Improvement of inner filter effect correction based on determination of effective geometric parameters using a conventional fluorimeter.

The most widely used correction of fluorescence intensities for inner filter effects in conventional (90 degrees ) fluorimeters fails at high absorbance values. We have critically examined this failure, which is caused by the difference between the geometrical parameters (GPs) of the excitation and emission beams in the typical instrument (focused beams) and in the theoretical picture on which the correction is based (collimated beams). We provide two types of experimental measurement of GPs and show that their substitution in the correction equations leads to significant improvements in the linear range of corrected fluorescence. We also demonstrate that mathematical optimizations give greater improvements and that the optimizations yield GPs consistent with experimental measurements. For solutions exhibiting primary inner filter effect only, we have extended the range of linearity of corrected fluorescence to a(ex) (absorbance per cm) up to 5.3; for systems with both primary and secondary inner filter effects we have achieved linearity for a(ex) + a(em) = 6.7. In all cases linear fits have slopes which agree well with the dilute limit. Different series of one- and two-solute solutions were used to demonstrate effectiveness of our correction methods. We also provide a rationale for the unexpected independence of GPs on excitation and emission bandwidths.