An Introduction to FIuorescence Spectroscopy 2 User Assistance PerkinElmer Ltd Post OIIice Lane BeaconsIield Buckinghamshire HP9 1QA Printed in the United Kingdom. Notices The inIormation contained in this document is subject to change without notice. PerkinElmer makes no warranty oI any kind with regard to the material, including, but not limited to, the implied warranties oI merchantability and Iitness Ior a particular purpose. PerkinElmer shall not be liable Ior errors contained herein Ior incidental consequential damages in connection with Iurnishing, perIormance or use oI this material. Copyright Information This document contains proprietary inIormation that is protected by copyright. All rights are reserved.No part oI this publication may be reproduced in any Iorm whatsoever or translated into any language without the prior, written permission oI PerkinElmer, Inc. Copyright 2000 PerkinElmer, Inc. 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PerkinElmer is a registered trademark oI PerkinElmer, Inc.An Introduction to Fluorescence Spectroscopy 3 Table of Contents Table oI Contents............................................................................................ 3 Luminescence and the nature oI light ............................................................. 5 Fluorescence ................................................................................................... 7 Phosphorescence........................................................................................... 11 Light scattering............................................................................................. 14 Rayleigh-Tyndall scattering .................................................................. 14 Raman scattering ................................................................................... 14 Quantitation .................................................................................................. 16 Sensitivity..................................................................................................... 18 Instrumental sensitivity ......................................................................... 18 Method sensitivity ................................................................................. 18 Absolute sensitivity ............................................................................... 18 Instrumentation............................................................................................. 21 General .................................................................................................. 21 Light sources ......................................................................................... 22 Wavelength selection............................................................................. 24 Detectors................................................................................................ 25 Read-out devices ................................................................................... 26 Sample holders ...................................................................................... 26 Problems oI high blank values...................................................................... 28 Cuvettes ................................................................................................. 28 Solvents and Reagents........................................................................... 28 Other contaminants................................................................................ 29 Working with dilute solutions ...................................................................... 30 General .................................................................................................. 30 Adsorption............................................................................................. 30 Photo-decomposition............................................................................. 31 Oxidation............................................................................................... 31 Factors aIIecting quantitative accuracy ........................................................ 32 Non-linearity.......................................................................................... 32 Temperature eIIects ............................................................................... 32 pH eIIects............................................................................................... 33 Inner-Iilter eIIects.................................................................................. 33 Quenching.............................................................................................. 33 ReIerences .................................................................................................... 34 An Introduction to Fluorescence Spectroscopy 4 An Introduction to Fluorescence Spectroscopy 5 Luminescence and the nature of light A hot body that emits radiation solely because oI its high temperature is said to exhibit incandescence. All other Iorms oI light emission are called luminescence.When luminescence occurs, the system loses energy and iI the emission is to be continuous, some Iorm oI energy must be supplied Irom elsewhere. Thus the radioluminescence emitted Irom a luminous clock Iace is supplied by high energy particles Irom the radioactive material in the phosphor and the electroluminescence oI a gas discharge lamp is derived Irom the passage oI an electric current through an ionised gas. Other such phenomena include chemiluminescence, derived Irom the energy oI a chemical reaction, and this is called bioluminescence when the reactions take place within living organisms, Ior example, glow-worms and IireIlies. When the external energy supply is by means oI the absorption oI inIrared, visible or ultraviolet light, the emitted light is called photoluminescence and this is the process that takes place in any Iluorimetric analysis. To account adequately Ior the processes oI absorption and emission oI light, it is necessary to assume that radiant energy can only be absorbed in deIinite units, or quanta. The energy, E, carried by any one quantum is proportional to its Irequency oI oscillation, that is Ehhc ergs

whereis the Irequency,the related wavelength and h Planck's constant (6.624 x 10-27 ergs/seconds). The energy oI a single quantum is too small Ior convenience and it is usual to talk oI the energy associated with N quanta (where N 6.023 x 1023 the number oI single molecules in a gram molecule), which is called an einstein. Thus, iI in a photochemical reaction one molecule reacts Ior each quantum absorbed, then the absorption oI one einstein is suIIicient energy Ior the reaction oI one gram mole. Since the amount oI energy per einstein is proportional to the Irequency oI the radiation, it varies enormously over the range oI the electro-magnetic spectrum, as shown in the Iollowing table. An Introduction to Fluorescence Spectroscopy 6 Approximate sizes of Quanta Radiation(cm) (typical values) Wave- number (m-) Size of quantum(electron volts) Size of einstein (kilogram calories) Absorption or emission of radiation involves Gamma rays10-101061.2 x 106 2.9 x 107Nuclear reactions X-rays10-81041.2 x 104 2.9 x 105Transitions oI inner atomic electrons Ultraviolet Visible 10-5 4 x 10-5 8 x 10-5 101 2.5 1.25 1.2 x 10 1 3.1 1.62.9 x 102 7.1 x 101 3.6 x 101 Transitions oI outer atomic electrons InIrared10-310-11.2 x 10-1 2.9Molecular vibrations Far inIrared10-310-21.2 x 10-2 2.9 x 10-1Molecular rotations Radar Long radio waves 101 105 10-5 10-9 1.2 x 10-5 1.2 x 10-9 2.9 x 10-4 2.9 x 10-8 Oscillation oI mobile or Iree electrons The ultraviolet and visible regions oI the spectrum are oI most interest in Iluorimetry and absorption in these regions causes the excitation oI the outermost electrons oI the molecule. The energy associated with radiation oI this Irequency is quite high, around 100 kilogram calories per einstein, and is sometimes suIIicient to break down the absorbing molecules, as Ior instance with the Iading oI dyes by the action oI sunlight. The absorption oI light results in the Iormation oI excited molecules which can in turn dissipate their energy by decomposition, reaction, or re-emission. The eIIiciency with which these processes take place is called the quantum eIIiciency and in the case oI photoluminescence can be deIined as: E einsteins emitted orNo. oI quanta emitted einsteins absorbed No. oI quanta absorbedand never exceeds unity. An Introduction to Fluorescence Spectroscopy 7 Fluorescence At room temperature most molecules occupy the lowest vibrational level oI the ground electronic state, and on absorption oI light they are elevated to produce excited states. The simpliIied diagram below shows absorption by molecules to produce either the Iirst, S1, or second S2, excited state (Figure 1). Figure Transitions giving rise to absorption and fluorescence emission spectra (10 10 sec) Vibrational Levels of second excited state Vibrational Levels of first excited state Vibrational Levels of ground state Rotational levels Fluorescence (10-9 10-6 sec) nternal (