Analyte detector module

Analyte detector module (UV VIS dual wavelength detector). 

Figure 1. Photograph and block diagram of a modem, automated modular HPLC analytical system (Kontron Instrumems-HPLC system 400). (1) The eluant delivery module (dual piston pumps) (2) sample application module (automated) (3) HPLC column module (4) analyte detector module (UV-VIS dual wavelength detector) (5) central control module (controls module 1 —4 and 6) (6) data output module (printer-plotter).

In this discussion only continuous HPLC monitoring or on-line devices will be considered since the discrete methods referred to earlier in the introduction involving sequential analysis of collected eluate fractions are laborious, time consuming and are not therefore used today. An on-line HPLC analyte detector module is defined as a carefully designed device, through which the eluate from the HPLC column flows, and which (in most cases) generates a continuous electrical output signal that is a function of the mass of the analyte or of the concentration of the analyte in the mobile phase. 

Because of the importance of this module a really vast amount of research and development time and energy has been spent on trying to develop an ideal universal detector—flow-through cell combination. The specifications for such an analyte detector module are as follows. 

In spite of the large expenditure of research and development time and energy on this topic by industry and by academia, the construction of an ideal universal analyte detector and flow-through cell module which fulfills all of these specifications still eludes us. Indeed the current instrumentation situation is that a very large number of different analyte detector modules are now available so that the chromatographer is presented with an additional problem—namely which one (or more) to choose. The names of these different detector modules are listed in Table 2, together with some comments concerning their applicability to particular problems. 

Table 2. A listing and commentaiy on some analyte detector modules which are commercially available.

These detectors, when used in conjunction with a well designed, low volume, flowthrough cell, continuously monitor the absorbance of aiQf UV (or visible) absorbing analytes in the HPLC column eluate at one or many wavelengths. This critique of those most popular analyte detector modules will be presented in relation to the previously listed specifications of an ideal detector. 

In multi-residue analysis, an analyte is identified by its relative retention time, e.g., relative to aldrin when using ECD or relative to parathion or chlorpyrifos when using a flame photometric detection (FPD) and NPD. Such relative retention times are taken from corresponding lists for the columns used. Further evidence for the identity of an analyte is provided by the selectivity of the different detectors (Modules D1 to D3), by its elution behavior during column chromatography (Modules Cl and C2) and in some cases even by the peak form in a gas chromatogram. In a specific analysis for only some individual analytes, their retention times are compared directly with the corresponding retention times of the analytes from standard solutions. 

Four of the entries shown in the comment column of Table 3 for macromolecules also indicate that problems exist. The subjects of the first, tenth and thirteenth entries in Table 3, namely sample pre-treatment procedures, choice of detector and collection of separated fractions, are all connected in that they arise from the complexity of the analyte mixtures, a subject discussed previously. The subject of the first entry constitutes the major, at present unsolved, problem in the separation of macromolecules. As a result it may be confidently predicted that much of the instmment manufacturers research and development efforts at the present time lies in this area of automated sample pretreatment devices suitable for mixtures of macromolecules because this area must be automated if the whole HPLC process is to be automated. The problem indicated in the tenth entry of Table 3, concerning the choice of detector for macromolecules was also discussed in the previous part. Therefore it is sufficient to note here that because of the lack of a universal, sensitive detector for macromolecules two or more of the available detector molecules, arranged in tandem, may need to be employed. Alternatively if a single detector module of the type already discussed in the previous section is used, then discrete fractions of the eluate must be collected for subsequent off-line analysis by say, gel electrophoresis, immuno- or bio-assay procedures. This alternative practice accounts for the optional entry number 13 in Table 3 regarding the provision of a fraction collector. 

Module 3, Column and Mobile Phase Design (CMP). This is the core module for ECAT. It can currently specify i) analytical column and mobile phase constituents for reverse phase chromatography of common classes of organic molecules ii) reverse phase, ion exchange phase and hydrophobic interaction chromatography of proteins and peptides iii) a limited set of specialty classes of molecules best treated by straight phase chromatography (e.g., mono- and disaccharides). The rules for selection of the HPLC detector are under development within Module 3. Some of the rules for detector mobile phase compatibility are already encoded. A set of rules for detector selection is ready but not yet encoded. 

In atomic absorption spectrometry (AA) the sample is vaporized and the element of interest atomized at high temperatures. The element concentration is determined based on the attenuation or absorption by the analyte atoms, of a characteristic wavelength emitted from a light source. The light source is typically a hollow cathode lamp containing the element to be measured. Separate lamps are needed for each element. The detector is usually a photomultiplier tube. A monochromator is used to separate the element line and the light source is modulated to reduce the amount of unwanted radiation reaching the detector. 

The optical source is a diode laser and fiber-emitter and fiber-detector coupling is accomplished using standard optical fiber connectors. The detector is a PIN photodiode connected to a transimpedance preamplifier and the signal is amplified and filtered using a lock-in amplifier that also tunes the modulation frequency of the laser source. The analytical signal is collected and treated by a PC. 

Microstructures and systems are typically fabricated from rigid materials, such as crystalline silicon, amorphous silicon, glass, quartz, metals and organic polymers. Elastomeric materials can be used in applications where rigidity is a drawback. We have demonstrated the concepts of elastomeric systems by fabrication of photothermal detectors, optical modulators and light valves. We believe that elastomeric materials will find additional applications in the areas of optical systems, micro analytical systems, biomaterials and biosensors. 

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