Monolithic Separation Media Synthesized in Capillaries and their

Applications for Molecularly Imprinted Networks

by

Julien Courtois

Department of Chemistry Analytical Chemistry

Umeå University

©Copyright, Julien Courtois 2006. All rights reserved ISBN 91-7264-207-6 Printed by: Print och Media, Umeå University, UMEÅ. Cover illustration: Antoine Moreau Distribution: Department of Chemistry – Analytical Chemistry Umeå University, SE-901 87 UMEÅ, Sweden. Tel: +46 (0) 90-786 5000 E-mail: julien@julien-courtois.com

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To my parents and sisters

To my beloved wife …

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Preface

During the time I did my PhD Thesis in Sweden, people were keeping on asking me “Why Umeå?” … and the only good answer I found was: “Why not…”. So, it might be late but let’s make this clear once for all! I have spent 4 months searching for jobs in 2002, and was not sure about what to look for (young people…). I searched for PhDs as well, and answered to a few open positions. I did it for Umeå, without really considering it (like many people do), and in fact did not even remember when my supervisor, Knut, sent me a mail to say that I was “one of the chosen”. I checked on a map where Umeå was situated, got freaked out, and finally, decided that it would be a nice experience. So, I came here and started my thesis … not more complicated than that.

I would like to point out something really important (at least for me): I came from a more organic chemistry background, and had to start on analytical and polymer chemistry. Even more, I started that project dealing with MIPs in capillaries using micro HPLC, and there was no real experienced person in any of these fields in the department. So it has been quite a lot of new things to initiate and work with. That is one of the reason why this thesis is dealing with many fields. It just represents the problems and solutions over my way during this thesis (thus the subtitle on the cover). So, instead of studying only and deeply molecularly imprinted polymers, I have preferred varying fields and this makes that thesis more like overall information on monolith synthesis, characteri-zations and applications. It is, thus, important for the reader to understand that it was on purpose. I did that thesis in order to get knowledge in many fields and not be restricted by my main thesis subject, and I feel having somehow reached my goal (your turn to decide now).

During that time, I also discovered a country rich in people (I will never forget that myth about Swedish girls is in fact almost true), food (the word that can summarize this experience may be “köttbullar”), and Swedish traditions1, etc…. I hope that this thesis is written in such way that not only MIPs people can understand it (even the cheerleaders in the back of the dissertation room … no, I have nothing against cheerleaders). I also hope that you will enjoy the “not-classical” way (probably more close to who I am, and less to the Swedish traditions) I wrote it. I wish you a good reading and thank you for your upcoming attention. 1 http://crap.dawnshadow.se/5_kompletter_wahnsinn.mpg

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Title: Monolithic Separation Media Synthesized in Capillaries and their Applications for Molecularly Imprinted Networks

Author: Julien Courtois, Department of Chemistry – Analytical Chemistry, Umeå University, SE-90187 Umeå, Sweden.

Abstract: The thesis describes the synthesis of chromatographic media using several different approaches, their characterizations and applications in liquid chromatography. The steps to achieve a separation column for a specific analyte are presented. The main focus of the study was the design of novel molecularly imprinted polymers.

Attachment of monolithic polymeric substrates to the walls of fused silica capillaries was studied in Paper I. With a broad literature survey, a set of common methods were tested by four techniques and ranked by their ability to improve anchoring of polymers. The best procedure was thus used for all further studies.

Synthesis of monoliths in capillary columns was studied in Paper II. With the goal of separating proteins without denaturation, various monoliths were polymerized in situ using a set of common monomers and cross-linkers mixed with poly(ethylene glycol) as porogen. The resulting network was expected to present “protein-friendly pores”. Chemometrics were used to find and describe a set of co-porogens added to the polymerization cocktails in order to get good porosity and flow-through properties.

Assessment of the macroporous structure of a monolith was described in Paper III. An alternative method to mercury intrusion porosimetry was proposed. The capillaries were embedded in a stained resin and observed under transmission electron microscope. Images were then computed to determine the pore sizes.

Synthesis of molecularly imprinted polymers grafted to a core mono-lith in a capillary was described in Paper IV. The resulting material, imprinted with local anaesthetics, was tested for its chromatographic performance. Similar imprinted polymers were characterized by microcalorimetry in Paper V. Finally, imprinted monoliths were also synthesized in a glass tube and further introduced in a NMR rotor to describe the interactions between stationary phase and template in Paper VI.

Keywords: bupivacaine, etching, fused silica capillaries, isothermal titration calorimetry, local anæsthetic, molecularly imprinted polymer, monolith, nuclear magnetic resonance, phosphorylated tyrosine, poly(ethylene glycol), silanization, transmission electron microscopy.

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List of Abbreviations

Chemicals BV Bupivacaine DMF Dimethylformamide DPPH Diphenylpicrylhydrazyl EDMA Ethylene dimethacrylate GMA Glycidylmethacrylate LA Local anæsthetic MAA Methacrylic acid MV Mepivacaine NMP N-methyl-2-pyrrolidone PEG Poly(ethylen glycol) PEGPEA Poly(ethylene glycol) phenylether acrylate PMP 1,2,2,6,6-pentamethylpiperidine RfBP Riboflavin binding protein RV Ropivacaine TEATFB Tetraethylammonium tetrafluoroborate TEGDMA Triethylene glycol dimethacrylate TRIM Trimethylolpropane trimethacrylate γ-MAPS 3-[(methacryloyl)oxypropyl]trimethoxysilane

Techniques AFM Atomic force microscopy BET Brunauer-Emmett-Teller nitrogen absorption/desorption CP Cross polarization HPLC High performance liquid chromatography HR/MAS High resolution/Magic angle spinning ITC Isothermal titration calorimetry NMR Nuclear magnetic resonance PLS Partial least square regression SEM Scanning electron microscopy STD Saturation transfer difference TEM Transmission electron microscopy UV Ultraviolet XPS X-ray photoelectron spectroscopy

Miscellaneous CLD Chord length distribution Hg-MIP Mercury intrusion porosimetry IF Imprinting factor MIP Molecularly imprinted polymer NIP Non imprinted polymer SA Surface area UV Ultraviolet

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This thesis is based on the following papers, referred in the text by their corresponding Roman numerals:

I. A Study of Surface Modification and Anchoring Techniques used in the Preparation of Monolithic Micro-columns in Fused Silica Capillaries Julien Courtois, Michal Szumski, Emil Byström, Agnieszka Iwasiewicz, Andrei Shchukarev and Knut Irgum Journal of Separation Science, 2006, 29, 14–24.

The author was responsible for the experimental work, except XPS and AFM, and for writing the paper.

II. Novel monolithic materials using poly(ethylene glycol) as porogen for protein separation Julien Courtois, Emil Byström and Knut Irgum Polymer, 2006, 47, 2603-2611.

The author was responsible for the experimental work and for writing the paper.

III. Application of Transmission Electron Microscopy for Direct Determination of the Macropore Structure in Monolithic Columns Julien Courtois, Michal Szumski, Fredrik Georgsson and Knut Irgum Analytical Chemistry, in press, 2006.

The author was responsible for the experimental work except image treatment and TEM acquisition, and for writing most part of the paper.

IV. Molecularly imprinted polymers grafted to flow through poly(trimethylolpropane trimethacrylate) monoliths for capillary-based solid-phase extraction Julien Courtois, Gerd Fischer, Börje Sellergren and Knut Irgum Journal of Chromatography A, 2006, 1109, 92-99.

The author was responsible for the experimental work and for writing the paper.

V. An Artificial Riboflavin Receptor Prepared by a Template Analogue Imprinting Strategy Panagiotis Manesiotis, Andrew J. Hall, Julien Courtois, Knut Irgum and Börje Sellergren Angewandte Chemie Internationale Edition, 2005, 44, 3902–3906.

The author was responsible for the part related to calorimetry and for writing the corresponding part in the paper.

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VI. Interactions of Bupivacaine with a Molecularly Imprinted Polymer in a Monolithic Format Studied by NMR Julien Courtois, Gerd Fischer, Siri Schauff, Klaus Albert and Knut Irgum Analytical Chemistry, 2006, 78, 580-584.

The author was responsible for the monoliths synthesis and for writing parts of the paper. Manuscript not presented in the thesis:

Chromatographic comparison of bupivacaine imprinted polymers prepared in different formats: crushed monolith, microspheres, silica-based composites and capillary monolith. Joakim Oxelbark, Cristina Legido-Quigley, Ersilia De Lorenzi, Carla Aureliano, Maria-Magdalena Titirici, Eric Schillinger, Börje Sellergren, Julien Courtois, Knut Irgum, Laurent Dambies, Peter Cormack, David Sherrington, Analytical Chemistry, submitted September 2006.

The author was responsible for the capillary monoliths synthesis and for writing the related part of the paper.

Paper I and V are reprinted with permission from Wiley (Copyright 2005-2006). Paper II and Paper IV are reprinted with permission from Elsevier (Copyright 2006). Paper III and Paper VI are reprinted with permission from American Chemical Society (Copyright 2006).

