Detection derivatization

Column Detection Derivatization Col. Temp.°C Detection Lim. Recovery Ref. 

Other more conventional detectors that might ostensibly outperform CD in selectivity are nmr and mass spectrometry, and in fact they do for the analysis of diastereomers, although quantitation is a much more difficult task. They cannot compete with chiroptical methods for the distinction between enantiomers. In nmr detection, derivatization to diastereomers is a prerequisite to enantiomer analysis, and chiral forms of lanthanide reagents can been used with good effect [16,17]. For the analysis of mixtures by either nmr or mass spectrometry, total chromatographic separation is a necessity, so the completeness of the baseline separation is the limiting step not the detector. In contrast CD can be applied to the analysis of enantiomers in mixtures in methods that require no prior separation. 

Derivatization is the preferred option for aminoacids (making use of the anomalous ORD or CD effects introduced with the chromophore) to move the Cotton bands to longer wavelengths and to lower the limits of detection. Derivatizations might include N-acetylation as in the case of aminoglycosides [67], dansylation [72], or binding to metal complexes [73,74], see later. On the other hand aminoacids have an auxiliary role to play in the analysis of other substances, where advantage is taken of their chiroptical properties. 

Thiols, as well as sulfide and sulfite, were determined in porewater samples by reversed phase high performance liquid chromatography (HPLC). The technique is based on precolumn derlvatization with an o phthalaldehyde/amine reagent (Figure 1) followed by HPLC and fluorometric detection. Derivatized porewater samples were Injected directly into the HPLC system the detection limit is 0.1 nM (for 100 ul injection). Details of the method are given in Mopper and Delmas (12). 

Remedy, highly specific detection, derivatization (see Section 19.8), coupling with spectroscopic methods (Section 6.10), optimized gradient elution (Sections 18.2 and 18.5) or column switching (multidimensional separation. Sections 18.3 and 18.4). 

Methods to detect cyanide exposure in human urine, saliva, and either serum or plasma have concentrated on thiocyanate. These methods include derivatization with HPLC/UV detection, derivatization with spectrophotometric detection or GC with BCD detection. Some urine methods have measured the urinary metabolite, 2-aminothiozoline-4-carboxylic acid. 

Cai, W.M. Hatton, J. Pettigrew, L.C. Dempsey, R.J. Chandler, M.H.H. A simplified high-performance liquid chromatographic method for direct determination of warfarin enantiomers and their protein binding in stroke patients. Ther.DrugMon.it., 1994,16, 509-512 [chiral fluorescence detection phen-procoumon (IS) LOD 8 ng/mL post-column reaction detection derivatization]... 

Native-fluorescent LIE has been used to detect proteins with high tryptophan levels and serotonin. However, most molecules are not fluorescent and must be derivatized with a fluorescent moiety in order to be detected. Derivatization has been achieved both with covalent attachments and affinity agents such as aptamers. The limitation for LIE as a detection technique for rapid separations is usually the chemistry of the derivatization reaction. First, the kinetics of the tagging reaction should be fast, especially for online analysis systems. Some fluorophores such as fluorescein... 

Precolumn derivatization offers numerous advantages (Tables 2 and 3, Figures 3-6). It requires less equipment and allows the evaluation of the derivatives in an easier way from the point of view of their selectivity, sensitivity, various means of detection, derivatization yield, stability, and storability. All of these phenomena can be controlled and improved by means of modern instrumental techniques and computerization, both individually and simultaneously. Potential disadvantages in precolumn derivatization procedures can be completely avoided, such as contamination from the reagents (due to their insufficient purity) and loss of analyte from incomplete interactions, undesirable side reactions, and sample handling losses. 

Tryptophan and tyrosine are intrinsic fluorophores that are present in many peptides, which then can be identified with fluorescence detection. However, most peptides have no native fluorescence, thus making derivatization a prerequisite for fluorescence detection. Derivatization has been described with naphthalene-2,3-dicarboxaldehyde-(S-mercaptoethanol for determination of substance P and its metabolites, fluorescamine, and 5-carboxyfluorescein succinimidyl ester for luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, and 3-endorphin. Kostel and Lunte compared various postcolumn reactor designs, whereas Advis et al. employed precolumn derivatization, among others. In order to improve sensitivity, laser light is frequently employed for exciting the fluorescent molecules referred to as laserinduced fluorescence (LIE) and provides a 500-1000 times improved sensitivity compared to UV detection. 

The determination of the herbicide glyphosate at low residue levels is difficult, due to its ionic character, low volatility, low mass, and the lack of chemical groups that might facilitate its detection. Derivatization with FMOC has been used to facilitate its chromatographic retention, allowing the determination of glyphosate and its main metabolite AMPA in water and soil [17,18]. This approach could be applied to food samples as well. 

Kaddoumi, A. Nakashima, M.N. Nakashima, K. Fluorometric determination of DL-fenfluramine, DL-norfenfluramine and phentermine in plasma by achiral and chiral high-performance liquid chromatography, J.Chromatogr.B, 2001, 763, 79-90. [fluorescence detection derivatization human rat LOD 10 nM ephedrine norephedrine]... 

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