through surface-attached monomers
TRANSCRIPT
GGrraaffttiinngg ooff PPoollyymmeerrss oonnttoo SSiiOO22 SSuurrffaacceess
tthhrroouugghh SSuurrffaaccee--aattttaacchheedd MMoonnoommeerrss
genehmigte
Dissertation
zur Erlangung des Doktorgrades
Doktor der Naturwissenschaften
der Fakultät für Angewandte Wissenschaften
der Albert-Ludwigs-Universität Freiburg im Breisgau
von
Diplom-Chemiker
Daniel Mädge
geboren am 01.01.1974 in Oldenburg (Oldb.)
Freiburg im Breisgau 2007
The present work was carried out from August 2000 to March 2005 at the Institute for
Microsystem Technology (IMTEK), Albert-Ludwigs-University of Freiburg, Germany,
at the department “Chemistry and Physics of Interfaces”, under the supervision of
Prof. Dr. Jürgen Rühe.
Dekan: Prof. Dr. Bernhard Nebel (FAW, Freiburg)
Vorsitzender: Prof. Dr. Eicke R. Weber (Fraunhofer ISE, Freiburg)
Erstgutachter: Prof. Dr. Jürgen Rühe (IMTEK, Freiburg)
Zweitgutachter: PD Dr. Thomas Hanemann (IMTEK, Freiburg & FZK, Karlsruhe)
Beisitzende: Prof. Dr. Margit Zacharias (IMTEK, Freiburg)
Datum der mündlichen Prüfung: 02. 08. 2007
The beginning of knowledge is the discovery of something we do not understand.
Frank Herbert (Dune series)
Table of Contents i
Table of Contents
1 Introduction....................................................................................................................................1
1.1 Modified Silicon Dioxide Substrates ..................................................................................1
1.1.1 Chromatographic Material...........................................................................................1
1.1.2 Immobilization of Biomolecules..................................................................................2
1.1.3 Catalyst Immobilization ...............................................................................................4
1.2 Fabrication of Silica Particles & Silicon ..............................................................................5
1.3 Surface Decoration of Silica, Glass Beads & Silicon .........................................................7
1.3.1 Functional Silanes & Silane Anchors ..........................................................................7
1.3.2 Polymer Layers by “Grafting-to” & “Grafting-from”..............................................9
1.3.3 Polymerizing in Presence of Surface-attached Monomers ....................................10
1.3.4 Polymer Micropatterning...........................................................................................13
1.4 Radical polymerization.......................................................................................................14
2 Goals & Strategy of the Work ....................................................................................................21
2.1 Goals......................................................................................................................................21
2.2 Strategy .................................................................................................................................21
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers ..............26
3.1 Silane Monomers .................................................................................................................26
3.2 Homopolymers on Silica ....................................................................................................31
3.3 Mechanism of the Grafting of Polystyrene onto Silica Surfaces through Surface-attached Monomers ..............................................................................................35
3.3.1 Structure Variation of the Immobilized Monomer.................................................35
3.3.2 Variation of the Polymerization Time ......................................................................37
3.3.3 Influence of the Silica Gel Concentration.................................................................40
3.3.4 Influence of Monomer Concentration in Solution ..................................................42
ii Table of Contents
3.3.5 Influence of Initiator Concentration......................................................................... 44
3.3.6 Variation of the Polymerization Temperature........................................................ 46
3.3.7 Surface Concentration of Polymerizable Silanes & its Influence on Building Polymer Monolayers .................................................................................................. 50
3.3.8 Polymerization in Two Steps .................................................................................... 56
3.4 Grafting onto Various Porous Substrates........................................................................ 57
3.4.1 Influence of Substrates and Surface-attached Monomer Silanes ......................... 57
3.4.2 Influence of the Polymerization Time for Aerosil300............................................ 59
3.4.3 Influence of the Polymerization Time for Differently Modified Glass Beads.... 62
3.4.4 Influence of Initiator Concentration for Different Substrates............................... 63
3.4.5 Influence of the Monomer Concentration for Glass Beads ................................... 66
3.4.6 Influence of the Glass Bead Concentration ............................................................. 68
3.5 Discussion............................................................................................................................ 70
3.6 Conclusion ........................................................................................................................... 76
3.7 Grafting of Poly(N,N-dimethyl acrylamide) onto Silica Surfaces through Surface-attached Monomers ............................................................................................. 79
3.7.1 Polymerization Time Influence................................................................................. 79
3.7.2 Variation of Initiator Concentration......................................................................... 82
3.7.3 Influence of Monomer Concentration...................................................................... 86
3.7.4 Discussion & Conclusion for P(DMAA) on Silica.................................................. 88
4 Grafting of Functional Copolymers onto Silica Surfaces ...................................................... 90
4.1 Polystyrene Copolymers on Silica Particles.................................................................... 92
4.1.1 Poly(styrene-co-N-acryloyl-N-methyl-propyl phthalimide) & Poly(styrene-co-N-acryloyl-N-methyl-propyl amine)........................................... 92
4.1.2 Poly(styrene-co-N-methacryloyl-β-alanine succinimide ester).......................... 100
4.1.3 Two-step Grafting Processes................................................................................... 103
4.2 Poly(N,N-dimethyl acrylamide) copolymers on silica particles................................ 107
Table of Contents iii
4.2.1 Poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-methyl-propyl phthalimide) & Poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-methyl-propyl amine) ........107
4.2.2 Poly(N,N-dimethyl acrylamide-co-N-methacryloyl-β-alanine succinimide ester) ....................................................................................................................................108
4.3 Discussion & Conclusion Copolymers...........................................................................111
5 Applications for Functional Copolymers on Silica Substrates............................................113
5.1 Enzyme Immobilization ...................................................................................................113
5.1.1 Glucose Oxidase Assay.............................................................................................114
5.1.2 Stability against Leaching ........................................................................................116
5.1.3 Influence of the Immobilization Time on the Conversion Rate of the Enzyme Modified Glass Beads ...............................................................................................118
5.1.4 Influence of the Immobilization Concentration on the Conversion Rate of the Enzyme Modified Glass Beads ................................................................................119
5.2 Biomolecule Immobilization............................................................................................122
5.2.1 Biotin Labeling...........................................................................................................122
5.2.2 DNA Immobilization & Hybridization ..................................................................125
5.3 Catalyst Immobilization ...................................................................................................127
5.4 Discussion & Conclusions Applications ........................................................................130
6 Polymer Systems on Silicon Wafers........................................................................................133
6.1 Grafting of Poly(N,N-dimethyl acrylamide) Layers onto Silicon Wafers through Surface-attached Monomers ............................................................................................133
6.1.1 Influence of Monomer Concentration ....................................................................133
6.1.2 Polymerization Time Influence ...............................................................................136
6.1.3 Variation of Initiator Concentration .......................................................................138
6.1.4 Discussion & Conclusion P(DMAA) on Wafers....................................................140
6.2 Microstructured Surfaces .................................................................................................143
6.2.1 Photoablation of Surface-attached Monomers Followed by Polymerization ...143
6.2.2 Patterning through Polymer Network Formation................................................145
iv Table of Contents
6.2.3 Discussion & Conclusion Micropatterning........................................................... 160
7 Outlook....................................................................................................................................... 162
8 Experimental I – Materials & Methods.................................................................................. 163
8.1 Materials ............................................................................................................................ 163
8.1.1 Material List............................................................................................................... 163
8.1.2 Material Preparation ................................................................................................ 164
8.2 Methods ............................................................................................................................. 166
8.2.1 Elemental Analysis ................................................................................................... 166
8.2.2 Infrared Spectroscopy .............................................................................................. 166
8.2.3 Gel Permeation Chromatography (GPC) .............................................................. 166
8.2.4 Ellipsometry & Imaging Ellipsometry................................................................... 166
8.2.5 X-ray Photoelectron Spectroscopy ......................................................................... 167
8.2.6 Nuclear Magnetic Resonance Spectroscopy ......................................................... 167
8.2.7 UV/vis Spectroscopy ................................................................................................ 167
8.2.8 Fluorescence Microscopy & Reading..................................................................... 167
8.2.9 Profilometry............................................................................................................... 168
8.2.10 Titration...................................................................................................................... 168
9 Experimental II – Polymerizations ......................................................................................... 169
9.1 Immobilization of Silanes ................................................................................................ 169
9.1.1 Immobilization of Trimethoxy Silanes onto Silica Gel ........................................ 169
9.1.2 Immobilization of Trimethoxy Silanes onto Glass Beads ................................... 171
9.1.3 Co-Immobilization of MPS and OS onto Silica Gel ............................................. 172
9.2 Homopolymerizations ..................................................................................................... 173
9.2.1 Synthesis of Poly(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl acrylate)...................................................................................................................... 173
9.2.2 Synthesis of Polystyrene & Poly(methyl methacrylate) ...................................... 173
Table of Contents v
9.2.3 Synthesis of Poly(N-isopropyl acrylamide)...........................................................174
9.2.4 Synthesis Poly(N,N-dimethyl acrylamide)............................................................175
9.2.5 Synthesis of Poly(methacrylic acid) ........................................................................175
9.3 Polystyrene – Polymerization Parameters .....................................................................176
9.3.1 Influence of Reaction Time.......................................................................................176
9.3.2 Variation of the Overall Concentration of the Solid.............................................177
9.3.3 Monomer Concentration Variation.........................................................................178
9.3.4 Initiator Concentration Variation............................................................................178
9.3.5 Variation of the Polymerization Temperature ......................................................179
9.3.6 Influence of MPS Surface Concentration on the Polystyrene Graft Density.....180
9.3.7 Variation of the Overall Concentration of Glass Beads .......................................181
9.3.8 Two-step Grafting .....................................................................................................181
9.3.9 Polymerization Reactions without Surface-attached Monomers (blind tests) .181
9.4 Poly(N,N-dimethyl acrylamide) – Polymerization parameters..................................182
9.4.1 Influence of Reaction Time.......................................................................................182
9.4.2 Monomer Concentration Variation.........................................................................183
9.4.3 Initiator Concentration Variation............................................................................183
9.5 Copolymerizations ............................................................................................................185
9.5.1 Synthesis of Poly(styrene-co-N-acryloyl-N-methyl-propyl phthalimide), P(S-co-AC3pht) ...........................................................................................................185
9.5.2 Synthesis of Poly(styrene-co-N-acryloyl-N-methyl-propyl amine), P(S-co-AC3amine) ......................................................................................................187
9.5.3 Synthesis of Poly(styrene-co-N-methacryloyl-β-alanine succinimide ester), P(S-co-MAC2ae) .........................................................................................................189
9.5.4 Synthesis of Poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-methyl-propyl phthalimide), P(DMAA-co-AC3pht) .......................................................................191
9.5.5 Synthesis of Poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-methyl-propyl amine), P(DMAA-co-AC3amine).............................................................................192
vi Table of Contents
9.5.6 Synthesis of Poly(DMAA-co-N-methacryloyl-β-alanine succinimide ester), P(DMAA-co-MAC2ae).............................................................................................. 193
10 Experimental III – Reactions with Functional Groups ........................................................ 195
10.1 Two Step Grafting on Functionalities ............................................................................ 195
10.1.1 Immobilization of Acryloyl Chloride as Monomer ............................................. 195
10.1.2 Immobilization of 2-Brom-Propionic Acid Bromine as Starter for ATRP ........ 195
10.1.3 Polymerization of Styrene with Polymer-attached Monomers.......................... 196
10.1.4 Atom Transfer Radical Polymerization of Styrene with Polymer-attached Initiators ..................................................................................................................... 197
10.2 Coupling of Dyes to the Functionalities of the Copolymer ........................................ 198
10.2.1 Coupling of Fluorescein Isothiocyanate ................................................................ 198
10.2.2 Coupling of DY-635-NH2......................................................................................... 199
10.3 Enzyme Immobilization .................................................................................................. 199
10.3.1 Activity Assay ........................................................................................................... 199
10.3.2 Washing Cycles......................................................................................................... 200
10.3.3 Varied Immobilization Time................................................................................... 200
10.3.4 Varied Initial Immobilization Concentration ....................................................... 201
10.3.5 Immobilization of Dyed Enzymes.......................................................................... 201
10.3.6 Hydrolysis of Immobilized Enzymes .................................................................... 201
10.4 Oligo Nucleotide Immobilization .................................................................................. 202
10.4.1 Immobilization Reaction.......................................................................................... 203
10.4.2 Hybridization ............................................................................................................ 203
10.5 Biotin assay........................................................................................................................ 204
10.6 Catalyst Immobilization .................................................................................................. 204
11 Experimental IV – Silicon Wafers ........................................................................................... 205
11.1 Silane Immobilization ...................................................................................................... 205
11.2 P(DMAA) Layers .............................................................................................................. 205
Table of Contents vii
11.2.1 Influence of the Polymerization Time ....................................................................206
11.2.2 Influence of the Monomer Concentration..............................................................206
11.2.3 Influence of the Initiator Concentration.................................................................206
11.3 Microstructured Layers ....................................................................................................206
11.3.1 UV Photoablation of Surface-attached Monomers ...............................................207
11.3.2 Grafting of Polystyrene.............................................................................................207
11.4 Combined Functional Layers & Networks ....................................................................208
11.4.1 Preparation of Copolymers for Network Formation............................................208
11.4.2 Coating & UV-cross-linking.....................................................................................210
11.4.3 Functional layers .......................................................................................................211
12 Summary.....................................................................................................................................212
13 Zusammenfassung ....................................................................................................................216
14 References ...................................................................................................................................220
viii Abbreviations & Symbols
Abbreviations & Symbols monomer
N-acryloyl-N-methyl-propyl phthalimide AC3pht
N,N-dimethyl acrylamide DMAA
3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecylfluordecyl acrylate
HDFDA
Methacrylic acid MAA
N-methacryloyl-β-alanine succinimide ester
MAC2ae
Methacryloyl-4-oxy-benzophenone MABP
Methyl methacrylate MMA
N-isopropyl acrylamide NIPAM
polymer
Poly(N,N-dimethyl acrylamide) P(DMAA)
Poly(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecylfluordecyl acrylate)
P(HDFDA)
Poly(Methacrylic acid) P(MAA)
Poly(Methyl methacrylate) P(MMA)
Poly(N-isopropyl acrylamide) P(NIPAAm)
Polystyrene PS
silane surface monomer
modification
3-methacryloylpropyl trimethoxysilane MPS -ME
N-methacryloyl-N-methyl-propyl trimethoxysilane
MNPS -MA
3-acryloylpropyl trimethoxysilane APS -AE
N-acryloyl-N-methyl-propyl trimethoxysilane
ANPS -AA
octyl trimethoxysilane OS -Oct
substrate
LiChrospher Si60 LC700
Aerosil 300 AR300
Glass beads 200 Å GB250
Glass beads 500 Å GB80
Glass beads 1000 Å GB40
Abbreviations & Symbols ix
Ai, Ap, At Arrhenius constants for initiation, propagation and termination Asp, Amod specific surface area before/after monomer immobilization ABTS 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) AIBN Azobis(isobutyronitrile)
ATRP Atom transfer radical polymerization
c concentration
CM, CS transfer constant, transfer to monomer and solvent respectively
δ, δmod amount of grafted silane or polymer δ chemical shift (nuclear magnetic resonance spectroscopy)
d distance or diameter
DCC N,Nʹ-dicyclohexyl-carbodiimide
DMF N,N-dimethyl formamide
DMSO Dimethylsulfoxide
DMT [1,1-bis(4-methoxyphenyl)-1-phenyl]methyl
DNA Deoxyribonucleic acid
DRIFT Diffuse reflection infrared Fourier transformation spectroscopy
DSC Differential scanning calorimetry
ελ extinction coefficient at wavelength λ
Eλ extinction at wavelength λ
Ei, Ep, Et activation energy for initiation, propagation and termination EA Elemental analysis
FITC Fluorescein-5-isothiocyanate
Freon® 113 1,1,2-trichloro-trifluoro ethane
FT Fourier transformation
Γ graft density of silane or polymer
GC Gas chromatography
GOD Glucose oxidase
GPC Gel permeation chromatography
HPLC High-performance liquid chromatography
I, I0 intensity of transmitted light through sample or blank cuvette
IR Infrared (spectroscopy)
[I] initiator concentration
ϕ percentage of functional units inside a copolymer
ki, kp, kt rate constants for initiation, propagation and termination
km, ks transfer rate constant to monomer and solvent respectively
KPS Potassium peroxodisulfate
x Abbreviations & Symbols
LC liquid chromatography
m mass
MC atomic weight of carbon
MF atomic weight of a molecule fragment
Mj dead polymer chain with j repeat units •jM growing polymer chain with j repeat units
Mn number average of the molecular weight distribution
Mw weight average of the molecular weight distribution
[M] monomer concentration
n amount of substance
ν kinetic chain length
NMR nuclear magnetic resonance (spectroscopy)
PD polydispersity
PEBr 1-Phenylethylbromine
PMDETA N,N,N’,N’,N”-Pentamethyl-diethylenetriamine
ppm parts per million
R gas constant
RCM Ring closing metathesis •R radical initiator fragment
rpm rounds per minute
[S] solvent concentration
SEC Size exclusion chromatography
SEM Surface electron microscopy
t time
T temperature
TEA Triethylamine
TEM Transmission electron microscopy
UV Ultraviolet
UV/vis Ultraviolet/visible light spectroscopy
v enzyme conversion rate
V volume
THF Tetrahydrofurane
nX degree of polymerization
XPS x-ray photoelectron spectroscopy
Z number of atoms in a molecule fragment
1 Introduction 1
1 Introduction
1.1 Modified Silicon Dioxide Substrates In the last decades there has been remarkable theoretical, experimental and practical
interest in the surface decoration of solid SiO2 substrates with molecules and polymers for a
broad range of applications such as analytics[1], chromatography for separation and
purification[2-4] or catalysis [5]. One of the most common substrates for immobilization of
complex molecules on a solid support are silica gels and glass beads. One general advantage
why these systems are widely used is the easy handling of such materials. Pouring them into
a chromatography column or reaction vessel and separate them after use from a liquid or
gaseous phase is quite simple. A description of applications and a discussion of advantages
of these materials compared to other materials such as variable size of the particles and
surface topology will be addressed in the following subchapters. There are many reports on
silica solids which have been physically or chemically surface modified to enhance their
properties towards the selected application and its needs.
1.1.1 Chromatographic Material
Separation or purification via chromatography is based on differences in partitioning
behavior of substances between a mobile and a stationary phase. The stationary phase is
always a solid and SiO2 substrates play an important role in such applications. The analyte
(together with solvent) travels as mobile phase through the stationary phase and interaction
with both phases results in specific retention of the analyte in the system. The interaction of
the analyte with the stationary phase can be based on relative solubility, adsorption or
interaction with charges and determines the retention of the analyte. Therefore it is
important to properly control the surface properties of materials used in chromatography.
Chromatography, especially the very common liquid chromatography (LC) has been of great
interest for industrial applications. In fact, many patents dealing with liquid chromatography
have been filed using silanes[6-9] or polymers[10-13] as surface modification. For example
for reversed phase liquid chromatography oxidic support material has to be “switched”
regarding its surface polarity. Yang et al. have bound octadecyl silane to silica (ODS-SiO2)
and polybutadiene on zirconia (PBD-ZrO2)[14]. Amino-silane modified silica is a very
versatile material because the reactivity of the amino groups allows many surface reactions.
2 1 Introduction
Based on such material Ascah et al. generated amide-functionalized reversed phase column
material[15], Jackson et al. used such modified silica in continuous flow reactors[16] and
Bruckner et al. separated amino acid enantiomers with column material prepared from
amino-modified silica[17]. Urea-functionalized materials were prepared by Silva et al. for
LC[18] and by Chen et al. for chiral LC[19]. Ruffing et al. basically changed the surface
properties of silica particles and capillaries by immobilizing polysiloxane using a platinum
hydrosilylation catalyst. Parallel, hydrosilylation was used for mounting chiral species to the
stationary phase to finally perform LC in organic liquids or supercritical fluids[20]. Based on
silica immobilized carbohydrate biopolymers Felix et al., Okamoto et al. and Wang et al.
performed chiral high-performance liquid chromatography (HPLC)[21-25]. The separation of
racemic mixtures into pure enantiomers is of great interest for pharmaceutical industries.
1.1.2 Immobilization of Biomolecules
Immobilization of biomolecules onto the surfaces of solid substrates is an area of great
interest for science and technology because the obtained biomaterials have numerous
applications in the fields of biotechnology, immunosensing and biomedical applications. At
the example of protein immobilization, especially enzyme immobilization, and
DNA/oligonucleotide immobilization the large application field of biomolecule
immobilizations is introduced.
A number of different pathways have been described which allow the immobilization of
proteins. They can be adsorbed tightly to the surface of insoluble materials[26, 27], covalently
bound onto carriers[28-30], entrapped within gels[31] or microcapsules[32, 33], or
crosslinked with bi- or multifunctional reagents[34]. Enzymes are proteins, which contain at
least one active center which catalyzes biotransformations. Immobilizing enzymes gives the
advantages of facile separation from the reaction mixture, minimized enzyme loss,
reusability and rapid termination of reactions. But it is crucial that enzymes do not lose their
activity after the immobilization process due to deformation of their structure or blocking the
active center. Proper immobilization techniques, adjusted to the particular reaction
conditions, have to be chosen for enzyme applications to maintain enzyme activities and get
the advantages of enzyme immobilization. That is the reason why enzyme engineering is a
vital and fast-growing application in pharmaceutical and food additive markets.
Among the many enzymes that have been immobilized in the past (Figure 1-1) are
dehydrogenase[35], isomerase[28], lipase[27], amylase[36], urease[29], oxidoreductase[37]
1 Introduction 3
and oxidase[30] just to name few that have been immobilized to silica supports. Recently
Kim et al. immobilized IgG-peroxidase species on polymer layers covalently bound to glass
substrates using a “grafting-to” method via polymers containing surface reactive amino
groups and a “grafting-from” method[38]. Nakamura et al. immobilized IgG-monoclonal
antibodies or glucose oxidase to silica via silane linker molecules[39]. Glucose oxidase is a
well known and intensively investigated system and therefore a good model compound for
immobilization as experiments in the past have shown[30, 40-42]. Biofunctionalization for a
wide variety of applications can be achieved by coating silica surfaces with biomolecules
such as lipids, proteins or enzymes. However, to decorate surfaces with such biomolecules a
surface optimization of the inorganic SiO2 with a silane or polymer coating is favorable in
many respects, e.g. to create a layer being capable to covalently bind the desired
molecules[43], to enhance adsorption specificity of the surface[44], or as support layers for
lipid membranes[45]. The modified materials may then be used for high-throughput
screening[46].
