Isocratic Anion Exchange Chromatography Research Articles

Competitive protein adsorption in isocratic anion exchange chromatography

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.

Separation and determination of fluorobenzoic acids using ion chromatography–electrospray mass spectrometry

A method for the trace analysis of fluorobenzoic acids (FBAs) via IC–MS based on solid-phase extraction (SPE) prior to isocratic anion exchange chromatography is described. Up to 23 different fluorobenzoic acids were enriched and determined simultaneously. Solid-phase extraction on hydrophilic–lipophilic-balanced reversed-phase cartridges containing a poly(divinylbenzene-co-N-vinylpyrrolidone) polymer allowed a 500-fold enrichment of the acids if 100mL sample was used with recoveries between 28% and 98%. The method enables the determination of FBAs down to the range of 16–210ngL−1, provides sample-preparation (pH-adjusting prior to SPE only) with no need of derivatization, uses low amounts of chemicals and is adaptable to larger or smaller sample volumes.

, -Alkyl Dicarboxylic Acids: Characterization by Isocratic Anion-Exchange Chromatography

A homologous series of 7 α,ω-alkyl dicarboxylic acids is characterized using isocratic anion-exchange chromatography at different mobile phase salt concentrations to determine the characteristic charge and the equilibrium ion-exchange constant (and the corresponding Gibbs free energy of ion-exchange). The characteristic charge agrees closely with the number of carboxylic acid groups in the samples, namely 2, and the ion-exchange constant seemed to remain almost unaffected by the number of methylene groups in the sample. Succinic acid, having 2 methylene groups between the terminal carboxylic acid groups, always produces 2 chromatographic peaks despite the proven chemical purity of the sample. The characteristic charge calculated for both peaks of succinic acid is approximately 2, as is that of the other homologues. The Gibbs free energy of ion-exchange calculated for the weakly retained peak of succinic acid is similar to that of the other samples, whereas that of the strongly retained peak is lower by 2.1 kJ/mol. The 2 peaks of succinic acid are explained in terms of an equilibrium between two conformers in solution, one binding the solution counterions tightly and the other loosely.

Carbohydrate-deficient transferrin in alcoholics with liver disease.

To assess the relationship between carbohydrate-deficient transferrin (CDT) and alcoholic liver disease, we measured the ratio of carbohydrate-deficient transferrin to total transferrin (rCDT) in 32 male alcoholics with liver disease (Child-Pugh class A, 8; B, 11; C, 13) and 14 male alcoholics without clinically evident liver disease. Twenty of 32 with liver disease and six of 14 without clinically apparent liver disease had recent abstinence. The 32 patients with liver disease were assessed, in addition to the Child-Pugh class, using a linear prognostic score, the Combined Clinical and Laboratory Index (CCLI). Transferrin and CDT were measured by isocratic anion exchange chromatography and a radio-immunoassay. When the total group (n = 46) was divided into those with recent abstinence (n = 26) and those without (n = 20), the rCDT was lower in the abstainers than non-abstainers (0.7 +/- 0.6 vs 2.9 +/- 2.4, P < 0.005). Similarly, abstainers with liver disease (n = 20) had a significantly lower rCDT than non-abstainers (n = 12) with liver disease (0.7 +/- 0.7 vs 3.5 +/- 2.8, P < 0.005). The rCDT in the 20 abstaining patients with liver disease did not differ significantly between Child-Pugh classes. Furthermore, there was no correlation between the CCLI and rCDT (r = 0.05). We conclude that the relationship between rCDT and alcohol abuse is not appreciably altered by the presence of clinically severe liver disease in male alcoholics.

Carbohydrate deficient serum transferrin in a new systemic hereditary syndrome.

Four patients with a new, inherited, complex developmental deficiency syndrome were studied. The syndrome affects the central and peripheral nervous system, and also the retina, liver, bone, adipose tissue, and genital organs. Abnormalities of glycoproteins, glycopeptide hormones, and lipids have been found in serum from these patients, the most pronounced being increased cathodal forms of transferrin. Isoforms of serum transferrin were therefore analysed qualitatively and quantitatively by isoelectric focusing and isocratic anion exchange chromatography, and the carbohydrate composition was determined in transferrin isolated by immune affinity chromatography. All the patients had about tenfold raised serum concentrations of isotransferrins with higher isoelectric points than normal. Similar findings, though less pronounced, were made in all the fathers and in one of the mothers. Half the transferrin in the patients was constantly present in two principal abnormal cathodal forms in approximately equal amounts. Carbohydrate determinations in purified transferrin showed quantitatively similar deficiencies of sialic acid, galactose, and N-acetylglucosamine, the mannose content being normal. The results suggest that either two or all of the normally four terminal trisaccharides in transferrin may be missing. A defect in the synthesis or catabolism, or both, of this trisaccharide, which is common to many secretory glyco-conjugates, is likely. Apart from providing a quantitative diagnostic method, the present findings may serve as a basis for further studies of the metabolic deficiency in this syndrome.

