Day 2 :
Keynote Forum
Dusan Berek
Polymer Institute of the Slovak Academy of Sciences, Slovakia
Keynote: Comprehensive molecular characterization of multi-component synthetic polymers with liquid chromatography
Time : 10:00-10:30
Biography:
Dusan Berek is employed at Polymer Institute, Slovak Academy of Sciences in Bratislava. He served as elected member of the Presidium of the Slovak Academy of Sciences, President of the Slovak Chemical Society, Chairman of the Czecho-Slovak and Slovak National Committee of Chemistry for IUPAC. He is the corresponding member of the Central European Academy of Sciences and member of the Learned Society of the Slovak Academy of Sciences. He is the author or co-author of two monographs and 250+ scientific papers in extenso published in refereed periodicals, proceedings and chapters of books, as well as 60+ patents (four of them were licensed) - cited more than 2,000x. He has presented over 110 invited plenary, key and main lectures, as well as over 900 regular lectures and poster contributions on symposia and conferences and during lecturing tours to over forty countries. He was elected as Slovak scientist of the year 1999 and “Slovak innovator of the year 2001”.
Abstract:
Many high-performance synthetic polymers contain macromolecular chains with distinct chemical structure (composition) or physical architecture. Typical examples represent multi-component polymers called complex polymer systems. Size exclusion chromatography, SEC is commonly used for determination of molar mass averages and dispersities of synthetic polymers. However, due to its limited both separation selectivity and sample capacity, as well as generally low detector sensitivity, SEC can hardly give quantitative information on molar masses of components of complex polymer systems. For example, SEC usually does not allow identification and molecular characterization of binary polymer blends, in which one (minor) constituent represents less than ten percent - even if the molar masses of constituents differ more than 100%. Moreover, SEC cannot recognize macromolecules of distinct chemical structure or architecture because both latter characteristics affect retention volumes together with molar mass. To characterize complex polymer systems, entropy based exclusion separation mechanism is to be combined with enthalpic retention mechanisms. Of such coupled methods of polymer liquid chromatography, the best known are liquid chromatography under critical conditions, LC CC, eluent gradient liquid chromatography, EG LC and temperature gradient interaction chromatography, TGIC. The common drawbacks of the latter methods are limited both sample recovery and capacity. LC CC allows only separation of two distinct sample constituents. Recently an alternative group of coupled LC methods were developed, namely liquid chromatography under limiting conditions of enthalpic interactions, LC LC. Similar to LC CC and EG LC, also in LC LC the molar mass effect is suppressed so that macro-molecules are separated exclusively according to their composition or architecture. LC LC exhibits remarkable separation selectivity, as well as high sample capacity and reasonable sample recovery. Moreover LC LC is highly robust, experimentally feasible and well repeatable. It was successfully applied to separation of chemically and physically similar macromolecules including low solubility polymers. It was demonstrated that the method allows reliable identification of very low (<1%) amounts of minor macromolecular admixtures in major polymer matrix. The backgrounds of LC LC will be presented in the contribution. For a comprehensive molecular characterization of complex polymer systems, different retention mechanisms are to be applied in two separate chromatographic systems. This is the basis of two-dimensional polymer liquid chromatography, 2D-LC. A flexible approach called sequential two-dimensional polymer liquid chromatography, S2D-LC was recently developed. It includes a combination of LC LC and SEC. The fractions produced by the LC LC column are in their entirety online transferred into the SEC column. The S2D-LC principle will be elucidated in detail and the typical examples of its application will be shown, especially separation and characterization of block copolymers that contain (small) amounts of their parent homopolymers.
Keynote Forum
Jorge Costa Pereira
University of Coimbra, Portugal
Keynote: Unsupervised component analysis: How to retrieve independent contributions
Time : 10:30-11:00
Biography:
Jorge Costa Pereira has completed his BSc, MSc and PhD from the University of Coimbra. He was a BSc Fellow, an Assistant Professor and is currently a Full Professor in the Department of Chemistry, Faculty of Science and Technology at the University of Coimbra. His main work is on Chemical Analytical Control field especially, related to Statistics, Analytical Method Validation and Chemometrics. He has 28 published works in well reputed international journals and is a Member of the Editorial Board of Portuguese Electrochimica Acta.