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Table of Contents 1. Introduction.......................................................................................................... 1 2. Miniaturization ..................................................................................................... 3

2.1. Introduction to monolithic capillaries columns.......................................... 3 2.2. Introduction to capillary pre-treatments ..................................................... 3

2.2.1 Variety of treatment............................................................................... 4 2.2.2 Ways of measuring validity ................................................................... 5 2.2.3 Results and future aspects ..................................................................... 6

3. Monolithic Material.............................................................................................. 9 3.1. Synthesis of monoliths ................................................................................ 10

3.1.1 Main goal.............................................................................................. 10 3.1.2 Improvements of the porous properties............................................. 11 3.1.3 Use of poly(ethylene glycol) as a pore template................................. 11

3.2. Optimization by chemometric means........................................................ 12 3.2.1 Basis of chemometry............................................................................ 12 3.2.2 Use of chemometry to understand monoliths ................................... 12

3.3. Applications of monoliths .......................................................................... 13 3.4. Characterization of the monolith porous properties ................................ 14

3.4.1 Mercury intrusion porosimetry .......................................................... 14 3.4.2 Transmission electron microscopy..................................................... 16

4. Molecularly Imprinted Polymers....................................................................... 19 4.1. Introduction to MIPs .................................................................................. 19

4.1.1 Basis of MIP formation ....................................................................... 19 4.1.2 Approaches........................................................................................... 20 4.1.3 Application areas ................................................................................. 20

4.2. Templates versus monomers ...................................................................... 21 4.2.1 The template ........................................................................................ 21 4.2.2 Monomers and cross-linkers............................................................... 21

4.3. Synthetic strategies ...................................................................................... 22 4.4. MIPs for bupivacaine and homologs ......................................................... 22

4.4.1 Properties of local anæsthetics ............................................................ 22 4.4.2 Choice of experimental procedures .................................................... 23 4.4.3 Retention properties of the MIPs for bupivacaine and homologs.... 24

4.5. Phosphorylated tyrosine ............................................................................. 27 4.5.1 Protein phosphorylation and its role in biology ................................ 27 4.5.2 Experimental MIP................................................................................ 27 4.5.3 Results obtained with the pTyr MIPs ................................................. 28

4.6. MIP evaluation and characterization techniques ...................................... 31 4.6.1 Isothermal titration calorimetry ......................................................... 31 4.6.2 Nuclear magnetic resonance ............................................................... 33

5. Concluding remarks and future aspects ............................................................ 39 6. Acknowledgments............................................................................................... 40 7. Literature Cited ................................................................................................... 42

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1. Introduction

When searching for MIP on the new world Bible named Google, the first answer given is “Minor In Possession” which Washington official state government mainly describes by the following “When a person age 13-17 signs a diversion agreement or is convicted of possession of alcohol or convicted of any offense involving a firearm, whether or not it is related to using a motor vehicle…”.

However, less people may know that MIP can also be the abbreviation for:

- Molecularly Imprinted Polymers

- Mercury Intrusion Porosimetry

Therefore I decided to study some aspects of these two other possibilities to prove that abbreviations can be source of misunderstandings! To really understand what these two MIPs are, it was needed to start from basics; that is all what science is about, right? So, here is the way to this under-standing and as Trinity said “Follow the white rabbit…”1.

Among all chromatography techniques, allowing the physical separation of components by their distinct distribution between two phases, liquid chromatography is one of the oldest separation technique2,3 that still has a major role in science. As described by its name, this technique employs a liquid phase to separate components. The broad area of chromatographic methods and the number of possible applications and needs have always required new improvements. As chromatographic systems have evolved, much work has been devoted to the synthesis of new stationary phases. With the increasing use of polymer chemistry in science, scientists decided to create their own columns based on organic synthesis. Among all these schemes, monoliths4 are among the most promising since studies have proven that their mass transfer can be superior to classical columns. These materials are normally synthesized by polymerization of various mono-mers mixed with one (or more) solvent called porogen, resulting in a one-piece macroporous structure with flow-through properties. The first use of monolith was actually to crush them and further pack them into columns, but the need of frits and the irregularity of the crushed pieces made in situ polymerized monolith an even more attracting possibility.5

With the demand in small scale analysis and environmental issues calling for reduction in the use of organic solvents as eluent, the monolithic for-mat was soon down-scaled to capillaries. However, this was not straight-

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forward as polymer phases does not attach naturally on pure silica walls. The first problem of this study was then focused on providing a good attachment of a monolith in a capillary.

With that problem solved, synthesis of various new monolithic stationary phases was possible. The need for a benign separation material for proteins in a more “friendly” way, with less folding, drove to the second step, allow-ing the stationary phase to be synthesized with poly(ethylene glycol)6 as porogenic solvent.

This study showed that characterization techniques other than chromato-graphy are needed in order to improve and optimize the polymerization conditions. Mercury intrusion porosimetry, commonly used by scientist in this field, became a good tool in these efforts, but it also showed its limita-tions. Thus, a new method had to be found to characterize monoliths in a better way and transmission electron microscopy was found to fulfil this role.

As the main goal of liquid chromatography is to accomplish separation of components, it is also necessary to point out that both efficiency (how well two components can be separated) and selectivity (how well one compo-nent or a family of components can be fished out from the solution) are sough by the chromatographers. The lack of high selectivity in common chromatographic media has lured scientists into a trendy scheme called molecularly imprinted polymers. This technology was revealed to play the role of man-made mimics7 of antibodies and two applications were created and successfully used with micro-HPLC.

But once again, the possibilities of characterization of these special mate-rials were limited and, as more powerful methods were available, our study led us to utilize nuclear magnetic resonance and isothermal titration calorimetry and good results were obtained.

The goal of this thesis is not to show the best and advanced applications of molecularly imprinted polymers, neither to describe the synthesis of a novel stationary phase or the utility of new visualization methods of capil-laries, but to show a negligible part of the complicated knowledge path that I took to create, improve, evaluate, understand and characterize a poly-meric material designed for analytical chemistry means.

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2. Miniaturization

In scientific and industrial settings where samples are more and more complex and often available in low quantities, it is necessary to size down the separation techniques. Szumski et al.8 gave a good overview on miniaturization of separation techniques showing advantages of micro systems compared to normal HPLC9 (e.g., less solvent use, better detection, facile coupling to MS, etc.). Microcolumns have been introduced in the late 70’s10,11 to fulfil this demand and are widely used in many applications nowadays.

2.1. Introduction to monolithic capillaries columns

Among problems related to packed capillaries are the difficulties to establish a homogeneous bed12 and to sinter retaining frits13 inside the capillary in order to prevent particles from being released into the detect-ion system. Therefore, continuous polymer rods, also called monoliths (further discussed in section 3.)14-16, are one of the best alternatives. Never-theless, compared to packed columns, porous polymer rods have a major drawback due to their shrinking proclivity.17

2.2. Introduction to capillary pre-treatments

One major issue in the manufacturing of monolithic capillary columns is the poor reproducibility.18,19 In fact, when comparing to silica particles synthesized on a large scale where packing is almost the only cause of irreproducibility of the finished columns, monoliths have to be prepared individually. Moreover, it is not only the monolith structure and surface properties that can be affected by polymerization conditions beyond control, also its attachment to the capillary can be source of problems (see Figure 1). If not properly attached to the silica wall, gaps can be formed between the continuous polymer rod and the wall. Thus, shrinkage of the monolith17 and formation of annular void channels20,21 should be avoided during polymerization by a solid attachment to the wall. Therefore, before considering the monolith itself, it was necessary to establish a reliable technique to ascertain a stable covalent attachment to the capillary wall.

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Figure 1. SEM showing problems observed in capillaries. On the left a monolith detached

from the wall. On the right a γ-MAPS polymer deposited on the wall. (From Paper I)

Surface properties of glassy materials have been studies for long22, and techniques for establishing covalent attachment of olefinic polymers are plentiful in the literature (see references cited in Paper I). A full survey leads to a relatively large number of procedures with large and sometimes surprising discrepancies among the methods. Collecting works that have been published over the years and classifying these methods to apply some challenging tests to each of them in order to find the preferred ones and discard the others was done in Paper I.

2.2.1 Variety of treatment

Surface treatment of siliceous glassy materials is a broad area covering fields like glass fabrics23, glass plates24, microchips25, soda glass26 or capilla-ries27. The variety of attachment mediators used for that purpose is there-fore also broad, and related to the combinations of material and applica-tion in each case. Focusing a study only on silica surfaces that are widely used for monolith synthesis and using the most common anchoring mole-cule was chosen in Paper I. Indeed, fused silica capillaries treated with the more common silanization reagent γ-MAPS (see Figure 2) gather this condition.

As described in Paper I, over 90 different procedures dealing with mono-lith synthesis in capillaries and using γ-MAPS as anchoring group on the silica surface were found. This set of scientific works (Table 1) describes variations within the etching and the silanization procedures. Some re-searchers did a non-exhaustive study of few of these procedures28, or only on the first step of etching29, with the purpose of increasing the surface area and the roughness of the capillary, but no study on the whole body of surface treatment was found.

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SiO

O

O

O

O

Si

O

O OH

O

Si OHO

O

Si

O

OHO

Si OHO

O

+

Si

O

O O

O

Si OO

O

Si

O

OO

Si OO

O

Si

Si

(CH2)3

(CH2)3

O

O

O

O

Figure 2. Principle of silanization by γ-MAPS

Whereas a lot of researchers cite the excellent work of Hjertén30 in 1985, they also often give a very different experimental section truly inconsistent with the original work. Moreover, mistakes were found to be perpetuated year after year because of inaccurate use of procedures from the literature, giving even more credit to the publication of Paper I.

2.2.2 Ways of measuring validity

When trying to test the attachment strength of a monolith to a capillary surface for further use in chromatography, Vidic et al.31 have shown that a common polymerization procedure producing a continuous porous poly-meric rod combined with simple measurements such as back pressure or retention of compounds were applicable tests. However due to the docu-mented difficulties of preparing polymer rods with high reproducibility19, the accuracy of the results could be subject to criticism.

An alternative way of testing this attachment was therefore used in Paper I to eliminate the variability inherent to monolith synthesis. A plug of rigid polymer was polymerized inside the capillary, and the pressure was mea-sured in a dead-end mode. The pressure build-up was recording up to a predetermined limit of 25 MPa (limited by the pump) or until the plug de-tached from the wall and started moving inside the capillary.

Wetting angle has for long been a common test28,32 to measure properties of glass surfaces.22 This fast and simple measurement based on the well-known contact angle equation applied to capillary rise has been proved to be reliable, and can be directly linked to the silanization process. Then, new surface characterization methods (XPS and AFM) are also available for understanding of glass surface, and composition. Finally, scanning electron microscopy (SEM) widely used to visualize monoliths (Figure 1) complete the set of techniques used in Paper I.

