Chromatography Forum

Has anyone ever experienced air bubbles getting caught in the flow cell (3 ul) of a fluorescence detector? I am using standard OPA/BME derivatization procedures for measuring amino acids, and no matter how much I try to minimize bubble formation during the mix method (degassing mobile phase, etc.), I end up getting a bubble trapped in the flow cell about every 2-3 samples. It causes cyclic noise and ruins all subsequent chromatograms. The only way to get rid of it is to "flick" the tubing near the flow cell to dislodge the bubble. Sure makes it a pain to have to keep checking the HPLC every 20 min.

Any possible solutions to this problem would be greatly appreciated...

folive

How are you degassing the mobile phase. Have you tried
sonicating the mobile phase as well?

Chemically - we always used IPA to flush the flow cell to
take out any air bubbles. But I assume that's not too
practical to do in the middle of your run!

Mobile phase is 0.1 M sodium phosphate and 25% methanol. I usually degas by stirring overnight and sparging with He. Sonicating might be a good idea too. Thanks!

Happens all the time. Since the detector is warmer than the solvent reservoir, dissolved gasses will come out of solution there. Degassing will prevent it. You can also add a backpressure regulator/restrictor to the waste line; 50-100 psi should do it.

We run a 5 uL cell, and have a 20 cm length of .005" ID tubing on the cell outlet. It helps.

How are you prepping the post-column reagents?

Evan Cooper

Also, if you sparge the post-column reagent with helium and keep it under static pressure of helium (about 2-5 psi) it will reduce the outgassing problem, and your reagent will last much longer.

A dirty flow cell is also more prone to bubble formation.

I really don't think I would want to be in a lab where someone was sparging a solution of mercaptoethanol. Mark, have you really done this? Aside from the stench, wouldn't the mercaptoethanol sparge out over time?

Evan Cooper