++ --
a) b) c) d)
e) f)
g)
Figure 1-1. Immobilization techniques: carrier binding: adsorption (a) [26, 27], binding via
electrostatic forces (b)[47], covalent binding (c) [28-30], biospecific binding (d); entrapment: lattices
(e)[31], microcapsules (f) [32, 33]; direct cross-linking (g) [34].
In biotechnology and clinical medicine modified beads and also planar systems are of
great interest for the generation of analytic chips and biosensors[48, 49]. Here especially
biotin- and DNA- or oligonucleotide immobilization[50-53] come into play. Composites of
4 1 Introduction
glass beads and polymers with immobilized biomolecules were developed to improve the
function of these supports[54-56]. In addition it was found that a polymer cushion of
functional hydrogels gives shear protection to biomolecules that were covalently bound by
activated ester groups within the hydrogel[31].
Various methods using polymer layers on planar substrates like plasma borne
polymers[57], dendrimers[58] or latex based polymer film[59] have been developed for
biomolecule immobilization. Patterned surfaces on planar systems with immobilized
biomolecules can be used in an even better way as analytical chips, e.g. DNA microarrays as
Freidank et al. and Oh et al. have shown[60, 61]. Protein chips for proteomics are a quite new
development. Snyder et al. examined the use of protein chips that have been generated by
just using densely packed wells[62]. Melnyk et al. immobilized peptides in a structured
fashion by using linker molecules[63]. Of course also combinations of the previously
mentioned polymer layer systems with common patterning methods are possible. Another
combination is the use of patterned planar systems with functional particles to immobilize
biomolecules[64, 65] as realized by the Frauenhofer Gesellschaft and Dickinson et al. The first
approach uses polymeric layers with reactive groups to bind the particles onto the surface,
the second is simpler by using pre-fabricated wells or well plates.
1.1.3 Catalyst Immobilization
As various industries use immobilized enzymes as biocatalysts there is also a large market
for immobilizing classic catalysts for chemical synthesis. Already 1975 rhodium catalyst
species for hydroformylation reactions were immobilized by a quite complicated method to
phosphine ligands contained within a polystyrene coating on silica gel by Arai et al. In
heterogeneous catalysis these substrates showed high efficiency[66]. Several approaches
within the last decades have been made on the immobilization of rhodium compounds and
use them to catalyze olefin hydroformylation[66-68] which is a very common industrial
reaction. Various other reactions have been performed by the use of functional silica
immobilized catalyst species, e.g. oxidation of alkanes and cyclohexanes with iron and
copper by Kurusu et al.[69, 70], or Heck re[71]action with palladium by Pan et al.[72]
Immobilization of catalytic compounds were also applied to the pyrogenic silica Aerosil.
Hultman et al. fixed the catalyst directly to the surface[73, 74] whereas Sarkany et al. used a
combination of polymer and [71]palladium[71].
1 Introduction 5
In the last years metathesis reactions grew considerably and techniques to immobilize
adequate catalyst species to solid supports evolved. Kingsbury et al. immobilized and used
olefin metathesis catalyst on particles[75], Buchmeiser et al. did the same on particles
equipped with a surface polymer layer[76]. Ring opening metathesis polymerization (ROMP)
with immobilized catalyst was performed by Mingotaud et al. to grow polymer layers on
silica nanoparticles constructing core-shell architecture[77]. Besides ROMP Krause et al. also
performed ring closing metathesis (RCM) reactions with immobilized Grubbs-Hermann type
catalysts[78].
1.2 Fabrication of Silica Particles & Silicon Silica is a material that offers a great variety of particle sizes, porosities and surface
topologies. Some variants exhibit high surface areas within a small amount of solid mass like
infusorial earth which is a naturally structured form of silica built by sedimentation of
diatoms and infusorians[79]. The detailed structure of synthetic silica depends on the
production process. The way it is generated determines particle size, particle agglomeration
and ageing respectively sintering. An important method of silica production is the sol-gel
process[80-82] where soluble silicates are converted into silicic acid that then condenses
building a large network of interconnected microscopic pores (Scheme 1-1a). Using alkoxy
silanes instead of sodium silicate, e.g. tetramethoxysilane or tetraethoxysilane, gives highly
pure and metal free silica (Scheme 1-1b).
a) Na2SiO3 + 2 H+ → SiO2 + H2O + 2 Na+
b) Si(OR)4 + 2 H2O → SiO2 + 4 ROH
Scheme 1-1. Reaction of sodium silicate (a) or alkoxy silanes (b) to silica by the sol-gel process[83, 84].
First a stable colloidal dispersion, the sol, is generated. During the condensation reaction
chain growth and network formation or particle growth can be influenced by parameters like
pH-value or salt concentration[85]. The viscosity rises until the gel point is reached. The gel
point is defined as the degree of conversion where a material is formed which exhibits an
elastic response when mechanical stress is delivered to the system[86]. The obtained
hydrogels are aged by moderate drying. Upon this evaporation process the pores collapse
resulting in socalled xerogels (Greek: ξερός, xerós = dried). In order to retain the original
pore structure and take advantage of the high specific surface of a silica network,
6 1 Introduction
supercritical drying is applied to the hydrogels[87] and the received material is called
aerogel (Greek: αέρας, aeros = air). These aerogels were developed as storage material for
other substances. Another advancement in the generation of silica materials is the Stöber
process[88] where large, well defined, monodisperse silica particles are obtained through
hydrolysis of alkoxysilanes.
Another major method for silica generation is hydrolysis of halide silanes within an
oxyhydrogen flame (Scheme 1-2). These pyrogenic silicic acids form very pure and compact
silica particles without pores. The primary particles may be sintered to larger agglomerates
creating pores between the primary particles. These aggregates have the advantage of a very
open structure with mainly macropores.
Scheme 1-2. Synthesis of pyrogenic silica: different stages of particle formation in an oxyhydrogen
flame (T > 1200 °C)[89].
An advantage of the sol-gel process is that already within the process hybrid materials are
accessible. “Hybrid materials” means a material consisting of clear regions where either
inorganic or organic structures dominate. Using silanes with organic substituents, instead of
pure silicates with four oxygen substituents, give particles with the chosen organic moieties.
Such particles with a low compressive modulus known as Tospearl have been used in
cosmetic formulations[90]. Other particles with a high compressive modulus and mesopores
have been developed by Cheng and Fisk for utilization in reverse-phase high-performance
liquid chromatography[91]. Another commercial species are “MQ resins”, particles whose
cores can be prepared by using tetraethoxy silane[92] or sodium silicate[93] from Q units
analog to the sol-gel process. The shell is then generated by using (functional) M units
(Scheme 1-3). The largest application for MQ resins is in pressure-sensitive adhesives
(PSA)[94, 95].
1 Introduction 7
Scheme 1-3. Nomenclature of siloxane units bearing different numbers of organic substituents (= R).
The combination of single letters in the name of a resin indicates the which kind units are used to
prepare the resin.
Another important substrate we use are silicon wafers. These planar substrates have a
silicon dioxide layer in the nanometer range and therefore adequate silanes may be attached
to the surface similarly as for the silica and glass substrates. Silicon wafers are mainly used in
applications of the semiconductor industry. But in the recent years miniaturization has
become an important aspect in many fields of technology. Especially the construction of
micro devices is interdisciplinary and combines amongst others semiconductor technology,
microelectronics, microoptics as well as surface chemistry[96, 97]. For example Jeyaprakash
et al. managed to reduce the surface conductivity of a gas sensor device by tailoring the
surface with a fluoropolymer film[98].
1.3 Surface Decoration of Silica, Glass Beads & Silicon
1.3.1 Functional Silanes & Silane Anchors
Silica, glass and silicon have in common that the surface of these substrates consists of a
silicon dioxide layer where reactive silanes can be attached to. As reactive silanes mostly
chloro- or alkoxy silanes are used. Already in the 70ies silanes were used to influence the
surface of solid substrates to get more specified interactions for liquid chromatography
(LC)[99-102]. Because of the great interest in such materials many basic investigations have
been carried out. Velasquez et al. gathered information about the surface free energy of
silane covered materials[103] whereas Tripp et al. had a closer view on the surface
attachment of chloro silanes[104, 105] and Kurth et al. investigated surface reactions of
surface-attached silanes[106]. Because of the good reactivity of amino groups special interest
has been set on amino-silanes and different measurements have been carried out concerning
8 1 Introduction
their surface adsorption and immobilization like calorimetry[107, 108], ellipsometry[109] or
x-ray photoelectron spectroscopy and magic angle spinning-NMR[110]. But also silanes with
other reactive groups have been immobilized, e.g. epoxy groups[111]. Another large field of
interest is the hydrophobization of silica substrates with long alkyl chain silanes like
octadecyl silane (ODS)[112] for chromatography purposes[14, 101]. This short overview
shows that the surface-attachment of various silanes with divers functionalities on solid
substrates has been intensively studied in the past.
The grafted amounts δ (in mol·g-1) of monomer in the composite material can be
determined from elemental analysis according to equations derived from calculations of
Berendsen and de Galan[113]:
ZM
g
C
Cmod ⋅
=δ equation 1-1
δmod is here the grafted amount of silane per gram modified silica (weight of silica
including the silane) and gC is the carbon content as determined by elemental analysis. Z
represents the number of carbon atoms in the immobilized unit and MC is the atomic mass of
carbon (12.01 g·mol-1). MF is the molecular mass of the part of the silane which is surface
attached (note: the methoxy groups are lost to surface anchoring or hydrolysis and are,
therefore, disregarded in this calculation). For a more detailed comparison in some cases it is
desirable to describe the grafted amount in relation to the pure silica (equation 1-2). However
to do so it has to be taken into account that the specific surface is altered by the surface
attachment (equation 1-3).
( ) 1F
Fmod
Fmod MM1
M −⋅
⋅−
⋅=
δδδ equation 1-2
( )( ) modunFmodmod AM1A ⋅⋅−= δ equation 1-3
δ is the grafted amount of silane per gram pure silica (weight of silica without the silane).
Aunmod and Amod are the specific surfaces (in m²·g-1) of the solid before and after silane
immobilization.
1 Introduction 9
1.3.2 Polymer Layers by “Grafting-to” & “Grafting-from”
The decoration of surfaces with thin layers of polymers is a wide spread approach for the
tailoring of surface properties of materials[114-117]. A huge number of different techniques
have been developed ranging from simple painting or solution casting processes to
sophisticated chemical approaches which allow for the generation of films of covalently
grafted polymers. The latter typically exhibit a outstanding stability against harsh
environments including treatments with solvents that would usually dissolve the coating.
Typical chemical strategies range from systems in which polymers with reactive groups
(either at the chain end or along the backbone) are attached to suitable groups at the
surfaces[118-121] (“grafting-to”; cf. Figure 1-2a) to more recent approaches that utilize
monolayers of initiator molecules to “grow” the chains at the surfaces[122-127] where also
living polymerization methods are available[128-131] (“surface-initiated polymerization”,
“grafting-from”, cf. Figure 1-2b). These two techniques have been thoroughly studied over
the past twenty years and books and reviews are available that describe the particularities of
each approach[132, 133]. As we will see in 1.3.3 some theoretical considerations and
calculations play an important role for understanding the behavior of the third grafting
technique using surface attached monomers.
Surface modifications on the substrates used in this thesis have been well studied. In the
70ies Negievich et al. already performed different graft polymerization techniques on Aerosil
material[134, 135]. A few years later Ivanchev et al. used surface-attached
methacryloyloxymethyl methyldiethoxysilane units for a graft polymerization of methyl
methacrylate on Aerosil[136] and in a “grafting-from” like approach polystyrene was grown
by initiation through the redox decay of immobilized hydroperoxides[137]. Grafted
polymethacrylate layers on Aerosil were investigated by Yushkova et al.[138]
Different types of LiChrospher, sol-gel borne particles, have been used for surface sensitive
applications in the past. Silica particles with a polyacrylate layer (LiChrospher Diol) showed
good separation properties in size exclusion chromatography (SEC)[139, 140] and in high-
performance liquid chromatography (HPLC) of raw sugars and polyols[141] as well as the
commercially available reversed phase material LiChrospher RP[142-144]. Guillaume et al.
coated LiChrospher successfully with a cross linked polymer film[145] and Kurganov et al.
built a thin polyvinylpyrrolidone film on a LiChrospher species[146] with pores of 30 to 50 nm
comparable to the glass beads used here. As we see the history of these solid supports and
the many surface modifications that have been delivered to them to get the desired
10 1 Introduction
properties, our choice of silica solids seems to be reasonable to explore the behavior of the
approach using surface-attached monomers.
1.3.3 Polymerizing in Presence of Surface-attached Monomers
Surprisingly, the third approach for the generation of monolayers at solid surfaces has not
received equal attention in the scientific community so far, even though it is frequently used
in industry as a so-called primer system for the modification of surfaces of inorganic
components of acrylate based formulations. As already mentioned above in this approach
(cf. Figure 1-2c) functionalized monomers are attached to the surfaces of the solid substrates
and the resulting surface-attached groups of the materials are then used as a “comonomer”
during a free radical polymerization. During this process all chains are initiated in solution
but some of the growing chains are captured by surface-attached monomers. From there they
can continue to grow away from the surface until a chain transfer or a termination reaction
ends growth of the individual chain.
Consequently the chains on the surface are not tethered to the substrate by one end but
with one or several anchor points somewhere along the chain. Among the first to study such
approaches were Hamann and Laible who used silane based systems on silica particles[147]
and Trachenko et al. who directly coupled acrylic acids to various oxide surfaces[148]. In
case of the silane based system the monomer is bound on the silica surface via siloxane
bridging links. Both systems were used for the copolymerization of the surface-attached
monomers with monomers in solution. Cohen et al have published the results of
experimental and theoretical work that to some extent describe the overall kinetics of the
process[149], but up to now there is no comprehensive study that elucidates the special
kinetic and mechanistic features of this approach. Delivering this information is the main
task of this thesis and the important targets as well as the strategy to reach them is described
in chapter 2. Furthermore it is important to establish our grafting technique for a wide
variety of silica because with the choice monomer, functionality and substrate it will be
applicable for many different purposes in a simple and successful way.
1 Introduction 11
Figure 1-2. Schematic depiction of three different techniques for the generation of surface-attached
polymer monolayers; attachment of functionalized polymers, “grafting-to” (a), surface-initiated
polymerization from immobilized initiators, “grafting-from” (b) and incorporation of surface-attached
monomer units into a growing chain during a solution-borne polymerization (c).
Before moving the focus towards applications we tackle the behavior of this grafting
system by comparing it (cf. chapter 3.3) with theoretical description of “grafting-to”
approaches of Chakrabarti et al.[150] and Binder et al.[151] which use Monte Carlo
calculations to explain kinetic details of the irreversible adsorption of polymer chains which
are uniform in length onto a surface with reactive sites. End-functionalized polymer coils
approach the surface because a growing polymer chain only attaches to the surface monomer
by its radical moiety at the growing end of the chain. Furthermore the polymer adsorption
process is irreversible because the growing polymer chain forms a chemical bond with the
surface attached monomer. One may draw parallels to a system of homopolymer adsorption
that is also discussed by Zajac and Chakrabarti[152]. There a polymer chain has several
12 1 Introduction
anchor points on a surface. This system may be realized such that the radical center moves
from surface monomer to surface monomer either building trains of directly connected
surface monomers or building loops with interconnected monomer units from solution. But
multiple attachment is not very favorable because even close to the surface the number of
free monomers that surround the active center of growing chains is much higher than the
number of surface attached monomers in close proximity[153]. Another important factor that
is taken into account by Binder et al. and used in the calculations of Chakrabarti et al. is
described by Ligoure and Leibler[154]. The early formation of a polymer layer attached to
the surface exhibits steric repulsion of polymer chains that approach the coated surface.
There are two successive regimes: First diffusion in solution governs the adsorption kinetics.
Later on adsorbed chains begin to overlap and penetration of this barrier controls the
kinetics. When assuming that an active center of a growing chain rapidly connects to a
surface attached monomer the grafting kinetics are controlled by diffusion in solution and
penetration of chains through the polymer layer protecting the surface. As this layer gets
thicker it becomes harder for polymer chains to permeate it. Shorter chains have then steric
and entropic advantages to penetrate the polymer layer and grow onto the surface. This
model, depicted in Figure 1-3, is important for the grafting system we describe because in
standard radical solution polymerization there are always growing chains of different
lengths as well as newly initiated chains present throughout the polymerization.
1 Introduction 13
Figure 1-3. The kinetic barrier built up by already surface attached polymer coils hinders larger
polymer radicals that approach the layer at a later point of time to reach the surface and get connected
to it (a) more effectively than smaller polymer chains that may have a better chance to get through to
an unreacted surface monomer and get linked to it (b).
1.3.4 Polymer Micropatterning
In the last decades different patterning techniques such as embossing[155], photoresist
technology, photolithography[156, 157], microcontact printing[158, 159] or ink-jet
printing[160, 161] have been used to give a surface a desired structure and also to deposit
various substances in a structured manner. Low molecular weight compounds that attach to
surfaces of glass and silicon like silanes or to surfaces of noble metals like thiols have been
used to tailor the chemical composition of the solid substrates with spatial resolution.
14 1 Introduction
Additionally there have been recent efforts to expand patterning to polymers because they
can deliver thicker layers and higher densities of functional groups. Various approaches with
destructive patterning of a homogeneous polymer layer like masking coupled plasma
treatment[162], UV-ablation, electron-beam lithography[163, 164] or even enzymatic
degradation[165] have been investigated. Different constructive approaches are possible
when using surface attached initiators to perform “grafting-from” polymerization like
polymerization of a pre-patterned initiator layer[166], photopolymerization of a
homogeneous initiator layer through a mask[166, 167], or even using a sequence of
photopolymerization and thermal polymerization to grow different polymer brushes from
the same initiator layer[168]. Another constructive approach is patterning by forming and
attaching a polymer network, also in multiple layers[60], through mask guided UV-cross-
linking of benzophenone units within a polymer. Direct photopolymerization of
poly(ethylene glycol) macromers with cross-linking compounds like di-, tri- or tetraacrylates
through a mask formed patterned hydrogels attached to a methacryloyl propyl
trichlorosilane modified substrate[169]. With direct thermal polymerization using pre-
patterned methacryloyl propyl trimethoxysilane modified substrates and UV-cross-linking of
benzophenone containing copolymers through a mask onto substrates with a homogeneous
MPS-layer we like to introduce a technique that offers robustness during the preparation
process as well as for the resulting product and the versatility of the previously mentioned
patterned polymer systems.
1.4 Radical polymerization Because radical polymerization is the main reaction in this work a short introduction to
this reaction collected from different textbook sources[170-172] will be given in the
following. Generally, in radical polymerization monomers containing a double-bond are
added to the radical end of a growing chain. The polymerization process can be divided into
three different stages: Initiation, propagation and termination. Before starting a growing
chain free radicals have to be formed. Special starter molecules, called initiators, are
decomposed by thermal or photochemical energy or generated by a redox process. In our
experiments we use azoisobutyronitrile (AIBN) as thermal initiator that decomposes into
two cyanopropyl radicals and nitrogen. The starting reaction is the addition of the first
monomer to the start radical. The addition of further monomers then is called propagation
reaction and it has a rate of around one monomer per millisecond although the rate may
vary dependent on the the used monomer. The growth is terminated through a termination
1 Introduction 15
reaction which has two different possibilities: Recombination is the addition of two radical
ends of growing chains and disproportionation is when a hydrogen is transferred from the
end of one radical chain to the end of another radical chain.
The description of the kinetics of a radical polymerization can be divided into the three
stages mentioned above. Equation 1-4 shows the decomposition of an initiator molecule I
into two radical fragments R• with an initiation rate constant ki followed by the starting
reaction where a monomer M adds to the radical fragment (equation 1-5).
equation 1-4 •→ R2I ik
equation 1-5 •• →+ 1k M-RMR s
The initiation, where the primary radicals are formed, is typically slower than the starting
reaction where the first monomer is added. Because of that the initiation is the rate
determining step and the reaction rate is defined as the decrease of initiator concentration [I]
with time (equation 1-6).
[ ] [ ]Ikdt
Idi ⋅=− equation 1-6
The propagation reaction is when a monomer molecule M adds to a growing chain Mj•
where j is the number of already added repeating units. The propagation rate constant is kp
(equation 1-7).
equation 1-7 •+
• →+ 1jk
j M-RMM p
Now the polymerization rate can be calculated as shown in equation 1-8 where the
decrease of the monomer concentration [M] with time is a product of the propagation rate
constant, monomer concentration and the sum of all growing chains with various lengths.
[ ] [ ] [ ]∑ •⋅⋅=− jp MMkdtMd
equation 1-8
After initiation the growing chain adds monomer units within a short time period
(milliseconds to seconds) as long as monomers are present and unless any termination
reaction takes place.
16 1 Introduction
Termination reactions are recombination (equation 1-9) and disproportionation
(equation 1-10) with the corresponding termination rate constants tc and td.
equation 1-9 •+
•• →+ kjk
kj MMM tc
equation 1-10 kjk
kj MMMM td +→+ ••
One termination reaction eliminates two undistinguishable radical chains. Now adding
both specific termination constants to one overall termination rate constant kt leads to
equation 1-11.
[ ] [ ]( 2
jtj Mk2
dtMd
∑ ••
=− ) equation 1-11
To simplify the kinetic model one presumes that during the reaction there is a steady state
where as many radicals are built as are consumed:
[ ] [ ]
dtMd
dtRd j
••
= equation 1-12
Using equation 1-11 and a combination of equation 1-6 and equation 1-4 we obtain for the
steady state
[ ] [ ]t
ij k
IkM =∑ • equation 1-13
Together with equation 1-8 the polymerization rate is calculated as
[ ] [ ] [ ] [ ] [ ]2
1
21
21
IMk
kkMMk
dtMd
t
ipjp ⋅⋅
⋅=⋅⋅=− ∑ • equation 1-14
If we define the kinetic chain length ν as the quotient of the propagation rate divided by
the initiation rate, we obtain for the steady state the following equation:
[ ]
[ ]( )21
Ikk2
Mkν
ti
p
⋅⋅
⋅= equation 1-15
1 Introduction 17
With the termination factor a = 1 for disproportionation and 2 for recombination we get the
degree of polymerization nX .
