FLD Wavelength accuracy and signal to noise not passing.


Recently Run the Wavelength accuracy and signal to noise at FLD detector for qualification purposes but it not passing. In wavelength accuracy test got Excitation value 350 it ok but in case of emission got 393 nm its failed required minimum 394. In Signal to noise got 367 required value minimum 400. Run Wavelength calibration from Labadvisor using Glycogen solution it passed but when try wavelength accuracy test from LA it aborted by system when scan emission. please help what should do next.

Note: I used double filter water and sonicate more than 1 Hour.


  • It would be good to see the intensity test result. This FLD more than five years old, so parts of the optic can show degradation. On the other hand, the FLD also passes WL calibration, so very likely the hardware is working. How and for how long did you flush after the calibration? Glycogen can be quite persistent. I usually flush with fresh water of best quality at a flow rate of 2ml/min for twenty minutes. I then reduce the flow to 0.25ml/min and run the WL verification. Usually I have a SST restriction capillary installed, to achieve some system pressure. Overnight flushing at flow rate of something like 0.1ml/min is a waste of time. If that doesn't work, try water from a different source, try a different degasser channel or combine two of them (I really hope that the fact you sonicated the water doesn't mean you don't have a degasser. Using an FLD without degasser is a no-go for me) and temporarily remove the solvent inlet frits or replace them with new ones.

  • Hi,

    Thanks for your reply.

    After wavelength calibration I flush the flow cell with 1.5 mL/min water for 10 minutes then reduce the flow to 0.25mL/min water and perform the test and then again flush the whole system with 10% IPA for whole night with 0.3 mL/min flow rate.. we have only one water source right now. I heard that mineral drinking water from market is very rarely works, is it safe for instument? can I try it?

    For your refference i am sharing with you the intensity result.

  • Thanks for sharing the intensity test. However, this is awful, I have to say. I'm surprised the WL calibration went through. If you examine the test, you will find that the absolute maximum is just below 3000 counts, where a brand-new FLD usually has ~15000 to 20000 counts. Furthermore, there is hardly any intensity below 450nm, but the WL accuracy test is using 350/397nm!

    Like I said, overnight flushing is mainly a waste of time. Please get yourself fresh(!) water of best(!) quality. Install a restriction capillary and flush at 2ml/min for 20 min, reduce the flow to 0.25 ml/min and try the WL accuracy test again. You need distilled or de-salted water of very good lab quality. Drinking water is a no-go! Make sure the system has a working vacuum degasser installed.

    In any case the optics of the FLD need an overhaul. I'm pretty sure the lamp house window, the EX condenser and the mirror show blind spots or are fully blind already; maybe there's even degradation on the EX grating. Window and condenser can be cleaned (but not dismantled), but should be replaced with new parts. Mirror and grating must not be touched, those have to be replaced as well, if degraded.

  • Hi,

    I understand your point and thanks for your suggestion. Can you please share the cleaning procedure of lamp house window and condenser as you said it can be cleaned. 

  • Apologies for the delay, but I was off for Christmas. There is no cleaning procedure. What I meant to say is that both, the lamp house window and the EX condenser (no dismantling!) could be cleaned with a lint free cotton cloth and IPA, but that would only help if there's dirt or dust. More likely there will be optical degradation, after constant bombardment with powerful UV light for years. Here cleaning does not help at all and the parts have to be replaced. No cleaning at all to be carried out on the mirror and the grating! Remember to re-calibrate the FLD, after you did any changes to the optical path, including removal of the cell. Do not carry out an intensity test before re-calibration.

Was this helpful?