= 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 Mechanism of Fluorescence

The majority of molecules exist at their lowest vibrational level in their ground state at room temperature. On exposure to electromagnetic radiation the molecules can absorb energy and, as a consequence, are promoted to an excited state. As a result of the energy absorption, the molecule can attain any of the vibrational sub-levels associated with each electronic state. Because electromagnetic energy can only be absorbed in discrete quanta it would appear that this should result in a series of absorption bands in the absorption Spectrum. However, superimposed on the various vibrational energy levels are a series of rotational energy levels, which result in so many absorption bands that it becomes practically impossible to resolve and, thus, depict all the individual bands in the Spectrum. As a consequence, most compounds excluding planar and aromatic compounds (where the rotation energy levels are limited) produce broad absorption spectra with little fine structure (see book 1 on UV and Visible Spectroscopy).

Having been excited and reached a high vibrational energy level, the vibrational energy is lost by molecular collision and the molecule is, thus, reduced to the lowest vibrational energy of its excited state. Subsequently the molecule will suffer internal conversion where there is an energy transfer from the lowest vibrational energy at the upper excited state to a higher vibration energy level at a lower excited state. This process will continue until the molecule reaches its lowest vibrational energy at its first excited state. From this condition the molecule can return to any vibrational level of the ground state with emission of a photon of energy in the form of Fluorescence.

If all the excited molecules return to the ground state the quantum efficiency is said to be unity, The return of the energy level from the first excited sate to the ground state is the reverse of the absorption process where a photon of radiation is absorbed and the molecule is raised from the ground state to the first excited state. It follows that the Fluorescence emission Spectrum could be expected to coincide with the absorption Spectrum at the first transition energy although the rest of the Spectrum to be of lower energy or at a longer wavelength. This in fact does not occur as depicted in figure 1.

Figure 1. Ideal Absorption and emission Spectra

In fact, this coincidence rarely occurs, as there is usually a small loss of energy due to interaction with the surrounding solute or solvent molecules.

It is clear that there will be three basic types of fluorescent spectra that can be taken:

(a) The Fluorescence intensity is measured while programming the excitation wavelength. This is called an excitation Spectrum.
The Fluorescence intensity is measured while programming the excitation wavelength but maintaining the excitation light at a constant intensity. This is called a corrected excitation Spectrum.
(c)The Fluorescence measured is taken over a range of wavelengths while employing a fixed excitation wavelength which is called an emission Spectrum.

Examples of a pair of spectra obtained by recording the Fluorescence intensity taken while programming the excitation wavelength and by recording the Fluorescence intensity over a range of wavelengths while employing a fixed excitation wavelength are given in figure 2.


Courtesy of the Perkin Elmer Corporation

Figure 2. Two Fluorescence Spectra: the First Monitored at 405 nm and the second Excited at 292 nm

The Spectrum on the left was obtained by monitoring the Fluorescence at 405 nm and programming the excitation light from 230 nm to 410 nm, and thus, provides an excitation Spectrum. The Spectrum on the right is obtained by fixing the excitation light at 292 nm and monitoring the fluorescent light from 360 nm to 480 nm, and thus, provides an emission Spectrum. Fluorescence spectra can be used for identifying substances by comparing them with reference spectra. However, they have rather limited use for the structure elucidation of a completely unknown substance.

It is seen that there is a considerable amount of fine structure present in both spectra, which means that if reference spectra are available compound identification could be confirmed with a fair degree of certainty.



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.

gamma rays