Fluid Stabilization during Isoelectric Focusing in Cylindrical and Annular Columns

Egen, Ned B.; Twitty, G. E.; Thormann, Wolfgang; Bier, M.

doi:10.1080/01496398708058406

Cited by 13 publications

(10 citation statements)

References 15 publications

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“…This agrees with the simulation data recently published by Thormann et al [23]. The result can be explained by the fact that while CA molecules approach more closely to their isoelectric states their electric charges continue dropping [26,36,37]. Once each CA component practically achieves maximal compression in its pI, no further conductivity change occurs with further voltage increase.…”

supporting

confidence: 93%

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

Stoyanov

Das²,

Fredrickson³

et al. 2005

Electrophoresis

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The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.

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supporting

confidence: 93%

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

Stoyanov

Das²,

Fredrickson³

et al. 2005

Electrophoresis

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“…It has been noted that there is 'no verification that the CAs segregate in a Rotofor in the same way as they do during IEF' and that, in fact, 'Rotofor fractionation is characterized with a lower resolution compared with IEF in capillaries and gels' [32]. This criticism is well taken, and we agree that the CAs' distribution about their pI might be wider in the Rotofor as compared with sharper peaks obtainable in gel IEF.…”

Section: Note Addedmentioning

confidence: 88%

Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results

et al. 2006

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In an attempt to prepare quasi-isoelectric buffers as BGEs for CE, carrier ampholytes (CAs) (Ampholine, pH 7-9; Servalyt, pH 7-9; Bio-Lyte, pH 8-10 and Pharmalyte, pH 8-10.5) have been subdivided with the Rotofor into 20 fractions, of ca. 0.1 pH unit span, whose composition has been studied by CZE-MS. The results have allowed identifying the number of different molecular mass compounds present in every commercial brand, as well as the number of isoforms (having identical mass, but representing positional isomers) associated with a given M(r) value. Ampholine is composed of 29 species, for a total of 85 different isoforms; Bio-Lyte is made of 43 compounds, for a total of 136 isoforms; Pharmalyte comprises 58 different M(r) chemicals, for a total of 102 isoforms and Servalyt is constituted by 65 species, for a total of 306 compounds (all of these values to be considered as minimum numbers, as detected by the present methodology). Surprisingly, and contrary to theory, a very large proportion (up to 70%) of these species are 'poor carrier ampholytes', in that they are unable to focus and are evenly distributed along the generated pH gradient in the electric field. Paradoxically, the pH gradient is created and sustained by the minority of species (30% for three brands, up to 50% for Pharmalyte) that appear to focus at their pI position into reasonably sharp zones. Even in the narrowest pI fraction, up to 20 different compounds can be detected. It is concluded that very few amines with different useful pK values are utilized for the synthesis and that a new generation of CAs with a more diversified population of amines with proper pK values within the given pH intervals should be sought. Ampholine, the poorest of the commercial brands, appears to be still made with the original synthesis devised by Vesterberg, i.e. by reacting a concoction of oligoamines with alpha,beta-unsaturated acids.

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“…Other electrokinetic devices that have evolved are based on the principles of IEF rather than exploiting size/mobility differences. Bier and associates have developed two systems based on IEF: one employs partial compartmentalilsation of the fractions by the use of screens to minimise the effects of convective mixing [5] while the other uses laminar flow to help create stable, focused protein zones [6]. The multicompartment electrolyzer, developed by Righetti and associates [7], is another IEF-based preparative technique.…”

Section: Multifunctional Apparatus For Electrokinetic Processing Of Pmentioning

confidence: 99%

Multifunctional apparatus for electrokinetic processing of proteins

et al. 1994

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New equipment (the "Gradiflow") has been designed and constructed to provide efficient large-scale preparative fractionation of macromolecules, based on charge and/or size differences, as well as the concentration of macromolecules and electrodialysis. Examples of its capability are the separation of a mixture of haemoglobin (50 mg) from bovine serum albumin (50 mg) within 15 min (based on charge differences at pH 6.8), the purification of phycoerythrin from a crude extract on the basis of size, and the fractionation of serum proteins into two discrete size classes.

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Fluid Stabilization during Isoelectric Focusing in Cylindrical and Annular Columns

Cited by 13 publications

References 15 publications

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results

Multifunctional apparatus for electrokinetic processing of proteins

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