Detection wavelength proper selection

The ultimate goal of an assay method is the separation and visualization of all components in a single chromatogram. Proper selection of detection wavelength is a critical part of method development. When choosing a detection wavelength, the following factors need to be taken into consideration ... 

The next stepping-stone to photoionization is finding the electronic levels of the neutral, because nonresonant ionization has rather low cross-sections that translate into poor ionization efficiencies along with high photon flux requirements. Resonant absorption of photons is more effective by several orders of magnitude [91]. Ideally, resonant absorption of the first photon leads to an intermediate state from where absorption of a second photon can forward the molecule into a continuum. This technique is known as 1 -i-1 resonance-enhanced multiphoton ionization (REMPI). From a practical point of view, the second photon should be, but not necessarily has to be, of the same wavelength (Fig. 2.20) [92]. Proper selection of the laser wavelengths provides compound-selective analysis at extremely low detection limits [90,91,93,94]. 

The confocal epifluorescent detection scheme we use is common among many who use this detection mode and features a cube-and-rail assembly system, specifically the microbench system from LINOS Photonics (Milford, MA) on an optical breadboard to maintain proper alignment of components (Figure 45.13). The excitation source is a multiline argon ion gas laser (model Reliant 150 m. Laser Physics, West Jordan, UT) that features user-selectable wavelengths (457,488, and 514 nm)... 

This chapter is devoted to a discussion of instruments and techniques that are of fundamental importance for the measurements of wavelengths and line profiles, or for the sensitive detection of radiation. The optimum selection of proper equipment or the application of a new technique is often decisive for the success of an experimental investigation. Since the development of spectroscopic instrumentation has shown great progress in recent years, it is most important for any spectroscopist to be informed about the state-of-the-art regarding sensitivity, spectral resolving power, and signal-to-noise ratios attainable with modern equipment. 

The infrared probe, which resembles a specific ion electrode, contains a sensitive element that is dipped into the sample. To operate the probe, (1) the user selects the proper wavelength by rotating a calibrated, circular variable filter (2) then adjusts the gain and slits to bring the meter to 100% (3) next, the probe is lowered into the sample. The meter indicates the absorbance. This value can be converted into concentration by reference to a previously prepared calibration curve. To detect the presence or absence of a particular fimctional group, the user scans through the portion of the spectrum where absorption bands characteristic of that group appear. 

While in most cases the color reactions used are not specific, numerous procedures can be made selective by the proper choice of pH and the addition of masking agents. Recent developments - in double-wavelength spectrophotometry have further increased the specificity of many direct methods. Separation and preconcentration techniques can be applied, but these impose a restriction on the detection limit based on the reproducibility and magnitude of the blank. Theoretical and practical considerations presently set the limit of detection on the most sensitive spectrophotometric methods at 5-20 ng. 

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