Synthesis and Modifications of Materials for

Separation Science

Anna Nordborg

AKADEMISK AVHANDLING

Som med vederbörligt tillstånd av Rektorsämbetet vid Umeå Universitet för erhållande av filosofie doktorsexamen framlägges till offentlig granskning vid Kemiska institutionen, Naturvetarhuset, sal N360.

Fredagen den 28:e mars 2008, klockan 10.00.

Fakultetsopponent: Ao. Univ.- Prof. Dr. Michael Lämmerhofer Christian-Doppler-Laboratory for Molecular Recognition Materials,

Institute of Analytical Chemistry, University of Vienna, Vienna, Austria

Title: Synthesis and Modifications of Materials for Separation Science Author: Anna Nordborg, Department of Chemistry, Umeå University, S-901 87 Umeå, Sweden Abstract: This thesis deals with the preparation of materials for use in separation science and their surface modification by grafting. The overall aim is the preparation of diverse materials by combination of a set of developed tools. Included in the thesis is the synthesis of monolithic media using non-traditional crosslinkers, the characterization of their porous properties and initial testing in reversed-phase chromatographic separation of proteins. The preparation of a library of short polymer chains, telomers, with varied functionality and their characterization is reported. Included in the characterization is the gradient polymer elution chromatography of selected telomers on a monolithic column in capillary format. The technique shows promise as a tool for monitoring of polymerization processes and for the separation of telomers with similar size but different functionalities or characteristics. Finally, the combination of polymeric support materials and the prepared telomer library is used in surface modification. Surface modification is performed onto activated surfaces via a “grafting to” approach. One example is shown, the surface modification of epoxy-modified divinylbenzene particles by attachment of telomer chains introducing ion-exchange functionality. The material is tested for the separation of proteins, in ion-exchange chromatography mode. Keywords: material synthesis, monolith, surface modification, telomer,

“grafting to”, gradient polymer elution, chromatography ISBN: 978-91-7264-528-8 Printed by: Arkitektkopia, Umeå

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”There’s no use trying,’ Alice said.’ One can’t believe impossible things’ “I daresay you haven’t had much practice,’ said the Queen. ‘When I was your age, I always did it for half-an-hour a day. Why, sometimes I’ve believed as many as six impossible things before breakfast.’” Alice's Adventures in Wonderland Lewis Carroll

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Till min familj.....

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Table of contents

List of Papers ................................................................................ ix Abbreviations, symbols and definitions .................................... xi Preface..........................................................................................xii 1. Introduction ................................................................................ 1 2. Materials Science ..................................................................... 2 3. Separation Science .................................................................. 3

3.1 Liquid Chromatography ................................................................. 3 4. Materials in Separation Science............................................. 6 5. Strategies in Material Characterization ................................. 7

5.1 Surface Imaging by Scanning Electron Microscopy ...................... 7 5.2 Porous Properties and Surface Area ............................................... 8 5.3 Elemental Composition and Functional Group Determination ...... 9

6. Strategies in Polymer Characterization............................... 11 6.1 Size-exclusion Chromatography and Mass Spectrometry ............ 12 6.2 Gradient Polymer Elution Chromatography ................................. 14

7. Synthesis of Monolithic Support Materials.......................... 15 7.1 The Monolith................................................................................ 15 7.2 Controlling Porous and Surface Properties .................................. 16 7.3 Introducing New Crosslinkers into Monolith Synthesis............... 18

8. Iniferter Mediated Polymerization ........................................ 21 8.1 Living Radical Polymerization..................................................... 21 8.2 The Iniferter Concept ................................................................... 22 8.3 Constructing a Telomer Library ................................................... 23 8.4 Gradient Polymer Elution Chromatography on Monolithic Capillary Column ............................................................................... 25

9. Surface Modification of Polymeric Supports ...................... 26 9.1 General Strategies for Surface Modification ................................ 26 9.2 “Grafting from” versus “Grafting to“ ........................................... 26 9.3 Grafting of Thiol-terminated Telomers onto Chromatographic Supports.............................................................................................. 27 9.4 Preparation of a Cation Exchange Material via “Grafting to” ...... 29

10. Concluding Remarks and Future Aspects........................ 31 11. References ............................................................................ 33 12. Acknowledgments ............................................................... 39

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List of Papers

This thesis is based on the following papers and manuscripts, which are referred to in the text by their Roman numerals.

I. Extending the Array of Crosslinkers Suitable for the Preparation of Polymethacrylate-based Monoliths

A. Nordborg, F. Svec, J. M. J. Fréchet, K. Irgum

Journal of Separation Science, 2005, 28, 2401–2406

II. A Library of Thiol-Terminated Methacrylate Telomers Prepared by Photo-Initiated Iniferter Mediated Polymerization for use in “Grafting to” Modifications of Chromatographic Surfaces

A. Nordborg, K. Irgum

Manuscript for Journal of Applied Polymer Science.

III. Gradient Polymer Elution Chromatography of Methacrylate Telomers on Monolithic Capillary Columns

A. Nordborg, E. Byström, K. Irgum

Manuscript for Journal of Chromatography A.

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IV. A Cation-Exchange Material for Protein Separations Based on Grafting of Thiol-Terminated Sulfopropyl Methacrylate Telomers onto Hydrophilized Monodisperse Divinylbenzene Particles

A. Nordborg, F. Limé, K. Irgum

Submitted to Journal of Separation Science.

Paper I is reprinted with permission from Wiley.

Other papers by the author not included in the thesis:

Polymer-based Monolithic Microcolumns for Hydrophobic Interaction Chromatography of Proteins

P. Hemström, A. Nordborg, F. Svec, J. M. J. Fréchet, and K. Irgum

Journal of Separation Science, 2006, 29, 25–32

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Abbreviations, symbols and definitions

BMA Butyl methacrylate BzMA Benzyl methacrylate DVB Divinylbenzene EMA Ethyl methacrylate GMA Glycidyl methacrylate HEMA 2-hydroxyethyl methacrylate MMA Methyl methacrylate ATRP Atom Transfer Radical Polymerization RAFT Reversible Addition-Fragmentation Chain Transfer ROMP Ring Opening Metathesis Polymerization GPEC Gradient Polymer Elution Chromatography HPLC High Performance Liquid Chromatography IEX Ion Exchange Chromatography RP Reversed Phase SEC Size Exclusion Chromatography MALDI Matris Assisted Laser Desorption/Ionization TOF Time-of-Flight SEM Scanning Electron Microscopy XPS X-ray Photon Spectroscopy IR Infrared (spectroscopy) NMR Nuclear Magnetic Resonance MS Mass Spectrometry EOF Electroosmotic flow pI pH of the isoelectric point.

pH at which a protein has no net charge Mw Weight average molecular weight Mn Number average molecular weight PDI Polydispersity Index

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Preface

Starting out on a thesis topic involving the studies of protein interactions with chromatographic separation materials, the synthesis of materials seemed likely to be an important but perhaps not a major part of the project. Continuing with studies on protein digestion and mass spectrometry, material synthesis was further afield and the end result presented in the following pages could surely have become something vastly different. Still, when aiming for fundamental and systematic studies of analyte- material interactions, the aim arose to find variety and control in the preparation of materials and surfaces. In joint efforts we went ahead to prepare new materials! And what is described ahead is what the project ended up becoming – numerous efforts into the synthesis of materials and their use in chromatographic applications. And although no detailed investigations into the protein-material interactions are included, the separations of proteins on prepared materials are.

