3.1 Calibration of the responsivity of the detection system for emission (EM) as a function of EM wavelength (?_EM), also referred to as spectral correction of emission, is necessary for successful quantification when intensity ratios at different EM wavelengths are being compared or when the true shape or peak maximum position of an EM spectrum needs to be known. Such calibration methods are given here and summarized in Table 1. This type of calibration is necessary because the spectral responsivity of a detection system can change significantly over its useful wavelength range (see Fig. 1). It is highly recommended that the wavelength accuracy (see Test Method E388) and the linear range of the detection system (see Guide E2719 and Test Method E578) be determined before spectral calibration is performed and that appropriate steps are taken to insure that all measured intensities during this calibration are within the linear range. For example, when using wide slit widths in the monochromators, attenuators may be needed to attenuate the excitation beam or emission, thereby, decreasing the fluorescence intensity at the detector. Also note that when using an EM polarizer, the spectral correction for emission is dependent on the polarizer setting. (2) It is important to use the same instrument settings for all of the calibration procedures mentioned here, as well as for subsequent sample measurements.

FIG. 1 Example of Relative Spectral Responsivity of Emission Detection System (Grating Monochromator-PMT Based), (see Test Method E578) for which a Correction Needs to be Applied to a Measured Instrument-Specific Emission Spectrum to Obtain its True Spectral Shape (Relative Intensities).

3.2 When using CCD or diode array detectors with a spectrometer for ?_EM selection, the spectral correction factors are dependent on the grating position of the spectrometer. Therefore, the spectral correction profile versus ?_EM must be determined separately for each grating position used. (3)

3.3 Instrument manufacturers often provide an automated procedure and calculation for a spectral correction function for emission, or they may supply a correction that was determined at the factory. This correction can often be applied during spectral collection or as a post-collection correction. The user should be advised to verify that the automated vendor procedure and calculation or supplied correction are performed and determined according to the guidelines given within this standard.