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5.1 Introduction
The scope of optical spectroscopy methods is very wide and encompasses many aspects
of life- and material sciences. Using optical spectroscopy one can analyze spectrochemical
events to monitor, for example, human health issues, food and water quality, environment
quality, materials for whitening agents, lighting, and light-emitting diodes. The instrumentation used comprises optical, mechanical, electrical as well as signal processing and data
analysis components. The data recorded are a convolution of the effects of each of these
components in addition to the sample and its behavior.
Fluorescence spectroscopy, although not a new technique, is still, compared to other
analytical methods, relatively immature in terms of standardization of measurement. As
mentioned in Chapter 1, the birth of fluorescence spectroscopy was marked by the work
of Sir George Gabriel Stokes, who in 1852 reported his studies on quinine bisulfate using
what today would be considered a filter fluorimeter arrangement, as shown in Figure 5.1.
More than 100 years later, commercial fluorimeter systems emerged owing to the
advancement in light sources, scanning monochromators, detector technology, and analog
signal recording mechanisms. In the last 50 years there have been considerable improvements in each electro-optical component used in fluorimeter systems but, equally, there has
also been a rapid advancement to computer-controlled, often termed “black-box,” technology. As a result, in recent decades less attention has been given to the measurement of
instrument performance and in particular to rigorous calibration and testing. The use of
quinine sulfate as a reference standard is widespread and therefore it is often not clear if the
measurements demonstrated by instruments are the true fluorescence spectra of the sample
(in terms of its behavior) or that of the measuring instrument (e.g., light source, intensity
of fluorescence, spectral features, etc.). More specifically, the observed measured spectrum
is the convolution of both the experimental system and the true fluorescence spectrum.
Some experimentalists who are not fully acquainted with measuring fluorescence spectra
may, understandably, take a black box approach to these important aspects of measuring
fluorescence spectra. Even so, it is important that we appreciate this convoluted system if
we are to gain a quantitative insight from the fluorescence measurements that is related to
the samples analyzed as opposed to the measurement system.
5
Optical Spectroscopy Instrumentation Design, Quality
Assurance, and Control: Bench-Top Fluorimetry
John R. Gilchrist and Darren M. Reynolds
5.1 Introduction
The scope of optical spectroscopy methods is very wide and encompasses many aspects
of life- and material sciences. Using optical spectroscopy one can analyze spectrochemical
events to monitor, for example, human health issues, food and water quality, environment
quality, materials for whitening agents, lighting, and light-emitting diodes. The instrumentation used comprises optical, mechanical, electrical as well as signal processing and data
analysis components. The data recorded are a convolution of the effects of each of these
components in addition to the sample and its behavior.
Fluorescence spectroscopy, although not a new technique, is still, compared to other
analytical methods, relatively immature in terms of standardization of measurement. As
mentioned in Chapter 1, the birth of fluorescence spectroscopy was marked by the work
of Sir George Gabriel Stokes, who in 1852 reported his studies on quinine bisulfate using
what today would be considered a filter fluorimeter arrangement, as shown in Figure 5.1.
More than 100 years later, commercial fluorimeter systems emerged owing to the
advancement in light sources, scanning monochromators, detector technology, and analog
signal recording mechanisms. In the last 50 years there have been considerable improvements in each electro-optical component used in fluorimeter systems but, equally, there has
also been a rapid advancement to computer-controlled, often termed “black-box,” technology. As a result, in recent decades less attention has been given to the measurement of
instrument performance and in particular to rigorous calibration and testing. The use of
quinine sulfate as a reference standard is widespread and therefore it is often not clear if the
measurements demonstrated by instruments are the true fluorescence spectra of the sample
(in terms of its behavior) or that of the measuring instrument (e.g., light source, intensity
of fluorescence, spectral features, etc.). More specifically, the observed measured spectrum
is the convolution of both the experimental system and the true fluorescence spectrum.
Some experimentalists who are not fully acquainted with measuring fluorescence spectra
may, understandably, take a black box approach to these important aspects of measuring
fluorescence spectra. Even so, it is important that we appreciate this convoluted system if
we are to gain a quantitative insight from the fluorescence measurements that is related to
the samples analyzed as opposed to the measurement system.
5
Optical Spectroscopy Instrumentation Design, Quality
Assurance, and Control: Bench-Top Fluorimetry
John R. Gilchrist and Darren M. Reynolds