Ion pair chromatography on reversed‐phase layers

Volkmann, D.

doi:10.1002/jhrc.1240021207

Cited by 15 publications

(4 citation statements)

References 4 publications

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“…An additional RP method of analysis based on ion‐pair RP‐HPLC (Volkmann, ) is also being used currently. Ion‐paired reversed‐phase chromatography has been described as “a versatile platform for the analysis of RNA” by Dickman ().…”

Section: Chromatographic Methodsmentioning

confidence: 99%

Analysis and Purification of Synthetic Nucleic Acids Using HPLC

Sinha¹

2015

CP Nucleic Acid Chemistry

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Chromatographic methods have been essential tools for analysis and purification of synthetic oligonucleotides since the 1970s. Significant developments in terms of instruments and stationary phases (media) have been made during the past several decades; among the latest are sub-micron to micron particles for the media, as well as ultra performance liquid chromatography (UPLC). Micron and sub-micron particles have increased product resolution. Applications of recently developed methods such as IP-RP-HPLC and LC-MS have been discussed for analysis, along with use of various methods for purification. Utilization of UPLC has decreased analysis time, increasing the throughput for analysis. Commonly used methods for analysis and purification of synthetic oligonucleotides have been described in this unit.

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Section: Chromatographic Methodsmentioning

confidence: 99%

Analysis and Purification of Synthetic Nucleic Acids Using HPLC

Sinha¹

2015

CP Nucleic Acid Chemistry

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“…In thin-layer chromatography, reversed-phase layers, made by chemical modification of silica gel, are well suited. Lepri et a/, separated amines [I], amino acids [2], and phenols [3] using silanized silica gel, Volkmann separated phenothiazine sulfoxides [4] and alkaloids [5] using RP2-, RP8-and RPI8 layers, which have recently become available. In most cases Lepri used dodecylbenzenesulfonate, dodecylpyridinium chloride, whereas Volkrnann preferred alkanesulfonic acids as ion pair formers.…”

Section: Introductionmentioning

confidence: 99%

Ion‐pair chromatography with UV absorbing counterions on reversed‐phase layers. Part 1: Chromatography with hydroxynaphthalenesulfonic acids

Volkmann

1983

J. High Resol. Chromatogr.

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SummaryThe results of ion-pair chromatography with UV-absorbing counterions are described. Hydroxynaphthalenesulfonic acids are used as ion pair formers, and alkaloids as test samples. The separationsare performed on silanized silica gel from Merck and KCIBF plates from Whatman. The influence of the counterion on the Rf-value is demonstrated. Some alkaloids exhibit characteristic fluorescence as ion pairs.

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“…Ion-pairing reversed-phase (IPRP) liquid chromatography is the reference purity method for chemically synthesized oligonucleotides, including 100-mer single-guide RNAs (sgRNAs) . IPRP often achieves a superior selectivity in comparison to hydrophilic interaction liquid chromatography (HILIC) for length-based separations and in particular for oligonucleotides modified with PS linkages. , Two main mechanisms dictate the IPRP separation with (i) the ion-pair formation occurring in the mobile phase and the neutralized pair subsequently adsorbed on the stationary phase and eluted based on the hydrophobicity and (ii) an unpaired IP agent from the mobile phase is adsorbed onto the stationary phase and allows the separation of charged analytes according to their charge. , In practice, both mechanisms occur. − …”

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confidence: 99%

“…11,12 Two main mechanisms dictate the IPRP separation with (i) the ion-pair formation occurring in the mobile phase and the neutralized pair subsequently adsorbed on the stationary phase and eluted based on the hydrophobicity and (ii) an unpaired IP agent from the mobile phase is adsorbed onto the stationary phase and allows the separation of charged analytes according to their charge. 13,14 In practice, both mechanisms occur. 14−16 However, the separation of impurities is hampered for fulllength RNA due to their length.…”

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Characterization of Impurities in Therapeutic RNAs at the Single Nucleotide Level

Goyon¹,

Nguyen²,

Boulanouar³

et al. 2022

Anal. Chem.

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The chemistry of guide RNA (gRNA) affects the performance of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technique. However, the literature is very scarce about the study of gRNA degradation and in particular at the single nucleotide level. In this work, we developed a workflow to characterize the impurities of large RNAs at the single nucleotide level and identified the residues prone to degradation. Our strategy involves (i) the reduction of RNA length, (ii) a chromatographic mode able to capture subtle changes in impurity polarity, and (iii) a streamlined data treatment. To illustrate the approach, stressed gRNA samples were analyzed by coupling an immobilized ribonuclease T1 cartridge to a hydrophilic interaction liquid chromatography (HILIC) column hyphenated with tandem mass spectrometry (MS/MS). Critical findings were made possible by the presented technology. In particular, the desulfurization of phosphorothioate (PS) linkages was the major degradation observed at the single nucleotide level while no change in purity profile could be observed when using conventional ion-pairing reversed-phase (IPRP) liquid chromatography. To our knowledge, this is the first time that several impurity types are screened for a large RNA molecule using an automated online digestion analysis approach.

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Ion pair chromatography on reversed‐phase layers

Abstract: SummaryThe results of investigations with ion pair chromatography on RPp, RP8 and RP18 thin layers are described. Heptane sulphonic acid is used as ion pair former. The best results are achieved on RP18-layers with the counter ion in the mobile phase.

Cited by 15 publications

References 4 publications

Analysis and Purification of Synthetic Nucleic Acids Using HPLC

Analysis and Purification of Synthetic Nucleic Acids Using HPLC

Ion‐pair chromatography with UV absorbing counterions on reversed‐phase layers. Part 1: Chromatography with hydroxynaphthalenesulfonic acids

Characterization of Impurities in Therapeutic RNAs at the Single Nucleotide Level

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