Question

A nonlinear relationship between analyte concentration and intensity during a fluorescence experiment. The function becomes exponential (tails off) as higher concentrations are analyzed. How would you modify the experiment or the instrument to combat this problem? Describe two solutions.

Answer 1

Ans. The following two solutions are possible-

I. Dilution: The linear relationship between analyte’s (fluorophore) concentration and intensity (or, absorbance, etc.) deviates most often when the concertation is relative higher.

Dilute the solution sufficiently to reduce the concentration. At lower concentrations, the intensity of the aliquots may become proportional to the analyte’s concertation, and a linear graph oh intensity vs concentration can be produced.

II. Baseline correction: Baseline correction is spectrofluorometer is similar to zeroing a UV-VIS spectrophotometer with suitable reagent blank.

The baseline correction is done by zeroing the intensity of the reagent blank at specified wavelength in the instrument. Recording intensities of the samples/ aliquots after baseline correction gives relatively lower intensity values compared to the same taken against distilled water or air as blank. Therefore, baseline correction may also yield a linear graph because it gives lower intensity of the aliquots.