Chromatography Forum

Greetings All,

I took a 4 or 5 month break from some method development work to do some survey work.

Upon returning, I discover that I had lost response and sensitivity on a fluorescence detection method, the sensitivity was also lost (i.e. the slope of the calibration curve).

I have ruled out bad preparation of the standards and reprepared them fresh from a stock solution. The stock solution was verified using UV-Visible Spectroscopy.

I have ensured that the gain was not inadvertently changed. I have performed the Raman S/N test.

The analyte I am looking at is Ochratoxin A with an excitation wavelength of 333 nm and an emission wavelength of 460 nm.

The detector is a Waters Acquity Fluorescence detector.

I will also mention the flowcell appears to have leaked at some point as there is encrusted material on the exterior, however it does not seem to be leaking now.

Our lab would rather not spend $4200 until we are sure that it is in fact the flowcell.

Would anyone have any suggestions as to how to test this flowcell or why the response and sensitivity were lost.

Cheers,

Tom

Well, the crust was most likely not put there by Gremlins. . . . .
A thorough cleanup and check of the cell and connections is in order here, I don´t know how to do this for a Waters detector.
How did the Raman test go?

Thank you very much for your response.

At an excitation wavelength of 365 nm, an emission wavelength of 415 nm with a gain of 135 the S/N of the Raman test was 1760.

However, upon further inspection, including removal of the flow cell from the detector, parts of the flowcell came off as well the flat black coating appeared to have dissolved in places.

These are all very good indicators that the flowcell requires replacement. I suspect that my supervisor shall be convinced that the flowcell needs replacement.

Regards,

Tom

It appears that the cell cracked, maybe due to a plug in the outlet from the cell. it is not a bad idea to put a safety valve ahed of such expensive flow cells (I think Upchurch had these?).
Incidentally, a Raman test without comparison of the data to a well tuned apparatus does´t tell me much.

Incidentally, a Raman test without comparison of the data to a well tuned apparatus does´t tell me much.

The result should be in the several/many thousands order. Anyway if carried out optimally i.e. 350 nm excitation and 397 nm emission.

Best Regards