An algorithm for effectively separating overlapping spectra of chlorophyll a and b in in vivo broadleaf leaves and the development of a low-cost non-destructive detector for their contents.

Chlorophyll a (Chla) and chlorophyll b (Chlb) in plant leaves play distinct physiological and ecological roles, but most portable chlorophyll meters, such as SPAD meters, mainly provide relative chlorophyll indices rather than the individual contents of Chla and Chlb in vivo leaf conditions. This limitation restricts the acquisition of component-specific pigment information from living leaves and reduces the usefulness of field measurements for validating satellite-derived vegetation biochemical parameters. One key difficulty is that the spectral responses of the two pigments are highly overlapped in vivo, making it difficult to quantitatively characterize their individual absorption contributions and determine suitable sensitive bands for low-cost optical detection. In this study, a Beer-Lambert-inspired pseudo-absorption framework was developed to characterize and effectively separate the overlapping absorption-related spectral responses of Chla and Chlb in leaves of broadleaf species under in vivo conditions. Unlike conventional spectral-index-based approaches, the proposed method provided a physically interpretable empirical basis for sensitive-band selection. Using leaf hyperspectral data, 700 nm and 650 nm were identified as the sensitive bands for Chla and Chlb, respectively, while 900 nm was selected as the reference band for optical path normalization. The algorithm achieved RMSE values of 3.001 μg·cm-2 for Chla and 1.805 μg·cm-2 for Chlb. Based on the selected bands and optimized channel bandwidths, a low-cost, non-destructive detector for leaf Chla and Chlb contents, named CabD, was developed by integrating controllable spectral generation from multi-band light sources with photoelectric detection. Validation showed that CabD achieved RMSE values of 1.743 μg·cm-2 for Chla and 0.871 μg·cm-2 for Chlb, respectively, with relative accuracy values of 94.746% and 92.789%. In situ validation using 84 intact leaves yielded RMSE values of 2.245 and 1.530 μg·cm-2, with corresponding relative accuracies of 93.86% and 90.24% for Chla and Chlb, respectively. These results demonstrate that the Beer-Lambert-inspired pseudo-absorption framework can support a physically interpretable empirical strategy for sensitive-band selection in low-cost in vivo chlorophyll detector design. The developed CabD provides a practical tool for non-destructive acquisition of component-specific chlorophyll information and can support field monitoring and remote sensing validation of vegetation biochemical parameters.