Synthesis and Modification of Monodisperse Polymer Particles

for Chromatography

Fredrik Limé

Akademisk Avhandling

som med vederbörligt tillstånd av Rektorsämbetet vid Umeå Universitet för avläggande av

filosofie doktorsexamen framläggs till offentligt granskning vid Kemiska institutionen,

sal KB3A9, Lilla Hörsalen, KBC-huset, fredagen den 23 januari 2009, kl. 10.00.

Fakultetsopponent: Prof. Harald D. H. Stöver, Department of Chemistry,

McMaster University, Hamilton, ON, Canada.

ii

Organisation Document type

Umeå University Doctoral thesis Department of Chemistry SE-90187 UMEÅ Date of publication 18 December 2008 Author Fredrik Limé Title Synthesis and Modification of Monodisperse Polymer Particles for Chromatography Abstract Liquid chromatography is an analytical technique that is constantly facing new challenges in

the separation of small molecules and large biomacromolecules. Recently the development

of ultra high pressure liquid chromatography has increased the demand on sturdy particles

as stationary phase. At the same time the particle size has decreased to sub-2 µm and packed

into shorter analytical columns. This thesis deals with the development of new ways of

preparing particulate polymer materials using divinylbenzene (DVB) as crosslinker. It

includes a novel procedure for synthesizing monodisperse polymer particles by

photoinitiated precipitation polymerization. A 150 W short arc xenon lamp was used to

initiate the polymerizations. The synthesized particles are monodisperse and have an

average particle size ranging from 1.5 to 4 μm depending on reaction conditions and have

subsequently been used as grafting templates. The surface of DVB particles contains residual

vinyl groups that serve as anchoring points for further functionalization via a variety of

grafting schemes. Copolymerization with incorporation of 2,3-epoxypropyl methacrylate

yielded pendant oxirane groups on the particle surface. Atom transfer radical polymerization

(ATRP) was used to graft methacrylates from the surface resulting in a core-shell type

material. A “grafting to” scheme was used to attach pre-made sulfopropyl methacrylate

telomers onto particles containing oxirane rings.

Keywords: Polymer particles; photoinitiation; precipitation polymerization; styrene;

divinylbenzene; atom transfer radical polymerization; liquid chromatography

Language: English Number of pages: 45 + 4 papers ISBN: 978-91-7264-709-1

iii

Synthesis and Modification of Monodisperse Polymer Particles

for Chromatography

Fredrik Limé

Kemiska institutionen

Umeå universitet

Umeå 2008

iv

Cover: Scanning electron micrographs of particles in Paper I-IV. Detta verk skyddas enligt lagen om upphovsrätt (URL 1960:729) ISBN 978-91-7264-709-1 Printed by: Print och Media, Umeå University, Sweden Distribution: Department of Chemistry Umeå University, SE-90187 Umeå, Sweden.Phone: +46 (0)90-786 5000 E-mail: fredrik.lime@chem.umu.se

v

Synthesis and Modification of Monodisperse Polymer Particles for

Chromatography

Author Fredrik Limé

Department of Chemistry, Umeå University, S-90187 Umeå, Sweden

Abstract Liquid chromatography is an analytical technique that is constantly

facing new challenges in the separation of small molecules and large

biomacromolecules. Recently the development of ultra high pressure

liquid chromatography has increased the demand on sturdy particles

as stationary phase. At the same time the particle size has decreased

to sub-2 µm and packed into shorter analytical columns. This thesis

deals with the development of new ways of preparing particulate

polymer materials using divinylbenzene (DVB) as crosslinker. It

includes a novel procedure for synthesizing monodisperse polymer

particles by photoinitiated precipitation polymerization. A 150 W

short arc xenon lamp was used to initiate the polymerizations. The

synthesized particles are monodisperse and have an average particle

size ranging from 1.5 to 4 μm depending on reaction conditions and

have subsequently been used as grafting templates. The surface of

DVB particles contains residual vinyl groups that serve as anchoring

points for further functionalization via a variety of grafting schemes.

Copolymerization with incorporation of 2,3-epoxypropyl

methacrylate yielded pendant oxirane groups on the particle surface.

Atom transfer radical polymerization (ATRP) was used to graft

methacrylates from the surface resulting in a core-shell type

material. A “grafting to” scheme was used to attach pre-made

sulfopropyl methacrylate telomers onto particles containing oxirane

rings.

Keywords Polymer particles; photoinitiation; precipitation polymerization;

styrene; divinylbenzene; atom transfer radical polymerization; liquid

chromatography

ISBN 978-91-7264-709-1

vi

Populärvetenskaplig sammanfattning på svenska

Vätskekromatografi är en analytisk kemisk teknik som ständigt står inför nya

utmaningar när det gäller att separera allt från små organiska föreningar till stora

makromolekyler. Denna avhandling beskriver tillverkning av polymera partiklar

med exceptionellt jämn storleksfördelning och ytmodifiering av dessa, för

användning som stationärfas i kromatografikolonner. Polymeriseringstekniken

som används är utfällningspolymerisering där lösningen UV-bestrålas av en 150 W

xenonlampa. Monomeren (byggstenen) löses tillsammans med en intiator i ett

lösningsmedel och efterhand som polymeriseringen fortskrider faller

polymerpartiklarna ut. Polymerpartiklarna är gjorda av monomeren divinylbensen

som fungerar som en tvärbindare, dvs att den länkar ihop flera kedjor till ett hårt

litet nystan. Partiklarna växte till en storlek på 1,5 till 4 µm under två till fyra dygn.

Efter tillverkningen är partiklarnas yta täckta av vinylgrupper som kan användas

för att fästa funktionella polymerkedjor. Genom att tillföra monomeren 2,3-

epoxipropylmetakrylat i polymeriseringen kunde man desutom få en partikelyta

som innehöll epoxigrupper. Epoxigrupperna användes för att fästa positivt laddade

polymerkedjor av bestämd längd. Materialet packades i en kromatografikolonn och

användes för att separera en testlösning bestående av fyra proteiner.

Partiklarna användes även som bas för ymppolymerisering där den vinyltäckta ytan

fått reagera med vätebromid. Detta gör att partiklarna blir stora makroinitiatorer

som kan användas för att på ett kontrollerat sätt låta polymerkedjor växa från ytan.

I en undersökning ympades 2,3-epoxypropylmetakrylat från ytan på partiklarna

och resultatet blev ett tjockt ytskikt. Epoxigrupperna kunde sedan hydrolyseras till

dioler vilket gjorde partiklarna mer hydrofila.

vii

This thesis is based on the papers and manuscript listed below, which are referred

to in the text by their corresponding Roman numerals.

I. Monodisperse Polymeric Particles by Photoinitiated Precip-

itation Polymerization

Fredrik Limé and Knut Irgum

Macromolecules, 2007, 40, 1962-1968

II. Polymerization of Divinylbenzene and Divinylbenzene-co-

Glycidyl Methacrylate Particles by Photoinitiated Precipitation

Polymerization in Different Solvent Mixtures

Fredrik Limé and Knut Irgum

Manuscript submitted to Macromolecules

III. Hydrobromination of Residual Vinyl Groups on Divinylbenzene

Polymer Particles Followed by Atom Transfer Radical Surface

Graft Polymerization

Fredrik Limé and Knut Irgum

Journal of Polymer Science Part A: Polymer Chemistry (Accepted for

publication 2008-11-08)

IV. A Cation-Exchange Material for Protein Separations Based on

Grafting of Thiol-Terminated Sulfopropyl Methacrylate

Telomers onto Hydrophilized Monodisperse Divinylbenzene

Particles

Anna Nordborg, Fredrik Limé, Andrei Shchukarev and Knut Irgum

Journal of Separation Science, 2008, 31, 2143-2150

Paper I is reprinted with permission from American Chemical Society (Copyright

2007) while Paper III and Paper IV are reprinted with permission from Wiley

(Copyright 2008).

viii

Also intresting reading by the author but not included in the thesis:

A. New Polymer Material Comprising Cross-Linked Spherical

Particles, A Method for Producing the Material and uses Thereof

Fredrik Limé and Knut Irgum

International Patent Application, Application No. PCT/EP2007/005096

WO-2007-144118

ix

Coulda Woulda Shoulda

x

Abbreviations

AN Acrylonitrile

AIBN 2,2′-azobis(2-methylpropionitrile)

ATRP Atom Transfer Radical

Polymerization

BET Brunauer-Emmett-Teller

BJH Barrett-Joyner-Halenda

BMA Butyl Methacrylate

BPO Benzoyl Peroxide

CRP Controlled/Living Radical

Polymerization

CV Coefficient of Variation

DA Dodecyl Acrylate

DC Direct Current

DMA Dodecyl Methacrylate

DMAEMA N,N-dimethylaminoethyl

methacrylate

DMF N,N-dimethylformamide

Dn Number-Average Diameter

Dw Weight-Average Diameter

DVB divinylbenzene (technical grade

80%)

EA Ethyl Acrylate

EDMA Ethylene Dimethacrylate

EMA Ethyl Methacrylate

FT-IR Fourier Transform Infrares

Spectroscoy

GMA 2,3-Epoxypropyl Methacrylate

HEMA 2-Hydroxyethyl Methacrylate

HIC Hydrophibic Interaction

Chromatography

HILIC Hydrophilic Interaction

Chromatography

HIPE High Internal Phase Emulsion

HPLC High Preformance Liquid

Chromatography

IEC Ion-Exchange Chromatography

IUPAC International Union of Pure and

Applied Chemistry

MA Methyl Acrylate

MAA Methacrylic acid

MMA Methyl Methacrylate

mCPBA meta-Chloroperoxybenzoic acid

NMP Nitroxide Mediated Polymerization

NPLC Normal Phase Liquid

Chromatography

PDI Polydispersity Index

PFA Perfluoroalkoxy (Teflon)

PMDETA Pentamethyl Diethylenetriamine

RAFT Reversible Addition Fragmentation

Chain Transfer

RPLC Reversed Phase Liquid

Chromatpgraphy

SEC Size Exclusion Chromatography

SEM Scanning Electron Microscopy

SFRP Stable Free-Radical Polymerization

SLS Static Laser Scattering

SPE 3-[N,N-Dimethyl-N-

(Methacryloyloxyethyl)

ammonium] propanesulfonate

SPM Sulfopropyl Methacrylate

St Styrene

TEGDMA Triethyleneglycol Dimethacrylate

TEMPO 2,2,6,6-tetramethyl-1-piperidinoxyl

THF Tetrahydrofuran

UHPLC Ultra-High Pressure Liquid

Chromatography

UV Ultraviolet

VBC Vinylbenzyl Chloride

XPS X-ray Photoelectron Spectroscopy

xi

Table of Contents

1. Introduction .....................................................................................................- 1 -

