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A large molecule will exhibit a range of adsorption lines in it’s UV Spectrum arising from both atomic and molecular orbitals. These lines will result from the different energy absorbing sources in the atoms, and combination of atoms, that will be unique to the structure of the compound. As already reported, (2πr) must be an integral number of wavelengths, i.e.,(n) Consequently, the absorption bands are frequently so close that they often cannot be observed individually and merely give a broad band in the UV adsorption Spectrum as shown for ethyl ethanoate in figure 4A. However, aromatic substances and unsaturated substances are example of substances that do give spectra with some fine structure and thus, can be used by the analyst for structure elucidation or compound identification (see figure 4B). All the same, most substances will absorb light in the UV range and, thus, although their spectra are mostly inadequate for analytical purposes, UV absorption can be used very effectively for detection in liquid chromatography.
Before discussing spectroscopic equipment, the use of UV absorption for the quantitative determination of substances in solution must be considered. Consequentially, the relationship between solute concentration and UV absorption has to be examined. The relationship between the energy of the light transmitted thorough a cell containing a solution of a UV absorbing substance was examined by Beer.
Beers law for Light Absorption
The relationship between the intensity of light transmitted through a cell (IT) and the concentration of solute in the cell, (c), is given by Beer's Law.
is the intensity of the light entering the cell,
is the path length of the cell,
If equation (3) is put in the form,
is termed the molar extinction coefficient.
Differentiating equation (4), (5)
It is seen that the sensitivity of the detector, as measured by the intensity of the transmitted light will be directly proportional to the value of the extinction coefficient (K) and the path length of the cell (l). Most modern UV detector sensors have path lengths that range between 5 and 20 mm and internal radii that range from about 1 to 10 mm. The UV spectrometer is a very sensitive instrument and, under suitable circumstances, spectra can be obtained from concentrations as low as 10-8 g/ml of sample. Measurements being made from a cell 8 mm long and 1 mm in diameter this would correspond to a mass of 0.63 ng
From equation (5), (6)
where (A) is termed the absorbance.
There are a number of situations where there can be serious deviations from Beers law. Such deviations usually arise from interactions between the solute itself or the solute and solvent in which it is dissolved. An example would be the light absorption by alcohol in an inert solvent such as isooctane . At low concentrations the alcohol molecules would exist as single molecules. As the concentration of alcohol was increased the molecules of alcohol would begin to associate with one another and the concentration of the monomer would be smaller than it should be. In some cases the solutes can interact with the solvent with the same effect. However, if solutions are kept sufficiently dilute and the solvent is carefully chosen not to interact with the solute, the deviation from Beers law can be maintained minimal.
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.