Postcolumn reactors

Figure 6. Chemiluminescent HPLC postcolumn reactor system.

Absorbance detectors are also commonly used in combination with postcolumn reactors. Here, most issues of detector linearity and detection limit have to do with optimization of the performance of the reactor. In a typical application, organophosphorus compounds with weak optical absorbances have been separated, photolyzed to orthophosphate, and reacted with molybdic acid, with measurement being performed by optical absorbance.58... 

Simple etching of the capillary end served to decouple the electrophoretic current from that of amperometric detection, permitting quantitation of attomole levels of catecholamines from brain microdialyzates.24 A postcolumn reactor using bromine generated electrochemically in situ has been used in the detection of peptide thiols, such as glutathione and cysteine, separated by capillary electrophoresis.25... 

Detection of Hydrogen Peroxide Generated in a Postcolumn Reactor... 

Figure 4 Schematic diagram of the postcolumn reactor developed by Wu and Huie. One arm of the tee contains the electrophoretic capillary, which is inserted in the reaction capillary (10 cm X 200 pm id X 400 pm od) situated at the opposite arm of the tee. The tee is connected to the detection cell via an adaptator and both the electrophoretic and reaction capillaries are inserted into the detection cell through the inner core of a PTFE tubing (400 pm id X 1.5 mm od). Two reagent capillaries (15 cm X 75 pm id X 144 pm od) inserted into the central arm of the tee are used to deliver the TCPO and H202 reagents into the mixing area through the small gaps that exist between the outer surface of the electrophoretic capillary and the inner surface of the reaction capillary. (From Ref. 78, with permission.)...

Obviously, the main purpose for the introduction of CL detection coupled to CE separations is inherent to the development and improvement of sensitive and uncomplicated devices to achieve a decrease of the band broadening caused by turbulence at the column end, together with the attractive separation efficiency of CE setups. With this purpose in mind, Zhao et al. [83] designed a postcolumn reactor for CL detection in the capillary electrophoretic separation of isoluminol thiocarbamyl derivatives of amino acids, because, like other isothiocyanates, isoluminol isothiocyanate has potential applications in the protein-sequencing area. 

Jacobson, S. C., L. B. Koutny, R. Hergenroder, A. W. Moore, Jr., and J. M. Ramsey. Microchip capillary electrophoresis with an integrated postcolumn reactor. Anal. Chem. 66, 3472-3476 (1994a). 

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... 

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. 

These reactions can occur ahead of the injection port, in a precolumn reactor, within the column, or in a postcolumn reactor. 

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.]...

For chemiluminescence measurements, a postcolumn reactor with a pulse-dampening filter was added to the HPLC apparatus. A 3-cm piece of narrow-bore tubing joined the pulse dampener to a Valeo l-/tl T chemiluminescent reagent with the chromatographic eluent. A C8 ECONOSPHERE (250 X 4.6-mm ID) column was used. Modifications were made with the... 

Fluri, K., Fitzpatrick, G., Chiem, N., Harrison, D.J., Integrated capillary electrophoresis devices with an efficient postcolumn reactor in planar quartz and glass chips. Anal. Chem. 1996, 68, 4285 1290. 

Fluorescence Detector. Typical of fluorescence in general, the fluorescence detector used in LC is about 100 times more sensitive and somewhat more selective than the UV detector. It is this sensitivity that accounts for its popularity and its incorporation into postcolumn reactors. The excitation sources used in LC instruments are as varied as those used in conventional... 

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. 

Serum cholinesterase (acetylcholine acylhydrolase, EC 3.1.1.8) refers to a family of at least 15 isozymes found in blood and many tissues of animals. In this assay, acetylcholine is used as the substrate, and a postcolumn reactor containing choline oxidase produces electrochemically active H2C>2 upon oxidation of choline. 

Fig. 2 Electrokinetically driven on-chip reaction in a CE-based system. Postseparation labeling of amino acids with OPA. Sample contained 200 /jlM each of phenylalanine and valine, and 10 ijlM of hydrolyzed dansyl chloride. The postcolumn reactor chip design is shown in the inset. [Reproduced from Electrophoresis 18 1733 (1997), with permission.]...

Sample Derivatization. For HPLC analyses, many analytes are chemically derivatized before or after chromatographic separation to increase their ability to be detected. For example, in automated amino acid analyzers, eluted amino acids are reacted with ninhydrin in a postcolumn reactor (see Chapter 20). The resulting chromogenic species are then detected with a photometer. Other examples include labeling amino acids or other primary amines with dansyl or fluorescamine tags either before or after the chromatographic step. 

Haginaka, J. Wakai, J. Yasuda, H. Liquid chromatographic assay of fl-lactamase inhibitors in human serum and urine using a hoUow-fiber postcolumn reactor. Anal.Chem., 1987, 59, 324-327 [serum urine post-column reaction]... 

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. 

HPLC analysis of APG is carried out with C8 or Cl8 columns by use of a refractive index detector or a conductivity detector after the addition of 0.3 mol/1 NaOH to the eluate in a postcolumn reactor. ... 

It might appear that the FIA flow channel has the same function as a postcolumn reactor, and therefore the flow injection systems could also be optimized solely by means of the predictive models developed for chromatography, simplified for zero retention, or by means of input-re-... 

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