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These techniques are briefly summarized below:

- Plug adhesion test: a 2 mm long plug of polymer was synthesized in the capillary and subjected to hydraulic pressure. The pressure curve was recorded, showing either a partial or total detachment after a few minutes, or a resistance to more than 25 MPa back pressure, considered as successful limit.

- XPS measurements: surface of the capillary was scrutinized under X-ray photoelectron instrument, and ratios of carbon to silica were measured to describe the proportion of γ-MAPS anchored at the surface.

- Wetting angle measurements: the treated capillary was immersed in water and rise of the liquid was recorded and related to its corre-sponding contact angle, according to the equations in Paper I.

- AFM measurements: 3D roughness of the capillary was recorded by atomic force microscope working in Tapping Mode™.

- SEM imaging: micrographs of monolith in the capillary and the etched surface of silica wall were recorded.

It is to notice that many of these techniques require an almost flat surface on the micrometer scale. Therefore, a large inner diameter capillary had to be used. Moreover, some of typical detachment behaviours may be directly visualized when the format is big enough to allow visual observation. Reproducibility also implies that as many external parameters as possible should be kept constant. Another recent study has also addressed this issue31, but their results were complicated by the use of various kinds of glass surfaces, whereas Paper I focused only on fused silica capillaries.

To ascertain reproducibility, a batch of 50 capillaries of 1 meter length and 1.0 mm i.d. was ordered from the company most widely cited in scientific literature as source of silica capillaries and used throughout this study.

2.2.3 Results and future aspects

Table 1 lists the procedures used in Paper I, chosen from literature for their pertinence and the number of times cited. As seen, some conditions are quite forceful (e.g., etching for 3 h in 1 M NaOH at 120 °C) whereas other are much more moderate (e.g., 30 min in 0.2 M NaOH at RT).

One major concern is the solvent used in the silanization step. Procedures using water were found to yield low attachment strength. Presence of water also increased the propensity for the silanization agent to polymerize (see Figure 1). It was also discovered that oligomerization took place during

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high temperature silanization procedures. DPPH is a polymerization inhi-bitor of the stable free radical type, capable of preventing this spontaneous polymerization of methacrylic groups. However, it is very tricky to handle, especially because of a very fast and strong aging. A joint visualization of the results from all three main characterization methods (XPS, adhesion test and wetting angle) is shown in Figure 3, enabling identification of the optimal conditions in a single glance.

Table 1. Summary of etching and silanization procedures used (From Paper I).

Etching [NaOH]a) Temperature Time HCl flushingb) Drying conditions Procedure (M) (°C) (min)

E1 1 23 30 0.1M, 30 min N2 gas at RT, 1 h E2 1 120 180 N Oven at 120°C, 1 h E3 0.2 23 30 0.2M, 30 min N2 gas at RT, 1 h

Silanization Solvent γ-MAPS Waterc) Acetic acidd) Temp. Time DPPHe) Waterf) Dryingg) Storageh) Procedure ( % v/v) (pH) (ºC) (h) (% w/v) rinse

SM1 MeOH 50 No - RT 24 – Yes N2 SM2 MeOH 50 No - RT 24 – No N2

SE EtOH 20 5% 5 RT 1 – No N2 24h at RT

ST1 Toluene 10 No - RT 2 – No N2 ST2 Toluene 10 No - 120 24 0.02 No N2

SW1 Water 0.4 Yes 3 RT 1 – Yes – In water SW2 Water 0.4 Yes 3 60 20 – Yes N2

SD1 DMF 50 No - 120 6 0.02 No N2 SD2 DMF 50 No - 120 6 0.02 No 120 °C Desiccator

SA1 Acetone 50 No - RT 24 – No 80 °C SA2 Acetone 50 No - RT 0.5 – Yes N2

a) NaOH concentration. b) HCl concentration, time of the acidic flushing step. c) Water was only present in the 95 % ethanol. d) Addition of glacial acetic acid to the pH. e) Concentration of inhibitor DPPH. f) Reagent was first flushed out with the solvent used in the silanization procedure, immediately followed by a rinse with water for 10 minutes. g) Default drying was flushing the capillary with dry nitrogen gas for 1 h; elevated temperature drying was done in an oven. h) Default storage conditions were in test tubes under nitrogen gas at RT, except where indicated. For citations, see the corresponding table in Paper I.

The plug adhesion test revealed that hydraulic conveying patterns were quite different for the plugs that detached during the test (see Paper I), depending on capillary pretreatment. If the plug behaviour under pressure is a function of silanization of the surface it is attached to, these patterns were also related to silica surface roughness. It has been shown that many ways of increasing roughness are available and especially formation of whiskers has been studied by Schieke et al.33,34 as a means of getting better coating efficiency in open-tubular columns for gas chromatography.

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Figure 3. Global comparison of all used procedures. Pressure is the detachment pressure

from adhesion test, with 25 MPa being the upper limit. Theta angles are from wetting angle measurements, and C/Si ratios from XPS data. (from Paper I)

However, as the different detachment modes shown in Figure 3 in Paper I cannot be directly related to special rough surfaces created after the etching process, it was intriguing to use AFM to visualize the topographies of the three-dimensional surfaces. AFM images presented in Paper I show clearly an increase of surface roughness for the treated capillaries. As the pheno-menon is linked to wetting angle35, roughness variations may introduce an unknown variable in the ranking of procedures considering their wetting angle, as well as relative back pressures during adhesion test.

Finally, wetting angles measured on sections cut from a single 1 m piece of capillary showed large variability, meaning that the neat and untreated fused silica surface was inhomogeneous over length. A cleaning step early in treatment procedure is therefore ineluctable.

Conclusion from the results we obtained was that a “soft procedure” using etching method E2 and silanization ST1 is the best combination of schemes taken from previous literature. ST2 shall be avoided due to the risks of auto-polymerization of silanization agent and difficulty to keep a capillary well sealed at elevated temperature.

It is important to notice that Paper I did not aim at finding the best condi-tions, but only at classifying existing ones in order to facilitate a rational choice. It will therefore probably have an impact in scientific community by saving time for new researchers working on the field of capillary mono-

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liths, who are easily confused by in the multitude of methods available on this subject. Thus, Paper I may serve as an advanced experimental review article in this field.

However, further studies proved that treatment needed to be related to capillary size (e.g., 25 µm i.d. capillaries treated with recipe E2-ST1 regu-larly become blocked by the treatment), and to column use. Therefore, if Paper I is a guide of the known procedures applied on 1.0 mm capillary, I would still recommend each researcher not to take the described method as granted for every application.

3. Monolithic Material

Švec rationalised the word “monolith” in his monography4 by its Greeks origin “μονολιηοσ” meaning “single stone”. In separation science, it de-scribes a porous one-piece polymer which is also often called “continuous polymer bed”36 or “continuous polymer rod”37. Preparation takes place by mixing a set of monomers, solvent and an initiator, leading after triggering of the initiator action to a growing single structural piece of polymer with a specific porosity linked to its components choice.

Kubìn et al.38 are regarded as the first researchers having published on this subject. However, it is only during the last two decades that this area really emerged with the works of Hjertén, Švec, Novotný, Horváth and many others.

The main advantage of these materials is their controlled39 macroporous structure, which is claimed to provide better mass transfer than in packed columns.40 This makes them useful for a broad range of applications (see 3.3). Monolithic columns may also be a valuable alternative to traditional packed columns because of packing difficulties, which is the main cause of band dispersion.41 Monolithic columns have further been manufactured in a wide variety of formats like disks42, tubes43, capillaries44, membranes45, and microchips46.

A problem that still remains is the relatively poor reproducibility of these polymer rods18,19 leading to slow commercialization and reluctance against implementation in methods that are to be validated, when compared to traditional packed columns. This reluctance has proven to remain even if test studies have shown that commercial monolithic columns can success-fully pass the reproducibility assessment.47

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3.1. Synthesis of monoliths

The first step in monolith design is to settle on the proper polymerization system (monomers, porogen, conditions) for the synthesis. These are chosen in relation to the desired material properties including intrinsic strength and chemical stability, meso- and macroporosity, surface area (which is closely related to porosity), and surface properties (inertness, presence of functional groups or reactive handles for further functionaliza-tion, hydrophylicity, etc).

3.1.1 Main goal

The polymerization mixture is the main determinant of the final stationary phase properties. As can be seen in Figure 4, presenting some of the mono-liths synthesized in Paper II, the macroporous structure of a material pre-pared from a hydrophilic set of monomers [poly(ethylene glycol) phenyl ether acrylate; second column of pictures] is remarkably different from one prepared from less hydrophilic monomer glycidyl methacrylate (third column).

Figure 4. SEM (from left to right) of M1 (GMA), M2 (PEGPEA), M9 (GMA and NMP) and M22 (as M9 with pulsed light) using a magnification of 3,000 (top) and 11,000 (bottom) from Paper II.

Thus, a screening is often done to choose right monomers and conditions to obtain the desired monolith. Most powerful parameters for controlling size and shape of pores are the choice of monomers and porogens48,49, their ratios in the mixture50, and the kind and intensity of initiation51,52. The latter is related to polymerization time, which is compounded to another essential variable, the polymerization temperature, when thermally initi-ated.53 For example, in Paper II photoinitiation gave a monolith with quite different macroporosity compared to the same mixture polymerized by thermal initiation (columns three and four in Figure 4).

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3.1.2 Improvements of the porous properties

Mass transfer and separation impedance are main optimization criteria to focus on when improving a monolith. Considering that both these proper-ties are related to material porosity, it is important to search for particular porosity. It was realized early on37 that a monolith with low flow resistance and good mass transfer properties would require a bimodal pore structure. However, one problem rising when using biological samples (proteins in the first place) is the stationary phase chemistry that might either unfold proteins irreversibly on the surface, or not attract them at all. This cannot be circumvented unless the stationary phase surface appears to be more “natural” for proteins.

