Significance and Use 4.1 When determining the limiting detectable concentration of a fluorescent substance, it is usually necessary to increase the readout scale of a photoelectric instrument to a point where noise (that is, random fluctuations of the system) becomes apparent. This noise will be superimposed upon the signal from the sample. 4.2 In molecular fluorescence spectroscopy, the limit of detection for the sample will be determined by the limiting signal-to-noise ratio, S/N, where the signal, S, is the difference between readings obtained with the sample and blank solutions, and N is the total root-mean-square (rms) noise. The limit of detection for the sample will be given by the instrument readings that give a signal equal to three times the rms value of the noise. Note 2: Factors other than noise affecting the sample concentration corresponding to the limit of detection include: the spectral bandwidths of the excitation and emission monochromators, the intensity of the exciting light that can be concentrated on the sample, the fraction of the fluorescence collected by the detection system, the response time of the detection system, and the purity of the solvent. The size and arrangement of the sample container with respect to the light beams are also important, as they affect both the desired signal and the extraneous signal that only contributes noise. Note 3: The value of rms noise (N) can be obtained by calculating the standard deviation of a series of readings of the signal from the sample at the peak emission wavelength at approximately 450 nm as follows: where: x = mean of the series of readings, x = value of the individual reading, and n = number of readings. Alternatively, rms noise may be estimated by noting the extreme differences between the members of a series of readings (peak-to-peak noise) and dividing by a factor that is usually taken to be 5.6, 7 Scope 1.1 This test method employs the signal-to-noise ratio to determine the sensitivity of a fluorescence measuring system in testing for the limit of detection (LOD) of quinine sulfate dihydrate in solution. The results obtained with quinine sulfate dihydrate in solution are suitable for specifying instrument performance on samples having excitation and fluorescence bands wider than 10 nm at or near room temperature. 1.1.1 This test method is not intended to be used as (1) a rigorous test of performance of instrumentation, or (2), to intercompare the quantitative performance of instruments of different design. Intercomparison of the LOD between instruments is commonly expressed as the ratio of the water Raman peak intensity to the root-mean-square (rms) noise as measured on a fluorometer using an excitation wavelength of 350 nm This test method uses the excitation and emission peak wavelengths for quinine sulfate dihydrate in solution, which are approximately 350 nm and 450 nm, respectively. 1.2 This test method has been applied to fluorescence-measuring systems utilizing non-laser, low-energy excitation sources. There is no assurance that extremely intense illumination will not cause photodecomposition2 of the compound suggested in this test method. For this reason, it is recommended that this test method not be indiscriminately employed with high intensity light sources. This test method is not intended to determine minimum detectable amounts of other materials. If this test method is extended to employ other chemical substances, the user should be aware of the possibility that these other substances may undergo decomposition or adsorption onto containers. 1.3 A typical LOD for conventional fluorometers using this test method is 1 ng of quinine sulfate per mL. 1.4 The suggested shelf life of a 1 mg/mL stock solution of quinine sulfate dihydrate is three months, when stored in the dark in a stoppered glass bottle. 1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.6 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
