Polymer wall coatings for capillary electrophoresis

Horváth, Judit; Dolnı́k, Vladislav

doi:10.1002/1522-2683(200102)22:4<644::aid-elps644>3.3.co;2-v

Cited by 131 publications

(179 citation statements)

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“…EOF is often eliminated by permanent coatings that are covalently bonded to the channel walls with neutral and hydrophilic polymers, but complex coating procedures and stability of the coatings at alkaline pH have been challenges. 24 Dynamic coating procedures that mix the soluble polymer in the buffer during operation are often more adaptable. A temporary layer of polymer adsorbs on the channel walls and suppresses the EOF as demonstrated for microfluidic glass, 25 PDMS26, 27 or plastic devices 28.…”

Section: Resultsmentioning

confidence: 99%

Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

et al. 2010

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Isoelectric Focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly (vinyl) alcohol (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370V/cm) efficient cooling maintains solution temperature inside the separation channel controllably in the range 2 -25 °C. Protein samples with a dynamic concentration range between µg/mL to mg/mL can be loaded into the micro device at a flow rate of 1 mL/hr and residence time of ~12 min. By using a protein complex of 9 proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device to device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10 to 20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The micro device has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches.

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Section: Resultsmentioning

confidence: 99%

Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

et al. 2010

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“…While these interactions are weaker than covalent bonds, multiple electrostatic interactions ensure a stable coating. Horvath and Dolnik [23] reviewed several polymeric coatings in capillary electrophoresis. Decher et al [24,25] introduced a multilayer procedure that employs electrostatic interaction between oppositely charged macromolecules.…”

Section: Introductionmentioning

confidence: 99%

Open‐tubular capillary electrochromatography using a polymeric surfactant coating

et al. 2003

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A stable polyelectrolyte multilayer (PEM) coating was investigated for use in open-tubular capillary electrochromatography (o-CEC). In this approach, the PEM consisted of the cationic polymer of a quaternary ammonium salt, poly(diallyldimethylammonium chloride) and the anionic polymeric surfactant, poly(sodium undecylenic sulfate). Both the cationic and anionic polymers were physically adsorbed to the surface of a fused-silica capillary by use of a simple coating procedure. This procedure involved an alternate rinse of the positively and negatively charged polymers. The performance of the PEM coating as a dynamic stationary phase was evaluated by use of electrochromatographic experiments and showed good selectivity for both phenols and benzodiazepines. Reproducibility of the PEM coating was also evaluated by calculating the relative standard deviations (RSDs) of the electroosomotic flow (EOF). The run-to-run and capillary-to-capillary RSD values of the EOF were less than 1.5%. The endurance of the coating was more than 100 runs. The importance of the PEM coating was illustrated by comparing separations on a bare uncoated capillary with the coated capillary. In addition, the chromatographic performance using o-CEC and micellar electrokinetic chromatography (MEKC) was compared for the separation of benzodiazepines.

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“…CE unfolding studies can be performed under alkaline buffer conditions notably for basic proteins (i.e., pI > 9), however this can lead to conformational changes not indicative of physiological pH conditions. Covalent and dynamic coatings [38,39] can be used to modify the capillary surface to be neutral and hydrophilic while masking the silanol charge density. For instance, Lucy et al recently reported the use of long-chain surfactants as semi-permanent self-assembled coatings for protein separa-tions in CE [40].…”

Section: Instrumentation and Experimental Considerationsmentioning

confidence: 99%

Subject Index To Volume 2

Publishers¹

2007

CAC

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Sensitive yet selective techniques for characterizing protein unfolding are important for assessing the conformational stability of wild-type and recombinant protein. This review presents a summary of recent developments in protein unfolding by capillary electrophoresis (CE). Relative to conventional spectroscopic methods, CE offers a versatile microseparation format for performing unfolding studies using small amounts of protein mixtures based on thermal or chemical denaturation. The fundamental theory of protein unfolding in CE, as well as correction factors required to normalize non-specific changes in apparent mobility are examined in this review. The integration of in-capillary ligand stripping with dynamic protein unfolding by CE provides a convenient way to characterize holoproteins without sample pretreatment. CE is also a useful format for resolving protein conformational intermediates involving multimeric proteins or oligomers that is relevant to understanding the dynamics of misfolded proteins. This review covers protein unfolding and conformational studies from 1991-2006 with emphasis on recent developments in CE that highlight its significance as a complementary biophysical tool for protein characterization.

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Polymer wall coatings for capillary electrophoresis

Cited by 131 publications

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Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

Open‐tubular capillary electrochromatography using a polymeric surfactant coating

Subject Index To Volume 2

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