Split peaks with injections of caffeine - HELP

GIACBE

<p>Hello everybody,</p><p style="height: 8pt"/><p>I'm having tailing problems with injections of caffeine (MeCN/H2O 1:9, BEH C18). I don't understand why this problem has appeared.</p><p>Pump is working correctly and I've changed everything from the sample needle to the UV inlet (sample needle, loop, injection valve cartridge, column inlet and outlet). All the connections look correct.</p><p>I've also tried with a second column (new) without succes...</p><p style="height: 8pt"/><p>I'm not a UPLC specialist, so if the solution is very simple, don't blame me too much...</p><p style="height: 8pt"/><p>Thanks forward for your answers...</p>

lizh

Hello:

This sounds like this has just appeared, but it is worth asking, is this a method that previously worked?

Usually bad peak shape is cuased by one of these three possiblities, once you ahve eliminated the column:

(A) bad connecton, is the ferrule at the column deformed?

(B) Could be a heating issue, is the the stabilizer set properley in the correct position and is the correct version installed

(C) Is the sample diluent too strong for the caffeirn. Please can you share what injection mode, sample diluent and initial conditions are used.

Let us know

Liz

dougm

Hello,

I have another question to add to Liz's excellent suggestions.

Does this tailing occur with ALL analytes or just caffeine? Could you inject other analytes and let us know what the peak shapes are?

--Doug

GIACBE

Hello,

thanks for your answers...

First of all, I'll precise a bit my question : I observe split peaks instead of tailing...

And here you are my answers :

- yes, this method worked previously. It's the method desribed and performed for the qualification of the UPLC... I've performed the "test injections" with succes ans suddently the problem appeared while doing the "System Precision Test".

- The ferrule at the column is not deformed and I checked all the connections (it looks OK). SInce the problem appeared, I've performed a total maintenance of the SM.

- Liz, I don't understand what you mean with stabilizer for the heating issue. I don't know if I have the correct version but the "Temperature Precision Test" was fine...

- I use what's desribed for the qualification :weak wash, strong wash, seal wash, A1 and A2 = MeCN/H2O 1:9; B1 and B2 = MeCN. For the injection mode, I've used initially "Partial Loop With Needle Overfil". Changing to full loop didn't help...

- Doug, I've tried also with other analytes, without succes... (For the caffeine, I'm using Waters standard solution).

Actually, I'm preparing a fresh MeCN/H2O solution, just in case...

Tahnks again for your help,

Fabrice

lizh

Hello:

Firstly, to answer your question, the column stabilizer is a pre-heater or heat exchanger it heats up the solvent to the column temperature, if it is missing or incorrectly installed the temp of the solvent is mismatched to the solvent and has an effect tailing, fronting on the peaks. Hence, my thought that this was the issue. Is the column seated in the correct position with the stabilizer seated a inch or so into the column heater pocket.I attached a photo.

If changing the injection mode did not help, and the fact that the separation is straightforward, I still think you have a fitting that is seated incorrectly, especially if the system has been worked on. I would check all the sample manager and the column management module connections. It is possible, it could be a kinked needle. However, I have sent this to our qualification experts and expect an answer tomorrow. The only other thought is what is the caffeine made up in, is it identical to the mobile phase. I am sure this is a long shot, but I have to ask.

I will get you further information shortly,

Liz

dougm

Hello,

Could you please tell me where you are located? The reason I ask is that if you do not mind, I would like to have your local Chemistry Operations support person get in touch with you.

Thanks,

--Doug

GIACBE

Hello,

Thanks again Liz and Doug for your attention...

Liz, yes the column stabilizer is correctly installed. Caffeine solutions are provided by Waters (700002642).

Doug, I'm also in contact with the Waters support here in Belgium, they are helping me a lot also...

Here you are the last test I've performed :

I've observed that the shape of the peak is different If I was using the "old" column or the new one.

So, I've decided to test a third one (C8 instead of C18, as I only have 2 C18).

And the problem disappeared...

I've also used the UPLC system with a HPLC column (after having changed the connections) and I didn't observed split peaks...

My conclusion is that I have troubles with these 2 C18 columns.

I'm still looking at the origin of this problem with my local support and with you if you have any explanation...

Thanks again for your help...

Best regards,

Fabrice

lizh

Very interesting, thank you for the update

Liz

GIACBE

Hello,

here you are the last results concerning this problem...

After having changed the frit and the nut (inlet) of the "new" column, everything is working perfectly...

We have done the same with the "old" column, without success (I've still split peaks but with a different shape).

It looks like I've had a contamination (bacterial? chemical?) in the system.

Hopefully, the "new" column hasn't been contaminated too much...

Thanks again for you help (and thanks also the my local support)...

Fabrice

STC

Try decreasing the amount/concentration of caffeine injected.

Tailing might sometimes be caused by overloading of column.