νXn ⋅= a equation 1-16
The number average of the molecular weight distribution Mn is the degree of
polymerization multiplied with the monomer weight M0:
0nn MXM ⋅= equation 1-17
Combining equations 1-15 to 1-17 and unifying all constant values to a constant b results in
[ ][ ]2
1
IMM n ⋅= b equation 1-18
Here we see that the number average molecular weight for a standard radical
polymerization is proportional to the monomer concentration and reciprocal to the square
root of the initiator concentration.
The polydispersity PD is defined as the quotient of the weight average molecular weight
divided by the number average molecular weight.
n
w
MMPD = equation 1-19
For the different termination modes the polydispersity differs. In case of pure
disproportionation the theoretical polydispersity (derived from Schulz-Flory distribution) is
PD = 2 because all chains keep their length upon termination. For a termination by a pure
recombination mode the theoretical polydispersity is PD = 1.5 because the recombination
process statistically reduces the number of different chain lengths and the molecular weight
distribution is narrowed.
The temperature influence on a radical polymerization can be derived from the Arrhenius
equations for the initiation, propagation and termination velocity constants. The general
equation for a reaction rate constant k is
⋅−
⋅= TRE
eAk equation 1-20
18 1 Introduction
where A is the frequency factor, E the activation energy of the reaction and R the gas
constant ( = 8.31451 J·K-1·mol-1). Using equation 1-20 with equation 1-15 and 1-16 delivers the
term for the temperature dependence of the degree of polymerization:
[ ][ ] 2
12
12
1IM
TR
E2E
2E
expAA2
AX
pti
ti
pn ⋅
⋅
−+⋅
⋅⋅⋅= a equation 1-21
Multiplied with the molecular weight M0 of the monomer (cf. equation 1-17) we get the
temperature dependence of the number average of the molecular weight:
[ ][ ] 0
pti
ti
pn M
IM
TR
E2E
2E
expAA2
AM
21
21
21 ⋅⋅
⋅
−+⋅
⋅⋅⋅= a equation 1-22
For batch reactions where defined amounts of initiator and monomer in the start reaction
mixture are given there is a time dependence of the molecular weight. Significant changes of
the reaction conditions, e.g. change of monomer/initiator concentration or viscosity, are the
reason for reaction stages a polymerization reaction runs through. Each stage may exhibit a
different distribution of the molecular weight of the polymer. Here conversion comes into
play as an important factor advancing with polymerization time.
1 Introduction 19
Figure 1-4. Different types of progress in conversion with advancing time are depicted. Curve a)
shows a typical conversion diagram for a polymerization in the bulk. In a relatively early stage the gel-
effect causes an auto-acceleration of the polymerization. The final conversion lies below 1 because the
glass-effect “freezes” the mixture, polymer segments lose mobility and monomers cannot diffuse to
growing chain ends. Curve b) shows a conversion diagram for a polymerization with a medium
monomer concentration. Auto-acceleration takes place but mobility of the ingredients is retained by a
certain amount of a solvent. Curve c) shows a conversion diagramm where no gel-effect occurs and the
conversion rate decreases with time because the monomer concentration decreases as well. In the latter
case full conversion can be reached in a longer period of time.
With ongoing conversion the polymer concentration increases in the polymerization
mixture and therefore the viscosity of the mixture rises. The higher the initial monomer
concentration and the higher the molecular weight of the formed chains the more
pronounced this effect is. The influence of this diffusion controlled process is reflected in
higher viscous polymerization systems show stronger gel-effects. Above a certain polymer
concentration the polymer coils begin to overlap and entangle. This impedes the diffusion of
polymer radicals and the termination rate decreases whereas the advancing initiator
decomposition generates constantly new radicals leading to an increase of radical
concentration and growing chains (of any length). Consequently the polymerization rate
increases (Figure 1-4).
20 1 Introduction
The decrease of the termination rate during the gel-effect causes an increase in kinetic
chain length. Therefore the degree of polymerization jumps up when the gel-effect starts
(arrow in Figure 1-5a). Polymer built during a gel-effect possesses a different molecular
weight distribution than the polymer built during the steady state. The distribution of the
degree of polymerization becomes broader.
Figure 1-5. Development of the average molecular weight with polymerization time in a
polymerization solution where the monomer content is high enough to lead to a gel-effect (a). The
arrow marks the beginning of this effect. In dilute polymerization solutions the average molecular
weight decreases parallel with the monomer concentration (b) as monomers are used up by polymer
formation. The grey area marks a region of linear molecular weight decrease where conversion increase
and monomer decrease are constant because of a steady state.
When the polymerization solution is as diluted as there is no gel-effect and the monomer
concentration decreases continuously with time because ongoing polymer growth consumes
monomer. When the initiation rate is held constant the average molecular weight will
decrease according to the monomer content within the solution (Figure 1-5b). As the
polymerization rate and conversion are constant in the steady state the molecular weight
decreases linearly (cf. Figure 1-5b). If significant amounts of initiator are consumed during
polymerization the decrease of the molecular weight may be compensated by a slight
increase of molecular weight due to lower initiator concentrations.
2 Goals & Strategy of the Work 21
2 Goals & Strategy of the Work
2.1 Goals Surface-attached monomers are well established and often used in polymerization systems
but there is only little comprehension of such systems in regard to mechanistic details.
Monomer-modified substrates enable us to graft polymer onto them by radical
polymerization in solution. Such a reaction will occasionally attack a surface-attached
monomer leading to surface-attachment of the whole chain. We like to investigate this
method of surface modification using surface-attached monomers. First goal of this work is
to improve the understanding of the mechanism of a graft polymerization in the presence of
surface-attached monomers. This is attempted to reach by varying important polymerization
parameters which influence the kinetic chain length and in consequence the amount of
grafted polymer and graft density of the attached chains. Further goals emanate from the
knowledge we obtain by reaching the first goal. One is to explore the versatility of this
method by using different monomers on the surface as well as in the surrounding media.
Furthermore we investigate if silica porosity and size influence the result of the investigated
graft polymerization as these substrate properties are important for the use in different
applications. With the gathered knowledge we will be able to tailor polymer grafted surfaces
in a desired way. This leads to an investigation plot where feasibility studies for possible
applications for such modified substrates are performed.
2.2 Strategy Good polymerizability has to be a premised property of the surface-attached monomer
because the grafting step where the radical end of a growing chain connects to a monomer
on the surface has to be a fast and irreversible step to ensure a good yield of surface-attached
polymer chains. 3-methacryloyl-propyl trimethoxysilane (MPS) is a commercially available
and widely used compound with a silane anchor group. Because the methacrylic moiety is
linked to the spacer via an ester group that may be hydrolyzed under certain conditions it is
attempted to synthesize a similar compound where the methacrylate forms an amide group
which is more inert to hydrolysis. Amides can be hydrolyzed or transamidated only under
drastic conditions. To complete the picture also the acrylic versions of the ester linked and
amide linked compound are used in the experiments.
22 2 Goals & Strategy of the Work
To get a deeper insight to graft mechanism of a polymerization using surface-attached
monomers, the influence of important polymerization parameters such as polymerization
time and temperature, concentration of monomer in solution and on the surface as well as
initiator concentration on the resulting material is investigated. The focus at this is on the
determination of grafted polymer amount via elemental analysis (EA), molecular weight
distribution of the built free polymer by gel permeation chromatography (GPC). From these
analytical results the graft density is calculated. The influence of the polymerization
parameters on both values, grafted polymer amount and graft density, is then evaluated.
Laible and Hamann already discovered differences in the grafted polystyrene amount on
MPS-modified Aerosil[147]. They found out that with higher styrene concentration during
polymerization the amount of the grafted PS increases. Similar graft amount determinations
were made by Browne, Chaimberg and Cohen to investigate the influence of polymerization
time on polyvinyl acetate grafting on vinyl triethoxysilane modified silica spheres[173].
Cohen et al. developed a complex kinetic model for free radical graft-polymerization based
on a conservational polymerization and molecular weight distribution numerical algorithm
to predict graft yield for polyvinylpyrrolidone (PVP) on material with polymerizable surface
sites[149]. These “kinetic modeling” considerations do not provide a clear picture or answers
to questions like what happens if the surface-attached monomer has a different reactivity
compared to the monomers in solution or how does the porosity of the substrate or the
viscosity of the surrounding media influence the polymer monolayer buildup. As already
mentioned in 1.3.3 the formation of a graft polymer layer limits the grafting of further
polymer in a certain way. It is important to know in what manner the polymerization
parameter influence the surface polymer layer formation and in consequence the control of
this process.
Besides the understanding of the mechanism of this method we investigate its versatility
by varying the polarity of the used monomers from a hydrophobic perfluoro monomer to a
hydrophilic monomer like methacrylic acid. We will investigate if with surface-attached
monomers these different monomers in solution form a chemically bound polymer layer on
the modified substrates. To underline and expand this versatility the introduction of
functional groups by copolymerization in solution with special tailor-made monomers will
be a suitable way. These monomers bear amino or activated ester groups which allow many
further reactions, e.g. with catalyst species or biomolecules. If successfully applied to our
system this may lead to valuable applications for the silica-polymer hybrid systems. As
examples we will carry out copolymerizations with the aforementioned monomers in
2 Goals & Strategy of the Work 23
solution in combination with styrene to form a hydrophobic layer, or in combination with
N,N-dimethyl acrylamide to form a hydrophilic layer on different silica substrates.
According to the strategy of this work, substrates with different particle and pore sizes are
used which are commercially available and used in a broad range of applications as we have
introduced in the previous chapter. Here the specific surface of the used substrates ranges
from 700 m2 per gram for the highly microporous LiChrospher Si60 down to 40 m2 per gram
for macroporous glass beads with 100 nm pores. All values were determined by the producer
via BET method[174].
LiChrospher Si60 is a silica gel consisting of monolithic particles with spherical shape, a size
of around 25 µm and micropores with a diameter of around 2 nm (Figure 2-1).
Figure 2-1. SEM images of LiChrospher Si60 before (a) and after 24 hours (b) of polymerization of
styrene.
24 2 Goals & Strategy of the Work
Aerosil300 is a pyrogenic silica gel and has a specific surface of 300 m2·g-1. Its body consists
of small sintered silica beads building up a three-dimensional network with concave pore
geometry (Figure 2-2).
Figure 2-2. TEM images showing Aerosil300 in a pure state (a) and after 24 h PS polymerization (b).
The glass beads involved in our studies have pore sizes of 20 nm, 50 nm and 100 nm
(“mesoporous” up to “macroporous”). Their specific surfaces cover with 250 m2, 80 m2 and
40 m2 per gram the regions of medium and lower specific surfaces (Figure 2-3).
Figure 2-3. A CCD image of GB80 before used for polymerization (a); TEM reveals mesopores on the
surface of the glass beads (b) that stay intact after been in polymerization solution for 24 h.
Furthermore we investigate in how far chemical microstructures can be formed by using a
system with surface-attached monomers. Flat substrates such as silicon wafers will be
2 Goals & Strategy of the Work 25
equipped with surface-attached monomers and chemically microstructured in two different
ways. The first method will use a mask on the surface and surface monomers will be
destroyed in the illuminated areas by UV ablation. For the second method a polymer layer
with incorporated benzophenone units will be deposited and subsequently cross-linked by
UV irradiation. In the latter method C-H-bonds of the propyl spacer of the surface-attached
monomers will be used as anchor points for the benzophenone. Additionally a second
polymerization using the graft process will be possible afterwards to form a polymer layer
with different properties which might be an interesting feature.
26 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
3 Grafting of Polymers onto Silica Surfaces through Surface-
attached Monomers
3.1 Silane Monomers For the system described here the first step towards the deposition of the polymer
monolayers is the modification of the silica surfaces with appropriate monomer units
(Scheme 3-1). To allow a deeper insight into the subsequent polymerization reaction we have
looked at monomers that differ somewhat in their structure. The silanes we used were
acrylates and methacrylates. All of them carried a trimethoxysilane anchor group separated
from the (meth)acrylic acid residue by a propyl spacer. This trimethoxysilylpropyl moiety
was linked to the methacrylic acid via a ester or amide linkage as it can be expected that the
amide might be less prone to hydrolysis compared to the esters in chemically harsh
environments. On the other side, the nature of the polymerizable group i.e. acrylic and
methacrylic tails, was varied to study the influence of the reactivity of the monomer, i.e. their
polymerization behavior, of these two groups during the subsequent free radical
polymerization process for the deposition of the polymers on the silica particles. The name of
the compounds are 3-methacryloyl-propyl trimethoxysilane (MPS), N-methacryloyl-N-
methyl-propyl trimethoxysilane (MNPS), 3-acryloyl-propyl trimethoxysilane (APS) and N-
acryloyl-N-methyl-propyl trimethoxysilane (ANPS). It is well-known that trifunctional
silanes like the ones used in this study tend to form not ideal monolayers but surface
attached networks with varying structure due to hard to control pre-condensation reactions
in solution prior to deposition on the surface[175-177]. An factor which is here especially
important is the presence of water. As we used a porous silica gel such processes could,
under certain reaction conditions, lead to situations in which the pores become clogged with
a rather thick disordered siloxane layer. In order to avoid such complications we decided to
deposit the monomer silanes under strictly inert conditions, meaning that all solvents and
reagents were rigorously dried by appropriate means and all deposition reactions were
carried out in an atmosphere of dry nitrogen.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 27
Scheme 3-1. Reaction scheme for the preparation of SiO2 substrates with surface-attached monomers.
Substrates modified with polymerizable groups are generated by immobilization of a trimethoxy silane
carrying a (meth)acrylic group (1). These modified substrates can then be used in a radical
polymerization to form ultrathin (co)polymer monolayers on the substrate surface (2).
The resulting layers were characterized qualitatively using diffuse reflectance FT-IR
spectroscopy (DRIFT). Due to the fairly similar chemical nature of the silanes, very similar
spectra were obtained for all monolayers. As an example, Figure 3-1 shows the spectrum of a
MPS layer in comparison to a spectrum obtained from the as-obtained, i.e. unmodified, silica
gel. Signals as the C-H stretching vibrations below 3000 cm-1, a strong carbonyl absorption
band at 1700 cm-1, and the C=C stretching band at 1636 cm-1 are a clear indication for the
presence of an MPS layer on the silica surface.
28 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
4000 3000 2000 1000
HH( C=C )ν
ν (5H Aryl)
ν
ν
ν
νν
(CH)
(SiOH)
(SiOSi)
(C=C)(C=O)
ν
(Aryl-H)
tran
smitt
ance
(a.u
.)
wavenumber (cm-1)
Figure 3-1. Diffuse reflectance FT-IR (DRIFT) spectra obtained from unmodified silica gel (1), SiO2-
MPS (2), and a silica gel carrying a polystyrene monolayer (3).
In order to obtain a more quantitative picture the materials were investigated by elemental
analysis which allows for the determination of the grafting density of the silanes on the
LiChrospher surfaces in first place. The carbon contents of all materials was determined by
elemental analysis. The values were all rather close to each other and ranged from 6-8% (cf.
Table 3-1). The other substrates such as Aerosil and the different glass beads come into play
later in this work. Surface attachment of the monomer silanes is fundamental for all
investigations so these results are the first to be discussed. From the carbon content values
the immobilized amount δ can be calculated according to equations 1-1 and 1-3. Using the
immobilized amount and the specific surface area of the LiChrospher (700 m2·g-1 according to
the BET method[174]) the surface concentration Γ0 of the polymerizable groups can be
calculated.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 29
Table 3-1. Carbon contents and graft densities on different SiO2 surfaces with different immobilized
monomers.
substrate silica material
+ specific surface (m2·g-1)
carbon*
(%)
Γ0
(µmol·m-2)
LC700-ME (MPS) LiChrospher Si60 (700 m2) 6.9 ±0.9 0.9 ±0.1
LC700-MA (MNPS) LiChrospher Si60 (700 m2) 7.9 ±0.8 0.9 ±0.1
LC700-AE (APS) LiChrospher Si60 (700 m2) 6.0 0.9
LC700-AA (ANPS) LiChrospher Si60 (700 m2) 6.4 0.9
LC700-Si-octyl† (OS) LiChrospher Si60 (700 m2) 5.5 ±1.3 0.7 ±0.2
AR300-ME (MPS) Aerosil300 (300 m2) 6.4 2.0
GB250-ME (MPS) glass beads (250 m2) 4.8 1.8
GB250-AA (ANPS) glass beads (250 m2) 5.2 2.1
GB80-ME (MPS) glass beads (80 m2) 1.96 ±0.02 2.27 ±0.03
GB80-MA (MNPS) glass beads (80 m2) 2.13 ±0.09 2.47 ±0.11
GB80-AE (APS) glass beads (80 m2) 1.72 ±0.06 2.24 ±0.08
GB80-AA (ANPS) glass beads (80 m2) 1.97 ±0.09 2.56 ±0.11
GB40-ME (MPS) glass beads (40 m2) 1.3 3.1
GB40-MA (MNPS) glass beads (40 m2) 1.9 4.4
* obtained from elemental analysis, † reference sample with surface-attached octylsilane monolayer
It can be seen that monomer graft densities around 0.9 µmol·m-2 are found for LiChrospher
systems. These values appear to be somewhat lower than that of other trifunctional silanes
on comparable surfaces. However, it was not our premier goal to deposit as many
polymerizable groups as possible, but we were solely interested in finding conditions that
allow for a preparation of these layers in a very reproducible fashion and to avoid extensive
network formation which might cause problems in the polymerization reactions.
Appropriate monomer silane units are immobilized on the other substrates as well. The
standard MPS was used for all substrates, LiChrospher, Aerosil & different glass beads. In
special cases of interest also MNPS, APS and ANPS were attached to the surface silanol
30 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
groups. For MPS the carbon amount after immobilization is proportional to the specific
surface of the substrates with pore sizes between 5 and 50 nm (AR300-ME, GB250-ME, GB80-
ME) and a resulting monomer graft density of around 2 µmol·m-2 (Table 3-1). LC700-ME
possess a lower graft density (Γ = 0.9 µmol·m-2) probably due to inaccessibility of its smallest
pores. GB40-ME shows a higher graft density (Γ = 3.1 µmol·m-2) because the larger sized
100 nm pores allows larger monomer silane clusters bind to the surface. Different monomers,
which have the same reactivity towards silica surfaces because of the same head group
architecture build monomer layers of similar graft densities on the same glass beads (GB80-
ME, GB80-AE & GB80-AA).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 31
3.2 Homopolymers on Silica Different polymers have been attached to the surface during formation and incorporation
of the surface-attached monomers. Regardless of their polarity all polymers formed a well
defined layer on the silica surface (Figure 3-2).
Figure 3-2. The pathways from a silane modified silica substrate to different immobilized polymers
layers are summarized.
32 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
The results of a series of different polymerization reactions are summarized in Table 3-2.
The P(HDFDA) achieved the highest polymer load for the given parameters, but molecular
weight and graft density could not be determined because the perfluoro polymer was not
soluble in any solvent which could be used for GPC. P(MMA) showed also loading values
for mid sized polymer coils above those of comparable PS graft polymerizations. NIPAAm
and MAA with high propagation rate constants formed homopolymers that exceeded the
measuring range of the GPC columns so that the molecular weight was estimated to be
higher than the highest calibration standard for the SUPREMA column (> 1,300,000 g·mol-1).
Probably these large polymer coils clog easily the micropores of the LiChrospher. Therefore
the difference in polymer loading between silica gel with high specific surface area and glass
beads with medium specific surface area is less pronounced and meets at a level of around
0.4 g polymer per gram silica. PS as standard system was well described in recent
publications and its loading is higher and the graft density is lower for the LiChrospher case
in comparison to the glass beads. The polymer load with P(DMAA) was for both substrates
quite low because the chosen monomer concentration was low to avoid gel formation and
lead to low molecular weight polymer (~18,000 g·mol-1). Higher monomer concentrations
caused gelation in early stages of the polymerization. It was shown that the polymer load can
be driven to much higher values in a controlled manner despite to a gel effect occurring at
high conversion[178]. In general the high surface LiChrospher silica allowed a higher polymer
load compared to the mesoporous glass beads whereas the glass beads show in general
higher graft densities.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 33
Table 3-2. Polymer loads and graft densities from polymerization reactions of different monomers on
different silica substrates.
product mʹpoly per g2SiO
*
(g)
nM #
(g·mol-1)
Γ0
(µmol·m-2)
LC700-AE-P(HDFDA) 1.04 - -
GB80-AA-P(HDFDA) 0.59 - -
LC700-ME-PS 0.46 36,000 0.018
GB80-ME-PS 0.14 32,500 0.054
LC700-ME-P(MMA) 0.78 76,500 0.015
GB80-ME-P(MMA) 0.36 80,000 0.056
LC700-ME-P(NIPAAm) 0.40 > 1,300,000‡
GB80-ME-P(NIPAAm) 0.32 > 1,300,000‡
LC700-ME-P(DMAA) 0.12 18,000 0.009
GB80-AE-P(DMAA) 0.09 18,000 0.061
LC700-ME-P(MAA) 0.46 > 1,300,000‡
GB80-ME-P(MAA) 0.42 > 1,300,000‡
* determined from elemental analysis, # determined from GPC analysis of the free polymer, ‡ GPC column exclusion limit reached
The various surface-attached homopolymer monolayers were first characterized by DRIFT
spectroscopy. P(HDFDA) showed adsorption bands of C=O stretching vibrations at 1738 cm-1
and C-F stretching vibrations at 1193 cm-1 although the latter band overlapped with the
broad Si-O-Si scaffold vibrations (1200-1000 cm-1). Bands due to aromatic C-H stretching
vibrations (3100-3000 cm-1, several bands ) were observed in case of the PS covered solids. In
the P(MMA) case there was a typical signal for C=O stretching vibration at 1730 cm-1.
