Separation of gangliosides by anion-exchange chromatography on Mono Q

A new type of strong anion-exchange resin, Mono Q, has been used in the separation of brain gangliosides. The resin consists of monodisperse particles (9.8 micron) and was used in prepacked columns with a bed volume of 1 ml. The gangliosides were separated into mono-, di-, tri- and tetrasialoganglioside fractions by a discontinuous gradient of potassium acetate in methanol. The separation was complete in a volume of 50 ml. The major advantages of the new procedure compared to conventional methods are the shorter separation time, higher loading capacity and recovery of separated ganglioside fractions in small solvent volumes. The procedure was applied to the separation of gangliosides from normal human and GM2-gangliosidosis brain.     

Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on macroporous anion-exchange resin

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a macroporous anion-exchange resin of 10 microm in particle diameter. We studied effects of flow-rate, gradient time (steepness of salt concentration gradient) and column length on resolution in wide ranges. It was found that very high resolutions are attainable at long gradient times with long columns. The resolution continuously became higher as the gradient time and the column length became longer except in some special cases. The dependence of resolution on gradient time was particularly great when the column was long and the gradient time for the change of 0-0.5 M NaCl was longer than 2 h. On the other hand, the effect of flow-rate on resolution was very small. Although the separations at long gradient times with long columns have not been popular in high-performance IEC and it takes several hours for one separation, such separations should be advantageous when very high resolutions are required like in proteomics research.     

Sorption of germanium from alkaline solutions on anion-exchange resin

Isotherm of OH^?/GeO ₃ ^2? ion exchange on a synthetic sorbent is studied. The limiting sorption of the germanate ion and the apparent constant and Gibbs energy of ion exchange are estimated.     

Separation of aromatic compounds on pellicular anion-exchange resins

Aromatic compounds including phenols, carboxylic acids and aldehydes, can be rapidly separated on pellicular anion-exchange resins in the acetate form by elution with a sodium acetate solution. Like the aromatic carboxylic acids, hydroxybenzaldehydes are held as counter ions in the resin. The affinity of the resin for corresponding anions as well as that for sorbed non-electrolytes is largely influenced by the aromatic ring and non-polar aliphatic substituents. Formation of hydrogen bonds between the resin and phenolic groups is less important.     

Adsorption equilibrium of fructosyltransferase on a weak anion-exchange resin

The adsorption equilibrium of a glycoprotein, fructosyltransferase from Aureobasidium pullulans, on an anion-exchange resin, Sepabeads FP-DA activated with 0.1M NaOH, was investigated. The adsorption isotherms were determined at 20 degrees C in a phosphate-citrate buffer with pH 6.0 using the static method. Sodium chloride was used to adjust the ionic strength in the range from 0.0215 to 0.1215 mol dm(-3) which provided conditions varying from a weak effect of salt concentration on protein binding to its strong suppression. The equilibrium data were very well fitted by means of the steric mass-action model when the ion-exchange capacity of 290 mmol dm(-3) was obtained from independent frontal column experiments. The model fit provided the protein characteristic charge equal to 1.9, equilibrium constant 0.326, and steric factor 1.095 x 10(5).     

Separation of glycoprotein-N-glycans by high-pH anion-exchange chromatography

A series of high-mannose and complex type glycoprotein-N-glycans was subjected to high-pH anion-exchange chromatography. The results revealed that this method represents a useful tool for analytical characterization of single oligosaccharides as well as preparative separation of complex mixtures of carbohydrate side-chains. On the other hand, it became evident that in several cases a combination of different chromatographic techniques is required for efficient separation of individual oligosaccharide species.     

Dielectric analysis of macroporous anion-exchange resin beads suspensions

Dielectric measurements were carried out for suspensions of D354 anion-exchange beads dispersed in electrolyte solutions at different concentrations, and distinct Maxwell-Wagner dielectric relaxations were observed around 10(6) Hz. Through fitting the experimental data we obtained the dielectric parameters of the suspensions, and then we calculated the phase parameters from the dielectric parameters and the measured volume fractions by Hanai's method. In light of the present understanding of the interfacial properties, and with the information obtained from the phase parameters, we satisfactorily interpreted the concentration dependences of the dielectric parameters. It is concluded that Hanai's method is an effective tool for obtaining the properties of dispersed particles; the properties of the electrical double layer, which are mainly decided by the properties of the electrolyte solution, predict the dielectric behavior of suspensions with conducting particles. The dielectric relaxation spectroscopy (DRS), based on the M-W mechanism, is also a very sensitive tool for probing the properties of the liquid/solid interface.     

