Separation of peptides and oligonucleotides using a monolithic polymer layer and pressurized planar electrophoresis and electrochromatography.

The rapid separation of mixtures of six peptides using porous polymer monolithic layers in electrophoresis and pressurized planar electrochromatography modes has been achieved. The separations in the former mode were performed on a generic hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) layer with no ionizable functionalities and required 2 min. This layer also enabled the separation of three oligonucleotides. The separation in the pressurized planar electrochromatographic mode was carried out using a negatively charged layer prepared via cografting of 2-acrylamido-2-methyl-1-propanesulfonic acid and 2-hydroxyethyl methacrylate on top of the generic hydrophobic monolith and was completed in 1 min.

Results with an apparatus for pressurized planar electrochromatography.

Pressurized planar electrochromatography (PPEC) is a fast and efficient planar chromatographic technique. The mobile phase is driven by electroosmotic flow, while the system is pressurized in a manner that allows heat to flow between the sorbent layer and the pressurizing medium. The reproducibility of solute retention was not satisfactory in the initial report describing PPEC. In the current report, this reproducibility is improved by better control of several experimental variables. The pressure at which PPEC is performed is now free of drift, and the temperature at which the layer is preconditioned is maintained to within +/-1 degrees C. The best reproducibility of retention is obtained when the plate is soaked in the mobile phase for a defined time before each run. In the original prototype, the temperature of the sorbent layer was not controlled. In the present apparatus, water, at a constant temperature between 3 and 60 degrees C, is circulated through channels in the two die blocks that pressurize the layer. The highest efficiency is obtained at an intermediate temperature. This behavior is ascribed to high resistance to mass transfer at the lower temperatures and increased diffusion at higher temperatures. Efficiency, as measured by the number of theoretical plates, increases with increasing migration distance. The height equivalent of a theoretical plate diminishes with increasing migration distance, and values as low as 0.0106 mm are obtained under appropriate conditions. This extrapolates to 94 000 plates/m. Manual spotting was used in this report. Evidence is presented that substantially better efficiency would be obtained if the initial spot size were smaller. The efficiency of PPEC in its current form is illustrated by a chromatogram showing the separation of nine solutes in 2 min. PPEC was also performed with TLC plates in a back-to-back configuration, and this doubles the number of samples that can be simultaneously separated.

Planar electrochromatography.

Recent developments in planar electrochromatography (PEC) in both the normal-phase and the reversed-phase modes, and at both atmospheric and elevated pressure, are reviewed. Other forced-flow techniques in planar chromatography are also briefly covered. Mobile phase migration in PEC is primarily due to electroosmotic flow, which is controlled by the applied electric field. Capillary mediated flow is an important secondary contributor to migration, and occurs because the layer is unsaturated as a consequence of liquid evaporating from the layer due to Joule heating. The magnitude of the electric field and the concentration of ions in solution are important variables that control both electroosmotic flow and Joule heating. Separations are faster and more efficient than those obtained by conventional planar chromatography, provided appropriate experimental conditions are selected. With inappropriate conditions, either mobile phase accumulates on the surface of the sorbent layer, or Joule heating causes excessive evaporation. The former results in poor spot shape, and the latter can cause the layer to dry. Good separations are obtained when there is a balance between these two effects. The problems associated with mobile phase accumulating on the surface of the sorbent layer, and with excessive evaporation of mobile phase, do not occur with pressurized planar electrochromatography. This technique is performed at high pressure, under conditions that allow heat to be removed form the sorbent layer. This allows the use of a substantially higher electric field than in PEC, and results in a high mobile phase flow rate.

Apparatus and initial results for pressurized planar electrochromatography.

Pressurized planar electrochromatography (PPEC) is a new planar chromatographic technique in which the mobile phase is driven by electroosmotic flow, while the sorbent layer is pressurized in a manner that allows heat to flow from the layer through an electrically insulating, thermally conducting, sheet of aluminum nitride ceramic. A prototype apparatus for performing PPEC is described. Separation by PPEC is faster than by conventional TLC, and an example is presented of a 24-fold enhancement in the speed of separation. PPEC was performed on both regular and high-performance C18 layers, and the latter yield substantially faster separation. The sorbent layer requires conditioning at elevated temperature before use, and solute migration velocity increases with this temperature. The flow rate increases in a linear manner with increasing voltage and diminishes in a nonlinear manner with increasing pressure. Both electrical current and Joule heating diminish with increasing pressure, and the diminution of flow at high pressure can be compensated by an increase in voltage. PPEC is more efficient than classical TLC. Theoretical plate heights diminish with increasing Rf and are in the range 29-21 and 55-27 microm for the high-performance and regular plates, respectively. PPEC retains the advantages of classical TLC but has the ability to separate a substantially higher number of samples simultaneously. An example is presented on the separation of nine samples in 1 min on a 2.5 cm x 10 cm sorbent layer.

Role of buffer concentration and applied voltage in obtaining a good separation in planar electrochromatography.

Planar electrochromatography is performed by applying an electric field across a thin layer chromatography (TLC) plate. In addition to electroosmotic flow in the axial direction, there is also flow to the surface of the TLC layer, and this can substantially degrade the quality of separation. This effect is offset by Joule heating which causes evaporation of liquid from the layer surface, and which under some conditions causes degradation of separation quality by excessive drying of the layer. It is shown that pH, buffer concentration, and applied voltage control the balance between liquid being driven to the surface and liquid evaporating from the surface due to Joule heating. Conditions are discussed which result in good separation quality, or in separations degraded by either excessive wetting or drying of the layer. The above separations were performed at constant voltage. A chromatogram is presented that shows that a good separation is also obtained at constant power, i.e. under conditions where there is a constant amount of Joule heating.