Gu Qun, Kenny Jonathan E
Department of Chemistry, Edinboro University, Edinboro, Pennsylvania 16412, USA.
Anal Chem. 2009 Jan 1;81(1):420-6. doi: 10.1021/ac801676j.
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.
在传统（90度）荧光计中，用于校正内滤光效应荧光强度的最广泛使用的方法在高吸光度值时失效。我们严格审查了这种失效情况，它是由典型仪器（聚焦光束）中激发光束和发射光束的几何参数（GPs）与校正所基于的理论模型（准直光束）之间的差异引起的。我们提供了两种类型的GPs实验测量，并表明在校正方程中代入这些参数会显著改善校正荧光的线性范围。我们还证明数学优化能带来更大的改善，并且优化得到的GPs与实验测量结果一致。对于仅表现出初级内滤光效应的溶液，我们将校正荧光的线性范围扩展到a(ex)（每厘米吸光度）高达5.3；对于同时具有初级和次级内滤光效应的系统，我们实现了a(ex) + a(em) = 6.7时的线性。在所有情况下，线性拟合的斜率与稀释极限吻合良好。使用不同系列的单溶质和双溶质溶液来证明我们校正方法的有效性。我们还为GPs与激发和发射带宽的意外独立性提供了一个理论依据。
