The best way to actually find the pKa is to do a first derivative plot.The first derivative isD(fluor intensity)/DpH.Dis the change between two values (i.e. subtraction).You then plot that as the y-value vs the average of the pH unit.Make a first derivative plot of the data.The peak will be the pKa value (i.e. the midpoint on the graph before).

B C D E F G H K L M N P Q R S T 1 PH 1.5 40 43 39 PH 2.75 710 690 688 PH 4 2087 1492 2134 PH 1.75 74 72 69 PH 3.0 1008 1012 1010 PH 4.25 1915 2195 2027 PH 2.0 133 135 143 PH 3.25 1324 1353 1380 PH 4.5 2045 2073 2062 PH 2.25 256 253 245 PH 3.5 1636 1646 1646 PH 2.5 583 580 573 PH 3.75 2224 2171 2151 PH average fluor intensity SUMMARY OUTPUT Fluorescence intensity vs pH 10 1.5 41 11 1.75 72 Regression Statistics 2500 12 2 137 MultipleR 0.970384 2000 13 2.25 251 R Square 0.941645 14 2.5 579 Adjusted R 0.93581 1500 15 2.75 696 Standard E 0.228374 Fluorescence Intensity 16 3 1010 Observation 12 1090 17 3.25 1352 18 3.5 1643 ANOVA 19 3.75 2182 of SS MS F ignificance F 20 1904 Regression 1 8.415954 8.415954 161.3655 1.71E-07 2 21 4.25 2046 Residual 10 0.521546 0.052155 PH 22 4.5 2060 Total 11 8.9375 23 24 Coefficientsandard Erre t Stat P-value Lower 95% Upper 95% ower 95.0W/pper 95.0% 25 Intercept 1.860588 0.119389 15.58423 2.42E-08 1.594573 2.126604 1.594573 2.126604 26 40.66667 0.001089 8.576-05 12.70297 1.718-07 0.000898 0.00128 0.000898 0.00128