Fluorescent, labeling post-column

At this point, the anaiyte may not be amenabie to UV, FL, or EC detection. In this case, the best course of action may be to choose LC/MS (see Section 4.2). However, one other option is to use a pre- " or post-coiumn derivatization step to increase the detectabiiity of the anaiyte with respect to FL or UV. Fluorescent or UV labels are available for carboxylic acids," amines, phenols, and thiols. The decision to use pre- or post-column derivatization is predicated upon the functionality of the analyte available for derivatization and the rate and extent of the reaction between each derivatizing agent and the analyte. 

HPLC analysis of furosine (-peak II) in hydrolyzates of non-exposed- (bottom), buffer-exposed (middle), and glucose-exposed (top) dentin samples. Dentin was not reduced prior to hydrolysis. Only the relevant parts of the chromatograms are shown. Amino acids are visualized after post-column labelling with a fluorescent dye. I lysine, II furosine. III homoarginine (internal standard). Column Merck Polyspher AA-NA 120 x 4.6 mm flow 0.2 ml/min gradient pH 5.0 -10.2 postcolumn reagent 0.2 ml/min fluorescence Xgx 330 nm, 440 nm 100-yl injections in buffer pH 2. 

Most BAs do not show fluorescence or UV adsorption and a labelling procedure is indispensable to allow their detection. One of the most frequently studied fluorescent derivatizing agents in both the pre- and post-column methods is o-phthalaldehyde (OPA). Despite the limited stability of derivatives, the main advantages of OPA are a very fast... 

Fig. 12 (a) Cross-sectional view of a microchip, heating element, and thermocouples, (b) Diagram of the microchip used for on-chip proteolytic reactions, separations, and post-column labeling for generating fluorescent moieties. The fluid reservoirs are (1) substrate, (2) enzyme, (i) buffer, (4) sample waste, (5) NDA, and (6) waste. Reproduced from [134]... 

In post-column derivatization (see also Chapter 15), the HPLC mobile phase must be miscible and compatible with the reaction mixture in the reactor so that it dose not cause precipitation or interfere with the derivatization reaction. The reagent itself must have significant differences in spectral properties from the products. Post-column derivatization reactions are usually rapid, and fluorescent rather than UV-absorbing labels are preferred. 

From an operational viewpoint, the post-column reaction of biotin with avidin labelled with fluorescein isothiocyanate (avidin-FITC) has become the simplest HPLC method for the quantitation of biotin and biocytin, and has allowed routine testing laboratories to generate quality control (QC) data (Lahely et al. 1999). Typical chromatograms are shown in Figure 24.1 of a standard and a milk powder extract. The analytical time is only a few minutes because of the absence of interfering fluorescent substances. Biocytin, if present, elutes in front of biotin and can be concurrently detected. 

Many chemicals are not detected using a fluorescence detection scheme unless they are chemically derivatized with a fluorescent label. A reaction may be done before a separation is performed, but there are advantages if it is done after the separation. In chromatography this is known as post-column reaction. This is a chemical reaction performed "on-the-fly" in the sample stream as it moves towards a detector, and it must create a fluorescent product only when sample is present. One of the most common reagent used for post-column derivatization is o-phthaldialdehyde (OPA), which reacts with primary amines to create a fluorescent product. In electrophoresis this process might be called an in-capillary reaction, since due to the electric field the separation... 

Ye, M., Hu, S., Schoenherr, R.M., and Dovichi, N.J. (2004) On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection. ILlectrophoresis, 25,1319-1326. 

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