Fluorescence detectors postcolumn reaction detector

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. 

In the chemiluminescence-based HPLC detection system, illustrated schematically in Figure 6, the oxalate ester and hydrogen peroxide are introduced to the eluent stream at postcolumn mixer Mj, which then flows through a conventional fluorescence detector with the exciting lamp turned off or a specially built chemiluminescence detector. The two reagents are combined at mixer Mj, rather than being premixed, to prevent the slow hydrolytic reactions of the oxalate ester. 

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. 

Fig. 2 Postcolumn derivatization scheme for aflatoxin analysis 1, mobile phase 2, HPLC pump 3, injection valve 4, precolumn 5. analytical column 6, derivatizing agent solution 7, auxiliary HPLC pump 8, T-valve 9, oil or water bath 10, reaction coil 11, fluorescence detector 12, waste 13, chromatographic data handling system.

Carbamate pesticides are best analyzed by HPLC using postcolumn deriva-tization technique. Some common carbamate pesticides are listed in Table 2.19.1. Compounds are separated on a C-18 analytical column and then hydrolyzed with 0.05 N sodium hydroxide. Hydrolysis converts the carbamates to their methyl amines which are then reacted with o-phthalaldehyde and 2-mercaptoethanol to form highly fluorescent derivatives. The derivatives are detected by a fluorescence detector. o-Phthaladehyde reaction solution is prepared by mixing a 10-mL aliquot of 1% o-phalaldehyde solution in methanol to 10 mL of acetonitrile containing 100 pL of 2-mercaptoethanol and then diluting to 1 L with 0.05 N sodium borate solution. 

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. 

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-... 

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. 

Liquid phase chemiluminescence detectors usually consist of a postcolumn reactor (section 5.8) connected to a fluorescence detector with its source disabled [104,137,138,143-145]. The column eluent is combined with one or several reagents that initiates the desired chemiluminescence reaction. The intensity of light emission depends on the rate of the chemical reaction, the efficiency of production of the excited state, and the efficiency of light emission from the excited state. The chemiluminescence intensity is sensitive to environmental factors such as temperature, pH, ionic strength, and solution composition. In addition, the detection system has to be designed to accommodate the time dependence of the chemiluminescence signal to ensure that adequate and representative emission occurs in the detector flow cell. 

High performance liquid chromatography methods are widely used to quantify the PSP toxins present in a shellhsh sample, but they can also serve to identify the toxin profile of a PSP bloom. PSP toxins have only a weak natural chromophore and must be modified before detection [16]. When oxidized in alkaline solution, they form a purine, which becomes fluorescent in acidic solution [6]. This derivatization reaction can be carried out precolumn or postcolumn, and the purines are monitorized in a fluorescence detector. 

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. 

Derivatives are used in LC primarily to improve the response of an analyte to a specific detector or, less frequently, to increase the stability of the analyte in a particular separation system or the chromatographic separation of a mixture yielding overlapping peaks [133,137]. Most derivatization procedures introduce chromophores or fluorescent groups into functionalized molecules of pesticides. The reagent most widely used to obtain fluorescent derivatives is o-phthaldehyde (OPA), which is employed in a postcolumn reaction with the pesticides [68,74]. 

Koshiishi and Imanari (42) used the postcolunm ascorbic acid oxidation with cupric acetate to DHA and o-phenylenediamine for derivatization. Ascorbic acid and DHA were assessed in a single run (Fig. 6). Their system consisted of a double-plunger pump for pumping o-phenylenediamine and cupric acetate for postcolumn reactions. The fluorescence detector was set at exitation wavelength of 345 nm and emission wavelength of 410 nm. They introduced the poly(ethyl-ene glycol) copolymer as separation material in the analytical column (see Sect. III.C). 

Figure 6 Chromatograms of ascorbate and dehydroascorbate in standard (A), human plasma (B), and human urine (C). Conditions GS-320H poly(ethylene glycol) copolymer column 0.1 M acetic acid containing 0.5 mM EDTA (1.0 mL/min) as mobile phase. The postcolumn reaction conditions were reagent 1, 0.1 M acetate buffer (pH 4.0) containing 20 mM o-phenylenediamine reagent 2, 0.1 M acetate buffer (pH 4.0) containing 5 mM cupric acetate reaction temperature 55°C reaction time 1 min sample volume 5 rL fluorescence detector (excitation 345 nm, emission 410 nm). (From Ref. 42.)...

Vitamin Bi (thiamin) is related to beriberi, a disease associated with a deficiency of this vitamin. In fact, thiamin is a coenzyme in different biochemical reactions. Pork, legumes, as well as liver and kidney products are regarded as excellent sources of this vitamin. Thiamin, as well as other water-soluble vitamins, is frequently found bound to proteins or carbohydrates or even phosphorylated. Therefore, prior to their analysis, a sample treatment to release the free forms of the vitamin is common. A t)q)ical extraction protocol for water-soluble vitamins includes autoclaving the sample with hydrochloric acid for the acid hydrolysis of the vitamin followed by an adjustment in the pH to values around 4.0—4.5, adequate for an enzymatic treatment. This vitamin can be, subsequently, separated by ion-pair RP chromatography and detected with a fluorescence detector after postcolumn oxidation to thiochrome. MS detection through electrospray ionization is also used, although the separation pH should be adjusted to maximize the ionization of the vitamin. 

OPA precolumn derivatization is probably the most commonly used precolumn method. OPA precolumn derivatized amino acids are detected with fluorescent detectors using the same wavelengths used for postcolumn OPA. The derivatized sample can also be loaded directly on the reversed-phase column, allowing automation of the derivatization reaction with an antosampler. The sample, however, does need to be evaporated to dryness after hydrolysis for removal of the acid. The removal of the acid needs to be complete, usually requiring redissolving and redrying or strongly buffered to an alkaline pH. 

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