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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 \u201ctime\u2010zero extrapolated\u201d heteronuclear single quantum coherence (HSQC<jats:sub>0<\/jats:sub>) 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 <jats:sup>1<\/jats:sup>H NMR, it is validated that the HSQC<jats:sub>0<\/jats:sub> 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\u2010resonance 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\u2010less organelles.<\/jats:p>","DOI":"10.1002\/cbic.202500401","type":"journal-article","created":{"date-parts":[[2025,7,24]],"date-time":"2025-07-24T09:14:43Z","timestamp":1753348483000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Quantification of Small Molecule Partitioning in a Biomolecular Condensate with 2D Nuclear Magnetic Resonance Spectroscopy"],"prefix":"10.1002","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-5706-723X","authenticated-orcid":false,"given":"Julie Maib\u00f8ll","family":"Buhl","sequence":"first","affiliation":[{"name":"Interdisciplinary Nanoscience Center (iNANO) Aarhus University  Gustav Wieds Vej 14 Aarhus C 8000 Denmark"},{"name":"Institute of Biochemistry Johannes Kepler University Linz  Altenberger Stra\u00dfe 69 4040 Linz Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sujata","family":"Mahapatra","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics Aarhus University  Universitetsbyen 81 Aarhus C 8000 Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7020-9366","authenticated-orcid":false,"given":"Magnus","family":"Kj\u00e6rgaard","sequence":"additional","affiliation":[{"name":"Interdisciplinary Nanoscience Center (iNANO) Aarhus University  Gustav Wieds Vej 14 Aarhus C 8000 Denmark"},{"name":"Department of Molecular Biology and Genetics Aarhus University  Universitetsbyen 81 Aarhus C 8000 Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9427-8406","authenticated-orcid":false,"given":"Frans A. 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