Peculiarities of gradient ion-exchange high-performance liquid chromatography of proteins.

Since the influence of column length on protein resolution in high-performance liquid chromatography (HPLC) is not clear, different viewpoints presented in the literature are analysed in detail. The influence of gradient steepness on the length of the working column part (X0) or the part of a column in which the quasi-steady state is attained was studied. The equation for estimating the X0 value was obtained for the general case of the retention model. It was shown that at steep gradients only a short part of the column is used as the working part on which all separation processes develop. The other part of a column is a ballast where the protein zone migrates in a regime of parallel transfer. These results form a theoretical basis for high-performance membrane chromatography. As was shown experimentally, this method makes it possible to perform protein separation at low gradient times with appropriate resolution, comparable with that of HPLC.

Peculiarities of zone migration and band broadening in gradient reversed-phase high-performance liquid chromatography of proteins with respect to membrane chromatography.

The peculiarities of zone migration and band broadening in the reversed-phase gradient HPLC of proteins were investigated. In the isocratic mode a critical composition of the mobile phase was found at which all proteins regardless of their molecular mass migrate with equal velocity and have a capacity factor equal to the phase ratio (VP/V0), i.e., the same capacity factor as a marker of total accessible volume would have in steric exclusion chromatography. It is shown that steric exclusion conditions are never achieved in gradient HPLC. In the first (adsorption stage) of gradient elution where the separation takes place the velocity of a protein increases until it becomes equal to the velocity of the desorbing solvent front at a critical distance X0 from column entrance. Strong broadening is characteristic of this stage. In the second (critical) stage the protein travels the remaining distance (L-X0) with the velocity of the solvent. A definition of X0 is given allowing one very simple calculation of the minimum permissible column length as a function of gradient steepness, mobile phase velocity and protein adsorption parameter. When x = X0 the protein zone has the smallest dispersion. Making L < X0 is especially disadvantageous, as it leads to anomalous bandspreading. The theory of gradient HPLC was refined on this basis and the usefulness of this approach in high-performance membrane chromatography is demonstrated.

Determination of molecular-weight distribution and average molecular weights of block copolymers by gel-permeation chromatography.

The problem of preparation of a block copolymer of precise molecular-weight distribution (MWD) and with heterogeneous composition on the basis of gel-permeation chromatography (GPC) data has been investigated. It has been shown that in MWD calculations the distribution f(p) of the composition p in individual GPC fractions should be taken into account. The type of the f(p) functions can be simultaneously established by an independent method, such as use of adsorption-column or thin-layer chromatography sensitive to the composition of the copolymer. It has also been shown that the actual f(p) may be replaced by a corresponding piecewise distribution, of simple form, without decrease in the precision of calculation of the MWD and average molecular weights of most known block copolymers.

[Determination of the kinetic association and dissociation constants of proteins]. / Opredelenie kineticheskikh konstant assotsiatsii i dissotsiatsii belkov.

On the basis of protein solution characterising by a reversible association--dissociation monomer--n-mer without intermediate products the statistic moments have been calculated. On the basis of these expressions and numerical experimental values one can compute the values of kinetic constants of the association--dissociation reactions.

[Gel-permeating chromatography of interacting multi-component systems. I. Study of protein isomerization by the method of gel-permeation chromatography. II. Method of determining the kinetic constant of isomerization]. / Gel'-pronikaiushchaia khromatografiia vzaimodeistvuiushchikh mnogokomponentnykh sistem. I. Izuchenie izomerizatsii belkov metodom gel'pronikaiushchei khromatografii. 2. Metodika opredeleniia kineticheskikh konstant izomerizatsii.

A technique is suggested of determining the isomerization constants by means of gel-permeation chromatography. Chromatograms of proteins and expressions for statistical moments of their distribution along the chromatographic column obtained in the authors previous work [1], are used. A necessary condition for optimal setup of the GPCh experiments is found. Fulfilment of this condition provides a maximal accuracy of determination of the isomerization constants.