Gel permeation chromatography with mixed eluents: Polymer solution as mobile phase

The GPC elution behaviour of a polymer was studied when a solution of another polymer in a liquid was used as an eluent. In ternary systems containing two polymers, GPC results are influenced by the thermodynamics of polymer incompatibility. The incompatibility manifests itself both in the slope of the dependence of elution volume upon the concentration of injected polymer and in the shift of elution volumes extrapolated to zero concentration. Experimental data for systems containing poly(styrene) and poly(methyl methacrylate) have been accounted for qualitatively by theoretical views on coil shrinkage resulting from polymer incompatibility. The concentration effects of the injected polymer and the polymer in the mobile phase on elution volume are compared; further parameters affecting the separation in the systems with polymer solution as eluent are discussed.     

Observations on the rapid size-exclusion chromatography of proteins

Summary The retention behaviour of reference proteins on commercial siliceous size-exclusion supports was studied. Sorption was observed on both surface modified and unmodified supports. When sodium dodecylsulfate was added to the aqueous mobile phase, normal elution patterns were found. With this system, proteins, such as those isolate from different alfalfa genotypes, may be compared rapidly. Comparisons were facillitated by use of on-line central data processing capability.     

Verhalten von ng- und μg-Mengen biochemisch interessierender Amine bei Trennverfahren,wie Ionenaustausch-Chromatographie,Mitfällung und Extraktion

The behaviour of ng- and μg-amounts of amines of biochemical interest is compared. ng-amounts are co-precipitated from aqueous solutions with the tetraphenyloborates of potassium, ammonium and creatinine. A separation from potassium chloride, ammonium chloride and creatinine is achieved by extraction of the amines with methanol. The elution volumes of about 50 amines during ion-exchange chromatography using a weakly acidic resin are compared. A procedure has been elaborated by which the amines can be collected from urine.     

Radiation stability of some thermally stable inorganic ion exchangers

Effect of various doses of gamma radiation on the ion-exchange capacity, distribution coefficient values, elution behaviour, pH titration and infrared spectra of some thermally stable inorganic ion exchangers has been studied systematically. No change has been observed in the ion-exchange capacity, elution behaviour and the infrared spectra of the materials irradiated up to a total dose of 3·10⁸ rad, while, a change has been observed in their pH-titration and distribution behaviour.     

Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments.

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.     

Anion exchange studies of indium(III) in malonate and ascorbate solutions separation from mixtures

Summary Systematic studies are presented on the anion-exchange behaviour of indium on Dowex 21K in malonic acid and ascorbic acid solutions. Hydrochloric, sulphuric, nitric and perchloric acids, also sodium and ammonium nitrate at different concentrations were tested as eluants in the two systems. Their efficiency was evaluated in terms of elution constants. Methods have been developed for the separation of indium from several elements in malonate as well as ascorbate media with the technique of selective sorption, selective elution or by gradient elution.     

Theoretical Aspects of Gradient Reversed-Phase High Performance Liquid Chromatography of Styrene – Butylacrylate Block Copolymers

Butylacrylate – styrene co-polymers prepared by atom transfer radical polymeratization were separated on an octadecyl silica column by gradient elution with tetrahydrofuran in water, up to the molar masses 10,000. In reversed-phase high performance liquid chromatography (RP-HPLC), the retention of macromolecules is affected very significantly even by change of a few tenths of per cent of the organic solvent in the aqueous-organic mobile phase. Therefore, gradient elution was used for the determination of the parameters of the equations describing the effects of the mobile phase on the retention behaviour of synthetic polymers. The retention parameters of homopolymers and copolymers were calculated from the gradient data using two retention models. The retention behaviour of the copolymers was described using the experimental gradient retention data for homopolymers.     

Anion-exchange chromatographic properties of alpha-lactalbumin eluted from quaternized polyvinylimidazole. Study of the role of the polymer coating.

The anion-exchange elution behaviour of alpha-lactalbumin was studied on cross-linked and quaternized polyvinylimidazole, deposited on various high-performance liquid chromatographic supports (porous silica and diol silica). The influence of the nature and thickness of the coating layer on the retention and band-width properties of the protein elution peak was examined by isocratic elution. The retention properties of alpha-lactalbumin were studied from the plot of log k' vs. log([NaCl]), where k' is the capacity factor and [NaCl] the displacer salt concentration in the aqueous phase. The retention depends on the amount of stationary phase deposited on the support, but an increased hydrophobic effect is found when the polymer films do not coat the chromatographic support uniformly. Band broadening of the elution peaks was studied in terms of plots of plate height vs. mobile phase velocity. An important mass-transfer contribution is found, which decreases with increasing k' and increases with the thickness of the coating layer. These effects reveal that the diffusion into the polymer layer is the controlling step of the ion-exchange process with non-uniform polymer layers of large mean thickness.     