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1. Introduction

This thesis summarizes a few of the efforts that I have been involved in aiming at new materials for separation science. Using polymerizations and chemical reactions in various forms, the aim is to find ways to vary separation materials – both in its bulk and surface properties – to get easily attainable materials with known and controllable properties. This thesis includes the synthesis of polymeric monolithic support materials by radical polymerization using a set of non-traditional crosslinkers. The construction of a telomer library using living radical polymerization is described as well as its characterization, including first attempts at the use of gradient polymer elution chromatography in a monolithic capillary column. Finally, an example of the use of the telomer library in the surface modification of a chromatographic support material is described. The presented materials are used for protein separations in reversed-phase and ion exchange mode.

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2. Materials Science

The world of materials science is a vast and diverse one, dealing with many aspects of both naturally occurring and man-made materials. A fair representation is given by the materials science tetrahedron, Figure 1, which contains anything and everything connecting the synthesis, structure, properties, performance and characterization of materials. In materials science aspects of chemistry, physics, and engineering join hands. Materials have always had an important role and the development of new materials or the search for new usage areas for already existing materials is an ever growing part of science. Materials are even a basis of which we define time (stone age, bronze age, iron age…) although it is not clear what the current time would be named, could it be the era of plastics? Optimization of manufacturing processes, modifications of biocompatibility or down-sizing are important goals, although not all materials can fit into the ‘hyped’ areas of nanotechnology or nanoscience.

Synthesis

Structure

Performance

Properties

Characterization

Figure 1. The materials science tetrahedron.

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3. Separation Science

The science of separations is important to consider and master in an analytical chemistry laboratory, both in academia and industry. Separations are most likely a part of the determination of purity of a product in a manufacturing industry, the determination of composition in samples of environmental concern or the isolation of protein probes.

3.1 Liquid Chromatography

Liquid chromatography[1] is a technique for separation of analytes dissolved in a liquid. The system set-up in high-pressure liquid chromatography (HPLC) typically consists of a pump, a solvent reservoir, an injector, a column, and a detector, as shown in Figure 2. In liquid chromatography, the key components to achieving separations are the column with its stationary phase and the liquid mobile phase, the eluent. The role of the eluent is to transport the analytes through the system and past the stationary phase while at the same time being in competition with the stationary phase for the analyte. In almost all chromatography modes, as the sample is transported through the system, analytes with an affinity for or capability of interaction with the stationary phase will be retained. The exception is size-exclusion chromatography were separations are based on size and, ideally, no interactions take place between analyte an stationary phase. In either case, different analytes will be retained to different extents and thus have different residence times in the system, resulting in different retention times. The analytes will, in an ideal case, elute and exit the system as individual bands, possible to detect in the detector.

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Figure 2. A schematic of a HPLC–set-up.

Liquid chromatography is performed in various modes, denoted by the kind of interaction that governs the retention and separation. Common modes in many analytical chemistry set-ups are reversed-phase (RP) and ion chromatography (IC). In protein purification, separations in ion-exchange (IEX) or hydrophobic interaction chromatography (HIC) have, when performed under suitable conditions, the advantage of preserving the structure of the isolated proteins. Other chromatographic modes include hydrophilic interaction chromatography (HILIC), ion exclusion chromatography (IEC) and size-exclusion chromatography (SEC).

3.1.1 Reversed-phase liquid chromatography

In reversed-phase liquid chromatography[2], RP, the stationary phase is hydrophobic and the mobile phase (eluent) is a mixture of an organic solvent (typically methanol, acetonitrile, or 2-propanol) and water. In this separation mode, separation is achieved due to differences in hydrophobicity. The higher the content of organic modifier in the eluent, the better it is at competing with the stationary phase for the analyte, i.e., the eluent is ‘stronger’. Reversed-phase chromatography is highly versatile and is a very common technique in many application areas. Through modifications of the column and eluent, including rather advanced additive schemes, a large range of analytes can be separated in this mode. Its usefulness in protein separation or other biomolecule separations is slightly limited depending on the application since the rather harsh conditions (high organic modifier content) are likely to

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damage the protein structure and cause partly or complete denaturing. This makes RP unsuitable in a protein purification scheme where the protein needs to retain its structure and activity, However, for a quantitative determination scheme there are less possible limitations in using RP and the possibility of on-line coupling with mass spectrometric detection is an advantage.

3.1.2 Ion exchange chromatography

Ion-exchange chromatography[3], IEX, is a technique that is common in protein purification due to its rather mild conditions and aqueous eluents, enabling the proteins to retain the structure and function after purification. This separation mode achieves separation based on the charge and charge distribution of the analyte and elution takes place in a gradient of increasing salt concentration, often sodium chloride. When used in protein separation, the buffered eluent keeps the pH constant and thus the charge of the proteins. Proteins with a pI (pH-value at which the protein net charge is zero) above the pH of the eluent will have retention in cation exchange mode whereas proteins with a pI below the pH of the eluent will have retention in anion exchange mode.

The stationary phases differ in support material composition and in the type of ionic groups at their surface. In cation-exchange chromatography, common sources of negative charge on the stationary phase are carboxylate functional groups for weak cation exchangers, and sulfonate functional groups for strong cation exchangers; for anion exchangers the strong variants typically involve quartery ammonium groups, whereas weak ion exchangers are usually based on secondary or tertiary amines. The terminology distinguishing between weak and strong ion exchangers relates to the pH interval over which the ion exchanger retains its charge[3]. Strong ion exchangers retain their charge and thus the retentive capacity over the entire aqueous pH interval, whereas the functional groups of weak ion exchangers undergo dissociation/protonation equilibria and therefore retain their charge only over a certain pH interal. In addition to its usefulness in protein separation, ion exchange chromatography can also be used in, for example, the separation of carboxylic acids[4].

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The use of both reversed phase and ion exchange chromatography is common for separation of proteins, with ion exchange mode utilizing the protein charge and its distribution on the surface of the proteins, whereas reversed-phase relies on differences in hydrophobicity. The soft characteristics make the use of ion exchange chromatography a more viable alternative in purification schemes[3].

4. Materials in Separation Science

When browsing the literature of analytical chemistry and chromatography or when attending conferences, it is evident that the world of materials science and separation science are closely linked. The material in the column in a liquid chromatography system is vital to its use and usefulness since the column material to a large extent provides for retention in the system and hence, the separation. In order to achieve elution or optimize separations, many modifications of eluent compositions can be made, including a number of different solvent mixtures or additives. And still, without a material capable of causing retention, no separation will be achieved.

Chromatographic separation materials or stationary phases come in many shapes, types and with different bulk and surface chemistries. They range from rigid particles with high mechanical and pressure stability [6], to soft agarose gels with comparatively low rigidity [7]. Silica particles are common support materials in commercially available columns, as are different types of polymeric particles. Other types of support materials are the monolithic materials which are not packed like particles, but made within the column, and also exist in both silica and polymeric variants [8,9]. Common for most materials is that they can be modified in a number of ways, the range of possible variations dependent on the surface chemistry of the support as well as the strategy chosen for functionalization or modification.

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5. Strategies in Material Characterization

Characterization is central to the development of new materials. In chromatographic applications both the bulk and surface properties of a separation material are of importance to the usefulness and suitability of a material for a certain application. These properties can be investigated using a number of different techniques. Factors such as pressure stability and chemical stability are important and related to the bulk of the material, while the porous and surface properties will influence retention characteristics. A more detailed examination of the surface chemistry and functional group content will yield vital information not only about interaction sites for retention but also possible sites of further surface modification.

5.1 Surface Imaging by Scanning Electron Microscopy

Scanning Electron Microscopy[10], abbreviated SEM, is a technique used to acquire images of micrometer sized samples and is highly useful for the visualization of surface structure. In particle analysis it can be used to assess particle shape, size, and polydispersity (size distribution). When applied to monolithic materials SEM can be used to examine uniformity throughout the monolith and estimate porositys as well as for verifying attachment of the monolith to the walls of a capillary column.

