Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial
With this work, we present a comprehensive tutorial for the analysis of oligonucleotides (ONs, 5 to 100 mer) using ion-pair reversed-phase liquid chromatography (IP-RPLC) on ultra-short columns (20 × 2.1 mm). We explore the impact of ion-pairing (IP) agents on ON retention and demonstrate that while...
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| Language: | English |
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Elsevier
2024-11-01
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| Series: | Journal of Chromatography Open |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772391724000744 |
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| author | Szabolcs Fekete Mateusz Imiołek Matthew Lauber |
| author_facet | Szabolcs Fekete Mateusz Imiołek Matthew Lauber |
| author_sort | Szabolcs Fekete |
| collection | DOAJ |
| description | With this work, we present a comprehensive tutorial for the analysis of oligonucleotides (ONs, 5 to 100 mer) using ion-pair reversed-phase liquid chromatography (IP-RPLC) on ultra-short columns (20 × 2.1 mm). We explore the impact of ion-pairing (IP) agents on ON retention and demonstrate that while IP agents significantly influence absolute retention, their effect on selectivity is often minimal. Our findings emphasize the utility of systematic method development, including software-assisted retention modeling, to optimize gradient steepness and temperature such that resolution can be optimized for both sequence and length variants. We recommend the use of low-adsorption column hardware to minimize nonspecific interactions, which is to the benefit of improving method robustness, peak shapes and recovery (especially of shortmer impurities). Our results confirm the utility of so-called ultra-short column formats as is demonstrated by way of the example, quick run time separations and high-throughput ON analyses. The study establishes practical guidelines for developing robust, reproducible, and high-efficiency IP-RPLC methods for ONs which will be of assistance to analysts working on new therapeutics and new sequencing and diagnostic reagents alike. |
| format | Article |
| id | doaj-art-7091a7b2a27c48a698281f30664c32bf |
| institution | Kabale University |
| issn | 2772-3917 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Chromatography Open |
| spelling | doaj-art-7091a7b2a27c48a698281f30664c32bf2024-12-14T06:34:13ZengElsevierJournal of Chromatography Open2772-39172024-11-016100187Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. TutorialSzabolcs Fekete0Mateusz Imiołek1Matthew Lauber2Waters Corporation, 1 Rue Michel Servet, Geneva 1211, Switzerland; Corresponding author.Waters Corporation, 1 Rue Michel Servet, Geneva 1211, SwitzerlandWaters Corporation, 34 Maple Street, Milford MA 01757, USAWith this work, we present a comprehensive tutorial for the analysis of oligonucleotides (ONs, 5 to 100 mer) using ion-pair reversed-phase liquid chromatography (IP-RPLC) on ultra-short columns (20 × 2.1 mm). We explore the impact of ion-pairing (IP) agents on ON retention and demonstrate that while IP agents significantly influence absolute retention, their effect on selectivity is often minimal. Our findings emphasize the utility of systematic method development, including software-assisted retention modeling, to optimize gradient steepness and temperature such that resolution can be optimized for both sequence and length variants. We recommend the use of low-adsorption column hardware to minimize nonspecific interactions, which is to the benefit of improving method robustness, peak shapes and recovery (especially of shortmer impurities). Our results confirm the utility of so-called ultra-short column formats as is demonstrated by way of the example, quick run time separations and high-throughput ON analyses. The study establishes practical guidelines for developing robust, reproducible, and high-efficiency IP-RPLC methods for ONs which will be of assistance to analysts working on new therapeutics and new sequencing and diagnostic reagents alike.http://www.sciencedirect.com/science/article/pii/S2772391724000744Oligonucleotide separationIon-pairingRetention modelingUltra-short columnPlatform method |
| spellingShingle | Szabolcs Fekete Mateusz Imiołek Matthew Lauber Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial Journal of Chromatography Open Oligonucleotide separation Ion-pairing Retention modeling Ultra-short column Platform method |
| title | Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial |
| title_full | Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial |
| title_fullStr | Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial |
| title_full_unstemmed | Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial |
| title_short | Ion-pair reversed-phase chromatography analysis of oligonucleotides using ultra-short (20 x 2.1 mm) columns. Tutorial |
| title_sort | ion pair reversed phase chromatography analysis of oligonucleotides using ultra short 20 x 2 1 mm columns tutorial |
| topic | Oligonucleotide separation Ion-pairing Retention modeling Ultra-short column Platform method |
| url | http://www.sciencedirect.com/science/article/pii/S2772391724000744 |
| work_keys_str_mv | AT szabolcsfekete ionpairreversedphasechromatographyanalysisofoligonucleotidesusingultrashort20x21mmcolumnstutorial AT mateuszimiołek ionpairreversedphasechromatographyanalysisofoligonucleotidesusingultrashort20x21mmcolumnstutorial AT matthewlauber ionpairreversedphasechromatographyanalysisofoligonucleotidesusingultrashort20x21mmcolumnstutorial |