Effect of steric exclusion on the separation of proteins by hydrophilic size-exclusion chromatography

On the basis of studying the retention and band broadening of proteins on the TSK SW column, diffusion coefficients (Ds) of solute in stationary phase were obtained which elucidate the hydrodynamic process of chromatographic resolution of proteins by hydrophilic size-exclusion chromatography (SEC). After calculating the correlation between Ds and the molecular weight of the solute, the molecular dimensions of proteins in the process of chromatographic separation can be predicted. Deviations in diffusion coefficient of a protein from the calculated value reflect differences of measured molecular dimensions from molecular volumes predicted from the calibration curve of the SEC column. This study illustrates a convenient method for estimating the purity of proteins by SEC. Deviations from 2 lambda dp (where dp is the particle diameter) in the intercept of the theoretical plate height (H) versus flow-rate (U) curve from the band broadening equation H = CsU + 2 lambda dp + f(alpha M)T (where CsU represents mass transfer resistance caused by solute diffusion in the stationary phase and f(alpha M)T an added term for polydisperse solutes as proposed by Knox and McLennan [Chromatographia, 10 (1977) 75]) reflect impurities in the proteins.     

Capillary electrochromatographic separation of proteins on a column coated with titanium dioxide nanoparticles

A TiO2 nanoparticle (TiO2 NP)-coated open-tubular column for the capillary electrochromatographic separation of proteins is described. The surface chemistry of the TiO2 NPs on the inner wall of the fused silica was significantly affected by the running buffer. By varying of the phosphate buffer pH, only cathodic EOF was indicated. The results showed that TiO2 NPs are existed as a complexed form with the buffer ligand. Good separation of conalbumin (ConA), apo-transferrin (apoTf), ovalbumin (OVA), and BSA could be achieved with phosphate buffer (40 mM, pH 8.0) and an applied voltage of 15 kV. Five peaks of glycoisoforms of OVA were observed under these conditions. In comparison with the retention behavior of the analytes on the bare fused-silica column, the new column's high resolving power seems to be predominantly derived from the ligand exchange of the analytes with the phosphate adsorbed onto the TiO2 NPs. The method was also used to separate egg-white proteins. Both acidic and basic proteins in egg white were separated in a single run. The microheterogeneities of OVA could also be found in it. The separation efficiency for the main peak of OVA in egg white was around 10,000 plates/m.     

Size-exclusion separation of proteins using a biocompatible polymeric monolithic capillary column with mesoporosity

Biocompatible poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared for size exclusion chromatography (SEC) of proteins in the capillary format using Brij 58P in a mixture of hexanes and dodecanol as porogens. The monolithic columns provided size separation of four proteins in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights. Compared to SEC monoliths previously synthesized using a triblock copolymer of polyethylene oxide and polypropylene oxide, an increase in mesoporosity was confirmed by inverse size exclusion chromatography. As a result, improved protein separation in the high molecular weight range and reduced column back-pressure were observed.     

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.     

High-performance size-exclusion chromatography of humic substances on the hydroxyethyl methacrylate column

HEMA, the copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, has proven to be a suitable stationary phase for the size-exclusion chromatographic (SEC) characterization of humic substances in a wide pH range. The SEC measurements were performed typically with alkaline borate-based mobile phase: 0.025 mol/l Na2B4O7, 0.025 mol/l NaCl, 0.001 mol/l Na4P2O7, pH 9.1. After calibrating with globular protein standards, apparent molecular weights of typical peat- and coal-derived humic acids were estimated. The average molecular weights determined with the aid of the HEMA column were quite comparable with the values obtained previously by the method of low-pressure gel-permeation chromatography on the Sephadex column.     

Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Effects of inner diameter of monolithic column on separation of proteins in capillary-liquid chromatography

Polymer monolithic columns with I.D. between 100 and 320 microm were prepared by in-situ polymerization of styrene and divinylbenzene in fused silica capillaries. The effects of monolithic column I.D. on the separation of proteins in reversed-phase capillary-liquid chromatography under gradient elution were systemically studied. The loading capacity was positively proportional to the volume of the stationary phase. It was found that the smaller diameter columns showed better performance for protein separation. The minimum plate height decreases from 34.99 microm (320 microm I.D. column) to 5.39 microm (100 microm I.D. column) for a retained protein. After studying the three parameters of the Van Deemter equation, it was interpreted that the smaller diameter can provide less flow resistance and the better performance may also be improved by the increasing of the effective diffusion. This conclusion was also supported by the data of separation permeability and breakthrough curves.     

高聚物型液相色谱柱在蛋白分离中的应用研究

评价了麦科菲高聚物型(苯乙烯-二乙烯基苯共聚物,PS-DVB)反相色谱柱(MKF-RP色谱柱)的柱压,分离柱效,化学和机械稳定性,并研究了梯度条件、色谱柱长等因素对4种常见蛋白(胰岛素、溶菌酶、牛血清白蛋白、卵清蛋白)分离性能的影响.结果表明,在对4种常见蛋白分离时,每米理论塔板数均可达到105以上,且150 mm的柱长优于250 mm及50 mm.以牛血清白蛋白为例,在0.2～20 μg范围内,其进样量与峰面积的线性关系良好(r=0.9997);最大载样量为80 μg左右.经强酸强碱清洗不会改变色谱柱的柱效.     

High-speed and high-performance size-exclusion chromatography of proteins on a new hydrophilic polystyrene-based resin

High-performance size-exclusion chromatography of some standard proteins, peptides and amino acids on a new hydrophilic packing material obtained by chemical transformation of a cross-linked polystyrene-divinylbenzene copolymer was studied. Columns filled with 4 and 7 micron particles were compared. The influence of the concentration of acetonitrile, isopropanol and trifluoroacetic acid in the mobile phase on the chromatographic performance was investigated. A good linear calibration graph covering the molecular weight range from 200 to 700,000, was obtained under the optimal conditions. The packing material can be used for separations, for molecular weight determinations and for the pre-fractionation of proteins. The high rigidity of the packing material allows relatively high pressures to be used and therefore fast separations to be achieved. The packing material was applied to the chromatography of proteins from beer, bones and milk.     

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXIV. Application of derivative spectroscopy to the study of column residency effects in the reversed-phase and size-exclusion liquid chromatographic separation of proteins

The influence of column residency times on the reversed-phase gradient elution behaviour of human and bovine growth hormones has been investigated using on-line photodiode array spectroscopic detection. Stationary phase induced effects on protein conformation were monitored by changes in the maxima to minima ratio of the second derivative spectrum of the eluted protein. Significant changes in the second derivative ratio of the unmodified and the fully reduced and alkylated protein were observed following long incubation times, i.e. t(dwell) greater than 15 min, at the stationary phase surface in the presence of 0.1% trifluoroacetic acid before elution with a 0-75% aqueous acetonitrile gradient. The application of multi-wavelength detection in the study of equilibrium unfolding of growth hormones in urea by size-exclusion chromatography was also investigated. On-line photodiode array instrumentation and derivative spectra rationing was employed to monitor tertiary and quaternary structural changes associated with protein denaturation during a chromatographic separation. These studies clearly demonstrate the powerful detection capabilities of such instrumental approaches for the on-line evaluation of both stationary phase surface and/or mobile phase mediated changes in protein conformation.     

Designs for size-exclusion separation of macromolecules by densely-engineered mesofilters

This critical review reports on the up-to-date development designs of mesofilters for molecular sieving of macromolecules. Tight mesoporous silica nanotubes (NTs) provide a promising candidate as a filter for the size-exclusion separation of large quantities of macromolecules within a short period. First, we focus on the formation of the mesoporous structures inside anodic alumina membrane (AAM) using nanolinker approaches. Second, we discuss the systematic design of the robust mesofilters based on densely-engineered mesopores of silica NTs, which are vertically aligned with the open surfaces of top-bottom ends inside the AAM. Third, we present the bench-top filtration method as a powerful tool for the size-exclusion separation of macromolecules. Significantly, the evaluation of the intrinsic properties of mesofilters (e.g., diffusivity, separation efficiency, long-term stability, and reusability) compared them with commercial, mature membranes used for separation of macromolecules.     