Abstract:
HPLC chromatography is a well-known standard instrumental analytical technique, widespread over all analytical labs around the world, very convenient to perform fast separation and simultaneous quantification of several analytes in several complex samples. Most convenient, reliable and roust detectors are usually related with UV-Vis detection. However, while analyzing real and complex samples, it is frequent to observe signal distortions related with analytes co-elution, spectral interference and column lack of selectivity. Is it possible to be aware of these situations and circumvent these problems? In a recent study we suggested how to retrieve independent component contributions in spectroscopic (synchronous fluorescence) analysis of environmental samples. In this work, independent component analysis (ICA) was used in order to retrieve spectral sources and respective component contributions. However, in order to preform ICA, it was necessary to previously determine the number of independent components responsible for data spectra. Using principal object analysis (POA) we showed how it is possible to find out system independent contributions in a robust and reliable manner. With this same concept we believe it is possible to determine: 1) independent contributions, 2) their specific chromatogram, and 3) respective contributions using same approach, especially when there are evidences for co-elution profiles in (UV/Vis)-HPLC and other non-specific instrumental methods. Some examples, practical and theoretical considerations will be addressed in order to maximize the ability of unsupervised component analysis (UCA) to evaluate chromatographic response in complex systems.
Keynote Forum
R Kapeller
Austrian Agency for Health and Food Safety, Austria
Keynote: Retention time correction in gas chromatography by modelling concentration related effects
Time : 11:00-11:30
Biography:
Rudolf Kapeller received his Dr. phil.in Chemistry from the University of Innsbruck. He worked as Analytical Chemist at the Bundesstaatliche bakt.-serol. Untersuchungsanstalt and since 8/1985 at the Austrian Agency for Health and Food Safety – Institute for Food Safety in Linz; from 5/1996 to 12/2015 as Head of the institute. He has been the author of various publications in the field of analytical chemistry and food safety. His research interests include Analytical Chemistry in the field of Food Safety; Chemometrics, Statistics, Method Validation and Automation (Author of Computer Programs in this field)
Abstract:
Highly reproducible retention times (tR) are a basic requirement for automation of peak assignment in complex gas chromatograms. Drifting retention times are handled best by frequent updates, based on reference peaks, even for every chromatogram, if a sufficient number of reference peaks is always present. The updates may be interpolated for peaks between the reference peaks. Some refinements of this concept have been reported as well as alternative concepts, including pattern recognition algorithms to achieve ultimate precision.
However, there are applications causing additional challenges requiring special measures. One is the effect of concentration on tR of individual peaks, becoming relevant, where - in addition to high complexity and variability in composition - a wide concentration range has to be covered. The analysis of fatty acid methyl esters (FAME) is such an application. A model describing concentration related effects on tR was drawn up and found to work best for this purpose. The precision of peak alignment could be improved by more than an order of magnitude, allowing reliable automation of routine analysis.
- Adsorption Chromatography
Location: Meeting Place 2
Chair
Jorge Costa Pereira
University of Coimbra, Portugal
Co-Chair
Wojciech Strzałka
Jagiellonian University, Poland
Session Introduction
Yun Fa
Chinese Academy of Sciences, China
Title: Multi-dimensional ion chromatography–mass spectrometry for simultaneous determination of extracellular metabolites in Clostridium thermocellum
Time : 12:00-12:20
Biography:
Yun Fa is an Associate Professor in key lab, Qingdao Institute of Bioenergy and Bioprocess Technology Chinese Academy of Sciences. She worded on ion-chromatography analysis for 7 years. She has published more than 15 papers in reputed journals.