The SEM imaging relies on scanning the sample surface with a tightly focused beam of high energy primary electrons. Their impaction results in release of secondary electrons and electromagnetic radiation, which is detected. Prior to being examined in the SEM microscope, the sample is typically coated with a nanometer thick layer of electrically conductive material, commonly gold or platinum, which increases image contrast. In a SEM micrograph, steeper regions of the surface will appear as brighter regions in the image, the result being images which appear to be three-dimensional. The emission of electrons and electromagnetic radiation from the surface is dependent on its atomic composition and density, also making it possible to distinguish one type of atom from another.

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5.2 Porous Properties and Surface Area

The porous properties and surface area of a material are important factors in separation science and the two factors are interconnected with materials of large (and hence easily accesible) pores having low surface area. The common techniques used for assessment of porous properties are mercury intrusion porosimetry[11,12], inverse size exclusion chromatography (ISEC)[13,14] and surface area determinations by gas adsorption at cryogenic temperatures [15].

Mercury Intrusion Porosimetry is based on the behaviour of non-wetting liquids (mercury) in capillaries; where a liquid does not spontaneously enter a small pore opening but can be forced to do so by an applied pressure[11,12]. In a pore determination by mercury intrusion porosimetry, a dried solid sample is placed in a small sample chamber and mercury is then applied to the chamber at small, constant volume increments, which causes the mercury to enter into smaller and smallar pores of the material. Depending on the pore size, different pressures are required to force the mercury into the pores. From the pressures/volume relationship, the pore size distribution and the total porosity of the material can be determined. The calculations are based on the Washburn equation[11,12] which describes the pressure required for mercury to enter a cylindrical pore of a certain size. Although pores in separation materials are seldom cylindrical, the equation is still used as an approximation and porosity values obtained by mercury intrusion porosimetry are typically used for comparative studies.

In inverse size exclusion, a series of standards of known composition is injected under eluent conditions where enthalpic interaction of the standard substance with the stationary phase is eliminated. Elution in size exclusion mode is performed and the elution volume of each standard is used to assess the pore size distribution in the material [14]. The major advantage of ISEC is that the porous properties are obtained in situ, as the column is used in actual separations.

The use of gas (typically nitrogen) adsorption in determination of surface area and porosity is based on theories of gas adsorption to a surface

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according to the Brunauer-Emmett-Teller (BET) principle [16]. In a typical determination, a dried sample submerged in liquid nitrogen is subjected to nitrogen gas at varying pressure, allowing equilibration and monitoring the amount of adsorbed gas[15]. As the amount of adsorbed gas at any given pressure will depend on the surface area and the pore size, both surface area and a pore size distribution can thus be determined.

5.3 Elemental Composition and Functional Group Determination

A number of techniques can be used to assess composition and existence of functional groups, including infrared (IR) spectroscopy[17,18], and NMR[19]. IR is a highly versatile technique that can be used for determination of solid samples as well as solutions and by which certain functional groups can be easily identified, such as OH-groups with their characteristic broad adsorption in the upper region of the IR-spectra. In addition, the disappearance of a signal for certain functionality can also yield valuable information, such as the disappearance of vinyl groups during an addition reaction. In simple titration set-ups, determinations of surface vinyl, epoxy, and hydroxyl groups can be performed[20],[21]. Information about ion exchange capacity can be useful for materials used in ion exchange media, where high ion exchange capacity is often a desirable feature, as well as in hydrophobic interaction media, where ion exchange capacity will counteract the use of hydrophobic interaction to achieve retention. Ion exchange capacity can be determined by fully charging the material with one type of ion followed by elution with another ion of the same charge (preferentially more strongly retained by the ion exchanger), followed by determination of the total amount of the ion initially charged in collected fractions. Determinations of electroosmosis at a surface yields information about surface charge and has been used to assess surface shielding, grafting density, and coating thickness [22].

5.3.1 Elemental analysis

Elemental analysis is used to determine the total element composition of a sample and can be used both as a quantitative and qualitative technique.

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The technique does not distinguish between different bonds between the atoms or in what form the atoms are present in the sample. It also does not distinguish between the surface and bulk, but gives valuable information about the overall or average composition of a material. In common applications, elemental analysis is used to identify compounds. When applied to materials for separation science, the data obtained from elemental analysis can be used to identify a component in a material, or to verify and assess grafting. For example, when grafting a carbon chain onto silica, the amount of carbon from elemental analysis will yield information about the amount of grafted chains per gram of silica. Similarly, if performing a series of surface modification reactions which should introduce two types of atoms in a known ratio, the success or yield of each step can be determined.

The way in which elemental analysis is performed varies. Gravimetric determinations are possible, but a combustion technique is commonly used; the sample is burned in excess oxygen and the formed gases are collected and detected. The gases formed; such as CO2, NO2, SO2, and water, are characteristic of elements in the sample, and hence, the weight percent of each element can be determined. The technique can be used to determine carbon, hydrogen, and nitrogen, as well as sulfur, halogens or other heteroatoms.

5.3.2 X-ray Photoelectron Spectroscopy

X-ray Photoelectron Spectroscopy, XPS, [23,24] is a surface-sensitive technique by which it is possible to determine type and concentration of elements in the top 1-10 nm surface layer of a material. The studied surface is subjected to a monochromatic X-ray beam and the photons are absorbed by atoms at, or just beneath the surface. The energy needed to cause emission of electrons from the surface depends on the kind of atom being excited and its environment, i.e., other atoms and bonds, that surround the atom and hence, information about atom and bond type is obtained. Since only the top 10 nm of the material is probed, XPS is a truly surface sensitive technique, and surface contamination is therefore a problem. When used for the characterization of separation material surfaces XPS verify surface composition, identify impurities, and verify

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introduction of new atoms, bonds or groups on the surface. XPS can thereby be used to verify covalent attachment and hence grafting. However, if the grafted layer is too thick, the anchoring group may be shielded from the X-ray beam and hence not produce a signal.

6. Strategies in Polymer Characterization

The characterization of prepared polymers typically has two main objectives, the determination of polymer composition and size. In addition, in the determination of polymer size, the distribution of sizes is also of importance. The resulting size of the polymer influences many of its characteristics, and the size distribution gives insight into the level of control in the preparation process.When determining polymer size either the weight average molecular weight (Mw) or the number average molecular weight (Mn) can be determined. The relation between these two averages is the polydispersity index, which for a completely monodisperse sample is 1, since then the majority of the polymer chains are of the average molecular weight. Different types of end group analysis can be performed to determine the number average molecular weight, including the use of NMR[25] or mass spectrometry [26], while ultracentrifugation or light scattering measurements [27] are commonly used for measurements of weight average molecular weight. The determinations of size distribution can be performed by fractional precipitation [28] but a more convenient technique that is commonplace in polymer analysis is size-exclusion chromatography, SEC[29].

The factors of interest in polymer composition include overall chemical composition, overall functionality, and the distribution of functionality within the polymer. Techniques such as elemental analysis or IR [30] can be used for characterization of the chemical composition of polymers, and end group analysis will yield information about functionality. In addition, different types of mass spectrometry can be used in polymer analysis [31], with MALDI-TOF MS and ESI MS being the most common. These techniques can be applied to studies of both the composition and the size of the polymers. The introduction of various

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fragmentation techniques in mass spectrometric determinations increases the information gained on polymer composition and functionality [32],[33].

Attaining simultaneous information about size and composition, or size and functionality, would be the ideal scenario, one which could be achieved by SEC [34] or gradient polymer elution chromatography (GPEC) [35].

