Post-column reaction detection

Figure 11.2 (a) LC-LC system with post-column reaction detection for the determination... 

Eskinja, M., Lamprecht, G., Scherer, G., and Schmid, E. R., Assay of S-ethyl-N-acetyl-L-cysteine in urine by high-performance liquid chromatography using post-column reaction detection, /. Chromatogr. B, 704, 159, 1997. 

Figure 11.2 (a) LC-LC system with post-column reaction detection for the determination of ampicillin in plasma (b) Chromatogram of plasma sample (collected 10 min after oral administration of 670 p,mol of ampicillin) containing 1.26 jlM ampicillin (amp). Reprinted from Journal of Chromatography, 567, K. Lanbeck-Vallen et al., Determination of ampicillin in biological fluids by coupled-column liquid chromatography and post-column derivatization, pp. 121-128, copyright 1991, with permission from Elsevier Science. 

A protein-binding assay (BA) coupled with hplc provided a highly sensitive post-column reaction detection system for the biologically important molecule biotin and its derivative biocytin, biotin ethylenediamine, 6-(biotinoylamino) caproic acid, and 6-(biotinoylamino)caproic acid hydrazide (71). This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay a conjugate of streptavidin with fluorescamine isothiocyanate (streptavidin—FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin—FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The hplc/BA system is more sensitive and selective than either the BA or hplc alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. 

Detection of weak acid anions is best by indirect conductivity detection or post-column 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. 

Cai, W.M. Hatton, J. Pettigrew, L.C. Dempsey, R.J. Chandler, M.H.H. A simplified high-performance liquid chromatographic method for direct determination of warfarin enantiomers and their protein binding in stroke patients. Ther.DrugMon.it., 1994,16, 509-512 [chiral fluorescence detection phen-procoumon (IS) LOD 8 ng/mL post-column reaction detection derivatization]... 

F. B. Bigley, Studies Using Flow Injection Analysis and Post-Column Reaction Detection Analysis with High Performance Liquid Chromatography. Diss. Abstr. Int. B, 45(8) (1985) 2528. 

HPLC analyzers based on post-column reaction detection. However PCR noise sources will not be addressed further In this report. 

Buchberger, W. (1988). Determination Of Iodide And Bromide By Ion Chromatography With Post-Column Reaction Detection. Journal of Chromatography A, Vol.439, No.l, pp 129-135, ISSN 0021-9673... 

In general, acid digestion is better suited to preparing samples which are to be analyzed for cations (e.g., transition metals and rare earth elements) using ion chromatography with post column reaction detection. 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

With the development of HPLC, a new dimension was added to the tools available for the study of natural products. HPLC is ideally suited to the analysis of non-volatile, sensitive compounds frequently found in biological systems. Unlike other available separation techniques such as TLC and electrophoresis, HPLC methods provide both qualitative and quantitative data and can be easily automated. The basis for the HPLC method for the PSP toxins was established in the late 1970 s when Buckley et al. (2) reported the post-column derivatization of the PSP toxins based on an alkaline oxidation reaction described by Bates and Rapoport (3). Based on this foundation, a series of investigations were conducted to develop a rapid, efficient HPLC method to detect the multiple toxins involved in PSP. Originally, a variety of silica-based, bonded stationary phases were utilized with a low-pressure post-column reaction system (PCRS) (4,5), Later, with improvements in toxin separation mechanisms and the utilization of a high efficiency PCRS, a... 

The application of the fluorescence derivatization technique in an HPLC method involves utilization of a post column reaction system (PCRS) as shown in Figure 3 to carry out the wet chemistry involved. The reaction is a 2-step process with oxidation of the toxins by periodate at pH 7.8 followed by acidification with nitric acid. Among the factors that influence toxin detection in the PCRS are periodate concentration, oxidation pH, oxidation temperature, reaction time, and final pH. By far, the most important of these factors is oxidation pH and, unfortunately, there is not one set of reaction conditions that is optimum for all of the PSP toxins. The reaction conditions outlined in Table I, while not optimized for any particular toxin, were developed to allow for adequate detection of all of the toxins involved. Care must be exercised in setting up an HPLC for the PSP toxins to duplicate the conditions as closely as possible to those specified in order to achieve consistent adequate detection limits. 

