Reversed-phase pH gradient thin-layer chromatography of biologically active substances with controlled developing solvent velocity.

For the first time, stepwise pH gradient thin-layer chromatograms of biologically active substances with controlled developing solvent velocity are presented and described in the paper. Change in buffer pH of the mobile phase solution influences retardation, selectivity, and shape of the separated substances' spots. The conducted research has confirmed that the mobile phase's pH gradient could be an essential factor to optimize the conditions of the separation of substances in reversed-phase high-performance thin-layer chromatography. The reproducibility of the gradient retardation factor values of separated substance zones is satisfactory.

Reversed-phase stepwise gradient thin-layer chromatography of test dye mixtures with controlled developing solvent velocity.

The new approach to the development of planar chromatogram with controlled developing solvent velocity was implemented for gradient reversed-phase chromatography of a test mixture of dyes. The developing solvent components were directly delivered onto the surface of the C18 type of silica adsorbent layer with controlled velocity by moving pipette combined with two syringe pumps. The eluent components were mixed onto the adsorbent layer, so in this way, there was no dwell volume of the developing solvent during gradient chromatogram development. The delivery of the developing solvent solution to the adsorbent layer was adjusted to its absorption rate by the adsorbent layer. Under such conditions, there was no excess of the eluent solution on the surface of the adsorbent layer, so the high performance of the chromatographic system could be obtained. In the paper, the results of the repeatability and reproducibility test of migration distances of selected separated solute zones are presented.

Thin-layer chromatogram development with a moving pipette delivering the mobile phase onto the surface of the adsorbent layer.

The new approach to the development of thin-layer chromatograms is presented. For the first time we show flexible mobile phase dosage onto the surface of the adsorbent layer by moving pipette combined with precise syringe pumps. The pipette is driven into movement by computer controlled 3D machine (modified 3D printer mechanism). Delivery of the mobile phase to the adsorbent layer is equal to or lower than that of conventional development. Therefore chromatograms can be developed with optimal mobile phase velocity, adjusted to its absorption rate by the adsorbent layer. Under such conditions there is no excess of eluent on the surface of the adsorbent layer so higher performance of the chromatographic system can be obtained. Moreover chromatograms can be developed with constant linear mobile phase velocity and therefore the relationships the plate height vs. mobile phase linear velocity obtained with planar chromatography driven by capillary forces are investigated and reported. In addition the contribution of starting spot variance in total peak variance and the influence of narrowing of starting spots on performance of the chromatographic system have been studied. The results confirm a very significant starting spot variance contribution to total peak variance and consequently considerable influence of starting spot size on plate height of the separation system, when chromatogram is developed on a short distance. In the paper the advantages and disadvantages of the prototype device and its possible application are discussed.

Influence of the Modifier Type and its Concentration on Electroosmotic Flow of the Mobile Phase in Pressurized Planar Electrochromatography.

The aim of this work was to find a relationship between electroosmotic flow (EOF) velocity of the mobile phase in pressurized planar electrochromatography (PPEC) and physicochemical properties like zeta potential, dielectric constant, and viscosity of the mobile phase as well as its composition. The study included different types of organic modifiers (acetonitrile, methanol, ethanol, acetone, formamide, N-methylformamide and N,N-dimethylformamide) in the full concentration range. In all experiments, chromatographic glass plates HPTLC RP-18 W from Merck (Darmstadt) were used as a stationary phase. During the study we found that there is no linear correlation between EOF velocity of the mobile phase and single variables such as zeta potential or dielectric constant or viscosity. However, there is quite strong linear correlation between EOF velocity of the mobile phase and variable obtained by multiplying zeta potential of the stationary phase-mobile phase interface, by dielectric constant of the mobile phase solution and dividing by viscosity of the mobile phase. Therefore, it could be concluded that the PPEC system fulfilled the Helmholtz-Smoluchowski equation.

A Modified Device for Pressurized Planar Electrochromatography and Preliminary Results with On-Line Sample Application.

Pressurized planar electrochromatography (PPEC) is a separating technique in which an electric field is applied to force the mobile phase movement through a porous media (electroosmotic effect). High separation efficiency, fast separations and changes in separation selectivity in comparison to liquid chromatography, especially thin layer chromatography (planar chromatography, TLC), are features of this technique. Constructional methodological challenges to PPEC are obstacles to its development and application in laboratory practice. In this article, an attempt to overcome the challenges related to device construction and sample application/injection is described. The introduced device enables both prewetting of the adsorbent layer and electrochromatogram development with a single PPEC device. It also enables simultaneous application/injection of six samples on a chromatographic plate in a stream of the mobile phase (on-line application/injection). In addition, the PPEC chamber was equipped with a thermostat. The device is characterized by an impressive throughput in comparison to the other planar technique, TLC/HPTLC. Although the developed device still needs improvement, it is, in our opinion, a considerable step toward possible automation of this planar separation technique.

Pressurized planar electrochromatography.

Theoretical backgrounds, development, examples of separations, constructional details and principle of action of devices of pressurized planar electrochromatography (PPEC) are presented. Development of the mode is described in respect of operating variables (composition of the mobile phase, pressure exerted on adsorbent layer, mobile phase flow velocity, temperature of separating system, etc.) influencing separation efficiency (kinetic performance, repeatability, separation time). Advantages of PPEC such as high kinetic performance, short separation time and different separation selectivities, especially relative to conventional thin-layer chromatography, are described. Examples of two-dimensional separations are demonstrated to show high separation potential of the mode when combined with conventional thin-layer chromatography (TLC). The PPEC mode is in infancy stage of development, so its challenges are presented as well.