Invited speakers

Erwin Rosenberg1, Maria Antoniadou1, Chrysoula Kanakaki1, Bernhard Klampfl1, Robert D. Müller1, Jürgen Kahr2
1Vienna University of Technology, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164 A-1060 Vienna, Austria
2AIT Austrian Institute of Technology GmbH, Center for Low-Emission Transport, Electric Vehicle Technologies, Giefinggasse 2, 1210 Vienna, Austria
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Gas chromatography is the most powerful technique to separate mixtures of volatile compounds; however, doing so normally requires a significant amount of time, and imposes thus limitation to the speed and the time resolution that can be achieved with chromatographic measurements. Many technologically relevant processes do, however, require measurements with a time resolution that is at the scale of one minute, or potentially even less. From the van Deemter equation it is obvious that it is impossible to maximise separation speed beyond the optimum separation velocity without losing efficiency [1]. A closer inspection of this equation reveals, however, various possibilities to gain separation speed. We will discuss various approaches to speed up chromatographic separation and to gain time resolution between measurements that we have developed and applied in response to the need of monitoring the volatile compounds formed by the degradation of the organic electrolyte of lithium ion batteries during various use conditions. As extreme operation conditions or misuse can lead to catastrophic degradation of the electrolyte in the lithium ion battery, fast responding chromatographic techniques are required [2]. These include the use of vacuum outlet conditions, multiplexing, and of fast (positive or negative) thermal gradients. Their individual benefits and limitations will be presented and critically discussed here, with a particular view to their practical applicability.

References
[1] J.J. van Deemter, F.J. Zuiderweg, A. Klinkenberg, Chem. Eng. Sci. 5 (1956) 271-289.
[2] Y.P. Stenzel, F. Horsthemke, M. Winter, S. Nowak, Separations 2019, 6(2), 26.

Acknowledgements
The authors gratefully acknowledge financial support of this work by the Austrian Research Promotion Agency (FFG) under Project numbers 835790 ("SiLithium"), 858298 ("DianaBatt") and 879613 ("OPERION").

Pharmaceuticals are micropollutants belonging to the main sources of water pollution due to their relative persistence and toxicity in the water body. These compounds are used to treat human and animal diseases and come in the surface waters from the industrial production, hospitals and incompletely metabolized drugs. During the last time an increased resistance of human and animal bodies to drugs was registered that leads to some unexpected health effects. This fact is caused by the uncontrolled input of pharmaceuticals in surface waters that determined the European Union to pay a special attention to this topic. Thus the European Parliament amending the Water Framework Directive (2000/60/EC) is preoccupied to introduce some anti-inflammatory drugs, namely diclofenac and 17beta-estradiol, in the list of emerging pollutants and to establish their maximum allowable concentration in surface waters. In this work, the authors present the results of one year monitoring (July 2014-June 2015) of some classes of pharmaceuticals (nonsteroidal anti-inflammatories, hormones, antibiotics, anticancers) widely used for the human and veterinary treatments, in Tisza River and some of its tributaries on Romanian territory. The isolation of studied pharmaceuticals from water matrices was achieved by solid-phase extraction (SPE) on Strata C18-U and Strata X cartridges. The pharmaceutical analysis was performed by high performance liquid chromatography coupled with diode array detector and/or mass spectrometer. The SPE-HPLC-DAD/MS procedures were used for the determination of the target compounds in river water samples collected from the Romanian Tisza River Watershed during four seasons. The developed procedures were validated and studies of statistical significance of the obtained results were done. Among the target pharmaceuticals found in the investigated river water samples were diclofenac and ibuprofenas anti-inflammatories; estrone and 17beta-estradiol as hormones and tetracycline, doxycycline, ceftazidime and ceftriaxone as antibiotics. The monitoring results show the importance of the study of pharmaceuticals in surface waters for a better risk assessment.

Acknowledgements: This work has been performed in the frame of NATO Science for Peace 984440/2014-2017 Project.

Presenting author:
Virginia Coman
Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
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Authors:
Mihail Simion Beldean-Galea - Babes-Bolyai University, Faculty of Environmental Science and Engineering, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
Florina Copaciu - Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
Miuta Filip - Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
Mihaela Vlassa - Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania

Paola Dugo1, Katia Arena1, Francesco Cacciola2, Luigi Mondello1
1University of Messina, Dipartimento CHIBIOFARAM, polo Annunziata, 98168 Messina, Italy
2University of Messina, Dipartimento BIOMORF, polo Annunziata, 98168 Messina, Italy
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Natural products and vegetable extracts are rich source of bioactive molecules such as flavonoids, alkaloids, saponins, and so on. Although many studies have been carried out on the chemical characterization of these samples, there is still much interest in the detection of novel bioactive compounds. Conventional one-dimensional liquid chromatography (1D-LC) does usually provide sufficient resolving power for their determination. However, in many cases, their complexity is so high that the separation capability offered by 1D-LC is not enough thus requiring more powerful analytical technologies for both their characterization and quantification. Comprehensive two-dimensional LC (LC×LC) involving the coupling of two or more orthogonal or quasi-orthogonal separation systems is an interesting alternative capable, through its selectivity and sensitivity, to detect minor components.
For the analysis of natural products and vegetable extracts, different LC×LC methodologies including NP×RP, RP×RP and HILIC×RP have been successfully investigated. In this contribution, applications of MicroLC×LC in the field of natural products are reported and discussed.

