= m
= eV
= Hz


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UV Light
Beer's Law
Diode Array
Deuterium Discharge Lamp
Low Pressure Zinc Discharge Lamp
High Pressure Mercury Discharge Lamp
Low Pressure Cadmium Lamp
Tungsten Halogen Lamp
Lens and Window Material in Spectrometers
Fluorescence Reagents
Diffraction Grating
Fourier Transform IR Spectrometer
Halide Disks
Mull Samples
Film Samples for IR Spectroscopy
Light Pipes
Attenuated Total Reflectance Spectroscopy
Multiple Internal Reflectance
External Reflectance
Specular Reflectance
Diffuse Reflectance
Photoacoustic Spectroscopy
Beam Splitter
Raman Scattering
Rayleigh Scattering
Raman Spectroscopy
Atomic Spectroscopy
Atomic Emission Spectroscopy
Atomic Absorption Spectroscopy
The Inductively Coupled Plasma Torch
The Helium Plasma Torch
Emission Spectrometer
Atomic Absorption Spectrometry
Flame Atomic Absorption Spectrometer
Flame AA
Hollow Cathode Lamp
Electrothermal Atomization
Graphite Furnace
L’vov Platform
Electron Paramagnetic Resonance
Zeeman Effect
Continuous Wave
Electron Paramagnetic Resonance
Pulsed EPR
Electron Spin Echo
Multple Resonance Spectroscopy
Magnetic Resonance Spectroscopy
Nucleus Spin Decoupling in NMR
Superconducting Magnets
NMR Microcells
Electron Impact Ionisation
Chemical Ionization
Inductively Coupled Plasma Ionization
Secondary Ion Mass Spectrometry
Fast Atom Bombardment
Plasma Desorption Mass Spectrometry
Laser Desorption Mass Spectrometry
Matrix Assisted Desorption mass Spectrometry
Field Desorption Ionization
Thermospray Ionization
Electrospray Ionization
Atmospheric Pressure Ionization
Particle Beam Interface
Permeable Membrane Interface
Sector Mass Spectrometer
Quadrupole Mass Spectrometer
Ion Trap Mass Spectrometer
Time of Flight Mass Spectrometer
Optical RotationCircular Dichroism
Circularly Polarized Light
Verdet Constant
Faraday Effect

The Fluorescence spectrometer in Practice

Figure 8 shows the layout of the basic Fluorescence spectrometer.

Courtesy of the Perkin Elmer Corporation

Figure 8. The Fluorescence spectrometer Detector

The excitation source (a deuterium or Xenon lamp) that emits UV Light over a wide range of wavelengths is situated at the focal point of an ellipsoidal mirror. This mirror is shown at the top left hand corner of the diagram. The consequent parallel beam of light falls onto a toroidal mirror that focuses it onto a Diffraction Grating. This is shown on the left-hand side of the diagram. Rotation of the grating allows the frequency (or wavelength) of the excitation light to be selected, or the whole Spectrum scanned which would provide an excitation spectra.

Light of the selected wavelength then passes first to a spherical mirror and then to an ellipsoidal mirror (shown at the base of the diagram) that focuses the light onto the sample. Between the spherical mirror and the ellipsoidal mirror, (in the centre of the diagram,) is a beam splitter that reflects a portion of the incident light onto another toroidal mirror. This toroidal mirror focuses the portion of incident light onto the reference photocell providing an output that is proportional to the strength of the incident light.

Fluorescent light from the cell is focused by an ellipsoidal mirror onto a spherical mirror (at the top right-hand side of the diagram). This spherical mirror focuses the fluorescent light onto a grating (situated at about the centre- right of the figure). This grating can select a specific wavelength of the fluorescent light to monitor, or scan the fluorescent light and provide an emission fluorescent Spectrum. Light from the grating passes to another photocell, which monitors its intensity. The instrument is rather complex and, as a result, rather expensive.

However, from the point of view of measuring Fluorescence spectra it is extremely versatile. Much less sample is required to produce useful Fluorescence spectra compared to UV spectra and, in general, Fluorescence spectra contain more fine detail and are, thus, more useful for solute identification where sample size is limited. It is seen that as both the wavelength of the excitation light and that of the fluorescent light can be exclusively selected, so the spectra are not liable to same errors as those of from the Diode Array as already discussed in the book on UV/viz spectroscopy.

