Day 3 :
Keynote Forum
Paweł K Zarzycki
Koszalin University of Technology, Poland
Keynote: Micro-planar chromatography as useful tool for low resolution metabolomics study and environmental samples assessment
Time : 10:30-11:00
Biography:
PaweÅ‚ K Zarzycki has his background in Pharmaceutical and Biomedical Analysis involving Chromatography, Supramolecular Chemistry and Chemometrics. His research interest includes “Development and optimization of effective extraction of biomarkers, temperature effects in liquid chromatography, thermochromic host-guest supramolecular complexes and biomedical analysis of low-molecular mass compounds from complex samples driven by simple separation protocols and multivariate data processing”.
Abstract:
The aim of this research communication is to demonstrate that relatively low-resolution separation techniques based on planar chromatography and combined with properly optimized samples pre-purification (involving e.g., liquid-liquid or solid phase extractions) and sensitive detection (e.g., fluorescence or colour staining) can be very helpful to investigate various complex problems. Described methodology may provide complementary information that can be used for fast samples screening, before quantification involving more accurate and sophisticated techniques. Using the own experimental works, several issues associated with low molecular mass metabolites, biomarkers and micro pollutants quantification from different complex samples, will be reported and discussed, particularly: Group-type separation of biological samples including spirulina containing food products and pharmaceutical formulations; screening of environmental samples: surface waters, treated and untreated sewage waters and activated sludge materials as well as soot dust originated from cold surfaces of biomass fuel, fossils-fired home heating ovens, truck exhaust systems and asphalt concrete samples; selective solid phase extraction (demonstrated for battery of key steroids with polarity ranging from estetrol to progesterone, for which the retention data are collected via breakthrough curves from C18 SPE tubes and compared with the retention of target analytes on micro-TLC wettable with water RP18 HPTLC plates) and; optimization of detection of target analytes separated by 1D and 2D micro-TLC based on sensitive methods like photo-thermal detection or DPPH derivatization for quantification of fullerenes and measurement of the total antioxidant potential of food samples, respectively.
Keynote Forum
Sergey N Krylov
York University, Canada
Keynote: Kinetic chromatography: A new approach to study kinetics of reversible protein-drug binding
Time : 10:00-10:30
Biography:
Sergey Krylov completed his PhD from Moscow State University. He has been Professor of Chemistry at York University in Toronto since 2000. He had held Canada Research Chair Tier II for a maximum term and is now York Research Chair in Bioanalytical Chemistry. He is a Founder and Director of the Centre for Research on Biomolecular Interactions. He is recognized internationally for his pioneering work in the fields of “Chemical cytometry, kinetic analysis of affinity interactions, and selection and applications of oligonucleotide aptamers”. He has authored over 160 peer-reviewed papers and his research contributions have been recognized with a number of distinctions including two awards from the Chemical Society of Canada.
Abstract:
Studying the kinetics of reversible binding between a protein and a small-molecule drug is a major challenge. The available approaches require that either the small molecule or the protein modified by labeling or immobilization on a surface. Not only can such modifications be difficult to do but they can also drastically affect the kinetic parameters of the interaction. To solve this problem, we developed kinetic chromatography with mass-spectrometry detection, a solution-based label-free approach. Our novel approach utilizes the ability of some modes of chromatography to separate any small molecule from any protein-small-molecule complex without immobilization and the ability of mass-spectrometry to detect a small molecule without a label. The rate constants of complex formation and dissociation are convoluted in the temporal pattern of small molecule elution measured with mass spectrometry at the exit from the chromatography column. We developed advanced algorithms and software to deconvolute the unknown constants from the temporal pattern by solving an inverse mathematical problem either analytically or numerically. In this lecture, the concept of kinetic chromatography with mass-spectrometry detection will be explained and a examples of its use for studying kinetics for protein-drug interactions will be presented. Prospectives of the utilization of kinetic chromatography with mass-spectrometry detection in the pharmaceutical industry and academic research will be discussed.
