July 1

1 Vienna University of Technology, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164 AC, A-1060 Vienna, Austria

2 Austrian Institute of Technology, Vienna, Austria, This email address is being protected from spambots. You need JavaScript enabled to view it.

Rechargable Li-ion batteries have become an indispensable element of our daily life, as they power up laptops and mobile phones, consumer electronics, medical devices, and also electric and hybrid vehicles [1]. For electrical transportation they are typically used in much larger aggregates. The larger scale of these batteries increases the hazard arising from malfunctioning or misuse significantly, and has thus prompted detailed studies regarding the safety of Li-ion batteries and particularly their electrolytes under extreme conditions. This electrolyte, consisting of a mixture of organic carbonates with the addition of a suitable lithium salt, degrades already under conditions of normal use, but even more under extreme conditions of temperature or overcharging/overdischarging, as well as under mechanical stress, thereby forming degradation products that represent both a fire or explosion hazard, and also may be toxicologically relevant.

We report here on the chromatographic characterization of the organic electrolyte and its volatile degradation products under conditions of normal use as well as after exposure to thermal and electrochemical stress. The great variety of reaction products formed under these conditions requires the application of different chromatographic techniques for their characterization [2]. In particular, GC/MS and HPLC/MS are advantageously used for the characterization of the volatile degradation products and the electrolyte, respectively. Both an off-line and an on-line approach for the sampling of the volatile emissions from the headspace have been investigated, the latter also allowing the dynamic behavior of the emissions to be studied. Various experimental set-ups have been developed and applied to this end, and their performance and analytical potential will be presented and critically discussed [3,4].

Financial support of this work by the Austrian Research Promotion Agency (FFG) under Grant No. 835790 (SiLithium) is gratefully acknowledged.

REFERENCES:

1. J.B. Goodenough, K.-S. Park, J. Am. Chem. Soc.2013, 135, 1167-1176.
2. G. Gachot, P. Ribière, D. Mathiron, et al., Anal. Chem. 2011, 83, 478-485.
3. A. Amon, Master Thesis, Univ. of Vienna (2014).
4. C. Kanakaki, PhD Thesis, Vienna Univ. of Technology (in preparation, 2015)

 1 National University of Science and Technology "MISIS", Leninskiy prospekt 4, 119049, Moscow, Russia

2 State Scientific-Research and Design Institute of Rare-Metal Industry "Giredmet", B. Tolmachevsky lane, Building 5-1, 119017, Moscow, Russia, This email address is being protected from spambots. You need JavaScript enabled to view it.

New family of heterochain N,S-containing sorbents has been synthesized by thiomethylation of polyamins. Applicability of the sorbents for extraction and concentration of noble metals (NM) and platinum group elements (PGE) was tested in the presence of macroscopic amounts of mineral and secondary raw materials for their further study by atomic-spectral methods. Quantitative information about PGE and NM was obtained by high resolution continuum source atomic absorption spectrometry (HRCS-AAS).The properties of the sorbents were studied depending on the composition and structure, the number of complex-forming groups, solvent composition, and concentration and nature of the secondary components. In order to achieve better understanding of the sorption mechanism EXAFS investigation at Pt LIII edge was performed using ANKA synchrotron source, Karlsruhe. Preliminary data suggest that Pt atoms are sulphur bonded. Kinetics of PGE, Au, Ag sorption was studied at static conditions at room temperature and on heating. Capacity of the sorbent was established. For example, for the sorbent (S - 29.40%, N - 11.89%, C - 44.28%, H - 7.36 %) the capacity is 3 g Ag per gram of the sorbent. The sorbents were developed and tested in the analytical laboratory practice “Separation and concentration in the chemical diagnosis of functional materials and objects of the environment” of NITU MISIS and the test analytical and certification center of Giredmet Institute.

The authors gratefully acknowledge the financial support of the Ministry of Education and SRF in the framework of Increase Competitiveness Program of NUST «MISiS»(№ К1-2014-026), financial support of RFBR (13-03-00440).