2. Particulate Stationary Phases in Liquid Chromatography .................- 1 -

2.1 Silica-based Materials .................................................................................. - 2 -

2.2 Polymer-based Materials ............................................................................. - 3 -

2.3 Specific Permeability for Packed Columns.................................................. - 4 -

3. Free Radical Polymerization ...................................................................... - 5 -

3.1 Polymerization Techniques in Polymer Synthesis....................................... - 7 -

3.1.1 Definition of Monodisperse Materials .................................................. - 8 -

3.2 Thermally Initiated Precipitation Polymerization ...................................... - 9 -

3.3 Distillation Precipitation Polymerization................................................... - 11 -

3.4 Photoinitiated Precipitation Polymerization..............................................- 12 -

3.4.1 Copolymerization with DVB as Cross-linker.......................................- 15 -

3.4.2 Cosolvents........................................................................................... - 18 -

3.4.2 Particle Pressure Stability ...................................................................- 19 -

4. Reactions with Residual Vinyl Groups .................................................. - 20 -

4.1 Hydrobromination ......................................................................................- 21 -

4.2 Epoxydation ................................................................................................- 21 -

5. Grafting Techniques ................................................................................... - 22 -

5.1 The “grafting to” Approach......................................................................... - 22 -

5.2 The “grafting from” Approach ................................................................... - 25 -

5.2.1 Nitroxide Mediated Polymerization (NMP) ....................................... - 25 -

5.2.2 Reversible Addition Fragmentation Chain Transfer Polymerization

(RAFT) ......................................................................................................... - 25 -

5.2.3 Atom Transfer Radical Polymerization (ATRP)................................. - 26 -

6. Materials Characterization....................................................................... - 29 -

6.1 Size and Morphology.................................................................................. - 30 -

6.2 Surface Area and Porosity...........................................................................- 31 -

6.3 Characterization of Elemental Composition ............................................. - 33 -

7. Concluding Remarks .................................................................................. - 36 -

8. Acknowledgment......................................................................................... - 38 -

9. References and Notes................................................................................. - 39 -

xii

- 1 -

1. Introduction

Analytical chemistry spans a wide range of techniques, where chromatography is an

important widely used tool because of the need to separate compounds in complex

samples before they can be presented to a detector. The term chromatography was

first mentioned over 100 years ago by Tsvett, as he separated plant pigments by

flushing them with aid of solvents through a column packed with natural porous

material.1 In liquid chromatography the sample containing the analytes is injected

into a continuously flowing mobile phase and then interacts with a stationary phase

which is packed inside a tubular column. The desired result from these interactions

is that an injected multicomponent sample emerges as individual analytes at the

column exit.

High performance liquid chromatography (HPLC) is one of the most used

techniques in an analytical laboratory today and relies on hydraulic pressure to

force the eluent through the packed column bed. It can be divided into several sub-

categories, depending on eluent and stationary phase polarity, and the kind of

intermolecular interaction that dominates the retention process. Normal Phase

(NPLC), Reverse-Phase (RPLC), Ion-Exchange (IEC), Hydrophobic Interaction

(HIC), Hydrophilic Interaction (HILIC) and Size-Exclusion (SEC) chromatography

are some of the most common sub-techniques of HPLC. They all need a solvent

delivering system in form of a pump, capable of handling high pressures ranging

from a few MPa up to more than 100 MPa. In this thesis the field of polymer

chemistry is used to develop new particulate materials for use in chromatography.

2. Particulate Stationary Phases in Liquid

Chromatography

It is estimated that two million analytical chromatography columns are consumed

worldwide every year.2 Much of the development within liquid chromatography has

been to synthesize new stationary phases, and a prevailing trend in packings for

liquid chromatography is that the particle size of the supports have continuously

become smaller and smaller. In the 1950s, particles had irregular shapes and were

in the size range of 50-100 μm. Today the particles are spherical, monodisperse and

sub-2 µm. The standard particle diameter in analytical columns has for many years

- 2 -

been 5 μm, introduced in the 1980s. In the last decade researchers have focused on

developing column material in the sub-2 μm range for fast or ultrafast separations.3

The pressure required to pump eluent through a column is inversely proportional

to the square of the particle size of the packing, meaning that a decrease in the

particles size by half causes an increases in the pressure by a factor of four. To

reduce the pressure over the column created by smaller particles, shorter columns

are used. Columns packed with small particles operated at high pressures with a

low viscosity mobile phase obtain the highest efficiency.

2.1 Silica-based Materials

Porous silica is the most common substrate in liquid chromatography and is made

by the sol-gel process. Classical sol-gel manufacturing hydrolyses sodium silicate

followed by polycondensation.4 Bonded silica is by far the most widely used

stationary phase in reversed phase chromatography, with the bonded material

most predominantly being octadecylsilane.5,6 Silica particles have a good

mechanical strength making them suitable as support in chromatography. A

drawback with the silica material is that it can only be operated at pH between 2

and 8. This is due to the hydrolysis of the siloxane bond used for attaching the

bonded phase at low pH, and dissolution of the silica particles at high pH. This can

however be partly overcome by introducing steric protecting groups.7

Jorgenson pioneered the use of small particles when he introduced non-porous

octadecylsilane modified silica particles with an average particle diameter of 1.5 μm

packed into 30 μm inner diameter fused silica capillaries.8 The capillary columns

were run under extremely high pressure (140 MPa) and were used to improve the

efficiency and shorten the analysis time for the separation of small molecules.

Figure 1. A representative SEM

micrograph of a state-of-the-art

spherical silica stationary phase,

Kromasil with an average particle

size of 5 µm.

- 3 -

It has proven difficult to optimize the manufacturing process for spherical porous

silica to yield a monodisperse particle distribution, and particles are therefore often

subjected to sizing in order to remove fines and larger particles until the desired

particle size distribution is achieved.2 Figure 1 shows a scanning electron

micrograph (SEM) of state-of-the-art commercial silica material.

2.2 Polymer-based Materials

Silica is the main support material in liquid chromatography but in areas where

silica is inadequate there is a need for polymeric supports. Polymeric stationary

phases are often used in ion exchange, hydrophobic interaction, and affinity

chromatography.9 Reasons for this are the stability of polymeric material in the

entire aqueous pH range,10 and the possibilities of a wide range of surface

functionalizations.11

Chemical cross-linking of the polymers confer strength and insolubility to the

polymers, and particles cross-linked with DVB have a good mechanical strength

and can withstand high pressures. Ng et al. reported non-porous DVB particles that

could withstand pressures up to 69 MPa.12 If there is a low degree of cross-linker,

such as 1-2% in particles prepared by dispersion polymerization (Chapter 3.1) the

particles have a tendency to swell.13 This has been alleviated by conducting a two-

stage dispersion polymerization where polystyrene microspheres was made first

and the cross-linker was added in the second stage, forming highly cross-linked

particles.14 For the material to be suitable as chromatographic support it needs to

contain more than 20% cross-linker.5

Wang et al.15 used staged templated suspension polymerization according to the

Ugelstad principle16,17 to achieve monodisperse macroporous particles of styrene

and DVB.18 In this technique, non-porous uniform particles prepared by emulsion

polymerization are used as templates and swollen together with solvents,

monomer, and cross-linker before polymerized. The monodispersity of the

template particles is retained, but the size increases, and the solvent creates a

macroporous structure.

Another group of polymers that has been successfully implemented as support in

chromatographic columns is particulate methacrylates, prepared by using ethylene

methacrylate as cross-linker. Coupek et al.19 developed hydrophilic particles from

- 4 -

2-hydroxyethyl methacrylate (HEMA) using ethylene dimethacrylate (EDMA) as

cross-linker similar to the commercial available Separon (Tessek, Prague, Czech

Republic) particles in Figure 2. These particles had controlled porosity, high

mechanical strength and chemical stability. Particles consisting of methacrylates

are typically made by suspension polymerization (Chapter 3.1) or seed suspension

polymerization in aqueous phase with an organic modifier acting as a porogen

creating a porous structure.20,21

The interactive processes leading to retention occur on the surface of the stationary

phase, and in the swollen layer of ligands attached to the surface. The porous

structure of the material allows small molecules to diffuse in and out of the

stagnant mobile phase.18 Retention is therefore more or less proportional to the

specific surface area of the packing. Non-porous particles consequently suffer from

low loading capacity due to their low surface area.22 This makes them less suitable

for separation of small molecules, but the absence of a tortuous pore system also

make such sorbents more favorable for fast separations of larger biopolymers such

as oligonucleotides, protein, peptides and DNA due to their slow diffusion rates.23

2.3 Specific Permeability for Packed Columns

An important property of particulate stationary phases is the homogeneity in size.

A column with a monodisperse particle bed results in a lower column pressure drop

and a better column efficiency.24 As the illustration in Figure 3 shows, the extra

column void is larger in a column with particles of different sizes. The hexagonally

close packed particles to the left, gives less voids and better efficiency. It is also

important for column reproducibility that the particles are homogeneous so that a

good packing density can be achieved.25

Figure 2. SEM micrograph of

Separon HEMA particles having

an average particle size of 10 µm.

- 5 -

Figur 3. Illustration of particles packed in a separation column. Left are particles of equal size

(monodisperse) which give a homogeneous packing and right particles with a broad size distribution

that give an inhomogeneous packing density.

The specific permeability of a chromatographic column, packed with particles

determines the pressure drop at a certain flow rate according to Darcy's law.6 It is

determined by measuring the flow velocity and pressure drop over the column.

Darcy's law is written as in equation 1:

L

dKPu

⋅

⋅⋅Δ=

η

2p0 (1)

where u is the superficial fluid velocity, ∆P the columns pressure drop, K0 the

specific permeability, dp the mean particle diameter, η the viscosity of the eluent,

and L the length of the column.26 The specific permeability for a packed column is

highly dependent on the size of the particles and the limiting factor in obtaining the

highest amount of theoretical plates is the column inlet pressure.27 The pressure

drop over the column can be reduced by using a low viscosity organic modifier.28

3. Free Radical Polymerization

Free radical polymerization consists of three steps; initiation, chain propagation,

and chain termination, where monomers (building blocks) are linked together. The

initiation is triggered by a radical initiator such as hydroperoxides, peroxides or azo

compounds either photolytically or thermally.29 One commonly used azo com-

pound is 2,2'-azobis(2-methylpropionitrile) (AIBN) which decomposes into

- 6 -

nitrogen gas and a pair of resonance stabilized 2-cyanopropyl radicals that can act

as initiator radicals (Figure 4).

Molecular oxygen is a diradical and acts as an inhibitor in the polymerization of

most vinyl monomers. Polymerization mixtures consisting of monomer, initiator,

and solvent are therefore usually purged with inert gas or subjected to several

freeze-pump-thaw cycles to remove dissolved oxygen from the solution.

Figure 5. Radical chain growth of the polymerization of polystyrene with the three steps; initiation,

chain propagation and chain termination.

Figure 4. Decomposition

of AIBN into two 2-

cyanopropyl radicals and

nitrogen gas.

- 7 -

The initiator radical reacts with the monomer in the initiation process and gives the

start of a polymer chain. Initiation is followed by chain propagation as shown in

Figure 5 for the conversion of styrene to polystyrene. The combination or

disproportionation of the radicals in two polymer chains terminates the growth

process.

Monomers with more than one vinylic group have the ability to combine or link

together two polymer chains, and are called cross-linkers. A scheme of styrene with

addition of the cross-linker, divinylbenzene (DVB-co-St) is shown in Figure 6. In

polymerizations where more than one species of monomer is used is termed

copolymerization.30

Figure 6. A cross-linked network of styrene and DVB where polymer chains are linked together. The

figure only show para-DVB isomer, but the technical grade DVB that is commercially available contains

a mixture of meta and para in a ratio 2:1.31

3.1 Polymerization Techniques in Polymer Synthesis

Polymer particles used as chromatographic separation material can be synthesized

in many ways resulting in different size and morphology. These polymerization

- 8 -

techniques include suspension31,32, dispersion33, precipitation34 and emulsion

polymerization35.

