If possible with your fluorescence spectrometer, first try an excitation emission scan (=EEM, also called "two-dimensional fluroescence"). this gives you the total overview for the fluroescence behaviour. It allows to identify separate fluorophors (if present) and their relative intensities. For later identification purposes, you can stick to this measurement because it has the maximal information content. Or else, you can quite easily decide, which excitation/emission wavelength gives you the best discrimination power.

A more precise answer is difficult, because you don't give the purpose of what you plan to do... Is it spectra discrimination of plant species, do you want to investigate the differences in composition by spectral investigations, or the influence of solvent on the spectral properties of plant extracts or perhaps something totally different...

you can check with broad range with colour change than you go small range ..

Use a UV-vis spectrometer. The max absorbance will generally be the excitation wavelength for a fluorescent compound- the light needs to be absorbed before anything can be emitted. The emission wavelength will be longer (generally 10 to 20 nm, sometimes more) than the absorption wavelength due to the Stokes shift.

If you are using a fluorescence spectrometer, run it so that the detector is opposite the light source so you can get the absorption wavelength. You can then place the detector orthogonally to the light source, set the light source to the absorbance wavelength, then run an emission scan to determine the emission wavelength.

If possible with your fluorescence spectrometer, first try an excitation emission scan (=EEM, also called "two-dimensional fluroescence"). this gives you the total overview for the fluroescence behaviour. It allows to identify separate fluorophors (if present) and their relative intensities. For later identification purposes, you can stick to this measurement because it has the maximal information content. Or else, you can quite easily decide, which excitation/emission wavelength gives you the best discrimination power.

A more precise answer is difficult, because you don't give the purpose of what you plan to do... Is it spectra discrimination of plant species, do you want to investigate the differences in composition by spectral investigations, or the influence of solvent on the spectral properties of plant extracts or perhaps something totally different...

I am not sure whether you are asking how to find the emission/excitation wavelengths for every plant extract of how to compare the fluorescence of the different extracts. If the question is the first one a synchronous spectrum will be helpful to find the excitation maximum. You can also run a 3D fluorescence spectrum. It is much more time-consuming, but you will fully characterize the fluorescence of your samples. 3D fluorescence of every extract in every sample will probably take several hours, but will be an optimal comparisson.

In any case, either if you run 3D or 2D spectra, please be sure that the emission and excitation slits are the same in all the experiments. Otherwise you cannot compare the intensities of the spectra.

If you are looking to quantify particular molecules present in your extract, you can simply use the UV-vis. The concentrations of the extracted solutions should be sufficiently dilute so that a notable amount of the light capable of being absorbed by the molecules in your extract solution is passing through the given solution inside the cuvet; typically this means keeping the absorption to below 1 for the highest peak. Also the solvent's absorption spectrum should be subtracted from the extract solution's spectrum (with that solvent); this way the solvent's role in absorption of light does not effect quantification of the molecules of interest.

I agree, it really depends on what you are looking to learn ... I would run both excitation and emission spectra on all your samples for comparison on your spectrofluorometer. If you only use UV-Vis, it will give you something equivalent to an absorption spectrum, so the fluorescence emission information will be missing. Are you simply trying to compare how much material each solvent extracts from each plant species?

I don't know which instruments you are using but the simplest procedure is as follows: Before going for any fluorescence measurements, check the UV-Vis absorption. Take the absorption of the solvent as baseline.Note the absorption maximum. Adjust the solute concentration so that the absorbance is below 1 (as Stuart suggests) and measure fluorescence in a normal fluorimeter. Start recording the fluorescence from a wavelength 5 to 10 nm red shifted than the excitation wavelength to avoid rayleigh scattering. You may adjust the slits and other parameters to get good intensity but keep these parameters same for the samples you want to compare. Once you get the fluorescencence peaks, record the excitation spectra by detecting fluorescence intensity at a fixed wavelength while scanning the excitation wavelength where the molecule absorbs (look at the absorption profile and decide the range). Adjust slit opening etc. accordingly. Note that with different solvents, position of absorption maximum and fluorescence behavior are expected to change.

1) You need to determine if your extracts are pure first. Why not stick a spot on a TLC and then separate it and look under a UV-lamp to see how many colored or fluorescent spots you have in each extract

2) You can also measure the excitation spectrum and compare it to the absorption spectrum to see if you are dealing with a simple emission from the main absorbing species and not an impurity. Note that the excitation spectrum may not be exactly the same shape as your absorption spectrum because of geometrical factors. If your sample absorbs the excitation light too strongly then you will get distortions because your excitation beam will be too weak when it gets to the middle of your cuvette, from where the spectrometer has been optimized to collect the light. You can even absorb all of the light on the front face of your cuvette (with respect to the excitation beam).

If the samples are impure or mixtures then best to try and purify them at this stage. The stronger your sample absorbs the more distorted your excitation spectrum will be. Try to stay at absorbances less than 0.3 (or less) at the excitation wavelength for most measurements. Remember more absorption can actually result in less emission due to the reasons of geometry stated above.