Separation of iron-52 from chromium cyclotron targets on the 2% cross-linked anion-exchange resin AG1-X2 in hydrochloric acid

Iron-52 can be separated from solutions of chromium cyclotron targets by eluting chromium, copper and radioactive impurities with 9.0M hydrochloric acid from a column containing 1.0 g of AG1-X2 anion-exchange resin. Iron-52 is retained and can then be eluted with 6.0M hydrochloric acid containing 0.05M hydrogen iodide or 0.05M sodium iodide. The separations are sharp and quantitative. Less than 2 microg of chromium will remain with the iron-52, from 2.0 g originally present.     

Separation of sugar derivatives by partition chromatography on anion-exchange resins

The separation of a number of sugar derivatives such as methyl, ethyl, benzyl, hydroxyethyl erners and various glycosides has been studied. An efficient separation of most species was obtained in 94% ethanol using an anion-exchange resin in its sulfate form. The eluate concentration was determined automatically using the orcinol method.     

Thermal decomposition of anion-exchange resin based on copolymer of 4-vinylpyridine

Formal kinetic model is suggested on the basis of experimental studies of the heat-release kinetics in decomposition of an anion-exchange resin, copolymer of N-methyl-4-vinylpyridinium chloride and divinylbenzene. The model makes it possible to determine the service life of the resin in the temperature range 100–170°C in optimization of the process in which monosilane is synthesized from trichlorosilane with the resin used as a catalyst.     

Separation of nucleotide oligomers by unitary anion-exchange.

This paper is the first report on the retention behavior of synthetic oligonucleotides and nucleotide oligomers on a continuous-bedmatrix, strong-anion-exchange column. The separation mechanism is predominantly an anion-exchange process, but hydrophobic interaction plays a role as well. The separation is based on the chain length of the oligonucleotide. Both the addition of organic mobile phase modifiers and changes in column temperature affect the retention of oligomers significantly. A volatile buffer system (e.g., triethylamine acetate) could be employed to purify oligonucleotides, and no desalting procedure would be required after the column separation step. The recoveries from the separation are 70% or higher. The maximum loading capacity of an analytical column (35 x 7-mm i.d.) was found to be more than 366 micrograms.     

Separation of rhodium and iridium by anion-exchange thin-layer chromatography

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Spectro photometric determination of thallium(I) and dimethylthallium compounds in small concentrations

     

Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography

Synthetic oligonucleotides (ONs) are often prepared for development of therapeutic candidates. Among the modifications most often incorporated into therapeutic ONs are phosphorothioate (PT) linkages. The PT linkage introduces an additional chiral center at phosphorus to the chiral centers in D-ribose (and 2-deoxy-D-ribose) of the nucleic acid. Therefore, modified linkages can produce a diastereoisomer pair ([Rp] and [Sp]) at each PT linkage. These isomers are of identical length, sequence, charge and mass, and are not reliably separated by most chromatographic approaches (e.g., reversed phase chromatography) unless the ON is very short. Further these isomers are not distinguishable by single-stage mass spectrometry. During chromatography of a purified anti-NGF (nerve growth factor) aptamer containing 37 bases with 2 PT linkages by monolithic pellicular anion-exchange (pAE) column, we observed four components. The four components were postulated to be: (i) distinct folding conformations; (ii) fully and partially athioated aptamers; or (iii) PT diastereoisomers. Fractionation of the components, followed by de- and re-naturation failed to produce the original forms by refolding, eliminating option (i). Mass spectrometry of the fractionated, desalted samples revealed no significant mass differences, eliminating option (ii). Oxidative conversion of the PT to phosphodiester (PO) linkages in each of the purified components produced a single chromatographic peak, co-eluting with authentic PO aptamer, and having the PO aptamer mass. We conclude that the components resolved by pAE chromatography are diastereoisomers arising from the two PT linkages. Hence, pAE chromatography further enhances characterization of ON therapeutics harboring limited PT linkages and having up to 37 bases.     

