Invited speakers

In the past ten years, LC on polar stationary phases with mobile phases containing organic solvent and less than 5–40% water (HILIC) has become preferred method for separation of polar compounds, namely pharmaceuticals or  biopolymers. On polar columns, sample interactions, retention and separation selectivity may strongly change across the full composition range of aqueous-organic mobile phases, HILIC retention mechanism predominating in the organic-rich, and reversed-phase (RP) mechanism in more aqueous media. We determined the accumulation of water from acetonitrile-rich aqueous-organic mobile phases in the water-rich diffuse layer filling the pores of eighteen polar columns, using frontal chromatography combined with Karl Fischer titration. The sorption can be described by Langmuir isotherm and it strongly depends on the type of stationary phase, ranging from less than one monomolecular water layer up to 6 - 10 water layers equivalents. Some water present in polar solvents is accumulated even on non-polar columns. Considering the actual concentration of water in calculations largely improves the accuracy of prediction of gradient elution data in HILIC systems. The dual retention mechanism enables using a single polar column alternatively in the HILIC and RP retention modes of various polar sample compounds such as phenolic acids and flavones. This approach enhances separation selectivity and peak capacity in single- and two-dimensional HPLC.

Presenting author:
Pavel Jandera
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, CZ-53210 Pardubice, Czech Republic
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Authors:
Pavel Jandera - University of Pardubice, Czech Republic
Jan Soukup - University of Pardubice, Czech Republic
Petr Janás - University of Pardubice, Czech Republic
Magda Staňková - University of Pardubice, Czech Republic

Optimization of the separation performance of fluid-based separation techniques, and especially of liquid chromatography (LC), is essential for analysis of the complex samples encountered in today’s research (omics, food, environmental, natural product, etc. samples). Around a decade ago, tremendous improvements were made in LC column technology (sub-2 µm porous particles, core-shell particles) and in instrumentation (pressures up to 1200 bar) opening new possibilities in terms of speed and resolution to LC practitioners for tackling complex samples. A now well-accepted metric of separation power in gradient elution, mandatory for samples of high complexity, is the peak capacity (nc). nc is the maximum number of peaks that can fit side-by-side between the first and last peak of interest with resolution 1. With state-of-the-art LC, peak capacities of ca. 600 (in ca. 1 h) in conventional one-dimensional LC (1D-LC) can routinely be obtained. It is, however, wishful thinking that such peak capacities are sufficient to separate very complex mixtures. Peak capacity should significantly outstrip the number of components in a sample; the statistical theory of peak overlap (developed by Giddings) indicates to resolve 98% of randomly distributed sample components, nc should exceed the number of components by a factor 100 ! This means that a nc value of 10.000 corresponding to ca. 1x108 theoretical is needed to “chromatographically” resolve a sample containing 100 components.  One straightforward approach to increasing nc is multidimensional LC, especially two-dimensional LC or 2D-LC. In 2D-LC the sample is subjected to two different separation mechanisms and this in an off-line or on-line way. On-line 2D-LC is divided in heart-cutting LC (LC-LC), multiple heart-cutting or parking LC (mLC-LC) and comprehensive LC (LC×LC). LC-LC and mLC-LC are applied to better resolve components in a selected retention time window or windows, while in LC×LC, the entire sample is subjected to two separation mechanisms. The recent developments in 2D-LC hardware design, software approaches and column configurations will be presented. The figures of merit of LCxLC will be discussed and illustrated with analysis of several complex matrices including some of QA/QC.

Presenting author:
Pat Sandra
Research Institute for Chromtaography, Kortrijk, Belgium
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Authors:
Pat Sandra - Research Institute for Chromatography, Kortrijk, Belgium

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

Typically, we controlled chemistry and through it selectivity of our monolithic columns by using: (i) direct copolymerization of functional monomers, (ii) preparation of parent monolith with reactive functionalities and its post-polymerization functionalization, (iii) thermally or photoinitiated grafting of functional monomers on the pore surface, and (iv) attachment of gold and silver nanoparticles to the pore surface followed by reaction with functional thiols.  Recently, a new approach was introduced that enables functionalization of pore surface of monoliths and involves application of metal-organic frameworks (MOFs). These frameworks are compounds consisting of metal ions or clusters coordinated to rigid organic molecules to form one-, two-, or three-dimensional structures that can be porous. We used two implementations: (i) admixing preformed MOF to the polymerization mixture followed by the thermally initiated free radical polymerization and (ii) forming the MOF within the pores applying layer-by-layer approach. The former technique will be demonstrated with the preparation of monolithic column designed for enantioseparation while the latter led to column for selective  preconcentration of phosphopetides and to column for the separation of native fatty acids according to their length and degree of unsaturation.

