Anna Klimek-Turek

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.

Pressurized Planar Electrochromatography as the Mode for Determination of Solvent Composition-Retention Relationships in Reversed-Phase Systems

Pressurized planar electrochromatography (PPEC) was introduced by Nurok et al. [1]. The mobile phase in PPEC is driven by the electroosmotic effect through the adsorbent layer of the chromatographic plate. A special plastic film or plate is pressed on to the adsorbent layer to eliminate the vapor phase and flow of mobile phase to the surface of the adsorbent layer. The paper mentioned above, and others [2–6], indicate that this method is characterized by high-efficiency separation, making it very attractive for application in laboratory practice. Contemporary applications have, however, been mainly restricted to separation of test solutes to show the advantages, practical possibilities, and efficiency of PPEC in comparison with conventional planar chromatography (TLC). At the current stage of PPEC development its performance is similar to that of HPLC [1–6]. Separation times in PPEC are reported to be much shorter than in TLC [1, 2, 4], in some circumstances by as much as a factor of 24 [1]. Pressurized planar electrochromatography experiments can be performed under equilibrated conditions [3, 4]. This is another very important advantage of PPEC compared with TLC. The adsorbent layer of the chromatographic plate is prewetted with the mobile phase for the time necessary for equilibration. After prewetting, the mobile phase is used to feed the adsorbent layer during separation process in pressurized planar electrochromatography. Otherwise, conventional planar chromatographic separation usually proceeds under non-equilibrated conditions with the exception of use of a pure solvent as mobile phase. TLC separations are, however, usually performed with mixed mobile phases. Then demixing of the mobile phase occurs during chromatogram development [7]. Even conditioning with mobile phase vapor followed by chromatogram development does not lead to full equilibration of the chromatographic phases.

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.

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.

Frontally eluted components procedure with thin layer chromatography as a mode of sample preparation for high performance liquid chromatography quantitation of acetaminophen in biological matrix.

A new concept of using thin-layer chromatography to sample preparation for the quantitative determination of solute/s followed by instrumental techniques is presented Thin-layer chromatography (TLC) is used to completely separate acetaminophen and its internal standard from other components (matrix) and to form a single spot/zone containing them at the solvent front position (after the final stage of the thin-layer chromatogram development). The location of the analytes and internal standard in the solvent front zone allows their easy extraction followed by quantitation by HPLC. The exctraction procedure of the solute/s and internal standard can proceed from whole solute frontal zone or its part without lowering in accuracy of quantitative analysis.

The influence of metallic impurities on the free silanol activity of commercial thin-layer chromatography adsorbents demonstrated by retention changes of basic/amphoteric compounds such as peptides

In our previous papers, we have mentioned some specific disruption of peptide zones shape and chromatogram distortion, when using mobile phase containing ion-pairing acids. This problem is investigated here. It concerns not only some specific separation conditions but also various separation systems with silica-based adsorbents and water—alcohol mobile phases. We show that the problem results from significant amount of metallic impurities present in the adsorbents investigated. Our results prove that these impurities strongly affect the activity of free silanol groups and thus the retention of basic or amphoteric compounds and the quality of the results obtained. The standard method of washing adsorbent layer with methanol is not effective against the impurities. Washing chromatographic plates with a solution containing an acid significantly reduces the amount of metallic impurities in the adsorbent, resulting in the reduction/elimination of these adverse effects. However, it also leads to the increase of heterogeneity of acidic groups activity and deterioration of separation system efficiency. Therefore, removing metal ions from the adsorbent may not always be advantageous. Avoiding of use of strong ion-pairing acids is also problematic and not always possible. Thus, the production of high-purity silica of homogenous activity seems to be the best and the most reliable solution of the problem described.

Thin-layer chromatography and pressurized planar electrochromatography of amino acids in systems with silica gel and water mobile phase

Thin-layer chromatography (TLC) and pressurized planar electrochromatography (PPEC) of amino acids in normal-phase system is presented. The results have been obtained for various normal-phase high-performance thin-layer chromatography (HPTLC) and PPEC systems with the mobile phase that comprised acetonitrile in the concentration ranges 40–90% and 20–90%, respectively, and HPTLC silica gel 60 F254s plates from Merck. The data obtained show differences in separation selectivity between HPTLC and PPEC systems. The respective separation selectivities have been obtained for HPTLC and PPEC systems with the mobile phase buffer pH in the range 2.6–9.0. The retention of amino acids in HPTLC systems has demonstrated minor dependence on buffer pH, while, in analogous PPEC systems, migration distances of the solutes have shown considerable changes. The differences of separation selectivity in HPTLC and PPEC systems are interpreted in terms of solute partition in the former and solute partition and electrophoresis in the latter.

Pressurized Planar Electrochromatography as a Supporting Tool for Qualitative Toxicological Chemical Analysis with Thin-Layer Chromatography and UV-VIS Spectrometry

ABSTRACT Pressurized planar electrochromatography (PPEC) was applied to support qualitative toxicological chemical analysis performed with thin-layer chromatography (TLC) and UV–Vis spectrometry. Based on retention/migration distance data of substances obtained in TLC and PPEC systems and database of their wavelength maxima of remission UV–Vis spectra as well, a combined fit factor was calculated for substance identification. The involvement of PPEC, TLC, and UV–Vis spectral data together in calculation of the combined fit factor lead to its lower values for substances, which were not identical with reference, in comparison with those when the combined fit factor was calculated using TLC and spectral data only. The results evidence that involvement of PPEC data in qualitative toxicological chemical analysis performed with TLC and UV–Vis spectrometry enhances reliability of it. GRAPHICAL ABSTRACT

A new semiautomatic device with horizontal developing chamber for gradient thin-layer chromatography

ABSTRACT A new semiautomatic device with two types of horizontal developing chamber for gradient thin-layer chromatography (TLC) that does not require special procedure of chromatographic plate preparation was developed. The new device was applied for separation of test mixture of 10 dyes showing general elution problem. In addition, a general equation for determination of relative position, , of solutes chromatographed under conditions of stepwise gradient elution with one void volume of mobile phase in TLC has been applied. A satisfactory agreement between calculated (by computer program) and experimental values of has been obtained. In the article, based on obtained results, the advantages and disadvantages of different devices for gradient elution in TLC are discussed.

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.