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Particle size is of great importance for the quantitative model of the NIR diffuse reflectance. In this paper, the effect of sample particle size on the measurement of harpagoside in  Radix Scrophulariae  powder by near infrared diffuse (NIR) reflectance spectroscopy was explored. High-performance liquid chromatography (HPLC) was employed as a reference method to construct the quantitative particle size model. Several spectral preprocessing methods were compared, and particle size models obtained by different preprocessing methods for establishing the partial least-squares (PLS) models of harpagoside. Data showed that the particle size distribution of 125–150 μm for  Radix Scrophulariae  exhibited the best prediction ability withR    pre    2= 0.9513, RMSEP = 0.1029 mg·g −1 , and RPD = 4.78. For the hybrid granularity calibration model, the particle size distribution of 90–180 μm exhibited the best prediction ability withR    pre    2= 0.8919, RMSEP = 0.1632 mg·g −1 , and RPD = 3.09. Furthermore, the Kubelka-Munk theory was used to relate the absorption coefficient  k  (concentration-dependent) and scatter coefficient  s  (particle size-dependent). The scatter coefficient  s  was calculated based on the Kubelka-Munk theory to study the changes of  s  after being mathematically preprocessed. A linear relationship was observed between  k / s  and absorption  A  within a certain range and the value for  k / s  was >4. According to this relationship, the model was more accurately constructed with the particle size distribution of 90–180 μm when  s  was kept constant or in a small linear region. This region provided a good reference for the linear modeling of diffuse reflectance spectroscopy. To establish a diffuse reflectance NIR model, further accurate assessment should be obtained in advance for a precise linear model.

Discovery of the Linear Region of Near Infrared Diffuse Reflectance Spectra Using the Kubelka-Munk Theory