Presenting author:
František Švec
The Molecular Foundry, E.O. Lawrence Berkeley National Laborator
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Authors:
Xin Wang - E.O. Lawrence Berkeley National Laboratory
Adeela Saeed - E.O. Lawrence Berkeley National Laboratory
Fernando Maya - E.O. Lawrence Berkeley National Laboratory
Alexandros Lamprou - E.O. Lawrence Berkeley National Laboratory
Hongxia Wang - E.O. Lawrence Berkeley National Laboratory

Although microsystems and miniaturized instrumental techniques, which can be considered key technologies for future progress in biochemistry, biotechnology, and medicine, have inspired many scientists toward further methodological developments, there continues to be a great need for analytical systems that enable dynamic and real-time monitoring of biological processes. Among many instrumental techniques and tools employed in exploring the role of biomolecular interactions in physiological and pathological phenomena, biosensors, capillary electromigration techniques, nano liquid chromatography and microscale thermophoresis enable small sample size, low reagent consumption, and  label-free analysis, all of great advantage relative to more traditional approaches such as ELISA and affinity chromatography. The strength of the interactions is determined as affinity constants, partition coefficients, retention factors, and  reduced mobilities. In this talk versatility of capillary electromigration techniques, including adsorption energy calculations, that allow the differentiation of even small differences in the binding processes, will be introduced. Furthermore the  exploitation of molecularly imprinted polymers in the isolation of human biomolecules will be demonstrated. Other techniques and approaches, such as microscale thermophoresis, quartz crystalline microbalance and molecular dynamics simulation calculations provide supportive and complementary insight into the interaction mechanisms.

[1] K. Lipponen, P. W. Stege, G. Cilpa, J. Samuelsson, T. Fornstedt, M.-L. Riekkola, Anal. Chem. 83 (2011) 6040.
[2] K. Lipponen, Y. Liu, P. Wanda Stege, K. Öörni, P. T. Kovanen, M.-L. Riekkola, Anal. Biochem.424 (2012) 71.
[3] K. Lipponen, S. Tähkä, M. Kostiainen, M.-L. Riekkola, Electrophoresis 35 (2013) 1106. [4] K. Lipponen, S. Tähkä, J. Samuelsson, M. Jauhiainen, J. Metso, G. Cilpa-Karhu, T. Fornstedt, M. Kostiainen, M.-L. Riekkola, Anal. Bioanal. Chem. 406 (2014) 4137.
[5] J. Witos, J. Samuelsson, G. Cilpa-Karhu, J. Metso, M. Jauhiainen, M.-L. Riekkola,” Analyst, 2015, DOI: 10.1039/C5AN00210A.

Presenting author:
Marja-Liisa Riekkola
Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
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Authors:
Marja-Liisa Riekkola - Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
Joanna Witos - Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
Katriina Lipponen - Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
Ning Gan - Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
Heli Sirén - Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
Jörgen Samuelsson - Department of Engineering and Chemical Sciences, Karlstad University, 651 88 Karlstad, Sweden
Torgny Fornstedt - Department of Engineering and Chemical Sciences, Karlstad University, 651 88 Karlstad, Sweden
Katariina Öörni - Wihuri Research Institute, Haartmaninkatu 8, FIN-00290 Helsinki, Finland
Matti Jauhiainen - National Institute for Health and Welfare, Public Health Genomics Unit, Biomedicum, Haartmaninkatu 8, FI-00251, Helsinki, Finland