The Fluorescence spectrometer can be used in a unique way in conjunction with the liquid chromatograph by programming the excitation wavelength and emission wavelength to provide the maximum sensitivity for each component of the mixture as it is eluted from the column. Thus, once the separation has been developed and retention times identified, the spectrometer is programmed to monitor each peak at the optimum excitation and Fluorescence wavelengths. This procedure provides the ultimate in sensitivity when using Fluorescence detection.

The principle of optimizing both the excitation and emission light wavelengths to obtain maximum sensitivity, however, can become quite complex as shown by the separation of some priority pollutants carried out on an early PE LC/FL tandem instrument and depicted in figure 9. The separation was carried out on a column 25 cm long, 4.6 mm in diameter, and packed with a C18 reversed phase. The mobile phase was programmed from a 93% acetonitrile, 7% water mixture to 99% acetonitrile, and 1% water mixture over a period of 30 minutes. The gradient was linear and the flow rate was 1.3 ml/min.

Table 2 The Fluorescence Excitation and emission Program


Time - seconds wavelength of Excitation light wavelength of emitted light
0 280 nm 340 nm
220 290 nm 320 nm
340 250 nm 385 nm
510 260 nm 385 nm
720 265 nm 420 nm
1050 290 nm 430 nm
1620 300 nm 500 nm


1 Naphthalene

9 Chrysene

2 Acenaphthene

10 Benzo(b)fluoranthene

3 Fluorene

11 Benzo(k)fluoranthene

4 Phenanthrene

12 Benzo(a)pyrene

5 Anthracene

13 Dibenzo(a,h)anthracene

6 Fluoranthene

14 Benzo(ghi)perylene

7 Pyrene

15 Indeno(123-cd)pyrene

8 Benz(a)anthracene

Figure 9. Separation of a Series of Priority Pollutants with Programmed Fluorescence Detection

All the solutes are separated and the compounds, numbering from the left, are given in the figure 9. The separation illustrates the clever use of wavelength programming to obtain the maximum sensitivity. The program is shown in Table 2.



About the Author
RAYMOND PETER WILLIAM SCOTT was born on June 20 1924 in Erith, Kent, UK. He studied at the University of London, obtaining his B.Sc. degree in 1946 and his D.Sc. degree in 1960. After spending more than a decade at Benzole Producers, Ltd. Where he became head of the Physical Chemistry Laboratory, he moved to Unilever Research Laboratories as Manager of their Physical Chemistry department. In 1969 he became Director of Physical Chemistry at Hoffmann-La Roche, Nutley, NJ, U.S.A. and subsequently accepted the position of Director of the Applied Research Department at the Perkin-Elmer Corporation, Norwalk, CT, U.S.A.
In 1986 he became an independent consultant and was appointed Visiting Professor at Georgetown
University, Washington, DC, U.S.A. and at Berkbeck College of the University of London; in 1986 he retired but continues to write technical books dealing with various aspects of physical chemistry and physical chemical techniques. Dr. Scott has authored or co-authored over 200 peer reviewed scientific papers and authored, co-authored or edited over thirty books on various aspects of physical and analytical chemistry. Dr. Scott was a founding member of the British chromatography Society and received the American Chemical society Award in chromatography (1977), the M. S. Tswett chromatography Medal (1978), the Tswett chromatography Medal U.S.S.R., (1979), the A. J. P. Martin chromatography Award (1982) and the Royal Society of Chemistry Award in Analysis and Instrumentation (1988).
Dr. Scott’s activities in gas chromatography started at the inception of the technique, inventing the Heat of Combustion Detector (the precursor of the Flame Ionization Detector), pioneered work on high sensitivity detectors, high efficiency columns and presented fundamental treatments of the relationship between the theory and practice of the technique. He established the viability of the moving bed continuous preparative gas chromatography, examined both theoretically and experimentally those factors that controlled dispersion in packed beds and helped establish the gas chromatograph as a process monitoring instrument. Dr. Scott took and active part in the renaissance of liquid chromatography, was involved in the development of high performance liquid chromatography and invented the wire transport detector. He invented the liquid chromatography mass spectrometry transport interface, introduced micro-bore liquid chromatography columns and used them to provide columns of 750,000 theoretical plates and liquid chromatography separations in less than a second. Dr. Scott has always been a “hands-on” scientist with a remarkable record of accomplishments in chromatography ranging from hardware design to the development of fundamental theory. He has never shied away from questioning “conventional wisdom” and his original approach to problems has often produced significant breakthroughs.

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