Chromatography Forum

Good morning girls and guys.
I want to know why this is happening.
I´ve developed a quantitation method with a Zorbax Eclipse Plus C18 RRHT column, 1,8 µm, 50 x 4.6 mm in an Infinity 1290 UHPLC. Pressure is not high, is about 230 bar. It is the only UHPLC in my lab.
So, I think, well, let´s try it in an HPLC as pressure is acceptable. Because is the only UHPLC we have, so if it brokes, HPLC may help.
When I did that, in a Shimadzu Prominence and in an Agilent 1260, A shuolder appeared. It is not the column, because another one new did the same, and so the same with mobile phase.
Solvent is HCL 0,1 N, 20 µL injection. Same happens with 15 or 10 µL.
The only thing that solves the problem, is to change solvent to water instead of HCl. So, I think pH is involved. But the question is, ¿Why this happen in HPLC but not in UHPLC?
Regards.

I can speculate that the HPLC system has a substantially larger volume of tubing "pre-column" which is increasing the effective injection volume. If you have no problem using water as the diluent, you can just keep doing that. I you want (and have time) to pursue the problem, check the tubing between the injector and the column (make sure that the tube end goes all the way down into the fittings; since the same problem occurs on two different HPLC systems, I'll admit that this is a long shot).
Also, what was the injection volume in the UHPLC system? What would happen if you added some relatively large diameter tubing between injector and column on the UHPLC system? Or if you used narrower tubing on the HPLC system?

In my opinion Tom is right that it is a extra column effect. So please check the tubings and dead volume. Good luck.

Whats UHPLC? IN any place i have worked, we always referred to them as UPLC instruments.

Whats UHPLC? IN any place i have worked, we always referred to them as UPLC instruments.

UPLC = UHPLC = vHPLC (v = very). UPLC is just a trademark of Waters. However, the UHPLC abbreviation is recognized by Waters as well:

https://www.waters.com/waters/en_US/UPL ... dCountry=Y

Another question to ask, is it isocratic or gradient elution?

If gradient, then are all instrument binary pumps or do the HPLCs have quatnary with mixing solenoids?

The different types of pump setups can result in the actual gradient getting to the column at different times, which can change the resolution of the peaks.

0.1N HCl mp for a C18 column?
If there's no organic component, there are going to be other problems as well...

0.1N HCl mp for a C18 column?
If there's no organic component, there are going to be other problems as well...

I read that as referring to the sample, not the mobile phase.

Outside tiny possibility: If a gradient method, is your equilibration time adequate? Edgy methods, with perfectly optimized equilibration times, as short as you can get away with, are sometimes vulnerable to being moved to other autosamplers, which take a different length of time to make an injection (thereby altering the overall reequilibration between runs).