Chemiluminescence-based HPLC detection

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. 

Luminol-based chemiluminescence methods have also been employed for detection of analytes in flowing stream analytical techniques such capillary electrophoresis (282), flow injection analyses, and hplc (267). AppHcations of the enhanced luminol methodology to replace radioassay methods have been developed for a number of immunological labeling techniques (121,283). 

Relative purity measurement and the relative purity-based reaction optimization have long been used in combinatorial synthesis. In order to make high-through-put purification a success, the yield-based optimization is essential. Chemiluminescent nitrogen detection (CLND) [4] with HPLC determines the quantitative yield after each reaction step during the library feasibility and rehearsal stages. The yield of each synthetic step provides guidance for the final library synthesis. 

Carbon-14 content is measured by specially designed gas proportional counters (7. Aerosol samples are first converted to CO2 by combustion in a macroscale version of the thermal evolution technique. A clam shell oven was used to heat the sample for sequential evolution of organic and elemental carbon under equivalent conditions. Due to the possibility of thermal gradients, conditions in the macroscale apparatus were adjusted to produce the same recoveries of total carbon (yg C per cm of filter area) as for the microscale apparatus. Carbon-14 data are reported as % contemporary carbon based on the 1978 1 C02 content in the atmosphere. Aldehyde data referred to in this paper were obtained by impinger sampling in dinitrophenylhydrazine/acetonitrile solution and analysis of the derivatives by HPLC with UV detection (12). Olefin measurements were made by a specially designed ozone-chemiluminescence apparatus (13) difficulties in calibration accuracy and background drift with temperature limit its use to inferences of relative reactive hydrocarbon levels. 

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... 

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. 

Most flow-based techniques coupled to chemiluminescence do not require separation before detection. However, for complex matrices, flow-based techniques are first coupled to a separation technique such as HPLC or CE (Table 10.1). The major drawback of HPLC is the additional need for pumps to deliver the postcolumn chemiluminescence reagent or for additional devices to mix the column eluate and chemiluminescence reagent. Furthermore, the analysis time is increased because of the necessary optimization of the separation parameters (mobile phase composition, flow rate) and chemiluminescence parameters (pH, temperature, catalyst, type of enhancer). On the other hand, CE coupled to chemiluminescence has the advantages of high resolution, reduced analysis time, and lower expense [28]. 

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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