Reversible entrapment of plasmid deoxyribonucleic acid on different chromatographic supports

Gabor, Boštjan; Černigoj, Urh; Barut, Miloš; Štrancar, Aleš

doi:10.1016/j.chroma.2013.08.075

Cited by 11 publications

(15 citation statements)

References 29 publications

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“…These sorbents include silica-based neutral or cationic particles and monoliths (i.e. bare silica gel, or silica gel with aminopropyl or quaternary ammonium groups) [3][4][5][6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation

Günal

Kip

Öğüt

et al. 2017

Artificial Cells, Nanomedicine, and Biotechnology

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Monodisperse silica microspheres with bimodal pore-size distribution were proposed as a high performance sorbent for DNA isolation in batch fashion under equilibrium conditions. The proposed sorbent including both macroporous and mesoporous compartments was synthesized 5.1 μm in-size, by a "staged shape templated hydrolysis and condensation method". Hydrophilic polymer based sorbents were also obtained in the form of monodisperse-macroporous microspheres ca 5.5 μm in size, with different functionalities, by a developed "multi-stage microsuspension copolymerization" technique. The batch DNA isolation performance of proposed material was comparatively investigated using polymer based sorbents with similar morphologies. Among all sorbents tried, the best DNA isolation performance was achieved with the monodisperse silica microspheres with bimodal pore size distribution. The collocation of interconnected mesoporous and macroporous compartments within the monodisperse silica microspheres provided a high surface area and reduced the intraparticular mass transfer resistance and made easier both the adsorption and desorption of DNA. Among the polymer based sorbents, higher DNA isolation yields were achieved with the monodisperse-macroporous polymer microspheres carrying trimethoxysilyl and quaternary ammonium functionalities. However, batch DNA isolation performances of polymer based sorbents were significantly lower with respect to the silica microspheres.

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“…These sorbents include silica-based neutral or cationic particles and monoliths (i.e. bare silica gel, or silica gel with aminopropyl or quaternary ammonium groups) [3][4][5][6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation

Günal

Kip

Öğüt

et al. 2017

Artificial Cells, Nanomedicine, and Biotechnology

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show abstract

“…Relatively complex solution is against our aim, which is improvement of pDNA separation on standard anion exchangers through altering mobile phase composition without using organic modifiers. In previous works, we found the correlation between decreased recovery of OC pDNA isoform and reversible convective entrapment in the channels of chromatographic supports, based on convective mass transport [17]. This effect was more pronounced with increasing pDNA size.…”

Section: Introductionmentioning

confidence: 64%

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Pavlin,

Černigoj,

Bavčar

et al. 2023

Electrophoresis

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High‐performance liquid chromatography (HPLC)‐based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min−1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC–LIN and LIN–SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in‐process control monitoring of pDNA production processes.

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“…), increased pressure drop might also be a consequence of a partial resin blocking due to a non‐specific adsorption or entrapment described to occur for convective media. Later process is reversible as it was demonstrated for very large molecules like plasmid DNA and viruses being dependent on the applied mobile phase velocity affecting Peclet number. In extreme case, when aggregates or molecules are comparable or even exceed pore size of convective media, they cause blocking of pores resulting in a constant increase of pressure drop , similar to compressible media beyond critical velocity .…”

Section: Pressure Drop Versus Mobile Phase Velocity Non‐linearitymentioning

confidence: 93%

Pressure drop in liquid chromatography

Podgornik

2018

J of Separation Science

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Measurement of pressure drop is routinely performed during chromatographic runs. In many cases this information is only used for regulation of mobile phase flow rate to keep pressure drop below defined limit. However, pressure drop can provide additional important information about performance of chromatographic process. In this review different parameters affecting pressure drop such as compressibility of chromatographic media, nature of applied sample and mobile phase flow regime are discussed. Detailed analysis correlating organization of particle based chromatographic media and pressure drop is presented together with its extension to convective media such as membranes and monoliths but also novel 3D printed media. Finally, estimation of layer thickness formed by adsorbed molecules based on a pressure drop data is presented and its applicability is discussed.

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Reversible entrapment of plasmid deoxyribonucleic acid on different chromatographic supports

Cited by 11 publications

References 29 publications

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Pressure drop in liquid chromatography

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