Keynote Forum
Sergey N Krylov
York University, Canada
Keynote: Kinetic chromatography: A new approach to study kinetics of reversible protein-drug binding
Time : 10:00-10:30
Biography:
Sergey Krylov completed his PhD from Moscow State University. He has been Professor of Chemistry at York University in Toronto since 2000. He had held Canada Research Chair Tier II for a maximum term and is now York Research Chair in Bioanalytical Chemistry. He is a Founder and Director of the Centre for Research on Biomolecular Interactions. He is recognized internationally for his pioneering work in the fields of “Chemical cytometry, kinetic analysis of affinity interactions, and selection and applications of oligonucleotide aptamers”. He has authored over 160 peer-reviewed papers and his research contributions have been recognized with a number of distinctions including two awards from the Chemical Society of Canada.
Abstract:
Studying the kinetics of reversible binding between a protein and a small-molecule drug is a major challenge. The available approaches require that either the small molecule or the protein modified by labeling or immobilization on a surface. Not only can such modifications be difficult to do but they can also drastically affect the kinetic parameters of the interaction. To solve this problem, we developed kinetic chromatography with mass-spectrometry detection, a solution-based label-free approach. Our novel approach utilizes the ability of some modes of chromatography to separate any small molecule from any protein-small-molecule complex without immobilization and the ability of mass-spectrometry to detect a small molecule without a label. The rate constants of complex formation and dissociation are convoluted in the temporal pattern of small molecule elution measured with mass spectrometry at the exit from the chromatography column. We developed advanced algorithms and software to deconvolute the unknown constants from the temporal pattern by solving an inverse mathematical problem either analytically or numerically. In this lecture, the concept of kinetic chromatography with mass-spectrometry detection will be explained and a examples of its use for studying kinetics for protein-drug interactions will be presented. Prospectives of the utilization of kinetic chromatography with mass-spectrometry detection in the pharmaceutical industry and academic research will be discussed.
- Spectroscopy
Location: Meeting Place 2
Chair
Sergey N Krylov
York University, Canada
Co-Chair
Jennifer E Claus
MilliporeSigma, USA
Session Introduction
Adéla Kotzianová
Contipro Pharma a.s., Czech Republic
Title: Raman spectroscopy based method for the evaluation of compositional consistency of nanofibrous layers
Time : 11:15-11:35
Biography:
Adéla Kotzianová graduated from the Czech Technical University in Prague in 2012, where she earned her Master’s degree in Instruments and Methods for Biomedicine. Currently, she is finishing her Doctoral studies of Physical Chemistry at the Masaryk University in Brno. Since 2012 she has been working as a researcher in the Department of Nanotechnology Device Development of the Contipro Holding. She has been specializing in spectroscopic methods since her studies. In recent years, her work was focused especially on the preparation and analysis of nanofibrous materials. She has published 7 papers in reputed journals and presented her work on international conferences.
Abstract:
Electrospinning is currently a very popular method used across a number of industries. Electrospinning enables the production of nanofibrous layers of various structures and compositions. The production of a multi-component nanofibrous layer may result in an uneven distribution of the individual components throughout the layer. Confocal Raman spectroscopy combined with statistical methods allows these layers to be analyzed by determining their chemical composition and thus provides feedback for the spinning process. This paper presents a method which combines Raman spectroscopy analysis and its subsequent evaluation with singular value decomposition (SVD). Automated measurement of Raman spectra makes it possible to gather extensive spectral data from a particular area selected on a sample; the spectra are measured from a specific volume and not from individual fibers. Samples require no preparation for the analysis and the non-destructive nature of Raman spectroscopy ensures their reusability. When spectra of the individual component materials are included for reference, the subsequent SVD analysis of the spectral data makes it possible to determine the chemical composition of the measured areas, thus providing the content percentages of the individual components, which can be displayed either in the form of a scattered plot or a Raman map.
Nguyen Khanh Hoang
Industrial University of HoChiMinh City, Vietnam
Title: Spectrophotometric determination of Cr(iii) and Pb(ii) using 5,11,17,23-tetra[(2-ethyl acetoethoxyphenyl) (azo) phenyl]calix[4]arene
Time : 11:35-11:55
Biography:
Le Van Tan obtained his PhD in Chemistry department in 1997 from Ha Noi National University, VietNam. He researched Post-doctoral in Seoul National University -2004-2005 (Korea) and Visiting Professor in Technical University Kaiserslautern- 2006, 2010 (Germany). He has published and presented 80 papers in International and National Conferences and journals. Currently he is working as the Professor of this University and Professor of Chemical Engineering, Industrial University of Ho Chi Minh City. More than 15 students completed their Master degree and four Candidates completed their PhD. under his supervision. He is a member of Vietnam Chemical Society, Vietnam Analytical Chemical Society, ASC member and also Technical Committee for many international conferences on chemistry and chemical engineering in Japan, Singapore, Hong Kong, Thailand and so on. He is an Editor Board of Vietnam Journal of Chemistry, Reviewer for the International Journal of Chemical and Biomolecular Science and many International Journals on Chemistry. His interested research areas are finding new organic reagent for analytical chemistry; advanced separations and environment; postharvest technology.