In the search for monolithic materials with “protein-friendly” pores, the idea of templating the porous structure with a molecule normally used in combination with proteins is quite obvious. In that case, two main advan-tages can be reached. First, this special molecule might be incorporated in the material, producing a surface with low tendency for irreversibly alter-ing biological materials. Second, this molecule will act as a template agent during pore formation (akin to molecularly imprinted polymers discussed in section 4); the polymerization solution will find the best arrangement to stabilize this template, and therefore, pores will have properties related to the acceptation of this molecule. Water may be necessary to express these properties and was therefore used as co-porogen for some applications.54 However, as polymerizations are often carried out at high organics load-ing, an alternative should be found.

3.1.3 Use of poly(ethylene glycol) as a pore template

Poly(ethylene glycol)s, initially studied by Lourenço55,56, are flexible non-toxic polyethers57 with the chemical structure HO-(CH2-CH2-O)n-H. Their hydrophilic heads and chains of intermediate polarity make them unique polymers with high solubility in water even at low temperature.58 At the same time, their polyether chains allow solubility in organic solvent and therefore in common polymerization mixtures.

PEG has been widely used for prevention of biofouling59 and in drug de-livery60,61. Commercial availability of a broad size range of PEGs (from 2-ethoxyethanol to > 30,000 g/mol) and ability for this molecule to fulfil the need for protein-friendly porogen are reasons for its major use in Paper II. However, a better surface also needs to be chemically “friendly” and “PEG-like” monomers and cross-linkers should be able to provide such a surface on the monolith. A screening of more than one hundred mixtures was done to find the best possible materials (shown in Figure 4). The best PEG chain in relation to its porosity and mechanical properties was found to

11

have an average molecular weight of 10,000. However, considering the need for a bimodal structure, a co-porogen was necessary. Thus, a chemo-metric model was used to identify the best set of solvents.

3.2. Optimization by chemometric means

The search for conditions that will produce a true bimodal pore structure is an arduous and time-consuming work, but it is simplified by a deeper understanding of the properties of co-porogens62 related to their effect on porous network. However, the range of porogen properties is relatively large, and it is probable that more than one property will cause changes in the material, and that these effects will be compounded. Thus, it is essen-tial to classify properties in relation to their effects on the porous structure.

3.2.1 Basis of chemometry

The International Chemometrics Society (ICS) gives the following defini-tion of this field: “Chemometrics is the science of relating measurements made on a chemical system or process to the state of the system via application of mathematical or statistical methods.”

This scientific domain has been used in a very broad number of fields63 due to its ability to simplify optimization by uncovering latent variables. It aids in reducing the number of variables to the more significant ones, and to compare results based on multiple responses and compounded variables.

3.2.2 Use of chemometry to understand monoliths

A monomer library screening was used in Paper II to accomplish a reliable monolith synthesis. However, it was hard to compare the effect of each co-porogen, based on the physico-chemical properties. Thus, when a chemo-metric model was used to draw relations between properties of solvent and resulting columns, it was possible to understand the important parameters.

Figure 5. Loading scatter plot of the PLS

model for three response factors (Paper II) Figure 6. Score plot of the PLS model with

two components (Paper II)

12

As shown in Figures 5 and 6, dipole moment and log P of the porogens are factors that contribute significantly in material porosity. Porogens with similar properties are also seen to be associated in the score plot in Figure 6. This study shows a way of systematically screening for porogens by identifying characteristic physical properties related to their ability in making pores.

3.3. Applications of monoliths

Whereas monoliths have often been used in electrochromatography64, other fields like capillary liquid chromatography65,66, solid phase extrac-tion (SPE)67 or microchip technologies46 have benefited from their deve-lopment. Among all uses, monolithic polymer rods have been employed for separation of peptides68, drugs69, small molecules70, or nucleic acids71. However, monoliths are still often used for fast separation of proteins72, 73 as developed in Paper II (see Figure 7).

Other possibilities of monoliths are their utilization as porous core material prior to a grafting step74 or surface modification. For example, Švec depicts some recent developments in enzyme immobilization on monoliths.75 Some other examples of surface modifications were de-scribed by Bergbreiter76, many of them using glycidyl methacrylate as the reactive handle. This monomer contains a hydrolysable oxirane which can open under acidic conditions to form a diol77, or be used as reacting group for the formation of, e.g., sulfonic acid78 or amine79,80 groups for ion exchange chromatography.

Figure 7. Chromatogram from separation of (in order of elution) Cytochrome C, lysozyme, ovalbumin, trypsin inhibitor, α-chymotrypsinogen and bovine serum albumin when using hydrophobic interaction chromatography. (from Paper II)

As columns synthesized in Paper II already gave good results for protein separation without further modifications (see Figure 7), it is likely that

13

applying some of the common modifications would have given useful ionic properties coupled to a protein-friendly porous network. It is also worth noticing that the use of PEG as porogen is closely related to im-printing process described below.

3.4. Characterization of the monolith porous properties

Among characterizations methods (SEM, AFM, ISEC, HPLC, …) used to determine the porous structure of monolithic materials, surface area and pore size distribution have for long been the most widely used properties measured for assessing the desirable bimodal porous structure.

Common instruments for measuring these parameters are mercury intrusion porosimetry and nitrogen sorptiometry. Measurements are normally fast, do not require optimization, and can be used in com-parative studies or reproducibility tests. However, both techniques are based on equations that have been discussed81-83 and neither technique is optimal for relatively soft xerogels like polymeric monoliths. It was thus of main interest to find an additional method that could be easily used and, eventually, give more information on the elemental structure of the material.

3.4.1 Mercury intrusion porosimetry

Mercury is a particular liquid that does not wet most surfaces, and there-fore needs to be forced into pores. Its behaviour has been first described by Washburn in 192184 who has lent his name to the main equation used to calculate pore sizes:

P

D)cos(4 θγ−

= [1]

, where D is the pore diameter, P the applied pressure, γ the surface tension (474 mN/m at 25 °C) and θ the contact angle (most often fixed at 130 °). This simple equation describes the relationship between the applied pressure and the pore diameter, assuming cylindrical pores. The instrument uses a penetrometer (Figure 8), where the sample is placed. The external void space is filled entirely by mercury in a so-called “low pressure” step. Higher pressure is then gradually applied stepwise and the amount of mercury injected in each applied pressure step is comput-ed to give the cumulative intrusion curve. From this, pore diameter plots as shown in Figure 9 are constructed by means of Equation 1.

14

Figure 8. Principle of Hg-MIP (reproduction with authorization from the Fraunhofer Institute for

Building Physics85)

Figure 9. Example of Hg-MIP plots for Chromo-lith material. Top: cumulative intrusion vs.

pressure. Bottom: log differential intrusion vs. pore size diameter.

As seen in Figure 8, the mercury is slowly forced stepwise into pores of the material. The maximum pressure that can be used in our instrument is 425 MPa, and it is thus likely that a material consisting of thin polymer structures can be subject to partial crushing by the extreme force applied. The result of the experiment would therefore be inconsistent in describing real sample porosity.

Another main drawback of Hg-MIP is that the calculation fundament, the Washburn equation, is valid only for cylindrical pores.86 This will produce biased results in case of pores of significantly different shape. Research has been done on intrusion into conically or spherically shaped pores87 and the conclusion is that the real pore size will be over- or underestimated. For example, when mercury enters a conical pore from a small orifice at the peak of the cone, the pressure needed will be equal to a cylindrical pore of the orifice diameter with the volume of the entire cone, i.e., as a narrower and deeper pore. The pore dimension will thus be underestimated. More complex models like an “ink bottle” were also studied88 but none of them approach the complexity of a monolith network. Since the Hg-MIP tech-nique is neither designed, nor particularly well suited for polymeric mono-liths, it was necessary to find a supplementary technique that is more de-voted to visualizing the macroporous structure of polymeric monoliths.

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3.4.2 Transmission electron microscopy

The idea of using imaging for structure assessment of a micro-element is not new. The first electron microscope was built by Ruska in 193389, and has grown into an indispensable tool in materials characterization. As the monolith field developed, it became very common to characterize these continuous porous polymeric structures by acquiring SEM images of their snapped cross-sections90,91. This has led to the discovery of problems such as difficulties in exact reproduction of the structures19 or failure to attach firmly to the wall (see Paper I). A SEM image presents a 2D image with the impression of a 3D structure and should therefore not be completely trusted for measurements of fundamental parameters like porosity.

The approach described in Paper III is to prepare an ultra-thin stained slice of resin embedded monolith and image it in an electron microscope. The inter-spaces between structural elements were then measured on these 2D images and thus, an assessment of the porosity was accomplished.

3.4.2.1. Experimental part

The experimental set-up in Paper III was directly derived from biological embedding.92,93 It is of major importance, when trying to cut a 100 nm thick section of monolith, to ensure that the structure does not to break or that the polymer “pops out” from the section. Monolith structures are so sparse that most structural elements would be detached from the continu-ous structure. It was therefore necessary to inject a resin inside the porous material to maintain its integrity during ultra microtomation. The resin should be able to “freeze” the structure without causing deterioration and should be fluidic enough to be pushed through the whole porous network. A “low viscosity” Spurr94 resin was used but care was taken to choose monoliths of low macro-porosity to avoid excessive back pressures.

A standard set-up for TEM of biological samples requires a staining (with heavy metal atoms, such as tungsten, osmium, ruthenium or uranium)95 of the sample itself to obtain contrast within continuous, non-porous bio-logical structures to identify different structural elements. This is not the case with polymeric monolith, since we were mainly interested in visualiz-ing the bulk structure, which consists of a relatively homogeneous material with void interspaces. A direct “positive” staining was attempted by adding a low amount of lead methacrylate to the monolith precursor mixture, but even percent amounts changed the porosity of the resulting monolith, and the approach was therefore discarded. A “negative” staining procedure was therefore used by adding lead methacrylate in the embedding resin. Thus the embedding resin should appear dark in the macropore space and the polymer constituting the monolith more transparent. The presence of lead

16

in the embedding mixture was not expected to affect the macropore pro-perties of the monolith significantly.

