Absorbance detectors UV/Vis

A UV/Vis absorbance detector can also be used if the solute ions absorb ultraviolet or visible radiation. Alternatively, solutions that do not absorb in the UV/Vis range can be detected indirectly if the mobile phase contains a UV/Vis-absorbing species. In this case, when a solute band passes through the detector, a decrease in absorbance is measured at the detector. 

The most commonly used detector for HPLC is still the UV-Vis absorbance detector. The amount of light transmitted through a solution of concentration c in a flow cell with path length ( is given by the Beer-Lambert law. 

UV/VIS absorbance detector (single wavelength or diode array) placed in-line between HPLC column and mass spectrometer for additional characterization of eluting chlorophylls (optional)... 

Optional Direct the eluate from the HPLC column through a UV/VIS absorbance detector prior to the mass spectrometer for additional sample characterization. 

Figure 4.8. A schematic of a UV-Vis absorbance detector (a) and a photodiode array (PDA) detector (b). Diagrams adapted from and reprinted with permission from Academy Savant.

Figure 10.4. A picture of a Waters 2487 UV/Vis absorbance detector with the front panel removed showing the position of the UV lamp (source) and the flow cell cartridge. Picture courtesy of Waters Corp.

Perhaps the most revolutionary development has been the application of on-line mass spectroscopic detection for compositional analysis. Polymer composition can be inferred from column retention time or from viscometric and other indirect detection methods, but mass spectroscopy has reduced much of the ambiguity associated with that process. Quantitation of end groups and of co-polymer composition can now be accomplished directly through mass spectroscopy. Mass spectroscopy is particularly well suited as an on-line GPC technique, since common GPC solvents interfere with other on-line detectors, including UV-VIS absorbance, nuclear magnetic resonance and infrared spectroscopic detectors. By contrast, common GPC solvents are readily adaptable to mass spectroscopic interfaces. No detection technique offers a combination of universality of analyte detection, specificity of information, and ease of use comparable to that of mass spectroscopy. 

The following guidelines are recommended for UV/Vis absorbance or photodiode array (PDA) detectors ... 

UV/vis absorbance detection is widely used in capillary electrophoresis. Absorptivity depends on the chromophore (light-absorbing part) of the solute, the wavelength of the incident light, and the pH and composition of the run buffer. A photodetector measures light intensities and the detector electronics convert this into absorbance [11]. 

A CE instrument comprises a high-voltage source and electrodes, containers for buffers, provision for holding and injecting samples, a thermostatted capillary housing and a detector. Most CE instruments use UV-vis absorbance detection, sometimes with spectral analysis capabilities. In instrumental separation sciences quantitative data is derived from the peak elution/mi-gration time and peak dimensions therefore any instrumental function which can affect these must be tested. 

A photodiode array detector (PDA), also known as a diodearray detector (DAD), provides UV spectra of eluting peaks while functioning as a multiwavelength IJV/Vis absorbance detector. It facilitates peak identification and is the prefered detector for method development. Detector sensitivity was lower in earlier models but has improved significantly in recent years. 

Microscale fluidic systems use small volumes so sensitivity of detection can be a challenge. Any detector for chip-based LC needs to be small and ideally have low power consumption. It is generally a problem of interfacing. Flow cell geometry is also a big factor, e.g. a U cell instead of linear flow cell can give a ten-fold increase in sensitivity for absorbance measurements. Electrochemical detection is very common, mainly ampero-metric and potentiometric, and very amenable to detection on chip. Fluorescence is more sensitive than UV-Vis absorbance and chemiluminescence is sensitive down to a single molecule, similar to LIF. 

In stopped-flow kinetics, small volumes of reagent solutions are mixed rapidly in a flow cell by injection from separate syringes (Figure 6.3). The flow cell is typically fitted with transparent quartz windows so that UV/vis absorbance or fluorescence can be followed as a function of time. Circular dichroism (CD) kinetics can also be measured in some instruments. The injection pulse is controlled by a stopping syringe which, when it hits the end stop, electronically triggers the detector to start recording. 

Organic compounds that possess an ultraviolet- or visible-absorbing chromophore obey the Beer Lambert or Beer-Bouguer law of spectrophotometry. In what is generally termed molecular absorption spectrophotometry, a cuvette (in the case of stand-alone UV-vis spectrophotometers) or a micro-flow cell (in the case of flow through HPLC UV-vis detectors). We now proceed to derive the fundamental equation that relates absorbance as measured on an UV-vis HPLC detector to concentration because this relationship is important to the practice... 

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