P(NIPAAm) and P(DMAA) showed both C=O stretching of the amide group (amide I) which
lied for the monosubstituted monomer P(NIPAAm) at 1650 cm-1; a few wavenumbers higher
than the disubstituted case P(DMAA) at 1641 cm-1. For P(NIPAAm) as a monosubstituted
amide there was an additional band due to a N-H stretching vibration (amide II) at 1530 cm-1.
34 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
A characteristic C=O stretching vibration band at 1710 cm-1 was observed for P(MMA) on the
solids.
For those homopolymer carrying substrates that were measured with x-ray photoelectron
spectroscopy (cf. Figure 3-3) the measured elemental composition of the surface-attached
polymer monolayer corresponds to values one expects from elemental analysis of free
polymer and silica combined (cf. Table 3-3). For the P(DMAA) monolayer the silicon signals
are quite intense and are reduced for the polystyrene layer on silica compared to the
P(DMAA) layer. Because in XPS experiments the depth from that photoelectrons can escape
from the material through the surface to be detected and analyzed is limited to a few
nanometers. Due to this fact stronger silicon signals indicate a thinner polymer layer. We
conclude that for the used polymerization conditions the P(DMAA) layer is thinner than the
PS layer. In case of the perfluoro polymer both silicon signals have almost vanished and we
got the here the thickest layer what is in agreement with the results of elemental analysis.
1000 800 600 400 200 00
50
100
150
200
c)
b)
a)
O KLLF KLL
F 2s
F 1s
Si 2pSi 2s
C 1s
N 1s
O 1s
coun
ts (1
000/
s) (a
.u.)
binding energy (eV)
Figure 3-3. X-ray photoelectron spectra show the surface composition of glass beads with different
homopolymer monolayers: (a) polystyrene, (b) poly(heptadecafluordecyl acrylate) and (c)
poly(dimethyl acrylamide).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 35
Table 3-3. Ratios of different elements on the surface of silica particles modified by various polymers.
product C*
(%)
N*
(%)
F*
(%)
O*
(%)
Si*
(%)
GB80-AA-P(HDFDA) 33.0 - 52.2 - 4.7
GB80-AA-PS 45.9 - - 35.6 18.5
GB80-AE-P(DMAA) 31.9 5.0 - 43.3 19.9
LC700-ME-PS 58.1 - - 27.0 14.9
* obtained from XPS and standardized
3.3 Mechanism of the Grafting of Polystyrene onto Silica
Surfaces through Surface-attached Monomers As substrate for the following detailed investigations the highly porous LiChrospher Si60
(LC700) was used because its large specific surface grants that significant amounts of
polymer bind to the silane monomers which had been attached in the first step. The behavior
of other substrates, especially various glass beads, upon monomer immobilization and
subsequent polymerization is described in 3.7.
3.3.1 Structure Variation of the Immobilized Monomer
To study the polymerization behavior of various surface-attached acrylate and
methacrylate monomers, styrene was used as the “free” monomer and polymerizations were
triggered using azobis(isobutyronitrile) (AIBN) as an initiator. The following conditions were
employed: cstyrene = 2.9 mol·L-1, cAIBN = 9 mmol·L-1, csilica = 10 g·L-1, temperature T = 60°C. The
polymerization time was set to 24 h. In two separate reference experiments unmodified silica
gel and silica gel treated with an alkyl silane (octyl trimethoxysilane) were used as substrates
for the polymerization mixture.
All gels were extensively washed after polymerization to remove any adsorbed, “free”
polymer from the surfaces (for details see 9.1.1 and 9.3). The so obtained solids were
characterized by FTIR spectroscopy (DRIFT) and elemental analysis. Figure 3-1 shows a
spectrum obtained from an MPS-SiO2 sample after it was exposed to a polymerization
reaction. The attachment of polystyrene is proven by the presence of adsorption bands
36 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
typical for polystyrene, especially in the region just above 3000 cm-1 which is indicative for
the presence of aromatic C,H groups in the sample.
The amount of polymer attached to the surfaces was determined by elemental analysis. For
the two reference samples which contained no immobilized monomer at the surface only
very small carbon contents of 0.5 – 3% (Γ0 < 1 nmol) were determined. Such values are often
found even on the surfaces of untreated silica gels due to airborne carbon containing
contaminants and are therefore not indicative for any attachment of polymers. For all other
silanes much higher carbon values of 28 – 33% (Γ0 = 15 - 18 nmol·m-2) were found except for
SiO2-MNPS where much lower polymer loadings (i.e. only one forth or one fifth of those of
the other monomers) were observed. All values are summarized in Table 3-4 together with
the molecular weights of the “free” polymers recovered from the respective polymerization
mixture.
Table 3-4. Carbon contents and graft densities of surfaces prepared from different immobilized
monomers and surface-attached polystyrene monolayers on LiChrospher Si60.
carbon*
(%)
nM #
(g·mol-1)
Γ0
(µmol·m-2)
substrate
monomer polystyrene monomer polystyrene
LC700-ME (MPS) 8.1 32.9 36,000 1.3 0.018
LC700-ME (MNPS) 8.2 15.0 36,000 1.2 0.004
LC700-ME (APS) 6.0 28.1 33,000 1.0 0.018
LC700-ME (ANPS) 6.5 31.5 36,000 1.0 0.017
LC700-Si-octyl† (OS) 6.5 7.0 37,000 0.9 <0.001
LC700 (unmodified) - 3.0 40,000 - 0.001
* obtained from elemental analysis, # determined from GPC analysis of the free polymer, † reference sample with surface-attached octylsilane monolayer
In order to obtain values for the graft density of the polystyrene on the various surfaces we
assumed that the molecular weights of the free and the attached polymers are similar. This
assumption seems to be reasonable because the molecular weight of a polymer prepared by
free radical polymerization is determined by the ratio of the rate constant of propagation to
that of the termination reaction. It is known from the corresponding kinetics of solution
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 37
polymerization that a significant change of the termination reaction only occurs at
conversions of 50% or above. Such segment densities, however, do not occur in the grafted
layers.
As we compare the graft densities of the polymers on the silica gels to the graft density of
the polymerizable groups we find that only about 3-5% of all surface-bound vinyl groups
were actually “caught” by a growing chain and thereby incorporated into the surface-
attached polymer, if we assume that each chain gets only attached by the incorporation of
one monomer anchored to the surface.
3.3.2 Variation of the Polymerization Time
In order to further elucidate this process for the modification of silica particles with
polymers and to explore the limits of the method in terms of available graft densities or
grafted amounts we performed a series of polymerizations in which all conditions were kept
constant and only the polymerization time was varied from 2 –72 h. The grafted amount and
the graft density of the resulting materials were derived from elemental analysis data and
from the molecular weights of the “free” polymers in the same way as described above
(Figure 3-4). The data depicted as a triangle in Figure 3-4 Figure 3-5 and Figure 3-6 were
obtained from a sample for which the polymerization mixture was renewed after a initial
polymerization time of 48 h. Details are explained in the text.
38 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
0 10 20 30 40 50 60 7005
101520253035404550
reaction time (h)
Mn (
103 g
/mol
)
Figure 3-4 Influence of the polymerization time on the number average of the molecular weight
distribution as obtained by GPC analysis of the free polymers
It can be seen that the molecular weight of the polymers stays constant for the entire range
of polymerization times as it is expected for a typical free radical polymerization.
Furthermore, the value of 36,000 g·mol-1 agrees well with the expected molecular weight
derived from the Mayo equation (equation 3-1) for the given set of polymerization
parameters[179, 180].
[ ][ ] 0
SMn X
1MSCC
X1
+⋅+= equation 3-1
With this equation the molecular weight can be calculated from the degree of
polymerization nX according to equation 1-17. The constant CM (= 1,67·10-4) is the quotient of
the propagation rate constant kp divided by the transfer rate constant km for a transfer
reaction from a growing chain to a monomer. The constant CS (= 8,3·10-4) is the quotient of kp
as dividend and ks the transfer rate constant for a transfer from a growing chain to a solvent
molecule as divisor. For the given polymerization mixture the styrene concentration [M] is
2.9 mol·L-1 and the toluene concentration [S] is 6.3 mol·L-1.The degree of polymerization in
absence of a solvent 0X (= 1920) we get from the experiments carried out by Bialk et al.[153].
The used initiator concentrations are same in the compared cases. All values put into
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 39
equation 3-1 give a calculated number average of the molecular weight of 41,200 g·mol-1.
More, in this simplified equation unconsidered, transfer reactions may reduce the molecular
weight further. Additionally the GPC measurement method implies an error of up to 10%.
Taking these two factors into account the theoretical value is close to the practical one.
0 10 20 30 40 50 60 700.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
graf
ted
poly
mer
(g P
S / g
SiO
x)
reaction time (h)
Figure 3-5. Influence of the polymerization time on the grafted amount of polystyrene onto a silica
surface.
The grafted amount in terms of g PS per g SiOx is initially a function of the polymerization
time but after about 25-30 h of polymerization no further polymer chains (or only minor
amounts) become attached to the particles. Accordingly, also the graft density initially
increases as the polymerization proceeds but then levels off after about 25-30 h at around
0.5 g PS·g-1 SiOx. This corresponds to an theoretical average layer thickness of 0.7 nm when
the total surface of the LiChrospher material is covered equally. If the molecular weight of the
polymer is taken into account that is a graft density of 20 nmol·m-2. It should be noted, that
this behavior is not caused by exhaustive conversion of initiator or monomer. This is
demonstrated in a control reaction in which styrene was firstly polymerized in the presence
of SiO2-MPS for 48 h, then the silica gel was recovered, washed and subjected again to a
polymerization solution for another 24 h corresponding to a total polymerization time of
again 72 h. The total grafted amount of 0.52 g PS / g SiO2 determined from this sample is very
similar to the value found for the sample which was directly polymerized for 72 h (cf. Figure
40 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
3-5 and Figure 3-6). The error of elemental analysis measurements (~1%) is too small to be
displayed as error bars in the diagrams that show grafted polymer amounts.
0 10 20 30 40 50 60 700.000
0.005
0.010
0.015
0.020
0.025
0.030
graf
t den
sity
(µm
ol/m
²)
reaction time (h)
Figure 3-6. Influence of the polymerization time on the graft density of the polystyrene chains on the
silica.
3.3.3 Influence of the Silica Gel Concentration
In another set of experiments we explored the practical limits of the described approach
and varied the overall amount of modified silica for a given set of polymerization conditions
(styrene/toluene 1/2 v/v, 15 mL, cAIBN = 9 mmol·L-1, T = 60°C, t = 24 h). The amount of
modified silica (SiO2-MPS) was varied from 0.15 g to 4.8 g, i.e. by a factor of 32. The highest
silica concentration used in this set of polymerizations poses an upper practical limit as the
solids are barely wetted by the monomer solution. Again, the resulting materials were
characterized in terms of the grafted amount, the molecular weight of the polymers and the
graft density as described above and the results are plotted as a function of the concentration
of the solid in Figure 3-7 and Figure 3-8. This plot demonstrates that the amount of solid
does not influence the overall procedure and graft densities of 19±1 nmol/m² were obtained
for all cases. However, it was found that the amount of “free” polymer which is formed
during the reaction diminishes if higher amounts of solid are added to the mixture.
Comparing the amount of isolated “free” PS from the 0.15 g and the 4.8 g sample the weight
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 41
is halved from 1.3 g to 0.6 g for 15 mL of polymerization solution (10 mL toluene, 5 mL
styrene) after a conversion of approximately 30 – 40%.
0 50 100 150 200 250 300 3500.0
0.1
0.2
0.3
0.4
0.5
0.6
graf
ted
poly
mer
(g P
S / g
SiO
x)
silica gel concentration (g/L)
Figure 3-7. Grafted amount of polystyrene as determined by elemental analysis for varied
concentrations of the monomer modified silica gel inside a polymerization dispersion.
0 50 100 150 200 250 300 3500.000
0.005
0.010
0.015
0.020
0.025
0.030
graf
t den
sity
(µm
ol/m
²)
silica gel concentration (g/L)
Figure 3-8. Graft density of polystyrene for varied concentrations of the monomer modified silica gel
inside a polymerization dispersion.
42 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
3.3.4 Influence of Monomer Concentration in Solution
The monomer concentration is an important parameter for free radical polymerizations
and how it effects the built-up of the polymer monolayer onto silica is explored in this set of
experiments. A change of the monomer concentration in solution by nearly a factor of ten
causes countable differences in polystyrene load. The higher the styrene concentration the
higher the final polymer load on LiChrospher. For polymerization in pure styrene (8.7 mol·L-1)
the grafted polymer achieved 0.49 g PS per gram silica gel as maximum. Diluting the styrene
with toluene down to 0.97 mol·L-1 (v/v 1/8) the polystyrene content drops down to 0.15 g PS
per gram of silica (Figure 3-9). The factor of dilution (x8) is higher than the decrease in
polymer load (x3).
0 1 2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5(1/0)
(1/1)(1/2)
(1/4)
(1/8)
graf
ted
poly
mer
(g P
S / g
SiO
x)
c(M) (mol/L)
Figure 3-9. Influence of the monomer concentration on the amount of grafted polystyrene as
determined by elemental analysis; values in brackets: ratio styrene/toluene (v/v)
The average molecular weight for the formed free polystyrene increases proportionally
(Figure 3-10). For a styrene concentration of 0.97 mol·L-1 the number average of the molecular
weight is 21000 g·mol-1. Up to a styrene concentration of 4.35 mol·L-1 and a number average
of the molecular weight of 114000 g·mol-1 the graft density drops linearly from 10 down to
4 nmol·m-2 (Figure 3-11).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 43
0 1 2 3 4 5 6 7 8 90
100
200
300
400 (1/0)
(1/1)
(1/2)(1/4)
(1/8)
Mn (
103 g
/mol
)
c(M) (mol/L)
Figure 3-10. Correlation between molecular weight of the free polymer and monomer concentration in
solution; higher concentration leads to higher molecular weights.
0 1 2 3 4 5 6 7 8 90.000
0.002
0.004
0.006
0.008
0.010
0.012
(1/0)
(1/1)
(1/2)
(1/4)
(1/8)
graf
t den
sity
(µm
ol/m
²)
c(M) (mol/L)
Figure 3-11. Graft density as a function of monomer concentration calculated from elemental analysis
and GPC results.
The molecular weight of the polystyrene created during the polymerization in pure
styrene does not follow this trend. In this case the molecular weight is higher than expected.
During polymerization this mixture got highly viscous. Caging effects of growing radical
chains due to reduced mobility of the polymer coils lead to higher molecular weights. This
44 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
effect occurs during polymerization at higher conversion and is known as Trommsdorf effect
[178]. So it is due to the long polymerization time of 48 hours for this sample. The molecular
weight was in this case 400000 g·mol-1 and a factor of 20 higher than for the lowest monomer
concentration. Therefore the graft density leaves the linear trend and is settled at 2 nmol·m-2.
3.3.5 Influence of Initiator Concentration
Molecular weight of radical polymerization reactions can be easily controlled by variation
of the initiator concentration. To elucidate how this parameter influences the polymer
monolayer while the monomer concentration is kept constant, the initiator concentrations
were varied in a range from 10-4 to 10-1 mol·g-1.
1E-4 1E-3 0.01 0.10.0
0.1
0.2
0.3
0.4
0.5
graf
ted
poly
mer
(g P
S / g
SiO
x)
c(I) (mol/L)
Figure 3-12. Influence of initiator concentration on the amount of deposited polystyrene as determined
by elemental analysis.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 45
0 20 40 60 80 1000
50
100
150
200
250
Mn (
103 (g
/mol
))
(c(I))-1/2 (L1/2/mol1/2)
Figure 3-13. The molecular weight as a function of the reciprocal square root of the initiator
concentration according to the dependence in equation 1-18.
1E-4 1E-3 0.01 0.10.00
0.02
0.04
0.06
0.08
0.10
0.12
graf
t den
sity
(µm
ol/m
²)
c(I) (mol/L)
Figure 3-14. Influence of the initiator concentration on the graft density as calculated from grafted
amount and average molecular weight.
With increasing initiator concentration the polystyrene load increases from 0.02 to 0.45 g
per gram silica (Figure 3-12). The molecular weight decreases from 225000 down to
6000 g·mol-1. As shown in Figure 3-13 the molecular weight increases linearly with the
reciprocal square root of the initiator concentration. For initiator concentrations above
46 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
1 mmol·L-1 the dependence of the molecular weight from the initiator concentration derived
from equation 1-18 is valid. Both effects combined result in a strongly raised graft density
which increases by a factor of 1000 from 0.1 nmol·m-2 to 100 nmol·m-2 (Figure 3-14).
3.3.6 Variation of the Polymerization Temperature
In several experiments the reaction temperature for the polymerization was varied in a
range from 60 to 90 °C. Two sets of polymerizations were performed: In the first set of
experiments polymerization reactions were carried out using polymerization times that
correspond to one half-life time of the initiator for each temperature. In the second set of
experiments the polymerization time was set to 21.5 h for each run regardless of the
temperature chosen (cf. Table 9-6).
330 340 350 360 370
0.1
0.2
0.3
0.4
0.5
21.50 h
16.05 h10.85 h
4.00 h
1.05 h
0.4 h
polymerization times
graf
ted
poly
mer
(g P
S / g
SiO
x)
T (K)
Figure 3-15. Temperature dependence of the grafted amount of polystyrene to a silica surface. All
samples were polymerized up to an initiator conversion of 50%. The polymerization time for each
sample is given next to the respective data point.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 47
330 340 350 36005
101520253035
Mn (
103 g
/mol
)
T (K)
Figure 3-16. Temperature dependence of the number averaged molecular weight of the free polystyrene
during polymerization of a monomer modified silica (LC700-ME), initiator conversion 50%.
330 340 350 3600.000
0.005
0.010
0.015
0.020
graf
t den
sity
(µm
ol/m
²)
T (K)
Figure 3-17. Temperature dependence of the graft density of polystyrene on a silica surface after an
initiator conversion of 50%.
The results obtained from the polymerizations in which the reaction times were set to one
half-life time of initiator decomposition are shown in Figure 3-15 to Figure 3-17. Both, the
48 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
grafted amounts and the molecular weight decrease with increasing temperature. The
dependence of the molecular weight of the obtained polymer on the reaction temperature
shows a behavior as it is expected on the basis of the standard description of the mechanism
of free radical polymerizations that predicts a decrease of the molecular weight with
increasing temperature. As we calculate the graft density of the resulting polymer
monolayers we find that this value does not depend on the polymerization temperature but
stays constant for the full range of temperatures under investigation. This result
demonstrates that the decrease of the grafted amount with increasing temperature is solely
caused by the decreasing molecular weight for this set of polymerizations.
A different picture was obtained from the experiments in which the polymerization time
was set to 21.5 h for all temperatures. The results of these experiments are shown in Figure
3-18 to Figure 3-20.
330 340 350 3600.0
0.1
0.2
0.3
0.4
0.5
0.6
graf
ted
poly
mer
(g P
S / g
SiO
x)
T (K)
Figure 3-18. Grafted amount of polystyrene obtained from polymerizations at different temperatures
for a constant polymerization time of 21.5 h.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 49
330 340 350 3600
10
20
30
40
50
60 Mn Mw
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
T (K)
Figure 3-19. Polymerization at different temperatures for a constant polymerization time of 21.5 h
gives for polystyrene the molecular weights as shown in Figure 3-18.
330 340 350 3600.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
graf
t den
sity
(µm
ol/m
²)
T (K)
Figure 3-20. Polystyrene graft densities from polymerizations where the polymerization temperature
was varied and the polymerization time was kept constant at 21.5 h.
Both the grafted amount and the graft density firstly decrease as the samples are prepared
at higher temperatures but the minimum is reached for a polymerization temperature of
65°C after which higher amounts of polymer can be grafted to the surfaces if higher
polymerization temperatures are employed. Also, a different trend for the molecular weight
50 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
as a function of temperature is observed. Initially, Mn decreases with increasing
polymerization temperature as it was observed for the first set of experiments. At moderate
temperatures up to 65°C the graft process and the polymer formation follow the expected
kinetics and the molecular weight distribution is similar to that of the experiment with
constant initiator conversion. Also the graft density for the lower temperature range can be
considered constant within the error margins. However, for temperatures of 70°C and higher
the value for Mn settles at a certain value while Mw increases sharply with increasing
polymerization temperature. At higher polymerization temperatures the rapid consumption
of initiator causes a significant drop of its concentration and formation of longer polymer
chains occurs at later polymerization stages. Therefore an increase for the polydispersity
index from initially 1.7 to values more than 2 is observed which is largely due to a more and
more bimodal distribution of the molecular weight distribution as evidenced in the GPC
traces. This is probably caused by the change of polymerization conditions at high monomer
conversions with low initiator concentrations. For higher polymerization temperatures the
polymer chains become shorter and the polymer formation is faster. So the yield of surface-
attached polymer coils becomes better again with higher polymerization temperatures when
polymerization time is kept constant.
3.3.7 Surface Concentration of Polymerizable Silanes & its
Influence on Building Polymer Monolayers
3.3.7.1 Co-Immobilization of MPS & OS
The initial step for attaching polymer monolayers to surfaces through the strategy
described here is the immobilization of a monomer unit on the silica. To get a better
understanding how the grafting process is influenced by the density of monomer molecules
on the surface, the concentration of the monomer MPS is varied by “diluting” the
immobilization mixture with octyl silane (Figure 3-21). Although it has a very similar
reactivity towards silica surfaces the latter trialkoxy silane cannot be incorporated into a
polymer.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 51
Figure 3-21. Co-immobilization of MPS and OS (a) to control double bond density on the silica gel
surface (b) and subsequent polymerization (c).
The overall concentration of the two silanes was the same for all immobilization solutions.
Only the ratio between MPS (monomer unit) and OS (aliphatic unit) was varied. Elemental
analysis of the dried LiChrospher samples after the immobilization process shows similar
levels of surface coverage with the two silanes, independent of the starting composition. The
samples had between 0.65 and 0.75 mmol silanes per gram of silica gel. The overall graft
density for all samples was around 1.0 µmol·m-2 ( Figure 3-22). Only for the immobilization
of pure OS a slightly lesser density of 0.8 µmol·m-2 is observed.