Modest Gertsiuk
State Institution “Institute of Environmental Geochemistry of the National Academy of Sciences of Ukraine”, Academician Palladin Avenue, 34а , 03142, Kyiv, Ukraine
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The city of Kalush, located in Western Ukraine, was a major center of the chemical industry in the former Soviet Union. In particular, at the production association "Chlorvinyl" were produced perchloroethylene, carbon tetrachloride by direct chlorination of hydrocarbons. Hexachlorobenzene was formed as a by-product of this synthesis, the content of which in production waste was over 90%. According to the Stockholm Convention, this compound belongs to the persistent organic pollutants, and waste with hexachlorobenzene - to the first class of danger. As there were no such waste disposal plants in the Soviet Union at that time, it was decided to bury of hexachlorobenzene waste at a landfill near the city of Kalush. The waste was placed in iron barrels with a capacity of 200 liters and buried in the trench. As of 2001, there were 11,352.5 tons of hexachlorobenzene wastes at the landfill. Over time, the barrels corroded and hexachlorobenzene migrated into soils, groundwater and surface water. This posed a great danger to the health of the population of Kalush and other settlements located nearby. In view of this, it was decided to remove waste from the landfill. During the work on the removal of waste, the placement of waste and the state of contamination of soils, groundwater and surface water were monitored. Sampling was performed both from the surface and from wells located on the landfill. It was found by gas chromatography that the concentrations of hexachlorobenzene in the soil exceeded the maximum allowable concentrations by hundreds of thousands of times. Soil contamination reached a depth of 12 m and the maximum levels of contamination were at a depth of 2-3 meters, where waste barrels were placed. Significant exceeding of the maximum allowable concentrations of hexachlorobenzene was observed in groundwater and surface water in the area of the landfill. A map of waste location was compiled and used for their removal.

Günther K. Bonn
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria. ADSI - Austrian Drug Screening Institute, 6020 Innsbruck
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Innovative approaches in analytical chemistry have become increasingly important for natural product research and new advances in separation science enable research into inaccessible areas of natural product isolation. Since herbal preparations like plant extracts are often multi-component mixtures with hundreds or thousands of small molecules at different concentrations, their separation and analysis is often difficult to perform. Thus, novel enrichment and purification methods based on advanced solid-phase extraction techniques have been developed to reduce the complexity of plant extracts, while HPLC has been used for separation, pre-concentration and fractionation. The ability to apply these techniques to robotic systems enables high throughput screening. Significant progress has been made in developing new stationary phases that can be tailored to a specific application and thus offer endless possibilities for optimizing selectivity. A further coupling to high-resolution mass spectrometry facilitates the identification and quantification of active ingredients in natural products. Additionally, the combination of separation science with spectroscopy offers the possibility to combine various technologies in phytopharmacy as well as food analysis. Near- and mid- infrared spectroscopy enable quick and simultaneous qualitative and quantitative analysis of raw plants and liquid extracts without destruction. Moreover, infrared imaging has been used to study the distribution of active substances in plant materials. All of these approaches offer new strategies for quality control in phytoanalysis and enable deeper insights into the biochemical background of medically relevant questions. In this presentation, novel approaches in analytical chemistry for various applications in the areas of phytopharmacy, phytocosmetics and phytonutrients are demonstrated and discussed.

Bogusław Buszewski1,2, Hussam Al-Saoud2,3, Michał Szumski2, Marek Potrzebowski4, Myroslav Sprynskyy2
1Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicu University in Toruń, Gagarina7 str. Pl-87 100 Toruń (Poland)
2Interdyscyplinary Center of Modern Techniology, Nicolaus Copernicu University in Toruń, Gagarina7 str. Pl-87 100 Toruń (Poland)
3Departament of Materiale Engineering and Production, Faculty of Mechanical Engineering, Białystok University of Technology, Wiejska 45c, Pl-15 351 Białystok (Poland)
4Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza str. 112, Pl-90 001 Łódź (Poland)
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Silica has always been the object of interest of specialists and used for many purposes. Particularly for over 100 years, it has been used in chromatography and other separation techniques as an adsorbent or a carrier for stationary phases. Due to the high demands placed on chromatographic stationary phases, synthetic silica is used to provide high column efficiencies and avoid many adverse interactions. Recently, bio-silica obtained as a result of biogenic processes carried out by the selected strains of algae turned out to be an alternative to the synthetic silica [1]. This material, in contrast to the synthetic one, is characterized by a micro- or nano-hierarchical structure. Biosilica is an inorganic polymer formed by organisms such as diatoms or siliceous sponges of orthosilicate units in which two silanol groups are joined together to one bond or siloxane. Its low manufacturing cost as well as high quality in terms of chemical composition, mechanical stability and resistance are guaranteed by a good biosynthesis reproducibility. In this paper, the conditions of bio-silica biosynthesis, used as an adsorbent and carrier of stationary phases for liquid chromatography and related techniques, will be presented. The characteristics of bare and modified materials using porosimetric techniques, microscopic imaging, surface architecture characteristics via spectral and spectroscopic methods as well as chromatographic assessment will be discussed. Potential application possibilities (first proposals in the literature) in chromatographic separation and sample preparation will also be presented.

Acknowledgements:
This work was financially supported by the Foundation for Polish Science co-financed by the European Union under the European Regional Development Found, project Advanced bio-composites for tomorrow’s economy BIOG-NET, FNP POIR.04.04.00-00-1792/18-00. The project is carried out within the TEAM-NET program.

[1] M. Sprynskyy, P. Pomastowski, M. Horonowska, A. Król, K. Rafińska, B. Buszewski, Naturalny organic functionalized 3D biosilica from diatommicroalge., Mater. Des., 132 (2017) 22-29.

