{
  "abstract": "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 (≤1.5 kbp) exhibited traditional on-off 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 (≥5.0 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.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, LT-03225Vilnius, Lithuania.",
    "Thermo Fisher Scientific Baltics, V. A. Graičiu̅no 8, LT-02241Vilnius, Lithuania.",
    "Thermo Fisher Scientific , Dornierstraße 4, 82110, Germering, Germany.",
    "State Research Institute Center for Physical Sciences and Technology, Saulėtekio av. 3, LT-10257Vilnius, Lithuania."
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 6,
  "authors": [
    {
      "affs": [
        0,
        1
      ],
      "name": "Simonas Balčiu̅nas",
      "orcid": "0009-0001-2511-2813"
    },
    {
      "affs": [
        1
      ],
      "name": "Audrius Žvirblis"
    },
    {
      "affs": [
        1
      ],
      "name": "Matas Damonskis"
    },
    {
      "affs": [
        2
      ],
      "name": "Frank Steiner"
    },
    {
      "affs": [
        0,
        3
      ],
      "name": "Evaldas Naujalis"
    },
    {
      "affs": [
        1
      ],
      "name": "Lukas Taujenis"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/1fbk9d7xnbwfr.bib",
  "cited_by": 0,
  "doi": "10.1021/acs.analchem.6c02096",
  "fulltext_url": "https://europepmc.org/articles/PMC13584626",
  "id": "1fbk9d7xnbwfr",
  "issns": [
    "0003-2700",
    "1520-6882"
  ],
  "landing_url": "https://europepmc.org/article/MED/42915313",
  "language": "eng",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1607|Analytical Chemistry and Chromatography"
    ]
  ],
  "openalex": "W7211946100",
  "pdf_url": "https://europepmc.org/articles/PMC13584626?pdf=render",
  "pmcid": "PMC13584626",
  "pmid": "42915313",
  "published": "2026-09-01",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/1fbk9d7xnbwfr.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:21:02Z",
      "native_id": "MED:42915313",
      "source": "europepmc",
      "url": "https://europepmc.org/article/MED/42915313"
    }
  ],
  "subjects": [
    {
      "field": "chemistry",
      "origin": "openalex:1607|Analytical Chemistry and Chromatography",
      "subfield": "physical"
    }
  ],
  "title": "Length-Dependent Behavior of Linear dsDNA in IP-RP-HPLC on Supermacroporous Polymer Resin.",
  "title_text": "Length-Dependent Behavior of Linear dsDNA in IP-RP-HPLC on Supermacroporous Polymer Resin.",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:21:02Z",
  "url": "https://research.draconitic.com/paper/1fbk9d7xnbwfr",
  "venue": "Analytical chemistry",
  "versions": []
}