Increasingly nanoscale particles, pores and surfaces, are being tailor-made to provide high performance separation and detection. New superficially porous (core shell) silica particles for liquid chromatography are a good example in separation science, where nanoscale control over shell thickness enhances column efficiency. Nanoparticle incorporation in the separation and/or sensing roles, can also be an attractive means of enhancing selectivity and detectability. In this work, sensitive and selective detection of disease indicating biomarkers is demonstrated using capillary electrophoresis with embedded gold and silica nanoparticles and amperometric detection at a boron doped diamond (BDD) electrode. The BDD electrode has emerged to play an important role in improving sensitivity in electrochemical detection. Luong et al.1 reviewed the application of the BDD electrode for sensitive detection in bioanalysis. Chen et al.2 reviewed the application of the BDD electrode for electrochemical detection in capillary electrophoresis.By coating the fused silica capillary with gold nanoparticles (AuNPs) embedded in a cationic polymer poly (diallyldimethylammonium) chloride (PDDA), efficient separation and sensitive detection was achieved for analysis of 3-indoxyl sulphate (IXS), vanillylmandelic acid (VMA), homovanillic acid (HVA) and tryptophan (TRP) in urine3. The application of this separation and sensing approach is further applied in the field of bacterial cell-to-cell communication in the analysis of cell signalling molecules in the Pseudomonas Quinolone Signal (PQS) quorum sensing system4. P. aeruginosa is best known as an antibiotic resistant human pathogen associated with hospital-acquired infections and is the primary cause of morbidity and mortality in cystic fibrosis sufferers.

References:
1. J.H.T. Luong, K.B. Male, J.D. Glennon, Analyst, 2009, 134, 1965.
2. G. Chen, Talanta, 2007, 74, 326.
3. L. Zhou, J.D. Glennon, J.H.T. Luong, Anal Chem, 2010, 82, 6895.
4. L. Zhou, J.D. Glennon, F.J. Reen, F. O’Gara, C.M. McSweeney, G.P. McGlacken, Chem Comm, 2011, 47, 10347.

Presenting author:
Jeremy D. Glennon
Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical &Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, Cork, Ireland
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Authors:
Jeremy D. Glennon - Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical &Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, Cork, Ireland
Alyah Buzid - Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical &Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, Cork, Ireland
Phyllis E. Hayes - Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical &Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, Cork, Ireland
Gerard P. McGlacken - Department of Chemistry and the Analytical & Biological Chemistry Research Facility (ABCRF), University College Cork, Ireland
John H.T. Luong - Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P2R2

Advanced synthetic polymers represent important group of modern, high-performance materials. Here belong complex polymers (COP) and complex polymer systems (CPS). COP exhibit more than one dispersity (distribution) in their molecular characteristics - molar mass, chemical structure and physical architecture. Their typical representatives are various kinds of copolymers and functional macromolecules. CPS comprise the two and multicomponent blends of macromolecules and block copolymers containing their parent homopolymers. Molecular characterization of both COP and CPS is an exacting analytical challenge because the analytes must be separated. At present, size exclusion chromatography/gel permeation chromatography (SEC/GPC) dominates the area. Though SEC/GPC is a marvelous method, it cannot give quantitative data on molecular characteristics of COP and of most CPS because the size of macromolecules in solution depends on all molecular characteristics of macromolecules to be separated. Moreover, the separation selectivity of SEC/GPC is fairly limited and both low detector sensitivity and sample capacity of SEC/GPC does not enable to characterize minor (solve the problem, coupled methods of polymer HPLC were introduced, which combine exclusion – entropic, and interaction – enthalpic retention separation mechanisms. Two of the coupled methods of polymer HPLC, namely liquid chromatography under critical conditions (LC CC) and liquid chromatography under limiting conditions (LC LC) of enthalpic interactions will be presented, compared and critically evaluated in the contribution with the conclusion that LC CC is preferable for oligomers and for binary blends of macromolecules with molar mass below about 10 kg/mol whereas LC LC has no upper molar mass limit but its performance may be not sufficient for low oligomers. The sample recovery represents an important limitation of LC CC of macromolecules with high molar mass. In contrast to LC CC, LC LC is robust and user-friendly. LC LC enables to separate even four-component CPS in one single step and can be easily engaged in the two-dimensional polymer LC.

