Decrease in signal during repeatability test

[lo8864324]

Hello, I'm new to HPLC, we have just installed a new chain of HPLC from Shimadzu recently; when we run a repeatability test, picking the sampel same 5 times, we observe the intensity of the pic decrease progressively (e.g. 100% down to about 50% after the 5th cycle). When we re try next day, same sample, same vial, same effect. How do we explain this phenomena? And how to make it constant? Thank you.

[tom jupille]

Additional information would help:

1. Is the method isocratic or gradient?

2. Are you measuring peak height (amplitude) or area?

3. How much of a decrease are you seeing (1%, 10% . . . )?

4. When you repeat on the second day, does the peak return to its original value, or does the trend continue where you left off (e.g., is it [Day 1: 10,9,8,7,6; Day 2: 10,9,8,7,6] or [Day 1: 10,9,8,7,6; Day 2: 6,5,4,3,2])?

5. Are you using a fresh batch of mobile phase on the second day, or still using the original batch?

6. Are the retention times and the dead time (t0) constant?

[laboratoriocqc]

I have the same problem. We are using an isocratic method with electrochemical detection.

The decrease of the peak high between the first and the second run is about 40% but is not constant in the further runs
We measure peak area
In subsecuent days we use the same mobile phase and the retention times are the same.

[tom jupille]

laboratoriocqc:

. . . but is not constant in the further runs

By "not constant", do you mean that it continues to decrease, or that it varies around the same (lower) average value?

And on the second day, does the value return to its original level or stay about where it left off at the end of the first day.

The point to all these questions is that decreasing peak area can be due to a short list of possible causes:
- change in column chemistry
- change in mobile phase chemistry
- change in temperature
- change in detector response

The fact that the retention times are remaining constant mitigates against the first three, so I'm focusing on the detector. Electrochemical detectors are notoriously subject to electrode poisoning, and the contaminants can come from either the sample or the mobile phase. If it were a mobile phase issue, I would expect the response to continue decreasing with exposure to the mobile phase.

[laboratoriocqc]

laboratoriocqc:

. . . but is not constant in the further runs

By "not constant", do you mean that it continues to decrease, or that it varies around the same (lower) average value?

And on the second day, does the value return to its original level or stay about where it left off at the end of the first day.

The point to all these questions is that decreasing peak area can be due to a short list of possible causes:
- change in column chemistry
- change in mobile phase chemistry
- change in temperature
- change in detector response

The fact that the retention times are remaining constant mitigates against the first three, so I'm focusing on the detector. Electrochemical detectors are notoriously subject to electrode poisoning, and the contaminants can come from either the sample or the mobile phase. If it were a mobile phase issue, I would expect the response to continue decreasing with exposure to the mobile phase.

laboratoriocqc:

. . . but is not constant in the

further runs

By "not constant", do you mean that it continues to decrease, or that it varies around the same (lower) average value?

And on the second day, does the value return to its original level or stay about where it left off at the end of the first day.

The point to all these questions is that decreasing peak area can be due to a short list of possible causes:
- change in column chemistry
- change in mobile phase chemistry
- change in temperature
- change in detector response

The fact that the retention times are remaining constant mitigates against the first three, so I'm focusing on the detector. Electrochemical detectors are notoriously subject to electrode poisoning, and the contaminants can come from either the sample or the mobile phase. If it were a mobile phase issue, I would expect the response to continue decreasing with exposure to the mobile phase.

Thank you for your answer

usually after the first day it becomes a large peak again but smaller the first of the first day.

We changed de column and the problem persists.

Yesterday I "cleaned" de detector but the problem appeared again today

May it be related to the sample preparation? The problem appears with plasma serotonin and urinary cathecolamines, so the preparation process is different...

[danko]

Let me get something clear here: When injecting the same sample the following day, where is the sample?
1. Exactly the same HPLC vial as the day before
2. Same sample preparation/solution but in a new HPLC vial
3. Same sample material but freshly dissolved and dispensed in new vials
In addition to the above questions:
1. On the first day, do you inject from the same HPLC vial every time or do you inject from different vial every subsequent injection?
2. Have you looked at the remaining content of the vials after injections and if so, is the solution clear or maybe cloudy?

Best Regards

[Kreall]

Maybe there is something wrong with the injector. Is there a same problem with unknow samples if you inject them more then once?
Maybe the solution is very thick so the injector parametrs needs to be adjusted accordingly.
Maybe from injection to injection the pressure in vial is getting lower and lower and the syring doesnt have enough power to overcome this pressure. Taking the septa of the vial could solve this problem.

Just shouting ideas. Maybe they will help you to find a solution to this problem.