Flow cells, detectors absorption

Schematic diagrams of flow cell detectors for HPLC using (a) UVA/is absorption spectrophotometry and (b) amperometry.

Radiation from a xenon or deuterium source is focussed on the flow cell. An interchangeable filter allows different excitation wavelengths to be used. The fluorescent radiation is emitted by the sample in all directions, but is usually measured at 90° to the incident beam. In some types, to increase sensitivity, the fluorescent radiation is reflected and focussed by a parabolic mirror. The second filter isolates a suitable wavelength from the fluorescence spectrum and prevents any scattered light from the source from reaching the photomultiplier detector. The 90° optics allow monitoring of the incident beam as well, so that dual uv absorption and fluorescence... 

The ultraviolet (UV) absorption HPLC detector is basically a UV spectrophotometer that measures a flowing solution rather than a static solution. It has a light source, a wavelength selector, and a phototube like an ordinary spectrophotometer. The cuvette is a flow cell, through which the column effluent flows. As the mobile phase elutes, the chromatogram traces a line at zero absorbance, but when a mixture... 

For ultraviolet and visible spectroscopic detectors, a standard solution of a compound whose molar absorption constant is known must be prepared, and placed in the flow cell. The absorbance obtained is then compared with the value measured by a standard spectrophotometer. 

The photodiode array detector (PDAD) measures absorption of light waves by a sample. This is considered the most powerful of the ultraviolet spectrophotometric detectors. The optical system focuses light from a deuterium source through the sample flow cell onto several photodiodes. These act as capacitators by holding a fixed amount of charge. When light strikes the photodiodes, they discharge a certain amount of current. 

Monochromatic detection. A schematic of a monochromatic absorbance detector is given in Fig. 3.12. It is composed of a mercury or deuterium light source, a monochromator used to isolate a narrow bandwidth (10 nm) or spectral line (i.e. 254 nm for Hg), a flow cell with a volume of a few pi (optical path 0.1 to 1 cm) and a means of optical detection. This system is an example of a selective detector the intensity of absorption depends on the analyte molar absorption coefficient (see Fig. 3.13). It is thus possible to calculate the concentration of the analytes by measuring directly the peak areas without taking into account the specific absorption coefficients. For compounds that do not possess a significant absorption spectrum, it is possible to perform derivatisation of the analytes prior to detection. 

Figure 3.14—Principle of the diode array detector. The flow cell is irradiated with a polychromatic UV/Vis light source. The light transmitted by the sample is dispersed by reflection on a grating and the reflected intensities are monitored by an array of photodiodes. Several hundred photodiodes can be used, each one monitoring the mean absorption of a narrow band of wavelengths (i.e. 1 nm).

Absorption and Fluorescence Instrumentation. Absorption spectra were obtained using a Princeton Applied Research Corp. (PARC) Model 1208 polychromator, a Perkin-Elmer 8 yL absorption flow-cell and a 50 watt deuterium light source. Fluorescence spectra were obtained using a Farrand Mark 1 Spectrofluorometer (previously described (13)) and either a 10 iiL Farrand micro flow-cell, or a Precision Cells, Inc. (Model No. 8830) 20 yL flow-cell. A PARC Model 1254 SIT detector, having a UV scintillator, was mounted on both the absorption polychromator and fluorescence spectrofluorometer. Spectral coverage in the absorption and fluorescence modes was 60 and 115 nm, respectively. All absorption and fluorescence spectra were obtained in one second, i.e., 32 scans of the SIT target. 

Ki, the detector response factor, describes the signal generated when particles are present in the eluant as it transits the detector flow-through cell. Detector response arises primarily from scattering of light by the latex particles (15), although a small contribution from light absorption by the sample may occur (16). Polystyrene latex standards of known size and concentration were used to determine Ki factors for conversion of detector response into mass concentration information. 

Many detection principles require a finite volume of eluent. For example, a UV absorption detector yields a signal that is directly proportional to the optical pathlength (Beer s law, see eqn.5.21). The volume of the detector flow cell is usually well-defined and its contribution to aejc, and hence its effects on the observed dispersion ctg, can be discussed in quantitative terms (see section 7.4.2). 

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