Precolumn derivatization

Precolumn derivatization with a solid-phase derivatizing precolumn has also been reported. Xie et al. (43) applied polymeric permanganate oxidations of alcohols and aldehydes for the production of UV-absorb-ing species. 

A. Precolumn Derivatization Precolumn derivatization tends to be the more sensitive of the two methods, but it requires additional steps prior to the chromatographic separation of the solutes in the sample. However, standard chromatography equipment can be used without modification. Because the precolumn derivatization requires additional time on the part of the chemist, precolumn derivatization is best suited for situations where derivatization is not the standard protocol. 

Among the more sensitive methods for the determination of A-nitrosamines are the ones based on the denitrosation of A-nitroso compounds by hydrobromic-acetic acid, and the subsequent detection of the liberated secondary amines via fluorescence derivatization. Precolumn or postcolumn derivatization has been used for the determination of A-nitrosamines. The most commonly used fluorescent derivatization reagents are listed in Table 12.4. 

Liquid Ghromatography/Mass Spectrometry. Increased use of Hquid chromatography/mass spectrometry (Ic/ms) for stmctural identification and trace analysis has become apparent. Thermospray Ic/ms has been used to identify by-products in phenyl isocyanate precolumn derivatization reactions (74). Five compounds resulting from the reaction of phenyUsocyanate and the reaction medium were identified two from a reaction between phenyl isocyanate and methanol, two from the reaction between phenyl isocyanate and water, and one from the polymerisation of phenyl isocyanate. There were also two reports of derivatisation to enhance either the response or stmctural information from thermospray Ic/ms for linoleic acid hpoxygenase metabohtes (75) and for cortisol (76). 

S. Emai a, H. Askal and T. Masujima, Rapid detemination of methoti exate in plasma by high-perfomance liquid clrromatography with online solid-phase exti action and automated precolumn derivatization , Biomerf Chromatogr. 12 338-342(1998). 

Heinrikson, R.L. and Meredith, S.C. 1984 Amino acid analysis by reverse-phase high-performance liquid chromotography Precolumn derivatization with phenylisothiocyanate. Analytical Biochemistry 136 65-74. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Bartok, T., Borsok, G., and Sagi, F., RP-HPLC separation of polyamines after automatic FMOC-C1 derivatization and precolumn sample clean-up using column switching, /. Liq. Chromatogr., 15, 777, 1992. 

Mitsui, A., Nohta, H., and Ohkura, Y., High-performance liquid chromatography of plasma catecholamines using 1,2-diphenylethylenediamine as precolumn fluorescence derivatization reagent, /. Chromatogr., 344, 61, 1985. 

Larsen, B. R. and West, F. G., A method for quantitative amino acid analysis using precolumn o-phthaladehyde derivatization and high performance liquid chromatography, /. Chromatogr. Sci., 19, 259, 1981. 

Cooper, J. D. H., Ogden, G., McIntosh, J., and Tumell, D. C., The stability of the o-phthaldehyde/2-mercaptoethanol derivatives of amino acids an investigation using high-pressure liquid chromatography with a precolumn derivatization technique, Anal. Biochem., 142, 98, 1984. 

Albin, D. M., Wubben, J. E., and Gabert, V. M., Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate,. Agr. Food Chem., 48, 1684, 2000. 

Arnold, U., Ludwig, E., Kuehn, R., Moeschwitzer, U., Analysis of free amino acids in green coffee beans, Part 1, Determination of amino acids after precolumn derivatization using 9-fluorenylmethylchloroformate, Z. Lebensm.-Forsch. 199(1), 22, 1994. (CA121 132515y)... 

Many times an analyte must be derivatized to improve detection. When this derivatization takes place is incredibly important, especially in regards to chiral separations. Papers cited in this chapter employ both precolumn and postcolumn derivatization. Since postcolumn derivatization takes place after the enantiomeric separation it does not change the way the analyte separates on the chiral stationary phase. This prevents the need for development of a new chiral separation method for the derivatized analyte. A chiral analyte that has been derivatized before the enantiomeric separation may not interact with the chiral stationary phase in the same manner as the underivatized analyte. This change in interactions can cause a decrease or increase in the enantioselectivity. A decrease in enantioselectivity can result when precolumn derivatization modifies the same functional groups that contribute to enantioselectivity. For example, chiral crown ethers can no longer separate amino acids that have a derivatized amine group because the protonated primary amine is... 

Guo, X., Fukushima, T., Li, F., Imai, K. (2002). Determination of fluoxetine enantiomers in rat plasma by precolumn fluorescence derivatization and column-switching high-performance liquid chromatography. Analyst 127, 480-484. 

Wen-Chen Z, Ling-Jun L, Xian-En Z et al (2008) Application of 2-(l l//-benzo[ i]carbazol-11 -yl) ethyl carbonochloridate as a precolumn derivatization reagent of amino acid by high performance liquid chromatography with fluorescence detection. Chin J Anal Chem 36 1071-1076... 

Prados P, Fukushima T, Santa T et al (1997) 4-/V,/V-Dimethylam inosu 1 lbnyl-7 -N-(2-aminoethyl)amino-benzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography. Anal Chim Acta 344 227-232... 

Jones, B.N., and Gilligan, J.P. (1983) o-Phthaldialdehyde precolumn derivatization and reversed phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J. Chromatogr. 266, 471-482. 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

S Einarsson, B Josefsson, P Moller, D Sanchez. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chlo-roformate and reversed phase liquid chromatography. Anal Chem 59, 1191, 1987. 

Lindroth, P., and K. Mopper. 1979. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Analytical Chemistry 51 1667-1674. 

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