Quantitative Raman Spectroscopy when the Signal-to-Noise is Below the Limit of Quantitation due to Fluorescence Interference: Advantages of a Moving Window Sequentially Shifted Excitation Approach

Abstract

Raman spectroscopy is a useful analytical tool. However, its application is often limited because shot noise from fluorescence obscures the Raman signal. In such cases, quantitative analysis is not possible when the signal-to-noise ratio (SNR) drops below two. A method is described for performing quantitative Raman spectroscopy that not only removes fluorescence backgrounds, but also results in a significant improvement in the SNR. The Raman data is extracted using a moving window sequentially shifted excitation algorithm. To demonstrate the capabilities of the method, a binary mixture of two analytes at varying concentrations is quantified in the presence of a highly fluorescent dye. Linear calibration plots were constructed and validated for the binary model using individual Raman peaks with SNR ranging from 0.073–12.6; r² values are greater than 0.96 in all cases, with all but the weakest peaks yielding values greater than 0.997. The presented method demonstrates a universal and autonomous approach for the quantitative analysis of highly fluorescent samples via Raman spectroscopy. The lower limit on the SNR ratio for quantitative Raman analysis with the described method is 0.1. In order to assess the effectiveness of the presented method, the entire set of experiments was also processed using the more common shifted excitation Raman difference spectroscopy (SERDS) approach. The advantages of the proposed method over SERDS are demonstrated for both the detection limit and the SNR of the processed spectra.