6.1 Size-exclusion Chromatography and Mass Spectrometry

6.1.1 Size exclusion chromatography

Size-excusion chromatography, SEC, is a chromatography mode that is commonly used for separation of biomolecules and polymers, and where separation is based on size, or more accurately hydrodynamic radius, of the analyte. The column material is a porous material with pores of a carefully controlled size distribution, which surfaces are completely wettable and thus filled by the eluent. Depending on the size of the analyte it will either be able to enter the pores of the material or be excluded. Small analytes will have access to a larger volume of pores while larger analytes will only be able to enter larger pores, or none at all. The smaller the analyte, the larger the volume of eluent available and the elution time is therefore determined by the fraction of time the analyte spends in the stagnant stationary phase in the particle interior and the mobile eluent in the interstitial volume. Small analytes thus take longer time to emerge at the column outlet, whereas molecules large enough to be complete excluded are eluted as a peak early in the chromatogram. SEC places other demands on the stationary phase than other chromatographic modes since in an ideal size exclusion scheme; no enthalpic interaction should take place between the analyte and the stationary phase. Instead the separation takes place by en entropic process, which allows or prevents access to the pore space dependent on the hydrodynamic diameter of the analyte. However, complete absence of enthalpic interactions is difficult to realize and interactions between the analyte and the pore surfaces often adds to the retention, distorting the

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size distribution determination.[36]. In addition, the size-separation is not always straightforward since the analyte typically behaves differently in different solvents[37]. Because all analytes elute in a window extending from the totally excluded first eluted peak and a fully imbedded t0 marker, the possibilities of obtaining resolution is limited. Gradient elution is also not an option because of the (ideal) absence of enthalpic interaction.

When using size-exclusion chromatography to determine the size of polymers, a set-up is calibrated with a set of standards of known and narrow molecular weight. Common calibration standards available are linear polystyrenes, poly(ethyleneglycol)s, and sugars. The usefulness of size exclusion chromatography is highly dependent on the resolution achievable with the column [38], the possible solvent systems, the mode of calibration, as well as the employed calibration standards and the mode of detection. Careful choice of calibration standards is important. Ideally the standards should be as similar as possible to the analyte to be analysed with regards to chemical properties and “shape”, but this is not always possible. Size-exclusion chromatography is also valuable as a tool for purification of polymer samples, or to monitor the consumption of monomer or some other starting reagent, providing they are detectable under the separation conditions.

Common detectors in SEC are the evaporative light scattering detector (ELSD), UV-absorbance, or refractive index (RI) detectors.[39]. Direct measurements of polymer size can be obtained by the use of a multiangle laser light scattering (MALLS) detector[40], which can also be directly included in a size exclusion system[41],[40]. In addition, on-line coupling of mass spectrometric detection to a liquid chromatographic separation is possible [42] and both on-line or off-line determinations of polymer size can be performed by mass spectrometric techniques such as MALDI or ESI-MS [31,43].

6.1.2 MALDI-TOF mass spectrometry

MALDI-TOF MS, matrix assisted laser desorption/ionization time-of-flight mass spectrometry[43,44], is a technique that was originally

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developed for the analysis of biomacromolecules, but has been extensively used also in polymer analysis. End group analysis and molecular weight determinations are routinely performed. In this technique, the sample is mixed with a solution of “matrix”, and irradiated by laser pulses of a wavelength absorbed by the matrix compound which cause simultaneous vaporization and ionization of the sample. The matrix is thus present to facilitate transport of energy from the laser to the sample. The formed ions are thereafter analysed and their mass-to-charge (M/z) ratios are determined by their flight times in the instrument flight tube. MALDI represents a ‘soft’ ionization technique, producing singly charged intact molecule ions. In MALDI-TOF MS, the key factors for attaining interpretable and reproducible spectra are the choice of matrix and solvent, as well as their concentrations and the matrix to analyte ratio[45]. The optimum analyte-matrix-solvent combination is typically found via a trial-and-error approach.

6.2 Gradient Polymer Elution Chromatography

In gradient polymer elution chromatography, GPEC, also known as precipitation-redissolution chromatography, the polymer is injected onto the column when pumped with a non-solvent eluent. This causes the polymer to precipitate onto the column. A gradient with an increasing proportion of good solvent is thereafter applied so that redissolution and elution of the polymer can take place as the critical point is traversed by the gradient. Separation thus takes place because of the steep dissolution/precipitation curves of polymers in solvents of intermediate quality (Θ-solvents), according to the Flory-Huggins theory[27,37].

Depending on the material used, a number of different retention effects can be exploited in GPEC. The solvent/non-solvent combination is key to retention but the mode of the separation material used will also influence the final separations. Unlike size-exclusion chromatography, where the material should ideally be completely inert to the analyte, interactions between analyte and material could be beneficial in GPEC. The support material can be involved in interactions with the polymer, a factor that is utilised in GPEC determinations of polymer functionality[35],[46]. The redissolution of the polymer will occur as a function of solubility which

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is typically a function of polymer size, releasing the polymer from the material. As the redissolved polymers are carried along the column, interactions with the material surface occurs and retention is based on, for example the occurance of any reversed-phase characterstics within the polymer. Utilising these factors enables the separation of polymers having equal elution volumes in a SEC-set up, if there are differences in chain and/or terminal functionality. In addition, the porosity of the material might in a similar way add another dimension of size dependent separations, which may or may not be beneficial.

Gradient polymer elution chromatography has, among other things, been used for separations based on functionality[35], and for studies of polymerization mechanisms [47].

7. Synthesis of Monolithic Support Materials

7.1 The Monolith

Monolithic materials are materials that are made in one piece (compare the greek translation “single stone”) and are in current separation science either polymeric or silica-based. Materials for separation science that are made in a single piece were introduced in the late 1980’s and early 1990’s with the development of the continuous bed of Hjérten[48], the rigid polymer rod of Svec and Fréchet[49], and the corresponding silica version introduced by (Nakanishi) and Tanaka[8]. The use of the term “monolith” to describe a separation material was introduced in the early 1990s’ by Svec and has come to be a common name for both polymer and silica based materials of this type. Since their introduction, monoliths have been used in various applications and many developments have been made in the field. Polymeric monolithic materials have been used as supports in liquid chromatography in various modes. Browsing the reviews in the area shows the large diversity in preparation and applications[9,50]. Monoliths can be made by free radical polymerizations induced by either heat or irradiation [51],[52], the use of

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ring-opening metathesis polymerization [53]. The use of nitroxyl stable free radicals [54] has also been reported in monolith synthesis.

When preparing and using a monolithic material there is no need for packing since a monolithic material can be made ‘in situ’ in virtually any confinement. The need for retaining frits is also eliminated since the monolith is typically secured to the wall of its confinement by modifying the surface by polymerizable groups. These factors make the materials advantageous, especially when a small size column is needed. Monoliths are therefore convenient in a capillary columns [55] or microchip channels [56].

7.2 Controlling Porous and Surface Properties

The chemical composition of the polymerization mixture and the polymerization process are important factors in monolith synthesis since they will influence both the porous properties and the chemical nature of the material, and thereby its usefulness in a chosen application.

In a monolithic material for applications in separation science, the porous properties are essential to its function. Large through-pores are needed to sustain hydraulic or electroosmotically driven flow through the monolith structure, and it is the large through-pores formed in the materials that give rise to the desirable convective mass transfer that is a distinctive feature of the monolith[57]. Polymeric monolithic materials are made from a polymerization mixture consisting of monomers (including a crosslinking monomer), an initiator and pore-forming solvents (porogens). The properties of the resulting monolith depend on the composition of the polymerization mixture and the conditions at which polymerization takes place [50]. The starting composition is homogeneous with both initiator and monomer being dissolved but as the polymerization proceeds, polymer chains are growing to a point where they can no longer remain dissolved. Phase separation then occurs. The monolith hence “precipitates” out and forms a structure consisting of joined solid entities which make up a one-piece column-shaped porous particle. Depending on how this process occurs and proceeds; the macroporous properties of the resulting material will be different.

