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Herein, a novel fully automatic reciprocating free\u2010flow isoelectric focusing (RFFIEF) system, a batch\u2010type of automated closed\u2010loop free\u2010flow isoelectric focusing (FFIEF), was developed, integrating a 24\u2010channel separation chamber, closed gas\u2013liquid circulation drive, and intelligent control system to achieve unattended standardized operation. Standard protein verification showed excellent flow stability and pH gradient reproducibility (pooled standard deviation [SD]\u00a0=\u00a00.19 pH units excluding ampholyte\u2010induced outliers). The system was applied to large\u2010scale separation of milk exosomes, which were effectively focused at pH 5.0\u20137.5. Exosome subpopulations were fractionated by surface charge: CD81\u2010positive subpopulations concentrated at pH 5.8\u20136.5, whereas CD63\u2010positive subpopulations were highly enriched at pH 6.9. This system breaks through the automation bottleneck of conventional devices, providing an efficient platform for large\u2010scale biological particle separation with broad industrial prospects.<\/jats:p>","DOI":"10.1002\/elps.70127","type":"journal-article","created":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T12:30:38Z","timestamp":1783686638000},"page":"894-907","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Novel Fully Automatic Free\u2010Flow Isoelectric Focusing System and Its Application in the Large\u2010Scale Isolation of Milk Exosomes"],"prefix":"10.1002","volume":"47","author":[{"given":"Yaoguo","family":"Wang","sequence":"first","affiliation":[{"name":"Faculty of Chemical Engineering and Energy Technology Shanghai Institute of Technology  Shanghai China"},{"name":"Joint Centre of Biochine\u2010Shanghai Jiao Tong University Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuai","family":"Qi","sequence":"additional","affiliation":[{"name":"Joint Centre of Biochine\u2010Shanghai Jiao Tong University Shanghai Jiao Tong University  Shanghai China"},{"name":"School of Automation and Intelligence Sensing Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Joint Centre of Biochine\u2010Shanghai Jiao Tong University Shanghai Jiao Tong University  Shanghai China"},{"name":"School of Automation and Intelligence Sensing Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengxi","family":"Cao","sequence":"additional","affiliation":[{"name":"School of Automation and Intelligence Sensing Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xu","family":"Xu","sequence":"additional","affiliation":[{"name":"Faculty of Chemical Engineering and Energy Technology Shanghai Institute of Technology  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,10]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/elps.200700725"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1039\/C7LC01105A"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/elps.200900771"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2015.03.023"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aau6977"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nri855"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0167\u20107799(97)01137\u20102"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ebiom.2025.105834"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ceb.2018.06.003"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.TM118.001192"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580\u2010022\u201000558\u20108"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1113\/jphysiol.2014.282665"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.2174\/1874325001610010241"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.snb.2024.136505"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.msec.2012.12.002"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2022.1009338"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.repbio.2017.09.007"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.3389\/fnut.2022.871346"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms232416106"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpcb.110"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0145686"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.8b00479"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules25235585"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12017\u2010021\u201008696\u20106"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-2550-6_15"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1002\/elps.202400071"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cej.2025.160850"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/elps.202400123"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1039\/D3LC00189J"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2017.06.008"},{"key":"e_1_2_9_32_1","doi-asserted-by":"crossref","unstructured":"D. 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