Chromatography Forum

Hi,

I would like to use our Waters 2475 fluorescence detector (FLD) with our Waters Aquity UPLC (UHPLC) system.
The FLD tolerates a pressure up to 145 psi (10 bar). When running UHPLC with an ordinary C18 column pressures up to about 800 bar are common, using acetonitrile gradients at flow rates of 0.2-0.4 mL/min. However, the pressure after the column (or the post-column pressure), that is, the pressure exposure to the detector, is much lower.
Do you think I can use the HPLC FLD together with the UPLC?
Should I check the pressure at the actual flow rate without a column - is that reliable, or will it overestimate the pressure over the FLD with a column connected? Other suggestions?

Thank you.

Regards,

Goran Karlsson
Pfizer
Sweden

Hi,

I would like to use our Waters 2475 fluorescence detector (FLD) with our Waters Aquity UPLC (UHPLC) system.
The FLD tolerates a pressure up to 145 psi (10 bar). When running UHPLC with an ordinary C18 column pressures up to about 800 bar are common, using acetonitrile gradients at flow rates of 0.2-0.4 mL/min. However, the pressure after the column (or the post-column pressure), that is, the pressure exposure to the detector, is much lower.
Do you think I can use the HPLC FLD together with the UPLC?
Should I check the pressure at the actual flow rate without a column - is that reliable, or will it overestimate the pressure over the FLD with a column connected? Other suggestions?

Thank you.

Regards,

Goran Karlsson
Pfizer
Sweden

Goran,

I suspect that post column pressures will not be a concern when using this detector. The major problem will be the data rate of the signal. The detectors available with the UPLC operate using a much higher data rate than standard detectors allowing integration of the relatively narrow peaks obtained from a UPLC assay. In addition their dead volumes and flow cells are much smaller. You may find that the 2475 with a standard tubing/flow cell size cannot generate meaningful data from the UPLC assay. The dead volume in the detector alone is probaly 5 X the rest of the system.

The detector cell only sees the pressure related to the waste tubing post-cell - the separation pressure has no relation to the pressure limits of the cell, so you only need to make sure the post-cell tubing isn't clogged, and isn't too narrow an internal diameter. JGKs comments are correct - the volume of the cell is going to be the limiting factor for this detector. If you have adequate sensitivity and enough resolution in your separation, you can use this detector, although it's kind of like having a Ferrari and only ever driving 100kph.