UPLC from Waters

[pve]

hy everybody,
we've now one year of experience in UPLC, and we are quite a little desapointed. To transfert an hplc application in UPLC is not si easy. When we increase the flow (and the pressure, the noise increases too), we loose the sensibility. We have also problems with the columns provided by Waters: it seems that the quality of the batches are not very reproductibles.
has anyone experience in that subject
regards

[juddc]

When we increase the flow (and the pressure, the noise increases too), we loose the sensibility....

Sorry to hear that...talk about unintended consequenses!

[AA]

I have been using this system for some time now and would like to comment:

1: I agree, transfer of methods can be difficult. Crappy HPLC methods transfer as crappy UPLC methods. I have had much better luck not bothering with the transfer, and just redelevoping the chromatography from the beginning.

2: Increasing the flow does not increase the noise, it is hard to say what is happening with you.

3: When I increase the flow, my peaks generally get narrower and provide a greater peak height. I have the same noise and therfore greater sensitivity (I am pretty sure you ment sensitvity and not sensibility). Again, what is happening with you is not clear.

4:In the begining, column quality was a bit of a problem, but it has gotten much much better. I would say that today, the batch to batch reproducibilty is as good as any HPLC column.

If you are truly unhappy, get on the phone and get Waters in there to help you. They have lot of techincal and application experts who will be more than willing to spend time with you in your lab showing you the ins and outs of that instrument.

[rnuijts]

I Totally agree with AA

[Newman768]

Method transfer is as difficult as one can expect when changing the column type and "downscaling" a method. Most likely the seperation (if it could be transfered) enhances.
We have right now 2 uplc systems and use them as often as possible - our Allicane systems are loosing their importance.
We are adapted to the ultra high lc and is it not easy to accept the slowness of normal hplc when we needs to use the "old" systems...

I don't want to miss them any more (and btw we can't do it without them any more)

[pve]

specialists from Waters in UPLC will be in our site next week. They will know my problems and I wish they will help me.

I try to explain my problem in the next paragraph:

when I use a column (particule size 1,7 µ - 10 cm x 2,1 mm) the flow is 0,2 ml/ mn and the pressure is about 5000 psi. at this flow we are not at the maximum efficiency of the column, and I think there is a lot of diffusion in the column and we loose a lot of plates
the solution would be to use a shorter column why a higher flow rate: particule size 1,7 µ - 5 cm x 2,1 mm - flow: 0,4 to 0,6 ml/ mn
I've tried to do that, but unfortunatly, I don't have the separation otained on a classical column (particule size 3,5 µ - 15 cm x 4,6 mm)

next week, I will explain my new results on the forum

[james little]

We have had our UPLC system for a while and I really like it. Using it with a Quattro Micro LC-MS/MS.

Very low dead volume, very reproducible retention times even with step gradients and very fast gradients.

However, must admit I haven't been using their columns. Initially, I tried their columns for doing some basic drugs, but got much better peak shape with a Monochrom C18 3 u column.

I need to try their columns again, especially their new polar embedded columns.

[Uwe Neue]

PVE:

If indeed you are working with small molecules, you probably want to increase the flow rate by about 50% or so. The system can run at up to 15000 psi, why do you want to stop at 5000 psi? If this is an isocratic method, you can measure the plate count and see how it changes when you increase the flow rate.

If you are using a different packing material with a different surface chemistry for your HPLC column and your UPLC column, the lack of resolution is probably not related to plate count, but to the differenc ein the surfac chemistry. If you give me some details, I can advise you what you could do.

[adam]

Uwe (or anyone who knows the answer)

This may be slightly off topic - but can you tell me if the pathlength for the UV detection cell used with the UPLC system is the same as the pathlength for the detection cell used with the "standard pressure" systems, like the Alliance?

Thank You
Adam

[AA]

The standard flow cells for both UV detectors (the PDA and TUV) have a path length of 1 cm, same as traditional detectors, and a volume of 0.5 uL, much lower than traditional detector flow cells.

[DR]

Then there's Thermo's PDA flow cell - 5cm.

[pve]

hello
after two days spent with experts from Waters in UPLC, my separation is now on the way to success.
they insisted that it's important to put the right flow on the column - for me, 0,5 ml on a 100 mm lenght column.
also it's important to use important slope in the gradient.
for all the future clients in UPLC, ask to the vendor to have a good training in UPLC, you will save time and money