Several intrinsically disordered proteins have been shown to undergo phase separation and it is highly relevant to know how small molecule metabolites distribute between these phases. It is shown here that the partitioning can be robustly obtained from 2D time-zero heteronuclear single quantum coherence nuclear magnetic resonance spectra recorded on the dilute and dense phases separately.

Several intrinsically disordered proteins have been shown to undergo phase separation into a dense and dilute phase and this process is intimately linked with the regulation of cellular processes. It is therefore highly relevant to know how metabolites partition between these phases. It is shown here that the partitioning of components in a complex mixture can be robustly obtained from a single set of 2D nuclear magnetic resonance (NMR) spectra recorded on the dilute and dense phases separately using “time-zero extrapolated” heteronuclear single quantum coherence (HSQC₀) spectroscopy. The spectral separation power of 2D NMR spectroscopy circumvents the need for physical isolation or workup of the mixture components in the two samples. Using quantitative 1D ¹H NMR, it is validated that the HSQC₀ approach effectively removes all the undermining effects that plague quantification in common 2D NMR experiments, including differential attenuation due to relaxation in the two phases, pulse imperfections, partial decoupling, off-resonance effects, and incomplete coherence transfer in case of scalar coupling variation. These results should be of widespread interest as partitioning into biomolecular condensates is crucial for the calibration of computational physicochemical models of phase separation and key to the further understanding of cellular biochemistry involving membrane-less organelles.