Post reaction - separation

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. 

As noted in the previous chapters, when an EC detector is used in a flowing system, such as HPLC, it is simply one type of post-separation reaction detector. As with all post-colunm reaction detectors some knowledge of the chemistry involved in the detection process is essential in order to be able to use such detectors successfully. For maximum sensitivity with an EC detector, it is necessary to optimise conditions, such as the pH and composition of the reaction medium, the energy input, the time allowed for the reaction and the nature of the catalytic surface. However, when ED is used in conjunction with chromatography, the EC reaction conditions usually have to be a compromise with the chromatographic conditions necessary to achieve optimum resolution of the analytes, in particular the pH and the composition of the chromatographic eluent, as well as the need to maintain column stability. 

Figure 6. Layout of post-separation reaction device with typical applied voltages indicated.

In most of today s FCC operations, the desired reactions take place in the riser. In recent years, a number of refiners have modified the FCC unit to eliminate, or severely reduce, post-riser cracking. Quick separation of catalyst from the hydrocarbon vapors at the end of the riser is extremely important in increasing the yield of the desired product. The post-riser reactions produce more gas and coke versus less gasoline and distillate. Presently, there are a number of commercially proven riser disengaging systems offered by the FCC licenser designed to minimize the post-riser cracking of the hydrocarbon vapors. 

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... 

Following the incorporation of a number of modifications, the fluorometric method was utilized in a post column reaction system (PCRS) with separation of the toxins by high pressure liquid chromatography (HPLC) (, ). While this system proved quite useful... 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

Amino acids (isoleucine, phenylalanine, arginine and alanine) have been analysed on a microchip with a post-channel reaction with amino acid oxidase reaction [144], Pre-channel derivatisation of amino acids with naphthalene-2,3-dicarboxyaldehyde (NDA) has been described for facilitating its amperometric detection [145]. Separation and direct detection of amino acids without derivatisation have also been achieved in microchips [89,109,122,132,146-148]. 

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. 

Amino acid analysis. There are some 20 amino acids found in proteins and these are released by overnight hydrolysis in 6M HCl. Plasma and urine contain an even larger number of amino acids or related compounds. At low pH, amino acids are cations and for 40 years have been separated by cation exchange column, chromatography. The problem with amino adds is that in general they possess no chromophores by which they can all be detected. In the traditional amino add analyser, their detection was accomplished by a post-column reaction with nin-hydrin which forms a purple colour on heating with an amino acid at pH 5.5. This colour, Ruhemann s purple, is formed with all primary amino acids and can be detected at 570 nm. Secondary amino acids such as proline form a yellow chromophore measurable at 440 nm. 

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... 

Fig. 8-2. Simultaneous analysis of weak and strong inorganic acids. — Separator column IonPac AS4A eluent 0.0017 mol/L NaHC03 + 0.0018 mol/L Na2C03 flow rate 1 mL/min detection (A) suppressed conductivity, (b) photometry at 410 nm after post-column reaction with sodium molybdate injection volume 50 pL solute concentrations 3 ppm fluoride, 4 ppm chloride, 10 ppm nitrite and bromide, 20 ppm nitrate, 10 ppm orthophosphate, 25 ppm sulfate, and 27 ppm orthosilicate.

Fig. 8-12. Analysis of heavy and transition metals in industrial waste water. — Separator column IonPac CS5 eluent 0.05 mol/L oxalic acid + 0.095 mol/L LiOH flow rate 1 mL/min detection photometry at 520 nm after post-column reaction with PAR injection 50 pL waste water (1 50 diluted).

An important constituent in copper pyrophosphate baths is nitrate, which enhances the maximum permissible current density [31]. Fig. 8-30 shows the respective chromatogram with the separation of nitrate and orthophosphate. The latter is the hydrolysis product of pyrophosphate that is formed during the plating process. The main component pyrophosphate may also be separated on a latexed anion exchanger. It is detected after complexation with ferric nitrate in a post-column reaction by measuring the light absorption (see Section 3.3.5.2). 

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