Suspension polymerization is conducted in aqueous solution utilizing a stabilizer

(often cellulose or polyvinyl alcohol) to prevent particle aggregation. Aggregation is

one of the major problems associated with suspension polymerization but can be

prevented by efficient stirring in addition to the stabilizer. The initiator is either

water soluble and added to the water phase, or soluble in the organic phase and

dissolved in the monomer solution. The resulting particles are usually in the size

range of 20 µm-2 mm and with wide polydispersity.31

Precipitation polymerization typically produces monodisperse particles in the size

range of 1-5 µm.34 It is carried out in a solvent (often acetonitrile) in which both the

monomers and initiators are soluble. As the polymer forms, it precipitates from the

solution. The monomer loading is very low, generally 1-4% of the solvent and a high

amount of the monomer mixture is a cross-linker. Due to the high amount of cross-

linker there is no need for any steric stabilizers.

Dispersion polymerization is very similar to precipitation polymerization but a

suitable stabilizer is added, which together with the monomer and initiator forms a

homogeneous solution. The solvent is usually an alcohol and the monomer

concentration can be as high as 40% of the solvent with less than 1% cross-

linker.36,37

Emulsion polymerization creates latex particles in nanometer range and is

conducted in a water phase that contains a surfactant and a water-soluble initiator.

When monomer is slowly added to the water phase the surfactant forms micelles in

which the polymerization takes place by encapsulating the monomer droplets.

Latex particles prepared by emulsion polymerization are too small for use in liquid

chromatography, but the two step swelling method developed by Ugelstad38

enables the preparation of monodisperse particles in the micrometer size range.

3.1.1 Definition of Monodisperse Materials

Monodispersed particles are defined as particles with a uniform distribution where

all particles have the same size, shape, and structure.39 If a group of particles has a

wide size distribution, it is termed polydisperse. Polydispersity index (PDI) is a

- 9 -

measure of the size distribution in a polymer sample. The combined diameters of

the particles are used to calculate the number-average diameter Dn (equation 2)

and the weight-average diameter Dw (equation 3). To calculate the PDI, (U), the

weight-average diameter Dw is divided with the number-average diameter Dn

according to equation 4. The PDI is always greater than 1.o and a sample is

considered as being monodisperse if the polydispersity index is between 1.0 and

1.1.40

i

iin N

DNDΣΣ

= (2)

3ii

4ii

w DNDND

ΣΣ

= (3)

n

w

DDU = (4)

Another way of reporting the distribution is by the coefficient of variation by taking

the standard deviation divided with the number-average diameter. There is no

clear consensus on how large the coefficient of variation can be for the sample to be

considered as monodisperse. Polymerizations are often classified as monodisperse

with coefficients of variation of at least below 10%, but sometimes as low as 2%.41

3.2 Thermally Initiated Precipitation Polymerization

Introduced by Stöver, precipitation polymerization produces spherical particles in

the micrometer range.34 It closely resembles dispersion polymerization in that the

particles become stable in an early stage. The monomer is soluble in the solvent

and forms a homogeneous mixture together with the initiator (AIBN). When the

polymer chain or particles are formed they phase separate from the solvent and

precipitate as insoluble polymers.42,43 A difference compared to dispersion

polymerization is the high amount of cross-linker. This high degree of cross-linker

makes the particles self-stabilizing with no need for surfactants or steric stabilizers.

The cross-linker is usually commercially available DVB of technical grade with 55

or 80% divinylbenzene isomers, but other cross-linkers have been used as well.44

The high amount of cross-linker gives rigidity inside the particle as well as at the

- 10 -

surface, with the surface stability preventing fusion between particles.45 The

amount of monomer used to produce monodisperse spherical particles is relatively

low, between 2-5% (v/v) of monomer compared to the solvent. Monomer

concentrations above 10% have been shown only to result in coagulum in a 24 hour

polymerization which is the time typically needed to reach diameters of about 3 µm

at lower monomer loading.34 Shorter polymerization time in the same batches gave

monodisperse particles and there are some reports of a monomer concentration as

high as 15% being utilized in particle synthesis.46

Copolymerizations of various monomers have been successfully implemented into

the precipitation polymerization scheme, yielding particles with different chemical

composition. Most work has been done on styrene and methacrylates such as

methyl methacrylate (MMA), butyl methacrylate (BMA), dodecyl methacrylate

(DMA), 2,3-epoxypropyl methacrylate (GMA), and 2-hydroxyethyl methacrylate

(HEMA).47,48 The solvent predominantly used is acetonitrile, which is a near-Θ

solvent for the polymer. That is, an ideal solvent for interactions of the polymer.

Some attempts with acetone and mixtures of acetonitrile and toluene have been

performed with good results.49-51 The initiation is accomplished by carrying out the

polymerization at elevated temperature, around 70 °C, with gentle agitation

arranged by tumbling or shaking the reaction flasks in a thermostatted water bath

or air chamber. By constantly tumbling the flasks, the particles that precipitate

from the solution can continue to attract oligomers from the solution and thereby

continue to grow.

A mechanism for how the particles are formed and how they grow in precipitation

polymerization has been proposed.42 It states that the formation probably starts by

aggregation of soluble oligomers to form a particle core. Growth then takes place

when soluble oligomer radicals are captured by the residual vinyl groups and form

the new self-stabilized particle surface (Figure 7). The investigation into the

mechanism was done by having a batch of particles divided into three equal sub-

batches. The surfaces of two of the batches were modified to alkyls by converting

the residual vinyl groups on the surface of the particles to hexyl and ethyl groups.

The three batches of seed particles were then dispersed in acetonitrile and a new

polymerization was started. Particles that had not been subjected to modification of

the vinyl groups grew substantially during the second polymerization, while the

- 11 -

modified seed particles were almost unchanged. Instead of increased particle size,

the second polymerization of the modified particles showed formation of new

particles. A similar mechanism was presented by the group of Choe but based on

observations of morphology, circularity and surface area.52

Figure 7. Schematic drawing of the mechanism suggested by Stöver et al. Adapted from reference.42

3.3 Distillation Precipitation Polymerization

The basic concept of this technique is precipitation polymerization carried out in a

round-bottom flask. This technique was first used for polymerization of DVB, and

is a process where the solvent (acetonitrile) is removed by distillation.53 A standard

distillation equipment is used, where the monomer, initiator, and solvent are mixed

in a round-bottom flask connected to a fractionation column and a Liebig

condenser. It is a fast polymerization where the mixture is heated to boiling point

and the solvent is distilled from the system within 1.5 hours resulting in

micrometer-sized particles with a yield above 35%. The particles formed in the

initial study were non-porous but in an attempt to increase the porosity, up to 25%

of toluene was added in the solvent. Since toluene has a higher boiling point

(108 °C) than acetonitrile (82 °C) this resulted in toluene remaining in the mixture

thereby creating porosity. Higher toluene fractions only resulted in the formation

of macrogel and soluble polymer.54

- 12 -

This polymerization method has also been used to produce copolymers with DVB

or EDMA as cross-linker.55 The non cross-linking monomers incorporated into

these particles have been vinylbenzyl chloride (VBC), HEMA, methacrylic acid

(MAA), and acrylonitrile (AN).56-59 Poly(MAA) particles in the range of 50-288 nm

have also been polymerized using this technique without the use of any cross-

linker.60 These type of MAA particles without cross-linker were used in a two-stage

distillation polymerization where N-isopropylacrylamide was polymerized in the

second stage to create core-shell particles. The MAA core was later removed to

form hollow N-isopropylacrylamide microspheres.61

Highly cross-linked DVB particles have been used numerous times as core particles

in a two-step distillation polymerization forming a core-shell material. After the

core was polymerized using the equipment mentioned above, or in a rotary

evaporator, the resulting particles were resuspended in acetonitrile. The DVB core

particles contain residual vinyl groups that can be further reacted and the

monomer to be used in the shell was added together with initiator to the core

fraction and the polymerization continued.62,63 Several monomers such as HEMA,

MMA, ethyl methacrylate (EMA), BMA, EDMA, trimethylolpropane

trimethacrylate (Trim), methyl acrylate (MA), ethyl acrylate (EA), i-octyl acrylate

(i-OA), dodecyl acrylate (DA), and triethyleneglycol dimethacrylate (TEGDMA)

were polymerized in the second stage to form the shell.64,65 It was found that

monomers having longer alkyl chains as substituents like BMA, i-OA and DA

formed coagulum in the second stage. In a polymerization with DVB and HEMA in

the second stage, it was found that too high concentration of HEMA led to

secondary nucleation and the new particles thereby formed resulted in increased

polydispersity.62

3.4 Photoinitiated Precipitation Polymerization

In Paper I photoinitiation was introduced as an alternative to thermal initiation

for producing monodisperse polymeric particles by precipitation polymerization.

Photoinitiation was achieved by irradiating the sample mixture with a focused

150 W short arc xenon lamp. The most commonly used initiator in precipitation

polymerization is AIBN, which can also be cleaved to form initiating radicals upon

UV-irradiation. The photoinitiated precipitation polymerization is a single-step

- 13 -

process that yields non-porous particles at close to ambient temperature. Figure 8

shows a schematic drawing of the setup where polypropylene or Teflon PFA flasks

were attached to an axis four at the time. The axis was powered by a geared down

DC motor with adjustable speed. The tumbling ramp of flasks was placed at a

distance of approximately 30 cm from the lamp. The proximity of the flask to the

lamp increased the temperature in the sample mixture to about 31 °C.

Figure 8. The custom made polymerization setup used in photoinitiated precipitation polymerizetions

with the custom made rotor. [Paper I]

The conversion rate for the UV-induced polymerization is slower than thermally

initiated or distillation precipitation polymerization which have a conversions of up

to 60% in 24 hours and 45% in 1.5 hours, respectively.34,53 The first photoinitiated

experiment using DVB as monomer was left to react for 24 hours resulting in a

conversion of only 3.4%, an average particle size of 0.39 µm and a polydispersity

index of 1.52. The way of improving the conversion, size and uniformity turned out

to be an increase in polymerization time.

In Paper II polymerizations of DVB were plotted size vs. time to show an almost

linear relationship. As evident from Figure 9, a continuation of the polymerization

made the particles grow further and at the same time the PDI decreased which

means the particles became more monodisperse. The increase in growth can also be

seen in the SEM micrographs in Figure 10.

- 14 -

Figure 10. SEM micrographs of particles polymerized with different polymerization time, (a) 48 h, (b)

88 h, (c) 112 h, and (d) 163 h. The other reaction conditions were the same using 4% (v/v) DVB and 2%

(w/w) of AIBN. Magnification 5000. [Paper I and II]

Even though photoinitiation has a positive effect on the heat-affected likelihood of

particle coalescence, through the slower Brownian motion of the growing particles

and a lower risk that the swollen surface layer is in a rubbery state, there are other

Figure 9. Graph showing the

increase in average particle

diameter with a concurrent

decrease in the polydispersity

index for DVB particles.

[Paper II]

- 15 -

factors playing an important part in the characteristics of the resulting particles.

The polymerization experiments were carried out in 250 mL Teflon PFA flasks

loaded to the brim with polymerization mixture and with 2% (w/w) of AIBN. This

resulted in an increase in particle diameter compared to the standard setup with

200 mL polymerization mixture in a 250 mL polypropylene flask. Rational

explanations to the faster growth could be that Teflon is a better material for UV

penetration, or that there was less oxygen in the flasks to inhibit the reaction.

