Aneta Hałka-Grysińska

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.

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.

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.

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.

OPTIMIZATION OF SOME VARIABLES OF ON-LINE INJECTION IN PRESSURIZED PLANAR ELECTROCHROMATOGRAPHY

Pressurized planar electrochromatography (PPEC) is a separating technique, in which an electric field is applied to force movement of the mobile phase through a porous media (electroosmotic effect). High separation efficiency, fast separation, and changes in separation selectivity in comparison to liquid chromatography, especially to thin-layer chromatography (planar chromatography, TLC) are features of this technique. Our group has recently introduced the prototype device, which enables simultaneous on-line injection of six samples on chromatographic plate in a stream of the mobile phase. In this paper influence of some operating variables of on-line injection mode on efficiency of PPEC separation system is shown and discussed.

Reversed-phase stepwise gradient thin-layer chromatography of 19 test dye mixtures with a single void volume of the mobile phase

Satisfactory separation of a test mixture of 19 dyes with a general elution problem was obtained by reversed-phase stepwise gradient thin-layer chromatography with single void volume of the mobile phase. An effect of the supplementary mobile phase flux to the surface of the adsorbent layer, observed previously, was eliminated. This improves the flow profile of the mobile phase and the shape of spots, and increases the repeatability of the results within the same plate and between studies. The experimental values of the relative zone/spot position, Rpg, showed good correlation with the results predicted by the computing calculation.

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 separation selectivity of purine derivatives in high-performance thin-layer chromatography and pressurized planar electrochromatography systems

Our article presents the comparison of two methods: high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC), implemented for the separation of a test mixture of purine derivatives. The two methods were compared in terms of separation selectivity and separation time. Our results show that PPEC enables the separation of the mixture (azathioprine, caffeine, theobromine, theophylline and acyclovir) which could not be efficiently separated in the HPTLC system, due to the different selectivities of separation. PPEC also enables to obtain a much faster separation, performed on the longer distance, in comparison to HPTLC. This makes PPEC a technique which can be useful in the analysis of purine derivatives and other drugs.

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.

Stepwise gradient thin-layer chromatography of chamomile anthodium essential oil with single void volume of the mobile phase

High-performance thin-layer chromatography (HPTLC) has a great potential, especially in screening analysis. On the other hand, the separation of multicomponent mixtures with large differences in the polarities of the various components usually requires the application of different types of gradient. Currently, gradient thin-layer chromatography (TLC) is performed almost exclusively in normal-phase system, using only one commercially available device so far, i.e., Automated Multiple Development (AMD) 2 system manufactured by CAMAG (Muttenz, Switzerland) [1]. AMD 2 has a lot of advantages. The most important ones are good performance and reproducibility. Moreover, this device is fully automated, so it could work without operator supervision. However, AMD 2 has some disadvantages, such as the long time of chromatogram development or the lack of adequate and simple optimization and method development model. Moreover, due to many stages of drying, there is a risk of decomposition of some of the analyzed components, so stability testing of samples should be performed. Therefore, our research group has been looking for alternative solutions for gradient mode in thin-layer chromatography [2–4]. The developed prototype devices are not automated; however, with these devices, it is possible to obtain reasonable time (e.g., 20–30 min) of gradient chromatogram development for 4–6 cm migration distance of the mobile phase in reversed-phase thin-layer chromatography. More over, a general equation could be applied for the determination of