Chemiluminescence-based HPLC detection system

In the chemiluminescence-based HPLC detection system, illustrated schematically in Figure 6, the oxalate ester and hydrogen peroxide are introduced to the eluent stream at postcolumn mixer Mj, which then flows through a conventional fluorescence detector with the exciting lamp turned off or a specially built chemiluminescence detector. The two reagents are combined at mixer Mj, rather than being premixed, to prevent the slow hydrolytic reactions of the oxalate ester. 

The last but not least part of an HPLC system is the detector. There are several ways to detect when a certain compound elutes from the colunm. Detection systems based on molecular absorption (UV, Visible, or DAD), fluorescence (FLD) or chemiluminescence, EC or MS are the most popular. It is worthy to highlight the high sensitivity and selectivity provided by detection systems based on fluorescence or chemiluminescence. 

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... 

Boyle and co-workers [27] undertook a study to determine whether an HPLC method based upon luminol chemiluminescence detection could be used for the analysis of Co(II) in seawater and freshwater samples at the 10 mol kg level. Their preliminary results showed weak chemiluminescent signals for those chromatographic systems in which cobalt eluted from the column in a complexed form chemiluminescence from the luminol reaction is dependent upon catalysis by free metal ions. It was also observed that the chemiluminescent signal was not severely depressed when moderately strong complexing agents were used in the mobile phase. These results ultimately led the researchers to use weakly complexing eluents in order to elute cobalt in a relatively uncomplexed form, and thus ensure maximum sensitivity from the chemiluminescence reaction. 

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