Pharmaceuticals are micropollutants belonging to the main sources of water pollution due to their relative persistence and toxicity in the water body. These compounds are used to treat human and animal diseases and come in the surface waters from the industrial production, hospitals and incompletely metabolized drugs. During the last time an increased resistance of human and animal bodies to drugs was registered that leads to some unexpected health effects. This fact is caused by the uncontrolled input of pharmaceuticals in surface waters that determined the European Union to pay a special attention to this topic. Thus the European Parliament amending the Water Framework Directive (2000/60/EC) is preoccupied to introduce some anti-inflammatory drugs, namely diclofenac and 17beta-estradiol, in the list of emerging pollutants and to establish their maximum allowable concentration in surface waters. In this work, the authors present the results of one year monitoring (July 2014-June 2015) of some classes of pharmaceuticals (nonsteroidal anti-inflammatories, hormones, antibiotics, anticancers) widely used for the human and veterinary treatments, in Tisza River and some of its tributaries on Romanian territory. The isolation of studied pharmaceuticals from water matrices was achieved by solid-phase extraction (SPE) on Strata C18-U and Strata X cartridges. The pharmaceutical analysis was performed by high performance liquid chromatography coupled with diode array detector and/or mass spectrometer. The SPE-HPLC-DAD/MS procedures were used for the determination of the target compounds in river water samples collected from the Romanian Tisza River Watershed during four seasons. The developed procedures were validated and studies of statistical significance of the obtained results were done. Among the target pharmaceuticals found in the investigated river water samples were diclofenac and ibuprofenas anti-inflammatories; estrone and 17beta-estradiol as hormones and tetracycline, doxycycline, ceftazidime and ceftriaxone as antibiotics. The monitoring results show the importance of the study of pharmaceuticals in surface waters for a better risk assessment.

Acknowledgements: This work has been performed in the frame of NATO Science for Peace 984440/2014-2017 Project.

Presenting author:
Virginia Coman
Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
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Authors:
Mihail Simion Beldean-Galea - Babes-Bolyai University, Faculty of Environmental Science and Engineering, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
Florina Copaciu - Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
Miuta Filip - Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania
Mihaela Vlassa - Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania

Dayoung Park, Daesup Geum, Young Ji, Yong-Nam Pak
Korea National University of Education, Taesung 250, 28173, Cheongju, Rep. of Korea
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It is very important to monitor the concentration of selenium in the body precisely because of its narrow range of necessity and toxicity. In order to obtain biologically meaningful information, Se speciation study must be carried out for its bio-activities. Human nail samples, as well as hair are useful biological samples that reflect long-term selenium exposure while breath can reflect for a relatively short-term.. Researches have been carried to find out the optimum extraction condition and accurate determination of Se species with Isotope Dilution HPLC-ICP-MS. Nails were treated with NaOH solution at 90 °C for 24 hours, the degradation efficiency was about 70%. Selenium species were separated using Anion exchange HPLC to detect SeCys and Se⁴⁺. The concentration of selenium species was analyzed SeCys species was shown in the range of 700-950 ppb for a year. When a selenium supplement was given (300 μg day^-1), it was found out that the selenium in nails increased within 2 months and depleted after while a regular supplement given (22 μg day^-1) did not show much effect. Hair samples showed around 200 ppb, higher values at root area. Breath samples were also analyzed and only a trace amount of DMSe was shown. This research shows that human samples such as hair, nails and even breath can be used to monitor Se species in our body effectively either in a long or short period of exposures.

References
1. Y. S. Zhu, H. B. Xu, K. X. Huang, W. H. Hu, M. L. Liu, Biol. Trace Elem. Res. 89 (2002) 155.
2. M. Navarro-Alarcon, H. L. G. de la Serrana, V. Perez-Valero, C. López-Martı́nez, Sci. Total Environ. 228 (1999) 79.
3. M. P. Longnecker, M. J. Stampfer, J. S. Morris, V. Spate, C. Baskett, M. Mason, , W. C. Willett, Am. J. Clin. Nutr. 57 (1993) 408.
4. J. Kaluza, M. Jeruszka, A. Brzozowska, Rocz. Panstw. Zakl. Hig. 52 (2001) 111.

Imre Molnár, Hans-Jürgen Rieger, Arnold Zöldhegyi Molnár-Institute, Schneegloeckchenstrasse 47, 10407 Berlin, Germany
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First of all, we are thankful to Danilo Corradini, a former important coworker of Csaba Horváth and the chairwoman Irena Vovk to organize the Csaba Horváth session. Csaba Horváth was not only a pioneer in the creation of a whole new revolutionary analytical technology, which he christened in 1967 High Pressure Liquid Chromatography or HPLC. He was also the first, using the computer in his research work. He purchased in the spring of 1976 a PDP-11 brand new computer which had the size of ca. 50x50x200 cm and looked like a cabin. He was developing research theories on band spreading with Fred Linn on this computer. Consecutively, Lloyd Snyder, John Dolan, Tom Jupille and myself started to develop a program to model HPLC separations in 1985, which we also exhibited at PittCon 1986. Lloyd named this software DryLab. In the last 36 years a vigorous development took place to model HPLC. The lecture will present some of the milestones in this work and report about the most recent achievement of this excellent technology.

Coral Barbas
Centre of Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28060 Boadilla del Monte, Spain
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Metabolomics, the comprehensive analysis of metabolites in a biological sample, is a huge analytical challenge due to the physico-chemical diversity of analytes, their concentration ranges, and properties of the biological matrices. Separation techniques greatly enhance the analytical capabilities of mass spectrometry (MS) where accurate annotation is vital for data interpretation; however, metabolite identification is still a major bottleneck in untargeted metabolomics.
Along this presentation, I will discuss different analytical challenges in metabolite identification that we have faced at CEMBIO and how we have approached them using CE-TOF-MS, LC-QTOF-MS, and LC-IM-QTOF-MS.
We have demonstrated the robustness of the in-source fragmentation (ISF) in CE-TOF-MS, which makes possible the annotation of compounds by means of their fragmentation pattern [1]. ISF together with the mechanisms of the major fragmentation reactions observed for 57 amino acid standard compounds allowed us to establish a workflow for targeted extraction of modified amino acids [2] with high biochemical value which led us to the identification of unknown epi-metabolites.
A more recent technique, ion mobility spectrometry (IMS) allows the separation of molecules on the gas phase based on their spatial configuration. Combined with LC-MS, stands out as an excellent tool to study different isomeric compounds. Oxylipins and esterified oxidized lipids are bioactive compounds that play crucial roles in physio-pathological processes, like infections, cancer, and Alzheimer’s. Their analysis, usually done by LC-MS, is hindered by the presence of multiple isomers which in some cases can be disease-specific. Therefore, its identification could reveal reliable diagnostic markers for the diseases they are involved in. We have used LC-IMS-MS to improve the annotation of this type of compounds both in standards and plasma samples of patients suffering different diseases. This allowed the confirmation of elevated oxidized lipids as well as the identification of new and previously unresolved species.

