Comparison of the Retention and Separation Selectivity of Aromatic Hydrocarbons with Polar Groups in RP-HPLC Systems with Different Stationary Phases and Eluents.

In this manuscript, the retention of aromatic hydrocarbons with polar groups has been compared for systems with various nonpolar columns of the types from C3 to C18 and different mobile phases composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The selectivity separation of the solutes in systems with different adsorbents, when one eluent modifier is swapped by another, has been explained, taking into account molecular interactions of the solutes with components of the stationary phase region (i.e., extracted modifier depending on the chain length of the stationary phase).

Some theoretical considerations on preparative separation with orthogonal pressurised planar electrochromatography.

In this work, separation of multicomponent mixtures containing components with the same and different electrophoretic mobility by using orthogonal pressurised planar electrochromatography is studied theoretically. Additionally, a simple way for determination of a maximum amount of mixture causing volume overload of flat/planar columns used in this technique is presented. In the next stage, effects of change in different parameters on process performance by simulation case studies are investigated. A comparative study of separation productivity of orthogonal pressurised planar electrochromatography with the continuous and periodic modes of mixture delivery and column chromatography is carried out.

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 solid-liquid microextraction approach from a spot in an adsorbent layer of a chromatographic plate.

A new approach to extracting substances from a spot on a chromatographic plate for subsequent liquid chromatography-mass spectrometry analysis is described. This method involves extraction in a solid phase (an adsorbent layer of a chromatographic plate) - a liquid system using a simple device. For a single extraction of six selected coccidiostats from the adsorbent layer on the chromatographic plate with silica gel, 50 µL of methanol was used for 5 min. The data from the extraction experiments and liquid chromatography-mass spectrometry measurements demonstrated a good correlation between the ratio of the peak areas of the coccidiostats to the internal standard and the concentration of the substances in the range of two orders of magnitude. The coefficients of determination for the mentioned correlations range from 0.962 to 0.999. Moreover, the repeatability and reproducibility, expressed as the percentage values of relative standard deviation, do not exceed 7.5 % for any of the coccidiostats.

Comparison of the Retention of Aliphatic Hydrocarbons with Polar Groups in RP-HPLC Systems with Different Modifiers of the Binary Eluent.

The retention of aliphatic hydrocarbons with polar groups has been compared in respect to the separation selectivity changes in reversed-phase high-performance liquid chromatography with C18 stationary phase type and binary water eluent composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The changes in separation selectivity when one modifier is replaced by another in the eluent is explained, taking into consideration molecular interactions of the solutes with components of the stationary phase region, i.e., extracted modifier, and ordering of the stationary phase by the modifier.

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.

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.

Optimization of the procedure of solvent front position extraction for preparation of multi-component sample for instrumental analysis.

Solvent Front Position Extraction (SFPE) procedure has been recently introduced as a novel concept for multi-component sample preparation. According to the procedure, thin-layer chromatography (TLC) is used to separate the compounds of interest from matrix components, and to focus them into a common zone from which the compounds are extracted and transferred to apparatus for instrumental analysis. In the paper, we investigate different adsorbent types of the chromatographic plates and various mobile phases, including pH of their buffers, in respect of optimization conditions of the SFPE procedure. The research was carried out using a test sample containing 9 compounds characterised by different chemical properties, hence the conclusions from the obtained results can be applied to other multi-component samples. Under the optimal conditions, all target compounds are separated from other compounds (matrix), and evenly distributed along a narrow strip, which is advantageous for their quantitation. The determination results are good, the percentage values of relative error and relative standard deviation do not exceed 6%.

Solvent front position extraction with semi-automatic device as a powerful sample preparation procedure to quantitatitation of tryptophan in human plasma.

In the paper the results of the tryptophan determination in human plasma samples prepared with the novel Solvent Front Position Extraction (SFPE) technique are presented. The SFPE procedure is used for preparation of real biological sample for the first time. The results obtained using SFPE are compared with those using the classical sample preparation procedure. Under the optimal conditions, tryptophan and its internal standard were separated from other plasma compounds (matrix) as a small common zone/spot on a chromatographic plate using semiautomatic device equipped with moving pipet, which distributed developing solvent on the adsorbent layer. Tryptophan and the internal standard were evenly distributed within the small common zone from that the both substances were extracted and the solution obtained was transferred to quantitation with LC-MS and MS techniques. The determination results are satisfactory, the percentage values of relative error and RSD relative standard deviation do not exceed 5%. The procedure is characterized by simplicity, high analysis throughput, very good sample purification and seems to be easy applicable to other biological samples with these advantages mentioned.

Retardation of some drugs in thin-layer chromatographic systems with impregnated silica gel plates with hen's egg white and bovine serum albumin.

The influence of impregnation the chromatographic plate adsorbent layer, silica, with hen's egg white albumin (OVA) or bovine serum albumin (BSA) on the retention of some popular medicines (paracetamol, aminophenazone, theophylline, caffeine, acetanilide, ciprofloxacin, tramadol, acetylsalicylic acid, acebutolol) is investigated. The effect of composition and buffer pH of the mobile phase on solute separation selectivity is also studied. The chromatographic systems with and without above mentioned albumins and their influence on investigated drug retention are compared. In general, it has been turned out that retention of tested medicines in systems with the sorbent impregnated with albumin significantly increase relative to those with non-impregnated.

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 RP18 W plates of 5x20 cm from Merck were used in the investigations. In the first dimension, a HPTLC process was performed using 5 cm length of the plate and in the second dimension PPEC separation was obtained applying plate of 20 cm 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 20 cm long chromatographic plate and the reservoir for prewetting the adsorbent layer were applied.

Progress in planar electrochromatography.

