Excitation-emission fluorescence spectroscopy

Any analytical data obtained by hyphenated instruments or by two-way spectroscopic techniques such as excitation-emission fluorescence spectroscopy are bilinear ones. The bilinear data matrix has a very useful property, namely the rank of such matrix obtained with any chemical mixture is equal to the number of chemical components in the mixture. Thus, theoretically, the rank of a data matrix of any pure chemical component is unit. It can be expressed by the product of two vectors ... 

Raman scatter, and excitation emission fluorescence spectroscopy (EEFS). They use interaction with radiation from different regions of the electromagnetic spectrum to identify the chemical nature of molecules. For example, absorption of UV and VIS radiation causes valence electron transitions in molecules which can be used to measure species down to parts per million concentrations for fluorophores (i.e., EEFS) determination can even go down to parts per billion levels. Whereas UV, VIS, and EEFS are limited to a smaller, select group of molecules, the NIR, IR, and Raman scatter spectroscopy techniques are probing molecular vibrations present in almost any species their quantification limits are somewhat higher but can still be impressive. The reader is referred to textbooks for further details on basic principles of these spectroscopic techniques [3]. 

As we will see further in this chapter, knowing the structure of the data plays a fundamental role when applying any multiway technique. For illustrating this, we will comment on the data collected from the two most popular instrumentations used in food sciences nowadays that are able to produce multiway data Excitation-emission fluorescence spectroscopy (EEM) and hyphenated chromatographic systems (i.e. gas chromatography connected to mass spectrometry—GC-MS). The benefit and drawbacks of both techniques in the framework of food analysis will be discussed in successive chapters. Here we will just focus on the stmcture of the three-way array. Figure 1 shows the final three-way structure that is obtained when several samples are analysed by both EEM and hyphenated chromatography. However, the inner structure of this tensor varies due to the different nature of the measurement... 

The physical basis of spectroscopy is the interaction of light with matter. The main types of interaction of electromagnetic radiation with matter are absorption, reflection, excitation-emission (fluorescence, phosphorescence, luminescence), scattering, diffraction, and photochemical reaction (absorbance and bond breaking). Radiation damage may occur. Traditionally, spectroscopy is the measurement of light intensity... 

Graphite furnace AAS Atomic fluorescence spectroscopy Inductively-coupled-plasma optical-emission spectroscopy Glow-discharge optical-emission spectroscopy Laser-excited resonance ionization spectroscopy Laser-excited atomic-fluorescence spectroscopy Laser-induced-breakdown spectroscopy Laser-induced photocoustic spectroscopy Resonance-ionization spectroscopy... 

Figure 32.1 (a) A schematic illustration of time-gated excitation-emission matrix spectroscopy, (b) A typical example of the 3D fluorescence data measured for Rhodamine 590 in ethanol... 

Sensor types ISE, QCM, SAW, single-X spectroscopy sensor arrays (QCM, SAW, ISE) multi-X spectroscopy, mass spectrometry, chromatography GC-MS excitation-emission- fluorescence... 

The comparison of detection limits Is a fundamental part of many decision-making processes for the analytical chemist. Despite numerous efforts to standardize methodology for the calculation and reporting of detection limits, there is still a wide divergence In the way they appear in the literature. This paper discusses valid and invalid methods to calculate, report, and compare detection limits using atomic spectroscopic techniques. Noises which limit detection are discussed for analytical methods such as plasma emission spectroscopy, atomic absorption spectroscopy and laser excited atomic fluorescence spectroscopy. 

Important applications of Af-PLS are in the area of multivariate calibrations for excitation/emission fluorescence spectrometry, for hyphenated analytical methods, such as HPLC/diode array detection and GC/MS, or for multidimensional separation techniques with or without coupling to spectroscopy. 

As indicated in Fig. 21.3, for both atomic absorption spectroscopy and atomic fluorescence spectroscopy a resonance line source is required, and the most important of these is the hollow cathode lamp which is shown diagrammatically in Fig. 21.8. For any given determination the hollow cathode lamp used has an emitting cathode of the same element as that being studied in the flame. The cathode is in the form of a cylinder, and the electrodes are enclosed in a borosilicate or quartz envelope which contains an inert gas (neon or argon) at a pressure of approximately 5 torr. The application of a high potential across the electrodes causes a discharge which creates ions of the noble gas. These ions are accelerated to the cathode and, on collision, excite the cathode element to emission. Multi-element lamps are available in which the cathodes are made from alloys, but in these lamps the resonance line intensities of individual elements are somewhat reduced. 