Abstract:
New complexes of 5, 11, 17, 23-tetra[(2-ethyl acetoethoxyphenyl)(azo)phenyl]calix[4]arene (TEAC) with Pb(II) and Cr(III) were prepared in basic solution with a mixture of MeOH and H2O as solvent. The ratio of TEAC and metal ions complexation was found to be 1:1 under investigated condition. The complex formation constants (based on Benesi–Hildebrand method) for TEAC-Pb(II) and TEAC-Cr(III) were 4.03×104 and 1.2×104, respectively. Additionally, the molar extinction coefficients were 5×104 and 1.42×104 for TEAC-Pb(II) and TEAC-Cr(III), respectively. The H-point standard addition method (HPSAM) has been applied for simultaneous determination of complexes formation of Cr(III)/Pb(II) and TEAC with concentration from 2:1 to 1:20 (w/w). The proposed method was successfully utilized to invest lead and chromium content in plating wastewater samples. The results for several analyzed samples were found to be in satisfied agreement with those acquired by using the ICP-MS technique.
Katarzyna Adamska
Poznan University of Technology, Poland
Title: Inverse liquid chromatography in surface characteristic of materials
Time : 11:55-12:15
Biography:
Katarzyna Adamska was graduated at Wroclaw University of Technology at Faculty of Chemistry in 1999. In 2002 she started PhD studies at Poznan University of Technology in Institute of Chemical Technology and Engineering. In 2007 she got the academic degree of Doctor. Her PhD thesis entitled “Determination of the solubility parameter and its components for materials used in the pharmaceutical industry” was related, mainly, to the use of inverse gas chromatography in studies of different excipients, applied in pharmaceutical formulations.
Abstract:
Examination of the physicochemical properties of different materials is important for their adsorptive or adhesive characteristics. Inverse Liquid Chromatography (ILC) technique differs from among generally known chromatographic methods, that it is not related to the separation of components of mixtures, but the interaction of test compounds with the investigated material, constituting the stationary phase of chromatography column. Interactions between the material and the test compounds affect the measured retention parameters and the shape of chromatographic peaks. Application of ILC seems to be useful to direct study of solid-liquid interactions in real conditions, which include: pressure, temperature and pH. This technique, in conjunction with proper mathematical models, allows evaluating the physicochemical characteristic of the biomaterials surface: its ability to various types of intermolecular interactions (e.g. capacity to donor-acceptor interactions), assessment of the impact of the number and type of functional groups on the surface activity. ILC can be also applied to characterize materials used in a separation process - mesoporous aluminosilicates (zeolites) for e.g. their ability to adsorption. The characteristic of the surface layer of such materials allows the analysis of the influence of the respective modifier’s ligand for adsorption or separation processes. ILC technique thus allows the detailed characterization of the surface, taking into account its ability to various types of intermolecular interactions. This knowledge is essential for an appropriate development and improvement of different solid materials.
Tony Q Yan
Pfizer Inc., USA
Title: Enantiomer separations of amino acids and derivatives on immobilized polysaccharide phases with extended range of solvents using supercritical fluid chromatography
Time : 12:15-12:35
Biography:
Tony Q Yan is currently working for Pfizer, Inc. (Groton, CT, USA) in the field of impurity isolation for structure elucidation in the department of pharmaceutical science. He has been working in pharmaceutical research and development in the area of chiral and achiral purifications, and impurity isolation for over 20 years since he graduated from the department of chemistry in University of Missouri in Rolla with PhD degree in 1995.
