Post-column reaction techniques

This post-column reaction technique is based on the fact that the carboxyl groups of eluting acids can be neutralized by the sodium salt of o-nitrophenol [44,45]. This yields free o-nitrophenol which may be measured by an increase in the absorption of the free... 

Kronkvist K, Lovgren U, Svenson J, Edholm LE, Johansson G. Competitive flow injection enzyme immunoassay for steroids using a post-column reaction technique. J Immunol Methods 1997 200 145-53. 

A Note on Post-Column Reaction Techniques A post-column reaction unit is an online derivatization system that supplies reagents to the column eluent into a heated chamber to convert the analytes into more chromophoric forms for higher sensitivity detection. Some common applications of post-column reaction systems are amino acid analysis using ninhydrin (with visible detection), and carbamate pesticide analysis using o-phthaldehyde (with fluorescence 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 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. 

Ion Chromatography. Ion chromatography (IC) is a mode of HPLC in which ionic analyte species are separated on cationic or anionic sites of the stationary phase. The detection techniques largely fall under three categories electrochemical, spectroscopic, or post-column reactions. In general, IC provides an orthogonal separation mechanism to traditional reversed-phase HPLC (RP-HPLC).54 63 This technique can be exploited to quantify ionic... 

A disadvantage of the pre-column reaction technique, compared to post-column reaction, is that side products formed in the derivatization procedure may give rise to interferences in the chromatogram. 

Morimoto et al. [ref. 56] demonstrated that the rapid detection of activated rat urinary kallikrein (Mw 44,000), which was obtained by the trypsin treatment of inactive kallikrein purified from rat urine, could be made by post column reaction. After the SEC separation, the enzyme was allowed to react with polyphenyl-alanylarginine-4-methylcoumaryl-7-amide. In this work, TSKgel G3000SW was used as a column. The results obtained showed that the rapid detection of activated enzyme could be carried out by this technique. 

High-performance affinity chromatography has recently been reported with trypsin-modified avidin supported on 5 pm silica. While the separations were successful and a wide range of foods were studied, elution times were 80 minutes and ADAM post-column reactions were still required (Hayakawa et al. 2009). However, such affinity columns within a solid-phase extraction (SPE) platform make realistic choices for sample preparation, whereby the biotin can be purified and concentrated prior to reversed-phase HPLC. R-Biopharm has recently developed a commercially available antibody-based immunoaffinity column to bind biotin from aqueous extracts, providing an excellent technique to clean up complex samples. 

Pre-column off-line derivatisation requires no modification to the instrument and, compared with the post-column techniques, imposes fewer limitations on the reaction conditions. Disadvantages are that the presence of excess reagent and by-products may interfere with the separation, whilst the group introduced into the molecules may change the chromatographic properties of the sample. 

Post-column on-line derivatisation is carried out in a special reactor situated between the column and detector. A feature of this technique is that the derivatisation reaction need not go to completion provided it can be made reproducible. The reaction, however, needs to be fairly rapid at moderate temperatures and there should be no detector response to any excess reagent present. Clearly an advantage of post-column derivatisation is that ideally the separation and detection processes can be optimised separately. A problem which may arise, however, is that the most suitable eluant for the chromatographic separation rarely provides an ideal reaction medium for derivatisation this is particularly true for electrochemical detectors which operate correctly only within a limited range of pH, ionic strength and aqueous solvent composition. 

Post-column on-line derivatisation is carried out in a reactor located between the column and the detector. With this technique, the derivatisation reaction does not need to go to completion, provided it can be done reproducibly, and the reaction does not produce any chromatographic interferences. The reaction needs to take place in a fairly short time at moderate temperatures, and the reagent should not be detectable under the same conditions at which the derivative is detected. The mobile phase may not be the best medium in which to carry out the reaction, and the presence of the reactor after the column will increase the extra-column dispersion. 

This approach has been used extensively for amino acid analysis using low-pressure ion-exchange chromatography and post-column ninhydrin reaction. Spraying, dipping and vapour-treatment techniques are well known as post-separation reactions in TLC, but these are considered only briefly since the majority of them are not quantitative. While the problems of pre-separation techniques are quite similar for TLC and HPLC, they differ considerably for post-separation reactions. 

The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

Post-column identification reactions constitute a special kind of ancillary GC technique [10] that will briefly be discussed in Section 3.1.2, p.34. 

Of the two detection techniques mentioned above, fluorescence is preferred in general because it suffers from fewer operational difficulties. Many compounds do not display a native fluorescence however, this can be overcome if the analyte can be converted by chemical reaction into a fluorescent compound. This process is known as derivatisation and can be accomplished by either derivatising the sample prior to injection (precolumn) or after chromatographic separation (post-column). 

Ions that can be analyzed by electrochemical detection include cyanide, sulfide, hypochlorite, ascorbate, hydrazine, arsenite, phenols, aromatic amines, bromide, iodide, and thiosulfate [53], nitrite and nitrate [54.55], cobalt and iron [46], and others. The list may be extended through the technique of post-column derivatization to include many more ions such as carboxylic acids, halide ions, alkaline earth ions, and some transition metal ions [57,58). An example of an electrochemical reaction to detect ions is shown by Eq. 4.8. 

ECL has also been used in detector cells in chromatography. These again involve the ECL of Ru(bpy)3, where detected species, such as amines, NADH, and amino acids, behave as the coreactant. In one method, post-column ECL detection, a solution of Ru(bpy)3" is steadily injected into the solution stream containing separated species coming from the HPLC column. The mixed stream flows into an electrolytic cell where the ECL reaction occurs and emission is measured (39). Detection of separated species at the picomole level is possible by this technique. Alternatively the Ru(bpy)3 can be immobilized in a film of Nafion on the working electrode (28), and the ECL signal results when the solution from the HPLC column contains a species that can act as a coreactant and produce emission by reaction with immobilized Ru(bpy)3 in the detector cell (40). Observation of ECL with flowing streams can also provide information about the hydrodynamics in the detector cell (41). 

The use of HPLC with post-column chemiluminescence (CL) detection permits more sensitive and direct analyses of hydroperoxides from oxidized complex lipids in biological samples. By this technique, the HPLC effluent is mixed with different CL cocktails at a post column tee and is monitored by a CL detector measuring light emitted by the reaction of hydroperoxides with a heme... 

---