The use of emission (fluorescence and phosphorescence) as welt as absorption spectroscopy. From these spectra the presence of as well as the energy and lifetime of singlet and triplet excited states can often be calculated. 

FBAs can also be estimated quantitatively by fluorescence spectroscopy, which is much more sensitive than the ultraviolet method but tends to be prone to error and is less convenient to use. Small quantities of impurities may lead to serious distortions of both emission and excitation spectra. Indeed, a comparison of ultraviolet absorption and fluorescence excitation spectra can yield useful information on the purity of an FBA. Different samples of an analytically pure FBA will show identical absorption and excitation spectra. Nevertheless, an on-line fluorescence spectroscopic method of analysis has been developed for the quantitative estimation of FBAs and other fluorescent additives present on a textile substrate. The procedure was demonstrated by measuring the fluorescence intensity at various excitation wavelengths of moving nylon woven fabrics treated with various concentrations of an FBA and an anionic sizing agent. It is possible to detect remarkably small differences in concentrations of the absorbed materials present [67]. 

Enzyme structure may be studied by fluorescence spectroscopy [238-244]. Excitation in the 280-310 nm absorption bands of proteins, usually results in fluorescence from tryptophan (Trp) residues in the 310-390 nm region. The fluorescence from the Trp residues is a convenient marker for protein denaturation and large decreases or red-shifts in fluorescence are observed when proteins are denatured. These changes are most often due to the exposure of the Trp residues that are buried in the protein and may be due to the changes in the proximities of specific residues that may act as fluorescence quenchers. Fluorescence emission characterization of the immobilized... 

The EEM fluorescence spectroscopy involved scanning and recording of 23 individual emission spectra (220-510 nm) at sequential increments of 10 nm of excitation wavelength between 260 and 490 nm. The spectra were recorded at a scan speed of 1000 nm/min using excitation and emission slit bandwidths of 10 nm. Analyses were performed at a constant laboratory temperature of 22 3 °C, and blank water scans were run between every 10-20 analyses using a sealed distilled water cell. 

Emission-Excitation Matrix (EEM) fluorescence spectroscopy as a nondestructive and sensitive analytical technique was successfully applied in this study to characterize DOM in landfill leachte. The DOM is composed of complex mixture of organic compounds with different fluorescence properties. In particular, the EEM profiles of DOM show two well-defined peaks at Ex/Em=320-350 /400-420 nm, Ex/Em=320-350 /420-450 nm reasonably due to the presence of two different groups of fluorophores. An additional and less intense band at Ex/Em=280-290 /320-350 nm can be assigned to aromatic amino acids and phenol-like compounds. 

Wakeham [14] has discussed the application of synchronous fluorescence spectroscopy to the characterization of indigenous and petroleum derived hydrocarbons in lacustrine sediments. The author reports a comparison, using standard oils, of conventional fluorescence emission spectra and spectra produced by synchronously scanning both excitation and emission monochromators. 

A collection of corrected excitation and emission spectra can be found in Miller J. N. (Ed.) (1981) Standards for Fluorescence Spectrometry, Chapman and Hall, London. Corrected emission spectra can also be found in Appendix 1 of Lakowicz J. R. (1999) Principles of Fluorescence Spectroscopy, Kluwer Academic/ Plenum Publishers, New York. 

Yamanouchi, K., Takeuchi, S., and Tsuchiya, S. (1990), Vibrational Level Structure of Highly Excited S02 in the Electronic Ground State. II. Vibrational Assignment by Dispersed Fluorescence and Stimulated Emission Pumping Spectroscopy, J. Chem. Phys. 92, 4044. 

The film reacted with adipoyl chloride followed by coupling with 7-hydroxycoumarin was subjected to methanolysis at 1 N HC1 and 60°C. The regenerated coumarin was assayed at pH 10 by fluorescence spectroscopy at an excitation wavelength of 329 nm and an emission wavelength of 455 nm. A Hitachi MPF-4 Fluorescence Spectrophotometer was used for all fluorescence measurements. 

Luminescence is a well-established class of analytical spectroscopic techniques where a species emits light after excitation. Emission is an elecnonic nansition from an excited state as opposed to the ground state as is the case in most other spectroscopies. Photoluminescence, or light-induced fluorescence (LIE), is the most common route to induce emission where sufficient incident photons of a particular energy excite the target species via absorption. Although less common, nomadiative excitation can also occur via a chemical reaction termed chemiluminescence. Unless otherwise stated, the terms luminescence and fluorescence within this review infers excitation by light induction. 

---