How to Collect National Institute of Standards and Technology (NIST) Traceable Fluorescence Excitation and Emission Spectra

Contemporary spectrofluorimeters comprise exciting light sources, excitation and emission monochromators, and detectors that without correction yield data not conforming to an ideal spectral response. The correction of the spectral properties of the exciting and emission light paths first requires calibration of the wavelength and spectral accuracy. The exciting beam path can be corrected up to the sample position using a spectrally corrected reference detection system. The corrected reference response accounts for both the spectral intensity and drift of the exciting light source relative to emission and/or transmission detector responses. The emission detection path must also be corrected for the combined spectral bias of the sample compartment optics, emission monochromator, and detector. There are several crucial issues associated with both excitation and emission correction including the requirement to account for spectral band-pass and resolution, optical band-pass or neutral density filters, and the position and direction of polarizing elements in the light paths. In addition, secondary correction factors are described including (1) subtraction of the solvent’s fluorescence background, (2) removal of Rayleigh and Raman scattering lines, as well as (3) correcting for sample concentration-dependent inner-filter effects. The importance of the National Institute of Standards and Technology (NIST) traceable calibration and correction protocols is explained in light of valid intra- and interlaboratory studies and effective spectral qualitative and quantitative analyses including multivariate spectral modeling.