Post Column Derivatization

Post-separation labeling was also achieved for separation of four human serum proteins (IgG, transferrin, al-antitrypsin, and albumin) using 0.2 mM of TNS. This is a virtually non-fluorescent reagent which, upon non-covalent association with proteins, produces a fluorescent complex (A= 325 nm, A = 450 

FIGURE 6.34 Schematic diagram of the post-separation labeling design of the chip. Thick lines were 256 pm wide, thin lines were 66 pm wide. Channels were 14 pm deep [657]. Reprinted 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 reaction to take place. This necessitates the insertion of a post column reactor between the column and the detector. 

Provide a source of reagent and a means of mixing it efficiently with the column eluent. 

Ensure reaction is complete before the derivatization product enters the detector. 

Minimize the dispersion that taikes place in the reactor so that the integrity of the separation that takes place in the column is maintained. 

It consists basically of three parts, a -reagent reservoir and pump, mixing T and a reaction chamber. 

Sometimes called secondary electrochemical detection, this technique is usefiil when the sample species of interest is not electroactive. A reagent is mixed with the column effluent to transform the sample, M, into an electroactive species. An example is shown by Eq. (4.8)  

The DTPA (diethylenetriaminepentoacetate) metal complex is added and the sample metal displaces the mercury ion. The measured current is from the reduction of mercury. There are many other schemes that can be appHed to a variety of ions [65-67]. 

00375 % arsenazo III solution was in 2 M ammonium hydroxide and 1 M ammonium acetate, and the arsenazo 1 solution was in 3 M ammonium hydroxide. Other work [44,45] was done with more dilute PAR solutions (4 x 10 M PAR with 3 M ammonium hydroxide and 1 M acetic acid). For lower detection limits (because of lower background signal), the PAR concentration can be reduced even more. But care should be taken not to overload the reagent with too high concentrations of sample ion. 

Imanari et al. has reported a spectrophotometric detection of many inorganic anions using a post-column reactor [42]. A stream of ferric perchlorate, which is essentially colorless, is mixed with the column effluent. The ferric perchlorate is colorless because perchlorate is a poor complexing anion, but most anions will complex the iron and form colored species that can be detected at 330-340 nm (Table 4.3). A similar detection method works for ions such as orthophosphate, pyrophosphate, nitrilo-triacetic acid (NTA), and ethylenediaminetetraacetic acid (EDTA) [46]. 

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EC, electrochemical detection Flu, fluorescence detection MS, mass specu-omeu-ic detection pre-Flu, fluorescence detection after pre-column derivatization post-Flu, fluorescence detection after post-column derivatization UV, UV absorbance detection. 

Derivatization techniques are divided into pre-column and post-column techniques. Post-column derivatization is especially useful to enhance the detection of compounds, whilst pre-column derivatization is the method of choice for enan-tioseparations via derivatization. 

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... 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

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. 

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... 

Fraser CA, Gardner GJ, Maxwell PS, Kubwabo C, Guevremont R, Siu KWM, and Berman SS (1995) Preparation and certification of a biological reference material (CARP-i) for polychlorinated dibenzo-p-dioxin and dibenzofuran congeners. Fresenius J Anal Chem 352 143-147. Gahr A, Huber N, and Niessner R (1998) Fluorimetric determination of bromate by ion-exchange separation and post-column derivatization. Mikrochrm Acta 129 281-290. 

At this point, the anaiyte may not be amenabie to UV, FL, or EC detection. In this case, the best course of action may be to choose LC/MS (see Section 4.2). However, one other option is to use a pre- " or post-coiumn derivatization step to increase the detectabiiity of the anaiyte with respect to FL or UV. Fluorescent or UV labels are available for carboxylic acids," amines, phenols, and thiols. The decision to use pre- or post-column derivatization is predicated upon the functionality of the analyte available for derivatization and the rate and extent of the reaction between each derivatizing agent and the analyte. 

A method to determine nabam by HPLC after acidic hydrolysis to ethylenediamine and post-column derivatization with o-phthalaldehyde-mercaptoethanol has also been reported. " ... 

Reversed-phase HPLC followed by post-column derivatization and subsequent fluorescence detection is the most common technique for quantitative determination of oxime carbamate insecticides in biological and environmental samples. However, for fast, sensitive, and specific analysis of biological and environmental samples, detection by MS and MS/MS is preferred over fluorescence detection. Thus, descriptions and recommendations for establishing and optimizing HPLC fluorescence, HPLC/ MS, and HPLC/MS/MS analyses are discussed first. This is followed by specific rationales for methods and descriptions of the recommended residue methods that are applicable to most oxime carbamates in plant, animal tissue, soil, and water matrices. 

A drawback to conventional amino analysis by chromatography is the need for pre- or post-column derivatization to improve sensitivity. Ninhy-drin, the reagent originally applied for detection, has been increasingly displaced by other reagents such as phenylisothiocyanate,71 9-fluorenylethyl chloroformate,72 and o-phthaldialdehyde (OPA). OPA allows fluorimetric detection, which offers the potential for greater sensitivity.73 A limitation of OPA is that it doesn t derivatize secondary amines, so an additional reaction must be added for proline detection. And, as noted for amine analysis in section A5.4.2, such derivatization adds to the analysis time and may yield unstable products. 

Homish, R. E. and Wiest, J. R., Quantitation of spectinomycin residues in bovine tissues by ion-exchange high-performance liquid chromatography with post-column derivatization and confirmation by reversed-phase high performance chromatography-atmospheric pressure chemical ionization tandem mass spectrometry, /. Chromatogr. A, 812, 123, 1998. 

Mallat, E. and Barcelo, D., Analysis and degradation study of glyphosate and of aminomethylphosphonic in natural waters by means of polymeric and ion-exchange solid-phase extraction columns followed by ion chromatography-post-column derivatization with fluorescence detection, /. Chromatogr A, 823, 129, 1998. 

Fluorescence detection can be up to four orders of magnitude more sensitive than UV absorbance, especially where laser induced excitation is used, mass detection limits being as low as 10-20—10 21 mole. Pre- and post-column derivatization methods are being developed to extend the applicability of fluorescence detection to non-fluorescent substances. Several types of electrochemical and mass spectrometric detector have also been designed. Detector characteristics are summarized in Table 4.21. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

In contrast to the post-column derivatization, the inherent sensitivity of this method is higher since the column effluent is not diluted. Adequate sensitivity can therefore be achieved by UV monitoring at 330nm. For higher sensitivity, fluorescence detection is often chosen, and emission is measured at wavelengths above 430nm. moreover, baseline quality is superior. 

The use of reverse-phase columns with pre-column derivatization of the amino acids offers an acceptable alternative to the dedicated instrumentation of an amino acid analyser or separation by HPLC followed by post-column derivatization. 

A study of residual analysis of thirty pesticides and their transformation products was based on SPE on-line with HPLC-UVD or post-column derivatization with o-phthalaldehyde (73) and fluorescence detection (FLD), according to EPA method 531.1 and others. The method allowed determination of many pesticides in river and well waters at 0.01 to 0.5 -ig/L levels195. An automatized procedure was proposed for determination... 

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