Presenting author:
Dušan Berek
Polymer Institute, Slovak Academy of Sciences, Dúbravská cesta 9, 84541 Bratislava, Slovakia
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Authors:
Dušan Berek - Polymer Institute, Slovak Academy of Sciences, SIovakia

In this lecture the advantages of comprehensive two-dimensional liquid chromatography (LC×LC) will be discussed. LC×LC may provide high peak capacities in a much shorter time than conventional 1D-LC. Moreover, LC×LC provides unique selectivity. It is an attractive option for separating the very complex mixtures that are encountered in many fields (life science, food science, material science, etc.). LC×LC is instrumentally straightforward, but the development and optimization of methods may be an obstacle. If method development in LC is a time-consuming task, then method development in LC×LC threatens to be a (time)2 consuming task. Some guidelines will be discussed to help understand LC×LC and to develop methods more efficiently.If we are able to “modulate” collected fractions from the first-dimension separation we may minimize the effects of the first-dimension eluent on the second-dimension separation and we may essentially optimize the two separations independently. This is demonstrated for the separation of peptides by a combination of ion-exchange chromatography and reversed-phase LC. After the first-dimension separation the analytes are focussed on a trapping column. This allows the second-dimension column to me much narrower than the first-dimension column and it provides an increase in sensitivity by several orders of magnitude. In addition, salt is removed from the eluent, which promotes the MS compatibility of the system. Finally, directions for further development of multi-dimensional LC methods will be discussed.

Presenting author:
Peter Schoenmakers
University of Amsterdam, Faculty of Science, Science Park 904, 1098 XH Amsterdam, The Netherlands
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Authors:
Peter Schoenmakers - University of Amsterdam

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

While traditional drugs are usualy small molecules, biopharmaceuticals, like proteins, plasmid DNA, viruses or virus-like particles, are large complex bioparticles, with heterogeneus physicochemical properties, sensitive to the environmental conditions (pH, I) and shear forces. Therefore, during manufacturing process of biological nanoparticles there are several issues which have to be considered. To satisfy the severe requirements placed on the purity of biopharmaceutical products demanding several-step purification processes have to be developed. An integral part of downstream processing is the chromatographic unit operation. To gain as high productivity as possible chromatographic resin should exhibit high dynamic binding capacity and high purification yield meaning high selectivity and low degradation of target macromolecule should be achieved preferably at high linear velocity and low pressure drop. Convection-based transport is an extremely important feature that accelerates the separation and purification process, especially of large biomolecules with low mobility. In recent years chromatographic monoliths, also called Convective Interaction Media (CIM), appeared as a resin of choice for biopharmaceutics’ purification. Their specific porous structure enables the convective mass transport and high surface accessibility to large molecules. Therefore they allow very fast separations and exhibit very high binding capacities for extremely large bioparticles. In this work key features of large biological nanoparticles that influence the downstream process of target compound will be discussed. The differences between classical matrixes for liquid chromatography and for biochromatography will be exposed and case studies of several biopharmaceuticals of different classes using special monolithic chromatography columns will be presented.

Presenting author:
Nika Lendero Krajnc
BIA Separations d.o.o., Mirce 21, Ajdovscina, SI-5270, Slovenia
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Authors:
Nika Lendero Krajnc - BIA Separations d.o.o., Mirce 21, Ajdovscina, SI-5270, Slovenia

The lecture sums up the modern tools of the radiochromatography in preclinical and clinical pharmacokinetic and drug metabolism research. The essential pharmacokinetic and drug metabolism information of different species (mouse, rat, dog, rabbit and human) contribute to the final drug registration process.
The high sensitivity (pg/ml, fg/ml, atg/ml) and highly 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.
Nowadays in the course of drug development the radioactive isotopes (beta and gamma single and/or double source) labeled (³H, ¹⁴C, ⁹⁹Tc, ¹³¹I) pharmacokinetic studies combined with the new generation of triple-quad MS techniques (LC, CE, OPLC) are essential. A number of related case studies will be presented.
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 the above 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.

Presenting author:
Imre Klebovich
Department of Pharmaceutics, Semmelweis University, Hőgyes Endre St. 7., H-1092, Budapest, Hungary
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Authors:
Imre Klebovich - Department of Pharmaceutics, Semmelweis University

For several reasons, there is interest to replace helium for a different carrier gas in gas chromatography. Hydrogen is the obvious choice, but there is a concern on safety and reactivity. It is also possible to use nitrogen, but this is often not considered because it has a low optimal flow and velocity.
By using method translation and chromatogram modeling it became clear that the loss of efficiency using nitrogen could be perfectly compensated by using a smaller ID capillary of a shorter length. By replacing a 30m x 0.25 for a 20m x 0.15mm, it is demonstrated that separations under nitrogen are exactly the same as obtained with Helium, in the same analysis time, while using exact the same conditions for oven programming. Even the inlet pressures are very close.
The only price that has to be paid, is a loss in loadability, which means that this concept will not work for every application, but for many, it will. Besides the guaranteed availability, using nitrogen offers a big advantage in the cost and consumption volume of carrier gas, meaning cost per analysis will also benefit significantly.

