Postcolumn reaction detection

Holland, L.A. and Lunte, S.M., Postcolumn reaction detection with dualelectrode capillary electrophoresis-electrochemistry and electrogenerated bromine, Anal. Chem. 71, 407, 1999. 

Many IC techniques are now available using single column or dual-column systems with various detection modes. Detection methods in IC are subdivided as follows [838] (i) electrochemical (conductometry, amper-ometry or potentiometry) (ii) spectroscopic (tJV/VIS, RI, AAS, AES, ICP) (iii) mass spectrometric and (iv) postcolumn reaction detection (AFS, CL). The mainstay of routine IC is still the nonspecific conductometric detector. A significant disadvantage of suppressed conductivity detection is the fact that weak to very weak acid anions (e.g. silicate, cyanide) yield poor sensitivity. IC combined with potentiometric detection techniques using ISEs allows quantification of selected analytes even in complex matrices. The main drawback... 

Precolumn derivatization is often inadequate for dirty samples. In these cases, application of a postcolumn reaction detection system will often suffice. Deelder et al. (44) and van der Wal (45) have examined different configurations for postcolumn reactors and defined optimal selections on the basis of reaction time and type and effect on resolution and sensitivity. Both studies preferred the packed-bed reactor to the open tubular reactors when conventional column geometries were employed for separation, that is, 4.6 mm i.d. X 15 or 25 cm. 

Nondek et al. (46) reported an innovative approach to the analysis of N-methylcarbamates in river water using postcolumn reaction detection. Separation of the underivatized N-methylcarbamates was carried out on a reversed-phase column hooked directly to a bed reactor packed with Aminex A-28, a tetraalkylammonium anion-exchange resin. The packed bed catalytically base-hydrolyzed the carbamates and... 

Monitoring a liquid chromatographic effluent by means of an immunoassay provides sensitive and se-leetive deteetion in combination with the separation of eross-reaetive compounds [1,2]. When implementing the immunoassay as a postcolumn reaction detection system after liquid chromatography, it is frequently referred to as immunodetection [3,4]. Automation and assay speed are the main advantages of immunodetection over off-line eoupling of immunoassays to liquid ehromatography by means of fraction collection [5,6]. 

The approach of implementing a biological assay as a postcolumn reaction detection system after liquid chromatography can not only be applied to antibody-based assays (immunoassays) but also to assays employing other affinity interactions with high association and low dissociation rate constants, such as receptors. Information obtained from such a detection system not only provides quantitative results but also indicates the biological activity of the detected compound. 

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. 

Postcolumn reaction detection was reported used by 9% of the respondents to the detector survey (47). The most popular LC detectors are solute property detectors. From a cursory glance at the popular detection techniques already discussed, it is apparent there are many classes of important compounds for which there is no sensitive solute-property detector. For this reason, many types of postcolumn chemistries have been devised to derivatize separated solutes to form a detectable species. Postcolumn reaction detection has been thoroughly reviewed (68,69). 

Singer et al. developed a specific method in which a postcolumn reaction detection system is used for HPLC. This system is useful for those compounds which can be hydrolyzed in a dilute acidic solution to give the nitrite ion. This method involves the use of the Griess reagent in the postcolumn reactor for production of chromophores from A-nitrosamines. The theoretical detection limit for this method was reported to be 0.5 nmol. However, owing to the slow reaction kinetics of some nitroso compounds, this technique requires both an air segmentation system and a high-temperature reactor. 

A matrix elimination IC with postcolumn reaction detection was developed for the determination of iodide in seawater (Brandao et al, 1995). A Dionex lonPac ASH anion-exchange column was used, and the mobile phase contained sodium chloride to remove interference of the... 

Proposed sample preparation scheme Knowledge of the physicochemical properties of the analyte can determine many of the techniques that can be used to develop the method, e.g., extraction using functional groups and interactions on the molecule and applicable detection systems. The processes involved in an automated assay should be as simple as possible. Complex automated assays requiring sophisticated online sample preparation and postcolumn reaction detection will take a long time to develop into routine and robust methods. 

Kappes, T. Hauser, P.C. Electrochemical detection methods in capillary electrophoresis and applications to inorganic species. J. Chromatogr. A, 1999, 834 (1-2), 89-101. Holland, L.A. Lunte, S.M. Postcolumn reaction detection with dual-electrode capillary electrophoresis-electrochemistry and electrogenerated bromine. Anal. Chem. 1999, 71 (2), 407. Kappes, T. Hauser, P.C. Potentiometric detection in capillary electrophoresis with a metallic copper electrode. Anal. Chim. Acta 1997, 354, 129-134. 

Detection of weak-acid anions is best by indirect conductivity detection or postcolumn reaction detection because the suppressed conductivity detection will not perform. Indirect conductivity detection is often used because the high pH used to separate the anions will also facilitate indirect conductivity detection of these anions. Chapter 4 describes a method of combining suppressed conductivity detection and nonsuppressed detection. 

Holland, L.A. Lunte, S.M. Postcolumn Reaction Detection with Dual Electrode Capillary Electrophoresis-Electrochemistry and Electrogenerated Bromine. Anal. Chem. 1999 71, 407-112. 

NG Hentz, LG Bachas. Fluorophore-linked assays for high-performance liquid chromatography postcolumn reaction detection of biotin and biocytin. Methods Enzymol 279 275-286, 1997. 

Przyjazny, A., Hentz, N.G., and Bachas, L.G., Sensitive and selective liquid chromatographic postcolumn reaction detection system for biotin and biocytin using a homogeneus fluorophore linked assay, J. Chromatogr, 654, 79-86, 1993. 

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