Universal detection for capillary isoelectric focusing without mobilization using concentration gradient imaging system

Wu, Jiaqi; Pawliszyn, Janusz

doi:10.1021/ac00026a024

Cited by 103 publications

(58 citation statements)

References 16 publications

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“…Here, the whole-column imaging detection technique [40][41][42][43][44] was first applied for direct visualization of the IEF concentration of protein BSA in the dynamic pH gradient. Because the etched capillary could not be simply applied in the commercial whole-column imaging CIEF instrument, a commercial cartridge originally for CIEF was modified ( Fig.…”

Section: Direct Observation Of the Ief Concentration At The Boundarymentioning

confidence: 99%

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Isoelectric focusing sample injection for capillary electrophoresis of proteins

Wu¹,

Zhang²,

Onoda³

2005

Electrophoresis

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Carrier ampholyte-free isoelectric focusing (IEF) sample injection (concentration) for capillary electrophoresis (CE) is realized in a single capillary. A short section of porous capillary wall was made near the injection end of a capillary by HF etching. In the etching process, an electric voltage was applied across the etching capillary wall and electric current was monitored. When an electric current through the etching capillary was observed, the capillary wall became porous. The etched part was fixed in a vial, where NaOH solution with a certain concentration was added during the sample injection. The whole capillary was filled with pH 3.0 running buffer. The inlet end vial was filled with protein sample dissolved in the running buffer. An electric voltage was applied across the inlet end vial and etched porous wall. A neutralization reaction occurs at the boundary (interface) of the fronts of H+ and OH-. A pH step or sharp pH gradient exists across the boundary. When positive protein ions electromigrate to the boundary from the sample vial, they are isoelectricelly focused at points corresponding to their pH. After a certain period of concentration, a high voltage is applied across the whole capillary and a conventional CE is followed. An over 100-fold concentration factor has been easily obtained for three model proteins (bovine serum albumin, lysozyme, ribonuclease A). Furthermore, the IEF sample concentration and its dynamics have been visually observed with the whole-column imaging technique. Its merits and remaining problem have been discussed, too.

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Section: Direct Observation Of the Ief Concentration At The Boundarymentioning

confidence: 99%

“…A short section of porous capillary wall was made near the injection end of the capillary. The IEF concentration and its dynamical process are first visually confirmed with the whole-column imaging detection technique [40][41][42][43][44]. Then, IEF sample injection-capillary zone electrophoresis (CZE) of proteins is carried out.…”

Section: Introductionmentioning

confidence: 99%

Isoelectric focusing sample injection for capillary electrophoresis of proteins

Wu¹,

Zhang²,

Onoda³

2005

Electrophoresis

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show abstract

“…In 1992, whole capillary imaging techniques to detect the focused proteins were developed to eliminate the mobilization step. 172,173 The method has been applied to the analysis of several biopharmaceutical glycoproteins.…”

Section: ·6 Separation Of Glycoproteins and Glycopeptidesmentioning

confidence: 99%

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

Suzuki

2013

ANAL. SCI.

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“…Many RI detection methods have been investigated for CE, including a concentration gradient method, which probes the on-axis optical perturbation produced by a transient solute band [83] and the use of a tapered fiber optic [85], which probes the reduction in transmitted beam intensity due to refractive index interfacial beam intensity coupling. While powerful, these approaches to RI sensing have limitations.…”

Section: Refractive Index Detectionmentioning

confidence: 99%

“…For example, the concentration gradient detector [83] is somewhat insensitive to thermal noise, but most suitable for detection schemes in capillary isoelectric focusing or for separation systems where post-column detection is acceptable.…”

Section: Refractive Index Detectionmentioning

confidence: 99%