UV-irradiation with photoinitiators like 2,2-dimethoxy-2-phenylacetopheneone

(DMPA) and 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropanone-1

(MMMP) have also been used.66 The experiment was setup in an incubator which

made it possible to control the temperature between 25 and 40 °C. It was found

that when an acetophenone type initiator was used, the size of the particles was less

affected by reaction conditions.

3.4.1 Copolymerization with DVB as Cross-linker

Cross-linked polystyrene is a common support in chromatography and probably

the most extensively studied polymer system, especially in suspension

polymerization.9,67 In Paper I copolymerizations of styrene and DVB in a 1:1 molar

ration was done creating non-porous particles with a surface area of only 5 m2/g.

Figure 11 shows the relationship between the monomer loading and the size of the

particles for DVB and copolymers of St-co-DVB.68

Figure 11. The growth of St-

co-DVB and DVB particles

polymerized for 95 hours at

different monomer loadings.

- 16 -

The monomer loading in the polymerization mixture was also investigated by

increasing the concentration up to 10% (v/v). For particles consisting only of DVB

it was possible to achieve monodisperse particles with that high concentration. For

the copolymerization with styrene a concentration of 10% (v/v) there were some

cauliflower like particles formed but they where imbedded in coagulum, shown in

Figure 12. This shows the importance of the cross-linker at high monomer loading.

Shim et al. was able to achieve cauliflower like particles without coagulum with a

concentration of 15% (v/v) using a molar ratio of 50% styrene.69

Figure 12. SEM micrographs of (a) DVB and (b) St-co-DVB particles polymerized in acetonitrile with a

monomer loading of 10% (v/v) in relation to solvent. [PaperI]

The incorporation of GMA in the polymerization mixture will yield particles that

contain oxirane groups on the surface. The introduction of such groups presents an

advantage in providing alternative functionalization handles to the residual vinyl

groups on pure DVB particles. There are only a few reports on preparing highly

cross-linked DVB-co-GMA particles by precipitation polymerization. The first

paper by Li et al. used a monomer concentration of 2% (v/v) compared to solvent

and achieved mono- or narrow disperse particles with GMA weight fractions from

0.1 to 0.4 with respect to DVB, with an increase in polydispersity at higher GMA

fractions.47 The group of Choe used a total monomer concentration of 5.2% (v/v)

but was only able to achieve monodisperse particles at GMA fraction of 0.1, or by

polymerization of GMA and DVB separately.70 Zhang et al. polymerized DVB-co-

GMA particles by dispersion polymerization without steric stabilizer in acetonitrile.

Since dispersion polymerization has a close relationship to precipitation

polymerization this is the same principal in a distillation precipitation

- 17 -

polymerization setup using a round-bottom flask, mechanical stirrer and

condenser. The polymerization was started with 4% (v/v) of DVB compared to

solvent and 2% (v/v) of GMA was added after 4 hours of polymerization. The

polymerization thereafter continued for an additional 16 hours and yielded

monodisperse particles in the size range of 3 μm.71

Ring-opening reactions with oxirane groups have been used to further functionalize

the material. One of the frequently used functionalizations of polymeric material

that contains an oxirane ring is the reaction with various amines or sulfonate.72

These can later be turned into quaternary amines for use as support in anion

chromatography, where they can be used in a wide pH range. The modification of

silica particles has also been performed using the same principle. For instance,

Choi et al. first grafted GMA to silica particles before reacting the oxirane with a

large number of amines.73,74

In Paper II GMA was polymerized under precipitation polymerization conditions

using DVB as cross-linker and AIBN as photoinitiator to produce rigid

monodisperse particles. The GMA fraction in the experiments was fixed at a molar

ratio of 1:1 that of DVB. The results were largely similar to the results of the groups

Figure 13. SEM micrographs of DVB-co-GMA particles polymerized in acetonitrile with a total

monomer concentration of (a) 2% and (b) 4% (v/v). [Paper II]

mentioned above, that a higher monomer loading resulted in increased

polydispersity. For a monomer concentration of 4% (v/v) the particles ranged

between 2.2 and 4.5 µm (U=1.193, CV=25.8). Reducing the monomer

- 18 -

concentration to 2% (v/v) led to monodispersity (U = 1.019) with a coefficient of

variation (CV) of 9.6%, but a simultaneous decrease in the average diameter to

2.7 µm as shown in Figure 13.

3.4.2 Cosolvents

For polymerization of particles that are to be used as stationary phases in liquid

chromatography it is common to add a solvent to act as porogen, thereby yielding

porous particles. The purpose of creating pores is to increase the surface area and

establish a pore system with dimensions that will allow molecules to diffuse in and

out of the pores, so that retention can be achieved. The most frequently used

porogen in polymerization of DVB and styrene is toluene, which have resulted in a

surface areas well above 800 m2/g.51,75

In Paper II DVB was polymerized in different cosolvents, among them toluene

with the result of an increased surface area. However, when toluene was added in

the mixture the resulting particles came out with a wide size distribution. Along

with toluene, other solvents such as 1-decanol, 1-octadecanol, and THF were tried

which had been used as porogens or were deemed as potential porogens. Only THF

and the ternary system with acetonitrile:1-decanol:THF gave monodisperse

particles. THF showed remarkable results when added to the solvent during the

polymerization. The particles grew approximately four times as fast in size and this

made it possible to shorten the polymerization time and still get particles in the size

range (2-5 µm) suitable for chromatography (Figure 14). A series of

polymerizations with different fractions of THF were done to see if the particles

would continue to grow faster with increasing fractions. The particles were

polymerized for 46 hours and grew larger in size with increasing amount of THF up

to 40%. Above 40%, only oligomers were obtained, nothing was precipitated out

from solution and no particles were formed.

Since polymerizations with DVB gave a large increase in particle size it was used in

copolymariztions with GMA as well. The polymerizations with DVB-co-GMA

achieved similar results in a faster growth rates allowing the polymerization time to

be reduced to half and still achieve the same particle size. The ternary solvent

system of acetonitrile:1-decanol:THF also resulted in monodispersed particles but

not with the same growth rate as for only THF.

- 19 -

Figure 14. SEM micrographs of DVB particles polymerized for (a) 48 h with no added cosolvent and (b)

46 h with THF as cosolvent. [Paper II]

3.4.2 Particle Pressure Stability

In Paper II DVB and DVB-co-GMA particles were packed into stainless steel

columns at a pressure of 60 MPa and when connected to an HPLC system the

pressure vs. fluid velocity relationship was linear and without hysteresis up to

30 MPa. Uracil is among the most frequently used void volume markers in reversed

phase chromatography and was used to measure the void volume in the system.76

The fluid velocity was also used in calculating the specific permeability for the

columns together with the measured pressure drop over the column. Figure 15

shows the specific permeability for DVB and DVB-co-GMA particles polymerized

Figure 15. Specific permeability for

DVB and DVB-co-GMA particles

polymerized with and without add-

ition of THF as cosolvent. [Paper II]

- 20 -

with and without addition of THF as cosolvent. The results show data grouped

together based on material composition and not solvent composition. This meant

that addition of THF as cosolvent did not make the particles more compressible.

The graphs showed no sign of the particles being compressed. Scanning electron

microscopy (SEM) micrographs of the particles recorded after they were removed

from the column (Figure 16) showed no evidence of cracks, dents, or deformation.

4. Reactions with Residual Vinyl Groups

The two vinyl groups on DVB have differences in their reactivity due to steric

hindrance.77 The result of the difference in reactivity is, that even if the particles are

highly cross-linked, the surface contains residual vinyl groups that subsequently

can be used in further reactions.42 This facilitates the combination of the rigidity of

DVB particles with a diverse range of surface chemistries. The group of Darling has

made extensive research on reactions with vinyl groups on commercially available

Amberlite XAD-4. These reactions involved conversion of the double bond to 1,2-

dibromoethyl, bromohydrin, epoxydation, and addition of thiols and disulfides.78-80

Particles prepared by precipitation polymerization have also been subjected to

hydrochlorination resulting in 1-chloroethyl groups on the particle surface.81 These

particles were later used as macroinitiators in order to graft styrene from the

surface via atom transfer radical polymerization (ATRP). The surface of the

particles prepared by photoinitiated precipitation polymerization contained

residual vinyl group as well. The amount of residual vinyl groups on the particles

was determined by converting the vinyl groups to 1,2-dibromoethyl, turning the

bromine into salt and titrating with silver nitrate.82

Figure 16. DVB-co-GMA particles

unpacked from a column packed at 60

MPa final packing pressure and

subjected to a Darcy plot. [Paper II]

- 21 -

4.1 Hydrobromination

In Paper III, the residual vinyl groups on the surface of the particles were

subjected to hydrobromination by an anti-Markovnikov addition, to get bromine in

the primary position on the alkane chain. The solvent plays an important role for

the yield of anti-Markovnikov product and should preferably be a non-polar

hydrocarbon with more than five carbon atoms,83 and n-heptane was therefore

used to suspend the particles. Benzoyl peroxide (BPO) was added to the reaction as

a source of free radicals, thereby giving free radical addition of bromine to the

primary position. FT-IR (Chapter 6.3) showed a peak at 1263 cm-1 corresponding to

bromine being added to the primary carbon of the vinyl group yielding 2-

bromoethyl.84,85

4.2 Epoxydation

In Paper IV the pendant vinyl groups were modified by turning them into oxirane

rings as shown in Figure 17.86 Commercially available m-chloroperoxybenzoic acid

(mCPBA) was used since it has a tendency to form intramolecular hydrogen bonds

that results in an electrophilic oxygen that can be added to the vinyl group.

Figure 17. The transformation of the vinyl group to an oxirane ring using mCPBA. [Paper IV]

Most often, polymer particles containing oxirane groups on the surface have been

prepared by copolymerizations with GMA87-89, similar to the material described in

Paper II (Chapter 3.4.1). However, the conversion of the vinyl groups is an

interesting alternative which combines the full benefit of a rigid cross-linked DVB

particle and the introduction of a new reactive site. The oxirane group can also be

subjected to hydrolysis to give a hydrophilic functionality in 2,3-dihydroxypropyl

groups. An oxirane group produced in this way is not subject to hydrolytic cleavage,

which is otherwise a risk with hydrophilic methacrylic esters.

- 22 -

5. Grafting Techniques

Grafting functional polymer brushes or telomers to different kinds of substrates has

become an important way of changing the surface properties of a material. Both the

“grafting to” and the “grafting from” approaches have the possibility to change the

properties of polymer particles. For HPLC, this means an advantage since a highly

cross-linked pressure stable core particle can be grafted with a softer and more

permeable layer around the core in a core-shell fashion.

5.1 The “grafting to” Approach

One of the most common ways of functionalizing particulate materials for use in

chromatography is the “grafting to” approach where the grafting of octadecylsilane

is a classical example.2,73,90 In this method an already pre-made polymer chain

(telomer) is used, that carries an active terminal group that can be reacted with an

active particle surface. The benefit of this method is that it gives good control over

the graft size and that telomers can be tailor made for specific purposes, preferably

by a controlled polymerization to yield a molecular weight distribution of low

polydispersity. Because telomers are sizeable molecules, the amount of grafts that

can be immobilized onto the surface might be low. This is due to the risk of steric

hindrance from already attached chains which could lead to low grafting

density.79,91 Unsal et al. showed one approach of “grafting to” where they first

attached 3-(trimethoxysilyl)propylmethacrylate to hydrolysed EDMA-co-GMA

particles to get pendant methacrylate groups on the surface.92 In a “grafting to”

fashion they attached various sulfonate or amino methacrylates onto the

methacrylated particles and the material was later used for ion-exchange

separations of proteins.