Abstract:
This presentation discusses immobilized polysaccharide stationary phases with extended range of solvents for enantiomer resolution of select amino acids and derivatives using supercritical fluid chromatography (SFC) and high performance liquid chromatography (HPLC). Baseline resolution is achieved for selected amino acids and derivatives using this approach. The combination of an extended solvent (normally an aprotic solvent) in the presence of methanol and a suitable additive such as water, acid and base are critical for retention and resolution of amino acid pairs on immobilized columns. The amino acid separations are also achieved on the coated polysaccharide phases with alcohol based solvents in the presence of acid and base. In addition, the use of cyclofructan and polysaccharide phases for amino acids and small peptides separations along with the use of reversed phase chiral stationary phase for very polar compounds, acids and diacids, are also discussed.
- Special Techniques in Chromatography
Location: Meeting Place 2
Chair
Sergey N Krylov
York University, Canada
Co-Chair
Jennifer E Claus
MilliporeSigma, USA
Session Introduction
Aschwin van der Horst
Nuplex, Netherlands
Title: Comprehensive two-dimensional critical chromatography a tool to understand the behavior of random and block copolymers in chromatography
Time : 13:35-13:55
Biography:
Aschwin van der Horst is a Principal Chemist Analytics in the Analysis, Materials and Instrumentation Laboratory at Nuplex, Bergen op Zoom, Netherlands. He holds an experience of 16 years as a Polymer Analytical Chemist and 11 years of experience in Coating Technology. He published various papers on the analysis and characterization of polymers by Multidimensional Liquid Chromatography and Pyrolysis-Liquid Chromatography. Besides his work at Nuplex, he is also a Guest Lecturer at the Avans Hogeschool Breda on Polymer-, Resin- and Paint Analysis and its characterization.
Abstract:
Random, gradient and block copolymers are behaving differently in commonly used solvents in liquid chromatography. Copolymers show two or more distributions, depending on their topology. Linear polymers and random copolymers can be fully characterized by combining their chemical-composition distributions (CCD) and molecular-weight distributions (MWD); block copolymers can be characterized by their block-length-distribution (BLD). Liquid chromatography at the critical point of adsorption (LC-CC or simply critical chromatography, CC) is a highly useful technique used in polymer LC. The mechanism of CC can be studied by coupling LC with size-exclusion chromatography (SEC) yielding CCxSEC. This technique is often used for the characterization of functionality-type-distribution (FTD) in combination with MWD. What we would like to demonstrate is another approach; two-dimensional critical chromatography (CCxCC). Using this approach, more insight in the composition of random-, gradient- and di-block copolymers is obtained. Also the possibility of obtaining relevant information from the CCxCC analysis of the latter copolymers is investigated. As a step towards a complete understanding of the behavior of block copolymers in liquid chromatography, we are showing the results of a comprehensive method for determining the mutually dependent block-length distributions of the blocks in di-block copolymers. When critical chromatography is used in two-dimensions of a two-dimensional liquid chromatography (LCxLC) set-up, all relevant distributions (the molecular-weight, chemical-composition and block-length distributions (CCD, BLD(A), BLD (B), MWD, conversion and degree of gradient distribution (DGD) can be determined in one analysis.
Michael John
Institute of Anorganische Chemie, Germany
Title: Chromatography in the NMR tube
Time : 13:55-14:15
Biography:
Michael John completed his PhD with Horst Kessler at the Technical University of Munich in 2004, and then spent 2 years with Gottfried Otting at the Australian National University, Canberra. Since 2007, he is Lecturer and Director of the NMR facility at the University of Göttingen. His list of publications includes more than 60 papers in peer-reviewed journals and 25 conference contributions.
Abstract:
Recent developments in nuclecar magnetic resonance (NMR) equipment permit the spatially resolved observation of NMR signals on routine instruments that are part of virtually every chemistry laboratory. Specifically, a series of thin (~ 1 mm) horizontal slices may be excited and recorded of the sample instead of the ~20 mm bulk volume within the rf coil. Through referencing with solutions of known concentrations, local concentrations can be determined. We have recently applied slice-selective NMR spectroscopy to a range of chemical problems: A first example was the monitoring of the anisotropic swelling of cross-linked polymers in organic solvents and determination of the homogeneity of anisotropy across the polymer. Further applications were a "single-shot" NMR titration, where the signal of the first component was resolved over a concentration gradient of the "titrated" component, and a reaction monitoring, where two reagents diffuse towards each other in the sample tube. Here, we present another application: “chromatography in the NMR tube” (a glass tube with Ø = 5 mm): Two compounds that may differ in molecular size or polarity diffuse downwards through a polymeric matrix (which may be cross-linked polystyrene or even silicone grease) at their individual rates and appear in the slices at the bottom with individual concentrations. Although no complete separation of the compounds is achieved, signal assignment is facilitated, and diffusion coefficients may be calculated as an alternative to the DOSY method.
