Qualification of a fluorescence spectrometer for measuring true fluorescence spectra
Qualification of a fluorescence spectrometer for measuring true fluorescence spectra
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DOI：
10.1063/1.2715952
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发表时间：
2007-03-01
影响因子：
1.6
通讯作者：
Kramer, Gary W.
中科院分区：
文献类型：
--
作者：
DeRose, Paul C.;Early, Edward A.;Kramer, Gary W.
使用荧光检测的新分析方法变得越来越定量，并且需要易于使用的材料标准来进行荧光计鉴定和方法验证。 NIST 正在通过开发和制定此类标准来满足这一需求。这里报告的是该过程的第一步，即验证研究级荧光光谱仪用于测量候选参考材料的真实荧光光谱。 “真实”光谱在此定义为在校准波长并应用激发强度和检测系统响应的校正后，具有根据需要的相对或绝对荧光强度以及以高精度和已知精度报告的波长的光谱。比较了使用基于校准源 (CS) 和校准检测器 (CD) 的方法在相对和绝对强度校正荧光光谱中确定的不确定性。基于 CS 的方法给出的不确定性通常约为 +/- 5%（相对光谱校正），大约是用于确定相对和绝对光谱校正因子的基于 CD 的方法的一半。可以使用任一方法确定绝对光谱校正因子，而无需了解仪器的光学几何形状。我们发现绝对光谱校正因子比相应的相对校正因子具有更大的不确定性，基于 CS 的方法的不确定性典型值为 +/- 10% 至 +/- 15%，+/- 20% 或更多并不罕见，特别是对于低于 400 nm 的激发和发射波长。还探讨了检测系统非线性和仪器偏振比引起的不确定性。
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.