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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: [email protected]
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 -
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