References
[1] M. Mamani-Huanca, A. Gilde la Fuente, A. Otero, A. Gradillas, J. Godzien, C. Barbas, Á. López‐Gonzálvez, J. Chromatogr. A, 1635 (2021) 461758.
[2] M. Mamani-Huanca, A. Gradillas, A. Gilde la Fuente, Á. López‐Gonzálvez, C. Barbas, Anal. Chem. 92 (7), (2020) 4848–4857.

Jeremy D. Glennon1, Alyah Buzid2, Phyllis E. Hayes1, Majidah Alsaeedi1, Huda Alghamdi1, F. Jerry Reen3, Gerard P. McGlacken1, John H.T. Luong1
1School of Chemistry and the Analytical & Biological Chemistry Research Facility (ABCRF), University College Cork, College Road, Cork T12 YN60, Ireland.
2Department of Chemistry, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa, 31982, Saudi Arabia.
3School of Microbiology, University College Cork, College Road, Cork T12 YN60, Ireland.
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Combining the nanomolar detectability at solid electrodes with high efficiency chromatographic media can provide selective multianalyte trace determination of key electroactive compounds. In this work, the coupling of electrochemical detection at a boron-doped diamond electrode (BDD) with core-shell chromatography (LC-BDD) is applied for the rapid nanomolar detection of targeted solutes including phenolics, neurotransmitters and microbial metabolites. Among the applications examined is the rapid detection of key catecholamine biomarkers elevated in traumatic brain injury (dopamine, epinephrine and norepinephrine) using hydrophilic interaction liquid chromatography (HILIC) on superficially porous (core-shell) and porous Z-HILIC columns [1]. Key microbial metabolites are also rapidly detectable by Halo C18 LC-BDD exemplified by signalling molecules or bacterial pheromones for cell-to-cell communication (quorum sensing (QS)) from Pseudomonas aeruginosa, an antibiotic-resistant human pathogen associated with chronic lung infections. Application is extended to the detection of guaiacol (2-methoxyphenol), a food and beverage spoilage metabolite indicative of contamination by the Gram-positive thermophilic Alicyclobacillus spp. bacterium [2]. Additionally, studies into the profiling of essential phenolic flavouring compounds in whiskey samples [3] and to selected pharmaceutical compounds are described [4].

References:
[1] M. Alsaeedi, H. Alghamdi, P. E. Hayes, A. M. Hogan and J. D. Glennon, Separations 8 (2021) 124.
[2] P. E. Hayes, A. Buzid, J. H. T. Luong and J. D. Glennon, Electroanalysis 33 (2021) 766 – 773.
[3] P. E. Hayes, J. H.T. Luong, E. S. Gilchrist, A. Buzid, J. D. Glennon, Journal of Chromatography A 1612 (2020) 460649
[4] H. Alghamdi, M. Alsaeedi, A. Buzid, J. D. Glennon and J. H. T. Luong, Electroanalysis 33 (2021) 1137 – 1142

Acknowledgements:
The authors wish to acknowledge the following funding bodies for project grants and individual scholarships: Irish Research Council, Enterprise Ireland, the Ministry of Education (Saudi Arabia) and the Cultural Bureau (Dublin) and previously Science Foundation Ireland.

Dušan Berek
Polymer Institute, Slovak Academy of Sciences, Dubravska cesta 9, 84541 Bratislava, Slovakia
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Block copolymers, BLC belong to the group of synthetic nonmetallic materials called complex polymers. They have valuable properties and found applications for example as flocculants, carriers of drugs and compatibilizers of polymer blends, etc. BLC are formed with two or several (only rarely with more than three) polymeric chains called blocks. Blocks have different chemical composition or physical architecture and they are mutually connected with a chemical bond. The structure of common diblock and triblock copolymers is schematically displayed as A-B, A-B-A or A-B-C. Most BLC contain their parent homopolymers, the “free blocks”, which may spoil their utility properties. Therefore, it is important to determine their amount and to adjust the synthesis of BLC to reduce their formation. The molecular characterization of BLC and determination of parent homopolymers is an analytical challenge. The limited separation selectivity of the most commonly polymer characterization method, size exclusion (gel permeation) chromatography, SEC/GPC is usually not sufficient to fulfill the task. Other, advanced methods of polymer liquid chromatography must be applied. Three of them will be presented in the contribution.
1. Liquid chromatography under critical conditions, based on unique mutual compensation of entropic partition (exclusion) and enthalpic interactions of macromolecules within column
2. Full adsorption of samples containing distinct macromolecules on a nonporous microparticulate sorbent, followed with their sectional, selective elution into an SEC/GPC column
3. Barrier methods, the emerging techniques, which are based on the action of slowly eluting, narrow liquid zones, barriers, which selectively decelerate fast elution of macromolecules of distinct nature.

In molecule optics, a matter wave of molecules is manipulated by a molecule-optical component made of external, typically radiative, fields. The molecule-optical index of refraction, n, for a nonresonant IR laser pulse focused onto a molecular beam can be obtained from the energy conservation and wave properties of molecules. Experimentally measured values of n for benzene and nitric oxide agreed well with the calculated values. Since n depends on the properties of molecules as well as those of the laser field, a molecule prism composed of the focused nonresonant laser field can separate a multi-component molecular beam into several components according to their molecule-optical refractive indices n. We obtained a chromatographic resolution of 0.90 for the spatial separation of a mixture beam of benzene and nitric oxide using a focused Nd:YAG laser pulse as a molecule prism. In addition, ways of improving the efficiency of optical forces will be discussed.

