General local‐equilibrium chromatographic theory for eluents containing adsorbing buffers

Frey, Douglas D.; Narahari, Chittoor R.; Butler, Christy D.

doi:10.1002/aic.690480313

Cited by 10 publications

(8 citation statements)

References 20 publications

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“…Formation of pH Gradients on a Strong-Base Ion Exchange Column Packing Using Polyampholytes Figure 1 illustrates calculations adapted from Frey et al (2002) performed using the numerical method of characteristics and using conditions representative of those commonly used for chromatofocusing on a weak-base anion-exchange column packing, except that the polyampholyte elution buffer is replaced by a mixture of five weak-acid buffering species (species A 1 to A 5 ) and one weak-base buffering species (species B 1 ). The figure specifically shows the effluent values for the pH and the total concentration of each buffering species (i.e., the sum of the concentrations of all forms of each buffering species) as a function of the dimensionless time.…”

Section: Resultsmentioning

confidence: 99%

“…Although chromatofocusing employing a polyampholyte buffer does not strictly conform to the above restriction that only inert ions are adsorbed, where an inert ion is defined to be an ion not participating in acid -base equilibrium, it was nevertheless shown theoretically by Frey et al (1995Frey et al ( , 2002 that linear or nearly linear pH gradients can be produced using a strong-base anion-exchange column packing having no buffering capacity, provided that the packing is first presaturated with an adsorbed buffering species (i.e., with a weak acid for the case of an anion-exchange column packing), and then eluted with a polyampholyte buffer. This further indicates that an ''eveness'' for the column packing buffering capacity is not required to produce a linear pH gradient in chromatofocusing.…”

Section: Introductionmentioning

confidence: 99%

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Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion‐exchange adsorbents

Kang

Frey

2004

Biotech & Bioengineering

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Although it is commonly believed that a column packing used for chromatofocusing must have an ''even'' buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong-acid cation-exchange column packing or a strongbase anion-exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak-acid or weakbase functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using chromatofocusing and a strong-acid cation-exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ionexchange chromatography using a salt gradient for this purpose. B

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion‐exchange adsorbents

Kang

Frey

2004

Biotech & Bioengineering

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“…The stoichiometric relationship may [1][2][3]6] or may not [7][8][9][10][11] be incorporated into the model to account for interactions between mobile species and resin fixed-charge sites. When the stoichiometric relationship is not included, the phase equilibrium is usually seen to be described by means of an equilibrium constant [7] or selectivity factor [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

“…The stoichiometric displacement model assumes a strict relationship between bound or absorbed counterions and mobile counterions diffusing into the resin phase [1][2][3][4][5][6]. The literal interpretation of this approach is that one ion of charge z is bound to z resin fixed sites.…”

Section: Introductionmentioning

confidence: 99%

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Buffer salt effect on pH in the interior of an anion exchange resin

Hardin

Ivory

2006

Journal of Colloid and Interface Science

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Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures

Shen

Bieberich

et al. 2008

Methods in Molecular Biology™

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Chromatofocusing has many potential applications in the field of proteomics, such as for the isolation and removal of major sample components to facilitate the analysis of low-abundance components, and for sample prefractionation prior to a subsequent separation using SDS-PAGE, narrow-pI-range 2D-PAGE, or additional chromatography steps. However, the chromatofocusing techniques that are most commonly used employ propriety polyampholyte elution buffers and highly specialized column packings, both of which limit the use of chromatofocusing in practice. To expand the range of application for this technique, this chapter considers chromatofocusing methods which employ common ion-exchange column packings and elution buffers which are simple mixtures of readily available buffering species. Of particular interest is the use of chromatofocusing with a multistep pH gradient for the fractionation of protein mixtures into narrow-pI-range fractions. The cross-contamination characteristics of these fractions using SDS-PAGE are also assessed.

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General local‐equilibrium chromatographic theory for eluents containing adsorbing buffers

Cited by 10 publications

References 20 publications

Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion‐exchange adsorbents

Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion‐exchange adsorbents

Buffer salt effect on pH in the interior of an anion exchange resin

Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures

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