The depth near-infrared (NIR) radiation penetrates into a sample during spectral acquisitions in NIR reflectance microscopy was investigated for pharmaceutical materials. Cellulose and its derivatives are widely used as excipients for pharmaceuticals and hence, were the basis for this study. The evaluation of the depth of sample contributing to the measured reflected radiation (information depth) was achieved using varying thicknesses of cellulose placed on top of a substrate. Analyzing the change in the absorption profile of the substrate showed the relationship between thickness and absorption to be exponential. The information depth was evaluated using the point where the substrate signal was reduced by 50%, termed the DP<sub>50</sub> value. The DP<sub>50</sub> value ranged from 39 to 61 μm at ~1675 nm, but was found to have an exponential relationship with wavelength. Longer wavelengths had less penetration into the sample; at 2380 nm the DP<sub>50</sub> was ~27 μm but this increased to ~180 μm at 1100 nm. The sample size was determined using the information depth and an approximate model for the contributing sample volume. Sample size was found to be within the range of 0.03-418 μg of sample per NIR spectrum depending on the wavelength used.

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