0.0 0.2 0.4 0.6 0.8 1.00.0
0.2
0.4
0.6
0.8
1.0
1.2
graf
t den
sity
(µm
ol/m
²)
molar part MPS on surface
Figure 3-22. Comparison of overall graft densities (MPS+OS) from the silane co-immobilization with
the effective contingent of the polymerizable MPS after analysis and calculation of elemental analysis
and FT-IR results. The circles are overall and the triangles particular MPS graft densities.
52 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
The infrared spectra in Figure 3-23 show clearly a change in transmittance bands with
changing surface composition of the two silanes, which are the result of the different
compositions of the immobilization solutions. The difference in the intensity of the C=O-
vibration band is huge, whereas the band of the C-H2-vibration reveals less but significant
changes. The intensity of carbonyl vibration bands increases with the MPS content on the
silica gel surface and the intensity increase of methylene vibration bands proves a higher
amount of OS on the surface.
4000 3000 2000 1000
100% OS
5% MPS, 95% OS
20% MPS, 80% OS
50% MPS, 50% OS
80% MPS, 20% OS
100% MPS
tran
smitt
ance
(a.u
.)
wavenumber (cm-1)
Figure 3-23. DRIFT spectra of LiChrospher with different mixtures of immobilized MPS and OS on
the surface
Of these two characteristic IR-bands the extinction ratio was calculated as the quotient of
the appropriate integrals ( Figure 3-24). The calibration curve results as the same calculation
was done for the liquid silane mixtures consisting of known volumes of MPS and OS
( Figure 3-24).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 53
0.0 0.2 0.4 0.6 0.8 1.005
101520253035
extin
ctio
n ra
tio (
ν(C
H2)
/ ν(C
=O) )
partial volume MPS0.0 0.2 0.4 0.6 0.8 1.005
101520253035
Figure 3-24. A quantitative IR-analysis of the surface-attached silane-mixture compared to the
calibration curve calculated by measured extinction coefficients with exponential fit functions
A comparison of the DRIFT results of the modified LiChrospher with the calibration curve
shows that MPS is at higher concentration at the surface than what to expect from the ratio in
the initial immobilization solutions. This shift is depicted in Figure 3-25 as a co-
immobilization diagram. It shows that more MPS than OS is attached to the surface as it can
be derived from the silane ratios at the start. Now we can compare the overall surface
densities ( ) in Figure 3-22 with the effective surface density of MPS only ( ). Although the
molar amount of MPS in the immobilization solutions was varied by a factor of 20 the
effective variation of the surface density is within a factor of 10.
54 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
0.0 0.2 0.4 0.6 0.8 1.00.0
0.2
0.4
0.6
0.8
1.0M
PS o
n su
rfac
e (m
olar
par
t)
MPS in mixture (molar part)
1.0 0.8 0.6 0.4 0.2 0.0
1.0
0.8
0.6
0.4
0.2
0.0
OS
on s
urfa
ce (m
olar
par
t)
OS in mixture (molar part)
Figure 3-25. Co-immobilization diagram of MPS vs. OS
3.3.7.2 Influence of Monomer Surface Density on PS Monolayer
Formation
The polystyrene load after polymerization shows no significant variation when the MPS
surface density on the silica gel surface changes by more than one magnitude of 10 from
1.07 µmol·m-2 down to 0.08 µmol·m-2 (Figure 3-26). The calculated polystyrene graft densities
settle constantly around 0.02 µmol·m-2 (Figure 3-27).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 55
0.0 0.2 0.4 0.6 0.8 1.0 1.20.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
graft density MPS (µmol/m²)
graf
ted
poly
mer
(g P
S / g
SiO
x)
Figure 3-26. Relation between amount of polystyrene and graft density of MPS; only at very low
MPS densities the polymer gets a bit less
0.0 0.2 0.4 0.6 0.8 1.0 1.20.000
0.005
0.010
0.015
0.020
0.025
graf
t den
sity
(µm
ol/m
²)
graft density MPS (µmol/m²)
Figure 3-27. Graft densities dependence between the immobilized monomer and polystyrene
56 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
3.3.8 Polymerization in Two Steps
For this set of experiments polystyrene grafted samples come into play. The polystyrene
attached to these silica was grafted with our herein described technique. The carbon content
gets higher while the length of the immobilized polymer chains gets smaller (Figure 3-28 and
Figure 3-13). These different substrates were placed under same polymerization conditions
for short polymer chains into a second polymerization batch for 24 h (Table 3-5). In order to
get relatively short polystyrene chains a high amount of AIBN (c = 50 mmol·L-1) and a
medium monomer concentration (c = 2.9 mol·L-1) was used. It turns out that after final
elemental analysis in all cases the amount of polystyrene is almost the same and lies between
0.50 and 0.55 g polystyrene per gram LiChrospher (Figure 3-28).
Table 3-5. Molecular weights and graft densities of the two-step experiments
sample nM PS (1st)
(g/mol)
nM PS (2nd)
(g/mol)
ΓPS (1st)
(nmol/m²)
ΓPS (2nd)
(nmol/m²)
PS1 230000 13000 < 1 51
PS2 100000 13500 2 38
PS3 60000 13000 7 29
PS4 32000 14000 17 15
PS5 6500 13500 98 9
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 57
PS1 PS2 PS3 PS4 PS50.0
0.1
0.2
0.3
0.4
0.5
0.6
previous amount new amount
graf
ted
poly
mer
(g P
S / g
SiO
x)
sample
Figure 3-28. Grafting of short chains ends up with almost the same amount of polymer regardless of
the starting amount of polymer
3.4 Grafting onto Various Porous Substrates Transferring the simple grafting technique to various silica substrates with varying specific
surfaces and surface topologies is necessary to demonstrate the versatility of the system
towards applications.
3.4.1 Influence of Substrates and Surface-attached Monomer
Silanes
Several radical polymerization reactions with styrene as monomer were carried out and
the results of the polymerization under “standard conditions” (or mostly used conditions)
are summarized in Table 3-6. There is a trend to higher polymer loading with higher specific
surface. It is shown by the graft densities in Table 3-6 that the amount of surface-bound
polystyrene is not proportional to this surface when considering all silica topologies.
Looking at the different glass beads with similar molecular weight of the PS the polymer
loading is proportional to the surface and graft densities are in the same range. High surface
silica types have a bit more polymer load due to the higher specific surfaces. But only
equipped with smaller pores the graft densities are somewhat smaller than those of the glass
bead types.
58 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
Very obvious is also a difference in copolymerization ability between the different
monomer silanes. Initially all monomer silanes have comparable graft densities after the
immobilization step (Table 3-1). From Table 3-6 one sees that under comparable
polymerization conditions not just specific surface makes the difference but with MNPS the
polymer loading and graft density are significantly smaller. In the first copolymer
experiments MNPS-modified substrates show the same behavior resulting in low polymer
loadings and graft densities. Therefore we discard these products in the further course of this
work. Further details on polymerization on substrates with different topologies are given in
the following paragraphs (3.4.2 to 3.4.4).
Table 3-6. Resulting carbon contents and graft densities after polymerization (cstyrene = 2.9 mol·L-1,
cAIBN = 9 mmol·L-1, T = 60°C, t = 24 h) with different immobilized silanes and according molecular
weights of the “free” polystyrene.
substrate & surface
modification
carbon*
(%)
Γ0
(µmol·m-2)
nM
(g·mol-1)
LC700-ME 32.9 0.018 36,000
LC700-MA 12.0 0.002 43,900
AR300-ME 29.8 0.038 35,300
GB250-ME 28.2 0.050 30,900
GB80-ME 12.8 0.054 32,500
GB80-AE 13.7 0.060 32,700
GB80-AA† 10.7 0.025 53,400
GB40-ME† 8.8 0.031 71,700
GB40-MA† 3.7 0.006 69,100
* obtained from elemental analysis, † cAIBN = 3 mmol
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 59
3.4.2 Influence of the Polymerization Time for Aerosil300
To get a better picture of the influence of the pore topography on the overall process of
grafting polymer monolayers on silica surfaces we used Aerosil300 in a polymerization time
dependence setup and compared it to already won data of LiChrospher. The polystyrene
loading for both silica substrates is very similar in amount and increase with time (Figure
3-29).
0 5 10 15 20 25 300.0
0.1
0.2
0.3
0.4
0.5
graf
ted
poly
mer
(g P
S / g
SiO
x)
polymerization time (h)
Figure 3-29. Comparison between LiChrospher ( ) and Aerosil300 ( ); for same periods of
polymerization time and same molecular weights the amount of grafted polymer is independent from
the topography of the silica gel
The highest polymer load increase is in the first 10 hours. Then the increase levels down to
nearly zero for a polymerization time below 30 hours. The final polymer loadings are around
0.4 g PS per gram silica.
60 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
0 5 10 15 20 25 300.00
0.01
0.02
0.03
0.04
0.05
graf
t den
sity
(µm
ol/m
²)
polymerization time (h)
Figure 3-30. The equal polymer amount results in graft density shift because of the specific surface:
LiChrospher = 700 m²/g ( ) & Aerosil300 = 300 m²/g ( )
The specific surface of Aerosil300 is lower than that of LiChrospher what means on same
polymer loading the final graft density of 38 nmol·m-2 is higher by a factor of two (Figure
3-30). Obviously the bigger concave pore structure of the Aerosil agglomerates, built from
primary particles, is more likely to build in polystyrene than the compact LiChrospher spheres
where the significant inside part of the surface area is only accessible through micropores.
Looking on how the Aerosil silica gel responds on two parallel changes of polymerization
parameters gives an interesting picture. On the one hand less initiator in solution leads to
longer polymer chains and the final polymer amount is reduced. On the other hand more
monomer in solution creates larger polymer coils and increases the polymer load analog to
the LiChrospher experiments. Both parameter changes combined then result in the same
grafted amount of polystyrene (Figure 3-31).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 61
0 5 10 15 20 25 300.0
0.1
0.2
0.3
0.4
0.5
graf
ted
poly
mer
(g P
S / g
SiO
x)
polymerization time (h)
Figure 3-31. Polystyrene deposition with time on Aerosil300 with two different molecular weights
( : 35000 g/mol, : 95000 g/mol) results in same amounts for each molecular weight
Because the molecular weight is driven to higher values by both parameter changes a
decrease of graft density is the result (Figure 3-32).
0 5 10 15 20 25 300.00
0.01
0.02
0.03
0.04
0.05
graf
t den
sity
(µm
ol/m
²)
polymerization time (h)
Figure 3-32. The graft densities of polystyrene differ due to two different molecular weights
( : 35000 g/mol, : 95000 g/mol) on Aerosil300
62 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
3.4.3 Influence of the Polymerization Time for Differently
Modified Glass Beads
The main aspect of the polymerization time experiments on glass beads with differently
modified surfaces is to find out if the mesoporous glass beads show a different behavior
from the already used silica gel systems. Having a look at the polystyrene load (Figure 3-33)
and at the resulting graft densities (Figure 3-34) similarities with the high specific surface
substrates LiChrospher and Aerosil300 are discovered: There is an increase in loading over the
first 24 hours of polymerization up to 0.11 g PS per gram glass beads and a decreasing
deposition rate.
0 5 10 15 20 25
0.02
0.04
0.06
0.08
0.10
0.12
0.14
graf
ted
poly
mer
(g P
S / g
SiO
x)
polymerization time (h)
Figure 3-33. Increase of the polystyrene amounts with polymerization time for glass beads modified
with different silanes (GB80-ME , GB80-AE , GB80-AA ). During elapsing time for every
silane monomer on the GB80 glass beads the mass of grafted polymer is nearly the same.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 63
0 5 10 15 20 250.00
0.02
0.04
0.06
graf
t den
sity
(µm
ol/m
2 )
polymerization time (h)
Figure 3-34. Increase of the polystyrene graft densities with polymerization time for glass beads
modified with different silanes (GB80-ME , GB80-AE , GB80-AA ). Graft densities have a
similar increase with time but differ in the total values because molecular weights are influenced by
polymerization parameters (here initiator concentration) whereas the overall process is not.
The average values for Mn are 56000 g·mol-1 for GB80-AA-PS and 49500 g·mol-1 for GB80-
AE-PS as polymerizations carried out with 3 mM initiator and 26000 g·mol-1 for GB80-ME-PS
as polymerization executed with 9 mM initiator. The molecular weight stays at the same
level during the whole polymerization time, so the development of the graft densities with
time is similar for all polymerization sets and depends only on the chosen initiator
concentration. A closer look on the influence of AIBN concentration is depicted in the
following paragraph.
3.4.4 Influence of Initiator Concentration for Different Substrates The strong influence of the initiator concentration on the molecular weight of “free”
polymer is for all substrates similar (Figure 3-36) because the polymerization takes mainly
place in solution. Because the molecular weight values vary over a larger scale than the
specific surface areas of the substrates the calculation of the graft densities are mainly
influenced by them. So the graft densities exhibit an indifferent behavior regarding the
different substrates with the main trend of getting higher graft densities with more initiator
in polymerization solution (Figure 3-37).
64 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
1E-4 1E-3 0.01 0.10.0
0.1
0.2
0.3
0.4
0.5
c(I) (mol/L)
graf
ted
poly
mer
(g P
S / g
SiO
x)
Figure 3-35. Polymer loading for different silica solids at varied initiator concentrations
(LC700-ME , AR300-ME , GB250-ME , GB80-ME , GB80-AE ).
1E-4 1E-3 0.01 0.10
50
100
150
200
250
300
Mn (
103 g
/mol
)
c(I) (mol/L)
Figure 3-36. Molecular weights for different silica solids at varied initiator concentration
(LC700-ME , AR300-ME , GB250-ME , GB80-ME , GB80-AE ).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 65
1E-4 1E-3 0.01 0.10.00
0.05
0.10
0.15
0.20
0.25
graf
t den
sity
(µm
ol/m
2 )
c(I) (mol/L)
Figure 3-37 Graft densities for different silica solids at varied initiator concentration (LC700-ME ,
AR300-ME , GB250-ME , GB80-ME , GB80-AE ). The development with increasing
initiator concentration of the molecular weight as major factor and the graft densities is similar for all
different silica solids. A significant influence of the substrate topography on the result is recognized
for the final polystyrene loading.
Interesting is a detailed picture of the polymer load on the different substrates for different
initiator concentrations as the volume ratio of styrene/toluene is 1/2 (Figure 3-35). Reverence
is the LC700-ME-PS system where the polymer loading varies strongly with initiator
concentration/molecular weight between 0.02 g and 0.45 g polystyrene per gram substrate.
The value for the AR300-ME silica gel generated under same monomer concentration is
equal to the corresponding LiChrospher polystyrene loading although the specific surface is
more than twice lower. The GB250-ME material with less specific surface than the AR300-ME
achieves under same conditions even a higher polymer loading than the two silica gel types.
Interestingly the increase of polystyrene load between the two chosen initiator
concentrations is much smaller than observed for LiChrospher. GB80-ME and GB80-AE
systems exhibit a relatively small increase in PS load with higher initiator concentration in
the polymerization solution as well. Here the absolute value is smaller due to the less specific
surface but in the case of 0.1 mM AIBN the polystyrene load on LC700-ME drops even below
the value for these glass beads. The mesoporous structure of the glass beads allows polymer
66 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
coils of all sizes grown to access the whole surface. The small micropores of the used silica
gels are more easily clogged by formation of a surface-attached polymer layer than the
mesopores of the glass beads. Therefore the surface areas of the particle core are more
effectively blocked which reduces the overall number of growing radical chains diffusing in
to free surface sites.
3.4.5 Influence of the Monomer Concentration for Glass Beads
Three experiments with varying styrene concentrations in the polymerization solution
with GB80 glass beads complement the picture we already got from the experiments with
LiChrospher Si60. Variation of the PS loading by variation of the styrene concentration in the
polymerization process is also possible when glass beads are concerned. The higher the
initial styrene concentration the more PS will be bound covalently to the glass bead surface.
Interesting is that after polymerization in bulk styrene the PS loading of more than 0.5 g PS
per gram substrate is as high as it is on LiChrospher Si60 the case (Figure 3-38).
0 1 2 3 4 5 6 7 8 90.0
0.1
0.2
0.3
0.4
0.5
0.6(1/0)
(1/1)
(1/2)
graf
ted
poly
mer
(g P
S / g
SiO
x)
c(M) (mol/L)
Figure 3-38. Amount of grafted polystyrene on GB80-ME glass beads after polymerization with
different initial styrene concentrations in toluene; values in brackets: styrene/toluene (v/v).
The number average and the weight average of the molecular weight distribution follow
the expected route: Higher styrene concentrations lead to significantly higher molecular
weights of the polymer (Figure 3-39).
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 67
0 1 2 3 4 5 6 7 8 90
100
200
300
400
500
600
(1/0)
(1/1)
(1/2)
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
c(M) (mol/L)
Mn Mw
Figure 3-39. Averaged molecular weights for PS after polymerization with different monomer
concentrations.
The increase in molecular weight is more pronounced than the increase of the polymer
load with higher initial styrene concentrations in the polymerization solution. Therefore the
graft densities are apparently lower for styrene concentrations above 4 mol·L-1 (Figure 3-40).
We observe a similar tendency in monomer concentration influence for glass beads like we
do for silica gel. Graft density on LiChrospher drops from 6.6 nmol·m-2 down to 1.8 nmol·m-2.
The smaller graft density decrease factor for glass beads (~2) and if compared to that of
LiChrospher (~3) maybe a hint for the theory where smaller pores are more efficiently blocked
by the surface-attached polymer layer than mesopores.
68 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
0 1 2 3 4 5 6 7 8 90.00
0.01
0.02
0.03
0.04
0.05
0.06
(1/0)(1/1)
(1/2)
graf
t den
sity
(µm
ol/m
2 )
c(M) (mol/L)
Figure 3-40. Graft densities of PS on glass beads (GB80-ME) after polymerization with varied
monomer concentrations.
3.4.6 Influence of the Glass Bead Concentration
When the practical limits of a technique are explored it is important to see if it is applicable
where a maximum output is needed. The overall amount of modified glass beads for a given
set of polymerization conditions (styrene/toluene 1/2 v/v, 12 mL, cAIBN = 3 mmol·L-1, T = 60°C,
t = 24 h) was varied within a region of 0.6 g to 4.8 g, i.e. by a factor of 8. For the highest solid
concentration used in this set the glass beads are barely wetted by the polymerization
solution and therefore a practical limit for a batch reaction is reached. Characterization of the
resulting solids (GB80-ME-PS) in terms of the grafted polystyrene load and graft densities
leads to the plotted graphs shown in Figure 3-41 and Figure 3-42.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 69
0 100 200 300 400 5000.000.020.040.060.080.100.120.140.16
glass bead concentration (g/L)
graf
ted
poly
mer
(g P
S / g
SiO
x)
Figure 3-41. Variation of the concentration of the modified glass beads (GB80-ME) during a grafting
polymerization reaction does not effect the grafted amount as determined by elemental analysis.
0 100 200 300 400 500
0.00
0.01
0.02
0.03
0.04
0.05
glass bead concentration (g/L)
graf
t den
sity
(µm
ol/m
2 )
Figure 3-42. Variation of the concentration of the modified glass beads (GB80-ME) during a grafting
polymerization reaction does not effect the graft densities.
The amount of glass beads does not influence the deposition of polystyrene and graft
densities of 12±1 nmol·m-2 are achieved. Parallel the forming of “free” polymer was
70 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
dramatically reduced using higher glass bead concentrations. When 0.6 g GB80-ME glass
beads were used in 12 mL polymerization solution (styrene/toluene, 1/2, v/v) about 600 mg
of free PS had been separated after polymerization by precipitation of the solution in
methanol. For the case where 4.8 g GB80-ME glass beads were used in the same amount of
polymerization solution about 150 mg free PS had been separated.
3.5 Discussion The surface attachment of the polymers is based on a copolymerization of surface attached
monomers with free monomers in solution. As the number of monomer units at the surface
is almost negligible in comparison to the number of monomers in solution, the majority of all
chains formed during the process are simply “free” homopolymers. However, the described
experiments show that the polymers can be readily separated from the modified silica gel by
simple washing procedures with suitable solvents. Furthermore, the fraction of free polymer,
as compared to the amount of polymer on the substrate, can be reduced significantly as it is
possible to run the polymerization reactions at fairly high solid concentration and thus limits
volume for generating free polymer without influencing the results of the polymerization,
i.e. the graft density and the molecular weight of the polymers on the surface. For substrates
with lower specific surface and therefore less surface-attached monomer units the same
behavior is expected and confirmed by the results of the experiments with mesoporous glass
beads (GB80) where the polystyrene loads and the graft densities stay at the same level for
all substrate concentrations. So the most important factor for use in advanced applications, a
good cover with a good polymer loading, applies for silica gel with small particle sizes as
well as for glass beads with particle sizes of several hundreds of microns.
Elemental analysis of all polymer modified silica gels and glass beads revealed that
relatively large amounts of polymer can be grafted to the silica gel for most monomer moiety
carrying silanes. For the studied polystyrene systems materials, silica gel as well as glass
beads, can be obtained with an overall polymer content of around 30 weight-% for
LiChrospher Si60. This amount is remarkable especially when it is considered that the
densities of the two components are rather different so that the material itself consists of
about equal volumes of organic and inorganic components. Based on the assumption that the
whole surface (~ 700 m2·g-1) is covered by a PS layer with constant thickness the calculated
theoretical layer thickness is ultrathin with around 1 nm. Because experiments with MPS-
modified silicon wafers show under comparable polymerization conditions layer thicknesses
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 71
up to 15 nm it is obvious that not the whole surface area of the silica is covered by a PS layer.
Especially surface areas in the small pores of the inner core of the silica particles may not be
reached because pores in the shell got clogged with polymer during the polymerization
process.
Reference experiments in which polymerizations were carried out in the presence of
unmodified silica or silica carrying an inert alkyl silane demonstrate that the reaction indeed
proceeds according to the scheme shown in Figure 1-2c. The silica gels recovered after these
polymerizations remained largely unaltered and within the experimental error no polymer
could be found on the surfaces after completion of the polymerization reactions.
It is shown that suitable silane monomer molecules can be delivered and attached to the
different surfaces we offered in a more than sufficient density. The graft densities of the
monomer units are not influenced by different surface topologies (microporous,
mesoporous) and are settled at 2±1 µmol·m-2 which is at least higher by a factor of 40 than
the expected polystyrene graft densities on the monomer modified silica substrates.
