Which model of a spectrofluorometer are you using?

LS 55 Fluorescence Spectrometer.

I'm using quinine sulphate samples with a concentration of few ppb.

I tried to change the method and also to shut off everything but after the first measure the software say "overflow"!

from Perkin Elmer, I forgot

Sorry, but I have no idea what may cause such an error message (we are using an older Perkin Elmer LS 50B model)

Thank you for trying!

Most probably this error is caused by an excessive illumination of the detector. In which conditions you see such an error?

No, vice versum. Excessive illumination in not possible in UVVis spectroscopy. I think you are just getting 3A+ absorbance. Try dilution, recheck wavelength an make additional precautions if working in 180nm-210nm area

Indeed, you should add information at what situations you get this message

Dear all,

i don't think that the error is due to excessive illumination because the instrument works for the first measure without any problems and after that it don't work and appear the error "overflow".

Someone said me that i need to rename the method with a short name, but i think that this is a not scientific solution! I worked almost in all condition (opening the slits or closing them in order to avoid signal saturation) but with no results. I don't think that is a problem of my samples but i think it's an instrument problem.

I worked in 200-500 nm area with an excitation wavelenght =350nm and emission wavelenght = 450nm. The slits were both set at 4.0

Irene, you can try to contact Perkin Elmer technical support:

http://shop.perkinelmer.com/Registration/CustomerSupportForm.htm?FormID=TechnicalSupport

I hope they will be able to help resolve your problem...

Greetings!

Dear Irene, this may be caused by a hardware overflow. Can you set the number of points, resolution or whatever increases the size of the data or the aquisition rate? This may be related to an overflow in the aquisition cards or analog to digital conversion hardware. After the first aquisition of a data set to big to processed a second try results in the overflow. Another suggestion would be an I/O error.

Hi Irene,

There's nothing wrong with your equipment or your samples... :)

PerkinElmer fluorometers have a fluorescence scale from 0 to 1000 arbitrary units. Every time your sample has a fluorescence above 1000, you will get an overflow message.

The solution is simple: you can (a) use a lower slit value (2.5 nm, for instance), (b) use a lower voltage of the photomultiplier, (c) both. If your sample is very, very fluorescence you can always dilute it.

If those options didn't work, you can select an interference filter. Keep in mind that you have to select one with a wavelenght between your excitation and emission wavelengths.

The last option is to select the filter called "1% transmitance". This is the very last choice because it makes all your readings very low and you will loose sensitivity. I don't recommend to use this one.

Very important note: since fluorescence increases exponentially with the voltage of the photomultiplier, ALL your readings have to be performed using the same voltage. If one of your samples exceeds the scale of the fluorometer, setup the equipment again as I explained before, and read all your samples again. Don't try to use conversion factors, equivalences, or other mathematical manipulation of data. They just won't work.

I hope this will help you.

Best wishes,

Sandra

I am using LS 45 and recently it started showing 'overflow' message before the scanning. I can not change slit value in LS 45, it is fixed at 10 nm. I tried using the minimum PMT voltage, but the problem persists. I tried with only distilled water, so no question of over concentration. Can anyone solve the problem.

One question: where the excitation and emission wavelengths too close? Did they overlap? I ask you this because if both wavelengths overlap the signal will be spurious and it will cause the "overflow" message.

I kept 20 nm gap between excitation and emission, so there was no overlapping. Should I try increasing the gap?

Hi Bidhan,

No, I don't think so.

You can try to check if your fluorometer is working OK using a very diluted alkaline solution of fluorescein (I don't recall now the exact concentration, but is in the nanomolar range).

Try running excitation and emission spectra of diluted fluorescein, adjusting the voltage of the photomultiplier. If you still have the error message, you have not choice but to ask PerkinElmer to send somebody to fix your fluorometer.

If fluorescein works fine, then the problem is the probe you are working with (which one is it, by the way? please, let me know because if I have some in my lab I can check it with my LS55).

Best luck!!

Hi Irene and Sandra,

I have the similar problems and errors that Irene mentioned above with Pelkin Elmer LS 55. First of all, we have purchased the LS 55 two weeks ago, it is pretty new, yet the technical support from the local service is inadequate. That's why I hope I could get help from you. Thank you in advance. Below is the list of the problems:

1. The measurements for fluorescence (in solution and thin film) without Ex. and Em. Monochromator filters, in particular, without 1% attenuator filter, give the “over flow” errors or the signal is saturated. The dilute 10-6M fluorescein in ethanol sample is recently used and the same errors appear as well. When the 1% attenuator is chosen (Sandra says 1% at. is very disadvantageous indeed), the instrument works for the pre-scan and for the first emission measurement without any problems, after that it doesn't work and the error "overflow" appears as Irene said, too. In other words, when the emission scan is measured after the pre-scan, it gives a logical peak for the first time, but afterwards, it gives the overflow error. Subsequently, the pre-scan is repeated, but, this time "motor stepped error" appears.

2. Moreover, for the measurements mentioned above; The slit sizes, excitation and emission wavelengths, filters, photomultiplier type and voltage are adjusted many times in order to understand where the problem is, so that we could optimize the measurements and define a configuration. However, still we experience the same errors...

3. Strangely, during some measurements, when the slit size increases, the intensity of the peak decreases; from 10 nm to 15 nm. If it is adjusted to 5 nm, this time intensity decreases as expected.

4. In some of our trials, during the measurements with the 1% attenuator filter, as the slit size is changed, the maximum wavelength peak is changed as well (Not the intensity of the peak, but its position on x-axis changes). This seems illogical. Even more illogical thing is another peak appears from nowhere with the same sample used sometimes. As stated before, without the filter, as it is Open, no measurements can be taken; it gives errors like over flow or the peaks are saturated in 1000 intensity. Even an extremely diluted sample gives this error without the filter.

5. The software FL-Winlab often crashes after the program becomes unresponsive for no reason. It just stops itself. For fast and practical experiments, it becomes a big waste of time.

Btw, the excitation and emission wavelengths are not close; they do not overlap much for the fluorescein trial. So, that should not be a problem.

Since the instrument has been just bought, I will be demanding Perkin Elmer's technical support as soon as possible in case I cannot figure out the solutions to the problems I have listed.

Thank you all again,

Regards,

Guler Kocak

It was 250-700 nm Roman

Roman

The standard one is used so far mostly. I have been trying the others recently. The region is 650-900 in the allowed pmt settings, yet I've checked the std pmt's performance and it is better to use the lower ones as you suggested. For the red emitted samples the other pmt type seems reasonable. Thanks

I have a problem with a spectrofluorometer LS55 PerkinElmer.

The SCAN FLWINLAB application program does not run. And an error message is displayed: DDE Setup: Client failled to connect to server Lamp September. FL = On.

I do not know how to fix this bug. 

Dear Melissa, you can try to turn off your Antivirus or Windows defender

Dear Melissa,

Yes, I have the same bug with a LS-55 Perkinelmer spectrofluorometer and the software FL Winlab v4.0. The bug is due to a software problem with the file names, if you use simple short names without dots, spaces, etc, to save your spectra you will avoid this bug.

Best regards