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2.4 FLUORESCENCE SPECTROPHOTOMETRY

      Fluorescence spectrophotometry is the measurement of the emission of light from a chemical substance while it is being exposed to ultraviolet, visible, or other electromagnetic radiation. In general, the light emitted by a fluorescent solution is of maximum intensity at a wavelength longer than that of the exciting radiation, usually by some 20 to 30 nm.

      The terms used with fluorescence spectrophotometry are defined as follows:

      Fluorescence intensity is an empirical expression of fluorescence activity, commonly given in terms of arbitrary units proportional to detector response. 

      The fluorescence emission spectrum is the relationship between the intensity of the emitted radiation and the wavelength and is frequently represented in a graphic form.

      The fluorescence excitation spectrum is the relationship between the maximum intensity of radiation emitted by an activated substance and the wavelength of the incident radiation and is frequently represented in a graphic form.

Apparatus

      Measurement of fluorescence intensity can be made with a simple filter fluorimeter. Such an instrument consists of a radiation source, a primary filter, a sample chamber, a secondary filter, and a fluorescence detection system. In most such fluorimeters the detector is placed on an axis at 90º from that of the incident beam. This right-angle geometry permits the incident radiation to pass through the test solution without contaminating the output signal received by the fluorescence detector. Filters are used to eliminate the incident radiation as a result of the inherent scattering properties of the solutions themselves, or if dust or other solids are present. The primary filter selects short wavelength radiation capable of causing excitation of the test substance, while the secondary filter is normally a sharp cut-off filter that allows the longer wavelength fluorescence to be transmitted but blocks the scattered excitated radiation.

      A fluorescence spectrophotometer differs from a filter fluorimeter in that filters are replaced by monochromators, of either the prism or the grating type.

      The cells used in fluorescence measurements may be rectangular cells similar to those used in absorption spectrophotometers, except that they are polished on all 4 vertical sides and on the bottom, or cells in the shape of round tubes with flat polished bottoms may be used. A convenient size is 2 to 3 ml, but some instruments can be fitted with small cells holding 0.1 to 0.3 ml, or with a capillary holder requiring even less solution.

      STANDARDIZATION Fluorimeters and fluorescence spectrophotometers should be standardized daily with a stable fluorophore to assure proper reproducibility of response. The changes are due to instrumental variables such as differences in lamp intensity and photomultiplier sensitivity. The fluorophore may be a pure specimen of the fluorescent substance under test or another readily purified fluorescent substance with absorption and fluorescence bands similar to those of the test substance. Quinine in dilute sulfuric acid is often a convenient fluorophore for blue fluorescence, sodium fluorescein for green fluorescence, and rhodamine for red fluorescence.

      The wavelength scale of the fluorescence spectrophotometer should be periodically calibrated. 

      METHOD Dissolve the substance to be examined in the solvent or mixture of solvents prescribed in the monograph. Solutions prepared for fluorescence spectrophotometry are usually 10 to 100 times less concentrated than those used in absorption spectrophotometry. Transfer the solution to the cell or the tube of the fluorimeter and illuminate with an excitant light beam of the wavelength prescribed in the monograph and as nearly monochromatic as possible.

      Measure the intensity of the emitted light at an angle of 90º to the excitant beam, after passing it through a filter which transmits predominantly light of the wavelength of the fluorescence.

      For quantitative determinations, first introduce into the apparatus the solvent or mixture of solvents used to dissolve the substance to be examined and set the instrument to zero. Introduce the standard solution and adjust the sensitivity of the instrument so that the reading is greater than 50. If the second adjustment is made by altering the width of the slits, a new zero setting must be made and the intensity of the standard must be measured again. Finally introduce the solution of the substance being examined, record the intensity and calculate its concentration.

      If the intensity of the fluorescence is not directly proportional to the concentration, the measurement may be effected using a calibration curve.

      In some cases, measurement can be made with reference to a fixed standard (for example, a fluorescent glass or a solution of another fluorescent substance). In such cases, the concentration of the substance to be examined must be determined using a previously drawn calibration curve under the same conditions. v