The results show that a fairly wide variety of different acrylate and methacrylate groups
can be successfully tethered to inorganic surfaces and used for the attachment of solution-
borne macroradicals. The stability of the resulting materials in acidic or basic environments
can be improved if the more stable amide derivatives are used instead of the esters. On the
other hand, the esters of these silanes are more readily (i.e. commercially) available which
might be interesting and advantageous for the use of such hybrid materials in less critical
applications.
A somewhat special case is the surface grafted monomer N-methacryloyl-N-methyl-propyl
trimethoxysilane (MNPS) which yields much lower grafted amounts than the other
monomers. No effort was made to elucidate this behavior in detail, but it is rather well
known that methacrylamides show only low propagation rates in free radical
polymerizations[181] and yield only low molecular weight polymer under the reaction
conditions employed here. This case, however, proofs that it is necessary to immobilize
molecules with decent polymerization properties for the described process and that the sole
presence of a double bond is not sufficient.
In various experiments it is shown that the diversity of different polymers grown by free
radical polymerization based on a huge variety of available monomers can be expanded to
the technique where surface attached monomers are included. As it is exemplarily shown for
72 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
styrene the parameters and conditions of the radical polymerization can be tuned towards
the desired loading. For further monomers in radical polymerization different
polymerization behavior has to be taken into account influencing the details of the described
general. To get a detailed picture of how the response of the system (polymer load or layer
thickness) towards the polymerization conditions is, every group of chemically similar
monomers has to be observed separately.
In a number of experiments in which selected important polymerization parameters were
systematically varied it was found that the technique of grafting polymers via
copolymerization of free and surface-attached monomers is a well-controllable process. The
amount of grafted polymer, its molecular weight and the density of the grafted chains on the
surface can be controlled over a reasonable range by adjusting proper polymerization
conditions.
Varying the polymerization time, for example, the amount of polymer which is grafted on
highly porous LiChrospher Si60 can be adjusted in a broad range up to 0.5 g PS/g SiO2 in a
fairly simple way. As it is expected for this free radical polymerizations a variation of the
polymerization time does not influence the molecular weight of the generated polymers.
According the grafted amount of polymer increases at first and then slowly levels off. Using
Aerosil300 as highly porous silica gel, built from sintered smaller primary particles, shows a
behavior similar to LiChrospher on time dependence but the concave formed, larger pores
compensate the smaller specific surface and lead to same polymer loads for both silica gels.
As on the silica gels the amount of polymer that is grafted onto the glass beads can be
adjusted in a fairly simple way via polymerization time up to an amount of 0.5 g PS/g SiO2.
The time kinetics on glass beads reveal also a slow increase of deposited polystyrene
although the larger mesopores do not limit diffusion of growing chains into them. The
practical results support the theoretical calculations made by Chakrabarti et al., Binder et al.
and Leibler et al. [150, 151, 154] where we first have diffusion controlled regime and a
polymer monolayer forms on the surface and when the surface is covered by polymer the
approaching growing chains have to overcome this polymer barrier in order to connect to
one of the surface-attached monomers and be irreversibly bound into the polymer layer.
Having a look at the polymer deposition as a function of time (Figure 3-43) we see that in a
relatively short period of time the grafted amount of polymer increases to a value that is the
lion’s share of the overall grafted polymer amount (green area). This is the time range where
the growing chains can reach quite easy the surface with the monomer sites. Then there is a
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 73
longer time period where this increase levels of so that we get nearly a plateau for the
amount of grafted polymer (orange area). Here we get less polymer grafted to the surface
because the polymer monolayer as steric barrier makes it more difficult for growing chains to
get through to the surface-attached monomers.
Figure 3-43. Exemplary time-polymer deposition curve for a radical polymerization in presence of
silica with surface-attached monomers.
Variation of monomer or initiator concentration in the polymerization solution shows that
by these two important polymerization parameters the polystyrene loading on LiChrospher
can be controlled as well as the graft density. The grafting process is influenced by the size of
the polymer coils that approach the surface and by the probability of an growing chain
linking to a surface monomer. In the case of monomer concentration these two factors act as
antagonists because with higher monomer concentrations in solution the probability of
attaching grows as well as the size of the polymer chains that than are able to block a larger
area on the substrate. The resulting cross interaction leads to higher loading with polymer
while the graft density decreases with higher monomer concentrations during
polymerization. For the variation of initiator concentration the attaching probability and the
coil size work hand in hand because with higher initiator concentrations in solution the
probability of getting connected to the surface increases because of a higher number of
growing chains as well as the polymer coils become smaller. The molecular weight is linear
to the reciprocal square root of the initiator concentration as to expect from a radical
74 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
polymerization in solution. Both factors combined lead then to an increase in polymer load
on higher initiator concentrations and to a more pronounced increase of the graft density.
With the Aerosil300 experiments an important insight is achieved that equal polymer
loading with different molecular weights can be achieved by choosing the right ratio
between the influence of the monomer concentration and the initiator concentration. This
result strengthens the statement that both parameters influence the grafting process from
different directions. The combination of the initiation rate, building new growing chains and
the molecular weight of the surface approaching chains is important as polymer load is
gained with a high number of chains of lower molecular weight or a fewer chains with a
higher molecular weight that soon crowd the surface of the substrate.
If the initial initiator concentration is varied, glass beads reveal the same trend as
microporous silica but the influence is less pronounced. This means that in the same
concentration range of the styrene the variation of graft density is larger for silica gel than for
glass beads. Although the glass beads show higher increase of the absolute value for the final
PS load attached to the surface due to better accessibility of the surface regions in mesopores.
The variation of initiator concentration has not such an impact on the polystyrene loading of
mesoporous glass beads as on the polymer load of silica with smaller pores. On the different
glass beads the polymer load is mainly influenced by the specific surface and insensitive
towards molecular weight affected by initiator concentration. Comparing the amount of
polystyrene on LiChrospher to those on the glass beads of varying specific surface one can
estimate the “effective surface area” of LiChrospher for certain molecular weights, e.g. about
80 m2·g-1 for a Mn around 100000 g·mol-1 referring to the value for an initiator concentration of
1 mM (Figure 3-35). Under the given conditions the modified glass beads with 20 nm pores
(GB250-ME) are the most effective substrate concerning the specific polymer load.
Varying the polymerization temperature a special behavior is found. As long as the
initiator conversion stays more or less the same (i.e. as long as the polymerization time is
adjusted according to the temperature dependence of the initiator decomposition) the
molecular weight of the resulting polymers follows closely the expectations for a free radical
process for which a decrease of the molecular weight with increasing polymerization
temperature is predicted. Interestingly, the graft density remains constant for all
temperatures investigated so that the grafted amount is decreasing with increasing
polymerization temperature solely due to a decrease of the molecular weight. Choice of the
appropriate polymerization temperature therefore offers a handle for the preparation of
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 75
silica-polymer hybrid materials with different molecular weights of the grafted
macromolecules but constant anchor density. At very high conversions of monomer (> 60%)
and initiator (> 70%) as it is the case for the polymerizations at temperatures above 70°C in
the second set of temperature experiments we get a different picture. For these
polymerization at higher temperatures there seems to be a phase where still shorter polymer
chains are built because there is still enough initiator left. These chains can penetrate through
the surface polymer layer and bind to a surface-attached monomer even after half of the
initiator is consumed (results see above) and before the initiator diminishes so that are larger
polymer coils are formed that will not get through the surface polymer layer and bind to a
surface-attached monomer. The polydispersity of the generated free polymer increases and
GPC analysis of the free polymer reveals a more and more pronounced bimodal distribution
where a second peak with higher molecular weights superimposes. This might because of
leaving the steady state kinetics far behind and polymerizing at the end with different
initiator and monomer concentrations compared to the start of the polymerization reaction.
A central role plays an experiment, where silica gel with different surface monomer
densities is used in the graft polymerization process to elucidate the influence of this factor
on the layer formation. In detail the controlled deposition of certain monomer densities on
the surface is an important point of interest. To handle different monomer concentrations
during the silane immobilization step we chose a co-immobilization technique (Figure 3-21).
Delivering the known MPS as monomer silane in the presence of the “inactive” aliphatic OS
to the silica surface in certain molar ratios works well as it can be seen by DRIFT
measurements (Figure 3-23). The increase/decrease of the vibrational bands for each silane
follows the trend given by the initial ratio. Both silanes are of nearly same molecular weight
and size. The reactivity towards silica surfaces is identical because of the trimethoxy anchor
architecture. Taking a closer look by quantitative analysis of the IR-extinction values, i.e. the
carbonyl/methylene ratio, and comparison with extinction ratios of pure silane mixtures of
known composition (Figure 3-24), reveals that it is more likely to immobilize MPS than OS
(Figure 3-25). This “co-immobilization diagram” is of significance because the silanes were
present in double excess in the immobilization solution in order not to run out of silanes
during this attachment step. Interestingly, the following polymerization step under standard
conditions (9.3.6) is not affected by a 10fold variation of the MPS surface density (Figure 3-26
& Figure 3-27). That is plausible because it has been shown that on a silica surface where
only MPS has been immobilized only 3-5% of these double bonds are used up under given
conditions. The rest can be regarded as being “active” beneath the PS monolayer.
76 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
The experiment where silica material grafted with polymer of different molecular weight
in different previous polymerizations is used in a second polymerization generating low
molecular weight polymer allows insights into the layer limited graft process. Using MPS-
modified silica in two subsequent polymerizations with different polymerization solutions
the effect of smaller vs. larger polymer chains comes into play. As we have shown already
the number of attached polymer chains is much smaller than the number of available
monomer units on the surface. After a first polymerization step where polystyrene
monolayers with varying molecular weights have been delivered to the modified silica gel
(see samples from the initiator variation) there are still many “active” double bonds on the
surface. It has also been shown that applying polymer coils of the same molecular weight
(> 30000 g·mol-1) in a second polymerization step ends up in a minimal increase of the final
polymer load (see polymerization time experiments: 48 h + 24 h). When relatively short
polymer chains via high initiator concentration are generated, they are able to be built inside
the existing polymer monolayer. A high number of growing radical polymer chains makes
sure that there are enough chances to connect to surface sites and “fill up” the polymer
monolayer. In the end we have nearly same polystyrene loading for all 2-step samples that
indicates a saturation with polymer on the surface.
3.6 Conclusion The strategy of grafting polymers to solid surfaces by copolymerization of surface-attached
monomers with monomers in solution is a simple and versatile strategy for the covalent
anchoring of polymers to solid substrates. Using different surface attached monomers shows
that not every double bond is suitable for this radical polymerization process and one has to
ponder which surface monomer may be well copolymerized during the polymerization. The
nature of the covalently bound polymer, from nonpolar to polar, can be varied widely giving
the substrates surface the desired physicochemical properties. Well defined polymer
monolayers can be obtained without the need to synthesize polymers with specific (end-
)groups, which allow surface attachment. It might be considered as a draw-back that besides
the polymer monolayer a significant amount of free polymer is generated that needs to be
removed from the surface. However, a variety of applications can be envisaged where this
does not pose a significant problem. Actually the circumstance that the graft process can be
driven to high material output filling the polymerization solution completely with solids
without losing in polymer loading is a big bonus. In fact, such systems are used for a number
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 77
of industrial applications in which silanes as the ones used in this study are used as
“primers” for filler materials in acrylic formulations.
Typical features of free radical polymerization reactions are still valid for this type of
surface capturing polymerization and can be used to control layer thickness, molecular
weight and graft density of the surface attached chains to some extent. Surprisingly, despite
the simplicity of polymer monolayer formation through surface-attached monomers, such an
approach has not attracted much attention in the scientific community and only little is
known about the exact mechanism and kinetics of layer formation. In several sets of
experiments we confirmed the simplicity and variability of controlling this method. The
polymer deposition is insensitive to a wide range of surface concentrations of the previously
immobilized monomer unit. Variations in the surface density that may occur in the initial
silane immobilization step do not hinder to get “standardized” polymer monolayers. In
consequence polymer load and graft density are independent of smaller fluctuations in
quality of the silane immobilization and the polymerization step fully controls the properties
of the polymer monolayer. Beneath polymerization time and temperature monomer and
initiator concentration have been approved to control polymer load (layer thickness) and
graft densities very well in different directions because size of polymer coils and number of
growing chains are factors that compete against each other for linkage to the surface
monomers. A special feature concerning the control polymer load via polymerization should
be mentioned: As shown in Figure 3-43 a control of the polymer load and of the layer
thickness is only possible at the early stage of the polymerization with small polymer
loadings. In the later polymerization stage where the increase is very low we get an almost
stable amount of grafted polymer and respectively polymer monolayer thickness over a large
period of time. After having crossed a certain point in time, the polymerization system is
almost insensitive to the duration of the polymerization regarding polymer load or layer
thickness. This robustness might be a big advantage in processing such modified substrates.
Stable process conditions can be found, which give highly reproducable materials. A
“grafting-to” system with end-functionalized polymer chains reaches a certain maximum
plateau for the polymer load (Figure 3-44a) according to the length (“monodisperse” case) or
the molecular weight distribution of the participating polymer chains quite fast. Our system
generates throughout the polymerization new growing polymer chains. These freshly
formed, shorter chains may diffuse easily through the polymer monolayer to attach to the
surface-bound monomer increasing the overall polymer load. This chance gets smaller when
the segment density of the polymer monolayer becomes higher with time and number of
78 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
attached polymer chains but there might always be a newly formed polymer chain that gets
through to the surface unless all monomers are consumed. Therefore this method does not
reach a limit like the “grafting-to” process and is able to generate higher polymer loads and
thicker polymer monolayers (Figure 3-44b). Smaller polymer coils are able to penetrate the
existing polymer monolayer barrier to catch the immobilized monomers behind. These
“filled up” polymer monolayers have a discrete loading value that depends on the specific
surface and topography of the solid substrate and the whole method reminds of a “grafting-
through” process. This discussion, however, is directed towards rather subtle effects. For
practical polymerization reactions, the key feature is that a nearly constant polymer loading
is obtained rather easy for longer polymerization times although at any point in time of the
polymerization growing polymer chains that are small enough can diffuse through the
surface-attached polymer monolayer and increase the total amount of polymer that is
covalently bound to the surface.
Figure 3-44. The amount of grafted polymer on a surface as a function of time. For a genuine
“grafting-to” system with end-functionalized polymer chains the load reaches a maximum with a
stable plateau (a). For the radical polymerization system using surface-attached monomers the load
increase levels off but does not reach a maximum plateau (b).
The used grafting method works in a good controllable manner for a great variety of silica
substrates which differ in size, specific surface and surface topography. This underlines the
great versatility of this technique towards a wide field of applications where solids with a
special surface finish are involved. Based on our results and knowing the specifications of a
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 79
silica substrate one is enabled to estimate and forecast the polymer loading when using this
grafting process.
After gaining these insights of a standard polymerization system like styrene the
knowledge has to be expanded to a counter player in polarity. With
N,N-dimethyl acrylamide as monomer we get hydrophilic polymers that may act as a
helpful base polymer in applications where swellability in polar fluids is a desired property.
The investigations on grafting this polar polymer using our approach are described in the
following chapter.
3.7 Grafting of Poly(N,N-dimethyl acrylamide) onto Silica
Surfaces through Surface-attached Monomers The set of model polymerizations with different monomers to generate homopolymer
monolayers on silica has shown that the polymerization properties of the monomers have a
strong influence on the build-up of the polymer layer even though the polymerization
conditions were the same. It is therefore necessary to investigate the relation between
polymerization conditions and obtained surface-attached monolayer for each monomer in
detail. Because the polystyrene based system so far investigated is basically hydrophobic, we
now focus on the poly(N,N-dimethyl acrylamide) system to have a hydrophilic complement.
3.7.1 Polymerization Time Influence
When the influence of the polymerization time on a GB250-ME-P(DMAA) system was
studied it was observed that already after 30 minutes the amount of grafted P(DMAA)
almost reached the final value of 0.4 g polymer per gram substrate (Figure 3-45). No further
P(DMAA) was covalently bound to the substrate during the following 24 hours.
80 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
0 4 8 12 16 20 240.0
0.1
0.2
0.3
0.4
0.5
graf
ted
PDM
AA
(g/g
SiO
x)
polymerization time (h)
Figure 3-45. The amount of covalently bound P(DMAA) for different polymerization times.
The results of GPC measurements of the polymers obtained after polymerization times
between 0.5 and 24 hours show almost constant molecular weights (Figure 3-46). The
apparent slight increase in molecular weight at short polymerization times might be due to
thermal equilibration. The number average molecular weight is around 25000 g·mol-1 and the
weight average around 110000 g·mol-1. The system P(DMAA) in DMF gels very fast and all
reaction mixtures got very viscous with time at an initial DMAA concentration of 3.2 mol·L-1.
0 4 8 12 16 20 240
20
40
60
80
100
120
Mw Mn
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
polymerization time (h)
Figure 3-46. The molecular weight averages for different polymerization times as obtained from GPC measurements.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 81
Shorter polymerization times than 30 minutes were not realized because on the one hand
thermal equilibration takes some time as we already see some effect on the molecular weight
of the P(DMAA) that increases the shorter the polymerization time gets. The average
temperature is lower for shorter polymerization times when heating up from ambient
temperature. On the other hand there it is unpractical to control the amount of deposited
polymer via polymerization time because handling is too difficult when polymerization
times are that short. The calculated graft densities are around 0.075 µmol·m-2 (Figure 3-47).
For polymerization times below 2 hours the graft densities are a bit lower because of higher
molecular weights of the P(DMAA) caused by insufficient thermal equilibration.
0 4 8 12 16 20 240.00
0.02
0.04
0.06
0.08
0.10
graf
t den
sity
(µm
ol/m
2 )
polymerization time (h)
Figure 3-47. The graft density of P(DMAA) on GB250-ME for different polymerization times.
Polymerization time experiments were not carried out in detail with the other substrates
but the same independence is true for glass beads with a lower specific surface (GB80) and
for highly porous silica gel (LC700) as a number of tests measurements have shown. The
immediate layer buildup is not influenced by differences in specific surface and porosity.
These properties themselves have an influence on the final amount of grafted polymer as it
has been shown and discussed in chapter 3.4.
82 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
3.7.2 Variation of Initiator Concentration
Detailed experiments with different initiator concentrations in the polymerization
solutions were undertaken with GB80 type glass beads and LC700 silica particles.
Surprisingly neither the drastic change in initiator concentration over four decades nor the
change in molecular weight have an influence on the P(DMAA) loading. As it can be seen in
Figure 3-48 and Figure 3-49 the amount of grafted P(DMAA) is the same for the chosen
initiator concentration variation range for each substrate regardless of the surface topology.
1E-4 1E-3 0.01 0.10.00
0.05
0.10
0.15
0.20
graf
ted
PDM
AA (g
/g S
iOx)
c(I) (mol/L)
Figure 3-48. The amount of grafted P(DMAA) on GB80-AE after polymerization with different
initiator concentrations.
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 83
1E-4 1E-3 0.01 0.10.0
0.1
0.2
0.3
0.4
0.5
graf
ted
PDM
AA (g
/g S
iOx)
c(I) (mol/L)
Figure 3-49. The amount of grafted P(DMAA) on LC700-ME after polymerization with different
initiator concentrations.
As shown by Figure 3-50 and Figure 3-51 the molecular averages within an initiator
concentration range from 0.1 mol·L-1 down to 0.1 mmol·L-1 are almost constant in both cases,
GB80-AE glass beads and LC700-ME silica. For the given initiator concentration range one
would expect a change in molecular weight by a factor of approximately 30 but the
molecular weight is not influenced by initiator concentration in a way it is the case for PS.
This result shows that transfer reactions are dominating. If the transfer reactions are strongly
dominating the polymerization the terms representing the transfer reactions in the Mayo
equation (cf. equation 3-1) get much larger than the term 0X (degree of polymerization in
absence of a solvent) that is directly dependent from the initiator concentration because that
value is proportional to the squareroot of the initiator concentration. In this case the degree
of polymerization nX and the molecular weight respectively is not affected by the initiator
concentration.
84 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
1E-4 1E-3 0.01 0.10
102030405060708090
Mw Mn
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
c(I) (mol/L)
Figure 3-50. The molecular weight averages of P(DMAA) from polymerizations on glass beads
(GB80-AE) with different initiator concentrations as obtained from GPC measurements.
1E-4 1E-3 0.01 0.10
10
20
30
40
50
60
70 Mw Mn
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
c(I) (mol/L)
Figure 3-51. The molecular weight averages of P(DMAA) from polymerizations on silica gel (LC700-
ME) with different initiator concentrations as obtained from GPC measurements.
Having a constant amount of grafted P(DMAA) on the substrates with varying initiator
concentrations is within the range of reasonable expectations. Now polymerizing in a
monomer-polymer-solvent system that is not, averaged over the long polymerization time,
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 85
in a steady state puts the initiator concentration influence aside. As above-mentioned
transfer reactions are dominant and initiation reaction and termination reactions that have to
be balanced to get a steady state do not play a role. Therefore the resulting graft densities as
shown in Figure 3-52 and Figure 3-53 are constant as well.
1E-4 1E-3 0.01 0.10.00
0.02
0.04
0.06
0.08gr
aft d
ensi
ty (µ
mol
/m2 )
c(I) (mol/L)
Figure 3-52. Graft density of P(DMAA) on glass beads (GB80-AE) after polymerization with
different initiator concentrations.
1E-4 1E-3 0.01 0.10.000
0.005
0.010
0.015
0.020
graf
t den
sity
(µm
ol/m
2 )
c(I) (mol/L)
Figure 3-53. Graft density of P(DMAA) on silica gel (LC700-ME) after polymerization with different
initiator concentrations.
86 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
Like for the polymerization time experiments a number of single experiments revealed the
initiator concentration independence for other substrates as it was observed here on the
GB80 glass beads.
3.7.3 Influence of Monomer Concentration
In the experiments with variation of the styrene monomer concentration the LC700 silica gel
was most sensitive to the changes. Therefore the highly porous substrate comes into play
again with the DMAA monomer. A set of three polymerizations was chosen to evaluate the
effect of DMAA monomer concentration. In Figure 3-54 it is shown that variation of
monomer concentration has an influence on the amount of covalently to the substrate bound
P(DMAA).
