荧光光谱分析方法及原理.ppt

参考书
赵南明,,高等教育出版社,
2000
Emission Spectroscopy, Chapter 11, in . van
Holde. . Johnson and . Ho eds
Principles of Physical Biochemistry, Prentice-Hall
1998
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——茧石 fluospa)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
Fluorescence is a
technique
for detecting biological materials
Barak and Webb demonstrated that
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
荧头
七辐射的波长比激发光浪长长,测量到的荧
光频率与入射光的频率不同;
荧光在各个方向上都有发射,因此可以在与
入射光成直角的方向上检测
这样,荧光不受来自激发光的本底的干扰,
灵敏度大大高于紫外可见吸收光谱,测量用的
样品量很少,且测量方法简便。
荧光光谱第二个特点是
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1015秒),但两
个过程中有一个时间延搁,大约为10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竟争。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
系间交连
三线态
(10°-1)
102s-)
电子