3.4.2.2. Results and discussion

As seen in Figure 10, it was possible, using the method described in Paper III, to achieve good sectioning of the capillaries, and to observe the sample with a fair contrast.

10µm

A B

C D

However, the contrast had to be converted to a dicho-tomous variable in order to run a direct computation of inter-space porosity. It was decided to treat the micro-graphs with a photo soft-ware to arrive at 1-bit (B&W) images.

Thereafter, the automatic and randomized measuring “spiders” were used to sum up diameters in each image (see Figure 11) and from these values a pore size re-partition graph was derived (see Paper III).

After calculation of the median pore diameter on the average of more than

150,000 diameters in each picture, measurements of porosity was done by mercury intrusion as described above. A plot (see Figure 12) was drawn to see the correlation between the two techniques.

Figure 10. Transmission electron micrographs of: A. Chromolith (scale 10 µm), B. TEM1 (5 µm), C.

TEM6 (10 µm) and D. TEM4 (5 µm). (Paper III).

It is important to keep in mind that Hg-MIP is based on a cylindrical pore model, and therefore, a TEM measurement cannot be directly related to a Hg-MIP measurement. Even more, studies of Gille et al.96 show that mea-surements done by TEM are not calculating a pore diameter as in Hg-MIP. Instead, TEM micrographs will yield a porosity characteristic called chord length distribution (CLD), which is linked to pore diameter by complicated mathematical expressions.96 Thus, the CLD only provides an alternative characterization of monolith porosity. It should only be compared directly to the values of Hg-MIP if a simple relationship can be found. Unfortuna-tely, it is not the case considering the complexity and heterogeneity of the porous network.

17

Figure 11. Treatment of micrographs (Chromolith on top and TEM3 at the bottom). Left to right: raw micrographs, after background correction, measuring spot (dash lines

represent discarded measuring diameters hitting the border).(from Paper III)

Figure 12. Correlation between mercury intrusion porosimetry and pore repartitions obtained from CLD measurements of TEMs for all the monoliths studied in Paper III.

Guan Sajon et al.97 also challenged Hg-MIP with other techniques like nitrogen sorptiometry, helium pycnometry, and inverse size exclusion chromatography and found that the results may differ substantially. It is

18

therefore of important to have gained access to TEM as a unique tool for comparison of porosity instead of challenging one method against another. Keeping in mind that the technique presented in Paper III has not been studied extensively, the following points should already be convincing:

- it is cleaner to the environment, even if using monomers and heavy metals,

- it allows the user to double check that no mistake was done,

- it allows the user to visualize the monolith and thus discover other aspects,

- it is possible to measure other parameters like the size and the shape of the growing particles, and their repartition in a cross-section, thus giving further insight on monolith formations,

- it is directly measured on the chromatographic format.

However, a major drawback is still the high viscosity of embedding resins, which does not allow intrusion in a monolith with through-pores below ~500 nm using the present method. Micropore observations (compared to nitrogen sorptiometry) are therefore not possible. Resin also needs to be forced slowly into the monolith in order not to break the structure and this requires some time. Finally if the staining is not well performed, observa-tions cannot be made. Using an alternative technique based on dehydra-tion/infiltration by ethanol or acetone98 might enable such studies, which were not the aim of the investigation in Paper III.

4. Molecularly Imprinted Polymers

Yan and Ramström give the following definition of molecularly imprinting materials99: “A technique of tailor-making network polymers for the recogni-tion of specific analytes molecules”.

4.1. Introduction to MIPs

First syntheses of molecularly imprinted polymers were reported in the 70’s100,101. To date, more than 2000 scientific papers have been published on the subject and the field is growing every year. While basic idea is still the same, approaches to imprinting have broadened, as have applications.

4.1.1 Basis of MIP formation

The prevailing principle is based on the use of a “target” or “template” molecule during synthesis of the imprinted media with the intention of creating cavities with matching shape and complementary interactive moieties (see Figure 13). The template is then released by cleavage or by a simple cleaning procedure and the material should then offer high selecti-

19

vity to the target. The term of “man-made mimics of antibody”7 can thus be well understood.

Monomers Template

Polymerization(e.g. UV, temp.)

Crosslinker

Washing

Figure 13. Main principle of molecularly imprinting for a non-covalent approach

4.1.2 Approaches

There are three main approaches to molecularly imprinting: covalent, non covalent and semi-covalent.

The covalent method, developed by Wulff102, uses polymerization of a preformed template-monomer assembly with an excess of crosslinker. Cleavage and extraction should lead to specific functionality in the mate-rial. However, conditions for the cleavage (often hydrolysis of a boronate or a Schiff base) are often harsh and template recoveries low, leading to a limited use for this technique.

The semi-covalent approach103 uses an imprinting method similar to that of the covalent approach, except that the rebinding is non-covalent.

The non-covalent approach is by far the most used one; it relies on inter-actions (of which ionic, ion-dipole, dipole-dipole and hydrogen bonding are the more important) between functional monomers and template. The method, developed by Mosbach and co-workers104, is presented above in Figure 13.

4.1.3 Application areas

The SPE cartridge105 is the most widely used format for MIPs, and seems to be the only MIP-based separation product commercialized yet. A few other fields have benefited from this novel technique. Among these, most

20

successful are sensors106 and membranes107. The field of drug delivery108 has also embraced MIP technologies in various formats. The inherent slow release/bleeding of template after being caught on the imprinted material, which is a drawback in chromatography, can be turned into an advantage in drug release applications.

4.2. Templates versus monomers

Choice of template and monomers is the primary concern for researchers working with MIPs99. Interactions, mixing properties and solubility issues should be considered and might call for the synthesis of new monomers, or to the design of more reactive templates.

4.2.1 The template

The templates that were utilized in this thesis are summarized in Annex 1. As can be seen, they are all amphiphilic molecules, i.e., they have some hydrophilic and some hydrophobic parts.

There are three major points to consider in the choice of the target: a reasonable solubility of the template in the polymerization mixture, its possible affinities with the monomers and/or the stationary phase already present, and its solubility in a “normal” elution solvent allowing complete cleaning of the column with no subsequent bleeding of target.

Excessive solubility in the organic polymerization mixture is often a sign of low solubility in aqueous media (interesting for analysis of valuable biolo-gical samples), and might also obstruct efficient imprinting of the target if its association with the polymer network is low during its formation. This leads to solubility issues in both the polymerization and characterization steps for most of molecules shown in Annex 1.

Finally, one additional criterion for the choice of the template is often the possibility to find similar challenging analytes that should prove the high selectivity of the material.

4.2.2 Monomers and cross-linkers

Cormack et al.109 listed an extensive selection of monomers and cross-linkers used in MIP synthesis and some of them are presented in Annex 2. These monomers can be acidic, basic, or neutral, and they can be soluble in organic or in aqueous media. Common to all is that they are supposed to have properties that should enable formation of “special bindings”.99 Some researchers also found that tuning their own monomers was a key to successful MIPs and thus novel cross linkers110 are now used.

21

4.3. Synthetic strategies

The oldest and most widely used strategy for imprinted polymer prepara-tion is “bulk polymer imprinting”111, consisting of first synthesizing a bulk imprinted polymer, then crushing the structure, and sieving the material to select an appropriate particle size for further experiments. More recently other strategies have been introduced. Among these, we can cite surface imprinting (e.g., grafting on silica112 or grafting on membrane113), scaffold imprinting114, imprinting in inorganic matrices115, hierarchically imprint-ing116 and use of controlled polymerization schemes such as atom transfer radical polymerization (ATRP)117. Combinatory approaches for small scale (like MiniMIPS or 96-wells plate) parallel synthesis of a large number of MIPs have also recently been developed to enable rapid monomer screen-ing and optimization.118

Among all these approaches, four techniques were thus further chosen to be used the AquaMIP European consortium, within which this thesis work was done. Spherical particles were prepared by precipitation polymeriza-tion at Strathclyde University (Scotland),119 bulk polymer and iniferters120 were synthesized by Dortmund University (Germany) and grafting on monoliths in capillaries was done by our group. The resulting collaborat-ion work was summarized in a joint manuscript (not appended with the thesis) where these different formats are compared.

4.4. MIPs for bupivacaine and homologs

Aside many tested templates (aspartame, riboflavine, Asp-Ala-βNA, 2,4-D, theophyllin) which did not always give successful results, one particular, bupivacaine, was chosen mainly for the possibility to get challenging analytes from AstraZeneca, a partner in the AquaMIP project.

4.4.1 Properties of local anæsthetics

As the medical world has a continual interest in pain relief drugs, local anæsthetics (LAs)121-123 are often encountered in separation sciences. The operation principle of LAs is based on inhibition of signal propagation along nerves by reducing Na+ influx.124 LAs are typically amphiphilic mole-cules, and the interest in these compounds stems from the desire to estab-lish imprinting in aqueous application. Their amphiphilicity affords rea-sonable solubility in partially aqueous organic solvent mixtures, allowing dissolution in the monomer mixtures.

22

AstraZeneca provided us with three LA homologs (see Figure 14) in order to challenge the selectivity of the MIPs, and to have one application of the more recent S-Ropivacaine125 that represents an interesting enantiopure template.

Figure 14. Local anæsthetics used in Paper IV

4.4.2 Choice of experimental procedures

The experimental set-up presented in Paper IV used a 100 µm i.d. UV transparent capillary, treated using the method developed in Paper I. A TRIM monolith optimized on the basis of the recipe of Viklund et al.126 was used as core material for a further grafting. To avoid the reproducibility problem of the monoliths expressed above, the columns were flushed by methanol and ranked by their back pressure. Only columns falling within a ±5 % range of the average back pressure were thus chosen.

