Chromatography Forum

Hello fine people,

We're having an issue here in our lab running an assay of L-Glutamic Acid on our agilent 1100 hplc. We're doing a gradient elution with mobile phases of sodium phosphate 0.033 molar with a pH of 7.8(mobile phase A) and an acetonitrile 45% methanol 45% and water 10% solution(mobile phase B).

Our gradient program:


We're just using a Zorbax eclipse AA column with dimensions of 4.6 x 150 mm packed with 5 micrometer beadlets.

The issue we're running into is that randomly through our a run one or more of the injections have a steep drop in pressure that's unexplained that recovers in about 30 seconds. It's throwing off our assay. We're trying to nail down a cause but we haven't had luck yet.

Here is a image of the ideal pressure curve and then two pressure curves that had unexplained drops:

Normal


Abnormal:



As you can see the pressure drop isn't consistent as to where occurs in the injection and it happens randomly to one or more of the injections. The first time it happened everything was running smoothly for the system suitability and then when we finally got to our sample it happened. A bit frustrating considering each injection takes over thirty minutes.

The column has been in use for about a year and we've probably done close to fifty runs on it, we cleaned out the pump and we're really drawing blanks on what the problem could be.

We were hoping to not have to replace the column, but we will if necessary. We just replaced the guard column after having peak splitting issues.

Additional information can be provided at request.

Thanks you very much for any advice provided!

The saw-tooth short term fluctuations look like symptoms of a bubble in the pump to me.

Peter

We came to the same conclusion at first, and we purged the lines multiple times. The strange thing is that this happened after many injections the first time. Maybe that's how long it took to work the bubble out?

You are using a quaternary pump? I would suspect check valves or GPV.

We came to the same conclusion at first, and we purged the lines multiple times. The strange thing is that this happened after many injections the first time. Maybe that's how long it took to work the bubble out?

Do you have a vacuum degasser, or do you purge your mobile phases continually ?. If not then air will gradually dissolve in them, and sooner or later you will get a bubble in the mixer which gets into the pump and disrupts the flow and pressure.

Peter

We have a degasser on the HPLC, we have other HPLC setups so we might end up switching out the degasser from one to the other considering the setups are identical. We will have to wait and see because we just replaced some of the seals in the pump and we're doing another run to see if that was the problem.

Nearly all is already said.
How are your pistons looking?????
I'm 100% confident it comes from the pump head/pistons.
Look on them with a magnifier.
Good luck

We cleaned out the pump. We just ran the analysis again and it seems like the issue was the pump seals needed to be replaced. Thanks for all your suggestions! You guys were right on the money probably. Air was probably leaking in through the faulty seals after the degasser. Thanks again.

Guy - also remember to purge your C and D channels too, even if only A and B are used in your gradient/Method.