Abstract:
In bio-energy and bioprocess research, quantitative understanding of bacterial metabolism and determination of metabolic flux data are necessary, especially for the strain improvement, gene function analysis, optimizing the cell system and the production process, fermentation experiment design. It takes long time for so many metabolites because different methods are needed for different types of metabolites. Although traditional reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS) covered a wide range of small molecule metabolites, but some ions and polar compounds in the conventional reversed-phase columns do not retain or keep very weak, such as organic acids, sugar, sugar phosphate, amino acids and other. These compounds are just one of the main objects in the analysis of metabolomics. We have built a multidimensional ion chromatography - mass spectrometry combined system, and achieved simultaneous analysis for the polar group of amino acids, sugars, alcohols, organic acids, some important cations. Further, the accuracy and precision of the method were investigated. And the new method was successfully used to determine the target metabolites in extracellular culture media of Clostridium thermocellum for not more than 2h. Using a combined system target to metabolomic analysis, we provided the more exact experimental data for designing control process. The establishment of multidimensional ion chromatography tandem mass spectrometry platform can be used for analysis of water soluble metabolites not only in bio-energy research, but also in the field of other areas, such as food, environment and life sciences applications.
Sena Yaman
Izmir Institute of Technology, Turkey
Title: Dendron-based stationary phases for hydrophobic interaction chromatography
Time : 12:20-12:40
Biography:
Sena Yaman has completed her MSc degree in Middle East Technical University and is currently working on her Doctoral studies in Izmir Institute of Technology, Department of Bioengineering.
Abstract:
Hydrophobic Interaction Chromatography (HIC) is a powerful technique used to purify proteins. It relies on the interaction between non-polar groups on the hydrophobic patches of the proteins and ligands on the HIC medium. This interaction varies according to the hydrophobicity of biomolecules, leading to the separation of proteins in a complex mixture. In this work, two novel stationary phases for HIC were synthesized by incorporating dendronitic structures, branched molecules. The branched nature of the dendrons allowed the attachment of high amount of hydrophobic ligands to the support material. Depending on the number of branches of the dendron used, ligand densities were calculated as 82.5±11 and 175.6±5.7 μmol ligand/ml resin for Sorbent 1 and Sorbent 2, respectively. UV-Vis absorption spectra of the modified sorbents exhibited a band at 287 nm corresponding to the aromatic ring present on the dendrons suggesting their incorporation onto the sorbents. FTIR analysis evidenced the aromatic and carbonyl groups suggesting the presecence of dendrons with hidrophobic ligands on the sorbents. Adsorption capacity of the sorbents was evaluated in static and dynamic mode using bovine serum albumin (BSA) under high concentrations of ammonium sulfate (AS). Increasing AS concentration from 1.5-2 M led to significant increases in adsorption capacity. Dynamic adsorption was influenced by flow velocity. This innovative design allowed to increase the ligand density and therefore the adsorption capacity of the sorbents. This technology may permit to reduce the amount of sorbent to be used in a bioprocess and thus use smaller columns resulting in faster chromatographic processes.
- Ion Exchange Chromatography
Location: Meeting Place 2
Chair
Jorge Costa Pereira
University of Coimbra, Portugal
Co-Chair
Wojciech Strzałka
Jagiellonian University, Poland
Session Introduction
Berkant Kayan
Aksaray University, Turkey
Title: Separation of drug active ingredients by using subcritical water chromatography
Time : 12:40-13:00
Biography:
Berkant Kayan is an Associate Professor of Chemistry, Aksaray University, Aksaray, Turkey. He has joined Aksaray University in 2009 as Assistant Professor and then quickly promoted to the Associate Professor in 2012, because of his publications (18) and Grants (Scientific and Technological Research Council of Turkey -TUBITAK, Aksaray University-ASU-BAP-2012-9, and Mersin University- MERSIN-BAP Research Projects). He joined the Department of Chemistry at East Carolina University in 2005 as visiting doctoral students for four months by supporting Mersin University. He visited Technological and Educational Institute of Crete, Chania, Crete, Greece (2013) and Université Paris-Est Marne-la-Vallée., Paris, France (2015) with European ERASMUS BILETERAL as guest academic researcher.