Developments in planar electrochromatography in open (PEC) and closed (PPEC) systems are reviewed. The discussion focuses on progress in chamber construction for planar electrochromatography, separating system performance, equilibration of the PPEC process, separation time and selectivity, and the general advantages, disadvantages and prospects of this separation mode.

The influence of pH on retention and migration of peptides in systems with octadecyl silica-based adsorbent by high-performance thin-layer chromatography and pressurized planar electrochromatography techniques.

In our paper we investigate the influence of pH of the mobile phase buffer, on retention of peptides in both: normal phase (NP) and reversed phase (RP) high-performance thin-layer chromatography systems (HPTLC), using C18 silica-based adsorbent. We also compare the influence of pH on retention and/or migration distance of peptides in separation systems of HPTLC and PPEC (pressurized planar electrochromatography) techniques. Our results show, that the change of pH of the mobile phase buffer can be used for two-dimensional HPTLC separation of peptides, but much stronger change of separation selectivity can be obtained by overall inversion of separation system type (NP/RP), as we have described before (Gwarda et al., 2016). In PPEC influence of the mobile phase pH on selectivity of peptide separation is very clear and strictly different than that in HPTLC (including overall inversion of direction of solutes migration), what results from the significant share of electrophoretic effect in overall mechanism of separation. Migration of peptides in two opposite directions results in better selectivity of separation by PPEC. In separation systems of both HPTLC and PPEC techniques, the change of pH clearly affects the efficiency of the separation system, so it can be used for optimization of separation conditions. Our results also show, that some modifications of PPEC equipment and materials could be useful for application of this technique in the field of peptide analysis.

The influence of addition of ion-pairing acid and organic modifier of the mobile phase on retention and migration of peptides in pressurized planar electrochromatography system with octadecyl silica-based adsorbent.

In our previous papers we have investigated the influence of the mobile phase composition on mechanism of retention, selectivity and efficiency of peptide separation in various high-performance thin-layer chromatography (HPTLC) systems with commercially available silica-based adsorbents. We have also investigated the influence of pH of the mobile phase buffer on migration and separation of peptides in pressurized planar electrochromatography (PPEC). Here we investigate the influence of concentration of ion-pairing additive, and concentration and type of organic modifier of the mobile phase on migration of peptides in PPEC system with octadecyl silica-based adsorbent, and with the same set of the solutes as before. We compare our current results with the results obtained before for similar HPTLC and PPEC systems, and discuss the influence of particular variables on retention, electrophoretic mobility of solutes and electroosmotic flow of the mobile phase. We show, that the final selectivity of peptide separation results from co-influence of all the three factors mentioned. Concentration of organic modifier of the mobile phase, as well as concentration of ion-pairing additive, affect the retention, the electrophoretic mobility, and the electroosmotic flow simultaneously. This makes independent optimization of these factors rather difficult. Anyway PPEC offers much faster separation of peptides with quite different selectivity, in comparison to HPTLC, with similar adsorbents and similar mobile phase composition. However, we also present and discuss the issue of extensive tailing of peptide zones in the PPEC in comparison to similar HPTLC systems.

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 sample application mode on performance of pressurized planar electrochromatography in completely closed system.

Three modes of sample application on the chromatographic plate are applied at present investigations of pressurized planar electrochromatography (PPEC) systems taking into special attention their influence on performance of the separating system. These modes are as follows: application of the sample solution directly on the chromatographic plate with microsyringe, deposition of sample solution on scrap of adsorbent layer followed by location oft this scrap on the chromatographic plate, application of the sample solution with commercially available aerosol applicator. These modes were combined with prewetting procedures of the chromatographic plates which lead to an accomplishment of equilibration of the stationary phase-mobile phase system. The plots of plate height versus linear flow rate of the mobile phase are presented for PPEC systems for the first time. The best separation performance has been obtained in PPEC system when prewetting of the chromatographic plate followed the sample application with commercially available aerosol applicator. The higher repeatability of migration distance of the solute bands has been obtained in PPEC experiments when the sample application was followed by prewetting the chromatographic plate in comparison to the experiments when these operations were performed in reversed order.

Preliminary results for interval feeding the orthogonal pressurized planar electrochromatography system with sample solution for its preparative separation.

The orthogonal pressurized planar electrochromatography (OPPEC) is an example of 2-D separation technique, in which two simultaneous and orthogonal processes of electrophoresis and chromatography are involved in the separation mechanism. In the case of preparative separation of substances characterized by different electrophoretic mobility, such separation system can be constantly fed with the sample solution and the separated components can be constantly collected at its outlet. In the paper, as opposed to the previous studies, we discuss the capabilities of OPPEC technique for preparative separation of substances characterized by the same electrophoretic mobility. According to the proposed solution, the separation system can be periodically fed with the sample solution and separated components can be collected alternately at its outlet. The advantages of this new approach over the column chromatography with regard to the separation of complex mixtures have been signaled.

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.

Influence of some operation variables on continuous separation process of orthogonal pressurized planar electrochromatography.

Orthogonal pressurized planar electrochromatography (OPPEC) is the new separation technique, in which two simultaneous and orthogonal processes of electrochromatography/electrophoresis and chromatography proceed under external pressure. It means in the OPPEC separation system a mobile phase solution hydrodynamically flows against adsorbent layer of the chromatographic plate and electric field is orthogonally applied to it. This technique can be applied to analytical and preparative separations. In the paper we report influence of some operating variables on continuous preparative OPPEC separation of test dyes in system with HPTLC RP-18W plates from Merck and methanol-buffer mobile phase. The following operation variables are discussed: external pressure exerted on the adsorbent layer, mobile phase flow velocity, and the ratio of flow velocities of the mobile phase to the sample solution, voltage applied to electrodes, modifier concentration in the mobile phase, concentration and pH of buffer.