Fluorescence postcolumn reaction

Wetai Ion Analysis. We have reported a sensitive trace-metal analysis based upon HPLC separation of p-aminophenyl EDTA chelates and fluorescence detection by postcolumn reaction with fluorescamine (23). An application of the pyridone chemistry already discussed leads to a fluorescent-labeled EDTA (VIII). 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Nunes, G.S., M.P. Marco, M. Farre, et al. 1999. Direct application of an enzyme-linked immunosorbent assay method for carbaryl determination in fruits and vegetables. Comparison with a liquid chromatography-postcolumn reaction fluorescence detection method. Anal. Chim. Acta 387 245-253. 

A variety of pre- and postcolumn sample derivatization schemes has been developed in CE, mainly for the attachment of fluorescent labels for detection purposes. Precolumn sample modification is usually less demanding than postcolumn reactions for reasons discussed in Sect. 3.2.4. 

Postcolumn Reactors. Another growing field is the use of postcolumn reactors to produce a species that can be measured by one of the standard detectors, such as UV/visible, fluorescence, or electrochemical. Probably the earliest example of the use of postcolumn reactions was in the determination of amino acids by colorimetry using ninhydrin as the reactant. See the section on derivatization in Chapter 11, as well as the paper in Analytical Chemistry,57 or the book edited by Krull58 for further details. 

The enzyme called GMj cleaves the terminal D-/3-galactose from glycolipids and glycoproteins. A deficiency of the enzyme in humans results in gangliosidosis. The assay described quantitates the fluorescent compound formed in a postcolumn reaction between galactose and arginine. 

Electrochemical detection is inherently a chemical rather than a physical technique (such as ultraviolet, infrared, fluorescence, or refractive index). It is, therefore, not surprising to hnd that many imaginative postcolumn reactions have been coupled to LC-EC. These include photochemical reactions, enzymatic reactions, halogenation reactions, and Biuret reactions. In each case, the purpose is to enhance selectivity and therefore improve limits of detection. While simplicity is sacrihced with such schemes, there are many published methods that have been quite successful. 

Requirements with respect to the label used to mark one of the immunoreagents are comparable to those in other postcolumn reaction detection systems [4]. The label should preferably allow sensitive and rapid detection and be nontoxic, stable, and commercially available. So far, mainly fluorescence labels have been employed (e.g., fluorescein), although, in principle, also liposomes, time-resolved fluorescence, and electrochemical or enzymatic labels are feasible. On the other hand, labels providing a slow response, including radioactive isotopes and glow-type chemiluminescence, are less suitable for immunodetection. 

Spectroscopic detection (using ultraviolet/visible (UV/vis) absorbance, refractive index, fluorescence, atomic absorption or atomic emission). Techniques based on postcolumn reactions. 

Problems are often found in many analytical methods due to the complex nature of the mixture and the lack of adequate detection means, thus leading to poor quantitation techniques. For the routine separation of a broad range of surfactants, high-performance liquid chromatography (HPLC) appears to be the most cost-effective [7-18]. Ultraviolet (UV) and fluorescence detectors are commonly used in HPLC analysis of surfactants because of their compatibility with separation techniques requiring gradient elution. However, these detectors have two inherent limitations (a) the detector response is dependent on molecular structure (i.e., degree of aromaticity and type of substitution) and (b) only species with a chromophore can be detected. To overcome those limitations, postcolumn reaction detectors, based on extraction of fluorescent ion pairs, were introduced for on-line detection of alkylsul-... 

Figure 7.11. HPLC analysis of carbamates according to U.S. EPA Method 531.1 using postcolumn reaction and fluorescence detection. Reprinted with permission from reference 25.

Many compounds are not sensitive to uv detection. Currently, two main methods are used to improve this sensitivity, both requiring either a pre- or a postcolumn reaction. The first method is to chemically add a highly absorptive group to the compound to be detected, and the second is to add a compound that fluoresces to the compound being detected. Once the compounds are through the column and have been separated, they then are reacted with other reagents in small-volume reaction chambers before they get to the detector. One postcolumn apparatus to derivatize compounds is the Pickering apparatus discussed later. 

Very few ions are directly detectable by uv/vis absorption spectroscopy. However, if a postcolumn reaction is performed to chelate a cation with a chromophore (color producing group), to add a fluorescing agent, or to simply react the compound with another compound, such as using ninhydrin with amino acids, then uv/vis spectroscopy can be used. An example of such a system is the separation of lanthanides shown in the cation section. 

Lee and Field reported a selective fluorescence detection method for the determination of some A -nitrosamines after a postcolumn reaction. The nitrosamines eluted from the column are first hydrolyzed to produce the nitrite anion, which is then oxidized with Ce" to give the fluorescent Ce. The detection limit for this method is at the ppb level. A more sensitive fluorimetric method has been developed based upon the reaction of nitrite with 2,3-diaminonaphthalene to form the highly fluorescent product l-(H)-naphthotriazole. About 10 nmol/1 of nitrite can be detected by this procedure. 

HPLC methods are rarely applied to the analysis of short-chain organic acids due to their poor UV-absorbance and their nonfluorescent character. Only high concentrations can be measured directly by HPLC in combination with UV, diode array, or fluorescence detection. To enhance method sensitivity, organic acids may be derivatized in pre- or postcolumn reactions. Although an abundance of derivatization methods for various compounds is available, especially for... 

Fluorescence detection is not nearly as widely used as UV detection since most carbamates possess no native fluorescence. However, the structure of these pesticides contains a V-methyl substituted urethane with variations in the ester moiety. The common methylamine functionality allows the detection of compounds via a two-stage postcolumn reaction. Carbamates in the column effluent are first hydrolyzed with NaOH at a high temperature to form methylamine, which is converted into a fluorophore compound by addition of 6>-phthaldehyde (OPA) and... 

Others (i.e., Patch clamp ) Fluorescence Absorbance Luminescence Precolumn reaction Postcolumn reaction ... 

The fluorimetric method of Bates and Rapoport [8], based on the oxidation of PSP toxins in alkaline conditions to form fluorescent derivatives, was incorporated into a detection method with the PSP toxins separated in a chromatographic column by Buckley et al. [17]. This method set the basis for the development of a high pressure liquid chromatography with postcolumn reaction system that was subsequently improved to achieve a better toxin separation and adequate sensitivity [18]. Sullivan et al. [ 19] evaluated its applicability to shellfish toxicity monitoring, by comparing the results obtained by the HPLC method and the standard Association of Official Analytical Chemists (AOAC) mouse bioassay. They found, in general, a good correlation between the two methods. However, Cl and C2 toxins could not be separated and individually quantified. Further improvements and modifications... 

In these systems, a high-energy intermediate excites a suitable fluorophore, which then emits its characteristic fluorescence spectrum consequently, they are termed indirect or sensitized chemiluminescence. The most common analytical application has been as a postcolumn reaction detector for liquid chromatography. Various fluorescent analytes (polycyclic aromatic hydrocarbons and polycyclic aromatic amines) and compounds derivatized using dansyl chloride, fluorescamine, or o-phthalaldehyde have been determined with sub-femtomole detection limits. 

Derivatization can be employed to advantage with BCD detection as weU. Both precolumn or postcolumn reactions may be anployed to produce electroactive compounds analogons to the formation of fluorescent derivatives. The UV-induced reaction chamber for ECDs described in Section 15.4 and the dual electrode detector oxidation-reduction series described at the end of Section 13.1.4.5 may be regarded as nonreagent instrumental forms of the principle of postcolumn derivatization. 

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