Qualification of a fluorescence spectrometer for measuring true fluorescence spectra

Abstract

New analytical methods using fluorescence detection are becoming increasingly quantitative and require easy-to-use material standards for fluorometer qualification and method validation. NIST is responding to this need by developing and producing such standards. Reported here is the first step in this process, which is to qualify a research-grade fluorescence spectrometer for measuring true fluorescence spectra of reference material candidates. "True" spectra are defined here as those with fluorescence intensity, either relative or absolute as required, and wavelength both being reported with high accuracy and known precision, after wavelength has been calibrated and corrections for excitation intensity and detection system response have been applied. The uncertainties determined in relative and absolute intensity-corrected fluorescence spectra using both calibrated source (CS)- and calibrated detector (CD)-based methods were compared. The CS-based method gave uncertainties, typically about +/-5% for relative spectral correction, that were about half that of the CD-based method for determining both relative and absolute spectral correction factors. Absolute spectral correction factors can be determined using either method without knowing the optical geometry of the instrument. The absolute spectral correction factors were found to have much larger uncertainties than the corresponding relative correction factors with uncertainties for the CS-based method of +/-10% to +/-15% being typical and +/-20% or more not being uncommon, particularly for excitation and emission wavelengths below 400 nm. Uncertainties arising from detection system nonlinearity and instrument polarization ratios were also explored.

Moving towards a technical specification for fluorescence excitation-emission mapping and absorbance analysis of colored dissolved organic matter

“…Three recent publications from NIST researchers have particular significance for EEM correction, including a reference to the recently released ASTM standard guide (E2719) for fluorescence calibration and correction [9], a reference to CDOM EEMs and all aspects of instrument and IFE correction, [2] and a reference to the validation of the Fluorolog ® as the true accurate fluorometer for its use in generating and validating a series of Standard Reference Materials [7]. Furthermore, a recent paper has been accepted outlining the results of a major international interlaboratory comparison for CDOM IHSS standard samples; this study was headed by researchers and the United States Geological Survey (USGS), and was the focused outcome of a recent 2008 Chapman conference sponsored by the American Geophysical Union [8].…”

“…The key issues surrounding analysis of the EEMs with PARAFAC are the importance of reproducible and accurate spectral and instrumental correction of the data [1,2,6,7,9]. A major required spectral-correction method discussed below involves the coordinated evaluation of the absorbance spectrum of the sample in order to correct for the optical processes known as "inner-filter effects" (IFEs) [10][11][12][13].…”

Determination of the photoluminescence quantum yield of dilute dye solutions (IUPAC Technical Report)

“…The spectral responsivity of the emission channel always depends on both wavelength and polarization. Both dependences can be accounted for by excitation and emission correction curves [36,48,55,56,[58][59][60]. Sample-related polarization effects reflect the size of the fluorescence anisotropy or (de)polarization of the sample [1].…”

“…3. As previously stated, this method can only be recommended for expert laboratories, which can obtain an excitation correction curve with minimum uncertainties [36,56]. Otherwise, the resulting uncertainties are too high compared to the measurement uncertainties achievable with the latter method.…”

Characterization of photoluminescence measuring systems (IUPAC Technical Report)

“…[15,16]. These demands enhance the need for internationally accepted procedures for instrument calibration and instrument performance validation (IPV) in conjunction with suitable standards [7,17,18]. However, with the exception of colorimetry or surface fluorescence [19,20], at present, there are only a few guidelines, recommendations, and technical notes for the characterization and performance validation of photoluminescence measuring instruments [13,[21][22][23][24][25][26][27][28][29] including choice of standards [1,6,10,11,17,21,24,[30][31][32][33], see also ref.…”