Whole column absorbance detection in capillary isoelectric focusing

Wang, Tiansong.; Hartwick, Richard A.

doi:10.1021/ac00039a021

Cited by 42 publications

(25 citation statements)

References 8 publications

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“…Hjertén applied his scanning rotating tube apparatus to IEF [17] and developed scanning equipment for stationary capillaries [18]. More recently, to overcome the drawbacks of single point detection in capillary IEF, instrumentation with optical whole column imaging, including approaches based upon pulling the separation capillary through the detection window of a UV absorbance detector [19], spatial scanning laser fluorescence detection with the capillary mounted on a precision translational stage that moves the capillary through the probe beam [20] and real-time imaging without moving parts along a short (about 5 cm), internally coated capillary [21][22][23][24] or microchannel [25] led to the following of the IEF process of amphoteric solutes. The latter approach with the coated fused-silica capillary and UV absorption detection has been commercialized and is now available as iCE280 instrument (Convergent Bioscience, Toronto, ON, Canada).…”

Section: Introductionmentioning

confidence: 99%

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

et al. 2004

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A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High-resolution focusing data of four hemoglobin variants in a convection-free medium are presented for pH 3-10 and pH 5-8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5-10 min, whereas at 600 V/cm separation is predicted to be complete between 2.5 and 5 min. The time interval required for focusing of proteins is demonstrated to be dependent on the input protein charge data and, however less, on the properties of the carrier ampholytes. The simulation data reveal that the number of transient protein boundaries migrating from the two ends of the column towards the focusing positions is equal to the number of sample components. Each protein is being focused via the well-known double-peak approach to equilibrium, a process that is also characteristic for focusing of the carrier ampholytes. The predicted focusing dynamics for the hemoglobin variants in pH 3-10 and pH 5-8 gradients are shown to qualitatively agree well with experimental data obtained by whole-column optical imaging.

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Section: Introductionmentioning

confidence: 99%

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

et al. 2004

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“…In imaging CIEF there is no need for mobilization because while the components are being focused in the capillary, a charge-coupled device camera images the entire capillary. Several kinds of whole-column detectors have been developed for CIEF: universal refractive index gradient imaging detector [24], optical absorption imaging detector [9,24,25], and fluorescence imaging detector [26,27]. See reviews on whole column detection methods in [23] and [28].…”

Section: Methodology Of Ciefmentioning

confidence: 99%

Recent applications of capillary isoelectric focusing

Kilár

2003

Electrophoresis

101

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After the advent of capillary isoelectric focusing (CIEF) in the 80's several approaches have been developed in order to use the technique in routine analyses. The recent years showed an extensive increase in the applications of this technique employing its exceptionally high-resolution power. Methodological improvements, as well as hyphenation with other electrophoretic and chromatographic separation procedures, proved the versatility of CIEF in studies of clinically important proteins, recombinant product, cell lysates and other complex mixtures. The combination of CIEF with mass spectrometry detection is one of the major challenges for studying proteomics. This review collected the recent applications of CIEF including innovations in the experimental setup, remedies for the presence of salts in samples, calibration of the pH gradient, carrier ampholyte-free isoelectric focusing, the progress in micropreparation, two-dimensional separations, etc.

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“…[2][3][4][5][6][7][8][9][10][11] In the most simple scheme of whole-column detection for cIEF, a transparent capillary is illuminated by an expanded beam. Transmitted light or fluorescence from the capillary is monitored for UV absorbance or fluorescence detection, respectively, by a photodiode array, CCD camera, or imaging sensor.…”

Section: Introductionmentioning

confidence: 99%

High-speed Analysis of Proteins by Microchip Isoelectric Focusing with Linear-imaging UV Detection

2009

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High-speed analyses of proteins by microchip isoelectric focusing (MCIEF) were investigated on straight channel chips. By employing a commercially available microchip electrophoresis instrument equipped with a linear-imaging UV detector, sample proteins focused in a separation channel could be detected without a mobilization step. Typically, standard proteins were focused within 210 s at different positions in a separation channel with its total length of 38.8 mm on the basis of their pI values. The linear relationship between the focused position of each protein and its pI value was observed in the pH range from 6 to 10. The formation process of a pH gradient in the microchannel and the effects of the applied voltage and the channel length on the MCIEF separation were also investigated.

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Whole column absorbance detection in capillary isoelectric focusing

Cited by 42 publications

References 8 publications

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

Recent applications of capillary isoelectric focusing

High-speed Analysis of Proteins by Microchip Isoelectric Focusing with Linear-imaging UV Detection

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