By Lilly on Thursday, February 13, 2003 - 07:18 am:
Hi All:
I'm using a fluorescence detector (Waters 474)at 225 nm EX and 316 nm Em, I'm collecting data for 8 min but the run is 15 min (gradient mobile phase (buffer) program. I'm observing a few chromatograms with no expected peaks. Some of the injections are from the same vials.
I used the same instrument and detector for a different method (organic solvents as mobile phase) and everything was fine.
Any ideas?
Lilly
By ananda on Thursday, February 13, 2003 - 02:48 pm:
Lilly,
I think we need more info. such as what kind of compounds you are trying to detect and what kind of buffer at what pH you are using for the detection. Did you check the literature or a fluorascence scan for your compound to pick the right exitation and emission wavelengths?
Ananda S.
By Anonymous on Friday, February 14, 2003 - 03:23 am:
What about a malfunctioning of a check valve which causes longer and/or variable RT?
Other possibility: air bubbles trapped in pump B.
By Anonymous on Friday, February 14, 2003 - 05:36 am:
One thing to try as a troubleshooting tool, collect data for the entire run and see if you get the same or very similar results for multiple injections. One other thing, what type of data system are you using?
By DelphineG on Tuesday, February 18, 2003 - 03:26 am:
Hello!
I have met a similar problem a few weeks ago using HPLC with fluorimetric detection (WATERS 470). Injecting the same vial several times, sometimes I collected a usual chromatogram but sometimes I had no peak or enlarged peaks at unsual retention times.
After a day of investigation, we have found that the problem did not come from the detector but from the injector (WATERS 717+)!!
Indeed, the needle was shifted and we had to recalibrate the injector in order to repositionne it.
This problem occured another time 2 weeks later : there was a leak at an inox tubing connection in the injector and we have to cut the tube to eliminate the leak.
That's what happened to us, maybe it will help you and anybody else.
Bye.
By Einar Pontén - SeQuant AB on Tuesday, February 18, 2003 - 02:32 pm:
Excitation at that low wavelength (Ex 225 nm) is a high energy radiation of the sample molecules. They may photodecompose.
I would check if there are any other suitable absorbtion wavelength for excitation at a higher wavelength, and if the emission shifts accordingly. Else, a Stokes shift of at least -50 nm of the excitation compared to the emission is required.