Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns

Biba, Mirlinda; Jiang, Eileen; Mao, Bing; Zewge, Daniel; Foley, Joe P.; Welch, Christopher J.

doi:10.1016/j.chroma.2013.06.050

Cited by 69 publications

(34 citation statements)

References 19 publications

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“…Retention discrepancy was observed for all the groups with same base composition but different base sequence, indicating retention of oligonucleotide is also related to the base sequence. Same phenomenon was observed in the work of Biba et al [47], in which six base-flip isomeric RNAs showed a modest separation under a same IP-RP-HPLC condition due to the subtle difference in conformation. Additionally, in our work, for oligonucleotides with same base composition, there was a longer retention when two "T" were both near the 5'-end (oligos 5 and 6) than one "T" was exchanged by "G" (oligo 4).…”

Section: Retention Behavior Of Oligonucleotidessupporting

confidence: 78%

Retention of nucleic acids in ion‐pair reversed‐phase high‐performance liquid chromatography depends not only on base composition but also on base sequence

Qiao

Liang

Wei

et al. 2016

J of Separation Science

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The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography.

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Section: Retention Behavior Of Oligonucleotidessupporting

confidence: 78%

Retention of nucleic acids in ion‐pair reversed‐phase high‐performance liquid chromatography depends not only on base composition but also on base sequence

Qiao

Liang

Wei

et al. 2016

J of Separation Science

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“…Some researchers focused on the retention mechanism of the analytes on MMC columns . In addition, there are also a number of reports about the application of MMC stationary phases in the analysis of complex bio samples and natural products . Therefore, the MMC materials are of great potentials for the separation of complex samples.…”

Section: Introductionmentioning

confidence: 99%

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

Wei

Cai

et al. 2016

J of Separation Science

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In this study, two mixed-mode chromatography stationary phases (C8SAX and C8SCX) were evaluated and used to establish a two-dimensional liquid chromatography system for the separation of traditional Chinese medicine. The chromatographic properties of the mixed-mode columns were systematically evaluated by comparing with other three columns of C8, strong anion exchanger, and strong cation exchanger. The result showed that C8SAX and C8SCX had a mixed-mode retention mechanism including electrostatic interaction and hydrophobic interaction. Especially, they were suitable for separating acidic and/or basic compounds and their separation selectivities could be easily adjusted by changing pH value. Then, several off-line 2D-LC systems based on the C8SAX in the first dimension and C8SAX, C8SCX, or C8 columns in the second dimension were developed to analyze a traditional Chinese medicine-Uncaria rhynchophylla. The two-dimensional liquid chromatography system of C8SAX (pH 3.0) × C8SAX (pH 6.0) exhibited the most effective peak distribution. Finally, fractions of U. rhynchophylla prepared from the first dimension were successfully separated on the C8SAX column with a gradient pH. Thus, the mixed-mode stationary phase could provide a platform to separate the traditional Chinese medicine in practical applications.

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“…A rare but noteworthy are attempts to determine the suitability of mixed-mode columns. An example is work of Biba et al [ 23 ] who evaluated several commercial columns consisting of ion-exchange and octadecyl ligands for RNA oligonucleotide analysis. The structural and positional isomers of studied biomolecules were separated due to the properties of these stationary phases, when typical anion-exchange mobile phases were used [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

The Effects of Stationary Phases on Retention and Selectivity of Oligonucleotides in IP-RP-HPLC

2014

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There is a growing demand for the separation and identification of short nucleic acid fragments, such as oligonucleotides. There were two main goals of the present investigation, namely, evaluation of the impact of stationary phase type and the influence of various ion-pair reagents on the retention behavior of oligonucleotides in ion-pair liquid chromatography. Three types of ion-pair reagents were studied: triethylammonium acetate, dimethylbuthylammonium acetate and mixtures of 1,1,1,3,3,3-hexafluoro-2-propanol and triethylamine. Two novel types of packing materials, namely, cholesterol and alkylamide were used for this purpose for the first time. The results indicate that the mechanism of oligonucleotides retention is determined by the hydrophobicity of ion-pair reagents and polar ligands localized on the surface of stationary phases. Oligonucleotides were most effectively separated with the use of alkylamide and cholesterol packings. These two stationary phases reduce the time of analysis in comparison with the octadecyl packing material. Moreover, separation was achieved under non-denaturating conditions.

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Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns

Cited by 69 publications

References 19 publications

Retention of nucleic acids in ion‐pair reversed‐phase high‐performance liquid chromatography depends not only on base composition but also on base sequence

Retention of nucleic acids in ion‐pair reversed‐phase high‐performance liquid chromatography depends not only on base composition but also on base sequence

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

The Effects of Stationary Phases on Retention and Selectivity of Oligonucleotides in IP-RP-HPLC

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