Abstract:
Drug active ingredients are containing one or more chemical substances that cause physiological changes in vivo and substances that are responsible for the beneficial health effects experienced by patients. Drugs are usually grouped according to their physiological effects and antidepressants one of the most frequently used of this group. In this study fluoxetine and sertraline as selected model molecules from antidepressant active agent, which have environmental as well as pharmacological and biological importance. For this purposes naphthylamine attached poly (HEMA-MAH)(PHEMAH) (poly(hydroxyethyl methacrylate-N-methacryloyl-(L-histidine)-methylester) microspheres, which have different particle sizes will be synthesized and used as the column filling material (stationary phase) in high temperature liquid chromatography which is the one of the green chromatography techniques and determination of the method development and optimization conditions for the synthesized column filler will be conducted by using chemometrics application of response surface methodology. Many analytical methods such as High Performance Liquid Chromatography, Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, Gas Chromatography-Mass Spectrometry are used in literature for the chromatographic separation of the active ingredients of antidepressants. Among the used these techniques, the high performance liquid chromatography (HPLC) shows the most effective separation technique. While RPLC (reversed phase liquid chromatography) is a popular analytical technique used today, organic solvents are required in traditional RPLC and an enormous amount of organic solvent is consumed worldwide for just chromatographic separation. The organic solvents is used in HPLC are very hazardous and dangerous for environmental and human health, as well as expensive in term of both purchasing and waste disposal costs. Industrial wastewater pollution is being one of the major and perhaps most importantly problem resulting from uncontrolled of technology in developing countries. Therefore, separation, purification and determination methods are being more important and “nature-friendly” products and dissemination of related researchers is great importance for use as solid/solid phase of determination methods due to environmental concerns. High temperature liquid water chromatography technique is used the minimum level of organic solvent in which worldwide attention recently. At the same time, in order to be successful of separation in HTLC, synthesized a new generation of stable and resistant filler column and is also an important application be so successful at elevated temperature conditions. The systematic studies are expected rapid, accurate, is expected to reach accurate and reliable results as the requirement of a scientific research.
Wojciech Strzałka
Jagiellonian University, Poland
Title: Implementation of ssDNA aptamers for imidazole-free purification of His3-tagged recombinant proteins
Time : 13:00-13:20
Biography:
Wojciech Strzałka has completed his PhD at the Jagiellonian University. He completed Post-doc at Salento University, Italy and Osaka University, Japan. Currently he is a group leader at the Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University. He is studying mechanisms of plant DNA replication and repair, as well as working on the development of new affinity chromatography systems for the purification of recombinant proteins.
Abstract:
The dynamic development of genetic engineering has opened new perspectives for the production of recombinant proteins which are currently offered by many biotech companies. In addition to the medical and industrial applications they are also extensively used in basic research studies. Recombinant proteins are often produced in heterologous expression systems, for example in E. coli cells. Before proteins find a final application, purification, a key stage of the production process, must be performed. Therefore affinity chromatography systems were developed for the fast and simple isolation of recombinant proteins. One of such systems is Immobilized Metal Ion Affinity Chromatography (IMAC), which is commonly used for the purification of His6-tagged recombinant proteins. Although it is a powerful system it is not free of disadvantages. Recently an alternative solution, which is free of IMAC drawbacks, was developed. It is based on a unique ssDNA sequence, called the H3T aptamer, which was selected for the purification of His3-tagged recombinant proteins. The binding of the H3T aptamer to His3-tag is controlled by sodium ion concentration. Based on this feature H3T aptamer resins can be successfully employed for the purification of His3-tagged recombinant proteins from E. coli total protein extracts using imidazole-free buffers. The purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with IMAC resins.
- Molecular Exclusion Chromatography
Location: Meeting Place 2
Chair
Romain Kapel
University of Lorraine, France
Co-Chair
Fabian Holenstein
Novartis Institutes for Biomedical Research, Switzerland
Session Introduction
Koray Åžarkaya
Düzce University, Turkey
Title: Separation of hydrophobic amino acid enantiomers in CEC system by molecular imprinting technique
Time : 14:50-15:10
Biography:
Koray Åžarkaya is PhD student from Hacettepe University and has done his PhD studies from Biochromatography and Biodiagnostics Research Group of Hacettepe University. He has been a Research Assistant at the Düzce University since 2009.
