Quantitative Measurements at Fixed Wavelength

The simpler fluorimeters are manual instruments operating only at a single selected wavelength at any one time. Despite this they are perfectly suitable for quantitative measurements, as these are almost always carried out at a fixed wavelength. The experiments listed at the end of this chapter have all been carried out at single fixed wavelengths. 

Figure 11.12—Schematic ant optica path of a single beam spectrophotometer equipped with electronic regulation (Hitachi U-1000). Measurements in solution are often carried out at a fixed wavelength after a calibration curve has been plotted. The use of higher performance double beam UV/Visible spectrometers is not necessary for these measurements in which the spectrum is not recorded. On the other hand, quantitative measurements from mixtures represent a different type of analysis.

In conventional fluorescence analysis the majority of quantitative measurements is made using fixed wavelengths for excitation and emission. At low concentrations, a plot of the fluorescent power of a solution vi. the concentration of the emitting species ordinarily is linear. As to the sensitivity of fluorescence procedures in analysis, it is convenient to separate the contributions from the properties of the fluorescent molecule itself absolute sensitivity), the performance of the instrument instrumental sensitivity) and the chemistry involved in the preparation of the sample method sensitivity). The absolute sensitivity is determined chiefly by the molar absorptivity and the fluorescence efficiency of the analyte molecule itself. High fluorescence efficiency is usually associated with some rigidity. The method sensitivity takes account of pre-concentration steps in the preparation of the sample on the one hand and the limitations imposed by the fluorescence of the blank on the other. The sensitivities and selectivi-ties attained by fluorescence, phosphorescence and chemiluminescence are hardly paralleled by other techniques. In many cases it is not required that the analyte be isolated from the matrix. 

The validity of the Beer-Lambert law should always be tested before using it for accurate quantitative analysis. To test the Beer-Lambert law it is necessary to measure absorbance ( 4) as a function of concentration (c) at a fixed wavelength (A) and cell (film) path (/) (Fig. 10.57). If the Beer-Lambert law is obeyed over the concentration range tested, a straight line should be obtained through the origin. Deviations from the law are designated as... 

Emission spectra can provide important information about the nature and energy of the emitting excited state and can, obviously, be used in analytical chemistry, both qualitative (the emission spectram is characteristic of the luminophore) and quantitative (the intensity depends on the concentration, as we will see below). To record an emission speetrum, the sample is excited at a fixed wavelength, at which it absorbs and, by moving the emission monochromator, we measure how... 

In this section we deal with quantitative steady-state luminescence intensity determination in solution, at a fixed emission wavelength, using a commercial spectrofluorimeter with right-angle excitation (perpendicular geometry). These kind of measurements are particularly important in analyte detection, titrations, quenching and sensitization experiments, photoreaction and photoluminescence quantum yield determination, and whenever a luminescence signal is used to monitor a chemical process. 

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