Analytical Chem istry - DePauw University

Chapter 10 Spectroscopic Methods619sourceexcitation wavelength selectorλ 1 λ2λ 3ormonochromator filtersamplesignal processormonochromator filterλ 1 λ2λ 3appropriate for quantitative work, although only a spectrofluorimeter canbe used to record an excitation or emission spectrum.The sample cells for molecular fluorescence are similar to those formolecular absorption. Remote sensing with fiber optic probes also can beadapted for use with either a fluorimeter or spectrofluorimeter. An analytethat is fluorescent can be monitored directly. For analytes that are not fluorescent,a suitable fluorescent probe molecule can be incorporated into thetip of the fiber optic probe. The analyte’s reaction with the probe moleculeleads to an increase or decrease in fluorescence.In s t r u m e n t s f o r Me a s u r i n g Ph o s p h o r e s c e n c eInstrumentation for molecular phosphorescence must discriminate betweenphosphorescence and fluorescence. Because the lifetime for fluorescence isshorter than that for phosphorescence, discrimination is easily achieved byincorporating a delay between exciting the sample and measuring phosphorescentemission. Figure 10.54 shows how two out-of-phase chopperscan be use to block emission from reaching the detector when the sampleis being excited, and to prevent source radiation from reaching the samplewhile we are measuring the phosphorescent emission.Because phosphorescence is such a slow process, we must prevent theexcited state from relaxing by external conversion. Traditionally, this hasbeen accomplished by dissolving the sample in a suitable organic solvent,usually a mixture of ethanol, isopentane, and diethylether. The resultingsolution is frozen at liquid-N 2 temperatures, forming an optically clearsolid. The solid matrix minimizes external conversion due to collisions betweenthe analyte and the solvent. External conversion also is minimizedoremission wavelength selectordetectorFigure 10.53 Schematic diagram for measuring fluorescenceshowing the placement of the wavelength selectorsfor excitation and emission. When a filter is used the instrumentis called a fluorimeter, and when a monochromator isused the instrument is called a spectrofluorimeter.fromsourceExcitating SampleopenblockedsampletodetectorMeasuring EmissionfromsourceopenblockedchoppersblockedsampleopentodetectorFigure 10.54 Schematic diagram showinghow choppers are used to prevent fluorescentemission from interfering with themeasurement of phosphorescent emission.