Adam Chomicki

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 5×20 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.

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 separation of some amino acids by HPTLC and pressurized planar electrochromatography

Two-dimensional (2D) separation of eight amino acids has been achieved by HPTLC in the first dimension and pressurized planar electrochromatography (PPEC) in the second, orthogonal, dimension. Separation selectivity in PPEC was very different from that in HPTLC. This attribute of these systems enabled complete separation of the amino acids. Chromatography was performed on reversed-phase plates with acetonitrile-buffer mobile phase. PPEC was performed after complete prewetting the plate, including the sample zones.

Equipment and preliminary results for orthogonal pressurized planar electrochromatography

We report combination of overpressured layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC) techniques into a single technique in which both OPLC and PPEC processes proceed simultaneously and orthogonally. The separation process with this new technique is performed in adsorbent layer of a chromatographic plate, which is equipped with special sealing margin on its whole periphery and closed under pressure in special chamber. We have named this separation technique as orthogonal pressurized planar electrochromatography (OPPEC). Examples of analytical and micropreparative (continuous) OPPEC separations are demonstrated.

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.

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.

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.

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

Abstract A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP+/NBT/PMS (β-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H37Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H37Ra PGI was 226 μg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H37Ra PGI inhibition by PEP.

Electrochromatography Methods: Planar Electrochromatography

Planar electrochromatography is a technique in which mixture components are separated in adsorbent layer of a chromatographic plate placed in electric field. In such separation system a mobile phase movement stems from electroosmosis phenomenon. Partition and electrophoresis mechanisms are involved in separation of mixture components with this technique. Two principal modes of planar electrochromatography are described: planar electrochromatography in an open system (PEC) and planar electrochromatography in a closed system (pressurized planar electrochromatography, PPEC). The development of both modes is presented beginning with the first paper on electrochromatography by Pretorius et al. in 1974 and finishing with the last papers by Dzido et al. in 2010. Constructional development of equipment to planar electrochromatography is provided and influence of operating variables on separation efficiency as well. The advantages and challenges of PPEC technique are especially discussed.