The fluorescence detector

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

This detector is of great utility in biochemical analysis. It can be used for compounds that have native fluorescence, e.g., indoles, catecholamines, porphyrins, or else derivatization can be used to produce or enhance fluorescence. This latter aspect will be discussed later in this chapter. 

The fluorescence detector is available as either a filter fluorimeter or as a continuous wavelength fluorimeter. The filter fluorimeters are less expensive, but in most cases low wavelength excitation is not possible with these instruments, and this makes, e.g., the determination of indoles and catecholamines by their native fluorescence impossible. The selectivity of the fluorescence detector is much better than that of the ultraviolet detector, and for favorable compounds the sensitivity may also be better. 

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The fluorescence detector, perhaps the most sensitive of the commonly used detectors in lc, is limited in its utiHty to the detection of materials that fluoresce or have derivatives that fluoresce. These detectors find particular use in analysis of environmental and food samples, where measurements of trace quantities are required. 

The pressure sensitivity of a detector will be one of the factors that determines the long term noise and thus can be very important. It is usually measured as the change in detector output for unit change in sensor-cell pressure. Pressure sensitivity and flow sensitivity are to some extent interdependent, subject to the manner in which the detector functions. The UV detector, the fluorescence detector and the electrical... 

The popularity of the UV detector, the electrical conductivity detector and the fluorescence detector motivated Schmidt and Scott (5,6) to develop a trifunctional detector that detected solutes by all three methods simultaneously in a single low volume cell. 

Chromatograms demonstrating the simultaneous use of all three detector functions are shown in figure 22. It is seen that the anthracene is clearly picked out from the mixture of aromatics by the fluorescence detector and the chloride ion, not shown at all by the UV adsorption or fluorescence detectors, clearly shown by the electrical conductivity detector. 

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

Detectors are composed of a sensor and associated electronics. Design and performance of any detector depends heavily on the column and chromatographic system with which it is associated. Because of the complexity of many mixtures analysed and the limitation in regard to resolution, despite the use of high-resolution capillary columns and multicolumn systems, specific detectors are frequently necessary to gain selectivity and simplify the separation system. Many detectors have been developed with sensitivities toward specific elements or certain functional groups in molecules. Those detectors that exhibit the highest sensitivity are often very specific in response, e.g. the electron capture detector in GC or the fluorescence detector in LC. Because... 

Data Presentation The raw electrophoresis data consist of a long file of the intensity trace recorded by the fluorescence detector (Fig. 15.4). The trace consists of a pseudoperiodic set of peaks, where successive peaks are generated by a component, which is transferred in several fractions from the first to the second... 

As with the UV absorption detector, the sample compartment consists of a special cell for measuring a flowing, rather than static, solution. The fluorescence detector thus individually measures the fluorescence intensities of the mixture components as they elute from the column (see Figure 13.10). The electronic signal generated at the phototube is recorded on the chromatogram. 

The advantages and disadvantages of the fluorometry technique in general hold true here. The fluorescence detector is not universal (it will give a peak only for fluorescing species), but it is thus very selective (almost no possibility for interference) and very sensitive. 

Quantitative analysis of APEO with the fluorescence detector in comparison with LC-MS... 

A high-performance liquid chromatography system can be used to measure concentrations of target semi- and nonvolatile petroleum constituents. The system only requires that the sample be dissolved in a solvent compatible with those used in the separation. The detector most often used in petroleum environmental analysis is the fluorescence detector. These detectors are particularly sensitive to aromatic molecules, especially PAHs. An ultraviolet detector may be used to measure compounds that do not fluoresce. 

The hydride generator is controlled by a DIO card in the computer and this triggers the measurements from the fluorescence detector, which are recorded by a BCD interface. 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

Elements such as As, Se and Te can be determined by AFS with hydride sample introduction into a flame or heated cell followed by atomization of the hydride. Mercury has been determined by cold-vapour AFS. A non-dispersive system for the determination of Hg in liquid and gas samples using AFS has been developed commercially (Fig. 6.4). Mercury ions in an aqueous solution are reduced to mercury using tin(II) chloride solution. The mercury vapour is continuously swept out of the solution by a carrier gas and fed to the fluorescence detector, where the fluorescence radiation is measured at 253.7 nm after excitation of the mercury vapour with a high-intensity mercury lamp (detection limit 0.9 ng I l). Gaseous mercury in gas samples (e.g. air) can be measured directly or after preconcentration on an absorber consisting of, for example, gold-coated sand. By heating the absorber, mercury is desorbed and transferred to the fluorescence detector. 

A third type of detector that has only limited use is the fluorescence detector. This type of detector is extremely sensitive its use is limited to samples containing trace quantities of biological materials. Its response is not linear over a wide range of concentrations, but it may be up to 100 times more sensitive than the UV detector. 

Figure B3.6.5 The inner filter effect. A cuvette (10 x 10-mm) is represented in plan view, with the collimated incident beam from the monochromator having intensity /0. As a result of absorption by the protein solution, the intensity of the beam through the cuvette will decrease steadily, emerging with intensity /. The values are illustrated for a solution having an absorbance at the excitation wavelength of 0.1. The optics of the fluorescence detector are focused so that only fluorescence originating from the volume depicted by the heavily shaded square is seen by the photomultiplier. Thus the observed normalized fluorescence intensity will be less than that expected from the protein at infinite dilution. The fluorescence passes through the protein solution on its way to the detector and will be further decreased in intensity if the solution absorbs at the wavelengths of the emitted radiation.

Detectors. Fluorescence and UV detectors are used in the HPLC analysis. The high sensitivity and specificity of fluorescence detection in tocopherols and tocotrienols make the fluorescence detector the first choice. The fluorescence detector is ten times more sensitive and has less background noise than the UV detector. Electrochemical detectors are also used in the analysis of tocopherols and tocotrienols (Murphy and Kehrer, 1987 Sanchez-Perez et al., 2000). As a high-polarity mobile phase is needed for the electrolytes when using an elec-... 

Thompson and Hatina (135) showed that the sensitivity of a fluorescence detector toward unesterified vitamin E compounds under normal-phase conditions was at least 10 times greater than that of a variable-wavelength absorbance detector. The relative fluorescence responses of the tocopherols at 290 nm (excitation) and 330 nm (emission), as measured by HPLC peak area, were a-T, 100 /3-T, 129 y-T, 110 and 5-T, 122. The fluorescence responses of the corresponding to-cotrienols were very similar to those of the tocopherols, and therefore tocotrienol standards were not needed for calibration purposes. The fluorescence detector also allows the simultaneous monitoring of ubiquinone derivatives for example ubiquinone-10 has been detected in tomato (136). 

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