UV/Visible Diffusion-Ordered Spectroscopy: A Simultaneous Probe of Molecular Size and Electronic Absorption

Based on concepts from nuclear magnetic resonance, we have developed UV/vis diffusion-ordered spectroscopy, which simultaneously probes the size and electronic absorption spectrum of molecules and particles. We use simple flow technology to create a step-function concentration profile inside an optical sample cell, and by measuring the time-dependent absorption spectrum in an initially solvent-filled part of the sample volume, we obtain the diffusion coefficients and UV/vis spectra of the species present in the sample solution. From these data, we construct a two-dimensional spectrum with absorption wavelength on one axis and diffusion coefficient (or equivalently, size) on the other, in which the UV/vis spectrum of a mixture with different molecular sizes is separated into the spectra of the different species, sorted by size. We demonstrate this method on mixed solutions of fluorescent dyes, biomolecules, and the UV-absorbing components of coffee, caffeine, and chlorogenic acid, all with concentrations in the μM range.