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To overcome these constraints, we developed an online microSPE-UPLC platform that enables fully automated, high-throughput peak fractionation. The system allows the injection of up to 1 mL of organic extract onto microSPE cartridges in a 96-well format for metabolite enrichment and cleanup. A dual-pump loading and refocusing strategy ensures quantitative transfer to UPLC, maintaining high chromatographic resolution while collecting narrow fractions on postcolumn microSPE. Fractions can be directly eluted with deuterated solvents for NMR analysis, eliminating drying steps and preserving volatile compounds. This approach enables milligram-scale isolation of natural products per compound within a single day. The workflow was validated using plant and fungal extracts, enabling the successful purification and characterization of several sesquiterpenoids and a diterpenoid.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c00842","type":"journal-article","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T12:02:05Z","timestamp":1789646525000},"page":"27885-27894","source":"Crossref","is-referenced-by-count":0,"title":["Fully Automated\nPurification of Small Molecules Using\nOnline MicroSPE-UPLC-SPE"],"prefix":"10.1021","volume":"98","author":[{"given":"Venkata Ganesh","family":"Jarubula","sequence":"first","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gerd","family":"Grewing","sequence":"additional","affiliation":[{"name":"Axel Semrau GmbH , Stefansbecke 42 , ,","place":["Sprockho\u0308vel, Germany, D-45549"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pauline","family":"Stark","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maximilian","family":"Frey","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]},{"name":"Heidelberg University , , ,","place":["Heidelberg, Germany, 69120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dario","family":"Esposto","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon","family":"Isfort","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marvin","family":"Hempel","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guillaume","family":"Patoine","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henriette","family":"Uthe","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9406-4245","authenticated-orcid":true,"given":"Alain","family":"Tissier","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0475-0672","authenticated-orcid":true,"given":"Gerd U.","family":"Balcke","sequence":"additional","affiliation":[{"name":"Leibniz Institute of Plant Biochemistry , , Weinberg 3 , ,","place":["Halle\/S., Germany, D-06120"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,17]]},"reference":[{"issue":"4","key":"2026092909091897900_cit1","doi-asserted-by":"publisher","first-page":"525","DOI":"10.1039\/c3np20106f","article-title":"Natural product isolation\u2013how\nto get from biological material to pure compounds","volume":"30","author":"Bucar","year":"2013","journal-title":"Nat. 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Agric.\nFood Chem."},{"issue":"4","key":"2026092909091897900_cit23","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1016\/0167-7012(91)90063-V","article-title":"Characterization\nand\nDetermination of Prehelminthosporol, a Toxin from the Plant Pathogenic\nFungus Bipolaris-Sorokiniana, Using Liquid-Chromatography Mass-Spectrometry","volume":"13","author":"Carlson","year":"1991","journal-title":"J. Microbiol Meth."},{"key":"2026092909091897900_cit24","unstructured":"Huber, U. M.; Ronald, E.\n          Principles in Preparative\nHPLC, 2007. https:\/\/warwick.ac.uk\/research\/rtp\/warwick-scientific-services\/polymercharacterisation\/analyticalresources\/prephplcprimer5989-6639en.pdf."},{"key":"2026092909091897900_cit25","unstructured":"Shreve, J. A..; Michael, R. 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Autom."},{"issue":"12","key":"2026092909091897900_cit28","doi-asserted-by":"publisher","first-page":"1457","DOI":"10.1007\/s44211-022-00190-8","article-title":"A review of the modern principles and applications\nof solid-phase extraction techniques in chromatographic analysis","volume":"38","author":"Badawy","year":"2022","journal-title":"Anal. Sci."},{"key":"2026092909091897900_cit29","doi-asserted-by":"publisher","first-page":"720955","DOI":"10.3389\/fmolb.2021.720955","article-title":"Recent Developments Toward Integrated\nMetabolomics Technologies (UPLC-MS-SPE-NMR and MicroED) for Higher-Throughput\nConfident Metabolite Identifications","volume":"8","author":"Ghosh","year":"2021","journal-title":"Front.\nMol. Biosci."},{"key":"2026092909091897900_cit30","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/j.chroma.2019.03.042","article-title":"Utility of dry load injection for an efficient natural\nproducts isolation at the semi-preparative chromatographic scale","volume":"1598","author":"Queiroz","year":"2019","journal-title":"J. Chromatogr. A"},{"key":"2026092909091897900_cit31","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1016\/j.chroma.2005.04.056","article-title":"Development and application of a new on-line SPE system\ncombined with LC-MS\/MS detection for high throughput direct analysis\nof pharmaceutical compounds in plasma","volume":"1080","author":"Alnouti","year":"2005","journal-title":"J. Chromatogr.\nA"}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c00842","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/38\/27885\/68540935\/acs.analchem.6c00842.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/38\/27885\/68540935\/acs.analchem.6c00842.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T13:52:12Z","timestamp":1790689932000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/38\/27885\/5431850\/Fully-Automated-Purification-of-Small-Molecules"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,17]]},"references-count":31,"journal-issue":{"issue":"38","published-online":{"date-parts":[[2026,9,17]]},"published-print":{"date-parts":[[2026,9,29]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c00842","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,17]]}}}