On-column derivatization

Oguri, S., Maeda, Y., and Mizusawa, A. (2004). On-column derivatization-capillary electro-chromatography with o-phthalaldehyde/alkylthiol for assay of biogenic amines. /. Chromatogr. A 1044, 271-276. 

Saito et al. (70) developed a method for the determination of eight BAs in fish. After a cleanup procedure, including acidification with HC104 and cation-exchange HPLC, the samples were subjected to on-column derivatization and HPLC separation on an Ashipak ODP-50 column. The eluent was an ACN-borate buffer containing o-phthaldehyde (PA) and /V-acetyl-L-cysteine (NAC) the fluorimetric detection was performed at 430 nm. The method was applied to canned fish such as tuna, salmon, crab, and clams. 

Analysis of single mammalian cells by capillary electrophoresis has been reported using on-column derivatization and laser-induced fluorescence detection (11). Dopamine and five amino acids were determined in individual rat pheochromocytoma cells after on-column derivatization. 

Normal-phase separations at -30°C enabled the determination of the impurity profile of a mesylated ester, which underwent in-column cyclization at room temperature, to be determined [86]. Subambient temperatures down to -10°C were used by LoBrutto et al. [87] to generate a single product from the on-column derivatization of an acetylenic aldehyde with diethylamine. 

R. LoBrutto, Y. Bereznitski, T. J. Novak, L. Dimichele, L. Pan, M. Journet, J. Kowal, and N. Grinberg, Kinetic analysis and subambient temperature on-line on-column derivatization of an active aldehyde, /. Chromatogr. A 995 (2003), 67-78. 

Gilman, S.D. Ewing, A.G. Analysis of single cells by capillary electrophoresis with on-column derivatization and laser-induced fluorescence detection. Anal. Chem. 1995, 67 (1), 58-64. 

Paleologos, E.K. and Kontominas, M.G. (2004) On-line solid-phase extraction with surfactant accelerated on-column derivatization and micellar liquid chromatographic separation as a tool for the determination of biogenic amines in various food substrates, Anal. Chem., 76, 1289-1294. 

A number of various derivatization reactions have been used, as shown in Table 14.25. The derivatization has been carried out as off-column or on-column derivatization, mostly in order to give morphine and related alkaloids with phenolic or alcoholic hydroxyl groups better gas chromatographic properties. Especially for quantitative determination of morphine in opium or biological material such derivatization was necessary. Street et al. found, however, that the problem of adsorption of a phenolic alkaloid, such as morphine, because of "active sites" on the solid support could be solved by deactivation of the solid support by treatment of it (diatomaceous earth) with benzoyl chloride in pyridine before coating it with the stationary phase. 

Anders and Mannering introduced on-column derivatization of morphine with propionic anhydride and obtained only one peak. They believed that it was dipropionylmorphine. Von Meyer et al. made use of the same derivatization reaction for the quantitative determination of morphine in blood, but they found that the monopropionyl derivatives of morphine and nalorphine (used as an internal standard) were found in the proportion 99 1, as compared with the... 

R.M. Smith, A.M. Butt and A. Thakur, Determination of lead, mercury, and cadmium by liquid chromatography using on-column derivatization with dithiocarbamates, Analyst, 110,35,1985. 

The derivatization can be carried out in solution as a last step in sample preparation. This allows for whatever temperature and reaction time may be necessary to form the derivative. This is the standard practice for forming GC derivatives, and is termed precolumn derivatization (or labeling). In GC we can sometimes avail ourselves of the very high temperatures in a GC injection port during flash vaporization to perform on-column derivatization (cf. Section 12.10). While this is not possible in HPLC, we have an even better option with this latter form of chromatography namely, post-column derivatization. Here, the labeling agent is added to the column effluent by... 

Derivatized with phthalic anhydride 10 mM on-column Derivatized with 800 p,L of a 1% (w/v) Dns-Cl in acetone solution... 

Zhang, Y., and Gomez, F. A., On-column derivatization and analysis of amino acids, peptides and aUcylamines by anhydrides using capillary electrophoresis. Electrophoresis, 21, 3305, 2000. 

Such agents function less instantaneously than diazomethane. They react faster at quite high temperatures. One way to achieve those has been to mix them with the injection solution. Upon flash vaporization in the hot injection port or the start of the column, the derivatization reaction is nearly instantaneous and complete. This procedure is known as on-column derivatization. Such a mode of introduction of this reagent also achieves the purpose of reacting with and deactivating the free -Si-OH, active site groups in the column, as described at the very end of Section 11.5. This treatment is called silanization. 

Source From Development of 3,5-Wi-(trifluoromethyl)benzyl-dimethylphenylammonium fluoride, an efficient new on-column derivatization reagent, in... 

Analogous to the on-column derivatization used in gas chromatography, an oxidative chemical transformation was carried out on a TLC plate by m-chloroperbenzoic acid. The oxygen analogs of fenthion, disulfoton, and phorate that were produced were separated after this derivatization (57) (Table 3). Another example of on-plate derivatization involves a reduction reaction carried out in order to determine the residues of methylparathion, parathion, and fenitrothion. Parent compounds and corresponding amino derivatives were also separated (58) (Table 3). 

Applications CE. In addition to its prevalent use in detection following HPLC separation, the biuret detection strategy has also been used for electrochemical detection of peptides following CE separation. A Cu(lI)-coated capillary has been used to eliminate the need for pre-colunm or on-column derivatization using a Cu(II)-containing buffer [161], Detection limits on the order of 0.7 jM were obtained for nonelectroactive peptides (di-, tri-, tetra-, and pen-taglycines). Other peptides including Pro-Leu-Gly-amide were also successfully detected with a 2 oM detection limit. 

Trichloracetyl and 4-carbethoxyhexafluorobutyryl chloride derivatives have been nsed to increase the molecular weight of the amphetamines. These particular derivatives consequently lengthen the elution times and help separate these compounds from potential interference (34,35). A-Monotrifluoroacetylated (TFA) derivatives of amphetamine analogues have been prepared by on-column derivatization with A -methylbis(trifluoroacetamide) (MBTFA) (36). A separation on a 12-m methylsilicone capillary column of four phenethylamines with closely related structures is shown in Figure 16.2 using this procedure. 

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