Also, sometimes I notice the reverse where weak excitation peak and strong emission peak. For the best of my knowledge, the solvent peak should be (
Generally one doesnot use solvents which are fluorescent. Most of the solvents are non-fluorescent. In some solvents there can be background emission, autofluorescence etc depending on its composition (e.g. buffers - especially in biology). Raman scattering will be seen in all the solvents.
Can you be more specific than being general? Can you attach some figures where it is seen. It is much easier to explain looking at the figures.
Raman scatter is the most likely explanation of the solvent peak. It is easily tested by looking for a shift in the peak wavelength in the same direction as a change in excitation wavelength. That said, there could also be fluorescent contamination of the solvent.
I mean when i use very diluted solution of studied compound (1uM) the solvent background becomes 100% not (
This can happen with the Raman peak or if there is an impurity in the solvent. On dilution the solvent content in the sample increases, so correspondingly the Raman peak or the impurity peak will also increase.
For UV excitation, solvent purity is important because all compounds absorb in the UV.
Raman peaks are narrow with a FWHM of ~ few nms whereas impurity peaks are broad with a FWHM of ~ tens of nms. As already said, Raman peaks change with excitation wavelength whereas impurity peaks don't change with excitation wavelength (generally) unless multiple species are present.
In fluorescence measurement, there are two types of scattering peaks:
- Rayleigh peaks are always at the excitation wavelength, scattered from dust particles, the sample container windows, and so on. Rayleigh scattering is always present, the intensity depends on the optical beam geometry. You'll have more Rayleigh scattering in a microplate reader compared to a cuvette spectrometer with 90° geometry. The relative intensity compared to the sample peak depends on the sample concentration: because the Rayleigh peak intensity itself is constant, it seems to raise with decreasing sample concentration.
- Raman peaks are also scattering peaks, but dependent on the solvent and always shifted away from the excitation wavelength. The "Raman shift" is a unique property of the solvent, it is given in wavenumbers(!), which means the distance in nm is varying with excitation wavelength. Raman peak intensity strongly depends on the solvent type.
The bandwidth for both types of scattering peaks depends solely on the spectrometer's excitation bandwidth. Best way for clear indentification would be measuring an EEM (excitation emission matrix), which shows the full information. You may also see 2nd order scatter peaks from both types.
I've attached an example for a blank EEM measurement, the first order Rayleigh peak was already left out by the spectrometer software. Display was created with Spekwin32, an optical spectroscopy software I maintain and develop.
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