Thiols have been grafted to material with residual vinyl groups in an anti-

Markovnikov addition reaction in the presence of a radical initiator.93 The reaction

time for adding thiols and disulfides to the vinyl groups has varied between 1 day

and 2 weeks.79 To test this reaction, 2-mercaptoethanol was grafted to the surface

of DVB particles.94 The particles were suspended in toluene together with AIBN

and heated to 70 °C for 24 hours. This resulted in 2 atomic% (at.%) of sulfur on the

surface as verified by XPS (see chapter 6.3). Mercier et al. grafted 2-

mercaptoethanol, 4-aminothiophenol and 2-aminoethanethiol to vinyl groups of

- 23 -

DVB polyHIPEs (High Internal Phase Emulsion) during 48 hours at temperature of

60-70 °C.85,95

In Paper IV thiol-terminated telomers synthesized from 3-sulfopropyl

methacrylate were grafted to mCPBA modified DVB particles. The telomers were

attached by nucleophilic addition of the thiol to the oxirane ring, and heating the

mixture at 70 °C during 96 hours gave particles with strong cation-exchange

functionality. After the grafting procedure the particles were slurry packed in an

analytical column and the remaining oxirane groups were hydrolyzed with 0.5 M

H2SO4. By hydrolyzing the remaining oxirane groups that had been protected from

the bulky telomers, as is presented in Figure 18, the particles were made more

hydrophilic.

Figure 18. Reaction path for grafting sulfopropyl methacrylate telomers from the surface of DVB

particles and splitting the remaining oxirane into a diol after the particles were packed in an analytical

column. [Paper IV]

The SEM micrographs in Figure 19 show circular markings in the surface of the

particles that is an indication of a grafted layer of telomers. It appears as the

particles have been sticking together during the grafting process and that part of

the layer has been transferred from one particle to another when they have

detached from each other. The grafted particles were analysed by X-ray

photoelectron spectroscopy (XPS) to see the amount of sulfur and oxygen. The

amount of oxygen increase from 3.9 at.% on epoxy-modified particles to 20.7 at.%

after grafting. The amount of sulfur on the grafted particles was 2.5 at.%.

- 24 -

Figure 19. SEM micrographs of the particles at different magnifications after they were grafted with

sulfopropyl methacrylate telomers. The surface show indents in the soft telomer shell from particles

sticking together. [Paper IV]

The column was evaluated for the separation of a test mixture of four standard

proteins: cytochrome C, lysozyme, myoglobin and ribonuclease A in cation-

exchange mode. The hydrophilic nature of the material was verified by the lack of

retention of the proteins on a non-grafted but hydrolyzed material. The grafted

particles were able to separate the test mixture due to the cation exchange groups

introduced and showed no hydrophobic interactions between protein and grafted

particles (Figure 20).

Figure 20. Separation of protein test mixture containing 0.2 mg/mL of myoglobin and lysozyme,

0.3 mg/mL of cytochrome C and 1 mg/mL ribonuclease A in cation-exchange mode using gradient

elusion. Eluent A: 20 mM phosphate buffer pH 7; eluent B: 1 M NaCl in 20 mM phosphate buffer pH 7.

Linear gradient: 0-100% B in 5 min, starting t=0. Flow rate 0.5 mL/min. Detection: UV 280 nm.

[Paper IV]

- 25 -

5.2 The “grafting from” Approach

Controlled/living radical polymerization (CRP) comprises several reaction schemes

where there is an equilibrium between the active chain and a dormant species, and

where unintentional termination is strongly reduced.96 This type of polymerization

is suitable for use in surface-initiated polymerization schemes, known as “grafting

from” since the chains start growing from an active initiator site bound to the

surface. Examples of these techniques that have been successful in grafting from

surfaces97,98 are nitroxide mediated polymerization (NMP)99, reversible addition

fragmentation chain transfer (RAFT) polymerization100 and atom transfer radical

polymerization (ATRP).101

5.2.1 Nitroxide Mediated Polymerization (NMP)

NMP is the most successful stable free radical polymerization (SFRP) and it uses a

stable nitroxide radical as controlling agent. The most extensively studied nitroxide

radical is 2,2,6,6-tetramethyl-1-piperidinoxyl (TEMPO).99 There are two ways of

carrying out the polymerization; thermal decomposition of an alkoxyamine into a

reactive radical and a stable radical, or by using a conventional radical initiator

such as AIBN or benzoyl peroxide (BPO) together with the nitroxide radical.102

NMP using TEMPO requires the use of high temperature and long reaction times,

and only styrene and 4-vinylpyridine polymerize with good control.103 It has been

difficult to achieve successful control in SFRP with methacrylates, resulting in low

conversion and high polydispersity. Substantial effort has been put into testing new

nitroxides, which has resulted in shortened reaction times and lower temperatures.

When it comes to grafting from surfaces, silica nanoparticles or planar substrates

are currently most common, and the surfaces have been modified to contain an

alkoxyamine or a peroxide.104,105

5.2.2 Reversible Addition Fragmentation Chain Transfer

Polymerization (RAFT)

Introduced in 1998, RAFT can be used with a wide range of experimental setups

both when it comes to monomers and solvents.100 In RAFT polymerization the

chain growth is controlled by reversible chain transfer initiated by a conventional

initiator in the presence of a chain transfer agent.106 The chain transfer agent is

- 26 -

typically a dithioester and it makes RAFT a living polymerization since it is equally

labile attached to the polymer as to the free chain transfer agent. Grafting

polymeric chains from different types of substrates using RAFT have been made on

materials such as polymer particles, silica, and planar silica wafers.98 In the

“grafting from” reactions, the chain transfer agent is typically attached to the

surface of the substrate. Barner et al. grafted styrene from DVB particles prepared

by precipitation polymerization.107 They also incorporated the chain transfer agent

1-phenylethyl dithiobenzoate into the precipitation polymerization resulting in

residual RAFT end groups on the surface of the particles. Grafting of a zwitterionic

monomer 3-[N,N-dimethyl-N-(methacryloyloxy-ethyl) ammonium] propane-

sulfonate (SPE) was done both from DVB particles immobilized with chain transfer

agent and on unmodified particles where the chain transfer agent was in

solution.108

5.2.3 Atom Transfer Radical Polymerization (ATRP)

ATRP or transition metal catalyzed controlled radical polymerization uses a

transition metal-halide as catalyst. The concept was published by two groups in

1995, Matyjaszewski101 who used copper(I) and Sawamoto109 using ruthenium(II)

as catalyst metal. A common complex to catalyze the reaction is that of Cu(I) and

2,2'-bipyridine as complexing ligand.101,110-112 A large number of catalyst metals such

as iron, manganese, rhenium, nickel, palladium, and titanium, have been used.113-119

Multidentate amines are becoming more popular as ligands due to faster

polymerization rates and lower cost.120,121 The initiator in the system is an alkyl

halide activated by an electron-withdrawing group and capable of producing a

stable radical.122 In order to graft from a surface, it has to be converted to a

macroinitiator.

Figure 21 shows the scheme for grafting from the particles surface, were the copper

complex attracts the bromine on the initiator generating a radical that can react

with the monomer that is to be grafted. The radical containing moiety is the active

species and in this process the copper is oxidized to Cu(II) as it accepts the

bromine. The process is continued by the active radical until it is end-capped by

bromine from the complex and becomes a dormant species. It is a constant

- 27 -

equilibrium between the active and the dormant species, all controlled by the metal

complex.123

Figure 21. Scheme showing the mechanism for ATRP of GMA from a particles surface with CuBr and a

ligand (L) as catalyst. The scheme is adapted from Ouchi et al.123

ATRP is in recent years the most applied method of controlled polymerization

initiated from surfaces.106 Many different types of surfaces have been used, ranging

from silica wafers, silica particles, nylon membranes, and flat nickel and copper

surfaces.124-128 When it comes to polymer particles, ATRP can be used to create

material in a core-shell fashion. Highly cross-linked DVB particles have been

converted to halogen containing macroinitiators by hydrochlorination and

hydrobromination of the residual vinyl groups.81,129 The particles were then used to

graft styrene from the surface. There are reports of several different approaches to

graft methacrylates from polymeric particulate supports. Copolymerized particles

of DVB-co-HEMA with a low degree of cross-linking were converted into

macroinitiator particles with bromopropionyl bromide. Grafting of acrylates and

methacrylates were successfully carried out when the particles were in a swollen

- 28 -

state. THF was used as solvent, which allowed growth within the particle at room

temperature.130,131 Jhaveri et al. used surfactant-free emulsion polymerization to

produce cross-linked methyl methacrylate nanoparticles of 500 nm diameter. They

incorporated 2-methacryloxyethyl-2'-bromoisobutyrate, a monomer with initiator

characteristics (an ‘inimer’). Butyl methacrylate and styrene was grafted from the

surface creating a shell with a thickness of 50-100 nm.132 The group of Tuncel used

hydrolysed porous glycidylmethacrylate-co-ethylene dimethacrylate (GMA-co-

EDMA) particles with covalently attached 3-(2-bromoisobutyramido)propyl-

(triethoxy)silane as initiator. From these particles, 3-sulfopropyl methacrylate was

grafted to result in a cation-exchange material similar to the material presented in

Paper IV. The material was used to separate four proteins but experienced high

back pressure at flow rates above 0.75 mL/min for columns packed with particles

of the highest grafting content.133

In Paper III the DVB particles, where the vinyl groups had been converted to 2-

bromoethyl groups, were used as initiator particles for grafting from the surface by

ATRP. The ligand used in the experiments was pentamethyldiethylenetriamine

(PMDETA) since it was in a liquid state and miscible with the used solvent. A

common ligand previously used was 2,2-bipyridyl but it proved almost impossible

to dissolve in any solvent together with the copper catalyst.

In the experiment GMA was grafted from the surface of the particles together with

EDMA as a cross-linker. The use of a cross-linker was to prevent the surface from

being too soft by new long tentacles of GMA that could promote aggregation of the

particles. However the cross-linker did not only create intra-particulate bonds but

also created inter-particulate bonds that made the particles fuse together and form

a single piece of polymer in a monolithic fashion. The following graft experiments

were therefore performed with GMA only, according to the reaction scheme in

Figure 22.