Cation-exchange behaviour of uranium(vi) on amberlite ir-120 : Separation from mixtures

Quantitative studies are reported on the cation-exchange behaviour of uranium(VI) at the milligram level with Amberlite IR-120. Hydrochloric, nitric, sulphuric, perchloric, acetic and citric acids were tested as cluants; 200–300 ml of 2 N hydrochloric, nitric or sulphuric acid suffice for quantitative elution of 17 mg of uranium(VI) from a 1.4 cm X 14 5 cm bed The efficiency of the elutmg agents is discussed in terms of their elution constants Uranium is separated from thorium by selective elution, from zirconium, cerium(III), copper and nickel by converting the latter into suitable anionic complexes and from phosphate just by passing the mixture through the cation exchanger.     

Atypic Gel Permeation Chromatography of Alcohols

Abstract

The elution behaviour of alcohols in the systems Bio Beads SX-8/dichloromethane and Sephadex LH-20/dichloromethane is investigated. On Bio Beads SX-8 the elution volumes are lower than expected for normal GPC behaviour, which is perhaps due to hydrogen bonding in solution. On Sephadex LH-20 adsorption takes place by means of hydrogen bonding, which can be used for very selective separations.     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

Wheat β-amylase behaviour regarding salt promoted adsorption processes

Summary The behaviour of wheat β-amylase from crude extracts in chromatography on agarose gels substituted with different ligand types was investigated. The enzyme displayed high salt promoted adsorption onto thiophilic gels provided with sulfone-thioether and 2-thiopyridine ligands. Quantitative recovery of the enzyme was easily accomplished by elution with buffer in the absence of Na₂SO₄. The 3-(2-pyridylthio)-2-hydroxypropylagarose (PyS-gel) also allowed elimination of pigments present in the wheat extract. These pigments showed no adsorption onto the gel, thus regeneration is easily achieved, allowing its re-use. The enzyme also displayed strong salt-dependent adsorption onto adsorbents provided with pyridyldisulfide moieties, but in this case enzyme binding was due to its thiol content since elution was achieved mainly through reduction with DTT. When the enzyme was chromatographed on a series of hydrophobic alkyl ligands in the presence of 0.5 M sodium sulphate, it was partially adsorbed on pentylagarose and quantitatively adsorbed on hexyl-agarose, elution being easily performed by sodium sulphate-free buffer. This behaviour was markedly different from that towards phenyl-Sepharose, to which the enzyme was strongly adsorbed and which required much more drastic elution conditions.     

Study of affinity supports based on reactive polymers immobilized on silica: affinity constant determination from isocratic zonal elution.

Polymers bearing benzamidine moieties have been prepared from reactive copolymer containing chloroformate functions and deposited on porous silica matrices. These high-performance affinity chromatography supports were characterized by quantitative methods, which analyse the zonal elution behaviour of trypsin in the presence of a soluble competitor (L-arginine). The column loading capacity for trypsin was measured by the zonal elution method in mass overload conditions. On the basis of a Langmuir isotherm, the influence of the protein capacity and the concentration of the soluble ligand on the elution volume was studied for the determination of the binding constants. The plate heights determined for silica supports of various porosities and particle diameters show that the strong affinity interactions between trypsin and p-aminobenzamidine are mainly responsible for the low efficiencies observed.     

Isolation by Means of Preparative Reversed-Phase Liquid Chromatography of Epimeric Alcohols Formed Upon Reduction of Pregnenolone and Progesterone

Abstract

A method is described which permits complete separation on a preparatory scale of the 20R and 20S epimeric alcohols obtained from lithium aluminium hydride and sodium borohydride reduction of pregnenolone and progesterone, respectively. The retention behaviour and resolution obtained on chromatography of the epimers on C-18 bonded phase material and elution with different acetonitrile/water and methanol/water mobile phases were studied. The order of retention is in both cases in accordance with ¹H-NMR chemical shift data which indicate a stable conformation with a more exposed 20-OH group in the 20S (=20α) epimer. Deviations from the elution order expected for true reversed-phase retention mechanisms were found on elution with mobile phase systems of reduced water content.     