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Efforts have been made into understanding the factors affecting both the pore-formation and resulting material characteristics in monolith synthesis [58], and some important factors to consider have been identified [50,59] such as the properties of the initiator, the mode of initiation, the amount of crosslinker, as well as the porogenic solvent mixture and the polymerization temperature.

The ratio of crosslinker to monomer will affect the rigidity and porosity of the monolith, as will the type of crosslinker. An efficient crosslinker or a large proportion of crosslinker will yield a highly crosslinked and dense material[50]. A highly crosslinked material will result in high rigidity and a material capable of withstanding high back-pressures. On the other hand, higher levels of crosslinking will influence the porosity and result in a material with less macropores and more mesopores, often yielding a monolith with back-pressures so high that their use in flow applications in practically impossible.

The pore-forming solvents, the porogens, chosen will naturally affect the resulting material. The porogen is typically a mixture of two or more solvents, one which is better at dissolving the polymer formed. The total amount of porogen as well as the ratio of the individual solvents in the porogen mixture will also influence the final porosity and hence need to be adjusted. Polymerization is typically induced either by heat or light, and as the solubility of the monomers will be affected by temperature, the choice of porogen will be altered depending on initiator-system. In the case of styrene-based monoliths made with stable-free radical providers such as carboxy-PROXYL, polymerization is performed at 130 °C [60] which naturally puts other demands on the system than a thermally initiated polymerization at 70 °C [61]or a UV-initiated polymerization at room-temperature [52]. Although the basis for finding the required solvent combination to produce a porous monolith and its exact composition is usually very much a trial and error approach, some information can be gained from solubility of monomer in various solvents. In addition, some idea about pore-forming solvent combinations suitable for a chosen monomer combination can be extracted from the synthesis of porous particles [62].

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7.3 Introducing New Crosslinkers into Monolith Synthesis

The monomers used in monolith synthesis will influence the physical properties, such as rigidity and porosity, of the material but are mainly chosen based on their influence on the final chemical properties of the material. The starting monomers will be a part of the final material and hence, the chemical characteristics of the monomer will influence the characteristics of the final material. Common chemistries used in the preparation of monolithic materials arise from the use of either methacrylate-[63],[64] or styrene-based [65] monomers and crosslinkers.

The crosslinker and monomer take up ~20-45 % (by weight) of the polymerization mixture. They form a part of the resulting monolith, and contribute to the bulk and surface chemistry of the resulting material. Therefore, careful choice of monomer and crosslinker combination is essential. Using hydrophilic monomers adds hydrophilic characteristics to the materials, while introduction of charged monomers has been used to produce materials for electrically driven separations[63]. Once a monolith has been made it can either be used as is with the existing surface-chemistry or be further functionalized. In addition to providing desirable surface properties, the monomers can also provide reactive handles for further functionalization such as epoxy groups for preparation of enzymatic reactors [66]

In monolith synthesis, the monomer and the crosslinker are typically from the same chemical family; such as styrene being combined with divinylbenzene. In a typical polymerization mixture, ~40-70 % of the monomer mixture is crosslinking monomer, and yet, in most modifications of monolithic materials, the non-crosslinking monomer is the factor being altered. In Paper I, some efforts into the investigation of crosslinkers for methacrylate based monolithic separation media were made, aiming at monolithic capillary columns for protein separations. In the synthesis of methacrylate-based monoliths, the original recipe [67] combining butyl methacrylate with ethylene glycol dimethacrylate, has had many followers. In our study, the non-crosslinking monomer, butyl methacrylate, remained and the crosslinker, Figure 3, was altered.

18

Pentaerythritol tetraacrylate

O

O

O

O

O

O

O

OOO

OO

O

O

OO

O

O O

OO

O

O

Ethylene dimethacrylate

Triethylene dimethacrylate

Diethylene dimethacrylate

Figure 3. Structure of crosslinkers used in Paper I.

Aiming at materials suitable for applications in protein separation, the choice of crosslinking monomer fell upon a set of more hydrophilic crosslinkers with oligoethylene glycol moieties; diethyleneglycol dimethacrylate and triethyleneglycol dimethacrylate, to be compared to the traditionally used ethylene dimethacrylate, Figure 3. The final crosslinker included is one which is known to form highly crosslinked networks.

For the monolithic materials prepared using the non-traditional crosslinkers, materials with porosites enabling flow through could be attained and the porosity, as determined by mercury intrusion porosimetry, could be altered by alterations in the porogenic solvent combination of 1-propanol and 1,4-butanediol. The pressure stability of the prepared materials was assessed by back-pressure determinations and the use of the materials in a HPLC set-up was demonstrated by the reversed-phase separation of four standard proteins. This shows the existence of flow through pores and a surface that interacts with the proteins. The conclusion can be made that the investigated crosslinkers could be used in monolith synthesis in methacrylate based media and

19

used in HPLC-application. In reversed-phase mode, the retention order for the chosen protein probes remains the same, indicating that the effect of the hydrophilic part of the crosslinker, the extended ethylene glycol chain, does not result in a hydrophilic material. The retention is a result of both the hydrophobicity of the polymer backbone and the butyl chains originating from the non-crosslinking monomer used.

In a continuation of the work, aiming at more hydrophilic surfaces and a material for the softer separation mode hydrophobic interaction chromatography (HIC) [68], investigations of the effect of two different porogenic solvent systems was carried out. The polymerization mixtures were based on the crosslinkers glycerol dimethacrylate, and 1,4-butanediol dimethacrylate, and in addition to altering the crosslinker, a portion of the butyl methacrylate had been replaced by the more hydrophilic monomer 2-hydroxyethyl methacrylate [68]. The materials produced could be used in HPLC applications, but the hydrophobic nature of the material remained. In a recent study, monoliths synthesised by a poly(ethylene glycol) acrylate crosslinker were used in IEX-separation of proteins and peptides [69]. This shows the possibility of using crosslinkers of this type in less harsh separation modes than reversed-phase. In these materials the biocompatible crosslinker was combined with a charged monomer introducing the ion exchange mode of retention. However, addition of acetonitrile was still used to improve peak shapes, indicating that the polymer backbone still has an influence on analyte-material interaction.

The preparation of monoliths is convenient in that materials can be made in any confinement; their chemistry is based on what is mixed in the polymerization mixture, and the porous properties can be altered by careful choice of polymerization parameters and typically convenientially adjusted by altering the porogenic solvents. However, complex relation between the crosslinker-monomer-initiator system and the porogenic solvents is also what introduces some of the disadvantages. For every new combination of monomers, there is a need to optimize the composition of the polymerization mixture.

20

Since both the monomer and crosslinker form an integral part of the monolithic material, alterations of both types of monomers could (and should?) be used in the development of new materials. In addition, the development of post-synthesis surface-modification schemes could be a viable alternative. If the possible alterations to a prepared and characterized material could be increased, material diversity could be achieved without the need for time-consuming continuous optimisations of polymerization mixture composition.

8. Iniferter Mediated Polymerization

8.1 Living Radical Polymerization

Traditional free radical polymerization has the disadvantage of limited control over progress and result of polymerization. The initiating radicals are typically formed in a zero order reaction and hence trigger the polymerization of individual chains at different times. When the rate of propagation is fast in relation to initiation, polymer chains of highly different length are produced. This is augmented by various termination reactions, the most important of which are inter-chain radical recombination and chain transfer. In a living radical polymerization, an active and reversible chain transfer mechanism is used for controlling initiation and polymerization. Since reversible termination applies also to the growing polymer chain, the growing terminals are open to monomer incoporation (and thus termination by inter-chain radical recombination) only a small fraction of the total polymerization time. Chain propagation kinetics is thus limited by the dissociation step of the reversible termination, rather than availability of monomer. A number of different living polymerization schemes are available that have been used for the synthesis of linear and branched polymers, including atom transfer (ATRP)[70], iniferter [71] and reversible addition fragmentation (RAFT) [72]. In addition, schemes involving living radical polymerization have been used for surface modification with initiation taking place at the surface[73].