Presenting author:
Doo Soo Chung
Department of Chemistry, Seoul National University, Seoul 151-747, Korea
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Authors:
Doo Soo Chung - Seoul National University

Milan Hutta, Róbert Góra
Comenius University in Bratislava, Faculty of Natural Sciences, Department of Analytical Chemistry, Ilkovičova 6, 84215 Bratislava, Slovakia
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Various natural substances are too complex analytes even for recent highly efficient analytical methods. One research approach is to characterize and analyze such complex systems is based on operational definition. Operationally defined analytes isolated from complex matrices resulted in development of novel combined multidimensional methods based on liquid chromatography separation with potential extension to combinations with electroseparation methods (ESM) and multi-detection (UV-VIS spectrophotometry, spectrofluorimetry, electroanalytical detection) and mass spectrometry (MS). Liquid chromatography working under extreme separation conditions (pH, ionic strength, temperature, additives, solvents etc.) as first dimension separation method is investigated. Usually, tough separation conditions are not directly compatible with the other methods in second separation dimension – some modes of HPLC, capillary isotachophoresis (ITP), capillary zone electrophoresis (CZE) (with various detection principles) and MS. Systematic study of orthogonality of novel combined techniques for characterization of operationally defined humic substances (HS) and/or lignin was done. Compatibility of used extreme extractive isolation and separation conditions in HPLC (ion exchange, reversed phase, size-exclusion) with common conditions of various end techniques was tested. The approach is valuable for solution of HS interferences elimination in soil samples containing pesticides. Comprehensive techniques of multidimensional liquid chromatography (under the conditions of extreme pH, I, temperature, large-volume injection, solid sample injection etc.) combined off-line or on-line with the other separation or identification methods are discussed. Application of methods combining extreme separation conditions in HPLC (pH within the range 0-2 or alternatively 10-14, ionic strength above 0.1 mol/l, temperature well above 40°C, non-common mobile phase solvents dimethylformamide) and normal separation conditions in ESM and MS in the field of environmental analysis, analysis of degradation products of soil humic substances.

The research was supported by the grant of projects APVV-17-0318, APVV-0462-20.

Koen Sandra, Liesa Verscheure, Gerd Vanhoenacker, Tom Merchiers, Shauni Detremmerie, Julie Storms, Isabel Vandenheede, Pat Sandra
RIC group, President Kennedypark 26, 8500, Kortrijk, Belgium
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Biotech drugs, once considered a risky and nearly impossible route to explore, are today dominating the pharma landscape. In the footsteps of the biotech pioneers walking this bumpy road, our chromatographic community rose to the challenge and sharpened the analytical toolbox. Throughout the years we have seen products become increasingly complex and witnessed analytics becoming more and more performant. This talk will reflect on the recent advances in the analysis of biologics; from the coupling of historical incompatible chromatographic methods with mass spectrometry, to the combination of ever more separation modes in multidimensional set-ups and the implementation of chemical and enzymatic reactors in the flow path for primary and higher order structural assessment.

Gérard Hopfgartner1, David Ruskic1, Patrick Mueller1, Ron Bonner2
1Life Sciences Mass Spectrometry, Department of Inorganic and Analytical Chemistry, University of Geneva, 24 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland
2Ron Bonner Consulting, Newmarket, ON, Canada
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LC-MS/MS-based global metabolomics or forensic investigations with electrospray ionization rely on exact mass determinations, collision induced spectra and statistical analysis for compounds detection and structural identification. The use of large MS/MS libraries, in silico fragmentation and LC retention prediction tools plays more and more an important role to achieve that goal. Beside large ionization response range, metabolites do often not generate fragment rich CID spectra for protonated and deprotonated precursor ions. In addition, adduct precursor ions (Na, K,...) also do not fragment well which leaves many potential hits unexploited. This calls for alternative ionization approaches and additional fragmentation techniques such as photodissociation (PD) or electron induced dissociation (EID). As the analysis needs to be keep short and sample is limited, multimodal approaches are becoming essential to gain as much as possible qualitative and quantitative information in a single LC-MS run. Recent instrumental improvements in high-resolution mass spectrometry (HRMS) have enabled data independent information acquisition (DIA) schemes, such as SWATH [1]. With SWATH a collision induced dissociation MS/MS spectra can be generated for every precursor ion. Comparison with MS/MS database or the use of in-silico fragmentation tools can further improve compound identification, but improved selectivity and MS/MS information is needed. Implementing differential mobility spectrometry (DMS) for multidimensionnal separations (LCxDMS/DMS or SFCxDMS/MS) into the analytical workflow as the use of electron induced dissociation (EID), can significantly improve the performance of the analysis on singly charged precursor ions including adducts and multimers. Furthermore, under specific conditions atmospheric pressure ionization with micro-LC enables the selective generation either protonated molecules or radical cations. As for EID, radical fragmentation is of high interest as it opens the use of electron impact libraires for compound ID in LC-MS.

[1] R. Bonner, G. Hopfgartner, TrAC - Trends Anal. Chem. 120 (2019) 115278; https://doi.org/10.1016/j.trac.2018.10.014.