Optimization of St John's Wort flavonoid separation by reversed phase liquid chromatography on a silica-based monolithic column

Summary An optimization procedure for the separation of flavonoids from St John's Wort by reversed-phase high-performance liquid chromatography on monolithic stationary phase is described. Three-component mobile phase systems are studied using experimental design methodology. The starting experimental domain is a triangle, each vertex of which is a pure component. From preliminary isocratic experiments, truncations in the domain are performed leading to a quadrilateral shape working domain with no symmetry. To cope with both separation and analysis time, desirability functions are used. Optimal conditions are finally given by binary systems and the four flavonoids are separated in less than seven minutes.     

Investigation of the tetramer-dimer equilibrium in haemoglobin solutions by high-performance size-exclusion chromatography on a diol column

High-performance size-exclusion chromatography on a diol grafted column was applied to study the tetramer-dimer equilibrium in haemoglobin solution. Human haemoglobin A, isolated alpha A and beta A subunits and haemoglobin variants with structural modifications located at the interface between subunits were used as models. The elution volume of the subunits was found to deviate strongly from that expected from only a gel permeation mechanism and therefore ionic interactions are likely to participate in the protein retention. Experimental results and computer simulation indicate that the individual elution bands corresponding to the discrete components (tetramer, dimer, monomer) can be resolved under certain conditions. In general both the equilibrium and kinetic interconversion parameters must be considered. Single tetramer elution bands were observed from haemoglobin in the concentration range measurable, although the influence of dimers could be seen in the shape and shift of the profile. beta Chains showed resolved peaks for the tetramer-dimer forms.     

High-performance size-exclusion chromatography of polypeptides on a TSK G2000SW column in acidic mobile phases

Polypeptides subjected to high-performance liquid chromatography on a TSK G2000SW column in acidic mobile phases of low ionic strength underwent a combination of size exclusion and ion exclusion from the matrix. The distribution coefficient (Kd) of each polypeptide was dependent upon the concentration of ion-pairing agent present and increased as the concentration of ion-pairing agent increased. The fractionation range of the column could thus be manipulated by altering the concentration of acid in the mobile phase. The nature of the ion-pairing acid, in terms of hydrophilic or hydrophobic ion pairing, also had an effect on the chromatography, such that hydrophobic ion-pairing agents lead to increased Kd values, especially in the case of the most basic polypeptides. To account for these results it is proposed that the TSK GSW matrix is cationic at low pH, and that ion pairing suppresses the ion exclusion experienced by cationic polypeptides under these conditions.     

Gas chromatographic separation of anaesthetic gases on a single column

Summary Gas chromatography has been employed in the separation of mixtures of air, carbon dioxide, nitrous oxide and a volatile anaesthetic (halothane, isoflurane or enflurane) on a single Chromosorb 101 column by temperature programming from room temperature. Calibration over the required range for the analysis of exhaled air, demonstrated good linearity with a repeatability for test mixtures of about 1%.     

Methane separation by a continuous membrane column

A continuous membrane column was employed to separate methane from gas mixtures of CO₂CH₄, CH₄N₂ and CO₂CH₄ N₂ at room temperature. Shell-side composition profiles along the column were taken experimentally and compared with the calculated results. The agreements were excellent, indicating that the model calculation is adequate for a ternary system. Combination of two columns was developed to separate the ternary mixture.     

Aqueous size-exclusion chromatography of humic acids on a sephadex gel column with diluted phosphate buffers as eluents

The elution behavior of humic acids on a Sephadex gel column is senstive to the composition, concentration, and pH of the eluent. the concentrations of the eluent in the literature are too high to obtain the correct molecular size distribution of humic acids. By reducing the concentration of phosphate buffer eluents to about a hundredth of conventional concentrations, the correct distribution is obtained. In the proposed method, 1 ml of 0.1% sample solution is introduced into a Sephadex G-50 column (2.2-cm diameter, 55 cm long) and humic acids are eluted with a 10^?3 M phosphate buffer solution (pH 7–9) at a flow rate of 1 ml min^?1.