Temperature profiling of polypeptides in reversed-phase liquid chromatography. II. Monitoring of folding and stability of two-stranded alpha-helical coiled-coils

The present study extends the utility of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor folding and stability of de novo designed synthetic two-stranded alpha-helical coiled-coils. Thus, we have compared the effect of temperature on the RP-HPLC retention behaviour of both oxidized (two identical five-heptad alpha-helical peptides linked by a disulfide bridge) and reduced coiled-coil analogues with various amino acids substituted into the hydrophobic core of the coiled-coil. We were able to correlate the RP-HPLC retention behaviour of the oxidized analogues over the temperature range of 10 to 80 degrees C with the stability of the analogues as determined by conventional thermal and chemical denaturation approaches. In addition, the contribution of a disulfide bridge to coiled-coil stability was highlighted by comparing the elution behaviour of the oxidized and reduced analogues. Overall, we demonstrate the excellent potential of "temperature profiling" by RP-HPLC to monitor differences in oligomerization state and protein stability.     

Reversed phase behaviour of high molar mass polystyrenes in acetonitrile with halogenated methane solvent mixtures

Summary The reversed phase behaviour of high molecular mass polystyrenes was investigated on a C18 bonded phase column using acetonitrile as the polar mobile phase component and dichloromethane, chloroform and carbon tetrachloride, as separate nonpolar components. Solvent solubility compositions, elution compositions and resolutions of the various molar mass polystyrenes were measured and compared for each of the three mobile phases. Anomalous behaviour of the polystyrenes was not observed in any of the three mobile phases and only minor differences in resolution were observed. By comparing these results with previously published work, it is suggested that any mobile phase component that is both nonpolar and a good solvent for the polymer can be combined with acetonitrile for effective separations of polystyrenes by mass and that it is the poor solvent component of the mobile phase that is responsible for the anomalous behaviour that is sometimes observed in this type of chromatography.     

Gradient elution in normal-phase high-performance liquid chromatographic systems

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.     

Statistical properties of the band-broadening function

Statistical properties of the band-broadening function calculated from the equilibrium and kinetic theories of Giddings and Eyring are compared with real liquid-chromatography elution curves of low-molecular-weight substances and with light-scattering elution curves of a high-molecular-weight polystyrene standard injected at several concentrations. The curves obtained by liquid chromatography agree in a large span of elution times with the theoretical ones and can be described by one value of the plate-height. The size exclusion chromatography (SEC) elution curves seem to approach the predicted statistical properties with decreasing concentration of injected polymer but, in general, a reliable prediction of band-broadening (BB) functions in SEC of polymers, especially of non-symmetric BB functions near the exclusion limit, seems not possible at this point. The relation of the theory with the characteristic-function approach developed by Dondi is also discussed.     

Adsorption and separation of proteins by a synthetic hydrotalcite

In this study, the potential use of a synthetic Mg/Al hydrotalcite (layered double hydroxide) as a novel chromatography material for protein purification was investigated. The hydrotalcite is present in its carbonate form and is characterized by an Al/Mg-ratio of 1.85. Zetapotential measurements confirm a positive surface potential up to pH 10 suggesting applicability as anion exchanger. The binding of model proteins covering a broad range of isoelectric points and molecular weights was performed at different pH-values under batch conditions to evaluate the binding behaviour of the hydrotalcite. Furthermore, static binding capacities were exemplarily determined for hemoglobin and human serum albumin. Additionally, the adsorption and elution of hemoglobin was studied under dynamic conditions. The binding behaviour of the hydrotalcite was compared to commercially available anion exchangers and was found to be a function of pH, depending on the model protein. Variant adsorption behaviour is explained by further interactions like hydrogen bonds and by an unequal charge distribution over the protein surfaces. The hydrotalcite reveals high adsorption capacities under static (260 mg/g) as well as under dynamic conditions (88 mg/g at 34 cm/h; 61 mg/g at 340 cm/h). With appropriate buffers like 500 mM carbonate (pH 10) the adsorbed proteins can be nearly completely desorbed making regeneration possible. Due to the binding and elution properties it is concluded, that the hydrotalcite can serve anion exchange material for chromatographic protein separations.     

Isolation of lanthanides from spent nuclear fuel by means of high performance ion chromatography (HPIC) prior to mass spectrometric analysis

Pure and complete fractions of neodymium, samarium, europium, gadolinium and dysprosium were isolated by means of high performance ion chromatography, using a cation exchange column and gradient elution with alpha-hydroxyisobutyric acid solutions (α-HIBA). Intermediate precision and robustness of the isolation method was investigated, identifying eluent pH as the most important parameter. Investigation of the elution behaviour of the most important fission and activation products and actinides indicated that preventing the accumulation of cesium on the cation exchange column required further isocratic elution with a higher concentrated α-HIBA solution after elution of the lanthanides. A sample matrix free of carbon was achieved by means of acid digestion, followed by UV photo-oxidation, resulting in samples suitable for mass spectrometric analysis.