Presenting author:
Jaap de Zeeuw
Restek Corporation, weerhaan 9, 4336 KT, Middelburg, The Netherlands
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Authors:
Jaap de Zeeuw - Restek Corporation, weerhaan 9, 4336 KT, Middelburg, The Netherlands
Jack Cochran - Restek Corporation, weerhaan 9, 4336 KT, Middelburg, The Netherlands

This presentation provides an overview of the use of the solvation parameter model for characterizing the separation properties of chromatographic and liquid-liquid distribution systems from a knowledge of the contribution of intermolecular interactions to two-phase distributions. The same process used for chromatographic systems can be applied to study the fate, transport and distribution of compounds in environmental systems providing a bridge between the two processes. Although we have developed tools for screening chromatographic databases to find models that emulate environmental processes this has proven to be equivalent to searching for a needle in a haystack. An alternative, and more successful approach, has been to build models of the environmental process directly, allowing the prediction of properties for other compounds with known descriptor values. The central role chromatographic an liquid-liquid partition methods occupy in the experimental determination of the six descriptors used in the solvation parameter model will described and illustrated for recent studies of polycyclic aromatic compounds, plasticizers, and organosiloxanes of environmental interest [1, 2]. The general approach is shown to be suitable for devising conditions for analysis (sample preparation and chromatographic separations), and the prediction of environmental properties without additional experimental effort.

(1) C. F. Poole, T. C. Ariyasena and N. Lenca. Estimation of the Environmental Properties of Compounds from Chromatographic Measurements and the Solvation Parameter Model. J. Chromatogr. A 1317 (2013) 85-104.
(2) T.C. Ariyasena and C. F. Poole. Determination of Descriptors for Polycyclic Aromatic Hydrocarbons and Related Compounds by Chromatographic Methods and Liquid-Liquid Partition in Totally Organic Biphasic Systems. J. Chromatogr. A 1361 (2014) 240-254.

Presenting author:
Colin Poole
Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA
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Authors:
Colin Poole - Wayne State University

Rapid detection and identification of microorganisms is a challenging and important aspect in many areas of our life, beginning with medicine, ending with industry. Unfortunately, classical methods of microorganisms’ identification  are based on time-consuming and labor-intensive approaches. Screening techniques require rapid and cheap grouping of bacterial isolates, however modern bioanalytics demands comprehensive bacterial studies on molecular level. The new  approach to the rapid identification of bacteria is to use the electromigration techniques, especially capillary zone electrophoresis (CZE). CZE is an important technique used in the analysis of microorganisms. However, the analysis of microbial  complexes using this technology still encounters several problems – uncontrolled aggregation and/or adhesion to the capillary surface. One way to resolve this issue is the CZE analysis of microbial cell with surface charge modification by bivalent metal ions (e.g. Ca2+aq, Zn2+aq). Under the above conditions, bacterial cells create compact aggregates, and fewer high-intensity signals are observed in electropherograms. The capillary electrophoresis of microbial aggregates approach with  UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) detection has been presented. The aim of this study was to characterize the charge and surface of the bacteria in order to determine their role in adhesion and aggregation phenomena during the electrophoretic separation. The use of experimental techniques, including electrochemical and electrophoretic allowed the description of the relationship between the acid-base properties of pathogens and their behavior. In this studies was performed the identification of bacterial via spectrometric techniques.

Acknowledgments:
This work was supported by the National Science Centre (NCN, Warsaw, Poland) projects No. 2013/08/W/NZ8/701 – Symfonia-1, Maestro-6 (2015-2017) and by European Social Found, Polish National Budget, Kuyavian-Pomeranian Voivodeship Budget (within Sectoral Operational Programme Human Resources) “Krok w przyszłość 2014-2015”