0 1 2 3 4 50.0
0.1
0.2
0.3
0.4
0.5
graf
ted
PDM
AA
(g/g
SiO
x)
c(M) (mol/L)
P(DMAA) PS
Figure 3-54. The amount of grafted P(DMAA) for the polymerization with different monomer
concentrations on silica gel (LC700-ME). The results from the experiments with PS (grey marks) are
added for comparison.
Monomer to solvent ratios of 1:9, 1:4 and 1:2 (v/v) give monomer concentrations of 1.0, 1.9
and 3.2 mol⋅L-1 DMAA. Within this range there is a gain in covalently bound P(DMAA) of
0.3 g polymer on 1 g LC700-ME which means an increase of 170 % in polymer load. This is a
significant increase of surface-bound P(DMAA) with increasing DMAA concentration in
DMF as solvent. Because of the analogy of these results with those of polystyrene the PS data
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 87
points were added in Figure 3-54 to Figure 3-56 for easier comparison. Figure 3-54 shows
that the increase of polystyrene amount on silica levels off with higher monomer
concentrations whereas the increase of P(DMAA) amount has a tendency for an amplified
increase at high monomer concentration.
Analysis of the “free” P(DMAA) molecular weight distribution exhibits a proportional
increase of the molecular weight averages with rising DMAA concentration (Figure 3-55).
The number average of the molecular weight distribution grows from 18300 g·mol-1 for the
lowest to 59400 g·mol-1 for the highest DMAA concentration. The weight average increases
from 51600 g·mol-1 to 190700 g·mol-1. So the initial DMAA concentration has a strong impact
on the molecular weight of the formed P(DMAA).
0 1 2 3 40
50
100
150
200 Mn Mw
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
c(M) (mol/L)
Figure 3-55. The molecular weight averages of the formed “free” P(DMAA) for the polymerization on
LC700-ME using different DMAA concentrations.
Again the early gelation of the polymerization system plays a major role. Where as the
movements of growing chain ends in a highly viscous solution are strongly hindered the
small monomer molecules keep their mobility until the system reaches the glassy state. This
difference arranges for the fact that the monomer concentration influences the molecular
weight as well as the amount of grafted P(DMAA) for our polymerization system and the
initiator concentration does not. A glassy state is not reached because the solvent portion
keeps the mixture at least gelatinous. The calculated graft densities for P(DMAA) on silica
88 3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers
gel are almost constant because the amount of grafted P(DMAA) increases nearly
proportional with the molecular weights. Only a slight decrease in graft density with longer
polymer chains for higher DMAA concentration is observed (Figure 3-56). This effect might
be due to the fact that the calculated theoretical P(DMAA) layer thicknesses on LC700-ME
are 0.5 nm and below and these thin layers do not repel larger P(DMAA) coils that
effectively. Then the monomer concentration influences the molecular weight but the impact
on graft density is small.
0 1 2 3 40.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
graf
t den
sity
(µm
ol/m
2 )
c(M) (mol/L)
Figure 3-56. The graft densities of P(DMAA) on LC700-ME are constant for different DMAA
concentrations.
3.7.4 Discussion & Conclusion for P(DMAA) on Silica
If the polymerization of N,N’-dimethyl acrylamide is compared using the same system
with styrene there are some differences to mention. The first difference is that P(DMAA)
reaches its final amount of covalently bound polymer on porous substrates very fast whereas
PS needs more than 24 hours to reach a somewhat constant polymer loading. This is due to
much higher propagation constants (~50x) for DMAA than for styrene under same
polymerization conditions with comparable termination and transfer constants[182-184]. The
result is consistent with the concept of fast growing polymer chains in the vicinity of the
surface catching quickly surface-bound monomer units. Once this nearly complete P(DMAA)
monolayer is formed on the silica surface the approach of further growing P(DMAA) coils is
strongly hindered. This lack of mobility is even intensified by increasing viscosity and finally
3 Grafting of Polymers onto Silica Surfaces through Surface-attached Monomers 89
gelation of the P(DMAA) solution in the case of higher initial monomer concentrations
(> 1 mol·L-1). Only mobile monomer molecules can diffuse through and add to growing chain
ends whereas the radicals at the growing chain ends are encaged. This is the reason why the
main influence factor on polymer loading and graft density is the monomer concentration.
For P(DMAA) in our polymerization system the molecular weight variation is small for
varying initiator concentrations. That is because of the high amount (~ 90%) of N,N’-
dimethyl formamide controlling the polymerization. This high dilution of the polymerization
mixture with solvent was necessary to keep it fluid but this measure did not prevent the
Trommsdorf effect. This gel effect was even more pronounced for the higher DMAA
concentrations (1.9 mol·L-1 and 3.2 mol·L-1) but because of the above mentioned monomer
mobility a higher monomer concentration contributes to higher molecular weights. Sufficient
monomer is present close to the substrate surface[150] and so polymer chains with low
mobility can be connected to the surface by monomers with higher mobility. Overall the
P(DMAA) load is almost proportional to the molecular weight of the generated “free”
P(DMAA) and the graft density dependency from the monomer concentration is much less
pronounced compared to the PS system.
The possibility of growing polymer monolayers of different polarities onto glass substrates
leads us to tailor-made copolymer systems where different “basic polymers” and functional
copolymers are covalently attached to silica substrates to perform special jobs under
individual conditions.
90 4 Grafting of Functional Copolymers onto Silica Surfaces
4 Grafting of Functional Copolymers onto Silica Surfaces
In the previous chapters it is shown that surface attached monomers enable one to create
covalently bound polymer monolayers of varying polarity to the surface of silica and glass
beads. By controlling the polymerization parameters the polymer loading and graft density
can be adjusted as the previous investigations in this work reveal. With polystyrene and
poly(N,N-dimethyl acrylamide) as protagonists the foundations are laid to expand our
system to radical copolymerization during copolymerization with surface attached
monomers. Combining in solution the “basic” monomers with highly sophisticated
monomers that are equipped with special functional groups copolymer monolayers can be
built. The desired monomer combination is used in simple radical polymerization in
presence of the monomer modified, solid substrates in order to create functionalized
copolymer monolayers. The functionalities within the copolymer layer can then be used for
further polymeranalog reactions towards the needed system for specific, custom-built
applications.
For the copolymerization styrene as hydrophobic and N,N’-dimethyl acrylamide as
hydrophilic monomer are used (Figure 4-1 1 & 2). As functional comonomers we use tailor-
made molecules developed and polymerized exemplarily by Murata et al.[185, 186]. Through
a two step synthesis the N-methacryloyl-β-alanine succinimide monomer (MAC2ae, Figure
4-1 3) is accessible. First β-alanine is reacted with methacryloyl chloride and then N-hydroxy
succinimide is coupled to the carboxylic moiety[186]. MAC2ae is a monomer bearing an
activated ester which is reactive towards amino groups under appropriate conditions. The
N-acryloyl-N-methyl-propyl phthalimide (AC3pht, Figure 4-1 4) unit is synthesized in a two
step process starting from N-methyl-1,3-diaminopropane reacting with phthalic anhydride
in the first and with acryloyl chloride in the second step[185]. The AC3pht monomer contains
a protected amino group which is deprotected by treatment with methylamine or hydrazine.
This pathway is necessary because (meth)acrylic compounds with amino moieties are
difficult to polymerize because they undergo Michael addition during polymerization[187].
This unwanted reaction type leads to loss of the amino functionalities. But creating primary
amino groups within a polymer this way gives us a copolymer reactive towards a broad
range of reagents diversifying the possibilities of our surface-modified materials. Forming
copolymer monolayers via radical copolymerization with this set of monomers we have a
4 Grafting of Functional Copolymers onto Silica Surfaces 91
starting point for a number of subsequent reactions which then might be used as a toolbox
for generating functional copolymer monolayers on solid substrates.
Figure 4-1. Monomers used in polymerization of functional copolymer monolayers on silica surfaces:
Styrene 1 as main monomer for hydrophobic systems, N,N-dimethyl acrylamide 2 as main monomer
for hydrophilic systems. Two bifunctional compounds are incorporated into the polymer chains via
their (meth)acrylic site: N-methacryloyl-β-alanine succinimide ester 3 reacts as “activated ester” with
primary amines at room temperature and N-acryloyl-N-methyl-propyl phthalimide 4 is a protected
amino group.
92 4 Grafting of Functional Copolymers onto Silica Surfaces
4.1 Polystyrene Copolymers on Silica Particles
4.1.1 Poly(styrene-co-N-acryloyl-N-methyl-propyl phthalimide) &
Poly(styrene-co-N-acryloyl-N-methyl-propyl amine)
Poly(styrene-co-N-acryloyl-N-methyl-propyl amine) (P(S-co-AC3amine)) and its precursor
poly(styrene-co-N-acryloyl-N-methyl-propyl phthalimide) (P(S-co-AC3pht)) is the copolymer
system that is polymerized the most and attached to the most different substrates including
silica gel and different glass beads in the course of our studies. It was prepared with different
compositions varying the molar part of the functional monomer unit in the copolymerization
solution from 5 % up to 20 %. Because of the relatively high number of different material
carrying surface attached P(S-co-AC3pht) the results will be listed in the following in tables.
The elemental analysis of all substrates with P(S-co-AC3pht) shows that polymer load and
graft density for different specific surfaces on the varying substrates changes like the
homopolymer PS would do under same polymerization conditions (Table 4-1). GPC
measurements in THF against PS standard samples show that the apparent molecular weight
of the copolymer coils gets larger with increasing phthalimide content. This apparent
molecular weight increase might be an artifact of the GPC measurement caused by the fact
that the calibration was carried out with a different polymer (PS) than the measured (P(S-co-
AC3pht)). 1H-NMR analysis of the free phthalimide copolymers reveals two peaks in the
region between 7.9 and 7.7 ppm caused by the four hydrogen atoms of the aromatic ring that
is connected to the imide ring to form the phthalimide moiety. The signals of the five
hydrogen atoms of the ring of the styrene moiety are localized between 7.3 and 6.3 ppm.
Because phthalimide and styrene signals are clearly separated at the NMR scale the integrals
of these peaks can be rationed to calculate the content of N-acryloyl-N-methyl-propyl
phthalimide in the copolymer. For three P(S-co-AC3pht) copolymers on glass beads the 1H-
NMR spectra are depicted in Figure 4-2 and the differences in height and area of the two
phthalimide peaks change significantly following the trend in a proportional way.
4 Grafting of Functional Copolymers onto Silica Surfaces 93
8 7 6 5 4 3 2 1 chemical shift (ppm)
CDCl3
20'
20 19'
19
17,18
15,1613,14 5'
3
6
5
4
7,8
10 - 124'
91,2
N
NO
O
O
co
co
(4)
(3)
(2)
(1)
c)
b)
a)
Figure 4-2. 1H-NMR spectroscopy of free polystyrene copolymers with different contents of
phthalimide groups.
In the 1H-NMR spectra of the P(S-co-AC3pht) copolymers the peaks in the aromatic region
Figure 4-2(1) belong to the four phthalic protons 19, 19’, 20 and 20’ and they diminish as the
content of the functional monomer within the copolymer is reduced from 20 mol% (Figure
4-2a), over 10 mol% (Figure 4-2b), down to 5 mol% (Figure 4-2c). The peaks in the aromatic
region Figure 4-2 (2) represent the five styrene protons 4-6, 4’ and 5’. The peaks in the
aliphatic region Figure 4-2 (3) are caused by methylene and methyl protons next to the
nitrogen atoms and also exclusive for the functional unit. The backbone protons and the
middle methylene protons of the propyl spacer give the peaks of region Figure 4-2 (4).
94 4 Grafting of Functional Copolymers onto Silica Surfaces
The results listed in Table 4-1 show that after a conversion of 30 to 40% the P(S-co-AC3pht)
copolymers have nearly the same composition of subunits as the monomers in the initial
polymerization solution. In most cases the percentage value of phthalimide units lies
minimal below the expected amount derived from the monomer mixture before
polymerization start.
Table 4-1. Elemental analysis results, polymer load and graft density of the styrene-co-N-acryloyl-N-
methyl-propyl phthalimide monolayer modified substrates. Molecular weight and content of
phthalimide units of the free copolymer.
product C*
(%)
H*
(%)
N*
(%)
mʹpoly#
(g)
nM †
(g·mol-1)
Γ0
(µmol·m-2)
ϕ‡
(%)
LC700-ME-PS-AC3pht(5%) 29.27 2.83 1.32 0.409 36,000 0.016 4.8
LC700-ME-PS-AC3pht(10%) 27.01 2.94 0.64 0.391 33,000 0.017 11.8
GB80-ME-PS-AC3pht(5%) 11.63 0.92 0.15 0.126 69,000 0.023 4.7
GB80-ME-PS-AC3pht(10%) 12.99 1.05 0.30 0.152 78,500 0.024 9.4
GB80-ME-PS-AC3pht(20%) 12.76 0.92 0.52 0.154 90,500 0.021 15.0
GB80-AA-PS-AC3pht(10%) 8.39 0.96 0.50 0.088 21,500 0.051 9.5
GB250-AA-PS-AC3pht(20%) 22.29 2.25 1.33 0.323 34,500 0.038 19.4
* obtained from elemental analysis, # per determined from elemental analysis, 2SiO
g † determined
from GPC analysis of the free polymer, ‡ calculated from 1H-NMR integrals of the free polymer
After having successfully generated covalently bound P(S-co-AC3pht) copolymer
monolayers the next step is the cleavage of the phthalimide to set the amino group free for
further reactions. 1H-NMR measurements of the free P(S-co-AC3amine) reveal that cleaving
the phthalimide with methylamine off from the amino group does not lead to complete
conversion. The two typical phthalimide peaks between 7.9 and 7.7 ppm are still present
with reduced intensity. A reaction of P(S-co-AC3pht) with hydrazine instead gives
quantitative cleavage confirmed by missing phthalimide hydrogen peaks in the 1H-NMR
spectrum (Figure 4-3 (1)) in combination with the presence of signals of methylene hydrogen
next to nitrogen atoms (3.7-2.5 ppm; H 10-14, 17, 18; Figure 4-3 (3)).
4 Grafting of Functional Copolymers onto Silica Surfaces 95
8 7 6 5 4 3 2 1
17,18
15,1613,14
10 - 12
97,8
65'5
4 4'
31,2
chemical shift (ppm)
NH2
NO
co
co
(3)(1)CDCl3
(4)
(2)
Figure 4-3. 1H-NMR spectrum of the phthalimide functionalized copolymer P(S-co-AC3pht(10%))
after reaction with hydrazine.
Elemental analysis after deprotection of the amino group reveals the loss of the phthalic
acid unit, because the carbon content drops, the nitrogen content raises and the overall
copolymer load is reduced during this polymeranalog step.
Table 4-2. Elemental analysis results and polymer load for styrene-co-N-acryloyl-N-methyl-propyl
amine monolayer modified substrates after polymer analogue reaction of the phthalimide containing
substrates with hydrazine.
product C*
(%)
H*
(%)
N*
(%)
mʹpoly#
(g)
δ(NH2)†
(µmol/g) EA UV
δ(NH2)‡
theor. (µmol/g)
LC700-ME-PS-AC3amine(10%) 25.60 3.43 0.53 0.350 - 20 370
GB80-ME-PS-AC3amine(5%) 11.00 0.86 0.22 0.115 - - 50
GB80-ME-PS-AC3amine(10%) 11.17 0.86 0.33 0.120 - 90 120
GB80-ME-PS-AC3amine(20%) 11.84 1.14 0.66 0.139 - 90 180
GB80-AA-PS-AC3amine(10%) 7.83 1.19 0.60 0.081 80 50-80 70
GB250-AA-PS-AC3amine(20%) 19.46 2.51 1.62 0.242 150 120-160 470
* obtained from elemental analysis, # per determined from elemental analysis, 2SiO
g † determined
from elemental analysis of coupled DMT-disulfide units[188] and UV spectroscopy of cleaved DMT[189], ‡ calculated from actual load and actual functional group content of P(S-co-AC3pht)
96 4 Grafting of Functional Copolymers onto Silica Surfaces
Coupling of a 13-bis(4-methoxyphenyl)-13-phenyl-4-oxo-8,9-dithia-5,12-dioxa-tridecanoic
acid unit (see Figure 4-4 for chemical structure) to the amino groups[190] within the
copolymer monolayer enables quantification of the thereby obtained amine functions via
elemental analysis of the sulfur of the disulfide linkage and additionally photometric
analysis after cleavage of DMT ([1,1-bis(4-methoxyphenyl)-1-phenyl]methyl group). To
ensure that there are no residual amino groups the Kaiser test, a colorimetric test for the
presence of amino groups, is done after the coupling step[191]. This test is based on the
reaction of ninhydrin with amino groups to form a blue adduct and therefore an incomplete
coupling leads to a positive Kaiser test.
NH2
OH
O
O
OS S
O
MeO
OMe
OMe
NH
O
O
OS S
O
MeO
OMe
OMe
silica particle
polymer monolayer
+
cleavage
UV analysis
connecting step:DCC, DMAP, TEA
cleaving step:HClO4 / EtOH
Figure 4-4. Detection and quantification of the obtained amino groups within the surface-attached
copolymer monolayer (PS-AC3pht).
It turns out that the photometric approach gives varying results (± 10 to 20%) on the
amounts of amino groups whereas the results of the elemental analysis are constant.
Regarding the amino loading values in Table 4-2 this has to be considered. GB80-ME-PS-
AC3pht(10%) & (20%) have been transformed into the amine using methylamine. As we
recognize for the free polymer this reaction is not quantitative and this explains the lower
4 Grafting of Functional Copolymers onto Silica Surfaces 97
measured values for δ(NH2) on GB80-ME-PS-AC3amine(10%) & (20%) compared to the
theoretical values. In addition it has to be mentioned that with smaller pores and thicker
copolymer layers and higher loading respectively (LC700-ME-PS-AC3amine(10%) &
GB250-AA-PS-AC3amine(20%)) the gap between the theoretically possible value for δ(NH2)
and the measured actual value becomes bigger.
FT-IR measurements of the functionalized solids as well as the free copolymers allow a
characterization of the obtained surface modified materials. The copolymers with PS as main
component show the specific aromatic C-H stretching vibrations between 3100 and 3000 cm-1
(Figure 4-5). Surface-attached copolymers containing the phthalimide unit give sharp bands
at 1772 and 1715 cm-1 that are specific for C=O stretching of a five ring imide system in
conjugation to an aromatic ring. Out of plane vibration of the four aromatic hydrogen atoms
leads to a band at 722 cm-1. After phthalimide moieties being converted into amine functions
the phthalimide vibrations bands are not present anymore. The N-H stretching vibration
band of the amino groups is too weak to be recognized in the spectra, so that full conversion
of the phthalimide can only be inferred from the absence of the phthalimide bands.
4000 3000 2000 1000
(pht)
ν
b)
a)(Aryl-H)ν
ν
ν
(SiOSi)
(C=O)
(CH)
ν
trans
mitt
ance
(a.u
.)
wavenumber (cm-1)
Figure 4-5. Fourier infrared measurements of PS-copolymer covered silica particles carried out in
diffuse reflection mode (DRIFT): PS-AC3pht (a) and PS-AC3amine (b).
98 4 Grafting of Functional Copolymers onto Silica Surfaces
1000 800 600 400 200 00
10
20
30
40
50
60
70
b)
c)
a)
O KLL
Si 2pSi 2s
C 1s
N 1s
O 1s
coun
ts (1
000/
s) (a
.u.)
binding energy (eV)
Figure 4-6. X-ray photoelectron spectra of a bare glass bead (a), a glass bead modified with a
N-acryloyl-N-methyl-propyl trimethoxysilane layer (b) and a poly(styrene-co-N-acryloyl-N-methyl-
propyl amine) covered glass bead (c).
Table 4-3. Ratios of different elements on the surface of silica particles modified by PS and PS
copolymer containing amino groups polymers derived from XPS measurements.
product C*
(%)
N*
(%)
F*
(%)
O*
(%)
Si*
(%)
GB80-AA-P(S-co-AC3amine(10%)) 38.5 4.1 - 38.9 18.5
GB80-AA-PS 45.9 - - 35.6 18.5
* obtained from XPS and the integral of the XPS signals (normalized with the efficiency factor)
The XPS measurements confirm the composition of the P(S-co-AC3amine) layer that was
attached to glass beads (Figure 4-6, Table 4-3). To test the amino functions within the surface-
attached copolymer monolayer FITC was applied to the glass beads and bound irreversibly
to functional sites as detected after intense extraction with various solvents by a CCD camera
and fluorescence microscopy (Figure 4-7). The confocal fluorescence microscopy shows a
strong fluorescence on the surface facets of the glass bead. Both visualizations suggest a
homogeneous coverage of the surface with amino groups respective copolymer monolayer.
4 Grafting of Functional Copolymers onto Silica Surfaces 99
Figure 4-7. CCD image shows that glass beads with a P(S-co-AC3amine) monolayers get colored after
treatment with FITC (a). A slice of the examination with a confocal fluorescence microscope is shown
and exhibits that glass beads with a surface-attached P(S-co-AC3amine) monolayer show fluorescence
after treatment with FITC (b).
400 450 500 5500.0
0.1
0.2
a)
511 nm
482 nm
458 nm
433 nm
PS
P(S-co-AC3amine(10%))
P(S-co-AC3amine(5%))
P(S-co-MAC2ae)
FITC
abs
(a.u
.)
wavelength (nm)
Figure 4-8. Different free copolymers in DMF were investigated with an UV/vis spectrometer after
treatment with fluorescein isothiocyanate (FITC, see reference extinction curve). FITC can only be
recognized when covalently bound to the polymer, here: when amino groups were present. PS and
P(S-co-MAC2ae) as negative references indicate no dye bands.
100 4 Grafting of Functional Copolymers onto Silica Surfaces
The results of the labeling experiments on copolymer modified substrates are confirmed by
reactions of different free copolymers with FITC as labeling dye. Whereas PS copolymers
equipped with amino groups is colored even after reprecipitation and washing, pure PS or
PS copolymers containing activated ester groups reveal no extinction bands of the
fluorescein after the same procedure (cf. Figure 4-8). There is a shift of the intensity of
extinction maxima of the polymer bound fluorescein to longer wavelengths compared to the
unreacted fluorescein dissolved in methanol. This is due to the missing solvent interaction of
the dye within the polymer. A color change from green to yellow to orange and red,
dependent from the fluorescein content in the copolymer, is the result.
