Length-Dependent Behavior of Linear dsDNA in IP-RP-HPLC on Supermacroporous Polymer Resin.
4 affiliations
- 1Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, LT-03225Vilnius, Lithuania.
- 2Thermo Fisher Scientific Baltics, V. A. Graičiu̅no 8, LT-02241Vilnius, Lithuania.
- 3Thermo Fisher Scientific , Dornierstraße 4, 82110, Germering, Germany.
- 4State Research Institute Center for Physical Sciences and Technology, Saulėtekio av. 3, LT-10257Vilnius, Lithuania.
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.
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BibTeX
@article{balciunas2026lengthdependent,
title = {{Length-Dependent Behavior of Linear dsDNA in IP-RP-HPLC on Supermacroporous Polymer Resin.}},
author = {Simonas Balčiu̅nas and Audrius Žvirblis and Matas Damonskis and Frank Steiner and Evaldas Naujalis and Lukas Taujenis},
journal = {Analytical chemistry},
year = {2026},
month = sep,
doi = {10.1021/acs.analchem.6c02096},
url = {https://europepmc.org/article/MED/42915313},
note = {CC BY},
}
RIS
TY - JOUR
TI - Length-Dependent Behavior of Linear dsDNA in IP-RP-HPLC on Supermacroporous Polymer Resin.
AU - Simonas Balčiu̅nas
AU - Audrius Žvirblis
AU - Matas Damonskis
AU - Frank Steiner
AU - Evaldas Naujalis
AU - Lukas Taujenis
PY - 2026
DA - 2026/09/01
JO - Analytical chemistry
DO - 10.1021/acs.analchem.6c02096
UR - https://europepmc.org/article/MED/42915313
AB - 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.
SN - 0003-2700
SN - 1520-6882
ER -