Liquid chromatography post-column derivatization methods

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. 

Post-column derivatization of carbohydrates has been described for liquid chromatography and UV detection by heat treatment [46], acid treatment [47] and reaction with phenol-sulfuric acid [48,49]. These methods have been applied to the analysis of sugars in body fluids and in wood products. The procedures are only suitable for low-speed liquid chromatography. 

Thiamine, a soluble vitamin, is an essential nutrient for humans and is important in carbohydrate metabolism, maintaining normal neural activity and preventing beriberi. Various analytical techniques have been reported for the determination of thiamine in pure form, in pharmaceutical preparations, or in biological fluids. Spectrophotometric methods suffer from poor sensitivity (mg/L detection limit). Spectrofluorometric methods usually involve the conversion of thiamine to thiochrome.2 High performance liquid chromatography requires a post-column derivatization step3 and instrumentation for electrophoresis-based methods is expensive.4... 

Many methods have been employed for the detection of these compounds such as high-performance liquid chromatography with pre or post column derivatization [22-24], calorimetry [25], fluorometry [20, 26], chemiluminescene [27], capillary electrophoresis [28, 29] and NMR spectrometry [30]. Electrochemical methods are other choices for their determination because they are inexpensive, simple, fast and sensitive. Experiments using mercury, mercury amalgam and a chemically modified electrode as the working electrode [31-33] have been performed for thiol-compound determination. However, mercury has restrictions due to its toxicity and the fast deterioration of the electrode response. Using a chemically modified electrode [34, 35] is not practical for use in flow injection analysis and liquid chromatography. Therefore, the BDD thin film electrode has been introduced it is known as a unique electrode material for a number of electrochemical applications [12, 36, 37]. 

Development of selective analytical method for the determination of artemisinin poses challenging problems because it lacks ultraviolet (UV) absorption or fluorescent chromophores and does not possess functional groups with potential for derivatization. This study reports the determination of artemisinin by liquid chromatography with on-line post-column UV irradiation and peroxyoxalate chemiluminescence (PO-CL) detection. A similar method as previously reported by us for the determination of organic peroxide. In this method, after artemisinin is eluted from the HPLC column, it is UV irradiated to generate hydrogen peroxide, which is determined by PO-CL detection. 

High-performance liquid chromatography (HPLC) methods have been comprehensively applied to the determination of biotin in pharmaceutical multivitamin preparations. Some of them have also been used to analyse more complicated matrices of samples, e.g. food material (Livaniou et al. 2000). Biotin detection by HPLC can be achieved with (either pre- or post-column) or without sample derivatization (Livaniou et al. 2000). 

Dye, D., East,T., and Bayne, W.F. (1984) High-performance liquid chromatographic method for post-column, in-line derivatization with o-phtha-laldehyde and fluorometric detection of phenylpropanolamine in human urine. Journal of Chromatography, 284,457-461. 

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