Imre Klebovich
Department of Pharmaceutics, Faculty of Pharmacy, Semmelweis University, Hőgyes Endre St. 7., H-1092 Budapest, Hungary
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The presentation gives insight to the modern tools of radiochromatography in preclinical and clinical pharmacokinetic and drug metabolism studies. The essential pharmacokinetic and drug metabolism studies of different species (mouse, rat, dog, rabbit and human) provides information to the final drug registration process. The highly sensitive (pg/ml, fg/ml, at/ml) and selective hyphenated techniques (LC/Triple Quad-Jet Stream-ESI-MS and GC/MS-MS, etc.) required for the pharmacokinetic studies had replaced the conventional methods of detections such as GC and HPLC. In the course of drug development radioactive isotope (beta and gamma single and/or double source)-labeled (³H, ¹⁴C, ⁹⁹Tc, ¹³¹I) pharmacokinetic studies combined with new generation of triple-quad and high resolution MS techniques (LC, CE, OPLC) are of significant importance. A number of related case studies will be introduced. The former Imaging Techniques (DAR, PIT) and the new generation of in vitro – in vivo Imaging Techniques (MALDI Imaging, nanoScan, PET/MRI in animal and human studies) will also be presented. A complex multi-step process will be illustrated from separation, purification, isolation to structure elucidation (GC-MS, LC-MS/MS, LC-NMR) of minor and major metabolites derived from animal and human biological matrices. The addition of these systems to the off-line and on-line separation and radioactivity detection possibilities of OPLC-DAR/PIT, OPLC-RD, HPTLC-DAR-MS and GC-RD, HPLC-RD and the combined multi-hyphenated techniques, OPLC-DAD-RD-MS/MS, OPLC-DAD-RD-NMR as well as LC-DAD-RD-MS/MS and LC-DAD-RD-NMR resulted in a new, flexible and rapid high-performance complex solution in the metabolism research.

Attila Felinger
University of Pécs
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It is known in chromatography that the separation process and the migration rate of sample components through a porous stationary phase are highly influenced by the adsorption of each component. For the system which contains more than one component at a sufficient concentration (multicomponent system), their adsorption on the stationary phase will be influenced by each other, so it is important to characterize their competitive behavior. In SFC, competition is expected to occur between the organic modifier and the additive molecules when a ternary mobile phase (CO₂ + modifier + additive) is used for the separation of polar solutes. We elucidating the adsorption process of the mixture of modifier and additive on polar stationary phases in SFC, because there is still lack of information in describing the adsorption behavior of their mixture. There have not been many applications for the use of water as additive in the modified mobile phase in SFC comparing to bases or acids, because in most of the cases the use of additives usually is required for the separation of analytes with basic or acidic forms. Even though there are some applications by adding water to the mobile phase in SFC, but the role of water in the elution process still needs a dedicated study. Our objective is to show how water or methanol adsorption can influence retention and efficiency in SFC. For that purpose, a number of approaches of isotherm adsorption determination will be studied: the single component adsorption isotherm of either methanol and water, and the competitive adsorption isotherm of the water–methanol mixture.

Danijela Ašperger1, Bruna Babić1, Dijana Cerić2, Tomislav Bolanča1
1University of Zagreb, Faculty of Chemical Engineering and Technology, Department of Analytical Chemistry, Marulićev trg 19, 10000 Zagreb, Croatia
2Bergman Labora, Karlsrovägen 2D, 182 53 Danderyd, Sweden
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Pharmaceuticals are used in human and veterinary practice and are so-called emerging contaminants. Their occurrence in the environment is still not law regulated, while certain number of published articles record their negative impact on human health. From the literature overview, pharmaceuticals were determined in different environment samples, but little bit less in here examined sediment. Sediment acts like a potential receiver for many hazardous substances including pharmaceuticals who can be easily emitted to drinking water reservoirs depending on the sediment characteristics [1]. In this work pharmaceuticals who belong to different structure groups were determined in sediment sample and following five extractions were developed and optimized: extraction by agitation (EA), ultrasound solvent extraction (USE), microwave assisted extraction (MAE), matrix solid phase dispersion (MSPD) and pressurized solvent extraction (PSE). Chosen pharmaceuticals were tylosine as macrolide antibiotic, albendazole, febantel and levamisole as antihelmintics, lidocaine and procaine as anesthetics and hydrocortisone and dexamethasone as glucocorticoids. Pharmaceutical concentrations extracted from sediment sample were determined by high performance liquid chromatography using diode array detector. After chromatographic analysis, all methods were validated and the following optimal parameters were determined for each extraction method: sediment mass, solvent, contact time between sediment and solvent, extraction duration, agitation frequency and extraction temperature (EA); solvent, extraction duration, power and temperature of ultrasonic bath (USE); solvent, extraction duration, temperature, solvent volume (MAE); sorbent, solvent, solvent volume, sediment/sorbent mass ratio (MSPD); solvent (PLE) [2].

[1] M. S. Díaz-Cruz, M. J. López de Alda, D. Barceló, TrAC 22 (2003) 340-551.
[2] D. Drljača, D. Ašperger, M. Ferenčak, M. Gavranić, S. Babić, I. Mikac, M. Ahel, Chromatographia, 79 (2016) 209-223.

Acknowledgment:
This paper was prepared as part of the project Advanced Water Treatment Technologies for Microplastics Removal (IP-2019-04-9661, AdWaTMiR).

Jean-Luc Wolfender, Adriano Rutz, Arnaud Gaudry, Luis Quiros Guerrero, Olivier Kirchhoffer, Louis-Félix Nothias, Emerson Ferreira Queiroz, Pierre-Marie Allard
School of Pharmaceutical Sciences, University of Geneva, CMU – Rue Michel Servet 1, 1211 Geneva 11, Switzerland
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The recent rapid innovations made in metabolite profiling and bioassays have begun to drive a change of paradigm in natural products research. Indeed, having at hand full or partial of structure of possibly all metabolites in given natural extract at different quantitative levels open the possibility to perform pharmacognosy studies from a more holistic perspective. The increasingly amount of accurate metabolome data that can be acquired on massive sample sets, notably through high resolution mass spectrometry data dependent MS/MS analyses (HRMS/MS), allows mapping of natural extracts at an unprecedented precision level [1]. While the acquisition of larger volumes of data is ongoing, contextualizing it is a lagging process and new initiative such as LOTUS contributes to this effort by linking chemical, taxonomical and bibliographical information [2] In this context we push forward our applications and further development of UHPLC-HRMS/MS Molecular network (MN) approaches [3] to provide enhanced annotation confidence level. We have recently built massive MN on natural extracts libraries, including thousands of plant and microorganism extracts. This allows the creation of virtual libraries of NPs through an advanced automated annotation pipeline. We are developing computational methods and structural-taxonomic libraries to navigate this massive chemical space in order to rationally identify valuable bioactive NPs to focus on. For their full chemical and pharmacological characterization, we then deploy an advanced workflow that allows precise targeted isolation based on metabolite profiling data. Different recent applications will illustrate these aspects and give future prospects in natural products research.