Figure 15. Scanning electron micrographs of non-grafted core monolith at magnifications 3,000 x (A) and 10,000 x (B), and of grafted BV-mMIP at magnifications 3,000 x (C) and

10,000 x (D). (from Paper IV)

23

Unreacted vinylic groups on the surface of the core material made grafting possible, and most common monomers (MAA and EDMA) cited above (4.2.2) were chosen to provide the imprinting effect.

The recipe used in Paper IV was a compromise settled in accordance with the other synthesis groups in the AquaMIP consortium in order to collect data for a joint format comparison manuscript (not part of this thesis).

Thus, a fixed cocktail consisting of target/MAA/EDMA in a molar ratio of 0.33:4:20 was chosen, and other parameters like time and initiation were optimized. Reference material, called mNIP (monolithic non-imprinted polymer) was synthesized in the same way as the mMIP, but no template was added in the mixture.

Scanning electron microscopy images (Figure 15) reveal that a major change took place by the grafting step, but no significant morphological difference was observed between grafted MIP and NIP.

4.4.3 Retention properties of the MIPs for bupivacaine and homologs

It was decided in Paper IV to prepare MIPs with a two-step approach using a grafting step instead of a crushed imprinted monolith. The main reason is that crushed imprinted monolith may produce imprinting sites that are enclosed in the monolith, inaccessible to the analytes. As the core monolith was already porous, a single imprinted layer was sufficient. However, this method may cause another problem; blocking the pores. The grafted layer needs to be thick enough to show imprinting effect, but thin enough not to obstruct the macroporous network. Surface area of the grafted material could not be tested because of the very low volume of material in the column and the difficulty to remove it from the silica capillary. The back pressure after grafting was instead compared to the one before grafting, and a reasonable increase of < 30 % was found.

As three template/homologs were available, imprintings using each of them or an equimolar mixture of them were tested, and the columns were challenged by bupivavaine and two homologs to determine the parameters commonly used for characterizing MIPs (see Figure 16 and Table 2).

Figure 16 shows the tailing typical of MIP materials, characteristic of site heterogeneity127,128. It is more of a surprise to see a small “shoulder” in most of the chromatograms. This has been subject to discussions and no defini-tive answer was found. As this shoulder only appeared when the injected solute was either the template or a shorter carbon chain template homolog, we were led to think that the phenomenon was due to a cohort of “high selective sites” with similar binding strength. These sites would be more abundant than the remaining sites of higher binding heterogeneity in the

24

material. Kinetics were not really in favour of such a pattern, but the small “hump” in most chromatograms found no other good explanation.

Figure 16. Chromatograms from Paper IV. Upper left: mNIP; upper right: BV mMIP;

bottom left: MV mMIP; bottom right: LA mMIP. The vertical bar in the middle indicates the y scale and represents 0.01 absorbance unit for the mNIP and 0.001 absorbance unit

for the mMIPs.

Table 2. Retention and imprinting factors of MV, RV and BV on mMIP and mNIP columns based on gravity centre of the peaks (from Paper IV)

Analyte mNIP BV-mMIP MV-mMIP RV-mMIP LA-mMIP K k SF IF K SF IF k SF IF k IF

MV 0.30 3.73 0.58 12 4.73 1 16 4.09 0.44 14 4.09 14 6.27 a 21 a 7.73 a 26 a 7.18 a 24a 5.73a 19a

RV 0.13 7 1.09 54 2.36 0.50 18 9.36 1 72 5.64 43 BV 0.13 6.45 1 50 2.91 0.62 22 4.45 0.48 34 3.73 29

7.64b 59b

a) Retention and imprinting factors calculated for the peak/shoulder when MV was injected. b) Retention and imprinting factors calculated for the peak/shoulder when BV was injected.

The reference material mNIP, was synthesized without any template, and its relevance can therefore probably be discussed (vida infra). Moreover, the use of imprinting factor (IF) being based on the mNIP retention makes the results even more inclined to discussion. For example, retention of MV in the mNIP is higher than BV, and leads to a lower IF than expected. That is why separation factors were added in Table 2. Focus should thus be on the retention orders of each of the analyte in its relative column, and in challenging columns.

25

Eluents richer in water were also tried, but no good results were obtained, probably because of water disrupting the fragile interaction between the template and the monomers. Thus, mMIPs were not useful for their pri-mary purpose as solid phase extraction sorbents in aqueous media.

The temperature plays a key role in the polymerization of the MIPs.129 The molecular dynamics of the template and the polymerizing system will be slowed down if the temperature is low. The likelihood of exceeding the activation energy needed to break the relatively weak interactions between the template and monomer will thus be lower. The complex then becomes more stable, which facilitates the formation of an imprinting cage. On the other hand a higher temperature means more molecular movement in the solution and therefore templates will interact with the monomers at a higher rate. The polymerization and hence the creation of imprinted sites will driven to completion faster. Nevertheless, the lack of reproducibility of the BV mMIPs prepared at different temperatures did not allow any firm conclusion to be drawn.

As part of the joint efforts within the AquaMIP consortium, capillary columns identical to those prepared in Paper IV were synthesized and tested by frontal analysis.130 This method developed in 1940131 is one of the most widely used techniques for achieving adsorption isotherms.132

The main objective of the joint manuscript was to compare various MIP formats. However, the composition of the MIP cocktail could not be kept exactly the same for all formats, making a fair and unbiased comparison difficult. Further, injection volume was not the same in relation to the amount of material and size of the column, when columns of “conventio-nal” size were compared to capillaries. Thus, as the amount of solutes loaded onto capillaries was five times higher in both frontal analysis and normal chromatographic evaluation, and considering the dependence of IF to injected sample, it might be considered fairly irrelevant to directly compare the results. Also, frontal analysis was done on capillaries using chromatographic systems not specifically designed for micro columns, which require delivery of the eluent at flow rates as low as 2-5 µL/min. Yet this work shows the importance of the polymerization technique in MIP synthesis and points out the difficulties in acquiring reproducible data when different chromatographic systems are utilized. It also demonstrates the difficulties encountered when trying to compare apples and pears and the compromises to be made in a wide collaboration (e.g., disagreements in the feasible polymer ratios for different formats).

26

4.5. Phosphorylated tyrosine

Phosphorylated targets, synthesized for the AquaMIP consortium, were our first insight in MIP synthesis for recognition of biological solutes.

4.5.1 Protein phosphorylation and its role in biology

Phosphorylation of proteins is a very well studied process (over 140,000 articles in ISI Web of Science refer to the subject) that only takes place on the hydroxylated amino acids serine, threonine, and tyrosine. As phospho-rylated polypeptides (e.g., MAP kinase cascades) have key roles in cellular processes and in signal transduction133, it has become of great interest to identify them efficiently and selectively for further studies such as for their purification. Only a few methods are currently available, like hydroxyapa-tite134, immobilized metal ion affinity chromatography (IMAC)135 or anti-body-based assays136. It remains very attractive to search for an inexpensive method with high selectivity for phosphorylated residues of polypeptides, which would work with biological samples, possibly without denaturating proteins. Tyrosine phosphorylation is a less represented biological pro-cess137 (compared to serine/threonine kinase) and the subsequent phospho-rylated proteins are purified through well established methods using anti-bodies138 or other affinity methods139. Nevertheless, the price of such purifi-cation makes alternative techniques very attractive, especially MIPs, as they are very facile and inexpensive.

4.5.2 Experimental MIP

Considering the solubility issues related to imprinting, it was de-cided to prepare a MIP targeting phosphotyrosine side chain by imprinting a protected phos-phorylated tyrosine (shown in Figure 17). A strategy similar to that used for the bupivacaine MIPs was chosen. As ureas have a tendency to bind to phospho-rylated proteins140, a urea mono-mer, described in the text by ME1, was synthesized to maxi-mize interactions with the cho-sen template Fmoc-pTyr-OMe, as described below in Figure 17.

O

P O-

O

-O

NH

O

OFmoc Me

N O

N CF3

CF3

H

H

NO

NF3C

CF3

H

H

Figure 17. Monomers/template arrangement

27

Interactions of similar moieties with phospho-groups have been studied,141 and the results indicate a 2:1 monomers/template interaction. In order to validate this assumption, NMR was used to track interactions between monomer and template. It was found that a high pH was necessary to completely deprotonate the phospho-group to enable a 2:1 chemistry. Thus, it was decided to use PMP as base during MIP preparation.

The MIP mixture was improved by chemometric means and challenging solutes (Fmoc-pTyr-OH, Fmoc-Tyr-OMe, Fmoc-pSer-OMe, Fmoc-Glu-OMe) were ordered. A chromatographic evaluation made by a first elution step, followed by a cleaning step with methanol, and a small re-equilibra-tion before next injection, was chosen. Different eluent mixtures were then tested on the same system.

4.5.3 Results obtained with the pTyr MIPs

Figure 18 presents the repeated injections of five solutes in a MIP column. The elution profile was done by a ten-minutes step with eluent A (10/90 acetonitrile/water + 120 ppm TEATFB) followed by 15 minutes of B (methanol) and re-equilibration by A for ten more minutes.

Figure 18. Retention of five solutes in a pTyr MIP column.

As expected, the template (Fmoc-pTyr-OMe) was not eluted with A and was released from the column during cleaning step. Same behaviour was observed for the non-methylated solute. Good behaviour of the column was proven by the low retention of Fmoc-pSer-OH and the non-retention of non-phosphorylated compounds (Fmoc-Tyr-OMe and Fmoc-Glu-

28

OMe). This result shows that the MIP column was apparently highly selective for phosphorylated tyrosine moieties.

As presented in Figure 19, the water content in the mobile phase was also examined. It unfortunately showed a strong tendency for retention of all compounds in highly aqueous phases.

Figure 19. Top-left: retention factor dependence to water content in the mobile phase

(acetonitrile/water) for the four solutes. Others: retention of Fmoc-pTyr-OH for different concentration of TEATFB: a) 0 ppm; b) 2.5 ppm; c) 50 ppm; d) 100 ppm; e) 215 ppm for

eluents containing 70 % (A), 40 % (B) and 20 % water (C).