 1 Babes-Bolyai University, Faculty of Environmental Science and Engineering, 30 Fantanele Street, 400294 Cluj-Napoca, Romania

2 Babes-Bolyai University, Faculty of Geography, 5-7 Clinicilor Street, 400006 Cluj-Napoca, Romania

3 Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania, This email address is being protected from spambots. You need JavaScript enabled to view it.

Priority substances are defined according to the Water Framework Directive (WFD) as the substances identified to present a significant risk to or via the aquatic environment. These substances include 45 organic compounds from different classes and heavy metals, their determination representing the base of the characterization of the chemical status of water body.

In the frame of NATO SfP 984440 Project the authors wish to develop a methodology able to identify the River Basin Specific Pollutants from Upper Tisza. For this purpose, the analysis of the priority substances in the water body is required as a starting point.

This paper contains the results of eight months monitoring of different classes of priority substances such as pesticides, halogenated compounds, polyaromatic hydrocarbons, phthalates and heavy metals performed on river water samples collected from the Upper Tisza and its Romanian Tributaries.

For the quantification of these compounds different analytical protocols based on solid phase extraction subsequent by gas and liquid chromatography were used.

The obtained results show that the priority substances found in the studied area are p,p’-DDT, chloroform, naphthalene, anthracene, fluoranthene and benzo(b)fluoranthene as organic compounds, and lead, cadmium and mercury as heavy metals. Except of p,p’-DDT and cadmium, the concentration of the other found priority substances do not exceed the Environmental Quality Standard (EQS) requirements. Other compounds not being included on this list such as acenaphthene, fluorene, phenanthrene and pyrene have also been found.

For a better estimation of the aquatic medium contamination with priority substances, analyses of sediment and biota were carried out. Specific methods for the extraction based on Soxhlet or ultrasound assisted methods followed by gas and liquid chromatography were developed. These analyses were focused on organochlorine pesticides, polyaromatic hydrocarbons and phthalates. The obtained results confirm the presence of these compounds in the analyzed samples.

Acknowledgements: This work is performed in the frame of NATO SfP 984440 Project, Science for Peace and Security Programme.

 University of Tasmania, Australian Centre for Research on Separation Science, Private Bag 75, Hobart, TAS 7001, Australia, This email address is being protected from spambots. You need JavaScript enabled to view it.

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.

 Department of Chemistry, Seoul National University, Seoul 151-747, Korea, This email address is being protected from spambots. You need JavaScript enabled to view it.

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.

 1 Babes-Bolyai University, Raluca Ripan Institute for Research in Chemistry, 30 Fantanele Street, 400294, Cluj-Napoca, Romania

2 Babes-Bolyai University, Faculty of Environmental Science and Engineering, 30 Fantanele Street, 400294, Cluj-Napoca, Romania, This email address is being protected from spambots. You need JavaScript enabled to view it.

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 ibuprofen as 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.

 Institute of Environmental Geochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine, This email address is being protected from spambots. You need JavaScript enabled to view it.

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.

 University of Pécs, This email address is being protected from spambots. You need JavaScript enabled to view it.

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.

 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, CZ-53210 Pardubice, Czech Republic, This email address is being protected from spambots. You need JavaScript enabled to view it.

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.

 Thermo Fisher Scientific, This email address is being protected from spambots. You need JavaScript enabled to view it.

One of the most successful approaches to the retention and separation of polar compounds is hydrophilic interaction liquid chromatography (HILIC). HILIC is not a straightforward technique and one of its most common drawbacks during method development is the difficulty in identifying the most suitable column to use. The stationary phases used in HILIC are quite diverse, and systematic studies of their chemistries are relatively limited. Kumar et al. [1] re-developed early characterisation studies, incorporating the effects of changing a range of experimental parameters as well as a characterisation study on the stationary phases. This approach has been previously applied in RPLC [2, 3], but novel in HILIC and represents a good practical guide to adjustments of conditions that effect the selectivity of a separation.

