Isoprotein analysis by ion-exchange chromatography using a linear pH gradient combined with a salt gradient

Kaltenbrunner, Oliver; Tauer, Christa; Brunner, J.R.; Jungbauer, Alois

doi:10.1016/0021-9673(93)83086-8

Cited by 36 publications

(16 citation statements)

References 9 publications

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“…The buffers commonly used in chromatofocusing also interact with proteins and are difficult to remove from them. Kaltenbrunner et al [86] developed a method generating a linear pH gradient by mixing borate buffers with mannitol. The chemical reaction between these two components liberates protons and in addition the pH gradient can be combined with an ascending or descending salt gradient.…”

Section: Progress In Bioseparation and Bioconversionmentioning

confidence: 99%

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology

Jungbauer¹,

Hahn

2004

J of Separation Science

161

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Monoliths have consolidated their position in bioseparation. More than 200 different applications have been reported in the past two decades and their advantages compared to conventional chromatography demonstrated. These include the high mass transfer efficiency due to the convective flow enabled by the macroporous character of the matrix. Recently plasmid DNA and viruses were separated with high efficiency and cryogels and monolithic superporous agarose were developed for capture of proteins from crude homogenates and separation of microorganisms or lymphocytes. Currently four companies manufacture monoliths mainly for analytical applications although monoliths with a volume of 0.8 liter are commercially available and 8 L are available as prototypes. A book entitled "Monolithic materials: preparation, properties and applications" was published in 2003 and became standard reference of the status of this area. This review focuses on the progress in monoliths that goes beyond the scope of this reference book. Less progress has been made in the field of bioconversions in spite of the fact that monolithic supports exhibit better performance than beads in enzymatic processing of macromolecules. It appears that the scientific community has not yet realized that supports for these applications are readily available. In addition, monoliths will further substantially advance bioseparations of both small and large molecules in the future.

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Section: Progress In Bioseparation and Bioconversionmentioning

confidence: 99%

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology

Jungbauer¹,

Hahn

2004

J of Separation Science

161

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“…While CF generally exploits the buffering capacity of the ion exchanger to obtain a retained intra-column pH gradient, the opposite is true for pH-gradient IEC [17]. A wide-range pH gradient using a mixture of 6 amine buffering species at 10 mM concentrations and 10% ethanol in both start and elution buffer was employed to fractionate saliva proteins into three pI fractions in the first dimension as shown in Fig.…”

Section: Fractionation and Separation Of Saliva Proteins By 2-d Capilmentioning

confidence: 99%

Fractionation and separation of human salivary proteins by pH‐gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

Pepaj¹,

Holm

Fleckenstein

et al. 2006

J of Separation Science

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Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometryIn the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.

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“…While CF generally exploits the buffering capacity of the ion exchanger to obtain a retained intra-column pH-gradient, the opposite should be stressed in pH-gradient IEC [12]. Recently, Liu and Anderson [13,14], and Shan and Anderson [15,16] used pH-gradient IEC, which they termed gradient CF, to overcome many of the shortcomings of CF.…”

Section: Introductionmentioning

confidence: 99%

Isoelectric point separation of proteins by capillary pH-gradient ion-exchange chromatography

Andersen

Pepaj

Trones

et al. 2004

Journal of Chromatography A

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Isoprotein analysis by ion-exchange chromatography using a linear pH gradient combined with a salt gradient

Cited by 36 publications

References 9 publications

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology

Fractionation and separation of human salivary proteins by pH‐gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

Isoelectric point separation of proteins by capillary pH-gradient ion-exchange chromatography

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