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The need for the traceable characterization of fluorescence instruments is emphasized from a chemists point of view, focusing on spectral fluorescence standards for the determination of the wavelength- and polarization-dependent relative spectral responsivity and relative spectral irradiance of fluorescence measuring systems, respectively. In a first step, major sources of error of fluorescence measurements and instrument calibration are revealed to underline the importance of this issue and to illustrate advantages and disadvantages of physical and chemical transfer standards for generation of spectral correction curves. Secondly, examples for sets of traceable chemical emission and excitation standards are shown that cover a broad spectral region and simple procedures for the determination of corrected emission spectra with acceptable uncertainties are presented. With proper consideration of the respective measurement principle and geometry, these dye-based characterization procedures can be not only applied to spectrofluorometers but also to other types of fluorescence measuring systems and even to Raman spectrometers.
The development of fluorescence applications in the life and material sciences has proceeded largely without sufficient concern for the measurement uncertainties related to the characterization of fluorescence instruments. In this first part of a two-part series on the state-of-the-art comparability of corrected emission spectra, four National Metrology Institutes active in high-precision steady-state fluorometry performed a first comparison of fluorescence measurement capabilities by evaluating physical transfer standard (PTS)-based and reference material (RM)-based calibration methods. To identify achievable comparability and sources of error in instrument calibration, the emission spectra of three test dyes in the wavelength region from 300 to 770 nm were corrected and compared using both calibration methods. The results, obtained for typical spectrofluorometric (0°/90° transmitting) and colorimetric (45°/0° front-face) measurement geometries, demonstrated a comparability of corrected emission spectra within a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based spectral correction when measurements and calibrations were performed under identical conditions. Moreover, the emission spectra of RMs F001 to F005, certified by BAM, Federal Institute for Materials Research and Testing, were confirmed. These RMs were subsequently used for the assessment of the comparability of RM-based corrected emission spectra of field laboratories using common commercial spectrofluorometers and routine measurement conditions in part 2 of this series (subsequent paper in this issue).