Columns post-column derivatization

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. 

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. 

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... 

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. 

Like dissolves like is the basic concept for the selection of solvents in the eluent for liquid chromatography. Controlling the solubility of analytes is the key to success. If the selected solvent or mixture of solvents does not interfere with detection, it is a good eluent. The selection of a suitable solvent for low-wavelength absorption detection and post-column derivatization detection is important to obtain highly sensitive detection. The selection of a volatile solvent is the key for preparative-scale liquid chromatography and for mass spectro-metric detection. 

Kostel KL, Lunte SM. 1997. Evaluation of capillary electrophoresis with post-column derivatization and laser-induced fluorescence detection for the determination of substance P and its metabolites. J Chromatogr B Biomed SciAppl 695(1) 27-38. 

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

Apparatus. The HPLC instrument used was a Water s Associates model 6000A pump for the solvent supply, a U6K septumless injector and a radial compression module with standard Radial Pak columns. Immediately after the column a low dead volume tee was inserted and another 6000A pump was used to deliver a solution of OPT for the post-column derivatization of histamine. Twenty feet of 9 thousandths (id) coiled stainless steel tubing was used as a mixing chamber and held at 60 C in a water bath. The reaction mixture then passed through a Water s 420 fluorescence detector which was connected to a recorder. The detector was equipped with a 340-nm excitation filter and a 440-nm emmission filter. 

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). 

Determination of Glyphosphate in Drinking Water by Direct-Aqueous Injection HPLC, Post-Column Derivatization, and Eluorescence Detection... 

---