References
[1] J.L. Wolfender, J.M. Nuzillard, J.J.J. van der Hooft, J.H. Renault, Anal. Chem. 91 (2019) 704-742.
[2] A. Rutz, M. Sorokina, J. Galgonek, D. Mietchen, E. Willighagen, A. Gaudry, J.G. Graham, R. Stephan, R. Page, J. Vondrášek, C. Steinbeck, G.F. Pauli, J.L. Wolfender, J. Bisson, P.M. Allard, bioRxiv (2021) 2021.2002.2028.433265, DOI : 10.1101/2021.02.28.433265.
[3] A. Rutz, M. Dounoue-Kubo, S. Ollivier, J. Bisson, M. Bagheri, T. Saesong, S.N. Ebrahimi, K. Ingkaninan, J.L. Wolfender, P.M. Allard, Front. Plant Sci. 10 (2019) 1329.

Acknowledgements
The authors are grateful to Green Mission Pierre Fabre for establishing and sharing the unique library of extracts and the Swiss National Science Foundation (SNF N° CRSII5_189921 / 1).

Václav Kašička, Jan Bílek, Dušan Koval, Petra Sázelová, Filip Teplý
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague 6, Czechia
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Recently, new diquats (DQs), derivatives of widely used herbicide diquat, were prepared [1]. These chiral N heteroaromatic dicationic compounds comprising 2,2’-bipyridine moiety are attractive for variable applications, e.g. as catalysts, dyes, redox indicators, DNA intercalators, and chiral selectors. For these applications, enantiopurity of (P)- and (M)-isomers of DQs has to be checked. For this purpose, a new capillary electrophoresis (CE) method was developed. CE analyses were performed on Agilent Technologies 7100 CE system (Waldbronn, Germany) in a hydroxypropylcellulose coated fused silica capillary (id/od 50/375 µm, total/effective length 385/300 mm), at –12 kV separation voltage and 25°C. The DQs (0.2 mM) were injected hydrodynamically (700 Pa × 10 s) and detected at 200 nm. (P)- and (M)-enantiomers of all 11 DQs were baseline or better resolved by CE using aqueous 22/35 mM sodium/phosphate buffer, pH 2.5, as background electrolyte (BGE) and 6 mM randomly highly sulfated α-, β- and γ-cyclodextrins (S-CDs) as chiral selectors. Interactions of dicationic DQs with highly sulfated CDs resulted in formation of strongly negatively charged fast migrating DQ-CD complexes [2] that were separated with high resolution within a short time of 5–8 min. In addition, strength of the complexes of DQ enantiomers with S CDs was evaluated. The apparent stability constants of the DQ-CD complexes were determined from the dependence of effective mobilities of (P)- and (M)-enantiomers of the DQs on concentration of S-CDs in the BGE by nonlinear regression analysis as described in [3]. The DQs enantiomers formed moderate to strong complexes with all three types of S-CDs with the apparent stability constants in the range (7.80–547.4) × 10^3 L/mol.

Acknowledgements
This work was supported by the GACR (project 20-03899S) and the CAS (project RVO 61388963).

References
[1] H.R. Talele, D. Koval, L. Severa, P.E. Reyes-Gutiérrez, I. Císařová, D. Šaman, L. Bednárová, V. Kašička, F. Teplý, Chem.-Eur. J. 24 (2018) 7601-7604.
[2] D. Koval, L. Severa, L. Adriaenssens, J. Vávra, F. Teplý, V. Kašička, Electrophoresis 32 (2011) 2683.
[3] S. Štěpánová, V. Kašička, J. Sep. Sci. 38 (2015) 2708.

Luigi Mondello, Gemma De Grazia, Lorenzo Cucinotta, Danilo Sciarrone
ChiBioFarAm Department, University of Messina, Vaile Annunziata, 98168, Messina, Italy
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The collection of analytes from natural sources is the goal of each preparative system. Conventional GC analysis for preparative purpose presents different limitations when highly pure compounds have to be collected at milligrams level in a reasonable time. Although wide-bore columns (0.53 mm I.D.) are commonly used, providing an enhanced sample capacity, an excess of on-column sample amounts will result in skewed peaks and decreased resolution. On the other hand, the collection of pure components requires the injection of lower amounts in order to avoid co-elutions on the wide-bore column; as a consequence, the collection of highly pure components, at the milligram level, requires an increased total collection time. The higher is the injection volume, the lower is the total time required to collect a specific compound, thus the highest injection volume should be always used. Aiming to improve the productivity of the system a multidimensional prep-GC instrument is presented with the goal to reduce the total collection time and to improve the purity of the components collected.
To improve the capability of the system, an on-line 4D chromatographic system (prep LC-GC-GC-GC) instrument can be adopted enabling the injection of higher sample volumes from the separated HPLC components by exchanging the dirty sample matrix with a pure HPLC solvent containing the molecules of interest and acting as an additional automated sample preparation step.

