Using water raman intensity to determine the effective excitation and emission path lengths of fluorophotometers for correcting fluorescence inner filter effect

Authors: ZHANG, DONGMAO - Mississippi State University; NETTLES, CHARLES - Mississippi State University; HU, JUAN - Depaul University

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2015
Publication Date: 4/12/2015
Citation: Zhang, D., Nettles, C.B., Hu, J. 2015. Using water raman intensity to determine the effective excitation and emission path lengths of fluorophotometers for correcting fluorescence inner filter effect. Analytical Chemistry. 87:4917-4924.

Interpretive Summary: Fluorescence and Raman inner filter effects (IFE) cause spectral distortion and nonlinearity between spectral signal intensity with increasing analyte concentration. Convenient and effective correction of fluorescence IFE has been an active research goal for decades. We found that fluorescence and Raman IFE can be reliably corrected using an improved equation. This water Raman-based method is easy to implement. It does not involve complicated instrument geometry determination or difficult data manipulation. This work should be of broad significance to physical and biological sciences given the popularity of fluorescence techniques in analytical applications, and will help us develop rapid and sensitive methods in the future for detection of food-borne pathogens in fish and other foods.

Technical Abstract: Fluorescence and Raman inner filter effects (IFE) cause spectral distortion and nonlinearity between spectral signal intensity with increasing analyte concentration. Convenient and effective correction of fluorescence IFE has been an active research goal for decades. Presented herein is the finding that fluorescence and Raman IFE can be reliably corrected using the equation Icorr/Iobsd = 10dxAx + dmAm when the effective excitation and emission path lengths, dx and dm, of a fluorophotometer are determined by simple linear curve-fitting of Raman intensities of a series of water Raman reference samples that have known degrees of Raman IFEs. The path lengths derived with one set of Raman measurements at one specific excitation wavelength are effective for correcting fluorescence and Raman IFEs induced by any chromophore or fluorophore, regardless of the excitation and emission wavelengths. The IFE-corrected fluorescence intensities are linearly correlated to fluorophore concentration over 5 orders of magnitude (from 5.9 nM to 0.59 mM) for 2-aminopurine in a 1 cm × 0.17 cm fluorescence cuvette. This water Raman-based method is easy to implement. It does not involve complicated instrument geometry determination or difficult data manipulation. This work should be of broad significance to physical and biological sciences given the popularity of fluorescence techniques in analytical applications.