Postcolumn Reaction Systems

The parabolic flow profile in an open-tubular reactor restricts applications to fast reactions if extensive band broadening is to be avoided [244]. The reaction time can be extended by using reactors prepared from optimally deformed capillary tubes [245-247]. The production of secondary flow, even at low flow rates, breaks up the parabolic 

For slow reactions (reaction times 5 min) an air or liquid segmented reactor is typically used. Segmented systems are also required for reactions that require separation of the reagent from the reaction product by solvent extraction, for example when ion-pair reagents are used to form the product for detection. The reaction products are usually of different polarity to the reagent, permitting their separation by extraetion 

Indirect detection is an alternative to derivatization for the detection of analytes with a weak detector response. It is commonly used in ion exchange (particularly ion chromatography) and ion-pair chromatography with absorbance, fluorescence or amperometric detection [168,254,255]. This requires the selection of an eluent ion with favorable detection properties to regulate the separation process and provide a constant detector signal. Detector transparent analyte ions cause displacement of eluent ions from the eluted band and a decrease in the detector response compared with the steady state signal for the mobile phase. The detected ion concentration is coupled to the retention mechanism, which can result in the appearance of additional system peaks in the chromatogram (section 4.3.3.2). These applications should be 

Cabaleiro and R. Cela, Trends Anal. Chem. 18 (1999) 392. 

---

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. 

When one is deciding what column geometry is optimal for trace analysis with unlimited sample volume, two additional points should be evaluated. First, to what extent does the analysis require accurate and reproducible injections Strict performance specifications may eliminate microbore columns from consideration. The accuracy and reproducibility of injection systems that deliver 0.1-, 0.2-, and 0.5-/xL samples have not been adequately characterized. Second, if the analyte of interest requires postcolumn derivatization, construction of a postcolumn reaction system that is compatible with the exceedingly small band volumes characteristic of microbore columns may be extremely difficult, but not impossible. Apffel et al. (28) developed and evaluated both packed-bed and open tubular postcolumn reactors for use with 1-mm i.d. analytical columns. Catecholamines were postcolumn derivatized with o-phthal-aldehyde and detected spectrofluorometrically. The 5-/zm particle... 

Biotin and biotin analogs Infant formula Protein precipitation using concentrated hydrochloric acid neutralization with 6 M NaOH lipid extraction with n-hexane Precolumn Microsorb C18 (15 X 4.6 mm, 5 jam Rainin). Analytical Microsorb C18 (250 X 4.6 mm, 5 /zm Rainin). Isocratic 100 mM phosphate buffer, pH 7.0 + methanol (80 + 20, v/v). 0.4 ml/min. Postcolumn reaction system UV absorbance at 220 nm followed by streptavidin-fluorescein isothiocyanate (2.0 mg/L) knitted open tubular reaction system (10.0 m x 0.5-mm ID) at a flow rate External standardization. 184 Linear range = 0.08-1.00 fjM biotin. LoD = 0.02 /zM or 97 pg biotin at SNR = 3. Repeatability CV 3.5% for biotin in infant formula. 

Liquid chromatographic systems for environmental pesticide analysis have been extensively reviewed in a previous paper. Nowadays, LC is the technique of choice for analyzing those pesticides which, being thermolabile, are not amenable to direct GC analysis, such as phenylurea and SUHs. LC methods of analysis also have the important advantage over GC methods in that online pre- and postcolumn reaction systems are compatible with LC instrumentation. Furthermore, the LC apparatus can easily be coupled online with the enrichment step using SPE on precolumns, thereby making the analysis fully automated. 

Clarkin, C.M., Minear, R.A., Kim, S. and Elwood, J.W. (1 992) An HPLC postcolumn reaction system for phosphorus-specific detection in the complete separation of inositol phosphate congeners in aqueous samples. Environmental Science and Technology 26, 199-204. 

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... 

Another approach uses a postcolumn reaction specific for the compounds of interest. For example, amines can be converted to the V-chloramines, and the chloramines treated with iodide to form triiodide, the absorbance of which can be monitored at 355 nm (203). Another postcolumn reaction system uses formation of the ion pair with Orange II, which will occur at low pH, where the amphoteric compound behaves as a cation. The colored complex is continuously extracted, and its absorbance monitored at 484 nm. Nonionic and anionic surfactants do not interfere (9). 

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... 

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

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. 

Figure 27.18 Common configuration for postcolumn reactors with electrochemical analysis. (A) LC-chemical reaction-EC. Postcolumn addition of a chemical reagent (for example, Cu2+ or an enzyme). (B) LC-enzyme-LC. Electrochemical detection following postcolumn reaction with an immobilized enzyme or other catalyst (for example, dehydrogenase or choline esterase). (C) LC-EC-EC. Electrochemical generation of a derivatizing reagent. The response at the second electrode is proportional to analyte concentration (for example, production of Br2 for detection of thioethers). (D) LC-EC-EC. Electrochemical derivatization of an analyte. In this case a compound of a more favorable redox potential is produced and detected at the second electrode (for example, detection of reduced disulfides by the catalytic oxidation of Hg). (E) LC-hv-EC. Photochemical reaction of an analyte to produce a species that is electrochemically active (for example, detection of nitro compounds and phenylalanine). Various combinations of these five arrangements have also been used. [Reprinted with permission from Bioanalytical Systems, Inc.]...

The first automated analyzer was developed by Moore, Stein, Spackman, and Hamilton in the 1950s. Hydrolysates were separated on an ion-exchange column, followed by postcolumn reaction with ninhydrin. Although this system remains the standard method, its major drawback is low sensitivity. Several methods have since been developed offering high sensitivity and faster analyses without sacrificing reproducibility... 

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. 

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. 

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. 

---