Chromatography Forum

Hi, everyone,

Today I performed a formulation's assay, unfortunately, the baseline droped periodically during the sequence running, which shocked me greatly:
The injection 1 shows a good baseline:



but the next injections puzzled me:









Frome above pictures it seems that the baseline drops and uprises gradually and the period is 15min approximately between two consecutive injections. Comparing with the method validation and historical data, this worst baseline is unique and first case, and I don't know how to do next, would anyone give me some suggestions. Thanks in advance!:)

Best regards

Austin

Hi!

First of all check the

1. presure signal
2. temperature signal
3. Solvent composition AND mixing (if any)
4. proper degassing
5. UV lamp (intensity)
6. try change column

Hi Austin,

By looking at the y-scale I can see an alarming inconsistency at the beginning of each run/injection. That makes me think of something happening later in the chromatographic run.
For a start, I would say; the column isn’t equilibrated adequately at the moment of injection. Try and inject your sample and collect data for much longer than you do now (f. ex. 50 – 60 min) and show us the data. I would like to see the pump/gradient? method as well.
Also, have you monitored the backpressure through the run?

Best Regards

Austin:

Garlarg and Danko are guiding you on the correct course. I had something somewhat similar with an Agilent MWD detector a couple months ago, but the actual cause was somewhat atypical, so I won't go into it (yet).

Outside the obvious, which would be pump issues (wildly inconsistent pressure readings, flow inconsistencies, etc,) or incomplete degassing, I would try the following:

Equilibrate your column, and then program in a long run with no injection (overnight).

If you still have baseline issues, remove your flow cell, wait for the baseline to normalize, and collect data for the same period of time (obviously, you don't need any flow through the column for this data collection period).

If you have no issues with either set of conditions, that might point to a problem with your injection system.

If you have baseline issues with your flow cell in place, but not when you have the flow cell pulled, then you might have a problem with bubbles in the flow cell.

If you still have the problem with the flow cell pulled, you may have an issue with the detector optics, electronics, or the lamp, and possibly the environment the detector is in (check for consistent temperature within the operating range specified by the manufacturer, and make sure the detector is not exposed to direct sunlight, A/C flow, or excess vibrations). If environment is ok, or hasn't changed relative to this detector issue manifesting itself, the easiest fix is to change the lamp, recalibrate the detector, and repeat the data collection without the flow cell. If that doesn't work, it may be a more difficult problem to solve.

Thanks for your replying!

The gradient method comprises of two mobile phases:
A:pH3.0 acetate buffer-ACN(70:30), B:pH3.0 acetate buffer-ACN(20:80),
Gradient:10%-20%-100%-100%B(0-15-18-23min)

The chromatographic parameters are kept constant in sequence running(pressure, temperature, flow). For degassing, some batches of mobile phase which weren't be degassed completely had no impact on the baseline drop in previous runs yet, and the equipment can degas automatically, so I think those parameters are not be suspected.

Then I tried the isocratic equilibration of the column(100%B) and the baseline dropped and then uprised:







This time the period became bigger and more regularly(about 30min), and I didn't think the pump malfunction is the source of this problem, so the left suspicious parts are the injection loop and detector, OK, what can I do next step?

BTW, the buffer solution preparation is suitable(1L of 10mM acetate solution added 8ml TEA and 5ml TFA, then adjust pH3.0 with 20% TFA)?
because the analyte is a polar compound and analyzed with IPC method in USP&EP, for slow column equilibration and expensive IPC reagent, so our lab developed the gradient method, but from now on it seems that this method is not suitable for the compound analysis, it may be optimzed.

That looks almost exactly like the issue I had with my Agilent MWD. I had to send it in for bench repair. They ended up replacing the main board to finally fix the issue.

To confirm this, try to run your same detector parameters without the flow cell installed in the detector. If the baseline drops and rises the same way, then it's completely related to the detector, and not the rest of your system.