The reaction was at first conducted at elevated temperatures as previously reported

in literature for ATRP graftings.134 For grafting of GMA from the surface of DVB

particles, this resulted in too fast grafting rate, which made it difficult to control the

reaction. If the particles are to be used as stationary phase in liquid

chromatography they must remain individual and free from fused particles in order

produce columns with good packing density at minimal backpressure. Conducting

- 29 -

the grafting at ambient temperature prevented the formation of a too thick and soft

GMA layer. The particles were dispersible in water even with the oxirane ring intact

as were reported for poly(GMA) particles prepared by dispersion polymerization.135

Figure 22. The synthesis route of functionalizing the pendant vinyl groups and growing GMA from the

surface of the particles by ATRP. [Paper III]

A zwitterionic monomer, 3-[N,N-dimethyl-N-(methacryloyloxy-ethyl) ammonium]

propanesulfonate (SPE) has been grafted from non-porous DVB particles.108,136

This proved challenging since it was difficult to find a solvent that would be

suitable to both disperse the hydrophobic particles well and dissolve the highly

hydrophilic monomer. Dimethylformamide (DMF) has been used as solvent for

grafting of methacrylates from silica particles with ATRP137 and is also a solvent

that disperses DVB particles. SPE dissolves in water and when mixed, the water

and DMF phases form a homogenous mixture. The grafting was carried out at

ambient temperature and produced particles with a surface hydrophilicity

sufficient to enable dispersion of the particles in water. The particles were slurry

packed into analytical columns to determine if it was possible to separate small

molecules. The system was run in hydrophilic interaction chromatography (HILIC)

mode but the retention was not sufficient. The causes could either be that grafted

layer was not thick enough, that the interactions were suppressed by the

hydrophobic core particle, or that a non-porous material is not suited for

separation of small molecules.

6. Materials Characterization

Careful characterization of the intermediates and the final materials is a very

important part in developing new materials. Various strategies are used to evaluate

the size, shape and composition of the surface of polymer particles. Important

parameters for support materials in chromatography are particle size, surface area,

pore size and morphology, and the chemical characteristics of the particle surface.

In this work, the characterization can be divided into three parts. The particle size

- 30 -

determination has included scanning electron microscopy (SEM) and static laser

scattering (SLS) to evaluate the monodispersity by calculating the number-average

diameter, as described in Chapter 3.1.1. The surface area and porosity are measured

using nitrogen adsorption/desorption according to the Brunauer-Emmett-Teller

(BET)138 principle. Elemental composition has been measured to compare the

material before and after functionalization or grafting either by conventional

elemental analysis of the bulk material or surface analysis by X-ray photoelectron

spectroscopy (XPS). Additional information on the chemical composition has been

derived from Fourier transform infrared spectroscopy FT-IR.

6.1 Size and Morphology

A way of determining the size of the particles is to scan a random population of

particles by SEM. In SEM the electrons are generated by an electron gun and

focused by magnetic lenses. These lenses focus the beam into a small spot that

scans the sample surface. When the electron beam hits the surface of the sample,

electrons are emitted from the sample and detected. The more electrons that are

detected, the brighter the area in the image becomes resulting in image contrast.139

Before the sample can be scanned it is fixed to a specimen stub. To prevent non-

conducting polymer particles from accumulating electrons at the sample surface

and acquire a static charge the material has to be coated with an electrically

conducting material. This is done by sputter coating a very thin layer of a

conducting material such as gold or carbon.

Counting of particles from SEM micrographs was used in Paper I-IV for

determination of the average particle size. From SEM micrographs with sufficient

amount of particles, 100 particle diameters were measured. The micrographs were

divided into four equally large portions and 25 particles were counted starting from

the upper left corner. The combined diameters of the particles were used to

calculate the number-average diameter Dn and the weight-average diameter Dw and

from that calculate the PDI, U according to the equations in chapter 3.1.1. To

investigate the convergence, 1000 particles were counted in one of the samples.

This concluded that even though there was a small change in PDI, counting 100

particle diameters gave almost the same result, as shown in Figure 23.

- 31 -

Figure 23. The convergence of the PDI to the number of particle counted. The PDI is constant between

1.006 and 1.008 for counting of 100 to 1000 particles. [Paper I]

In Paper I the particle diameter was measured by static laser scattering (SLS) as a

complement to counting particles from SEM micrographs. To measure the particle

diameter with a commercially available instrumentation is probably the most

common and accepted way of determining monodispersity of a polymer sample.

The particles are dispersed in a solvent with known optical properties and

irradiated with monochromatic light as the particles sediment. When a photon hits

a particle it scatters, and the scattering angle and intensity is depending on the size

of the particle and the photon energy (wavelength). A detector constantly measures

the light intensity and scattering angle according to the Mie theory.140 The results

from SEM and SLS in Paper I are similar when compared, and it is difficult to say

if one method is better than the other. Manually counting particles from SEM

micrographs is very time consuming while SLS analyses are very fast. The difficulty

in using SLS for DVB particles was to find a recirculation liquid that prevented

particle aggregation.

6.2 Surface Area and Porosity

The IUPAC definition of pore widths distinguishes between three diameters;

micropores < 2 nm, mesopores between 2-50 nm, and macropores > 50 nm.141

Sorption of nitrogen gas on a porous sample submersed in liquid nitrogen is one of

the most frequently used methods for characterization of a wide range of porous

material. Based on a model of multilayer adsorption, the Brunauer-Emmett-Teller

(BET) theory138 is the standard procedure for determining the surface area of

- 32 -

materials. The BET equation (5) is often presented in the linear form and the

theory is only valid for relative pressures between 0.05-0.30, where it forms a

straight line. The equation is written as follows

⎟⎟⎠

⎞⎜⎜⎝

⎛−+=

− 00a

11)( p

pCV

CCVppV

p

mm

(5)

Where p is the experimental pressure, p0 is the saturation pressure, Va the quantity

of gas absorbed at pressure p, Vm the quantity absorbed gas when the surface is

covered with a monolayer, and C is a constant.

The adsorption isotherms are characterized in six different classes (Types 1-6)

depending on the shape of the Va vs. P/P0 curves.142 The material presented in

Paper I and II resulted in isotherms of Type 1 and 2, and are schematically shown

in Figure 24.

Figure 24. Typical adsorption isotherms for micro-porous material (Type 1) and non-porous (Type 2)

materials.143

Microporous materials with a small external surface give a Type 1 isotherm where

the steep rise is caused by the completion of pore filling.144 In the linear form of the

BET equation there is a constant, C, which is the enthalpy of the first layer

adsorption. If the value of C is negative or higher than 200, it is an indication of the

presence of micropores.145 Particles polymerized with 80% technical grade DVB

- 33 -

contain micro pores, which results in both negative and high C values. The BET

surface area calculated from this material should only be used as guidance.

The initial steep rise is due to single layer adsorption associated with micropores.

This is in Type 2 isotherms followed by multilayer adsorption and bulk

condensation, which occurs when the line is turning upwards for the second time.

The isotherm becomes horizontal again when all pores are filled.142,146 The pore size

is calculated using the Barrett, Joyner and Halenda (BJH) scheme.147 This

calculation is performed when all the pores are filled, which occurs at a relative

pressure of 0.95.

6.3 Characterization of Elemental Composition

IR spectroscopy is a technique suitable for measuring functional groups such as

hydrocarbons, hydroxyl, carbonyl, halogens as it measures the rotation and

vibration of atoms in a molecule.

Figure 25. Overlaped FT-

IR spectra where (a) is the

bare DVB particles, (b) after

subjected to hydrobromina-

tion and (c) after GMA have

been grafted from the

surface. [Paper III]

- 34 -

The residual vinyl groups of DVB particles have been measured and characterized

by IR. The bands at 990, 1015, 1410 and 1630 cm-1 in the IR spectrum corresponds

to stretching of the C-C bond in vinyl groups.

In Paper III the material was measured before and after being subjected to anti-

Markovnikov hydrobromination to get a qualitative verification. The particles were

also measured after grafting of GMA to verify the carbonyl stretch. The spectrum in

Figure 25 shows a peak at 1263 cm-1 for the 2-bromoethyl group and decreasing

bands for the vinyl stretch. If there would be no radical initiator added in the

hydrobromination, a peak would appear at 1183 cm-1 for 1-bromoetheyl groups.84

After grafting of GMA by ATRP, the spectrum showed a large carbonyl stretch at

1730 cm-1 verifying a grafted layer.

XPS is a semi-quantitative way of determine the elemental composition within

10 nm of the surface. The sample is irradiated by X-ray from a known source

(aluminum or magnesium anode) and the kinetic energy of electrons ejected from

the surface of the material are detected.148 The photon energy of the X-ray is

sufficient to excite electrons from all core levels. It can distinguish between

different elements since the binding energy is characteristic for every element, and

also between bond types which give rise to different electron configurations.

In the same way as with FT-IR, the materials were analyzed after reactions with the

surface with XPS to see the ratios of oxygen and sulfur to carbon on the surface. For

the grafting of SPM telomers in Paper IV, the particles were first reacted with

mCPBA to generate oxirane rings. XPS data showed a oxygen content of 0.5 at.%

before reaction and an increase to 3.9 at.% with C 1s signal for C–O at 286.7 eV in

Figure 26. After grafting of the SPM telomers an additional signal at 289.4 eV was

seen as expected from the methacrylate and S 2s and S 2p signals for sulfur.

- 35 -

Figure 26. A series of XPS spectra with wide-scans in the left column and expansion of C 1s band on

the right It follows the material grafted with sulfopropyl methacrylate telomers with (a, b) are plain DVB

particles, (c, d) after epoxidation and (e, f) after grafting of the telomers. [Paper IV]

For particles grafted by ATRP in Paper III the oxygen signal from GMA was much

stronger than from the grafting to of SPM telomers. The amount of oxygen on the

surface assigned to O 1s line was above 22 at.% for all samples. The peaks in the

C 1s spectrum correspond to C–C,H at 285.0 eV, C–O at 286.6 eV and C–OO at

- 36 -

289.0 eV in Figure 27. This shows that the particles have been successfully coated

with a layer of GMA.

Figure 27. XPS spectrum of GMA grafted particles using ATRP with a wide-scan (left) and an

expansion of C 1s band on the right. [Paper III]

7. Concluding Remarks

Non-porous and microporous monodisperse particles have been synthesized by

precipitation polymerization in acetonitrile, aided by the irradiation of a 150 W

short arc xenon lamp. In Paper I the method was first introduced by

homopolymerization of DVB and copolymerization with styrene using DVB as

cross-linker (St-co-DVB). It was possible to increase the monomer concentration to

10% (v/v) for DVB but the incorporation of styrene gave less percentage cross-

linker and thus higher tendency for agglomeration. It also showed a linear increase

in size with the polymerization time up to the 163 hours used for the longest

polymerizations.

Incorporation of a second monomer was taken one step further in Paper II when

copolymerizations of DVB and GMA, (DVB-co-GMA) were carried out. This

produced monodisperse particles when polymerized with a monomer

concentration of 2% (v/v). It was possible to achieve monodisperse particles with a

concentration of 4% (v/v) when THF was added as a cosolvent. Adding THF also

gave an increase in growth rate making it possible to reduce the polymerization

time. For particles consisting of DVB the addition of THF made the particles grow

to an average particle size 3.5 µm in 46 h compared to 0.8 µm in 48 h by only using

acetonitrile as solvent.

- 37 -

In Paper III and IV the particles were subsequently used as templates for grafting

functional groups or telomers on the surface. Before grafting, the residual vinyl

groups covering the surface were converted to suitable anchoring points via

hydrobromination or epoxydation. The material containing bromine was used as

macroinitiator for grafting GMA from the surface by ATRP. This created a

hydrophilic layer around the hydrophobic core particle making it possible to

disperse the particles in water. Particles modified to contain oxirane rings on the

surface were used to attach sulfopropyl methacrylate telomers. This created a

positively charged surface and the particles were packed into a short analytical

column and evaluated by separating a protein test mixture in cation-exchange

mode.

