Kasparek Adam, Smyk Bogdan
Department of Physics and Biophysics, University of Warmia and Mazury in Olsztyn, Oczapowskiego 4, 10-719 Olsztyn, Poland.
Department of Physics and Biophysics, University of Warmia and Mazury in Olsztyn, Oczapowskiego 4, 10-719 Olsztyn, Poland.
Spectrochim Acta A Mol Biomol Spectrosc. 2018 Jun 5;198:297-303. doi: 10.1016/j.saa.2018.03.027. Epub 2018 Mar 13.
The fluorescence technique is very popular and has been used in many fields of research. It is simple in its assumptions but not very easy to use. One of the main problems is the inner filter effect (IF) I and II which takes place in the cuvette. IF type I is permanently present, but IF type II occurs only when absorption and fluorescence spectra overlap. To avoid IF type I, absorbencies in the cuvette should be smaller than 0.05, which is however very difficult to obtain in many experiments. In this work we propose a new method to solve these problems in the case of a Cary Eclipse fluorimeter, having horizontally-oriented slits, based on old equations developed in the middle of the last century. This method can be applied for other instruments, even these with vertically-oriented beams, because we share scripts written in MATLAB and GRAMS/AI environment. Calculations in our method enable specifying beam geometry parameters in the cuvette, which is necessary to obtain the correct shape and fluorescence intensity of emission and excitation spectra. Such a specific fluorescence intensity dependence on absorbance can, in many cases, afford possibilities to determine the quantum yield (QY) using slopes of the straight-lines, which was demonstrated with the use of Tryptophan (Trp), Tyrosine (Tyr), and Rhodamine B (RhB) solutions. For example, assuming that QY=0.14 for Tyr, the QY determined for RhB reached QY=0.71±0.05, although the measurement for Tyr and RhB was performed at a completely different spectral range.
荧光技术非常受欢迎，已被应用于许多研究领域。其假设简单，但使用起来并非十分容易。主要问题之一是比色皿中会出现的内滤光片效应（IF）I和II。IF I型始终存在，但IF II型仅在吸收光谱和荧光光谱重叠时才会出现。为避免IF I型，比色皿中的吸光度应小于0.05，然而在许多实验中这很难实现。在这项工作中，我们基于上世纪中叶提出的旧方程，针对具有水平狭缝的Cary Eclipse荧光计，提出了一种新方法来解决这些问题。该方法可应用于其他仪器，甚至是具有垂直光束的仪器，因为我们共享了在MATLAB和GRAMS/AI环境中编写的脚本。我们方法中的计算能够确定比色皿中的光束几何参数，这对于获得发射光谱和激发光谱的正确形状及荧光强度是必要的。在许多情况下，这种特定的荧光强度对比色吸光度的依赖性使得利用直线斜率来确定量子产率（QY）成为可能，这通过使用色氨酸（Trp）、酪氨酸（Tyr）和罗丹明B（RhB）溶液得到了证明。例如，假设Tyr的QY = 0.14，尽管对Tyr和RhB的测量是在完全不同的光谱范围内进行的，但RhB测定的QY达到了QY = 0.71±0.05。