As the best performance was obtained for 10 % of water, especially in the presence of an ion-pairing reagent (TEATFB), it was decided to investigate retention for different concentrations of reagent. Results presented in Figure 19 demonstrate the importance of adding TEATFB. For the three water contents tested, the same trend was observed showing a higher retention of Fmoc-pTyr-OH at higher TEATFB content. This might be explained by the effect of TEATFB on the stationary phase global charge. TEATFB might also affect the phospho-group by aiding it to interact more easily with the stationary phase. However, this is quite speculative.

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A comparison with the reference material NIP is the most common way of assessing MIPs. However, the definition of what a “non imprinted” mate-rial should be remains problematic. In general cases, a reference material is synthesized following the same recipe as the MIP, omitting the template in the polymerization mixture. Thus, considering the formation principle of a polymer, it is disputable to consider the resulting material as a real refe-rence against which a comparison can be made.

As mixtures of PMP and Fmoc-pTyr-OMe were added to create a MIP, it was decided to test four different kind of NIPs synthesized as follows: All contained the basic mixture, and the additional components were none in NIPa, PMP in NIPb, Fmoc-Tyr-OMe in NIPc, and a combination of PMP and Fmoc-Tyr-OMe in NIPd. Chromatographic evaluations of these mate-rials are presented in Figure 20.

Figure 20. Injections of the four solutes in the four kinds of NIP.

Elution conditions: 15 min with 90/10 acetonitrile/water plus 120 ppm TEATFB, 10 min of methanol, 5 min re-equilibration.

It is proved that the choice of the mixture in the reference material synthe-sis had a great importance. Retention of all solutes on NIPb and NIPd was observed, meaning that the presence of PMP in the polymerization mix-ture had a major impact on the material itself. Introduction of a “fake” template not containing a phospho-moiety was apparently not affecting the separation.

From this test, we can conclude that the mechanism of specific sites syn-thesis in the MIPs was most probably also affected by the presence of PMP,

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which was primarily added to deprotonate the phospho-group. It high-lights one more time the lack of knowledge in the imprinting mechanisms, and the need for careful considerations in the choice of components that should be included in the preparation of the NIP.

Finally, this test casts a shadow on the possible retention mechanism in MIP columns, and the curious effect of TEATFB. Moreover, unsuccessful attempts to establish retention for tri-peptides and proteins containing phosphorylated side chain amino acids (phosphorylated and non-phos-phorylated angiotensin II) also show the actual limitations of the material, especially when it comes to complex moieties in partially aqueous solution.

4.6. MIP evaluation and characterization techniques

Among all characterization techniques used to study MIPs, batch inter-action studies are probably the assessment most widely used to check whether the material is imprinted or not. Additionally, many other tech-niques are used to characterize either physical properties142 (gas sorption, SEM, solvent swelling, gravimetry, elemental analysis, solid state NMR, IR), or imprinting properties (chromatographic assays, UV/fluorescence spectroscopy, chemical probes, etc.). In this thesis, studies were focused on use of HPLC (as described previously), isothermal titration calorimetry, and NMR.

4.6.1 Isothermal titration calorimetry

Isothermal titration calorimetry (ITC), mainly designed by Ramsay et al. in the 80’s143, is a technique for determination of basic thermodynamics of interaction between two entities in solution (for example a protein and a small molecule, or an antibody and an antigen).

4.6.1.1. Principles of ITC

Interaction between two species generates a characteristic heat exchange with the surrounding medium, which is directly related to the binding constant through the Gibb’s free energy of the interactive process. By measuring the amount of heat released to (or taken from in the case of an endothermal reaction) a solution, thermodynamic parameters can be determined and binding constants thus inferred. A typical ITC system144 is composed (see Annex 3) of:

- a reference cell which should be filled with the same solvent as the rest of the system, but will not be affected during titration

- a sample cell, which is normally filled with the “macromolecule” species (e.g., the antigen)

- a syringe, that is filled with the ligand to be titrated (e.g., the antibody)

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The tip of the syringe which duals as a stirring element is rotated at a speed between 200 and 600 rpm, providing fast mixing of the solution.

Provided ΔH is different from zero, each aliquot of ligand added to the cell will result in a thermal change (endothermic or exothermic) which will be recorded. Thus, thermodynamic constants may be calculated.145 ITC has mainly been used in studies of partitioning of solutes into lipid mem-branes146, but it has also recently been successfully used to characterize MIPs147-149. However, the body of literature on ITC of MIPs is still very limi-ted because of the difficulties of applying microcalorimetry to systems presenting a broad heterogeneity in binding sites.

4.6.1.2. ITC on riboflavine

Paper V describes the use of a common ITC instrument from MicroCal (Sweden) to assess imprinting in crushed monolith. The material was im-printed with riboflavine Rf (also called vitamin B2). The template was in the syringe and polymer particles suspended in the cell. From the number of binding sites per gram of polymer, it should have been possible to make a complete titration curve showing two plateaus. However, poor solubility of the template in many solvents and especially in water150 combined with high propensity of the relatively heavy particles to settle down quickly and a relatively low binding enthalpy made it impossible to record a complete normal binding experiment.

Therefore, a direct comparison of binding energies was used between MIP and NIP in the range of high specific binding. The material was also challenged with another analyte (uracil) as control experiment (Figure 21).

Figure 21. ITC profiles for energy released (q) vs. total conc. of the titrant in the cell (Ctot) for titration of MIP (dashed), NIP (solid), or blank (dotted) with Rf (lower

three profiles) or uracil (upper three profiles)(from Paper V)

Figure 22. Energy released vs. injection number for titration of Rf ( ) or uracil

( ) by RfBP. (from Paper V)

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The template bound more strongly to the MIP than to the NIP. Further, uracil reacted in the same way on both the MIP and the NIP, verifying that the interaction was specific to Rf. As shown in Figure 22, riboflavin binding protein (RfBP) was also challenged with either Rf or Uracil, and its binding constant was compared with its synthetic mimic, the Rf MIP.

The imprinted polymer appeared to have a binding constant close to that of the protein. However, whereas the protein worked perfectly in aqueous environment, this was not the case for the MIP which showed more un-specific binding.

Solubility issues and difficulties in reproducing measurements were some of the problems experienced there. Even stirring of the material might have been the cause of ghost signals which were appearing in some titrations and were attributed to heat formation due to chock or breaking of some particles. The obtained profiles were anyway insufficient to calculate all thermodynamic parameters for the Rf MIP and to enable a direct com-parison with RfBP.

4.6.1.3. ITC on phosphorylated tyrosine

The same set-up was used to track down binding properties of pTyr MIPs synthesized by Dortmund University. Crushed monoliths prepared with the same cocktail described in section 4.5 were suspended in various eluents, and Fmoc-pTyr-OMe (the MIP template) was used as titrand.

However, ITC was unable to reveal any difference between MIP and NIP, showing limitation of this technique:

- Particles need to be almost mono-disperse,

- Solubility of the template needs to be complete,

- The number of free binding sites needs to be exactly known,

- The specific binding should have a binding energy that is very different from the non-specific one.

If these conditions are not met, the experiment may fall outside the titra-tion range, or the signal may simply be too low. Moreover, a major draw-back is the formation of bubbles in the cell that may be due to the pores in the material. Extensive degassing cannot be used unless the solvent is non-volatile and bubble formation leads to unreliable results. Conclusions to the pTyr study may thus question the veracity and the reliability of the results found previously with riboflavin.

4.6.2 Nuclear magnetic resonance

Nuclear magnetic resonance is a complex and interesting technique which can provide observation of intricate interactions at atomic resolution.

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Since Prof. Dr. Albert from Tübingen University, specialist in NMR studies on stationary phases, was involved in the AquaMIP network, we were granted the possibilities of testing a brand new technique for the charac-terization of interactions in imprinted materials.

4.6.2.1. Principle of NMR

NMR, first described by Bloch151 and Purcell152 in the 1940’s, studies the properties of nuclei (mainly 1H and 13C) possessing a magnetic moment. Alignment of these magnetic nuclei can be achieved by applying a homo-genous and powerful external magnetic field.

Perturbation of the alignment is achieved by applying a strong external radio frequency pulse of a specific frequency. Observation of the return of the perturbed system towards equilibrium produces the characteristic NMR spectra exhibiting resonance line at frequencies specific for nuclei in different chemical environments. NMR can be performed in liquid state (more common in organic chemistry), in solid state, or in suspended state. It can also be coupled to other instruments like HPLC.153

4.6.2.2. Solid-state 13C CP/MAS NMR

The technique used in Paper VI is cross polarization/magic angle spinning NMR on 13C nuclei (13C CP/MAS NMR), introduced by Pines and Waugh.154 This combination of cross polarization and magic angle spinn-ing allows a major increase in the sensitivity, essential in observing dilute nuclei with a low magnetic moment such as 15N or 13C, especially in solid material which is not freely tumbling compared to molecules in an iso-tropic liquid state. As described in Figure 23 below, the major aim of cross polarization is to transfer polarization from an abundant high γ nucleus to a dilute and less sensitive spin. Thereafter, acquisition of the signal is drastically improved.

Figure 23. Principle of cross polarization NMR

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However chemical shift anisotropy and dipolar interactions are normally increasing the signal noise, leading to complicated spectra. The geometric dependence (by a factor 3cos2θ-1) of these interactions enabled one of the most known “tricks” in NMR called magic angle spinning, introduced by Lee and Goldburg in 1965.155 Thus, by spinning the sample, as shown in Figure 24, with a fixed angle of 54.74°, it is possible to get a clean spectrum (see example in Figure 25) also from anisotropic materials such as a suspended molecularly imprinted polymer.

Figure 24. Magic angle spinning principle. Figure 25. 31P Spectra showing effect of

MAS NMR on lipid membranes composed of different phospholipids.

