Ion-Exchange Chromatography

Walton, Harold F.

doi:10.1007/978-1-4684-1830-9_4

Modern Methods of Geochemical Analysis 1971

DOI: 10.1007/978-1-4684-1830-9_4

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Ion-Exchange Chromatography

Harold F. Walton

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2008

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“…However, these reports have not led to widespread use of ion-exchange chromatography interfaced to MS because the volatile ions have relatively weak displacing power [20,21]. This necessitates the use of high concentrations of displacer ions in the mobile phase that not only suppress the MS signal but also limits the applications to smaller, more readily displaced analyte molecules.…”

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Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

Lian

Hribar

Zhou

et al. 2008

J. Am. Soc. Mass Spectrom.

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A new analytical technique, gradient chromatofocusing-mass spectrometry (gCF-MS), was developed employing ion-exchange high-performance liquid chromatography (HPLC) interfaced to an electrospray-quadrupole mass spectrometer in the determination of proteins. There have been few reports, if any, of a HPLC-MS technique for proteins in which the ion-exchange column is directly interfaced to the mass spectrometer. The employment of a linear pH gradient elution scheme directly interfaced to mass spectrometry is also unique in the present work. The technique was demonstrated by the separation of six proteins (carbonic anhydrase II, enolase, ␤-lactoglobulin A, lactoglobulin B, soybean trypsin inhibitor, and amyloglucosidase) employing a descending linear pH gradient from pH 9 to 2.6 on a 50 mm ϫ 2.1 mm DEAE HPLC column using volatile buffer components. A signal enhancement solution consisting of 8% formic acid in acetonitrile was pumped post-column and was mixed 1:1 with column effluent and then directed on-line into the mass spectrometer. Molecular masses of the proteins were determined within Ϯ0.010% to 0.033% (Ϯ100 to 330 ppm) with peak height total ion current detection limits of 4 to 78 pmol of injected amounts (S/N ϭ 3). This technique is applicable to the analysis of proteins and other charged molecules. T here is great interest in developing a mass spectrometry (MS)-compatible high-performance liquid chromatography (HPLC) technology which performs charge-based separations, a principal dimension in the 2D-gel electrophoresis technique, for application in proteomic and protein characterization studies. Development of such a HPLC technique would be progress towards addressing the limitations of the 2D-gel electrophoresis technique, which has been hampered by its design incompatibility for direct interfacing with MS (requiring excision of each protein band from the gel), limitation in quantitative dynamic range, inability of determining small proteins (5-8000 Da), high labor intensity, long sample run times, as well as other disadvantages [1,2].Development of charged-based HPLC techniques directly coupled to mass spectrometry (MS) has been a difficult challenge. This is because the mobile phase salts commonly used in ion-exchange chromatography, the most used charge-based HPLC technique, suppress the MS signal. The most utilized approach incorporating ion-exchange chromatography with mass spectrometry has been to use a 2D ion-exchange/reversed-phase HPLC technique, directing the ion-exchange fractions to a reversed-phase column for further separation and removal of salt before on-line mass spectrometry. This has been applied to proteomic studies in the analysis of proteins [3] and protein digests [4,5]. A "stair step" gradient is employed for the ion-exchange dimension. This first dimension thus serves as a crude initial separation step, and is not the stage that is directly interfaced to the mass spectrometer.The application of two-dimensional chromatography using the combination of chromatofocusing and reversed-p...

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mentioning

confidence: 99%

Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

Lian

Hribar

Zhou

et al. 2008

J. Am. Soc. Mass Spectrom.

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Ion-Exchange Chromatography

Cited by 1 publication

References 68 publications

Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

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