Linearity and baseline issues
In dilute solution, the fluorescence intensity (If) for defined values of λex and λf is related linearly to molar concentration (c), according to the approximate relationship:
If=Kc at λex, λf (15.24)
under constant instrumental conditions. Sensitivity can be increased by working at high excitation powers to give larger signal-to-noise ratios. Since the source intensity can change from time to time, fluorescence signals are not measured as absolute parameters. They are expressed rather in terms of relative fluorescence. All measurements are made relative to reference standards of known concentration. All readings must be corrected for background fluorescence.
A necessary condition is that the total absorbance (=ɛlc) of the system should not exceed 0.05 absorbance units; otherwise progressively greater negative deviations from linearity are observed. At high drug concentrations, fluorescence intensity reaches a plateau. Beyond this, fluorescence intensity actually decreases with increasing concentration, because of inner filter effects (self-quenching), in which ground state molecules absorb the fluorescence emitted by excited molecules.
It is essential to establish the range of linearity of the calibration curve of If versus c, using at least five standard solutions, for which the condition that absorbance at the wavelength of maximum excitation is <0.05 absorbance units holds. Samples are usually analysed by single point bracketing, taking a standard conveniently close to the anticipated sample value and calculating the result by simple proportion.
The sequence of standard measurements before and after measuring the sample permits any baseline drift to be compensated. For additional assay security, two-point bracketing can be employed, in which two standard solutions, one higher and the other lower than the concentration observed for the sample, are used in bracketing sequence. Two-point bracketing becomes too onerous when large sample numbers are to be analysed, as may be the case in HPLC-FL. In this situation, standards are analysed first, followed by samples, with a standard analysed after approximately every 10 samples. This compensates for any baseline drift whilst maintaining a reasonable time for analysis.
Instrumental limitations caused by instability of the radiative source can be overcome by the ratio mode operation. A small fraction of the exciting radiation is directed to a reference photodetector, which is chosen primarily for wide wavelength response. The output signal is used as a monitor and, as the excitation radiation increases or decreases in power because of fluctuations in the source, there is a corresponding increase or decrease in relative fluorescence.