Separation of acidic compounds by strong anion-exchange capillary electrochromatography

Separation of the acidic compounds in the ion-exchange capillary electrochromatography (IE-CEC) with strong anion-exchange packing as the stationary phase was studied. It was observed that the electroosmotic flow (EOF) in strong anion-exchange CEC moderately changed with increase of the eluent ionic strength and decrease of the eluent pH, but the acetonitrile concentration in the eluent had almost no effect on the EOF. The EOF in strong anion-exchange CEC with eluent of low pH value was much larger than that in RP-CEC with Spherisorb-ODS as the stationary phase. The retention of acidic compounds on the strong anion-exchange packing was relatively weak due to only partial ionization of them, and both chromatographic and electrophoretic processes contributed to separation. It was observed that the retention values of acidic compounds decreased with the increase of phosphate buffer and acetonitrile concentration in the eluent as well as the decrease of the applied voltage, and even the acidic compounds could elute before the void time. These factors also made an important contribution to the separation selectivity for tested acidic compounds, which could be separated rapidly with high column efficiency of more than 220000 plates/m under the optimized separation conditions.     

Separation of oxidized variants of a monoclonal antibody by anion-exchange

Monoclonal antibodies are subject to a variety of degradation mechanisms, therefore orthogonal techniques are required to demonstrate product quality. In this study, the three individual antibodies comprising a multi-antibody drug product, XOMA 3AB were evaluated by both cation-exchange (CEX) and anion-exchange chromatography (AEX). In contrast to CEX analysis which showed only a single, broad peak for the force-oxidized antibodies, AEX analysis of Ab-A (pI=7.6) revealed two more basic peaks. Ab-B (pI=6.7) bound but exhibited only a single major peak while Ab-C (pI=8.6) flowed through. Peptide mapping LC/MS analysis of the isolated Ab-A fractions demonstrated that the basic peaks resulted from oxidation in a complementary determining region (CDR). Differential scanning calorimetry (DSC) analysis of the oxidized Ab-A species showed a decrease in the Fab melting point for the oxidized species consistent with unfolding of the molecule. Greater/lesser surface exposure of ionic residues resulting from a conformational change provides a likely explanation for the dramatic shift in retention behavior for the Ab-A oxidized variants. Peptide mapping analysis of the Ab-B antibody showed, in contrast to Ab-A, no detectable CDR oxidation. Hence, the lack of separation of oxidized variants in Ab-B can be explained by the absence of CDR oxidation and the associated changes in secondary/tertiary structure which were observed for oxidized Ab-A. In summary, anion-exchange HPLC shows potential as an orthogonal analytical technique for assessing product quality of monoclonal antibody therapeutics. In the case of the XOMA 3AB drug product, two of the antibodies bound and one, Ab-A, exhibited separation of CDR oxidized variants.     

Determination of uranium isotopes in urine samples from radiation workers using 232U tracer, anion-exchange resin and alpha-spectrometry

Bioassay technique is used for the estimation of actinides present in the body based on the excretion rate of body fluids. For occupational radiation workers urine assay is the preferred method for monitoring of chronic internal exposure. Determination of low concentrations of actinides such as plutonium, americium and uranium at low level of mBq in urine by alpha-spectrometry requires pre-concentration of large volumes of urine. This paper deals with standardization of analytical method for the determination of U-isotopes in urine samples using anion-exchange resin and ²³²U tracer for radiochemical recovery. The method involves oxidation of urine followed by co-precipitation of uranium along with calcium phosphate. Separation of U was carried out by Amberlite, IRA-400, anion-exchange resin. U-fraction was electrodeposited and activity estimated using tracer recovery by alpha-spectrometer. Eight routine urine samples of radiation workers were analyzed and consistent radiochemical tracer recovery was obtained in the range of 51% to 67% with a mean and standard deviation of 60% and 5.4%, respectively.