Presenting author:
Boguslaw Buszewski
Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry & Centre for Modern Interdyscyplinary Technology Nicolaus Copernicus University, Gagarin 7, 87-100 Toruń, Pola
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Authors:
Bogusław Buszewski - Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry & Centre for Modern Interdyscyplinary Technology Nicolaus Copernicus University, Gagarin 7, 87-100 Toruń, Pola
Viorica Railean-Plugaru - Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry & Centre for Modern Interdyscyplinary Technology Nicolaus Copernicus University, Gagarin 7, 87-100 Toruń, Pola
Paweł Pomastowski - Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry & Centre for Modern Interdyscyplinary Technology Nicolaus Copernicus University, Gagarin 7, 87-100 Toruń, Pola

A priori knowledge of the retention time of a given analyte simplifies the determination of separation conditions therefore quantitative structure retention relationship (QSRR) modelling might be considered a reasonable selection.  The first problem in QSRR modelling is to select the most informative descriptors from among a large number of mutually correlated descriptors, while the second one is to build the core model of isocratic and/or gradient elution retention. A lot of conventional methods have been elaborated that are mainly based on different types of regression and simple variable selection methodology (i.e. principal component analysis), showing rather questionable prediction ability. This work  reveals recent results on development of artificial intelligence (AI) hybrid methodology implementing all three AI paradigms: artificial neural networks, genetic algorithms and fuzzy logic. The developed models were fully optimized and validated with  external set of compounds showing significant improvement of generalization ability. Furthermore, recent demands for increasing the productivity using the gradients, in combination with ever growing complexity of analyzed samples, are  introducing an additional request on the analytical system – beside being fairly separated, the peaks are required be as “smoothly” shaped as possible to ensure their precise quantification. In other words, the analysts are becoming interested in  peak shapes and peak shape modelling as well. This work also discusses recent developments is peak shape modelling based on QSRR modelling. The developed models are based on generalized logistic distribution and hybrid AI systems. The  external validation results show promising predictive ability, but still indicate that there is much to be done before QSRR based optimization strategy could be efficiently built into a useful commercial software.

Presenting author:
Tomislav Bolanča
University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000 Zagreb, Croatia
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Authors:
Tomislav Bolanča - University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000 Zagreb, Croatia
Šime Ukić - University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000 Zagreb, Croatia
Mirjana Novak Stankov - University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000 Zagreb, Croatia
Marko Rogošić - University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000 Zagreb, Croatia

State pollution of rivers and other surface water reservoirs in Ukraine is under the regular control of the State. This control is carried out by the Ministry of Environment, Ministry of Health, other governmental departments and agencies. In Ukraine developed an extensive regulatory documentation, which determine the procedures waters monitoring and methods of measurement. Monitoring of water quality include the following mandatory chemical parameters as pH, the presence of ammonia nitrogen, heavy metals, content of acid residues as nitrites, nitrates, chlorides, sulfates, phosphates, oxygen content, chemical, and biological oxygen demand, phenols, surfactants. The control of organic components, such as pesticides, pharmaceuticals and others, conducted in areas where there is a threat of such pollution. Chromatographic methods primarily used in this case. Assessment of surface water pollution is carried by a combined index - an index of pollution IWP, which is calculated by the formula : IWP = ∑(C / MAC)/n
C – actual concentration
MAC - Maximum allowable concentration
n – number of indications
By this indicator are set 5 levels of contamination: allowable, reasonable; increased; high; very high. Most of the surface waters in Ukraine have a reasonable level of pollution. A high and very high levels of surface waters pollution is determined in the south and east of Ukraine. This is due to the presence in these areas, the mining industry and intensive agriculture. In the western regions of Ukraine, in particular in the Transcarpathian region the level of surface waters contamination is allowable. These areas belong to the recreational areas and the number of enterprises which pollute the environment on this territory is insignificant.

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

Presenting author:
Mоdеst Gertsiuk
Institute of Environmental Geochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine
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Authors:
Mоdеst Gertsiuk - Dr
Georgiy Lysychenko - Dr

Last year the Nobel Prize for Chemistry was granted to scientists, who were modeling physico-chemical interactions in biochemistry. Although we are modeling since 1985, the recognition of the importance of modeling for the whole Life Science is a great motivation for the Molnar-Institute to continue in finding the best column and the best separation in HPLC, by modeling a large number of possible chromatograms. It is 40 years ago, that Csaba Horváth discovered in 1975 the reason for the success of Reversed Phase Chromatography at Yale – changes in the structure of water as part of the eluent. Complex calculations were required, but computers were just starting to emerge. He used a PDP11 “cabin” to describe, how acids dissociation might be modeled by equations. Today we are modeling separation choices in a multi-variable Design Space to the most robust separation for industrial drug production. The talk will demonstrate on case studies, how modeling software can work together with modern instruments to produce the best separation instead in months in only as fast as in a few hours. With a computer generated separation model at hand, one can calculate the influence of various experimental parameters on the separation and, therefore, also model the robustness of a separation. This is extremely useful if a method should be used for a long time where one has to take into account day-to-day variations in method conditions.

