Post-column derivatization systems

Derivatlzatlon reactions are carried out In various types of detectors. Open reactors (straight, colled, knotted) are used with fast reactions, while packed reactors or air-segmented systems are to be preferred for slower reactions. The use of reactors packed with an active material (enzymatic or non-enzymatlc reactor) taking part In the derlvatizatlon is becoming more and more frequent. Post-column liquid-liquid extraction is basically used to remove the excess of derivatlzatlon reagent which might Interfere with the detection of the reaction product. 

The Insertion of a derivatlzatlon module between the column and the detection system in HPLC can be approached In three ways  

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A fraction collector and a post-column derivatization system were included (Figure 2.1) for a comprehensive and multi-purpose instrument. However, the fraction collector is needed only when collecting components from the effluent, and is generally not included in an analytical system. The post-column derivatization system is connected only when required for the selective and sensitive detection of specially targeted compounds. Usually, most compounds are directly detected by an on-line spectroscopic or other detector. 

Fig.4.35. Diagram of the post-column derivatization system for analysis of cyclohexanone.

Figure 13.14 Block diagram of HPLC post-column derivatization system. (Katz et al., used with permission.)...

In post-column derivatization systems, the derivatlzation reagent is continuously mixed with the column effluent consequently, the presence of trace amounts of amino acids or interfering components such as ammonia in the column eluent limits the ultimate sensitivity attainable. Figure 2 shows the effects of buffer contaminants on the baseline of a typical high sensitivity analysis (12). The basic contaminants are held up on the column until elution by the final buffer. Detection of the basic amino acids below lO-picomole levels is difficult using this approach. The use of a shorter second column with isocratic elution for the analysis of just the basic amino acids is a good solution to the problem but does require the use of a second aliquot of sample. 

Chemical derivatization can be carried out before the separation (pre-column derivatization) or after the separation and before detection (post-column derivatization). If derivatization is carried out prior to separation, then a phase system must now be selected to separate the... 

In post-column derivatization the chromatographic system is modified to allow the reagent to mix with the column eluent, give the reaction mixture sufficient time to complete and finally pass the reaction mixture to the detector. 

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. 

Fluorescence detection was selected to increase sensitivity and selectivity. Histamine has no natural fluorescence and a post-column derivatization with OPT was found to be facile. The OPT reaction with histamine or any primary amine will only occur in an alkaline medium. The derivatization reagent, pumped into the system after the mixture has been separated on the column, must be strongly basic to neutralize the acid in the mobile phase. The structure of the OPT adduct has been found to be dependent upon the pH at which the reaction is carried out as wel1 as the sol vent system (15). 

Unlike post-column derivatization, no restrictions are imposed by the solvent system used as mobile phase. The reaction conditions can therefore be chosen freely. 

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. 

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. 

Post-column derivatization does not merely require the selection of the most appropriate reagent to react with the solute to render it detectable, but also involves the modification of the chromatographic system to allow the reaction to take place prior to entering the detector. This necessitates the insertion of a post column reactor between the column and the detector. Such a reactor can easily interfere with the resolution obtained from the column and consequently the reactor system must be designed with some care to... 

The fluorescence detector is a specific and concentration-sensitive detector. It is based on the emission of photons by electronically excited molecules. Fluorescence is especially observed for analytes with large conjugated ring systems, e.g., polynuclear aromatic hydrocarbons and their derivatives. In order to extend its applicability range, pre-column or post-column derivatization strategies have been developed [9]. 

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