- 38 -

8. Acknowledgment

It has been four stimulating years of research with great friends. I would like to

take this opportunity to acknowledge the people that that I have worked with and

in one way or another helped me in my research.

My supervisor Knut Irgum for accepting me to your research group, and giving me

many interesting projects.

Tobias Jonsson and Bertil Eliasson, my assistant supervisors for help and interest

in my research.

Peter Cormack, Panagiotis Manesiotis and Laura Jankowska at Strathclyde

University for the collaboration.

The senior co-workers at the department, Erik Björn, Svante Jonsson, Wolfgang

Frech, Anders Cedergren, Lars Lambertsson, Mickael Sharp, Svante Åberg,

Solomon Tesfalidet, Olle Nygren, Roger Lindahl, Lars Lundmark, Ann-Helén

Waara and Anita Öystilä.

All the fellow phD student and postdocs over the years, Tom Larsson, Josefina

Nyström, Nguyen Van Dong, Yvonne Nygren, William Larsson, Sofi Jonsson,

Daniel Goitom and Maximilian Popp.

The past and present group members, Julien Courtois, Erika Wikberg, Nguyen Anh

Mai, Gerd Fischer, Michal Szumski, Bui Thi Hong Nhat, Jeroen Verhage, Ida

Fredriksson, Jonas Persson, Emma Hallberg, Dinh Ngoc Phuoc, Ryan Chadwick,

Phan Duong Ngoc Chau, Nguyen Thanh Duc, Tran Thi Thuy, Emil Byström, Anna

Nordborg and Petrus Hemström.

All the people that helped me with analysis, Per Hörstedt for SEM micrographs,

John Loring with FT-IR, Andrei Shchukarev for XPS data, Mats Ullberg and Håkan

Carlö at CIAB for SLS measurements.

The people at Merck SeQuant, Camilla Viklund, Patrik Appelblad, Wen Jiang, and

Einar Pontén.

My friends, family and especially Elaine.

- 39 -

9. References and Notes

1. Tsvett, M. S. Ber. Deut. Botan. Gessel. 1906, 24, 322-327.

2. Unger, K. K.; Skudas, R.; Schulte, M. M. J. Chromatogr. A 2008, 1184, 393-415.

3. Majors, R. E. LCGC Europe 2006, 19, 352-362.

4. Hench, L. L.; West, J. K. Chem. Rev. 1990, 90, 33-72.

5. Poole, C. F., The Essence of Chromatography. Elsevier: Amsterdam, 2003.

6. Unger, K. K., Porous Silica. ed.; Elsevier: Amsterdam, 1979; 'Vol.' 16, p.

7. Buchmeiser, M. R. J. Chromatogr. A 2001, 918, 233-266.

8. MacNair, J. E.; Lewis, K. C.; Jorgenson, J. W. Anal. Chem. 1997, 69, 983-989.

9. Lloyd, L. L. J. Chromatogr. 1991, 544, 201-217.

10. Lee, D. P. J. Chromatogr. 1988, 443, 143-153.

11. Zhu, Y.; Yongxin, C.; Mingli, Y.; Fritz, J. S. J. Chromatogr. A 2005, 1085, 18-22.

12. Ng, J.; Froom, D. Can. Chem. News 1998, 50, 24-26.

13. Penner, N. A.; Nesterenko, P. N.; Ilyin, M. M.; Tsyurupa, M. P.; Davankov, V. A.

Chromatographia 1999, 50, 611-620.

14. Cao, M. L.; Tong, B.; Shen, J. B.; Dong, Y. P.; Zhi, J. G. J Appl. Polym. Sci. 2008,

109, 1189-1196.

15. Wang, Q. C.; Hosoya, K.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1992, 64, 1232-1238.

16. Ugelstad, J.; Berge, A.; Ellingsen, T.; Schmid, R.; Nilsen, T. N.; Mørk, P. C.; Stenstad,

P.; Hornes, E.; Olsvik, Ø. Prog. Polym. Sci. 1992, 17, 87-161.

17. Ugelstad, J.; Mørk, P. C. Adv. Colloid Interface Sci. 1980, 13, 101-140.

18. Svec, F.; Frechet, J. M. J. Science 1996, 273, 205-211.

19. Coupek, J.; Krivakov.M; Pokorny, S. J. Polym. Sci. Symp. 1973, (42), 185-190.

20. Smigol, V.; Svec, F. J. Appl. Polym. Sci. 1992, 46, 1439-1448.

21. Benes, M. J.; Horak, D.; Svec, F. J. Sep. Sci. 2005, 28, 1855-1875.

22. Mazzeo, J. R.; Neue, U. D.; Kele, M.; Plumb, R. S. Anal. Chem. 2005, 77, 460A-

467A.

23. Huber, C. G.; Oefner, P. J.; Bonn, G. K. Chromatographia 1993, 37, 653-656.

24. Hosoya, K.; Teramachi, M.; Tanaka, N.; Kobayashi, A.; Kanda, T.; Ohtsu, Y. Anal.

Chem. 2001, 73, 5852-5857.

25. Stanley, B. J.; Foster, C. R.; Guiochon, G. J. Chromatogr. A 1997, 761, 41-51.

- 40 -

26. Guiochon, G. J. Chromatogr. A 2006, 1126, 6-49.

27. Cramers, C. A.; Rijks, J. A.; Schutjes, C. P. M. Chromatographia 1981, 14, 439-444.

28. van der Wal, S. Chromatographia 1985, 20, 274-278.

29. Stevens, M. P., Polymer Chemistry an Introduction. 3rd ed.; Oxford University Press:

New York, 1999.

30. Elias, H. G., An Introduction to Polymer Science. ed.; VCH: Weinheim, 1997.

31. Sherrington, D. C. Chem. Commun. 1998, 21, 2275-2286.

32. Lewandowski, K.; Svec, F.; Frechet, J. M. J. J. Appl. Polym. Sci. 1998, 67, 597-607.

33. Jayachandran, K. N. N.; Chatterji, P. R. J. Macromol. Sci., Polymer Reviews 2001, 41,

79-94.

34. Li, K.; Stöver, H. D. H. J. Polym. Sci. Part A: Polym. Chem. 1993, 31, 3257-3263.

35. Mouaziz, H.; Larsson, A.; Sherrington, D. C. Macromolecules 2004, 37, 1319-1323.

36. Kawaguchi, S.; Ito, K. Adv. Polym. Sci. 2005, 175, 299-328.

37. Li, K.; Stöver, H. D. H. J. Polym. Sci. Part A: Polym. Chem. 1993, 31, 2473-2479.

38. Ugelstad, J.; Kaggerud, K. H.; Hansen, F. K.; Berge, A. Makromol. Chem. 1979, 180,

737-744.

39. Sugimoto, T., Monodispersed particles. Elsevier: Amsterdam, 2001.

40. Liu, J.; Chew, C. H.; Gan, L. M.; Teo, W. K.; Gan, L. H. Langmuir 1997, 13, 4988-

4994.

41. Hong, J.; Hong, C. K.; Shim, S. E. Colloids Surf. A 2007, 302, 225-233.

42. Downey, J. S.; Frank, R. S.; Li, W.-H.; Stöver, H. D. H. Macromolecules 1999, 32,

2838-2844.

43. Chaitidou, S.; Kotrotsiou, O.; Kotti, K.; Kammona, O.; Bukhari, M.; Kiparissides, C.

Mater. Sci. Eng. B 2008, 152, 55-59.

44. Goh, E. C. C.; Stöver, H. D. H. Macromolecules 2002, 35, 9983-9989.

45. Shim, S. E.; Yang, S. H.; Choi, H. H.; Choe, S. J. Polym. Sci. Part A: Polym. Chem.

2004, 42, 835-845.

46. Shim, S. E.; Yang, S.; Jin, M. J.; Chang, Y. H.; Choe, S. Colloid Polym Sci 2004, 283,

41-48.

47. Li, W. H.; Stöver, H. D. H. J. Polym. Sci. Part A: Polym. Chem. 1999, 37, 2899-2907.

48. Li, W. H.; Li, K.; Stöver, H. D. H. J. Polym. Sci. Part A: Polym. Chem. 1999, 37,

2295-2303.

- 41 -

49. Yan, Q.; Bai, Y. W.; Meng, Z.; Yang, W. T. Acta Polym. Sin. 2007, 11, 1102-1104.

50. Yan, Q.; Bai, Y. W.; Meng, Z.; Yang, W. T. J. Phys. Chem. B 2008, 112, 6914-6922.

51. Li, W.-H.; Stöver, H. D. H. J. Polym. Sci. Part A: Polym. Chem. 1998, 36, 1543-1551.

52. Shim, S. E.; Yang, S. Y.; Choe, S. J. J. Polym. Sci. Part A: Polym. Chem. 2004, 42,

3967-3974.

53. Bai, F.; Yang, X. L.; Huang, W. Q. Macromolecules 2004, 37, 9746-9752.

54. Bai, F.; Huang, B.; Yang, X. L.; Huang, W. Q. Polymer 2007, 48, 3641-3649.

55. Bai, F.; Yang, X. L.; Huang, W. Q. Eur. Polym. J. 2006, 42, 2088-2097.

56. Li, S. F.; Yang, X. L.; Huang, W. Q. Chin. J. Polym. Sci. 2005, 23, 197-202.

57. Lu, X. Y.; Huang, D.; Yang, X. L.; Huang, W. Q. Polym. Bull. 2006, 56, 171-178.

58. Bai, F.; Li, R.; Yang, X. L.; Li, S. N.; Huang, W. Q. Polym. Int. 2006, 55, 319-325.

59. Bai, F.; Yang, X. L.; Li, R.; Huang, B.; Huang, W. Q. Polymer 2006, 47, 5775-5784.

60. Bai, F.; Huang, B.; Yang, X. L.; Huang, W. Q. Eur. Polym. J. 2007, 43, 3923-3932.

61. Li, G. L.; Yang, X. Y.; Wang, B.; Wang, J. Y.; Yang, X. L. Polymer 2008, 49, 3436-

3443.

62. Bai, F.; Yang, X. L.; Zhao, Y. Z.; Huang, W. Q. Polym. Int. 2005, 54, 168-174.

63. Qi, D. L.; Yang, X. L.; Huang, W. Q. Polym. Int. 2007, 56, 208-213.

64. Qi, D. L.; Bai, F.; Yang, X. L.; Huang, W. Q. Eur. Polym. J. 2005, 41, 2320-2328.

65. Bai, F.; Yang, X. L.; Huang, W. Q. J. Appl. Polym. Sci. 2006, 100, 1776-1784.

66. Joso, R.; Pan, E. H.; Stenzel, M. H.; Davis, T. P.; Barner-Kowollik, C.; Barner, L. J.

Polym. Sci. Part A: Polym. Chem. 2007, 45, 3482-3487.

67. Liang, Y. C.; Svec, F.; Frechet, J. M. J. J. Polym. Sci. A: Polym. Chem. 1997, 35,

2631-2643.

68. Limé, F. Highly cross-linked polymer particles as chromatographic separation

material prepared by photoinitiated precipitation polymerization, Analysdagarna,

Gothenburg, Sweden, 2006.

69. Shim, S. E.; Yang, S.; Jin, M. J.; Chang, Y. H.; Choe, S. Colloid Polym. Sci. 2004,

283, 41-48.