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8.2 The Iniferter Concept

The concept of iniferter mediated polymerization was introduced by Otsu in the early 1980’s [74] [75] and has been used for the preparation of linear and branched polymers, including block copolymers, in both photoinitiated and thermally initated processes and in surface-initiated polymerizations[76]. In iniferter mediated polymerization, the control over polymerization depends on the iniferter used. An iniferter is a compound which combines the role of initiator, transfer agent and chain terminator in one. The polymerization proceeds through a series of steps, as illustrated in Figure 4.

Dissociation

Polymerization

Dormant speciesX

Y

X-Y X Y

X MX

Y

+

X +

+

Figure 4. The progress of iniferted polymerization

At initiation, the iniferter is cleaved by irradiation or heat, and a radical pair is generated. Reaction of radical with monomer occurs and the monomer is incorporated into what is now the growing polymer chain. The radical pair, one member which has now become the growing polymer chain, recombines to form a dormant species. Continued irradiation or heat causes this dormant species to be repeatedly cleaved with incorporation of new monomer, and the reaction thus continues. The formation of the dromant species is essential to the livingness of the polymerization, since if the recombined dormant species is not formed, free radicals will exist that allow the polymerization to proceed as a traditional radical polymerization, with less control.

22

A number of factors have been shown to influence the progress of the polymerization in iniferter living polymerization, the extent of its livingness, and thereby the control over the end product. These factors include; the iniferter structure[77], the monomer [78], and the solvent [78] used. Steric effects contribute to the effect on polymerization by the iniferter and monomer while solubility is the factor combining the effect of monomer, iniferter, and solvent used.

Since the iniferter itself is incorporated into the growing/propagating polymer chain during polymerization, desirable end group functionality can be incorporated into the polymer[71].

8.3 Constructing a Telomer Library

Bearing in mind the aim of diversity of surface functionalized support materials for chromatographic applications, a library of telomers, Paper II, for attachment onto surfaces of chromatographic supports was constructed and characterized. Aiming at chains of controllable length, the use of living polymerizations was chosen and, more specifically, the use of a photoiniferter-mediated polymerization scheme. The iniferter chosen was isopropylxanthic disulfide, which is a photoiniferter and hence both its initiation and termination is triggered by light [79]. Isopropylxanthic disulfide has been used for the preparation of block copolymers [80] under living conditions and its reactivity compared to other photiniferters was described in some detail by Lalevée[79]]. For the purpose of attaching of the formed telomers onto a surface, a suitable end-group is needed. The chosen iniferter yields a thiol-terminated end to a polymer upon hydrolysis [81], which suits this purpose well.

23

A) D)

B) E)

C) F)

O

O

O

O

O

O

O H

O

O

O

O

O

O

O

A) BMA, B) EMA, C) MMA, D) HEMA, E) GMA, F) BzMA

Figure 5. Structures of monomers used in the initial telomer library

The monomers, Figure 5, were chosen based on obtainable diversity with the resulting polymer chains differing in hydrophobicity/hydrophilicity. The size of the resulting telomers could be altered by adjustments of the ratio of monomer to iniferter in the polymerization mixture. It was concluded that telomers of short chain length (<30 units) of the non-charged monomers could be readily prepared in solvents such as THF, DMSO, NMP and DMF. Preparations of longer chains of 2-hydroethyl methacrylate proved more difficult with precipitation occuring in both THF and DMSO. The observed linear relation of conversion versus time and polymer growth versus conversion indicates a living process.

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8.4 Gradient Polymer Elution Chromatography on Monolithic Capillary Column

Combining the use of monolithic materials with telomer characterization, Paper III, presents the synthesis and characterization of a styrene-divinylbenzene based monolithic material made by stable free radical polymerization and its use in gradient polymer elution chromatography in the capillary format. Telomers of methyl methacrylate (MMA), ethyl methacrylate (EMA) and 2-hydroxyethyl methacrylate (HEMA) were chosen as initial analytes and the selected combination of non-solvent and good solvent was water and methanol. The three tested telomer types elute at different times and their retention is different from that of their corresponding monomers and separation of monomer and telomers was thus readily achieved. This makes the experimental set-up suitable for determinations of monomer conversion which is an important factor in evaluation of polymerization processes. Unfortunately, under the employed conditions, there was no significant differences in retention for the HEMA telomers of different size in the range 10-30 units, which was attributed to overlapping distributions. In the elution profile of HEMA, a duplet peak was observed for the telomer, which was not due to overloading the column, as was originally suspected. Its origin could be determined as non-hydrolysed and hydrolysed telomer. The final step in the synthesis of the telomers is the hydrolysis, forming the thiol-terminated telomers suitable for attachment onto surfaces. Hydrolysis can be verified by adopting methods used for determinations of free thiols[82], as in Paper II, but a more convenient strategy would be a chromatographic determination.

1) Monomer and telomer elute at different times, enabling the determination of monomer and telomer concentration in mixed solutions and hence monitoring of polymerization progress.

2) Hydrolysed and non-hydrolysed telomers elute at different retention, enabling the monitoring of hydrolysis progress.

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9. Surface Modification of Polymeric Supports

As mentioned earlier, the surface characteristics are important in materials for separation science. The surface of a material depends on its preparation, and some characteristics can be controlled during initial material preparation. If the initial surface is not suitable for the intended application, a surface modification scheme could provide the answer.

9.1 General Strategies for Surface Modification

The chemical characteristics of a material surface can be altered in a number of ways, including coating strategies, grafting procedures, or reactions of surface groups with various reagents. A reaction of surface groups such as hydroxyl, epoxy, or vinyl groups is a common way to change the surface functionality. For materials with surface vinyl groups, these unsaturated moieties are excellent handles for further functionalization [83] such as bromination and subsequent addition/substitution reactions, or Friedel-Craft alkylations[84]. Using radical initiation vinyl grous can take part in a number of addition reactions. The reaction of hydroxyl groups with succinic anhydride introduces carboxylate functionality [85], while reaction with phosphoryl chloride [86] results in phosphonate groups.

Coating procedures involve either dynamically coating [87] or covalent attachment[88], but are not the only possible strategies for obtaining a layered structure. A layer of some thickness of functionality can be achieved by grafting strategies attaching monomers or polymer chains, either by “grafting from” or “grafting to”[89,90].

9.2 “Grafting from” versus “Grafting to“

Surface groups such as epoxy or vinyl groups can also be used as handles for attachment of larger groups, or as anchor points in grafting schemes. The attachment of a functional group to a surface is called grafting and can typically be divided into two subgroups – ‘grafting to’ and ‘grafting from’. Both strategies have proven useful in the modification of surfaces.

26

In ‘grafting to’[91], a pre-prepared chain is attached to the surface and the reaction can either take place directly, or aided by an external initiator. In grafting from[89], initiation takes place at the surface and the grafted chain grows from the surface. ‘Grafting to’ has the advantage that the chain to be attached can be both characterized and purified prior to attachment. In addition, the possibility of sequential grafting is an advantage. “Grafting to” has been shown to result in a more evenly grafted layer but sometimes suffers from a limited surface density. The limited surface coverage in “grafting to” might be due to sterical hindrance or diffusion limitations, if the attached chains are bulky, the first attached chain might present a sterical hindrance to the continued attachment of additional chains. To alleviate this problem, sequential grafting has been proposed, where dilute solutions are used and more graftable chains are added over time[[91]]. Grafting from has the disadvantage of the size of the attached chains not being known and controlled, however, if using living polymerizations in surface-initiated grafting the control over the thickness of the attached layer is increased[91].

