{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T01:41:22Z","timestamp":1791855682989,"version":"4.3.4"},"reference-count":17,"publisher":"Wiley","issue":"2","license":[{"start":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T00:00:00Z","timestamp":1771891200000},"content-version":"vor","delay-in-days":23,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,2,1]],"date-time":"2026-02-01T00:00:00Z","timestamp":1769904000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100003246","name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["ENPPS.TA.019.001"],"award-info":[{"award-number":["ENPPS.TA.019.001"]}],"id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003246","name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["KICH1.ST01.20.041"],"award-info":[{"award-number":["KICH1.ST01.20.041"]}],"id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J of Separation Science"],"published-print":{"date-parts":[[2026,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Hydrophilic interaction chromatography (HILIC) is an attractive separation mode for the analysis of therapeutic oligonucleotides (ONs). ONs are very polar compounds that are commonly dissolved in highly aqueous media, whereas HILIC eluents often comprise a high percentage of organic solvent. This solvent mismatch can cause breakthrough and peak splitting. In this study, we investigated the effects of the sample solvent composition and injection volume on the HILIC separation of nucleobases and ONs, and to what extent an in\u2010line mixer between injector and column can mitigate breakthrough and peak splitting. Using isocratic HILIC with nucleobases as medium\u2010polar, less\u2010retentive test compounds, we illustrated that an injection solvent of 90% water results in peak broadening, which deteriorates with increasing injection volume, leading to serious peak deformations and asymmetries. Here, in\u2010line mixing did not improve peak shapes, as the mixer volume adds to band broadening and the homogenization of the injection solvent with the HILIC eluent does not lead to on\u2010column focusing conditions. When analyzing an ON mixture (15\/16\/17\u2010mer) dissolved in a weak solvent (30% water) by gradient HILIC, injection volumes could be increased up to 20\u00a0\u00b5L (5.7% of the column volume) without losing separation performance. However, when the ON mixture was dissolved in 100% water, injection volumes above 2\u00a0\u00b5L caused severe peak distortion and extensive analyte breakthrough in gradient HILIC. When injecting the ONs in 100% water with a 100\u2010\u00b5L in\u2010line mixer, no analyte breakthrough and peak splitting were observed for injection volumes up to 40\u00a0\u00b5L, while maintaining baseline separation of the ONs. The usefulness of in\u2010line mixing in HILIC was demonstrated by the analysis of a pharmaceutical antisense ON (ASO). Large\u2010volume injection of this ON in 100% water allowed for the separation and detection of the main compound and adjacent minor impurities, without breakthrough or severe peak distortion.<\/jats:p>","DOI":"10.1002\/jssc.70372","type":"journal-article","created":{"date-parts":[[2026,3,8]],"date-time":"2026-03-08T15:53:33Z","timestamp":1772985213000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Enabling Large\u2010Volume Injections in Hydrophilic Interaction Chromatography of Oligonucleotides Through In\u2010Line Mixing"],"prefix":"10.1002","volume":"49","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5862-6036","authenticated-orcid":false,"given":"Joshka","family":"Verduin","sequence":"first","affiliation":[{"name":"Department of Chemistry and Pharmaceutical Sciences Division of BioAnalytical Chemistry Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam  Amsterdam Netherlands"},{"name":"Centre for Analytical Sciences Amsterdam (CASA)  Amsterdam Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-1348-7183","authenticated-orcid":false,"given":"Luca","family":"Tuti\u0161","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Pharmaceutical Sciences Division of BioAnalytical Chemistry Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam  Amsterdam Netherlands"},{"name":"Centre for Analytical Sciences Amsterdam (CASA)  Amsterdam Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-9955-1958","authenticated-orcid":false,"given":"Antonia","family":"Kritsima","sequence":"additional","affiliation":[{"name":"Centre for Analytical Sciences Amsterdam (CASA)  Amsterdam Netherlands"},{"name":"Analytical Chemistry Group Van \u2018t Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam  Amsterdam Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3361-7341","authenticated-orcid":false,"given":"Andrea F. G.","family":"Gargano","sequence":"additional","affiliation":[{"name":"Centre for Analytical Sciences Amsterdam (CASA)  Amsterdam Netherlands"},{"name":"Analytical Chemistry Group Van \u2018t Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam  Amsterdam Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4200-2015","authenticated-orcid":false,"given":"Govert W.","family":"Somsen","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Pharmaceutical Sciences Division of BioAnalytical Chemistry Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam  Amsterdam Netherlands"},{"name":"Centre for Analytical Sciences Amsterdam (CASA)  Amsterdam Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,2,24]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.apsb.2024.05.008"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41565\u2010021\u201000898\u20100"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.3389\/fphar.2023.1245809"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1089\/nat.2023.0068"},{"key":"e_1_2_9_6_1","unstructured":"European Medicines Agency Guideline on the Development and Manufacture of Oligonucleotides(European Medicines Agency 2024)."},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2023.464327"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.2c04681"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2025.466486"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.2c03572"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2025.344218"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.0c00114"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2017.11.071"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2023.464006"},{"issue":"6","key":"e_1_2_9_15_1","first-page":"368","article-title":"Mixing and Mixers in Liquid Chromatography, Part III: Solutions for Problems With Sample Diluents","volume":"37","author":"Stoll D. 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