actual peak collection before the PDA peak --??

[e]

I was checking the lag time between the fraction collector and Water's Empower software peak to set the wait time for the fraction collector. I noticed the seemingly impossible. I found the bulk of the injected standard at minute 13 (with hand collection or with a fraction collector), and the PDA software peak at minute 14.

The equipment being used is a Water's PDA 996 and a Water's 600 pump and controller.



This "pre-collection" occurs under only specific conditions and on one of 2 HPLCs: Silica column, 4%IPA-96%hexane, 1.7ml/min. isocratic. Other conditions tried with this std. and another compound actually gave a lag between the PDA software peak and the collection peak as expected.

When I pre-mix the 4-96 mobile phase myself, the situation doesn't occur.

On another lab computer with the same equipment, the situation doesn't occur. Also, switching columns between the two HPLC set-ups gives a retention time unique to the HPLC, not the column. In other words, Column A on HPLC A give a RT of 14 min. for my cmpd. Column B gives a RT of 11.2 min on HPLC B. However, Column A on HPLC B gives a RT of 11.2 min. And Column B on HPLC A gives a RT of 14 min.

The flow rate on both systems is identical and constant. The Water's technician checked the mixing on the HPLC and found no trouble.

Does anyone have any ideas? I am trying to develop a method involving peak collection which can be used on more than 1 HPLC system.

Thanks, E

[JA]

pretty confusing post

presumably, you do not have the detector in series (before) the fraction collector. Instead, you are using a tee/split and collecting/detecting in parallel. If so, I would suspect you have an insufficient split flow to the detector. Suitable split flow rate is going to be determined, or limited by 1) ensuring you detect your analyte before it reaches the collector 2) flow cell volume 3) something sensible so you don't waste too much analyte.

Depending on the tubing used, and therefore the backpressure generated, post-detector, the simplest way is to plumb the fraction collector in series after the PDA. This could be more pertinent at analytical-scale due to the necessary split flow to satisfy the detectior/collection timing criterion, above. In other circumstances, a make-up flow may be necessary.

I can't explain why your premixed mobile phase would work with the split flow situation.

I don't think it uncommon for changing retention times to be system specific rather than column specific. Assuming the same pumps and (if applicable) mixers it is probably down to differences in pre- and post- column volumes.