The aim of this work was to develop these areas of critical understanding by testing a wider range of experimental parameters to determine the interactions between them and how these parameters affect the chromatography of a series of test compunds. Five main classes of HILIC columns were investigated in this study:

Bare silica, neutral bonded ligands, charged ligands, zwitterionic phases and mixed mode phases. A preliminary column characterisation investigation was performed. This highlighted fundamental differences in the column chemistries. In addition the following experimental factors were studied: effect of solvent content, effect of buffer concentration and effect of buffer pH. The work was finally summarised in a convenient work flow which describes in detail a generic approach to method development within the HILIC mode of chromatography.

[1] Kumar A., Heaton J.C. and McCalley D.V., 2013, J. Chromatogr. A, 1276, 33.

[2] Neue U. D., O’Gara J.E. and Mendez A., 2006, J. Chromatogr. A, 1127, 161.

[3] Neue U. D. and Mendez A., 2007, J. Sep. Sci., 30, 949.

 1 Thermo Fisher Scientific, Tudor Road, WA7 1TA, Runcorn, UK

2 University of Liverpool, Oxford Street, L69 7ZD, UK, This email address is being protected from spambots. You need JavaScript enabled to view it.

In recent years there has been a renewed interest in particle technology for both HPLC and UHPLC, due to the introduction of solid core technology. Prior to this there was very little novel development in HPLC particle morphology The patents associated with solid core technology have resulted in manufacturers and researchers having a much greater level of interest in the basic manufacture of silica particles, particularly since the current manufacturing approach is known to be a fairly involved process.

This has led to the development of a completely new type of particle which is manufactured using a one-step synthesis delivering near monodispersed particles and core shell morphology. The morphology of the particle has been designed to deliver the real advantages of the core-shell particles, with a range of parameters being varied to ensure the robustness of the manufacturing.

Careful investigation of some of the morphologies produced revealed that the structure of the particle produced resembled very closely a true fractal structure. Fractal structures have been associated with chromatographic media for some time, however they have been more associated with the concept of being a fractional dimension rather than having a degree of self similarity. These new particles demonstrate, not only a fractional dimension, but also a degree of self similarity. This opens up a possibility of having a surface topography which is essentially homogeneous to solute molecules, thus eliminating one of the issues associated with traditional porous structures in which molecules effectively do not experience the same surface topography resulting in different retention times, the so called ‘c’ term in the van Deemter equation. This is particularly pertinent for large molecules where diffusional processes related to the different pore depths can result in poor peak shape, either through equilibration effects or because molecules effectively get stuck between the walls of the pores.

Examples will be given of the different morphologies that can be manufactured and also how these can be applied to the analysis of large protein molecules.

Boreskov Institute of Catalysis SB RAS, Novosibirsk, Russian Federation, Pr. Lavrentyeva

Novosibirsk state University, 630090, Pyrogova st., 2, Novosibirsk, Russia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Comprehensive two dimensional gas chromatography (GCxGC) is a high-effective technique for analysis of complex multicomponent mixtures.

An important advantage of GCxGC is that it allows analyzing of complex samples by separating the components in groups according to chemical classes. Frequently those samples contain high-boiling fractions of compounds. The examples of those samples are crude oil, products of petrochemistry, coal chemistry and etc. It is known that in order to obtain the best separation power in GCxGC one should use as polar phase as possible for one of the dimension. However, the thermal stability of common polar and high polar stationary phases is not sufficient to analyze high boiling complex samples.

Recent advances in the development of new liquid phases made it possible to use some classes of ionic liquids (ILs) as polar stationary phases [1]. There are works devoted to research of columns with imidazolium and phosphonium ILs for GCxGC in the literature [2]. However, the possibility of using ILs for high temperature GCxGC separations (up to 300°C) is not closely investigated. Our work is devoted to the research of imidazolium and pyridinium ILs as stationary phases columns for GCxGC. In our earlier studies we have shown that these classes of ILs allow to work at the temperature up to 300°C [3] and possess different range of selectivity [4]. Therefore, we consider that their use in GCxGC will allow to obtain the high selective separations at high temperature. In our work the series of columns with pyridinium and imidazolium ILs were prepared and were investigated as columns for GCxGC. These columns were shown to work at the temperature 280°C-300°C with good separation characteristics. The number of separation of multicomponent high boiling samples was obtained. Among them: crude oil and oil products, natural bitumen, high boiling mixtures of biological origin.

