Chromatography Forum

koen, I sure would like to see how much plate loss was involved while the resolution increased.

please send an e-mail to kh@shimadzu.de and I can give you an nice example.

OK, but you can give the numbers here?

My peaks where almost co-eluting at ambient temperature. When I increased the temperature resolution increased dramatically because the I got a different alpha value.
The loss of plates was not significant compared to the gain in selectivity. I didn’t calculated the resolution of my critical pair when it was co-eluting (no use).
Plate number neither, since I was working in gradient mode.

Hollow:
There are several possibilities for split peaks. The first question is if you used the solvent preheater tube (nicknamed the Uwe tube) in this experiment. Second question: what was the wash solvent in the injector? Third question: I assume you used the standard mixer in the system, and did not remove the mixer for some reason.

Uwe:
Thank you for your reply.
To answer your questions:
1) No, the "Uwe-tube" was not installed.
most used temperature was 30°C but with 40/50/60°C, no or not much improvement was found.

2) For this trial period we used ACN/H2O 80/20 for both weak and strong wash.

3) Yes, this trial-system came with standard mixer. No modifications were done by us.

Maybe some additional infos:
Injection volume was 1 ul, which gave heights between 60 mAU (acetone) and 26 mAU (butyrophenon, unsplited) resp. 8 mAU (splited)
Linearity was fine from 0.1-5ul injected, even if the peak shapes tends to more tailling (somewhat like a volume overload phenomena? (Peakfront is identically for all volumes, but peakend drifts to the right, and the "height vs. V" plot shows some kind of a logarithimc curve). I set a question mark to volume overload cause the sample solvent is equally with the mobile phase... Could volume overload still occure if sample is dissolved in mobile phase?
Nevertheless the peaksplit occurs also with 0.1ul

Detector was the UPLC-PDA, sampling rate was 80 Hz, detection @ 254nm

thanks and regards

My bet is on the use of the same strong solvent for the strong and the weak needle wash. If you do that, you inject your sample with some amount of the strong solvent, which can cause peak splitting.

Another possiblility is a lack of temperature equilibrium at the column inlet, which is why I asked for the Uwe tube. At 30 degrees, I don't think that this is the issue, but it will very definitely cause problems at higher temperature. You would not be the first one who has experienced something like this after removing the Uwe tube.

Thank you for this fast answer.

I'll forward your suggestions to Waters CH cause they allready have the UPLC back but would re-test my issue.

If the wash solvent was in fact the problem, would you then say: "Welcome to UPLC!.
That this is the real advantage of UPLC, that one can see effects like this respectively this is the region of resolution which wasn't achieved before"?

If the wash solvent was indeed the problem, I would say that we have not communicated well enough with you. But let us wait and see.

You can also contact me at my e-mail address below for further discussions, if you so choose to do.

thank you Uwe. I'll keep you updated.

Just for information to the others.

Waters CH found that the peak splitting was caused by a void volume somewhere between injector and column. After proper (re-)connecting the UPLC performed well as excpected.

@Uwe: did you get my email?

Hollow: No, sorry, I did not get an e-mail from you. - Uwe
Besides the address below, you can try also at Uwe_Neue@Waters.com.

Mattias wrote "and it is still impossible to run acetate or formate buffers due to a terrible baseline. Waters has changed almost every part of the instrument without any change."


We run acetate (just finished a nasty one with 100 mMol ammonium acetate) and formate buffers all the time on our ACQUITY systems, with few issues. If I were to make a list of issue we have had, "horrible baselines" with those buffers would not even majke the list. The question is, what are you guys doing that causes the horible baslines because it is not the instrument.

Jasco also makes and sells the HPLC/UPLC. I remember a sale rep telling me that he could reduce a run from 15-20 min to 2 min. And good resolution too.
Check this out: www.jascoinc.com

Alfred.

Forgive me for not being impressed with the Jasco promotional applications; I don't think they are trying hard enough. I saw their system at PittCon, and there is no reason why their examples should be so ordinary. I have done equal or better with my UltiMate 3000 system http://www1.dionex.com/en-us/lp35613.html. For example, check out this tutorial on Fast LC http://www1.dionex.com/en-us/webdocs/48 ... olumns.pdf

I was reluctant to join this thread, since they had already decided not to buy my product. But if we are going to add other vendors to the discussion, I had to say something.

Dear Mark Tracy:
I should have stated (a disclaimer) that I don't work for Jasco (past + present)! Actually, I have never used a Jasco HPLC/UPLC! If I am correct, Jasco was the OEM supplier for Varian and Waters, and now Jasco wants to market its products under its brand.

When comparing systems, we also need to look into the features (high end vs low end) and cost (initial cost & maintenance cost). I believe that Jasco offers much lower price than other brands (Perkin-Elmer, Agilent, Waters have price tag $55-65K. Gilson and Dionex are also higher. Shimadzu is cheaper, but I am reluctant to recommend it). If 'Hollow' has the fund, then he shoud not concern with the price tag.

Alfred.