High-Efficiency Detection Systems

The setup illustrated on the right uses a confocal principle. The laser is directed into the euvette by a diehroie mirror, D, and focused by a lens. The fluorescence light is colleeted by this lens, sent through a set of filters, focused by a second lens, and detected by the PMT module. The setup can be built with a high numeri-eal aperture and a eorrespondingly high collection efficiency. 

By using a field stop in front of the detector, light from outside the focus can be suppressed. This can be useful to suppress fluorescence of the cuvette walls, fluorescence of dye moleeules bound to the cuvette walls, or distortions by scattering or reabsorption. Confocal detection can also be used to reduce the daylight-sensitivity of the detection system. 

Simple systems such as that shown in Fig. 5.11 yield an excellent optical efficiency, and are almost free of pulse dispersion and wavelength-dependent pulse shift. Another benefit is that the detection path is almost free of polarisation effects compared to monochromator-based systems. The high numerical aperture of the light eollection system further reduces the influence of the rotational relaxation see Fig. 5.9. With aspheric lenses an NA of around 1 can be achieved, and at an NA this high the polariser in the detection path can often be omitted. In the setup in the left graphic of Fig. 5.11, residual depolarisation effects can be removed by slightly tilting the polarisation direetion of the laser. 

The downside of the compact design and the high numerieal aperture is that the systems are vulnerable to optical reflections. Reflections are particularly likely between the PMT cathode and an interference filter, or between an interference filter and another flat optical surface (see Sect. 7.2.6, page 285). Replacing an interference filter with an absorptive filter solves most reflection problems, but often causes problems by filter fluorescence. 

In the setup shown in Fig. 5.11, right, fluoreseenee from the lens and the di-chroic mirror ean eause problems. Moreover, exeitation light scattered at the di-chroic mirror and reflected at the lens can cause false prepulses when the IRF has to be reeorded. These problems can be largely avoided by using a small laser beam diameter, a small mirror instead of the dichroie beamsplitter, and a hole in the centre of Lens 1. 

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Dichroic beam splitters (or filters) use the same principle as interference filters. They are placed in a collimated beam, typically at a 45-degree angle. There are long-pass, short-pass and bandpass versions. They are extremely suitable for building high-efficiency optical systems. Figure 7.17, left, shows how a laser is coupled into a high-NA fluorescence excitation and detection system. 

Fig. 7.17 Use of dichroic beam splitters in high-efficiency optical systems. Left Coupling a laser into a high NA fluorescence system. Right Splitting fluorescence light into several wavelength channels for simultaneous detection with individual PMTs...

As stated earlier, taste, chemicals that initiate a gustatory response, and smell, chemicals that function as odorant molecules have been extensively studied, first with arthropods and more recently with vertebrates. Since the development of very efficient separation techniques and high sensitive detection systems, we are no longer limited to taste panels and threshold odor detection by humans. Table I shows some examples of the behavior response as shown by certain species for a specific chemical. 

With the development of HPLC, a new dimension was added to the tools available for the study of natural products. HPLC is ideally suited to the analysis of non-volatile, sensitive compounds frequently found in biological systems. Unlike other available separation techniques such as TLC and electrophoresis, HPLC methods provide both qualitative and quantitative data and can be easily automated. The basis for the HPLC method for the PSP toxins was established in the late 1970 s when Buckley et al. (2) reported the post-column derivatization of the PSP toxins based on an alkaline oxidation reaction described by Bates and Rapoport (3). Based on this foundation, a series of investigations were conducted to develop a rapid, efficient HPLC method to detect the multiple toxins involved in PSP. Originally, a variety of silica-based, bonded stationary phases were utilized with a low-pressure post-column reaction system (PCRS) (4,5), Later, with improvements in toxin separation mechanisms and the utilization of a high efficiency PCRS, a... 

Also, subcritical (hot/liquid) water can be used as a mobile phase for packed-column RPLC with solute detection by means of FID [942]. In the multidimensional on-line PHWE-LC-GC-FTD/MS scheme, the solid sample is extracted with hot pressurised water (without the need for sample pretreatment), and the analytes are trapped in a solid-phase trap [943]. The trap is eluted with a nitrogen flow, and the analytes are carried on to a LC column for cleanup, and separated on a GC column using the on-column interface. The closed PHWE-LC-GC system is suitable for many kinds of sample matrices and analytes. The main benefit of the system is that the concentration step is highly efficient, so that the sensitivity is about 800 times better than that obtained with traditional methods [944]. Because small sample amounts are required (10 mg), special attention has to be paid to the homogeneity of the sample. The system is... 

The multichannel coulometric detection system serves as a highly sensitive tool for the characterization of antioxidant phenolic compounds because they are electroactive substances that usually oxidize at low potential. The coulometric efficiency of each element of the array allows a complete voltammetric resolution of analytes as a function of their oxidation potential. Some of the peaks may be resolved by the detector even if they coelute (Floridi and others 2003). 

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