Fluorescence of Dyes in Solutions with High Absorbance. Inner Filter Effect Correction
Figure 2
The dependence of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance at high concentrations of fluorophore, as recorded by a Cary Eclipse spectrofluorimeter.
Curves 1, 2 and 3 are the same as in Figure 1. Curves 4 and 5 represent the fluorescence intensity recorded with the use of microcells (5×5 mm cross section) and in triangular cell. The absorbance was determined using a Hitachi U-3900H spectrophotometer in cells with different optical path lengths. Inset: The corrected and normalized fluorescence excitation and emission spectra of NATA. The fluorescence excitation and emission spectra were recorded by a Cary Eclipse spectrofluorimeter at λem = 350 nm and λex = 280 nm, respectively. The correction was performed according to Eq. 4. The red, blue and green curves correspond to A280 = 60, 1.5 and 0.2, respectively. The black curve is the absorption spectrum.