9.3 Grafting of Thiol-terminated Telomers onto Chromatographic Supports

The construction of a telomer library for use in surface modification schemes of chromatographic separation materials relies also on the availability of suitable support materials that could be modified through the reactive telomer terminal. Commercially available support materials available in bulk range from polymeric particles to silica. In addition, in-house made polymeric particles[92] or monoliths[93],[94] could be possible starting materials in a surface modification scheme.

Depending on the chain to be attached, grafted, to the surface of a material, different surface groups might be more or less suitable as attachment points. In the attachment of thiol-terminated chains, suitable handles for “grafting to” onto polymeric supports are groups such as vinyl groups, an epoxy group, or any other electrophilic moiety of sufficient reactivity (Figure 6). A thiol end group can be used in an addition reaction to vinyl groups using radical initiators [95,96] or to

27

epoxy groups using bases to promote formation of the highly reactive thiolate anions[97],[98].

+ HS-R

O

O

OH

SRO

Figure 6: “Grafting to” epoxy group

If a suitable handle for functionalization is not present at the surface it might be possible to introduce one. For silica materials, different silylation reagents can be employed in simple procedures to introduce functionality, examples including reagents such as γ-MAPS[99], introducing a vinyl group. In an initial grafting test, silica particles were activated by glycidoxypropyl trimethoxysilane introducing epoxy group functionality and thereafter grafted with a butyl methacrylate telomer. The surface structure of the grafted particle and the non-grafted particle differ, as visualised in Figure 7. Elemental analysis and XPS verify the introduction of sulfur and hence grafting, but no chromatographic evaluation has been performed.

2µm2µm

a)a) b)b)

Figure 7: SEM Micrograph of non-grafted and grafted silica particles

28

9.4 Preparation of a Cation Exchange Material via “Grafting to”

An example of a ‘grafting to’ scheme, presented in Paper IV, is the synthesis of an ion exchange material for protein separation. The prepared material is based on grafting of thiol terminated telomers onto highly crosslinked divinylbenzene particles which were prepared in-house by precipitation polymerization [92].

The photoinitiated precipitation polymerization, that has been developed by a fellow PhD student of mine, yields polymeric particles of high rigidity in a size range suitable for HPLC-applications [92]. The high rigidity and chemical resistance of these polymeric particles make them suitable for HPLC applications and their pH-resistance give them a distinct advantage over silica particles. However, their aromatic composition results in a highly hydrophobic surface which limits their use without prior surface modification, especially if aiming for soft separations of proteins. In order to increase the diversity of possible applications, the surface modification scheme was started by an attempt to increase the hydrophilicity of the surface. This was achieved by oxidation of vinyl groups into epoxy-groups, by an adaptation of the procedure of Fréchet[100]. The oxidation of vinyl groups also represents an activation step by which reactive epoxy functionality is introduced onto the surface. These epoxy groups present a convenient handle for subsequent surface modification due to their reactivity towards amines, hydroxyl groups, and thiols[101]. The epoxy-groups are themselves of moderate hydrophobicity but subsequent hydrolysis yields a highly hydrophilic 2,3-dihydroxypropyl functionality. In the “grafting to” step, a thiol-terminated telomer of sulfopropyl methacrylate was grafted to the surface epoxy groups by nucleophilic addition of the corresponding thiolate anion. This resulted in the attachment of the grafted chain, and simultaneous ring opening of the epoxy group. In the final step of the synthesis, remaining epoxy groups were hydrolysed to yield diol functionality, thus increasing the hydrophilicity of the unreacted surface. Thus, the preparation relies on polymeric supports with known characteristics and a series of surface modification steps to introduce the desired surface functionality.

29

The introduction of sulfur could be verified by elemental analysis and XPS shows the introduction of sulfur at the particle surface, as well as the presence of carbonyl functionalities, which was expected from the telomer structure. Verifying the introduction of functional groups is essential, but more important is the end result, the chromatographic evaluation. Starting with a hydrophobic particle we expected substantial residual hydrophobic interactions of proteins with the final material. Fortunately, this is not the case over a wide range of salt concentrations. In combination with the separation of four proteins in ion exchange chromatography mode, in accordance with their individual pI, and the eluted protein peak showing no signs of denaturing upon incubation on the column, we can conclude that the surface modification scheme has resulted in;

1) increased hydrophilicity of the polymeric particle surface and sufficient reduction in the hydrophobic interaction between protein and material surface,

2) introduction of ion exchange functionality to the surface via the “grafting to” procedure, and

3) a material that shows promise as separation materials for soft separations of proteins.

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10. Concluding Remarks and Future Aspects

The column material or chromatographic support is essential in liquid chromatographic separations and the worlds of material synthesis and separation science are therefore highly interconnected. In designing new materials both the properties of the bulk materials and the surface are central and new strategies for obtaining materials with controllable properties and tools for their characterization are always needed.

The results from the preparation of monolithic materials where non-traditional crosslinkers were used exemplify the possibility of extending a known recipe by simple means and thereby gain new (and improved?) materials. The need for optimization for each new set of monomer and crosslinker is however a drawback of ‘single-pot’ monoliths and the combination with surface modification schemes are therefore of interest.

In the development of new materials, attaining a wide range of surface properties from the same support is an advantage and can be attained by exploiting various grafting or surface modification schemes. The surface grafting of materials via “grafting to” of short polymer chains, telomers, could result in complex materials and surface compositions, but the success relies both on control in the preparation of the telomers, and on reliable characterization of both the telomers and the grafted surface.

For the characterization of the prepared telomers, the use of gradient polymer elution chromatography could be further exploited in the characterization, especially in the characterization of co-telomers, synthesized from two different monomers in a sequential manner, which have been prepared. but not fully characterized in this work.

In the continuation of the project, extension of the library, further developments into its characterization and the attachment onto different chromatographic supports would be of interest.

The use of ‘grafting to’ in surface modification is by no means the only possibility to get new materials - but the initial examples given in this thesis shows its usefulness. The use of living radical polymerization

31

schemes enables the control over polymer chain length and functionality and in combination with proper characterization and careful choice of attachment chemistry, a wide range of materials can be prepared.

“There is no end to the possibilities!?”

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“Don’t make fun of graduate students. They only made a bad career choice “ Marge Simpson

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12. Acknowledgments - mitt djupt kända tack! Slutligen är den då här, den mest lästa delen i avhandlingen! Och definitivt inte den lättaste att få ned på pränt. Om jag i den allmänna förvirringen glömt någon....... förlåt....... De senaste månaderna har inte varit roliga. Jag vill inte ens tänka på hur många timmar jag tillbringat inom universitetets väggar den senaste tiden för att få den här skriften att bli klar - och då har jag ändå inte latat mig under de tidigare åren heller. Sannerligen en prövning. Men, det blev en bok till slut. Och, så sant som det är sagt: ”Det som inte dödar härdar”.1 Så.... härdad är jag. Och så fick jag citera Nietsche också...... inte varje dag man gör det. Det är många människor man träffat på under de här åren och som på något vis påverkat det man gjort och inte gjort, och många är det som jag vill passa på att tacka; familjen, vännerna, arbetskamraterna....... Många tack vill jag ge till tidigare och nuvarande doktorander inom det som tidigare kallades ‘analytisk kemi’: Erik (se, du kom först!;)), Johanna, William, Josefina, Tom, Lars, Daniel, Dong, Yvonne, Sofi, samt givetvis platsknuttarna: Tobias, Wen, Julien, Petrus, Erika, Fredrik, Emil, Mai och Nhat. And also- thank you - to past and present postdocs; Meenakshi, Michal, Gerd, and Jeroen. A special “Tack så mycket” to Gerd for all the fun times in Umeå and everywhere - remember the word? “Tryna”. To Jeroen - for NMR help and for your patience in reading this thesis (and various other texts) – the only one to read other parts than the acknowledgments? Tack också till den f.d plastknutten – Patrik: handledare för ”strutsprojektet” för att du visade hur handledning kan (och ska?) gå till. TACK Anita och Ann-Helén - för all hjälp genom åren med det administrativa – särskilt tack till Anita - du är en klippa! Tack också till veteranerna: Michael, Anders, Wolfgang och Svante Å. Och ’nykomlingarna’ på plan 5: Roger och Olle. Tack Solomon: för att du någon gång under våren 1998 öppnade mina ögon för att det här med kromatografi – det är roligt det!, Svante J –tack för all hjälp med

