Highly sensitive detection derivatizing reagents, 7

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

Niacin can be detected by UV, ED, or FLD. UV is a widespread technique but it needs a longer preparation step and it does not reach high sensitivity. The FLD is more sensitive but it needs a pre or postcolumn derivatization to make niacin fluorescent. Krishnan et al. [599] describe a postcol-umn derivatization using cyanogens bromide and p-aminophenol, but this method involves toxic reagents. Mawatari et al. [600], instead, propose a fast, highly specific derivatization procedure, which involves UV irradiation at 300 nm in the presence of hydrogen peroxide and copper(II) ions. 

To enhance analytical sensitivity, some HPLC procedures incorporate precolumn derivatization with fluorescent and chemiluminescent reagents, thereby achieving detection limits in the femtomole range. Completely automated analyses of serotonin have been described, and some systems incorporate direct injection and online solid phase extrac-tion. For laboratories that may not have the equipment, staff, or experience to measure serotonin by HPLC, alternative technologies based on RIA and EIA are available as commercial test kits. These procedures are highly sensitive and... 

In post-column derivatization systems, the derivatlzation reagent is continuously mixed with the column effluent consequently, the presence of trace amounts of amino acids or interfering components such as ammonia in the column eluent limits the ultimate sensitivity attainable. Figure 2 shows the effects of buffer contaminants on the baseline of a typical high sensitivity analysis (12). The basic contaminants are held up on the column until elution by the final buffer. Detection of the basic amino acids below lO-picomole levels is difficult using this approach. The use of a shorter second column with isocratic elution for the analysis of just the basic amino acids is a good solution to the problem but does require the use of a second aliquot of sample. 

K. Shimada, M. Tanaka and T. Nambara, Sensitive derivatization reagents for thiol compounds in high-performance liquid chromatography with electrochemical detection. Anal. Chim. Acta, 1983, 147, 375-380. 

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