This procedure uses the measurement of the intensity of the fluorescent light emitted by the substance under examination in relation to that emitted by a given reference standard.
A simple filter fluorimeter or a more sophisticated spectrophotofluorimeter may be used but the latter is superior for analytical purposes on account of wavelength selectivity, accuracy, precision and convenience.
Operate the instrument strictly in accordance with the manufacturer's instructions.
Dissolve the substance under examination in the solvent prescribed in the individual monograph, transfer the solution to the cell or the tube of the spectrofluorimeter or fluorimeter and illuminate it with an excitation light beam of the nominal wavelength prescribed in the monograph and as nearly monochromatic as possible.
Measure the intensity of the emitted light at an angle of 90° to the excitation beam, after passing it through a filter which transmits predominantly light of the wavelength of the fluorescence.
For quantitative analysis, introduce into the apparatus the solvent or the mixture of solvents used to dissolve the substance under examination and set the instrument to zero. Introduce the prescribed standard solution and adjust the sensitivity of the instrument so that the reading is close to the maximum. If the 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 under examination and record the intensity of fluorescence. Calculate the concentration, Cx of the substance in the solution to be examined, using the
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 such as a fluorescent glass or a solution of stated concentration of quinine in 0.05 M sulphuric acid or of fluorescein in 0.1 M sodium hydroxide. In such cases, the concentration of the substance under examination must be determined using a calibration curve previously prepared under the same conditions.
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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