Abstract:
Capillary electrochromatography (CEC), first described by Pretorius, is a rapidly evolving hybrid technique between CE and LC. Analytes may be separated in CEC by the combined action of partitioning between stationary phase and mobile phase (chromatographic interactions) and by their difference in electrophoretic mobility (movement of solutes by electrical forces). Therefore, CEC usually possess higher efficiency and selectivity as compared with classical CE and LC. The use of CEC for chiral analysis has become popular in recent years due to high separation efficiency. Separation of enantiomers is very important because they may show different biological activities. Many single enantiomers of amino acids were also used for the synthesis of biologically important compounds. In this work, enantiomeric separation by CEC was extensively carried out using chiral monolithic capillary column. In this presentation, we will describe about the monolithic column, which is based on N-methacryloyl-(L)-phenylalanine methyl ester (MAPA) as hydrophobic monomer in CEC system.
Sheng Tang
Wuhan Institute of Technology, China
Title: N-Acylated chitosan Bis(arylcarbamate): A class of chiral separation materials with powerful enantioseparation ability and high tolerability to organic solvents
Time : 15:10-15:30
Biography:
Sheng Tang has completed his PhD in Analytical Chemistry from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences. He serves as a teaching and research fellow at Wuhan Institute of Technology. He has published more than 10 papers in reputed journals.
Abstract:
Due to the fact that cellulose and amylose derivatives tend to dissolve in some organic solvents, the related polysaccharide derivatives-based CSPs prepared by coating manner can only work in a limited range of mobile phases, thus, restricting their widespread application in enantioseparation. Chitin, as one of the most abundant optically active biopolymers, is similar to cellulose in primary structure. Much lower solubility of chitosan derivatives was observed compared with the derivatives of cellulose and amylose, enabling the corresponding chitosan derived CSPs to be possibly worked in a wider range of mobile phases. Herein, in order to develop new types of chitosan derivatives-based CSPs, which not only are capable of excellent enantioseparation performance, but also bear a desirable tolerability, we introduce a class of coated-type CSPs, which are based on chitosan bis(arylcarbamate)-(amide) (CACA). The N-acylated chitosan derivatives were synthesized by the reaction of chitosan with carboxylic acid anhydrides in water/methanol in the presence of the corresponding carboxylic acids. CACAs were prepared by further derivatizing N-acylated chitosan with aryl isocyanates. Fig. 1 shows the structures of the prepared CACAs, which were then coated onto 3-aminopropyl silica gel, affording the corresponding CACAs-based CSPs. When the substituents introduced on acyl group at 2-position and on aryl group of phenylcarbamates at 3- and 6-positions were perfectly coordinated, the prepared N-acylated chitosan bis(arylcarbamate) would possess powerful chiral recognition and enatioseparation abilities, and meanwhile exhibited a desirable tolerability towards a wider range of mobile phases, consequently resulting in a new class of promising chiral separation materials.
Hudson Polonini
Federal University of Juiz de Fora, Brazil
Title: Permeation profiles of hormones and NSAIDs through vaginal mucosa using Pentravan cream
Time : 15:30-15:50
Biography:
Hudson Polonini has completed his PhD at 2014 from Federal University of Juiz de Fora. He works in a pharmaceutical company (Ortofarma) and gives classes for two Pharmacy Colleges (Suprema and Unipac). He studies: analysis and control of medicines and related products, pharmaceutical and cosmeceutical technology, biopharmacy, natural products and (nano)ecotoxicology. He has published more than 40 papers in reputed journals and he has two patents, and he also has some awards in innovation competitions.