Presenting author:
Imre Molnár
Molnár-Institute for Applied Chromatography, Schneeglöckchenstr. 47, 10407 Berlin, Germany
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Authors:
Imre Molnár - Molnár-Institute for Applied Chromatography
Hans-Jürgen Rieger - Molnár-Institute for Applied Chromatography

The study of the details of mass transfer in liquid chromatography is of central interest. We apply the microscopic (or molecular dynamic) model of chromatography to study the reversed phase separation of small and large molecules. The microscopic theory of chromatography describes the evolution of a chromatographic peak as the random migration of the molecules along the column combined with adsorption–desorption processes that occur at random, too. The molecular dynamic model is rather straightforward to comprehend and it can furnish direct answers when one tries to understand the development of chromatographic peaks. We show that the microscopic model can be rather simply used to estimate the fundamental characteristics of the separation process. We can estimate the rate a molecule is adsorbed on the surface of the stationary phase while it migrates along the column. When combining the general rate model with the molecular dynamic model, one can consider and compare the kinetics of the transfer of solute molecules between the flowing and stagnant zones of mobile phase, the pore diffusion, etc. We analyze the peak shapes recorded under linear conditions, and can characterize the heterogeneity of the surface of the stationary phase. With a peak shape analysis that is based on the molecular dynamic model of chromatography, we can identify the presence of heterogeneous mass transfer or adsorption kinetics. We can, furthermore calculate the amount of retention due to the individual adsorption sites. The general rate model of chromatography, which is a macroscopic model, offers the most detailed description of the separation process. We compare the results provided by both microscopic and macroscopic analysis of the peak shapes and statistical moments. In this paper we discuss the influence of wide pore-size distributions. We present results obtained on nonporous, fully porous, and fused-core particles. Furthermore, mass transfer in supercritical fluid chromatography and reversed-phase liquid chromatography are compared.

Presenting author:
Attila Felinger
University of Pécs
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Authors:
Attila Felinger - University of Pécs

Plant secondary metabolites are organic compounds produced by plants, which are not involved in the growth, photosynthesis, reproduction and other primary functions of the plants but are believed to have a role in defence mechanisms, such as protection against herbivores, pests and pathogens. Phenolic compounds are one of the main classes of secondary metabolites and because are widely distributed in the plant kingdom form an integral part of human diet. In addition to confer specific sensorial characteristics to plant-derived foods and/or beneficial effects on human health, phenolic compounds are also biomolecules with pharmacological activity employed in phytotherapeutic medicine. The identification and quantification of these target compounds in plants tissues and agro-food matrices is a challenging task, continuously requesting the development of more robust, efficient and sensitive instrumental analytical techniques. This communication discusses fundamental and practical aspects of both reversed phase high performance liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE) tailored for the analysis of plant secondary metabolites. The communication reports the results of our recent studies carried out to evaluate the influence of various operational parameters and experimental conditions employed in CZE and in RP-HPLC on the separation performance of phenolic compounds and other secondary metabolites in plant tissues and agro-food matrices. Because both RP-HPLC and CZE require the use of a liquid phase, whose composition plays a fundamental role on the separation mechanisms and performance, most of our investigations has been focused on the selection of the proper composition of the liquid phase, which, in the case of RP-HPLC, has been optimized by a quality by design approach.

