The UV-VIS detector

The ultraviolet-visible spectrophotometer is the most widely used detector for HPLC. The basis of UV-VIS detection is the difference in the absorbance of light by the analyte and the solvent. A number of functional groups absorb 

Absorbance detectors are also commonly used in combination with postcolumn reactors. Here, most issues of detector linearity and detection limit have to do with optimization of the performance of the reactor. In a typical application, organophosphorus compounds with weak optical absorbances have been separated, photolyzed to orthophosphate, and reacted with molybdic acid, with measurement being performed by optical absorbance.58 

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It is being recognized increasingly that regulation can have a positive impact on laboratory productivity.36 System suitability testing has been proposed as superior to and supplemental to calibration in the UV-VIS detector.37 Large variations in both response factor and in relative response factors were observed on different instruments. Even on the same instrument, UV-VIS spectra can be extremely dependent on solution conditions, as was observed in a separation of hypericin, the antidepressant extract of St. John s wort.38... 

The UV/VIS detector has a broad linear dynamic range. However, as illustrated in Example 4.11, it does not offer the selectivity required for analysis of complex environmental matrices. Data obtained from UV/VIS detectors should be confirmed by other methods. Neither does the UV/VIS detector offer the sensitivity required for many DQOs, since a selected fixed wavelength does not necessarily have the maximum absorbance for all target analytes. 

Restart the flow on the HPLC in the equilibration conditions. Zero the UV/Vis detector Load the crude product solution onto the column at 190cm3-min 1 through Pump A. All eluent is to be collected. Record the following ... 

Carbon dioxide is transparent to below 190 nm, making it an ideal fluid for use with ultraviolet (UV) detectors. It can be used with acetonitrile above 195 nm and with methanol above 205 nm. The UV-vis detectors used in SFC are standard HPLC detectors in every way except for high pressure flow cells. Double tapered windows (45° bevels with matching seals) balance the stress and allow cells to be built that withstand more than 600 bar. The operator needs to be sure that the data collection rate is set fast enough to not round off the faster peaks experienced in SFC. 

Neither the MS or the UV-vis detector is very good at predicting the amount of material present. A third detector that is mass dependent is sometimes included to quantify the peak of interest. One common detector used in both SFC and FIPLC is the evaporative light-scattering detector or (ELSD) (5-8). There are a number of different ELSDs on the market. It is important to use one that is compatible with SFC and is capable of operating during steep composition gradients. 

The liquid adsorption and diffusion measurements were carried out in a BECKMAN HPLC system, which consists of one model 421 system controller, two model 110 solvent metering pumps, one solvent mixer and one model 210 sample injector with a 20 yl sample loop. A Hitachi model 100-40 UV-Vis Spectrometer was used as the detector. To increase the pressure in the UV-VIS detector cell, a back pressure regulator was connected to the effluent stream from the detector to avoid formation of air bubbles due to vaporization in the detector cell. Figure 1 shows the schematic of the HPLC system used in the experiments. 

For some experiments, an evaporative light-scattering detector (ELSDII, Varex Corporation, Burtonsville, MD) was connected in series with the UV-vis detector of Flow I. The operating conditions were those recommended by the instrument manufacturer. 

The UV-VIS detector is the most frequently used detector in HPLC (Fig. 10). It is simple to handle, concentration sensitive, selective, and nondestructive. The latter also makes it suitable for preparative chromatography. 

These techniques when coupled with detectors, the most popular being the UV-Vis detector for HPLC, may detect a separated compound, by matching the retention time to that of a known standard compound and pattern of UV-Vis scan. In addition to univariate identification, e.g., retention time in chromatography, and wavelength/frequency in spectrometry, the use of mass spectrometry is an excellent tool to define the chemical identification. 

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