1 Ecce Homo, Friedrich Nietzsche, 1888

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fixande och prylbyggande, Lasse - tack för hjälp med det elektriska och för trevligt småprat om Laos. De exjobbare som jag haft nöjet att handleda och jobba tillsammans med vill jag också säga tack till – ni har fått mig att lära mig mycket. Framför allt vill jag tacka de mycket smarta, trevliga, starka och ambitiösa tjejerna - Annamaria och Ida – jag önskar er all lycka i framtiden! Jag tror på er! Särskilt tack också till Ida för all peppning i Belgien och de roliga slagsmålen på IKSU. Snart dag sför en match igen? Tack också till övriga studenter, MFSare och andra, som passerat labbet genom åren och på ett eller annat sätt fått stå ut med handledning av mig - Av att (försöka) lära ut lär man sig själv mer än man anar. Mentorskapsprojektet: övriga adepter för många intressanta diskussioner och reflektioner samt för de lunchträfffar som blev av. Alla mentorer som deltog och framförallt ett stort och varmt TACK till min mentor Britt Inger Högberg för alla givande diskussioner. Det var en lyx att ha dig som min mentor. Tack också till alla andra jag träffat på under åren och som bidragit med glada skratt, gott humör, instrumenthjälp, träningssällskap, korrekturläsning av diverse alster, lunchsällskap, timmar på rött, och annat. Mina f.d kursare som gjorde Umeå till en bra plats att vara på första, andra och tredje gången – Katrin, Anna, Ulrika, Åsa G, Lisa och Karin. Mina fd arbetskollegor på STFI som gjorde att jag trivdes så bra och inte minst Anna och Olof mina handledare på exjobbet och sedermera kollegor. Ni fick upp mina ögon för att det här med forskning – det är ju kul det! Tack för att jag fick testa det ett tag hos er. Ola- det har varit kul och lärorikt att kämpa mot ’överheten’ – kul att du gjorde mig sällskap! Ett jättestort TACK till de härliga tjejerna på våningen ovanpå – Åsa, Hanna och Malin – det har varit kul att lära känna er! Åsa: Tack för träningssällskap och allmän supportverksamhet! Hanna och Malin – verkligen jättetack för matlådorna – ni anar inte hur mycket jag uppskattar det! Vad som än händer – jag tror på er! Tack också: Andrey Schukarev; for never-ending patience with XPS-measurements and interpretations, John Loring; for all the help with

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titration set-ups and programming, och Per Hörstedt; för alla SEM-bilderna. Tack vill jag säga också säga till Knut, min huvudhandledare. Tack för att du dök upp den där dagen på STFI i Stockholm och på något vis lyckades övertyga mig om att det där med att vara doktorand i Umeå, det kanske var en bra idé trots allt. Det har varit lärorikt. Ibland hemkst, men också hemskt lärorikt. Tack för att du anställde mig som doktorand i ett intressant projekt och för finansiellt stöd genom denna utbildning. Och speciellt för den välbehövliga lilla förlängningen på slutet. Tack också för att du nästan alltid tyckte att det var en bra idé att låta mig åka på konferens! Och stort tack för att du fick mig att åka till Berkeley – det gav mig mycket! Jonas Bergquist, Uppsala Universitet, min biträdande handledare, tack för stöd och hjälp och för trevliga stunder i både Uppsala och Umeå! Tack också till resten av Uppsala-gänget: framförallt Björn Arvidsson, Lotta Hagman och Per Håkansson. För hjälp med MS-körningar, trevligt sällskap i bunkern på Ångström och i Uppsala i övrigt, och såklart även i Umeå, och tack för att jag fick leka med FT-ICRn. Och till Björn –Lycka till i april! Tack också till Camilla och Tobias för bra input på slutet – tänk vad en galen idé en måndag på lab kan ge upphov till.... Tack också till Camilla för att du ställde upp och läste det allra sista när Knut försvann.

A special thank you is due to Prof. Frantisek Svec and Prof. Jean M. J. Fréchet, University of California, Berkeley and Lawrence Berkeley National Laboratory, for welcoming me into your labs and for giving me support through what for a long time seemed like the most productive 7 months of my PhD studies. Thank you to the rest of the Berkeley troops: Emily, Dominic, Dean, Quanzcho, Tim, Dewey, Kelly and Bas. Working with you all was a great experience! And let’s not forget the girlz at campus: Sarah, Kathy, and Carine. All of you, in your own way, made my stay in the Bay area a pleasant and memorable one. And I did learn some chemistry….

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Lite finansiellt stöd har också varit bra att ha under åren, inte minst för utbyten och konferensdeltagande: Under min andra termin som doktorand stack jag till USA: tack till Teknisk-Naturvetenskaplig fakultet, Umeå Universitet, för stipendium under utbytesterminen, vårterminen 2003, vid University of California, Berkeley, som gjorde det hela möjligt. Teknisk-naturvetenskapliga fakulteten, för bidrag från Stiftelsen J C Kempes Minnes Akademiska fonder I och II – till resebidrag för konferensen HPLC 2006, San Francisco, USA. The HPLC 2006 Symposium, is gratefully acknowledged for financial support given covering the registration fee and a travel award for the conference HPLC 2006, San Francisco, USA. Rectors Wallenbergmergmedel vid Umeå Universitet, för resebidrag för konferensen HPLC 2007, Ghent, Belgien. Ett särskilt extra stort TACK till de underbara människorna: Josefina: för peppning och välbehövliga kaffepauser. Ser fram emot chokladfabriken!! Petrus (truly ruggedly handsome) - min årsbroder som doktorand - för allt - inte minst stödet på lab och för galenskaper i Berkeley och annars, och naturligtvis för god chili! Sofi: tack för allmän crazy-dancing och trevligt sällskap inklusive lördagspizza i ett grått vinter Umeå. Och såklart för fruktkorg och annan välbehövlig support. Jag kommer tillbaka om sisådär 3-4 år och gör detsamma för dig?! Tobias: tack för den mycket välbehövliga axeln, stödet och sanningarna på slutet. Du är för snäll du! Och slutligen, sist men inte minst – mina hjältar - Fredrik och Emil. TACK och alla bamsekramar i världen till er! Utan er hade jag ärligt talat inte klarat det här. Och haft väldigt väldans tråkigt på vägen. Kul är det att jobba med er och jag gör det gärna igen. Vi kanske kan göra något om nitrobensener? Vissa människor är bara så himla bra – och ni är sådana människor! Er kommer jag VERKLIGEN att sakna!

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Alla andra vänner, bekanta och släkten – tack för att ni finns och visar att kemi verkligen inte är allt.... Slutligen att stort TACK och en bamsekram till min familj: Marie, Sten och Daniel Nordborg: Lill-mamma och storpappa och plutt-Daniel. Tack för att ni är ni, att ni gjort mig till den jag är och för att ni alltid, alltid funnits och finns där. Världens bästa föräldrar och världens bäste lillebror! Jag älskar er!

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Säg ett ord som blir bevingat Sätt en flagga på en topp Spring på under 3 min Ett 1500-meters lopp Vi kommer aldrig att dö

Bo Kaspers Orkester, Amerika

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