Chromatography Forum

Hi,

We are using a Poroshell 120, EC-C18, 4.6 x 50 mm, 2.7 µm column on our new Agilent 1260 HPLC. Our mobile phase is 60%MeOH/40% Water. Run parameters are 1.5 ml/min flow rate with a 5 ul injection.

The backpressure after a newly inserted column is around 175-180 bar. This pressure consistently increases by about 3 bar every 100 injections or so. The peaks begin to broaden/tail once the pressure hits around 200 bar.

We were able to get about 3 months out of our first column (roughly 100 injections a day/5 days a week.) Once we noticed the peak shape starting to go bad, we replaced it with a new column. This one lasted about a month. We replaced that one with our current column and it has only lasted 1 week.

I am starting to believe there is another underlying issue here besides the column, yet when a new column is installed and calibrated, the peaks look great and pressure returns to normal (175-180 bar.)

Any other ideas what we may be looking at here?

Thanks!

Hello

I'd recommend simple experiment:
-install guard column, run your method and monitor: pressure and peak shape
-when it's getting bad, remove guard column and run sample only with analytical column
it will tell you if your problem is linked with column only.
The you need to consider using guard columns or filtering your sample before injection.

Regards

Tomasz Kubowicz

What is in your sample prep and sample matrix?

I personally would have absolutely no concerns, when a column survives >500 injections. Why do you think there is an issue?

What is in your sample prep and sample matrix?

1 ml of acetonitrile is used as the desorption liquid and then 1 ml of K Acetate buffer is added. This is then filtered a .45 um PVDF filter.

Klaus I is absolutely right. I reckon you're getting 6000 injections out of your column. That is actually very good indeed, and if you divide the price of the column by the number of injections, it's probably costing you a lot less than the sample vials.

Klaus I is absolutely right. I reckon you're getting 6000 injections out of your column. That is actually very good indeed, and if you divide the price of the column by the number of injections, it's probably costing you a lot less than the sample vials.

I agree - I think that management and accountants see a $600+ charge for a column and that shocks them, where the small stuff adds up (sample filters, vials, cost of other disposables, glasswashing) seems less significant but actually is more $$$ per year. We use disposable containers instead of volumetric flasks a lot, but they have a cost and environmental impact, but so does more solvent (purchase and disposal) and glassware washing/drying if we use volumetric flasks. So complex issues.

Klaus I is absolutely right. I reckon you're getting 6000 injections out of your column. That is actually very good indeed, and if you divide the price of the column by the number of injections, it's probably costing you a lot less than the sample vials.

I agree - I think that management and accountants see a $600+ charge for a column and that shocks them, where the small stuff adds up (sample filters, vials, cost of other disposables, glasswashing) seems less significant but actually is more $$$ per year. We use disposable containers instead of volumetric flasks a lot, but they have a cost and environmental impact, but so does more solvent (purchase and disposal) and glassware washing/drying if we use volumetric flasks. So complex issues.

I'm sorry, I must not have made my initial post clear.

6000 injections is very good indeed, however, we were experiencing the peak broadening and increased pressure after only 3 days of running samples ( roughly 400 some samples.)

Use a filter with smaller pores to filter the sample - 0.45 um that you use currently passes particles that are larger than the gaps between the particles in the column (and a lot bigger than the pores through the column frit) so your flow path is progressivey getting blocked as you inject more samples.

Peter

Beside of the possible particles, the backpressure can also raise if the first part of the column is clogged by organic contamination. To pump several mL of isopropanol will often help in this case.

Have you also observed peak-splitting?

What about focusing on the sample preparation?
If the pressure rise correlates with the number of injections it is obvious that something in the sample stays in the column.
Make sure your sample solution is absolutely clear and remember to mix it with your mobile phase and make sure it stays clear.
Best Regards

"We were able to get about 3 months out of our first column (roughly 100 injections a day/5 days a week.) Once we noticed the peak shape starting to go bad, we replaced it with a new column. This one lasted about a month. We replaced that one with our current column and it has only lasted 1 week."

So things started well, got a bit worse and are now really bad? So what changed?

Do you change the water in the mobile phase reservoir (bottle) on a regular basis? (I'm assuming you mix the mobile phase on the pump and are not using pre-mixed solvent). Left in the bottle for too long the water can get algae/bacteria growing in it - and this can then contaminate your system. One way to check is this might have happened is to remove the PTFE filter from the purge valve on the pump - is it discoloured (greenish perhaps)?

"We were able to get about 3 months out of our first column (roughly 100 injections a day/5 days a week.) Once we noticed the peak shape starting to go bad, we replaced it with a new column. This one lasted about a month. We replaced that one with our current column and it has only lasted 1 week."

So things started well, got a bit worse and are now really bad? So what changed?

Do you change the water in the mobile phase reservoir (bottle) on a regular basis? (I'm assuming you mix the mobile phase on the pump and are not using pre-mixed solvent). Left in the bottle for too long the water can get algae/bacteria growing in it - and this can then contaminate your system. One way to check is this might have happened is to remove the PTFE filter from the purge valve on the pump - is it discoloured (greenish perhaps)?