Chromatographic behaviour of oxidised porous graphitic carbon columns

Törnkvist, Anna; Markides, Karin E.; Nyholm, Leif

doi:10.1039/b303076h

Cited by 36 publications

(40 citation statements)

References 17 publications

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“…We suspect that this decrease arises, at least in part, from an increase in the concentration of oxygen groups on the packing surface. 35 Characterization of the packing, after use in temperature experiments for several days, by Xray photoelectron spectroscopy indicated that the concentration of surface oxygen groups had reached a level as high as 0.9% (atomic) as a consequence of extended use. It is therefore evident that the stability of the packing at elevated temperatures needs to be improved in order to fully exploit the advantages of operation at elevated temperatures.…”

Section: Retention Stability At Elevated Temperaturesmentioning

confidence: 99%

High-Speed Electrochemically Modulated Liquid Chromatography

Ponton

Porter

2004

Anal. Chem.

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The performance advantages of carrying out electrochemically modulated liquid chromatography (EMLC) at elevated temperatures and mobile-phase flow rates are investigated. EMLC has the unique ability to manipulate analyte retention and enhance separation efficiencies through changes in the potential applied to a conductive stationary phase. Operation of high-performance liquid chromatography systems at elevated column temperatures also provides pathways to improve chromatographic performance by enhancing analyte diffusivity and facilitating the use of higher mobile-phase flow rates than conventionally attainable. The results show that performing EMLC separations at elevated temperatures (e.g., 100 °C) reduces the analysis time of a mixture of aromatic sulfonates in a mixed mobile phase by more than a factor of 20. Moreover, use of higher operating temperatures enables the separation of this mixture with an entirely aqueous mobile phase in less than 2 min. This paper investigates the advantages of performing separations using electrochemically modulated liquid chromatography (EMLC) at elevated column temperatures. EMLC is a unique chromatographic technique that manipulates retention through changes in the potential applied (E app ) to a conductive stationary-phase-like porous graphitic carbon (PGC). This capability is realized by fashioning a high-performance liquid chromatography (HPLC) column into an electrochemical cell and utilizing the packing as both a chromatographic stationary phase and as a working electrode. As a consequence, a change in E app alters the effective surface composition of the stationary phase and, in turn, retention. 1 Several laboratories, 2-5 including our own, 6-10 have demonstrated that EMLC can be utilized for the separation of a wide range of mixtures (e.g., aromatic sulfonates, 10 monosubstituted benzenes, 9 protonated pyridines and anilines, 11 corticosteroids, 7 benzodiazepines, 6 short-chain alkanoic acids, 12 and metal ion complexes 13 ).Recent studies have shown that elevated column temperatures can also be used to manipulate retention in HPLC by significantly reducing analysis times while maintaining an effective separation. 14-16 This observation was theoretically predicted several years ago 17,18 and comes about because of three different temperature-dependent phenomena. First, retention in most reversed-phase separations is an exothermic process. 19-21 An increase in column temperature therefore leads to a decrease in elution time. Second, the viscosity of most mobile phases decreases with increasing temperature. As a consequence, the back pressure of the HPLC system decreases, enabling operation at higher flow rates. Third, higher column temperatures increase analyte diffusivity and desorption kinetics. Enhanced diffusivity increases the efficiency of a separation by lowering the C-term in the van Deemter equation, 22 which counters the loss in efficiency that arises at higher mobile-phase flow rates.

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Section: Retention Stability At Elevated Temperaturesmentioning

confidence: 99%

High-Speed Electrochemically Modulated Liquid Chromatography

Ponton

Porter

2004

Anal. Chem.

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“…Oxidation reactions of nucleobases in DNA is hypothesized to play a major role in mutagenesis and cancer, and as many as 30 modifications of cytosine have been characterized from studies of the nucleobase, the nucleoside, and DNA [32]. PGC has known redox characteristics and has been shown to be oxidizing in its native state [17,37]. Reduction of PGC is usually achieved by adding sodium sulfite to the eluents, and PGC has been used for on-column electrochemical redox derivatization to enhance separation selectivity [38,39].…”

Section: Resultsmentioning

confidence: 99%

A Nano-Chip-LC/MSⁿ Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

Giessing

Scott

Kirpekar

2011

J. Am. Soc. Mass Spectrom.

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LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nanochip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ×150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MS n identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh 5 C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh 5 C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh 5 C was readily identified by its MH + m/z 260 and MS n fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.

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“…Unintentional alteration of retention on PGC coupled to mass spectrometry was also reported, caused by leakage current from electrospray ionization (ESI) source [11,12]. Similar effects were generated by chemical means [13]. Oxidizing and reducing agents were shown to affect the retention of aromatic sulfonates, while the retention of benzene was only marginally impacted.…”

Section: Introductionmentioning

confidence: 90%

Effects of the redox state of porous graphitic carbon on the retention of oligosaccharides

Melmer

Stangler

Premstaller

et al. 2010

Journal of Chromatography A

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Chromatographic behaviour of oxidised porous graphitic carbon columns

Cited by 36 publications

References 17 publications

High-Speed Electrochemically Modulated Liquid Chromatography

High-Speed Electrochemically Modulated Liquid Chromatography

A Nano-Chip-LC/MSⁿ Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

Effects of the redox state of porous graphitic carbon on the retention of oligosaccharides

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Chromatographic behaviour of oxidised porous graphitic carbon columns

Cited by 36 publications

References 17 publications

High-Speed Electrochemically Modulated Liquid Chromatography

High-Speed Electrochemically Modulated Liquid Chromatography

A Nano-Chip-LC/MSn Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

Effects of the redox state of porous graphitic carbon on the retention of oligosaccharides

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A Nano-Chip-LC/MSⁿ Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase