June 30

Chayanis Sutcharitchan¹, Worakorn Wiwatcharakornkul¹, Somchai Keawwangchai², Tasawan Kaewwangchai², Sornkanok Vimolmangkang¹, Suchada Sukrong¹, Wanchai De-Eknamkul<sup>1

1</sup>Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
²Department of Chemistry, Faculty of Science, Mahasarakham University, Mahasarakham 44150, Thailand

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Thailand is known to be one of the world’s best sources of cannabis strains, and thus has a potential to be a cannabis hub in Asia. Thai strains of cannabis commonly refer to 100% sativa strains native to Southeast Asia. Each strain contains different ratios of the psychoactive substances, tetrahydrocannabinol (THC) and cannabidiol (CBD), which are known to have medical benefits. Three major cannabis chemotypes (THC dominant, intermediate, and CBD dominant) and two minor ones (CBG dominant and cannabinoid-free) have been categorized for chemotaxonomic discrimination. So far, very little has been known about the chemotypes and genetic backgrounds of the diversified Thai cannabis strains and hybrids. Therefore, the techniques of HPTLC and DNA profilings were introduced to observe the relationship between their metabolic and genetic levels. Almost 100 samples of dried flowers obtained from various strains of Thai cannabis and their hybrids were collected and prepared for HPTLC and DNA analyses. The solvent system used for the HPTLC separation of cannabis extracts was modified from the previous report (1). The results showed that there was a high variation in the chemotypes among the Thai cannabis samples. For the DNA analysis, tetrahydrocannabinolic acid-synthase (THCA-synthase) and cannabidiolic acid-synthase (CBDA-synthase) genes (2,3) were used as the chemotype markers of various cannabis samples. The preliminary results obtained by PCR and DNA sequencing techniques suggested that there was a good correlation between the HPTLC chemical profiles and the presence of functional forms of THCA-synthase (drug-type) and CBDA-synthase (fiber-type). Therefore, the sequence of both genes can be used for early prediction of the cannabinoid profiled in Thai cannabis plants.

Acknowledgements
This work was supported by Chulalongkorn University's Natural Product Biotechnology Research Unit (for WD) and Center of Excellence in DNA Barcoding of Thai Medicinal Plants (for SS).

References
[1]  CAMAG Application Note A-108.1 (2017).
[2]  C. Onofri, E. de Meijer, G. Mandolino, Phytochemistry 116 (2015) 57–68.
[3]  A. R. Garfinkel, M. Otten, S. Crawford, Genes 12 (2021) 228. https://doi.org/10.3390/genes12020228

Bernd Spangenberg

University of Applied Sciences, Offenburg, Badstrasse 24, 77652 Offenburg, Germany

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A light-emitting diode (LED) is a semiconductor light source emitting light when current flows through it. Since the Nobel Prize-winning invention of high brightness blue LEDs in 1993 by Shuji Nakamura, LEDs with excitation wavelengths below 400 nm have been suitable illumination sources for fluorescence measurements. In high-performance thin-layer chromatography (HPTLC), these devices open up a new field in planar chromatography because their long-term stable light intensity makes them a valuable light source for fluorescence analysis directly on the plate. Examples of fluorescence measurements in HPTLC using LEDs of different wavelengths in combination with diode-array planar chromatography will be presented. Some reasons are given why fluorescence measurements should be preferably performed on RP-18 plates and not on uncoated silica gel plates.

References:
Spangenberg, C.F. Poole, C. Weins. Quantitative Thin-Layer Chromatography: A Practical Survey, Springer, 2011

Matthias Guggenberger¹, Josua T. Oberlerchner¹, Heinrich Grausgruber², Thomas Rosenau¹, Stefan Böhmdorfer<sup>1

1</sup>University of Natural Resources and Life Sciences, Vienna (BOKU), Institute of Chemistry of Renewable Resources, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria
²University of Natural Resources and Life Sciences, Vienna (BOKU), Institute of Plant Breeding, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria

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A reliable, chemometric evaluation of HPTLC chromatograms for identification and classification would be most useful to screen larger datasets for unusual samples, or to verify the identity of unknown samples with low operator load. Typically, linear models are used for these tasks, with limited success due to the high level of noise in visual detection and inadequate plate-to-plate reproducibility.
A Self Organizing Map (SOM) is an iterative, unsupervised classification algorithm. We applied it to two sets of HPTLC data (495 analyses of essential oils, 50 analyses of wheat anthocyanins; Vis detection). The sample sets were sorted correctly (97% of the essential oils); grouping by plate was never observed. Misplaced chromatograms were either distorted by a volatile component of the neighboring track or contained an admixture. The impaired samples provided insight as to method development and validation. In the anthocyanin set, misplaced samples were always indicating wheat samples with unusual provenance.
To classify unknown samples, two extensions were successfully used: using a global SOM of all training samples, or training one SOM for each class. Both cases allow rating the confidence of the classification by a quality factor. This can also be used to test robustness, as deficient plates will show a lower match quality.
Data selection (color channels, color spaces) and preprocessing was investigated. The method proved sensitive to variable migration distances, thus chromatography had to be conducted meticulously.
Thus, self-organizing maps permit an automated classification of HPTLC chromatograms with a safeguard, as match quality is indicated. They also allow to efficiently select unusual samples from a larger data set for a more detailed (and costly) analysis.

Acknowledgments
We are grateful for the essential oils provided as a gift by Sonnentor Kräuterhandelsgesellschaft mbH and for the support of the Austrian Biorefinery Center Tulln (ABCT).

Reference
[1] M. Guggenberger, J. T. Oberlerchner, H. Grausgruber, T. Rosenau, S. Böhmdorfer, Talanta 233 (2021), 122460.

Danica Agbaba¹, Darija Obradović¹, Marija Čarapić<sup>2

1</sup>Department of Pharmaceutical Chemistry, Faculty of Pharmacy-University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia
²Medicines and Medical Devices Agency of Serbia, Vojvode Stepe 450, 11000 Belgrade, Serbia

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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.

 

Irena Malinowska, Marek Studziński

M. Curie Skłodowska University, M. Curie Skłodowska Sq. 3, 20-031 Lublin, Poland

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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.

Eliza Łata, Agnieszka Fulczyk, Mieczysław Sajewicz, Teresa Kowalska

Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland

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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.

Radosław Gwarda, Rafał Gajos, Michał Żmuda Trzebiatowski, Adam Chomicki, Tadeusz Dzido

Department of Physical Chemistry, Medical University of Lublin, Chodźki Street 4a, 20-093 Lublin, Poland

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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.

Pierre Bernard-Savary

Club de CCM, 89 chemin de PREFANTON, 38340 La Sure en Chartreuse, France

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After 30 years of a professional contribution to HPTLC, I would like to take the opportunity of the kind invitation by the committee, to share my experience and views.
This contribution will review what are, in my opinion, the most important achievements and weaknesses of this liquid chromatographic technique. During all these years I have been installing hundreds of HPTLC instruments and training hundreds of people, who are now having great results in the field. I saw where they started and why they succeeded.
What are the key elements which make this happen? What is the role played by the manufacturers, of plates and of instruments, together with analytical academic research to the impact on HPTLC dissemination and recognition worldwide in analytics? How this had an impact or not on the behaviour of private company laboratories, when they must address an analytical question mark? Prof. Poole introduction to HPTLC’03 in Lyon’s Symposium advertisement sheet was self-explaining: “We are enthusiastic to learn how many analysts are now confronted with situations where HPTLC is a suitable solution to their problems and is favoured over better known and more widely used analytical methods.”
Besides HPTLC, other chromatographic techniques are having many developments, which are pushing their limits further. But at the same time HPTLC also reached unexpected areas and levels in terms of results.
Based on this ascertainment, this lecture will try to setup a few ideas to help HPTLC having its place in the analytical field worldwide. Training at all levels remains a must of course, as HPTLC has this weakness not to be very present in the programs of universities and high schools. But also manufacturing improvements of the instruments currently available, in cooperation with the users’ community remains a real key aspect for its recognition. And, finally, meetings of our community of fans, from students to famous professors specialized in HPTLC, is always a good occasion to make progress, sharing achieved works and ideas for future tasks.