The on-line measurement of reducing capacity can be performed with either a single or a series of electrochemical detectors, and linear correlations have been demonstrated between total antioxidative activities determined by the electrochemical detection and those determined by DPPH- reduction or by the ORAC assay (Guo et al, 1997 Peyrat-Maillard et al, 2000). The reducing capacity must also be quantified by post-column reactions, either with DPPH- or by the reduction of phosphomolybdenum complexes followed by UV-VIS-detection (Bandoniene and Murkovic, 2002 Cardenosa et al, 2002). A combination of HPLC and semi-automatic ORAC analysis has also been described (Caldwell, 2001). 

FIGURE 14.8 Overlay of Mn(II), Co(II), Cd(II), and Zn(II) chromatograms obtained on a lysine modified monolith. Eluent 3mM KCl, pH 4.5 Flow rate 2mL/min. Detection post-column reaction with PAR, absorbance at 495 nm. (From Sugrue, E. et al., J. Chromatogr. A, 1075, 167, 2005. Copyright 2005. With permission from Elsevier.)... 

The detection technique which perhaps has the most potential for trace analysis is post-column derivatization. This is based on the formation of reaction products immediately after column elution and prior to detection. The advantage of such a system is that the samples can be chromatographed directly without the need for prior reaction. Post-column reactions can be very selective, permitting only certain solutes to form derivatives for analysis. These derivatives usually absorb strongly in the UV-visible region or they fluoresce. 

The cerium(IV) oxidation reaction of many organic acids provides a sensitive and selective method for HPLC analysis of these compounds [116,117]. The oxidation of specific classes of organic compounds with cerium(lV), and the effects on the reaction of temperature, acidity, anion and catalyst, have been studied extensively [118-120]. The reaction produces cerium(HI) which is fluorescent and can be measured spectrofluori-metrically. The method has been applied successfully to the post-column reaction and detection of nmole amounts of organic acids by HPLC. 

Flaten AK, Lund W. 1997. Speciation of aluminum in tea infusions studied by size exclusion chromatography with detection by post-column reaction. Sci Total Environ 207 21-28. 

Ratanathanawongs and Crouch [19] have described an on-line post-column reaction based on air-segmented continuous flow for the determination of phenol in natural waters by high performance liquid chromatography. The reaction used was the coupling of diazotised sulphanilic acid with the phenol to form high coloured azo dyes. The detection limit for phenol was 17pg L 1 which represents a 16-fold improvement over determination of phenol with ultraviolet detection. 

Miles and Moye [171] have shown that several classes of nitrogen containing pesticides responded to a high performance liquid chromatography post-column reaction detector that employed ultraviolet photolysis with optional reaction with o-phthalicdicarboxaldehyde-2-mercaptoethanol followed by fluorescence detection. It was applied to the determination of jV-methylcarbamates, carbamoyl oximes, carbamethoic acids, dithiocarbamates and phenyl ureas, phenyl amides and phenyl carbamates in groundwater. See also Table 4.3. 

While ionic strengths as low as 1 mM have been used with the cell illustrated in Figure 1, most LCEC experiments are carried out with a minimum of 0.05 M buffer salts in the mobile phase. Postcolumn mobile phase changes (pH, ionic strength, solvent content) and post-column reactions (redox cross reactions, derivatiza-tions, enzyme catalyzed reactions) can expand the utility of electrochemical as well as other detectors. These subjects have recently been treated in some detail (9). Suffice it to say that direct detection, without post-column chemistry, is always preferable (more reliable, more sensitive, less expensive). 

Wehr, C. T. (1991). Post column reaction systems for fluorescence detection of polypeptides. In High-Performance Liquid Chromatography of Peptides and Proteins Separation, Analysis, and Conformation (C. T. Mant and R. S. Hodges, eds.), pp. 579-586. CRC Press, Boca Raton, FL. 

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