Tadeusz H. Dzido

Pressurized planar electrochromatography, high-performance thin-layer chromatography and high-performance liquid chromatography—Comparison of performance

Kinetic performance, measured by plate height, of High-Performance Thin-Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Pressurized Planar Electrochromatography (PPEC) was compared for the systems with adsorbent of the HPTLC RP18W plate from Merck as the stationary phase and the mobile phase composed of acetonitrile and buffer solution. The HPLC column was packed with the adsorbent, which was scrapped from the chromatographic plate mentioned. An additional HPLC column was also packed with adsorbent of 5 microm particle diameter, C18 type silica based (LiChrosorb RP-18 from Merck). The dependence of plate height of both HPLC and PPEC separating systems on flow velocity of the mobile phase and on migration distance of the mobile phase in TLC system was presented applying test solute (prednisolone succinate). The highest performance, amongst systems investigated, was obtained for the PPEC system. The separation efficiency of the systems investigated in the paper was additionally confirmed by the separation of test component mixture composed of six hormones.

Preliminary results for 2-D separation with high-performance thin-layer chromatography and pressurized planar electrochromatography.

Preliminary results of 2‐D separation of test dye mixture using high‐performance thin‐layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) are demonstrated. The advantage of 2‐D HPTLC/PPEC separation is based on different separation selectivities obtained in both HPTLC and PPEC systems. HPTLC RP18u2009W plates of 5×20u2009cm from Merck were used in the investigations. In the first dimension, a HPTLC process was performed using 5u2009cm length of the plate and in the second dimension PPEC separation was obtained applying plate of 20u2009cm length. PPEC process followed prewetting the chromatographic plate with sample zones on it, which were partly separated after first dimensional (HPTLC) separation. In the experiments, the modified version of PPEC device for 20u2009cm long chromatographic plate and the reservoir for prewetting the adsorbent layer were applied.

Comparison of retention of aromatic hydrocarbons with polar groups in binary reversed-phase high-performance liquid chromatography systems.

The retention of aromatic hydrocarbons with polar groups has been correlated as log k1 versus log k2 for reversed-phase high-performance liquid chromatography systems with different binary aqueous mobile phases containing methanol, acetonitrile or tetrahydrofuran as modifiers. Distinct changes in separation selectivity have been observed between tetrahydrofuran and acetonitrile or methanol systems. Methanol and acetonitrile systems show lower diversity of separation selectivity. The changes in retention and selectivity of aromatic hydrocarbons with various polar groups between any two chromatographic systems with binary aqueous eluents (tetrahydrofuran vs. acetonitrile, tetrahydrofuran vs. methanol and methanol vs. acetonitrile) have been interpreted in terms of molecular interactions of the solute with especially one component of the stationary phase region, i.e. extracted modifier, and stationary phase ordering. The ordering of the stationary phase region caused by modifier type influences the chromatographic selectivity of solutes with different molecular shape.

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.

Two-dimensional high-performance thin-layer chromatography of tryptic bovine albumin digest using normal- and reverse-phase systems with silanized silica stationary phase.

Among many advantages of planar techniques, two-dimensional (2D) separation seems to be the most important for analysis of complex samples. Here we present quick, simple and efficient two-dimensional high-performance thin-layer chromatography (2D HPTLC) of bovine albumin digest using commercial HPTLC RP-18W plates (silica based stationary phase with chemically bonded octadecyl ligands of coverage density 0.5μmol/m(2) from Merck, Darmstadt). We show, that at low or high concentration of water in the mobile phase comprised methanol and some additives the chromatographic systems with the plates mentioned demonstrate normal- or reversed-phase liquid chromatography properties, respectively, for separation of peptides obtained. These two systems show quite different separation selectivity and their combination into 2D HPTLC process provides excellent separation of peptides of the bovine albumin digest.

Simultaneous determination of acetaminophen, propyphenazone and caffeine in cefalgin preparation by pressurized planar electrochromatography and high-performance thin-layer chromatography

Pressurized planar electrochromatography (PPEC) and high-performance thin-layer chromatography (HPTLC) have been involved in separation and analysis of active components of cefalgin tablets: acetaminophen, propyphenazone and caffeine. A separation system comprised acetonitrile-buffer mobile phase and chromatographic plates with an adsorbent layer of the C18 type. The PPEC separation process, applying polarization voltage 1.8 kV, was 2.7 times shorter than that of HPTLC. Total time of the separation procedure by PPEC mode (6 min) is considerably shorter than that of HPTLC (20 min). Resolution of sample bands and performance of the PPEC separating system are more favorable for quantitative analysis than those of HPTLC under the same chromatographic conditions (qualitative and quantitative composition of the mobile and stationary phases). Under established experimental conditions, satisfactory sensitivity of the method was achieved. The LODs ranged from 0.06 to 0.16 μg of component per spot depending on the method and substance. Procedures were validated for selectivity, precision, linearity and accuracy. Investigations show that both PPEC and HPTLC modes were accurate, precise, sensitive, linear and specific. Furthermore, PPEC is promising mode, which, in our opinion, can be applied in routine pharmaceutical analysis in the near future.

Effect of Temperature on the Retention of Aromatic Hydrocarbons with Polar Groups in Binary Reversed-Phase TLC

RM (log[(1 - RF)/RF]) values at different temperatures have been compared for aromatic hydrocarbons with different functional groups in reversed-phase thin-layer chromatography with aqueous binary mobile phases containing methanol, acetonitrile, or tetrahydrofuran. Considerable retention and selectivity variations were observed among the systems investigated. Separation of the solutes at different temperatures can be advantageous, because of these selectivity changes and the shortening of the development time. A horizontal DS-chamber has been developed for performing chromatogram development with temperature control.

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.

Inversion of type of separation system in planar chromatography of peptides, using C18 silica-based adsorbents.

Our previous results show, that C18 silica-based adsorbents used in high-performance thin-layer chromatography (HPTLC), provide complex retention mechanism basing on various polar and nonpolar interactions. Here we present, that in chromatography of peptides, due to mixed-mode properties of these adsorbents, there is a simple way to obtain inversion of separation system type (from reversed-phase, RP, to normal-phase, NP, and vice versa). The results presented provide detailed information how to obtain inversion mentioned and reflect the extent (the type and concentration of organic solvent, the type and concentration of ion-pairing reagent in the mobile phase) of this phenomenon. We show, that the system type inversion results in significant change of selectivity of separation, which may be especially useful in 2D separation of complex samples of basic/amphoteric compounds such as peptides. This results from the fact, that C18 silica-based HPTLC adsorbents, may be used in hydrophilic interaction chromatography (HILIC) or RP chromatography, in dependence on mobile phase composition.

Influence of carboxylic ion-pairing reagents on retention of peptides in thin-layer chromatography systems with C18 silica-based adsorbents

One of the main problems related to chromatography of peptides concerns adverse interactions of their strong basic groups with free silanol groups of the silica based stationary phase. Influence of type and concentration of ion-pairing regents on peptide retention in reversed-phase high-performance liquid chromatography (RP-HPLC) systems has been discussed before. Here we present influence of these mobile phase additives on retention of some peptide standards in high-performance thin-layer chromatography (HPTLC) systems with C18 silica-based adsorbents. We prove, that due to different characteristic of adsorbents used in both techniques (RP HPLC and HPTLC), influence of ion-pairing reagents on retention of basic and/or amphoteric compounds also may be quite different. C18 silica-based HPTLC adsorbents provide more complex mechanism of retention and should be rather considered as mixed-mode adsorbents.