Confocal Raman Microscopy for in Situ Measurement of Octanol–Water Partitioning within the Pores of Individual C₁₈-Functionalized Chromatographic Particles

Abstract

Octanol-water partitioning is one of the most widely used predictors of hydrophobicity and lipophilicity. Traditional methods for measuring octanol-water partition coefficients (K(ow)), including shake-flasks and generator columns, require hours for equilibration and milliliter quantities of sample solution. These challenges have led to development of smaller-scale methods for measuring K(ow). Recent advances in microfluidics have produced faster and smaller-volume approaches to measuring K(ow). As flowing volumes are reduced, however, separation of water and octanol prior to measurement and detection in small volumes of octanol phase are especially challenging. In this work, we reduce the receiver volume of octanol-water partitioning measurements from current practice by six-orders-of-magnitude, to the femtoliter scale, by using a single octanol-filled reversed-phase, octadecylsilane-modified (C18-silica) chromatographic particle as a collector. The fluid-handling challenges of working in such small volumes are circumvented by eliminating postequilibration phase separation. Partitioning is measured in situ within the pore-confined octanol phase using confocal Raman microscopy, which is capable of detecting and quantifying a wide variety of molecular structures. Equilibration times are fast (less than a minute) because molecular diffusion is efficient over distance scales of micrometers. The demonstrated amount of analyte needed to carry out a measurement is very small, less than 50 fmol, which would be a useful attribute for drug screening applications or testing of small quantities of environmentally sensitive compounds. The method is tested for measurements of pH-dependent octanol-water partitioning of naphthoic acid, and the results are compared to both traditional shake-flask measurements and sorption onto C18-modified silica without octanol present within the pores.

Lacritin proteoforms prevent tear film collapse and maintain epithelial homeostasis

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Confocal Raman Microscopy Investigation of Self-Assembly of Hybrid Phospholipid Bilayers within Individual Porous Silica Chromatographic Particles

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Vibrational Spectroscopy for the Determination of Ionizable Group Content in Ionomer Materials

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“…In the experimental work presented, to compensate for variation in hydration levels and density among a set of samples, spectral intensities were normalized 27,36,37 relative to the total optical signal integrated across the wavenumber range investigated. Therefore, it was desirable to derive R in Eq.…”