3) Next you may want to measure the emission maxima for each pure extract. Actually if you have no impurities then it does not usually really matter where you excite on the absorption envelope, either in the S0-S1 bands or the S0-S2 bands. In most cases emission comes from the S1V0 level any way due to efficient vibrational cascade. But do leave a reasonable gap between your excitation wavelength and your desired emission wavelength measurement range. The necessary gap depends on your excitation slit widths, which will determine how broad your excitation beam is in nm and hence how much it will spill into your emission spectrum. Do use filters to stop artifacts. A bandpass filter for the excitation and a long pass filter that will cut scattered excitation beam without distorting you emission spectrum. Thus you can compare your emission maxima and spectral shapes.

4) Next you may want to compare the emission yields. To do this you need to excite the samples at the same wavelength (preferably with narrow slit width 5nm or less). The samples should have as much as possible exactly the same absorption at the excitation wavelength. You may also compare the samples to a reference Rhodamine or well know fluorophore with a known quantum yield. Then you can get the quantum yield of your sample. Try to stick to the same solvent or you will have to consider a refractive index correction as light will bend differently coming out of the cuvette in to air depending on the solvent refractive index. If you cant use the same solvent (or solvents with the same refractive index) then you need to apply a correction. You can find this on the web somewhere.

Make a plot of absorbance against emission for your samples compared to the standard reference and then by comparison of the slopes and knowing the quantum yield of your reference standard you can get your absolute quantum yields.

So as many people said, your wavelength choices depend very much on what information you want to get out.

Hope this helps

Which instrument you are using- a UV-Vis Spectrophotometer or a Spectrofluorophotometer?

If you are simply asking how to fix an emission and excitation wavelength then your system will require at least 2 monochromators working in the desired range and of course a surce at the excitation wavelengths you would like to measure. This means you will need a spectrofluorometer and to follow the suggestions above. What may have been missed above is that you will need some filters (Long Pass) to remove harmonics of the diffractive optics which may affect your measurments. Furthermore a calibration factor must be applied to account for the non-unity transmission.

1- take absorbance spectrum from 200 to 800 nm

2- peak with maxumam od coud be your potential excitation wave lengh

3- take emission spectrum using the potential excitation wavelengh range between your excitation WL and 800 nm, most important here to identify your filter as cutoff the pkea which gives you the most appropriate enhancement will be your emission peak, taken into account enhancement in your blank could be water should be approximately close to zero

4- make dilution test and watch the enhancement, if it consistant (increase with increasing concentration) this might be a good excitation emission wavelength.

5- you could improve your excitation wavelengh by taking excitation spectrum, if maximum peak is similar to your previous excitation wavelengh then you already had your right combination, if not try the new maximum peak in your excitation spectrum as excitation wavelengh and repeat the above process

You should use the scan function of the instrument to get the excitation and emission wavelengths for your test sample. If your sample has fluorescent compounds, the instrument will scan the working wavelengths. This function of the instrument is useful for unknown compounds. I think you should succeed.

Regards

check the scan function of your instrument. you will get it. Please refer to the SOP of the same.

@Shreetama Karmakar Well said. Follow the procedure probably u will get the result. you used the terms "three different plant species" and "four different solvents". Each solvent may have shown its own emission upon excitation if solvent showed florescent.

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Thanks & best wishes.

Actually, it sounds as if you are looking for a short-cut, by trying to select a couple of variables and sticking to them.

You should not do this, especially with plant extracts, which are probably mixtures of co-eluted compounds coming off your column.

It is more involved if you really want to understand anything about your “plant materials” because each compound in your co-eluted fractions can have its own emission and absorption spectrum.

What you should do is to measure the emission spectrum exciting at many different excitation wavelengths and see if the emission spectral shape changes. If it does then you have a mixture.

Next select several different emission wavelengths, concentrating your efforts on the spectral regions which change the most with excitation wavelength and run excitation spectra to try to estimate roughly what the absorption spectrum of each component is. Keep in mind that you will get spectral overlaps and co-excitation so you may need to deconvolute in order to get a reasonable excitation spectrum.

This will only really be possible for a maximum of three co-eluted compounds in each fraction. But you could have many more than that.

Keep your excitation and emission slits narrow (2.5nm) and accumulate with a very slow scan speed. This also helps to not decompose your delicate plant chromosphere.

If you have not put your extracts down a column, then you really should do that first otherwise the complexity will be unmanageable, because raw plant extracts are very crude with many components.

Assuming you separated the extracts on a column, then you would need to measure at least 20 spectra per fraction in order to have a good chance of interpreting what is present in each fraction. So 3 plants, 4 solvents and lets guess 4 fractions per solvent = 48 samples. Around 20 spectra per sample (OK maybe you can cut that a lot if things look clean and simple) so you are looking at at least 5 and up to 20 excitation/ emission spectra per fraction. ~250 to 1000 spectra.

You can try to simply as you appear to want to do .... but it will not produce meaningful data.

PS don’t forget the cut off and band pass filters in the emission measurement. And the cut off filter in the excitation.