Svetlana M Krylova
York University, Canada
Title: Aptamer-based affinity chromatography as a rapid, single step method for purification of native proteins
Time : 14:15-14:35
Biography:
Svetlana M Krylova completed her PhD from the Russian Academy of Sciences. She has over 10 years of research leadership experience in the area of Medical Diagnostics and Drug Development in biotechnology and pharmaceutical companies in Canada. She has been a contract faculty member at York University in Toronto since 2008. She is leading research projects in the area of Bioanalytical Chemistry as a Senior Research Associate in the Centre for Research on Biomolecular Interactions at York University.
Abstract:
Isolation and purification of recombinant proteins is one of major tasks of modern biotechnology. Isolation of enzymes and antibodies requires conditions that could preserve biological activities of proteins. Often fusion of proteins with His-, GST-, and MBP-tags is used to facilitate their isolation by affinity chromatography. However, the tags, may interfere with the application of the protein while there removal is often accompanied by protein’s loosing its biological activity. We developed aptamer-based affinity chromatography allowing isolation of the recombinant proteins from the crude cell lysate as a quick method yielding native biologically active enzymes. DNA aptamers to AlB protein were developed and characterized by Kinetic Capillary Electrophoresis (KCE). Synthetic DNA aptamers with Kd values in the nanomolar range were used for selective binding and isolation of AlkB from the cell lysate. Specifically, gold (DE3) bacterial culture of cells, expressing E. coli AlkB protein was loaded on aptamer-modified magnetic beads (immobilized though a biotin-streptavidin link). The unwanted components of the cell lysate were removed by washing the beads. AlkB was eluted using different solutions with high ionic strengths. The results were compared with the activity and yield of the enzyme purified using standard tag-based methods of protein purification. Our new method was also succesfully repeated for isolation and purification of MutS protein. In my presentation, I will discuss the CE based aptamer development technology, and I will demonstrate the potential of using aptamers for purification of enzymes from cell lysates in a single simple step, providing biologically active pure recombinant proteins.
Jennifer E Claus
MilliporeSigma, USA
Title: Removal of lipid interferences using zirconia-based solid phase extraction (SPE) and QuEChERS sorbents
Time : 14:35-14:55
Biography:
Jennifer E Claus has graduated from Lycoming College in 2001 with a Bachelor of Science in Chemistry and Biology. She has spent six years working at Merck & Co. in Rahway, NJ as a Medicinal Chemist. While at Merck, she attended Rutgers University and completed her Master of Science in Chemistry. She has been with MilliporeSigma for the past nine years, initially working as a Chiral Applications Chemist for the first four years of her MilliporeSigma career. She has been in her current marketing position as a Product Manager for Sample Preparation, for the past four years.
Abstract:
Although lipids are essential to life processes, they pose an obstacle in both bioanalysis and food analysis. In addition to producing inaccurate detection limits and contaminating chromatographic systems, interfering lipids can ultimately lead to the shortened lifespan of columns and instruments. Traditional solid phase extraction (SPE) cleanup techniques often provide insufficient lipid matrix removal. Therefore, the use of zirconia-based sorbents has been developed for selective lipid removal, and consequently, better analyte determination. In addition to hydrophobic interactions, zirconia-based sorbents utilize Lewis acid/base interactions to selectively retain undesirable lipid interferences. In SPE and/or dispersive SPE (QuEChERS) formats, these zirconia sorbents may be combined with traditional phases like C18 to further improve inference removal. Compared to traditional cleanup sorbents, these innovative sorbents have been shown to remove more lipid matrix interferences, including di, tri-, monoglycerides and phospholipids. A comparison of zirconia-based sorbents to traditional cleanup sorbents for lipid removal from various food and biological matrices will be demonstrated. Background removal, analyte recovery, and reproducibility of the different cleanup techniques will be compared in this presentation.