Step 2: Optimize Limits of Detection and Selectivity

To achieve optimum limits of detection and selectivity, analysts must find out about the fluorescent properties of the compounds of interest. Excitation and emission wavelengths can be selected for optimum limits of detection and best selectivity. In general, fluorescence spectra obtained with different instruments may show significant differences depending on the hardware and software used.

Achieving the best detection levels requires checking for the optimum excitation and emission wavelengths for all compounds. A good approach is to acquire online spectra for all compounds during a run. Two runs are sufficient for optimization.

During the first run, one wavelength is chosen for the excitation wavelength using a value found in literature or one in the low UV range (220 - 260 nm) and a spectral range for the emission side. Most fluorophores show strong absorption at these excitation wavelengths and the quantum yield is high. Excitation is sufficient for collecting emission spectra. Examining the recorded spectra will give the best wavelength for emission. With that value in mind, the method gets modified for excitation scan with the emission value found entered as the emission wavelength. For the excitation scan range a suitable spectral range should be selected. Examining the second recorded set of spectra will give the best wavelength for excitation. With the two values found, the method should be modified for single wavelength.

For best sensitivity, due to the distinct Hg-lines of the light source, methods, which got developed on a G1321A/B/C or G7121A/B may need to be reevaluated, mainly in terms of EX wavelengths.