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In spectrophotometry, often a cut off optical filter is used. With reference to a long-pass filter, which rejects any wavelengths outside a predetermined interval: low transmission in short wavelengths range and high transmission in long wavelength ranges.

Some are orange, orange-yellow, dark orange, may appear similar to a novice if not labelled well.

How can a beginner see/detect if they have used a wrong filter by looking at the spectra before they continue using it? Practical troubleshooting tips and knowledge?

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    $\begingroup$ Unless the filters are labelled in some way, e.g., Maker and number, it is not always easy to visually distinguish filters. I have a box of filters, including long pass non-fluorescent filters, and the only good way to know what they will do spectrally is to get a transmittance or absorbance spectrum. Quick and dirty: use a beam of sunlight and a diffraction grating to project a rainbow on a white wall or white paper. Then look at the rainbow using the filters. Obviously no good for UV or IR and it is very crude: a spectrometer is far better in every way. $\endgroup$
    – Ed V
    Oct 7, 2021 at 2:14
  • $\begingroup$ I don't understand the question. Are you near the spectroscopy set up or what? $\endgroup$
    – Alchimista
    Oct 7, 2021 at 9:47
  • $\begingroup$ @Edv appreciate your insights and recommending your colleague MFarque to comment. Well, with regards to protein assays (spectrophotometer: fluoroscopy), the cut off filters are sensitively crucial. According to your insights, if incorrect filter is used, there doesn't seem to be a way detect it unless actual recheck of the equipment is done. $\endgroup$
    – bonCodigo
    Oct 9, 2021 at 0:01
  • $\begingroup$ @Alchimista please see my comment. Any feedback? $\endgroup$
    – bonCodigo
    Oct 9, 2021 at 0:01
  • $\begingroup$ Afraid so. Filters are also problematic because of parasitic fluorescence: a long pass filter may block short wavelength light, but fluoresce, thereby ruining the measurements. This is not so easy to check for because the filter would be tested for its spectral properties while in the sample compartment of the spectrophotometer. Then unwanted fluorescence might well go unnoticed. This is one reason to use solution filters, e.g., potassium nitrate solution. But I do not know of a list of such solution filters and adding another cuvette to a sample compartment may not be feasible. Very annoying. $\endgroup$
    – Ed V
    Oct 9, 2021 at 0:52

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As Prof. Ed advised, take the transmission spectrum of each optical filter on a good quality double beam or single beam scanning spectrophotometer with the shortest possible wavelength step size (Caution: this could be slow, but better in terms of reliability). Keep the filter vertical. Use air as a blank. A cut-off filter is not as good as an interference filter. Note that Beer's law will not apply if you use cut-off filters. It is valid for a single wavelength. Unfortunately, there is no other way to characterize the optical filter. You do need a scanning spectrophotometer.

Edits: The instrument you are using is not called a spectrophotometer but it is a fluorometer. It uses a very bright xenon arc lamp and it needs two filters. One is called excitation filter and the other is called emission filter. Before you do fluorometry you should know

  1. Excitation spectrum of your analyte or absorption spectrum (shapes are same for simple molecules)

  2. Emission spectrum of your analyte.

  3. Transmission spectrum of your excitation filter

  4. Transmission spectrum of your emission filter.

Ideally there should be no overlap in transmission wavelengths of the two filters.

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  • $\begingroup$ Please see my comment to Ed V. It's a scanning spectrophotometer. So if that's the case, looking at the scan/spectra, can one detect/identify if correct filter is being used for their assays? You see, if a biomolecule based assay is continued, it can go on till the end and wouldn't be so beneficial to realize in the middle to at the end that filter was wrong. $\endgroup$
    – bonCodigo
    Oct 9, 2021 at 0:05
  • $\begingroup$ Sorry, I still do not understand the reason for using a filter with a scanning spectrophotometer. Could you describe your instrumentation please? $\endgroup$
    – AChem
    Oct 9, 2021 at 0:23
  • $\begingroup$ Spectrofluorometers and spectrophotometry are completely different instruments. $\endgroup$
    – AChem
    Oct 9, 2021 at 0:23
  • $\begingroup$ Is there a chance that both Spectrofluorometers and spectrophotometry functions are combined into one? $\endgroup$
    – bonCodigo
    Oct 16, 2021 at 7:16
  • $\begingroup$ @bonCodigo, spectrophotometry is not done with cut off filters. $\endgroup$
    – AChem
    Oct 16, 2021 at 12:46

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