Snezana Agatonovic-Kustrin^1,2, David Morton^1,2
1Department of Pharmaceutical and Toxicological Chemistry named after Arzamastsev of the Institute of Pharmacy, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow 119991, Russia
²Department of Rural Clinical Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia

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Olea europaea L. or European olive, is one of the oldest known agricultural fruit trees cultivated to produce olives and olive oil. Cultivation of olive trees for extraction of olive oil generates large quantities of olive leaves as a by-product. Due to their high polyphenolic content, olive leaves present an important raw material. It has been reported by many researchers that olive leaf extract has antimicrobial activity due to its high phenolic content. The aim of this study was to analyse the effect of spontaneous microbial maceration on the release and extraction of the flavonoids and phenolics from olive leaves, and to identify compounds responsible for the antimicrobial activity of olive leaf extracts via thin layer chromatography - direct bioautography (TLC-DB). Results show that fermentation significantly enhances extraction of phenolic compounds and flavonoids. Polyphenolic content was increased from 6.7 µg GAE (gallic acid equivalents) to 25.5 µg GAE, antioxidants from 10.3 µg GAE to 25.3 µg GAE, and flavonoid content from 42 µg RE (rutin equivalents) to 238 µg RE per 20 µL of extract. Increased antioxidant activity of fermented ethyl acetate extracts was attributed to the increased extraction of flavonoids and phenolic terpenoids, while increased antioxidant activity in fermented ethanol extract was due to increased extraction of flavonoids. This increase in extraction may be due, in part, to in situ generated lactic acid based natural deep eutectic solvents (NADES) from the plant sample. Lactic acid that is released during fermentation and glycine present in the olive leaves form a Natural Deep Eutectic Solvent (NADES) with significantly increased solubility for flavonoids. TLC screening and effect-directed analysis of olive leaf extracts revealed two zones that inhibit the growth of different Enterococcus and Staphylococcus bacterial strains. The active compounds were further identified as maslinic and oleanolic acid by comparison of the R_F values of the bands with the standard reference materials and confirmed with ATR-FTIR and NMR spectra after isolation with flash chromatography.

Md Khairul Islam, Tomislav Sostaric, Lee Yong Lim, Cornelia Locher

Division of Pharmacy, School of Allied Health, University of Western Australia, Crawley, Western Australia, Australia

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Honey, a natural sweetener produced by honeybees mainly from flower nectar, has been used for thousands of years not only for its nutritional value but also for its medicinal properties. As a highly priced natural product, quality control, including the authentication of the honey’s predominant nectar source, is important but can prove challenging, especially for honeys collected from botanically rich areas, such as the pristine natural forests of Western Australia. With the specific case example of Western Australian Marri (Corymbia callophylla) honey, this study explores the use of High-Performance Thin Layer Chromatography (HPTLC) as a novel analytical tool for honey authentication and quality control, using methods for (1) the authentication of its predominant floral source via HPTLC fingerprinting of its organic extract; (2) the detection and quantification of Marri honey constituents that might contribute to its antioxidant activity via a novel, validated HPTLC-DPPH analysis; and (3) the qualitative and quantitative analysis of its major sugars using a new validated HPTLC assay. HPTLC based analytical methods are also used as part of a comprehensive short- and long-term stability study to detect any temperature induced changes to Marri honey’s chemical composition and antioxidant activity, including the quantification of hydroxymethyl furfural, a potentially toxic, heat induced sugar degradant.
While presented in the context of a specific Western Australian honey, the findings of this study demonstrate the general value of using HPTLC analysis for comprehensive honey testing, including not only the authentication of a honey’s predominant nectar source, but also the analysis of a range of other honey quality parameters (e.g. honey composition, HMF content) and the ability to monitor potential impacts of handling, storage and processing on honey quality.

Acknowledgements
This work was funded by Cooperative Research Centre for Honey Bee Products Limited (CRC HBP), Perth, Western Australia, Australia.