Quasi-linear pH gradients for chromatofocusing using simple buffer mixtures: local equilibrium theory and experimental verification

Bates, Ronald C; Frey, Douglas D.

doi:10.1016/s0021-9673(98)00420-8

Cited by 46 publications

(17 citation statements)

References 15 publications

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“…22) Bu ers containing organic salts are generally avoided in LC-MALDI as well because they hinder the ionization of samples in MALDI.…”

Section: Chromatofocusing-maldi-msmentioning

confidence: 99%

“…25) eir linear pH gradient was ideal, but the binary elution program for producing the ideal linear pH gradient was extremely complex. Nonetheless, a method for constructing a linear pH gradient using commonly used bu ers in LC has been reported, 22,26,27) and the desired pH gradient is readily constructed (Fig. S-14, supporting information).…”

Section: Chromatofocusing-maldi-msmentioning

confidence: 99%

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Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

et al. 2014

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Mass spectrometry (MS) is a highly sensitive analytical technique that is o en coupled with liquid chromatography (LC). However, some bu ering salts used in LC (e.g., phosphate and tris(hydroxymethyl) aminomethane (Tris)) are incompatible with MS since they cause ion-source contamination and signal suppression. In this study, we examined salt tolerance of MALDI and applied a matrix additive methylenediphosphonic acid (MDPNA) to reduce salt-induced signal suppression. MDPNA signi cantly improved the salt tolerance of MALDI-MS. Using ammonium formate bu er at pH 5.0, the e ective range of bu ering salt concentration in MALDI-MS using MDPNA was estimated up to 250 mM. MDPNA reduced signal suppression caused by bu ering salts at pH 4.0 to 8.0. We observed that MDPNA e ectively worked over a wide range of bu er conditions. MDPNA was further applied to hydrophilic interaction chromatography (HILIC) and chromatofocusing-MALDI-MS. As a result, the analytes in the eluent containing high-concentration salts were detected with high sensitivity. us, our study provides simple and fast LC-MALDI-MS analysis technique not having strict limitation of bu ering condition in LC by using matrix additive MDPNA.

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“…22) Bu ers containing organic salts are generally avoided in LC-MALDI as well because they hinder the ionization of samples in MALDI.…”

Section: Chromatofocusing-maldi-msmentioning

confidence: 99%

Section: Chromatofocusing-maldi-msmentioning

confidence: 99%

Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

et al. 2014

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“…Frey and colleagues further developed the theory of CF advancing numerical simulation methods for the calculation of the internal pH [61,62] and creating simple buffers systems to generate a quasi-linear pH-gradient in the column [63]. During CF, proteins are separated according to differences in isoelectric points.…”

Section: Chromatofocusingmentioning

confidence: 99%

Chromatographic and electrophoretic characterization of protein variants

Ahrer

Jungbauer

2006

Journal of Chromatography B

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“…Under certain conditions, although a step change is applied at the column entrance, a smooth, reproducible pH gradient can be formed within the column. Since these gradients travel at a rate slower than the mobile phase velocity, individual species are focused at specific pH values along the traveling wave (8,9).…”

Section: Introductionmentioning

confidence: 99%

Predicting Retention and Resolution of Protein Charge Variants in Mixed-Beds of Strong and Weak Anion Exchange Resins with Step-Induced pH Gradients

Vetter

Ferreira²,

Robbins³

et al. 2014

Separation Science and Technology

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A model is developed to predict protein retention and resolution in mixed-bed columns containing a mixture of a small-pore weak base resin and a large-pore strong base resin. Using amine buffers, the weak base resin generates smooth pH gradients when the column is subjected to pH steps, while the strong base resin interacts with the proteins effecting the separation. The model describes simultaneously ion exchange interaction on the weak base resin and protein adsorption and transport on the strong base resin. The separation of a monoclonal antibody (mAb) from a charge variant whose pI is about 0.2 pH units lower is studied experimentally with the anion exchange resins AG 4-X4 and SOURCE 30Q as the weak and strong base components, respectively. The properties of each resin are characterized independently and model parameters are obtained experimentally for each. Model predictions of pH gradient-based separations in columns containing different proportions of the two resins and at different buffer concentrations are found to be in agreement with experimental results. The model can thus be used as a process design tool to predict combinations of buffer and mixed-bed compositions, column lengths, and mobile phase velocities that would yield a desired separation performance.

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Quasi-linear pH gradients for chromatofocusing using simple buffer mixtures: local equilibrium theory and experimental verification

Cited by 46 publications

References 15 publications

Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

Chromatographic and electrophoretic characterization of protein variants

Predicting Retention and Resolution of Protein Charge Variants in Mixed-Beds of Strong and Weak Anion Exchange Resins with Step-Induced pH Gradients

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