{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T16:04:14Z","timestamp":1789488254027,"version":"build-2803163510"},"reference-count":52,"publisher":"American Chemical Society (ACS)","issue":"36","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) is widely employed for the characterization of therapeutic oligonucleotides and mRNA vaccines. However, the retention behavior of linear double-stranded DNA (dsDNA), especially very large fragments, is not well-defined under IP-RP-HPLC conditions. This work systematically evaluates separations of dsDNA from 35 bp to 20 kbp using a monodisperse supermacroporous polystyrene-divinylbenzene polymer resin. Various parameters, including column length, flow rate, temperature, and ion-pairing reagent were investigated under fixed and varied gradient profiles, to determine their impact on dsDNA retention. A size-dependent behavior emerged, with a practical threshold near 1.5 kbp. Smaller fragments (\u22641.5 kbp) exhibited traditional on\u2013off IP-RP-HPLC behavior. Under fixed gradients, higher flow rates together with shorter columns and shallow gradients yielded the highest resolution and peak capacity values. Stronger ion-pairing reagents and elevated temperatures further increased retention and improved resolution in this range. Beyond this length threshold, behavior became less uniform, and the largest fragments (\u22655.0 kbp) showed the opposite trend: longer columns, steep gradients, and lower flow rates improved resolution. In contrast to smaller fragments, weaker ion-pairing reagents provided better separations for large dsDNA, with pair-specific optima that depended on temperature and flow. Additionally, very large fragments exhibited measurable retention and resolution even in the absence of IP-RP-driven retention. This supports the presence of a secondary retention mechanism, which is concurrently present during IP-RP-HPLC separation of large dsDNA. These results demonstrate size-dependent duality of dsDNA and provide practical guidelines for IP-RP-HPLC method optimization for both small and large dsDNA molecules.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02096","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T11:01:49Z","timestamp":1788865309000},"page":"26474-26485","source":"Crossref","is-referenced-by-count":0,"title":["Length-Dependent\nBehavior of Linear dsDNA in IP-RP-HPLC\non Supermacroporous Polymer Resin"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2511-2813","authenticated-orcid":true,"given":"Simonas","family":"Balc\u030ciu\u0305nas","sequence":"first","affiliation":[{"name":"Vilnius University , , Naugarduko 24 , ,","place":["Vilnius, Lithuania, LT-03225"]},{"name":"Thermo Fisher Scientific Baltics , V. A. Graic\u030ciu\u0305no 8 , ,","place":["Vilnius, Lithuania, LT-02241"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Audrius","family":"Z\u030cvirblis","sequence":"additional","affiliation":[{"name":"Thermo Fisher Scientific Baltics , V. A. Graic\u030ciu\u0305no 8 , ,","place":["Vilnius, Lithuania, LT-02241"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matas","family":"Damonskis","sequence":"additional","affiliation":[{"name":"Thermo Fisher Scientific Baltics , V. A. 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Mass Spectrom."},{"issue":"5","key":"2026091511090527900_cit33","doi-asserted-by":"publisher","DOI":"10.1002\/bmc.5045","article-title":"Evaluation of Alkylamines\nand Stationary Phases to Improve LC-MS\nof Oligonucleotides","volume":"35","author":"Bartlett","year":"2021","journal-title":"Biomed. Chromatogr."},{"issue":"21","key":"2026091511090527900_cit34","doi-asserted-by":"publisher","first-page":"22619","DOI":"10.1021\/acsomega.3c10533","article-title":"Quantification\nof SsDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography","volume":"9","author":"Silva-Santos","year":"2024","journal-title":"ACS Omega"},{"key":"2026091511090527900_cit35","doi-asserted-by":"publisher","first-page":"465523","DOI":"10.1016\/j.chroma.2024.465523","article-title":"Understanding\nthe Fundamentals of\nthe On-off Retention Mechanism of Oligonucleotides and Their Application\nto High Throughput Analysis","volume":"1739","author":"Lardeux","year":"2025","journal-title":"J. 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Chem."},{"issue":"17","key":"2026091511090527900_cit38","doi-asserted-by":"publisher","first-page":"2959","DOI":"10.1021\/ac960037v","article-title":"Detection of Partial\nDenaturation in AT-Rich DNA Fragments\nby Ion-Pair Reversed-Phase Chromatography","volume":"68","author":"Huber","year":"1996","journal-title":"Anal.\nChem."},{"key":"2026091511090527900_cit39","volume-title":"Introduction to Modern\nLiquid Chromatography","author":"Snyder","year":"2010","edition":"3"},{"issue":"1\u20132","key":"2026091511090527900_cit40","doi-asserted-by":"publisher","first-page":"139","DOI":"10.1016\/j.chroma.2007.09.005","article-title":"Peak Capacity in\nGradient Reversed-Phase Liquid Chromatography\nof Biopolymers","volume":"1169","author":"Gilar","year":"2007","journal-title":"J. Chromatogr. 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Chromatogr.\nA"},{"issue":"28","key":"2026091511090527900_cit46","doi-asserted-by":"publisher","first-page":"7127","DOI":"10.1007\/s00216-014-7959-5","article-title":"Evaluation\nof Ultrahigh-Performance Liquid Chromatography\nColumns for the Analysis of Unmodified and Antisense Oligonucleotides","volume":"406","author":"Studzin\u0301ska","year":"2014","journal-title":"Anal. Bioanal. Chem."},{"key":"2026091511090527900_cit47","doi-asserted-by":"publisher","first-page":"466457","DOI":"10.1016\/j.chroma.2025.466457","article-title":"Dual Retention\nMechanisms in DNA\nSeparation: Relevance of Slalom Chromatography in Anion-Exchange Gradients","volume":"1763","author":"Gritti","year":"2025","journal-title":"J. Chromatogr. 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