70. Jin, J. M.; Lee, J. M.; Ha, M. H.; Lee, K.; Choe, S. Polymer 2007, 48, 3107-3115.

71. Zhang, S. H.; Huang, X. A.; Yao, N. S.; Horvath, C. J. Chromatogr. A 2002, 948, 193-

201.

72. Bondar, Y. V.; Kim, H. J.; Lim, Y. J. J. Appl. Polym. Sci. 2007, 104, 3256-3260.

- 42 -

73. Choi, S.-H.; Hwang, Y.-M.; Lee, K.-P. J.Chromatogr. A 2003, 987, 323-330.

74. Choi, S.-H.; Hwang, Y.-M.; Ryoo, J. J.; Lee, K.-P.; Ohta, K.; Takeuchi, T.; Jin, J.-Y.;

Fujimoto, C. Electrophoresis 2003, 24, 3181-3186.

75. Li, W.-H.; Stöver, H. D. H. Macromolecules 2000, 33, 4354-4360.

76. Majors, R. E. LCGC North Am. 2006, 24, 16-21.

77. Bartholin, M. Makromol. Chem. 1981, 182, 2075-2085.

78. Hubbard, K. L.; Finch, J. A.; Darling, G. D. React. Funct. Polym. 1999, 42, 279-289.

79. Hubbard, K. L.; Finch, J. A.; Darling, G. D. React. Funct. Polym. 1999, 40, 61-90.

80. Hubbard, K. L.; Finch, J. A.; Darling, G. D. React. Funct. Polym. 1999, 39, 207-225.

81. Zheng, G.; Stöver, H. D. H. Macromolecules 2002, 35, 6828-6834.

82. Nyhus, A. K.; Hagen, S.; Berge, A. J. Polym. Sci. A: Polym. Chem. 2000, 38, 1366-

1378.

83. Martan, M., U.S. Pat. 4,228,106.

84. Faber, M. C.; van den Berg, H. J.; Challa, G.; Pandit, U. K. Reactive Polym. 1989, 11,

117-126.

85. Mercier, A.; Deleuze, H.; Mondain-Monval, O. React. Funct. Polym. 2000, 46, 67-79.

86. Fréchet, J. M. J.; Eichler, E. Polym. Bull. 1982, 7, 345-351.

87. Gong, B. L.; Zhu, J. X.; Li, L.; Qiang, K. J.; Ren, L. Talanta 2006, 68, 666-672.

88. Okubo, M.; Okada, M.; Miya, T.; Takekoh, R. Colloid Polym. Sci. 2001, 279, 807-

812.

89. Kuroda, H.; Osawa, Z. Eur. Polym. J. 1995, 31, 57-62.

90. Brambilla, R.; Pires, G. P.; dos Santos, J. H. Z.; Miranda, M. S. L.; Chornik, B. J.

Electron. Spectrosc. Relat. Phenom. 2007, 156, 413-420.

91. Xia, R. K.; He, W. D.; Pan, C. Y. Colloid Polym. Sci. 2002, 280, 865-872.

92. Unsal, E.; Irmak, T.; Durusoy, E.; Tuncel, M.; Tuncel, A. Anal. Chim. Acta 2006, 570,

240-248.

93. Gao, J. P.; Morin, F. G.; Darling, G. D. Macromolecules 1993, 26, 1196-1198.

94. Limé, F.; Irgum, K. Preparation of Non-porous Polymer Particles by Precipitation

Polymerization and Their use as Grafting Templates for Proteins Separation

Materials, HPLC 2005, Stockholm, Sweden.

95. Mercier, A.; Deleuze, H.; Mondain-Monval, O. Macromol. Chem. Phys. 2001, 202,

2672-2680.

- 43 -

96. Davis, K.; Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1-169.

97. Tsujii, Y.; Ohno, K.; Yamamoto, S.; Goto, A.; Fukuda, T. Adv. Polym. Sci. 2006, 197,

1-45.

98. Rowe-Konopacki, M. D.; Boyes, S. G. Macromolecules 2007, 40, 879-888.

99. Georges, M. K.; Veregin, R. P. N.; Kazmaier, P. M.; Hamer, G. K. Macromolecules

1993, 26, 2987-2988.

100. Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne,

R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H.

Macromolecules 1998, 31, 5559-5562.

101. Wang, J. S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614-5615.

102. Moad, G.; Rizzardo, E.; Thang, S. H. Acc. Chem. Res. 2008, 41, 1133-1142.

103. Odian, G., Principles of polymerization. 4 ed.; Wiley: Hoboken, 2004; 'Vol.' p.

104. Husseman, M.; Malmstrom, E. E.; McNamara, M.; Mate, M.; Mecerreyes, D.; Benoit,

D. G.; Hedrick, J. L.; Mansky, P.; Huang, E.; Russell, T. P.; Hawker, C. J.

Macromolecules 1999, 32, 1424-1431.

105. Kasseh, A.; Ait-Kadi, A.; Riedl, B.; Pierson, J. F. Polymer 2003, 44, 1367-1375.

106. Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. Rev. 2004, 33, 14-22.

107. Barner, L.; Li, C.; Hao, X. J.; Stenzel, M. H.; Barner-Kowollik, C.; Davis, T. P. J.

Polym. Sci. Part A: Polym. Chem. 2004, 42, 5067-5076.

108. Wikberg, E. Zwitterionic Sulfobetaine Polymers as Stationary Phases for Liquid

Chromatography. Thesis, Umeå University, Umeå, 2008.

109. Kato, M.; Kamigaito, M.; Sawamoto, M.; Higashimura, T. Macromolecules 1995, 28,

1721-1723.

110. Wang, J. S.; Matyjaszewski, K. Macromolecules 1995, 28, 7572-7573.

111. Wang, J. S.; Matyjaszewski, K. Macromolecules 1995, 28, 7901-7910.

112. Xia, J. H.; Matyjaszewski, K. Macromolecules 1997, 30, 7692-7696.

113. Koumura, K.; Satoh, K.; Kamigaito, M. Macromolecules 2008, 41, 7359-7367.

114. Uchiike, C.; Terashima, T.; Ouchi, M.; Ando, T.; Kamigaito, M.; Sawamoto, M.

Macromolecules 2007, 40, 8658-8662.

115. Kabachii, Y. A.; Kochev, S. Y.; Bronstein, L. M.; Blagodatskikh, I. B.; Valetsky, P.

M. Polym. Bull. 2003, 50, 271-278.

- 44 -

116. Moineau, G.; Minet, M.; Dubois, P.; Teyssie, P.; Senninger, T.; Jerome, R.

Macromolecules 1999, 32, 27-35.

117. Uegaki, H.; Kotani, Y.; Kamigaito, M.; Sawamoto, M. Macromolecules 1998, 31,

6756-6761.

118. Lecomte, P.; Drapier, I.; Dubois, P.; Teyssie, P.; Jerome, R. Macromolecules 1997, 30,

7631-7633.

119. Kotani, Y.; Kamigaito, M.; Sawamoto, M. Macromolecules 1999, 32, 2420-2424.

120. Patten, T. E.; Matyjaszewski, K. Adv. Mater. 1998, 10, 901-915.

121. Xia, J. H.; Matyjaszewski, K. Macromolecules 1997, 30, 7697-7700.

122. Tang, W.; Matyjaszewski, K. Macromolecules 2007, 40, 1858-1863.

123. Ouchi, M.; Terashima, T.; Sawamoto, M. Acc. Chem. Res. 2008, 41, 1120-1132.

124. Topham, P. D.; Howse, J. R.; Crook, C. J.; Parnell, A. J.; Geoghegan, M.; Jones, R. A.

L.; Ryan, A. J. Polym. Int. 2006, 55, 808-815.

125. Matyjaszewski, K.; Miller, P. J.; Shukla, N.; Immaraporn, B.; Gelman, A.; Luokala, B.

B.; Siclovan, T. M.; Kickelbick, G.; Vallant, T.; Hoffmann, H.; Pakula, T.

Macromolecules 1999, 32, 8716-8724.

126. Chen, R. X.; Zhu, S. P.; Maclaughlin, S. Langmuir 2008, 24, 6889-6896.

127. Xu, F. J.; Zhao, J. P.; Kang, E. T.; Neoh, K. G.; Li, J. Langmuir 2007, 23, 8585-8592.

128. Edmondson, S.; Huck, W. T. S. J. Mater. Chem. 2004, 14, 730-734.

129. Jankova, K.; Nguyen, K. C.; Chen, X. Y.; Kops, J.; Johannsen, I.; Batsberg, W. Polym.

Prepr. 1998, 392, 480-481.

130. Zheng, G. D.; Stover, H. D. H. Macromolecules 2003, 36, 1808-1814.

131. Zheng, G. D.; Stöver, H. D. H. Macromolecules 2002, 35, 7612-7619.

132. Jhaveri, S. B.; Koylu, D.; Maschke, D.; Carter, K. R. J. Polym. Sci. A: Polym. Chem.

2007, 45, 1575-1584.

133. Unsal, E.; Elmas, B.; Caglayan, B.; Tuncel, M.; Patir, S.; Tuncel, A. Anal. Cham.

2006, 78, 5868-5875.

134. Min, K.; Hu, J. H.; Wang, C. C.; Elaissari, A. J. Polym. Sci. A: Polym. Chem. 2002,

40, 892-900.

135. Horak, D.; Shapoval, P. J. Polym. Sci. A: Polym. Chem. 2000, 38, 3855-3863.

- 45 -

136. Limé, F.; Nordborg, A.; Irgum, K. Functionalization of Polymer Particles by

"Grafting to" and "Grafting from" in the Synthesis of Polymeric Stationary Phases for

HPLC, HPLC 2008, Baltimore, MD, USA, 2008.

137. Hemström, P.; Szumski, M.; Irgum, K. Anal. Chem. 2006, 78, 7098-7103.

138. Brunauer, S.; Emmett, P. H.; Teller, E. J. Am. Chem. Soc. 1938, 60, 309-319.

139. Hogmark, S.; Jacobson, S.; Kassman-Rudolphi, Å., Svepelektronmikroskopi i praktik

och teori. 7 ed.; Ångströmlaboratoriet, Uppsala universitet: 1998.

140. Webb, P. A., A Primer on Particle Sizing by Static Light Scattering. ed.; Micromeritics

Instrument Corp. Norcross, GA, USA: 2000.

141. Sing, K. S. W.; Everett, D. H.; Haul, R. A. W.; Moscou, L.; Pierotti, R. A.; Rouquerol,

J.; Siemieniewska, T. Pure & Appl. Chem. 1985, 57, 603-619.

142. Webb, P. A.; Orr, C., Analytical Methods in Fine Particle Technology. ed.;

Micromeritics Instrument Corp. Norcross, GA, USA: 1997.

143. Brunauer, S., The Adsorption of Gases and Vapors, Physical Adsorption. ed.;

Princeton University Press: 1945, p.150.

144. Sing, K. Colloids Surf. A 2001, 187, 3-9.

145. Buchmeiser, M. R., Polymeric Materials in Organic Synthesis and Catalysis. Wiley-

VCH: 2003.

146. Macintyre, F. S.; Sherrington, D. C. Macromolecules 2004, 37, 7628-7636.

147. Barrett, E. P.; Joyner, L. G.; Halenda, P. P. J. Am. Chem. Soc. 1951, 73, 373-380.

148. Karlsson, L. En introduktion till fotoelektronspektroskopi; Fysiska institutionen,

Uppsala universitet: 1992.