Pathlength independent spectrophotometric measurement of hemoglobin in solution

A spectrophotometric method that provides a quantitative estimation of hemoglobin concentration without explicit knowledge of the optical path length is described. Transmission measurements were made at six discrete wavelengths (476, 503, 661, 751, and 1015 nm) on solutions with hemoglobin content ranging form 3 g/dL to 14.8 g/dL, at path lengths from 100 μ to 1 mm. The resulting six-wavelength spectra were decomposed into contributions from both hemoglobin and water. The ratio of computed hemoglobin absorbance to the computed water absorbance is indicative of hemoglobin concentration, and is independent of the measurement path length. Application to noninvasive hematocrit monitoring is discussed