Session 7

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Thin Layer Chromatography (TLC) is one of the oldest chromatographic techniques which has widely been used in research on small molecules of natural and synthetic origin with physiological or pharmacological importance. Regulatory requirements for monitoring of quality and safety of substances which remain in pharmaceutical use throughout their whole life cycle (i.e., not only in human body, but also in the environment) become increasingly more strict. This trend is accompanied by development of instrumentation to establish novel and better conditions for drug analysis. The Quality by Design (QbD) methodology is going to be implemented by pharmaceutical industry in production and quality control of active pharmaceutical substances (APIs) and dosage formulations, although it still is not an obligatory regulatory demand. Nowadays, classical yet DoE-supported, or eco-friendly TLC/HPTLC methods are used for qualitative and quantitative pharmaceutical analysis, which includes identification, purity and chiral purity testing, dissolution, and content and uniformity of content with APIs as raw materials and in the dosage form.

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Chromatography is one of the most popular and most commonly analytical method. Another role of chromatography is basic research. Combination of these two aspects of chromatographic studies allows application of the method in biomedical research. In biomedical investigation chromatography is used in the following area:as analytical method for determination of composition of drugs, metabolism products herbal extracts,

• determination of biological activity of separated compounds – TLC bioautography,
• nature or synthesized by use of different kind of chromatographic method,
• investigations of different factors influencing on biological activity of compounds,
• determination of free drug concentration in blood.

In biomedical research often HPLC method is used, but TLC can be used in many cases as an alternative method or in some cases as only possible chromatographic method.
In presented investigations application of TLC for determination of biological activity of compounds in some chromatographic systems imitating biological environmental, used SBF as component of mobile phases and obtained results are compared with results obtained in “classical” RP system for determination of permeability of substances through biological barrier. Influence of presence of ionic liquid (increasingly used in medicine), magnetic field (often used in different therapies) on descriptors of biological activity of substances were investigated used TLC systems. TLC method can be successfully used in determination of free form of drug in system with blood protein. Some results obtained by TLC method are compared with results obtained by HPLC. Presented research shows that TLC can be successfully used in many aspects of biomedical research.

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Effective diffusion is one of these phenomena which on an everyday basis take place in chromatographic systems, yet usually tend to be overlooked in an everyday analytical practice. Unlike in the column chromatography, in planar chromatography effective diffusion is two-dimensional [1]. On some rare occasions, horizontal direction of effective diffusion can proof beneficial. It happens, when certain compounds of a very similar chemical structure (and/or molecular weight) cannot be separated vertically without applying special separation conditions. So far, however, an exact physicochemical mechanism of horizontal separations remains rather obscure. It can only be assumed that the chirality of silica gel (as confirmed by the circular dichroism (CD) spectroscopy [2]) is one reason of horizontal separations. In combination with horizontal effective diffusion, it can result in spectacular yet rare horizontal separations. Moreover, it was documented upon numerous examples that horizontal separations in the TLC/HPTLC systems most likely occur with the rod-shaped molecules additionally equipped with asymmetry centers [3] and with the pairs of enantiomers [4]. The mechanism of such separations can be by the auto-driven propeller-like motions of the analytes themselves, the Magnus forces-induced motion, or by still another yet unknown mechanism [3]. In this talk, a very spectacular example of horizontal separation is presented of the rod-shaped red betacyanin pigments equipped with asymmetry centers, which are derived from the red beet (Beta vulgaris L.) juice [5].

References
[1]  T. Kowalska, K. Kaczmarski, W. Prus, Theory and mechanism of thin-layer chromatography. In: J. Sherma and B. Fried (Eds.), Handbook of Thin-Layer Chromatography, 3^rd Ed. Revised and Expanded, Marcel Dekker, New York, 2003; pp. 47-80.
[2]  M. Sajewicz, H.-E. Hauck, G. Drabik, E. Namysło, B. Głód, T. Kowalska, J. Planar Chromatogr. 19 (2006) 278-281.
[3]  J. Polanski, M. Sajewicz, M. Knas, A. Zywocinski, M. Weloe, T. Kowalska, Chemistry Today, Monographic Supplement “Chiral Technologies” 30 (2012) 18-21.
[4]  M. Sajewicz, R. Piętka, A. Pieniak, T. Kowalska, Acta Chromatogr. 15 (2005) 131-149.
[5]  E. Łata, A. Fulczyk, T. Kowalska, M. Sajewicz, J. Chromatogr. Sci. 58 (2020) 5-15.

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For the first time, planar electrochromatography in an open system was introduced to the scientific literature in 1974 [1], and in a closed system in 2004 [2]. The latter technique was called pressurized planar electrochromatography (PPEC). Inspired by the results of the papers mentioned, our team also conducted research in this area. In this presentation, we provide a retrospective review of developments in pressurized planar electrochromatography and present the latest research that documents further progress in this separation technique, and two other techniques, which have been evolving because of investigations based on the former:

• pressurized planar electrochromatography, in which the flow of the mobile phase is generated by the action of an electric field (electroosmotic effect);
• orthogonal pressurized planar electrochromatography, in which the flow of the mobile phase is hydrodynamic, and an electric field is applied perpendicular to the direction of this flow;
• high-pressure layer electrochromatography, in which the flow of the mobile phase is hydrodynamic, and the electric field is applied parallel to the direction of this flow.

We also discuss the advantages and problems that should be the subject of further research on the techniques presented.

Acknowledgements
This work was supported by National Science Centre, Poland, Agreement No. UMO 2016/23/B/ST4/02877.

References
[1]  V. Pretorius, B. J. Hopkins, J. D. Schieke, J. Chromatogr. A 99 (1974) 23-30.
[2]  D. Nurok, J. M. Koers, A. L. Novotny, M. A. Carmichael, J. J. Kosiba, R. E. Santini, G. L. Hawkins, R. W. Replogle, Anal. Chem. 76 (2004) 1690-1695.