Session 6

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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.

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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.

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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.