Živoslav Tešić1, Milica Nešović2, Stevan Blagojević2
1Faculty of Chemistry, University of Belgrade, Studentski trg 12 – 16, 11000 Belgrade, Serbia
2Institute of General and Physical Chemistry, Belgrade, Studentski trg 12 – 16, 11000 Belgrade, Serbia
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In recent years, the botanical and geographical origin of food has become an important topic in the context of food quality and safety, as well as consumer protection, in accordance with international standards. In the case of honeys, Melissopalynology analysis is used as the method of choice for assessing botanical origin. When collecting nectar, bees add a certain amount of pollen to it, which is used to assess the botanical origin. By counting pollen particles of appropriate plant species, botanical origin is attributed to such honey. The problem arises when honey contains a large number of pollen particles from nectarless plants. In such cases, it is necessary to apply some additional methods to assess the botanical origin. Polyphenols, secondary metabolites of plants, can provide important information about the origin. In this study, we analyzed buckwheat honey with different pollen content, which varied from 5 to 40% of pollen, nectar as well as pollen using the UHPLC-DAD system connected to triple-quadrupole mass spectrometer. The 31 polyphenols were quantified. Dominant content is found for propolis-derived flavonoids such as chrysin and pinocembrin. The significant contribution was also attributed to p-hydroxybenzoic acid and p-coumaric acid as others discovered and suggested as markers for the botanical origin of buckwheat honey. An excellent correlation was found between nectar and honey polyphenols regardless of the content of pollen particles [1]. In the cases of buckwheat honey with low pollen content, comparing the polyphenolic profile of honey and nectar provides important additional data necessary for assessment of the botanical origin.

Reference
[1] M. Nesovic, U. Gasic, T. Tosti, N. Horvacki, N. Nedic, M. Sredojevic, S. Blagojevic, Lj. Ignjatovic and Z. Tesic, RSC Adv. 11 (2021) 25816 – 25829.

Virginia Coman1, Simion Beldean-Galea2
1Babeş-Bolyai University, Raluca Ripan Institute for Research in Chemistry, Fântânele 30, 400294 Cluj-Napoca, Romania
2Babeş-Bolyai University, Faculty of Environmental Science and Engineering, Fântânele 30, 400294 Cluj-Napoca, Romania
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The extraction stage of the compounds from a matrix is very important for the results of the final analysis because it can generate many errors, waste and health problems. Therefore, the principles of green chemistry in analytical chemistry aim to significantly reduce the waste generated by the analyses [1]. Due to the toxicity of solvents, various extraction methods have been developed in recent years to significantly reduce or completely remove the amount of solvents used to extract the compounds from the matrix [2]. The miniaturized extraction (microextraction) methods offer certain advantages: low amounts (microliters) of solvents, small amount of sample to be analysed, reduced extraction time, high enrichment factor (order of hundreds), and high selectivity and precision. Due to these benefits, these methods have developed rapidly and are applied to various classes of organic and inorganic compounds in different liquid or solid matrices [3]. This review is a state-of-the-art of the applications of the liquid-phase microextraction (LPME) in the analysis of pharmaceuticals (anti-inflammatories, antidepressants, antibiotics, hormones, anti-cancer drugs, etc.) in biological fluids (serum, urine, saliva, etc.) and environmental (waters, sediments) samples. Our work is focused on the theoretical aspects of LPME and the applications of dispersive liquid-liquid microextraction, dispersive liquid-liquid microextraction based on solidification of floating organic droplet and hollow fiber microextraction. Aspects related to the analysis techniques (gas chromatography, liquid chromatography, capillary electrophoresis) coupled with different detectors following the microextraction stage are also presented. A critical comparison is made between microextraction techniques and extraction techniques used for routine analysis to provide a viable and greener alternative.

References
[1] A. Spietelun, Ł. Marcinkowski, M. de la Guardia, J. Namieśnik, Talanta 119 (2014) 34-45.
[2] F.R. Mansour, N.D. Danielson, Anal. Chim. Acta 1016 (2018) 1-11.
[3] N. Campillo, K. Gavazov, P. Viñas, I. Hagarova, V. Andruch, Appl. Spectrosc. Rev. 55 (2020) 307-326.

Danilo Corradini
CNR, Institute for Biological Systems, Area della Ricerca di Roma 1, Via Salaria Km 29,300 - 00015 Monterotondo Stazione (Rome), Italy
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Plant secondary metabolites are not only an important part of plant defense system against pathogenic attacks and environmental stresses, but also a useful array of natural products with remarkable biological activities. Most of them are the therapeutic agents occurring in medicinal plants and play important roles in disease prevention and health-promoting effects of edible plants and plant-derived food products and dietary supplements. Moreover, a variety of plant secondary metabolites confer specific sensory characteristics to plant-based foods, processed or not, whose botanical and geographical origin may be related to their occurrence and amount. Thus, these compounds are also potential chemical markers for the evaluation of botanical and geographical origin of plant-derived food products and dietary supplements. This communication describes the development of computer-assisted RP-HPLC methods for the separation, identification and quantification of phenolic compounds, which are a large class of plant secondary metabolites with health-promoting properties comprising a great number of heterogeneous structures. The study has been conducted by a Design of Experiments (DoE) approach that allows the simultaneous optimization of gradient time (tG), column temperature (T) and binary or ternary mobile phase composition on the basis of the retention times and peak areas of selected phenolic compounds, obtained by a restricted number of experiments. The resulting RP-HPLC methods have been used to investigate the occurrence and content of phenolic compounds in leaves and fruits of olive trees (Olea europaea), in monovarietal extra virgin olive oil of different cultivar and geographical origin, and in olive mill waste water produced by processing the same selected batches of olive fruits by a laboratory olive oil press machine. The different phenolic compound profiles and content determined in samples differing for botanical and geographical origin and/or for the technological transformation process are illustrated and discussed.

Acknowledgements:
This work was supported by MSCA-RISE Action of the H2020 Programme, Project 734899 - Olive-Net.