High-performance liquid chromatography of amino acids, peptides and proteins : LXXXVIII. Calculation of the average distance between protein solutes and the stationary phase during isocratic anion-exchange chromatography

This investigation deals with protein retention behaviour in high-performance anion-exchange chromatography in terms of the average distance of approach between the protein solute and the positively charged anion-exchange stationary-phase surface. The theoretical treatment is based on a modified Debye-Hückel theory for spherical impenetrable ions, where the electrostatic potential energy has been related to the chromatographic capacity factor, k′. Results are presented for three globular proteins, eluted isocratically from a Mono-Q strong anion-exchange resin with sodium choride as the displacer salt by a mobile phase with pH in the range 5.50-9.60. Analysis of experimental retention data indicates that topographically predefined, charged regions on the protein surface, called ionotopes, control the orientation and approach distance of the protein solute.

High-performance liquid chromatography of amino acids, peptides and proteins : LXXXVII. Comparison of retention and bandwidth properties of proteins eluted by gradient and isocratic anion-exchange chromatography

The high-performance ion-exchange gradient-elution behaviour of a range of globular proteins has been investigated, using a strong anion exchanger as the stationary phase and sodium chloride as the displacer salt. Deviations were observed between the Z c values obtained from isocratic experiments and from gradient experiments with varied gradient time and varied flow-rate. These results indicate that theoretical treatments which relate gradient and isocratic elution processes do not adequately describe the retention behaviour of protein solutes separated by ion-exchange methods. Furthermore, the experimentally observed bandwidths deviated significantly from values predicted on the basis of plate theory for low-molecular-weight molecules. The significance of these results is discussed in terms of the influence of experimental parameters on the ability of particular electrostatically interactive areas on the surface of protein solutes to control the thermodynamic and kinetic properties of these polyelectrolyte molecules during ion-exchange chromatographic processes.

High-performance liquid chromatography of amino acids, peptides and proteins : LXXXVI. The influence of different displacer salts on the retention and bandwidth properties of proteins separated by isocratic anion-exchange chromatography

The influence of different displacer salts on the retention behaviour of seven globular proteins ranging in moleclar weight from 12 000 to 69 000 was investigated using the Mono Q anion-exchange resin as the stationary phase. Isocratic retention data were collected using several different alkali metal halides as the displacing salt, thereby systematically varying the anion and cation species in the series F −, Cl − and Br − and Li +, Na + and K +. The different anions were found to reduce protein retention in order of their decreasing hydrated ionic radii. Protein Z c values were found to be lower for fluoride and bromide than for chloride. It was demonstrated that the cationic co-ions also influence solute retention properties with this anion-exchange resin through, inter alia, preferential interactions with the protein solute. Protein band-broadening was found to systematically vary with the choice of displacer salt. These changes were related to known Hofmeister effects on protein aggregation kinetics and solubility and the degree of ion penetration at the double layer of the stationary phase-mobile phase interface. These studies now provide a rapid comparative basis for evaluating the mechanism of co- and counter-ion interactions with proteins in high-performance ion-exchange chromatographic systems.

Micro anion exchange chromatography of carbohydrate-deficient transferrin in serum in relation to alcohol consumption (Swedish Patent 8400587-5).

A new simplified and rapid method for detection and quantitation of "carbohydrate-deficient transferrin" in serum is described. The method is based on isocratic anion exchange chromatography of isotransferrins in disposable microcolumns followed by a double antibody transferrin radioimmune assay. This technique, which separates all transferrin components isoelectric above pH 5.65, showed a very good reproducibility and accuracy with a coefficient of variation between 5 and 9%. 77 alcoholic patients could be clearly separated from 80 healthy "normal consumers" and 33 total abstainers with a specificity of 100% and a sensitivity of 91%. The values were significantly correlated to the amount of alcohol consumed during the latest month, and declined in abstaining alcoholics with a mean biological half-life of 17 days. Elevated levels occasionally appeared in healthy individuals after daily consumption of 60 g of ethanol during a 10-day period. In a sample of 187 patients with nonalcohol-related conditions only 2% false-positive values were found. This method is suggested as a potential tool for detecting and monitoring alcohol abuse.