【课件】荧光谱分析方法及原理.ppt

荧光光谱;参考书; 某些物质被一定波长的光照射时，会在一定时间内发射出波长比入射光长的光，如果这个时间比较短，这种光就称为荧光。荧光由一种能发荧光的矿物 ?? 萤石(fluospar)而得名。 我们这里要介绍的荧光，是指物质在吸收紫外光和可见光后发出的波长较长的紫外荧光或可见荧光。 除了紫外光和可见光可能激发荧光外，其它的光如红外光、X射线也可能激发出荧光，因此除紫外荧光或可见荧光外，还有红外荧光、X射线荧光等。;?In discussing absorption we have been concerned entirely with the excitation of a molecule from its ground state to a higher energy level and have given no consideration to the subsequent fate of the excited molecule. ? In many cases the sequel is not very interesting; the energy is transferred as heat to the surroundings. ? In some cases light is emitted by the sample either as fluorescence or phosphorescence. ? We will discuss the general characteristics of emission and ignore the exceptions.;荧光光谱的特点;Barak and Webb demonstrated that as few as 30 fluorescent dye molecules bound to low density lipoprotein molecules can be detected on cell surfaces by microscopy using a sensitive video camera. With even more sophisticated methods others have moved toward detection of single fluorophore molecules in a laser-excited flow stream. Because this sensitivity can combine with ease of use and the potential for multiparameter analysis, fluorescence has become widely used in biology and medicine. ;荧光光谱灵敏度高的原因;荧光光谱能提供较多的参数，例如激发谱、发射谱、峰位、峰强度、荧光寿命、荧光偏振度等。 荧光光谱还可以检测一些紫外-可见吸收光谱检测不到的时间过程过程。紫外和可见荧光涉及的是电子能级之间的跃迁，荧光产生包括两个过程：吸收以及随之而来的发射。每个过程发生的时间与跃迁频率的倒数是同一时间量级(大约10-15秒)，但两个过程中有一个时间延搁，大约为10-8秒，这段时间内分子处于激发态。激发态的寿命取决于辐射与非辐射之间的竞争。由于荧光有一定的寿命，因此可以检测一些时间过程与其寿命相当的过程。例如，生色团及其环境的变化过程在紫外可见吸收的10-15秒的过程中基本上是静止不变的，因此无法用紫外可见吸收光谱检测，但可以用荧光光谱检测。 ;基本原理;Fluorescence ;Fluorescence;EMISSlON LlFETlMES;仪器结构;FLUORESCENCE INSTRUMENTATlON;Filter Fluorometers Instruments in which wavelength selection is accomplished with filters are generally referred to as fluorometers, whereas monochromator-based instruments capable of spectral scanning are called spectrofluorometers. The simplest, least expensive, fluorometers are single beam filter instruments with halogen lamp sources, phototube or PMT detectors