Absorbance fluorescence monitor with

Typically, sample detection in electromigration techniques is performed by on-column detection, employing a small part of the capillary as the detection cell where a property of either the analyte, such as UV absorbance, or the solution, such as refractive index or conductivity, is monitored. This section briefly describes the major detection modalities employed in capillary electromigration techniques, which are accomplished using UV-visible absorbance, fluorescence spectroscopy, and electrochemical systems. The hyphenation of capillary electromigration techniques with spectroscopic techniques employed for identification and structural elucidation of the separated compounds is also described. 

Schematic of a fluorescence monitor in series with an absorbance detector in HPLC... 

Pesticides Separated on a CN-Bonded Polar Phase. Figures 3 and 4 contain the chromatograms obtained for several of the pesticides listed in Table 2. Various mobile phases were used to facilitate separation on the CN-bonded polar phase. Detection is shown in both absorbance and fluorescence modes. It would appear that fluorescence detection is more sensitive for some pesticides, while absorbance detection is more sensitive for others. However, comparisons of one manufacturer s absorbance monitor with another manufacturer s fluorescence monitor could be misleading. (I will refer to this under Instrumental Parameters.) Furthermore, the ultimate useful comparison is obtained when practical samples are chromatographed since these... 

A fluorescence monitor can conveniently confirm and support data obtained with an absorbance detector. However, any comparison of the relative sensitivity/selectivity of the absorbance mode vs the fluorescence mode depends on the spectrochemical nature of both the pesticide itself, and the co-extracted co-elutants found in the agricultural product extracted. A judicious selection of mobile phase is required to optimize separation of the pesticide and co-extractives on a CN-bonded polar stationary phase. 

Alternatively, the same reaction can be assayed if adenosine is replaced by formycin A (FoA) (Fig. 4.14), a fluorescent analog. With this substrate, one product of the adenosine kinase reaction would be FoMP, the fluorescent analog of AMP, while AMP formed directly from ATP would not be fluorescent. Therefore, by monitoring both the fluorescence and the ultraviolet absorbance, using equipment arranged as shown in Figure 4.15, the analyst could follow both the kinase reaction and any secondary reactions. 

The Model UA-5 Is a sensitive, reliable, easy to use absorbance/fluores-cence monitor with built-in calibration standards and recorder. Automatic scale expansion keeps oversized peaks on scale. A selection of thirteen wavelengths (one or two at a time), sixteen different flow cells, and an optional fluorescence optical unit provide Impressive versatility. Absorbance peaks can be automatically deposited into separate test tubes when the Model UA-5 is used with an ISCO fraction coflector, making it possible to recover peaks with the least dilution possible. 

The process of identifying the products of the interaction between the enzyme and alternate substrate depends a great deal on the inhibitor itself. If the compound contains a chromophore or fluorophore, changes in the absorbance or fluorescence spectra with the addition of enzyme can be monitored and used to identify products (Krantz et al., 1990). For multiple product reactions, single turnover experiments can be used to determine relative product distribution. Stoichiometric quantities of enzyme and inhibitor can be incubated for full inhibition, followed by the addition of a rapid irreversible inhibitor of the enzyme, such as an affinity label. This will act as a trap for enzyme as the enzyme-inhibitor complex breaks down. Analysis of the products will determine relative... 

Another very sensitive method directly monitors the absorbed energy rather than relying on a difference measurement (Ij -Ij). The energy IgaxA absorbed per second in a volume V = Ax can either be converted into fluorescence energy and monitored with a fluorescence detection system excitation spectroscopy) or it can be converted by collisions into thermal energy with a resultant temperature and pressure rise, which is monitored by a sensitive microphone photoacoustic spectroscopy). 

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