[1] С.A. Shamsi, N.D. Danielson, J. Sep. Sci. 30 (2007) 1729.

[2] С. Ragonese, D. Sciarrone, J. Chromatogr. A 1255 (2012) 130-144.

[3] M.V. Shashkov, V.N. Sidelnikov, Anal. Bioanal. Chem. 403 (2012) 2673-2682.

[4] M.V. Shashkov, V.N. Sidelnikov, J. Chromatogr. A 1309 (2012) 56-63.

Polymer Institute, Slovak Academy of Sciences, Dúbravská cesta 9, 84541 Bratislava, Slovakia, This email address is being protected from spambots. You need JavaScript enabled to view it.

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.

BIA Separations d.o.o., Mirce 21, Ajdovscina, SI-5270, Slovenia, This email address is being protected from spambots. You need JavaScript enabled to view it.

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.

1 Bioprocess Engineering Laboratory, School of Engineering and Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan

2 Faculty of Chemistry and Chemical Technology, University of Ljubljana,1000 Ljubljana, Slovenia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Numerous techniques have been developed over the past decades to enhance the in-vivo stability of therapeutic proteins. In particular, PEGylation, known as the covalent binding of PEG chains to proteins, has become a technique of choice to extend their in-vivo circulation half-life. However, the side directed PEGylation reaction needed for human treatment still represents a challenge, due to consecutive competitive reactions with activated PEG, native and PEGylated protein. As a consequence, PEGylation produces positional isoforms and isomers which have different PEG chain number and further purifications steps are needed to isolate target positional isoform. For efficient development and production of PEGylated protein drugs, it is therefore essential to have fast analytical method for monitoring of PEGylation reaction either during development phase (QbD) or as a part of production process (PAT) where high frequency data are required for process control. As PEGylation alters surface protein charge distribution, ion-exchange chromatography seems to be a method of choice. Among different types of stationary phases monoliths were selected due to predominant convective transport providing efficient separation in short time at low pressure drop for such large protein molecules as PEGylated proteins.

In this work we developed different chromatographic methods for separation of PEGylated protein positional isoforms and isomers using a strong cation exchange SO3 CIM disk monolithic column (diameter =12 mm, thickness = 3 mm, volume 0.34 mL) and implementing either salt or pH gradient. While optimal separation using salt gradient was achieved with citric buffer pH 4.5, for pH gradient carbonate buffer from pH 10 to 12 provided optimal results. Despite different elution mechanism both methods provided separation of mono, di and tri PEGylated proteins as well as positional isomers but differing in resolution and elution order. Under optimal conditions, the analysis was completed within 3 min at flow rate of 10 mL/min and pressure drop below 1 MPa (10 bar).

University of Maribor, Faculty of Chemistry and Chemical Engineering, PolyOrgLab, This email address is being protected from spambots. You need JavaScript enabled to view it.

Emulsion templating method is frequently used for the preparation of macroporous polymeric monoliths (1). While the morphology of so prepared polymers (termed polyHIPEs) is generally composed of spherically shaped primary pores several micrometer in diameter and a number of secondary pores connecting primary pores, there are some methods enabling the manipulation of mesoporous and microporous structure of the polymer film in the monoliths. The lecture will focus on presentation of a combination of emulsion templating and post polymerisation hypercrosslinking in order to produce polyHIPE monoliths with a distinct bimodal porous structure. Furthermore, examples of chemical functionalization and solid phase extraction from aqueous sources will be given.

 1 I. Pulko, P. Krajnc, Macromol. Rapid Commun. 2012, 33, 1731