4.6.2.3. Suspended-state STD HR/MAS NMR

Suspended-state NMR was introduced in the late 70’s156 but mainly used and developed in the 90’s by Albert.157-159 As it is suggested by its name, this technique involves a solid (often some analytical stationary phase) being suspended in a liquid. Interactions are observed and recorded.

The term STD HR/MAS NMR means saturation transfer difference high-resolution/magic angle spinning NMR. Saturation transfer difference (STD)160 is a method that has been widely used in protein binding studies161 and drug discovery162. In this technique, an abundant proton in the host molecule is saturated and some of the spin energy can then be transferred to a proton with a different shift in the guest molecule, provided that it is in intimate contact (interacting) with the host. A signal increase in the guest protons will therefore give an indication about binding (Figure 26).

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Figure 26. Principle of STD HR/MAS NMR experiment

4.6.2.4. NMR on Bupivacaine MIP monolith

In Paper VI, a monolith was synthesized in a 3 mm i.d. borosilicate glass tube following the procedure in Paper IV. After drying and detachment from the glass holder, the monolith was fitting exactly into a 4 mm CP/MAS NMR rotor as shown in Figure 27.

This sample was then subject to the magnetic field of a 300 MHz NMR spectrometer, spinning the rotor at 10 kHz. The obtained 13C CP MAS NMR spectrum for MIP and NIP were compared to see if the two materials were identical (granted that the MIP and NIP should have the same composition but only a difference in the shapes of the cavi-ties providing special interactions only in the imprinted materials). Indeed, the spectra in Figure 28 are nearly identical.

Figure 27. From top to bottom: column holder; glass tube used as reactor/column

for polymerization; 4 mm solid state CP/MAS NMR rotor; monolith mMIP.

A 5.0 mg piece of monolith (either MIP or NIP) was then suspended in 80 µL of stock solution (acetonitrile and template) in the 4.0 mm double bearing rotor of a 400 MHz spectrometer spinning at 4.5 kHz.

36

Figure 28. 13C CP/MAS NMR spectra of mMIP and mNIP from Paper VI

1H MAS NMR experiment: as described previously in Figure 26, during the on-resonance step, only the magnetization of the monolith protons was affected. During the off-resonance experiment, the sample was irradiated, with the same reference power and duration, far from resonance for the monolith protons. A difference spectrum was generated by subtracting the on-resonance 1H NMR spectra from the off-resonance spectra.

The nuclear magnetization of the template gets partially built up by interaction with the monolith, with some of the spin energy from the partly saturated nuclei being transferred to the template. The relative amount of this saturation transfer is determined by the NOE (nuclear Overhauser effect). Thus, as can be seen in Figure 29, interaction of the template with MIP and NIP was recorded and relative intensities were calculated as in Table 3.

The signals called a), b) and r) were normalized for MIP and NIP as they should remain unchanged.

Table 3. Relative intensities of the 1H signals from suspended-state STD HR/MAS NMR spectra (Fig. 29) when the 1H signal a) is set to 1 in both mMIP and mNIP. (Paper VI)

-NH- Aromats a) b) r) -CH3

mMIP 5 8.5 1 2 4 1

mNIP 2.5 5 1 2 6 2

37

Figure 29. Suspended-state STD HR/MAS NMR spectra of the mMIP (upper) and the mNIP (lower) from Paper VI.

Then, the relative intensities of the other peaks was measured, and it was clearly shown that the template interacted with its amine and aromatic groups, which are considered to be the specific groups involved in the im-printing. On the other hand, for the case of non-specific interactions, the template interacts strongly with the NIP through its methyl group. These observations are in accordance to the described imprinting mechanism163,164 normally based on strong electronic donors.

An unsuccessful attempt was earlier made within the AquaMIP consort-ium to verify template binding by STD HR/MAS NMR. Those experiments were done on crushed monoliths but failed to show specific binding. The principle of grinding MIP monoliths may thus have caused higher amount of non-specific binding and corresponding lower signal difference in STD experiments, which made the measurements impossible.

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5. Concluding remarks and future aspects

This thesis has treated a wide spectrum of aspects related to separation materials, some of which have a direct bearing on molecular imprinting. It is therefore interesting to give forecast of what insights that can be gained from the most relevant findings in the future.

While Paper I provides an experimentally supported overview of possible silica treatments, it remains obvious that no optimal condition was found and that further studies are needed. Even more, the choice of etching and silanization techniques is closely related to the material used and to the application, thus it remains necessary to choose wisely in consideration of all parameters.

The use of a protein-friendly porogen in the synthesis of columns for protein separation has proven to be effective but no bimodal structure was created during the study in Paper II and the monolithic capillary columns were not utilized further. Thus, it is still possible to functionalize these columns, or expand this synthesis scheme with other porogens.

Moreover, the novel method of pore characterization by TEM imaging opens a new aspect to monolith understanding and thus, a probable solu-tion to poor reproducibility. The data, collected by visualizing the cross sections of monoliths, will definitely be valuable in future attempts to optimize the synthesis of porous separation materials.

Finally, the body of literature on molecularly imprinted polymers now encompasses almost 3,000 published papers. Yet, the promises and expec-tations often used to advocate their use have still not been fulfilled. There are still tough challenges remaining. Imprinting of bigger templates such as proteins might have been presented165 but most of the applications are still with small molecules. Even worse, the realms of operation in aqueous environments are still not crossed very often. Thus the goals and dreams remain the same: Creation of a perfect polymeric antibody at a very low price and infinitely re-usable.

“Children still believe in Santa Claus, so why should we stop believing in MIPs…” (Julien Courtois, December 2006)

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6. Acknowledgments

During that thesis, I met many people, and worked with many others. Thus I would like to thank all the ones that will not find their name in the list after (yes, you too).

I would like to thank my two co-supervisors Börje Sellergren and Peter Cormack for their help in the MIP project.

And what would be AquaMIP without its team workers …. Eric, Jeroen, Ersilia (“thanks mommy!!”), Joakim, Klaus, Amaia, Andrew, Christina, Cristiana, Rainer, Magdalena, Zoe, Tanja and all others that I forget to mention…

Thus I would like to also thank my AquaMIP roomy Panos and wish him good luck at the Greek border … (In case things turn wrong there, call me!)

I would like also to thank all people I met in labs in Groningen (especially Nicolas and our good discussions on life or so…).

I thank all the people from the RPAC group, Anna, Erika, Emil, Fredrik, Emma, Mai, Christer, Gerd, Thuy, Duc and Petrus, my old but still extremely good-looking roommate, and the rest of analytical chemistry department. A special thank to Svante without who the analytical department would not be.

I would like to thank Tobias, Patrick, Einar and Camilla who helped me in the lab when I came and the people who helped me in my thesis and manuscripts, Andreï, Lenore, Fredrik, Agnieszka… and the others. A very special thank to Wen who was really there for me when I came, helped me a lot with administration, lab, life in Umeå and more. Also thanks to Andreas for your help (see, I did not forget).

I would also like to thank Per Hörsted who made great pictures and who helped me a lot understanding my materials. And a special thank for my two exam-workers Anja and Prashant.

I would like to thank all the French Mafia from Umeå, Nathalie, Nadia, Christine, Laurent, Tatiana, Delphine, Rozenn, Gaia, Mathieu, Edouard and all other…

A special thank for Marco and Bud for our fruitful discussions on “meules”.

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I would also like to thank my friends Muriel, Jaana, Audrey and Dodie, who were there when I was feeling alone.

Also a deep thought to my best friend Olivier for trying to hire me in his company and to his wife Adeline.

How can I acknowledge people without thanking Knut, my supervisor, who took me to his home and family when I came (he would maybe reconsider this proposition now that he knows me). Thanks for your support during the thesis and the load of things I learnt with you.

Thanks for Antoine who made the nice cover of my thesis.

I would like to thank my wife Charleen who gave me all what I was waiting for during that time, and Makrill and Mai Rùa for their help home to move the plants. An extra thought to Rat-clette, Tartiflette, Rat-gnagna and Shaki-rat.

I would like to thank all my family. I hope you will understand maybe a word of all this … Merci …

Finally I would like to thank every single girl from Umeå who contributed to make days feel better when the weather was not good and when the weather was good.

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47

Annex 1: Templates used for imprinting during the thesis.

O

HOO

Cl

Cl

2,4-Dichlorophenoxyacetic acid

NNH

O

N

OH

OH

OH

OH

N O

vitamin B2 - Riboflavine

OP

O

OH

OH

NH

O

O

Fmoc-PTyr-OMe

O

O

H3C

NH

NN

O

N

O

theophyllin

NH2 O

O

HONH

OHN

H-Asp-Ala-BetaNA

O

O

NH

H2N

HO

O

O

aspartame

N

O

HN

Bupivacaine

N O

HN

(S)-Ropivacaine

N

O

HN

Mepivacaine

48

Annex 2: Main monomers and cross linkers used in MIP synthesis.

O

OH

methacrylic acid

O

HOp-vinylbenzoic acid

O

OHacrylic acid

N

4-vinylpyridine

N

NH

4-(5)-vinylimidazole

H2N

allylamine

O

N

HN

N,N'-diethyl aminoethyl methacrylamide

N

O

N-vinylpyrrolidone

O

NH2acrylamide

O

NH2

methacrylamideO

OOH

2-hydroxyethyl methacrylate

Nacrylonitrile

O

Omethyl methacrylatestyrene

O

HN

ONH

N,N'-ethylene bismethacrylamide

OO

O

O

O

O

triethylen glycol dimethacrylate

p-divinylbenzeneO

OOO

ethylen glycol dimethacrylate

NN

O

O

1,4-diacryloylpiperazine

O

O

O

O

O

O

trimethylolpropane trimethacrylate

OH

O

O

O

O

O

O

pentaerythritol triacrylate

49

Annex 3: Schematic of the MicroCal ITC instrument

50