Presenting author:
Danilo Corradini
National Research Council, Institute of Chemical Methodologies, Area della Ricerca di Roma 1, 00015 Monterotondo, Rome, Italy
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Authors:
Danilo Corradini - National Research Council, Institute of Chemical Methodologies, Area della Ricerca di Roma 1, 00015 Monterotondo, Rome, Italy
Isabella Nicoletti - National Research Council, Institute of Chemical Methodologies, Area della Ricerca di Roma 1, 00015 Monterotondo, Rome, Italy
Imre Molnár - Molnár-Institute Schneegloeckchenstrasse 47, 10407 Berlin, Germany

Our aspiration was to design portable LC as a widely available ‘open platform’, moderate-pressure (over 100 bar), and importantly highly flexible modular system, based almost entirely on commercial off-the-shelf low-cost components. In this work, a modular portable LC has been designed, characterised and used, weighing under 2 kg (plus data acquisition). The backbone of the system was a breadboard assembled modular flexible microfluidic system (LabSmith), complemented with other off-the-shelf components, including an injection valve and on-capillary detectors, all operated through a PC. The system can accommodate six syringe pumps with 80, 20 and 5 µL syringe options, and maximum backpressures of up to ca. 150 bar, with two used for each A and B mobile phase in gradient operation and another pump for sampling and one in reserve for post-column derivatisation. The flow rate (typically 1 µL/min), which exhibited substantial pulsations due to the individual steps of the step motors driving the syringe pump, could be well dampened by using microbubbles of low-solubility inert gas (nitrogen, helium), automatically aspirated in each mobile phase draw cycle. Each of the two pairs of syringe pumps for mobile phases A and B were connected with microfluidic switching valves (two 4-port valves), and the A and B streams joined together with a Y-connector and a microfluidic pressure sensor, thus providing a system for low hold-up volume gradient formation. The mobile phase was led to a nano-LC sampling valve and to a capillary LC column. The detection was on-column (100 µm i.d. PTFE-coated fused silica) with an LED-based deep-UV-vis-NIR photometric detector, and end-column with an electrochemical detector (amperometric and/or potentiometric). The performance of the system was evaluated including the pumping accuracy, linearity of gradient formation, separation performance and sensitivity of detection. Typically analysis runs used 12-20 µL of solvent with overall reproducibility of 1 5 % and 3 6% RSD for retention times and peak area in gradient mode, respectively. Future avenues for miniaturisation of the data acquisition and system control are discussed.

Presenting author:
Mirek Macka
University of Tasmania, Australian Centre for Research on Separation Science, Private Bag 75, Hobart, TAS 7001, Australia
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Authors:
Yan Li - University of Tasmania
Milos Dvorak - University of Tasmania
Nantana Nuchtavorn - University of Tasmania
Pavel N. Nesterenko - University of Tasmania
Roger Stanley - University of Tasmania
Mirek Macka - University of Tasmania

Analysing metabolomes from various natural sources is a task that requires methods providing high chromatographic resolution for detailed metabolite profiling or high throughput for rapid fingerprinting for metabolomics [1,2]. Furthermore these methods should give on-line spectroscopic information for the identification of each individual metabolite for dereplication purposes. In this respect the introduction of UHPLC has allowed a remarkable decrease in analysis time and increase in peak capacity, sensitivity and reproducibility compared to conventional HPLC. In complement to this powerful chromatographic method, the introduction of benchtop high resolution MS instruments that provide sensitive detection and accurate MS and MS/MS information for dereplication has been key for metabolomics. Recently the development of neural network mining methods for metabolite identification have revolutionized conventional MS/MS database search for establishing link between metabolites having similar fragmentation pattern that greatly help metabolite annotation. For unambiguous de-novo identification, LC-MS targeted isolation together with at-line microNMR approaches provide low microgram level of metabolites sufficient for acquiring a full set of 1D and 2D NMR spectra. Such information complements well the search in MS and chemotaxonomic data bases for dereplication. In addition to these methods, HPLC activity-based profiling provides information on the bioactivity of metabolites of interest, directly at the analytical scale, speeding up the drug discovery process in natural product research. The impact of these technologies in NP research studies and future trends will be discussed. This will be illustrated by examples or our latest metabolomics and bioactivity-guided isolation studies on plants and microorganisms.
1.    Wolfender JL, Rudaz S, Choi YH, Kim HK. Plant metabolomics: from holistic data to relevant biomarkers. Curr. Med. Chem. 2013 20: 1056-90.
2.     Wolfender J-L, Marti G, Thomas A, Bertrand S. Current approaches and challenges for the metabolite profiling of complex natural extracts. J Chromatogr A 2015 1382: 136-164.

Presenting author:
Jean-Luc Wolfender
School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, quai Ernest-Ansermet, CH-1211 Geneva, Switzerland
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Authors:
Jean-Luc Wolfender - School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, quai Ernest-Ansermet, CH-1211 Geneva, Switzerland