Experimental correction for the inner-filter effect in fluorescence spectra

Abstract

Recorded fluorescence intensity is in general not proportional to sample concentration owing to absorption of the incident and emitted light passing through the sample to and from the point inside the cell where the emission is detected. This well known inner-filter effect depends on sample absorption and on instrument geometry, and is usually significant even in samples with rather low absorption (the error is about 8% at an absorbance of 0.06 in a 1 cm square cell). In this work we show that a particular experimental set-up can be calibrated for the inner-filter effect from the absorption and fluorescence excitation spectra of a suitable standard. The calibration takes only a few minutes and provides correction with sufficient accuracy for most practical situations.