Abstract:
Vaginal delivery is currently considered to be an important route for poorly-absorbed, rapid-metabolized oral drugs, and it also provides both local and systemic drug delivery. Drug absorption studies are compulsory to establish vaginal permeation kinetics, and in this work we evaluated the possibility of using Pentravan® (Fagron) as a vehicle to deliver drugs by this route. For this purpose, we used gestrinone, progesterone, testosterone, nimesulide and piroxicam creams using Franz diffusion cells with porcine vaginal mucosas. The vehicle was able to deliver approximately 88.17, 7.70, 22.87, 8.34, and 95.71 µg of gestrinone, progesterone, testosterone, nimesulide and piroxicam (respectively) per cm2 of skin by the end of the experiment, when considering only the drug that reached the receptor medium. We also evaluated resveratrol vaginal permeation. For that, we used a previously validated method and tested it with three different stationary phases: a commercial C18 column and two laboratory-made chromatographic columns containing poly(methyloctadecylsiloxane) (PMODS) thermally immobilized onto zirconized silica (Zr-PMODS) or titanized silica (Ti-PMODS). The transdermal vehicle used was also Pentravan®. The permeation experiments showed that resveratrol presented a high rate of retention within the vaginal mucosa, which suggests a local use rather a systemic one. This creates the hypothesis that the formulations with resveratrol, progesterone, nimesulide and piroxicam would be suitable for local vaginal treatments that could benefit from the diverse biological effects of these substances. We also highlight the potential of gestrinone and testosterone to act systemically when compounded using Pentravan®, making the route a viable alternative for other traditional routes.
Fabian Holenstein
Novartis Institutes for Biomedical Research, Switzerland
Title: Automated harvesting and 2-step purification of eight 1-L unclarified mammalian cell-culture broths containing antibodies using a novel configuration on ÄKTATM pure
Time : 15:50-16:10
Biography:
Fabian Holenstein holds an Engineering degree in Biotechnology and a Master in Pharmaceutical Biotechnology. He works as a senior scientist in the section of protein production and antibodies at Novartis Institute for Biomedical Research in Basel/Switzerland.
Abstract:
Therapeutic antibodies represent one of the fastest growing segments in the pharmaceutical market. The growth of the segment has necessitated development of new efficient and cost saving platforms for the preparation and analysis of early candidates for faster and better antibody selection and characterization. We report on a new integrated platform for unattended harvesting and 2-step purification of antibodies expressed transiently in HEK293T-cells at the 1-liter scale. The system consists of two bench-top preparative chromatography instruments connected to a central unit with eight disposable filtrations devices used for loading and filtering the crude biomass feeds. Our end-product QC analysis demonstrates that the quality of the material prepared by the 2-step automated purification is fully comparable to the material purified by standard manual 2-step purification. Average recoveries were also comparable to those obtained by manual purification, indicating that this automated system allows the cost-efficient preparation of therapeutic antibodies in the 20-200 mg range, and covers the requirements for early in vitro and in vivo profiling of drug candidates.
Romain Kapel
University of Lorraine, France
Title: Size-exclusion HPLC as a sensitive and calibrationless method for complex peptide mixtures quantification
Time : 16:10-16:30
Biography:
Romain Kapel is a third year PhD student from University of Lorraine, France. In 2013, she graduated as a chemical and process engineer. She is now working on the fractionation of protein hydrolysate during her PhD program. She will present an original methodology to quantify peptide concentration in complex hydrolysates by size-exclusion HPLC.
Abstract:
Nowadays, protein hydrolysates and fractions are of great interest because of their nutritional or bioactive properties. The quantification of total peptide concentration is of a crucial importance in order to establish mass balance of fractionation processes. This is commonly done either by Kjeldhal analysis, or by colorimetric assays, whose are laborious and time-consuming. This work describes an original methodology to quantify complex peptide mixtures by size-exclusion high-performance liquid chromatography (SE-HPLC), already used to characterize the molecular weight distribution of hydrolysate. In the proposed methodology, each point of the complex mixture chromatogram is regarded as a mixture of peptides sharing same molar extinction coefficient and molar weight, estimated from its retention time and the hydrolysate aminogram. This allows a conversion of absorbance into concentration (using Beer-Lambert law) and the integration of the overall signal gives the peptide concentration of the analysed fraction. The methodology was first tested on simulated elutions of peptide mixtures and a good estimation of the total peptide concentration was observed (error less than 10%). Then 30 fractions obtained by ultrafiltration of hydrolysates from two different sources were titrated by Kjeldahl or BCA analysis and analysed by SE-HPLC for an experimental validation of the methodology. Very good matchs between methods were obtained (error less than 15%). Moreover, the presence of organic solvents or salts in samples does not impact the accuracy of the methodology contrary to common quantification methods.