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Understanding the determinants of permeability and selectivity in biological channels serves two general purposes. It provides fundamental biophysical insights and allows the evaluation of new membrane proteins and artificial channel designs for the development of biomimetic separation membranes. This understanding relies on accurate ways to quantify unitary (single channel) solute and solvent permeabilities. However, the current research in biomimetic and bioinspired membranes and in protein biophysics tends to focus on relative permeabilities. Further, many methods and approximations currently used result in erroneous permeability values which hinder and complicate the comparison between channels. Combined, these gaps in the available measurements obscure the biophysical insights and impede the development of novel high-performance channels. In this review, we summarize in vitro model membrane systems useful to functionally characterize artificial as well as biological channels. We also critically discuss biophysical techniques capable of extracting permeability values and membrane channel densities, which are fundamental parameters required to determine accurate unitary permeability values. Pertinent examples are provided, along with advantages, disadvantages, and potential improvements needed for specific techniques to acquire accurate unitary permeability values for a diverse set of small molecules, including water, ions, weak acids and bases, neutral molecules, and protons. [Display omitted] • Membrane proteins and their artificial mimics are important for biology and engineering. • Accurate unitary solute and solvent permeabilities are important benchmarks. • Biophysical quantification methods can help to improve membranes for separations. • Artificial channel performance can be benchmarked to model membrane proteins. [ABSTRACT FROM AUTHOR]

The endeavors to understand the determinants of water permeation through membrane channels, the effect of the lipid or polymer membrane on channel function, the development of specific water flow inhibitors, the design of artificial water channels and aquaporins for the use in industrial water filtration applications all rely on accurate ways to quantify water permeabilities ( P f ). A commonly used method is to reconstitute membrane channels into large unilamellar vesicles (LUVs) and to subject these vesicles to an osmotic gradient in a stopped-flow device. Fast recordings of either scattered light intensity or fluorescence self-quenching signals are taken as a readout for vesicle volume change, which in turn can be recalculated to accurate P f values. By means of computational and experimental data, we discuss the pros and cons of using scattering versus self-quenching experiments or subjecting vesicles to hypo- or hyperosmotic conditions. In addition, we explicate for the first time the influence of the LUVs size distribution, channel distribution between vesicles and remaining detergent after protein reconstitution on P f values. We point out that results such as the single channel water permeability ( p f ) depend on the membrane matrix or on the direction of the applied osmotic gradient may be direct results of the measurement and analysis procedure., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)