Chromatography Forum

Anybody experience any problems or what problems can occur when using a UPLC as HPLC?

Most problems I think will come from the difference in dwell volume and mixer size between UPLC and HPLC.

The dwell volume can be important for gradient methods. An old Alliance system can have a dwell volume of over 1 ml, a new UPLC does hardly have any dwell volume. (If you run a gradient method at 0.2 ml/min on an old instrument you actually start with a 5 minute isocratic step on the column)

An UPLC has usually a very small mixer (and a low overall dwell volume), meaning that the mobile phases are not mixed as well as on an old instrument. This can give a higher detector noise. Premixed mobile phases can solve this.

The Acquity system has some other nice-to-know features like:
Be careful what you use a weak needle wash, must be weaker than your mobile phase. Don't use partial loop injection for quantitative work. This is not specific for HPLC vs UPLC, but things that the technicians usually do not know.

I second Mattias' comment about the less effective mixers often used in UHPLC systems. SSI has recently developed some effective mixers for the purpose, of low dead volume.

Another difference from a regular HPLC: The backpressure against which the pumps can work is inversely proportionate to the cross-sectional area of the piston. Accordingly, UHPLC systems tend to have smaller pistons and smaller pump head volumes than do regular HPLC pump heads. That makes them more vulnerable to losing prime if an air bubble enters the pumping chamber. We've found that to be a moderately annoying problem in our use of UHPLC systems at any pressure.

...and another thing that might be considered: Because of the lower system volume, UHPLCs are more prone to peak deformations because of sample solvent/eluent discrepancies, e.g. using a solvent with higher elution strength than the initial mobile phase.
And, depending on the UHPLC, you might have the simple practical problem of squeezing a 250mm HPLC column into the column oven, if you have to use such a monster. Some vendors didn't seem to think that someone might use such a large column in their system.

I can only speak for the Acquity UPLC.
Waters sold us an UPLC with an 30cm column oven to be used as HPLC en UPLC.
But we had some troubles due to the differences between the injector of the Alliance and the injector of an UPLC. Recoveries were sometimes different for certain APIs.
Waters told us that this was by using mobile phases with ionpair reagents, but we also had some troubles with buffered mobile phases.

Also in the lower part of our validation (eg 0.001mg/ml) we sometimes see some serious recovery issues which do not occur with the HPLC.

But maybe the new injector with the flow through needle may overcome this problems as it's more like a regular Alliance injector.

Ace

I never was able to get any HPLC method to run on an Acquity system as an HPLC method without problems.

To fix the problem, I generally had to make changes to the method that would require additional validation work to be performed. This simply translated into the final push to transfer those methods not just to an UPLC instrument, but actually make the methods UPLC methods.

Keep your HPLC methods on your HPLCs. Use the UPLCs for what they were truly intended for. Transfer your HPLC methods to UPLC methods quickly