have built up. Thanks anyway for your suggestion. It's good to know that we are not the only ones who haven't transfered seamlessly into UPLC.

Just for the record, what is the P-max during your analysis?

The system will build pressure up and up until it eventually overpressures and stops the flow automatically. The p-max is usually about 15,200psi but obviously anything above 15,000 psi on the Acquity is an overpressure alert.

What meant is, if you take a fresh column what is the pressure at the maximum on your UPLC system?

When a new column is fitted at our gradient start conditions the psi is 6,900 psi which is almost exactly what the literature states it should be for our running conditions

This does not is extreme high.

Do you use a guard column?

I agree 6,900 psi is not extremely high but this is the starting pressure. You will see from my other comments that after we inject samples the pressure quickly builds up to over 15,000 psi!

I have ordered one of the new columns that developer1974 recommended and hope that this helps solve our problem - fingers crossed.

I agree that the best solution to such problems is to catch the sample debris with a guard column. I also know that not everybody agrees with me...

How old is your UPLC (i.e. are you one of the early adopters)? If yes, you might want to change your valve rotor...

We could try the guard column idea but does anyone know of any that are pressure tested for the Acquity. Waters UK haven't yet introduced any although they say it is coming this summer.

Our UPLC is very new - only been in service for around six months

When the engineer came to install our Aquity we were told to never backflush the waters UPLC columns, has anyone tried it, and do the columns survive? (we've been scared to try)

Also we run biological extracts (blood / tissue etc) on our UPLC and have seen a similar pressure buildup - as you might expect without a guard column however the buildup occurs over the first 50 injections or so, and then seems to stabilise.
A new column has pressure of roughly 8,000 and bulds up to about 13,000 and then is stable for 500 injections and counting... dont know why this should be??? maybe lower your flow rate by 0.05ml/min if possible and see if you get a stable pressure??

James