4.1.2 Poly(styrene-co-N-methacryloyl-β-alanine succinimide ester)
Poly(styrene-co-N-methacryloyl-β-alanine succinimide ester) (P(S-co-MAC2ae)) is a
copolymer with active ester units. The conversion of N-succinimide activated ester groups
with reactive functional groups, especially primary amines, is well known and widely used
in preparations and analytics, e.g. cell labeling with dyes[192], biotinylation[193, 194],
biomolecule immobilization on polymers[195, 196], or patterned surface
functionalization[197]. IR measurements of surface-attached copolymer monolayers reveal
the specific C=O stretching vibration at 1826, 1788 and 1732 cm-1 of the succinimide (Figure
4-9).
4000 3000 2000 1000
(ae)ν (Aryl-H) νν
ν
(SiOSi)
(C=O)(CH)
ν
tran
smitt
ance
(a.u
.)
wavenumber (cm-1)
Figure 4-9. DRIFT survey of silica particles with P(S-co-MAC2ae) layer display indicative C-H- and
C=O-stretching bands.
4 Grafting of Functional Copolymers onto Silica Surfaces 101
8 7 6 5 4 3 2 1
6
NHO
co
co
O O
NO
O16',17'
16,17
14,1512,13
9-117,8
5'5
4 4'
31,2
(1)
chemical shift (ppm)
(3)
CDCl3
(2)
Figure 4-10. 1H-NMR spectrum of P(S-co-MAC2ae) the active ester units.
In the 1H-NMR spectrum of P(S-co-MAC2ae) the methylene hydrogen located next to
carbonyl groups (14-17’) show up with peaks within region (2) in Figure 4-10. Best for the
determination of the MAC2ae content from the peak integral ratios in the “free” P(S-MAC2ae)
copolymer are the four succinimidyl hydrogen protons. In a 1H-NMR spectra they appear
between 2.9 and 2.6 ppm. MAC2ae copolymerized with styrene gives a spectrum where no
other peaks interfere with the succinimidyl proton peaks (Figure 4-10, region 2). 1H-NMR
analysis of the free copolymers generated during the polymerizations shows that a content of
2.1% MAC2ae was obtained for LC700-ME-P(S-co-MAC2ae) and 1.6% MAC2ae for GB80-ME-
P(S-co-MAC2ae). 95 % styrene and 5 % MAC2ae were used as monomers in solution. The
succinimidyl content of the analyzed copolymer is lower than expected from the initial
comonomer ratio in the polymerization solution due to insufficient solubility of the MAC2ae
constituent. LC700-ME-P(S-co-MAC2ae) has a loading of 474 mg copolymer per gram silica
and with 2.1 % active ester groups the functionality load is 93 µmol·g-1 and we have
22 µmol·g-1 of activated esters on GB80-ME-P(S-co-MAC2ae) glass beads. The activated ester
groups were tested after the polymerization procedures by binding dyes with primary
amines at room temperature. The primary amino groups of the dye react with the activated
ester groups by forming an amide group and N-hydroxysuccinimide is leaving the reaction.
Figure 4-11 shows a successfully dyed copolymer monolayer on glass beads dyed by
102 4 Grafting of Functional Copolymers onto Silica Surfaces
DY-635-NH2 (for chemical structure see Figure 4-13). For the free P(S-co-MAC2ae) labeling
with DY-635-NH2 was successful as shown by UV spectroscopy (Figure 4-12).
Figure 4-11. Glass beads with a grafted P(S-co-MAC2ae) monolayer were labeled with blue
DY-635-amine.
500 550 600 650 7000.0
0.1
0.2
DY-635-NH2
P(S-co-MAC2ae(5%))
648 nm
663 nm
wavelength (nm)
abs
(a.u
.)
Figure 4-12. For the case of active ester functions the dye DY-635 equipped with an aliphatic primary
amine function (DY-635-NH2) was bound and found.
4 Grafting of Functional Copolymers onto Silica Surfaces 103
N O+
NNH2
NH
O
SO3-
Figure 4-13. Chemical structure of DY-635-NH2. The primary amino group readily reacts with
activated esters at room temperature.
4.1.3 Two-step Grafting Processes
To get an impression of the impact of functionalities inside the ultrathin copolymer layer on
following polymeranalog reactions, a second generation grafting reaction were performed
with glass beads with an polystyrene-amine layer (GB80-AA-P(S-co-AC3amine(10%)), Figure
4-14 a). Two different systems were used to investigate the grafting onto the first polymer.
The first system for a second grafting step was based on acrylic moieties. The acryloyl
chloride reacted with the amino groups to introduce new double bonds on the copolymer
chain (Figure 4-14 b). The C=C vibration bands of these attached monomers were clearly
visible in DRIFT experiments. The thus obtained acrylic moieties took then part in a radical
polymerization of styrene to form PS branches on the copolymer layer (Figure 4-14 c).
104 4 Grafting of Functional Copolymers onto Silica Surfaces
Figure 4-14. Two step polymerization on amino-polymer modified glass beads leading to surface
attached comb polymers.
For the second system 2-brom-propionic acid bromine was added to the first layer amino
groups. This allows to obtain covalently attached units that can start a “grafting-from”
reaction via atom transfer radical polymerization (ATRP) mechanism (Figure 4-14 d). In a
subsequent ATR polymerization of styrene, polystyrene chains start to grow from the
copolymer layer (Figure 4-14 e). The results obtained from elemental analysis and GPC
measurements show that both second generation polymerizations add a significant amount
of polymer-grafted polymer.
The ATRP case, as a grafting-from technique results in much higher amounts of grafted
polymer because the monomer diffusion is less restricted than the diffusion of growing
polymer coils and also the probability of a monomer hitting the grafting-from radical is
much higher than a polymer coil radical hitting a grafted double bond.
4 Grafting of Functional Copolymers onto Silica Surfaces 105
Table 4-4. Resulting polymer amounts and graft densities before and after a second generation
polymerization with styrene on glass beads with different functionalized PS-monolayers.
modified substrate PS*, overall
(g·(g SiO2)-1)
PS*, 2nd
(g·(g SiO2)-1)
Γ0, surface
(µmol·m-2)
Γ0, polymer
(µmol·m-2)
nM 2nd
(g·mol-1)
GB80-AA-P(S-co-AC3amine(10%)) 0.082 - 0.051 - -
GB80-AA-P(S-co-AC3amine(10%)) + PS
0.086 0.004 0.052 - 32,500
GB80-AA-P(S-co-AC3AA-PS) 0.100 0.018 §0.052 0.007 33,000
GB80-AA-P(S-co-AC3PA-PS) 1.230 1.148 0.051 0.871 †16,500
* calculated from elemental analysis, † free polymer from an ATR polymerization that was started with starter molecules in solution under same conditions (cf. 10.1.4), § estimation from the blind test
Compared with the initially used functional beads (GB80-AA-P(S-co-AC3amine)) a slight
increase of surface-bound polymer can be found by just dispersing the amino-functionalized
glass beads during a radical polymerization of styrene (GB80-AA-P(S-co-AC3amine(10%)) +
PS, blind test) although the amino functionalities are not polymerizable. Due to remaining
double-bonds from the MPS-layer below the polymer layer there is a small amount of
polymer that increases the overall amount of grafted PS little compared to the
GB80-AA-P(S-co-AC3amine(10%)) before (cf. Table 4-4). This behavior is consistent with the
results shown in 3.3.8. The acrylic double-bonds along the primarily grafted PS-chain give an
additional increase in covalently bound polymer (GB80-AA-P(S-co-AC3AA-PS)) as the
additional polymer amount lies 18 mg per gram·SiO2 PS above that of the starting material
and 14 mg per gram·SiO2 PS above that of blind test (cf. Table 4-4). The best performance
gives the “grafting-from” approach with PS-chains branching out from the first polymer
layer (GB80-AA-P(S-co-AC3PA-PS)). The PS loading is with 1230 mg per gram·SiO2 PS
roughly twelve times higher than with the acrylic function within the polymer layer (cf.
Table 4-4).
Comparing the overall graft densities of the blind test and the two different approaches
one becomes aware that the blind test with 52 nmol·m-2 has an insignificantly higher graft
density than the glass beads with just the polystyrene-amine layer (51 nmol·m-2). The
polymerization that incorporates the monomer functions within the first polymer layer
106 4 Grafting of Functional Copolymers onto Silica Surfaces
shows an additional graft density of 7 nmol·m-2 on the polymer, whereas the surface bound
portion should increase like it is the case for the blind test, because below the previously
formed monolayer are also surface-attached monomer present. Due to the smaller molecular
weight of the generated PS the ATRP approach delivers a graft density of 871 nmol·m-2 onto
the first polymer layer where the initiating sites are. The surface graft density stays constant
because in this experiment no polymer chains are started in solution that could diffuse
through the polymer monolayer and connect to the surface-attached monomers beneath.
When the density of amino functions, as determined before, is around 90 µmol·g-1 there are
1125 nmol amino groups per square meter surface. Compared with the final ATRP graft
density there is an efficiency of 77% totalized for all synthesis steps. This means
approximately three of four amino functions grew into a PS-chain whereas the rest did either
not connect to an ATRP-starter molecule, did not start during ATR polymerization or just
terminated already at lower molecular weights due to sterically hindered functionalities or
lack of space for propagation of the polymer chain.
4 Grafting of Functional Copolymers onto Silica Surfaces 107
4.2 Poly(N,N-dimethyl acrylamide) copolymers on silica
particles
4.2.1 Poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-methyl-
propyl phthalimide) & Poly(N,N-dimethyl acrylamide-co-N-
acryloyl-N-methyl-propyl amine)
The copolymers with P(DMAA) as main component on the silica solids exhibit aliphatic C-
H stretching vibrations in the range between 3100 and 2800 cm-1 and a characteristic carbonyl
band at 1642 cm-1 (Figure 4-15).
4000 3000 2000 1000
(pht)
b)
a)
ν
ν
ν
(SiOSi)(C=O)
(CH)
ν
tran
smitt
ance
(a.u
.)
wavenumber (cm-1)
Figure 4-15. DRIFT spectra of (a) P(DMAA-co-AC3pht) and (b) P(DMAA-co-AC3amine), the
elimination of phthalimid carbonyl vibration bands after conversion into an amine shows the extent of
the reaction.
In the 1H-NMR spectra of the “free” poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-
methyl-propyl phthalimide) (P(DMAA-co-AC3pht)) copolymer the four phthalic hydrogen
show a broad peak between 7.8 and 7.5 ppm (Figure 4-16 (1)). The integral of this peak is
used to quantify the amount of the functional monomer in the copolymer monolayer when
compared to the integral of the methylene hydrogen in the backbone between 2.1 to 1.0 ppm
(Figure 4-16 (4)). The signals of the N-methyl hydrogen show up between 3.4 to 2.6 ppm
108 4 Grafting of Functional Copolymers onto Silica Surfaces
(Figure 4-16 (2)) together with the peaks of the AC3pht propyl spacer between 3.7 to 2.1 ppm.
After cleavage of the phthalic group with hydrazine the 1H-NMR spectra of the resulting
poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-methyl-propyl amine) (P(DMAA-co-
AC3amine)) show no phthalimide hydrogen signals. This indicates a successful
polymeranalog conversion of the phthalimide into amino groups as it was shown for the
styrene copolymer. For amino group quantification of GB80-AA-P(DMAA-co-AC3amine) the
DMT-disulfide linker[190] was used as described for the experiments in 4.1.1. As result of the
quantification from elemental analysis of sulfur we get 90 to 100 µmol amino groups per
gram glass beads.
8 7 6 5 4 3 2 1
13 - 15
23'
2322'
22
20,21
18,1916,17
1210,11
7 - 9
4 - 6
31,2
N
O NNO
O
O
co
co
(2)(1)(4)
(3)
chemical shift (ppm)
D2O
Figure 4-16. In case of 1H-NMR spectra of the phthalimide co-units the aromatic phthalic hydrogen
are located in region (1) whereas the peak ranges of the hydrogen next to the amide groups (2) and the
N-methyl hydrogen of the dimethyl acrylamide (3) are closely connected.
4.2.2 Poly(N,N-dimethyl acrylamide-co-N-methacryloyl-β-alanine
succinimide ester)
Substrates with poly(N,N-dimethyl acrylamide-co-N-methacryloyl-β-alanine succinimide
ester) (P(DMAA-co-MAC2ae)) have a hydrophilic, water swellable polymer layer combined
with a functional group that reacts well with amino groups. As amino groups are present in
many biological molecules, this opens interesting perspectives. In a copolymer with
P(DMAA) the succinimide C=O stretching gives slightly shifted signals at 1817, 1786 and
1740 cm-1 (Figure 4-17).
4 Grafting of Functional Copolymers onto Silica Surfaces 109
4000 3000 2000 1000
(ae)
ν
ν
ν
(SiOSi)(C=O)
(CH)
ν
tran
smitt
ance
(a.u
.)
wavenumber (cm-1)
Figure 4-17. DRIFT spectrum of grafted P(DMAA-co-MAC2ae) on LC700-ME.
The content of functional groups cannot be unambiguously obtained by 1H-NMR analysis
of the “free” P(DMAA-co-MAC2ae) copolymer because the N-methyl hydrogen of P(DMAA)
give a broad and intense signal between 3.0 to 2.6 ppm that superimposes the succinimidyl
hydrogen peaks between 3.1 to2.8 ppm (Figure 4-18).
8 7 6 5 4 3 2 1
NNHO
co
co
O
O ON
O
O19',20'
19,20
17,18 15,16
12-1410,11
4 - 6
7 - 9
31,2
(1) (3)
(2)
chemical shift (ppm)
DMSO
Figure 4-18. 1H-NMR spectrum of free P(DMAA-co-MAC2ae); the results show that the functional
succinimide ester units cannot be distinguished from the dimethyl acrylamide units because the peaks
of the active ester (1) have nearly the same shift as the N-methyl proton peaks of the main subunits (2).
110 4 Grafting of Functional Copolymers onto Silica Surfaces
Figure 4-19. Fluorescence detection (detection wavelength from 671 nm to 693 nm) on P(DMAA-co-
MAC2ae) monolayers and P(DMAA) monolayers on glass beads after treatment with DY-635-NH2.
If the fluorescence of the different samples is recorded, the difference between a reactive
polymer monolayer functionalized with activated ester groups and an inert homopolymer
monolayer becomes evident. Both monolayers on the same substrate are exposed to a
solution of DY-635-NH2. After filtering off and washing the solids the glass beads with the
P(DMAA) homopolymer layer give no fluorescence response, only some reflection from the
glass surface (Figure 4-19b). The P(DMAA-co-MAC2ae) copolymer layer, however, reacts
with the amino dye, binds it covalently (Figure 4-19a), which leads to a strong fluorescence
of the resulting material if illuminated with light of an appropriate wavelength.
4 Grafting of Functional Copolymers onto Silica Surfaces 111
4.3 Discussion & Conclusion Copolymers As a first example, four different copolymer systems have been used for monolayer
formation on silica substrates to demonstrate the potential of this grafting system. We have
the ability to incorporate functionalities into the surface-attached polymer monolayer by
using appropriate comonomers in the polymerization process. To characterize these
copolymer systems the substrates with the surface-attached copolymer monolayer as well as
the free copolymers have been examined by various techniques. Basic monomers and
functional monomers in the copolymer layers generate specific FT-IR bands what gives an
easy handle to distinguish between all systems presented here. Elemental analysis and XPS
of the polymer loaded substrates and 1H-NMR spectroscopy of the free monomer give an
idea of the composition of the copolymers and the loading of functionalities can be
calculated. Statistical distribution of the different repeat units inside the generated
copolymers is generally recognized for styrene acrylic copolymers[198-201] and for systems
built from acrylic or methacrylic monomers[60, 202] always assuming monomers without
extraordinary steric or electronic hindrances. General accessibility and activity of the desired
functionalities within the copolymer layer have been successfully proven by different means,
qualitively as well as quantitively. The layer composition concerning the subunits can
controlled by the monomer quantities in the initial polymerization solution. When realizing
copolymer monolayers insufficient monomer solubility can be a limiting factor for the
incorporation of certain monomer species.
Somewhat delicate is the polymeranalog step converting the phthalimide into a primary
amine. With the right choice of reagent and conditions as identified with experiments with
free polymer and 1H-NMR analysis this conversion can be carried out quantitatively using
hydrazine (9.5.2). We identified that there are differences between theoretical and actual
value of the amine loading δ(NH2) for substrates with a high polymer load and small pores.
There are two possible explanations that were not explored any further: First the
deprotection of the amine group may be incomplete because the agent is not able to diffuse
into pores that got clogged with polymer. The second reason may be that because of clogged
pores the quantification of amino groups with the DMT-disulfide label is not completely
possible.
That a relatively high number of functionalities, amino groups as well as activated ester
groups, within the surface attached copolymer monolayer are accessible and reactive is
112 4 Grafting of Functional Copolymers onto Silica Surfaces
shown by the various experiments with different colored dyes and fluorescence labels
analyzed by (fluorescence) microscopy.
The amino functions in the copolymer monolayer are successfully used for binding
monomers or ATRP starters in order to perform a second polymer grafting step
subsequently as radical solution polymerization or surface polymer started ATRP. These
experiments have shown that the ATRP as a “grafting-from” technique boosts the graft
density by a factor of 20 compared with our standard copolymerization with immobilized
monomers. Besides this advantage the drawback of the ATRP is the problem of getting rid of
unwanted green color of the silica substrates due to copper complexes. Nevertheless both
polymerization paths give us the ability to graft a second polymer layer onto the first. This
second layer may be of different chemical nature or bear different functional groups. The
ATRP approach is of interest when the first polymer layer just acts as compatibilizer and has
to be masked by the second polymer layer for the final usage.
In a next step application possibilities have to be realized by using the final functionalized
spherical substrates in different chemical experiment sets and environments. Of great
interest are solutions for carrier materials for catalyzed chemical reactions and
biotechnological processes where the reactive sites are located on solid substrates.
5 Applications for Functional Copolymers on Silica Substrates 113
5 Applications for Functional Copolymers on Silica
Substrates
5.1 Enzyme Immobilization Target of the set of experiments with glass beads having a monolayer of copolymer with
active ester units (GB80-AA-P(DMAA-co-MAC2ae(10%))) and glucose oxidase (GOD) is to
explore the immobilization behavior of a common enzyme onto these tailor-made substrates.
The active ester units on the supports surface are capable of linking covalently to the amino
group of lysine units within the amino acid sequence of the protein. Different aspects of the
enzyme immobilization like washing stability, immobilization time and enzyme
concentration during immobilization were subject of the following investigations.
Figure 5-1. Three steps for the enzyme experiments: a) preparation of the support material, b)
immobilization of the enzyme (yellow) and c) measurement of the substrate (blue) conversion with an
assay procedure.
Generally the experiments on enzymes can be divided into three steps: The first step is to
prepare the glass beads according to 4.2.2, to get the functionalized support material (Figure
5-1a). Afterwards the important immobilization step of the enzymes takes place in aqueous
media (Figure 5-1b).
During this reaction step procedure variations are made to investigate the immobilization
behavior of GB80-AA-P(DMAA-co-MAC2ae(10%)) and GOD. Right after the immobilization
the GOD assay is carried out to determine the enzyme activity (Figure 5-1c). To make the
important enzyme immobilization step visible, at least qualitatively, for one immobilization
114 5 Applications for Functional Copolymers on Silica Substrates
experiment dyed GOD was used and analyzed by a fluorescence reading device (Figure 5-2).
DY-635-NH2 reacted with the carboxyl groups in the enzyme structure of GOD forming the
dyed species.
Figure 5-2. Fluorescence labeled glucose oxidase immobilized on glass beads with P(DMAA-co-
MAC2ae) copolymer monolayer visualized by a biochip fluorescence reader.
5.1.1 Glucose Oxidase Assay
The first task while observing enzymes is to track their activity using a quantitative
visualization method. These so-called assays allow to quantify the enzymes activity. It is
important that activity of the enzymes is the limiting factor in the assay and that there is
enough substrate for the enzyme to convert. In our case we use glucoseoxidase. It is a
dimeric protein with a molecular weight of around 160000 g·mol-1 containing the cofactor
flavin adenine dinucleotide (FAD). Each enzyme unit has two FAD-sites which are not
covalently bound to the enzyme. GOD transforms β-D-Glucose into D-Glucono-1,5-lactone
and forms hydrogen peroxide. The FAD cofactor produces from oxygen present in the
aqueous (buffer) solution via an intermediate the hydrogen peroxide. 1 mole hydrogen
peroxide is used by the horseradish peroxidase, which is also present in excess, to oxidize 2
moles of 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) dye (ABTS) from the colorless
ABTS2- species into the colored ABTS- species (Figure 5-3) which has an absorption
maximum at 405 nm[203-206].
5 Applications for Functional Copolymers on Silica Substrates 115
N
SS
NN
SS O
OO
O
O
O
SSO
O
O
NN
N
S SO
O
O
O
OHOH
OH
OHO
OHOH
OH
OH
O2H2O2
-2 OH
ABTS 2- (reduced, colorless)
ABTS .- (oxidized, dark green)
β−D-Glucose D-Glucono-1,5-Lactone
peroxidase(POD)2
2
Glucoseoxidase(GOD)
N+
O
N
OH
Figure 5-3. Chemical pathway for the visualization of the GOD conversion rate with peroxidase and
ABTS.
As long as all participants except for GOD of the assay are used in excess the velocity
determining step is the conversion carried out by GOD. Thus the activity is displayed
indirectly by the absorption of the dye. The absorption maximum of ABTS- is at a
wavelength of 405 nm with an extinction coefficient of ε405 = 36.8 L·mmol-1·cm-1 [207-209].
According to Lambert-Beer’s law (equation 5-1) the chronological change in concentration of
the colored dye is directly proportional to the change in extinction (equation 5-2) recorded by
a UV/Vis spectrometer at the wavelength of the absorption maximum. The resulting graph of
the extinction as a function of time is linear. The slope represents the activity of the enzyme
and can be calculated with equation 5-3.
116 5 Applications for Functional Copolymers on Silica Substrates
dεcII
logE λ0
λ ⋅⋅=
−= equation 5-1
dε
1dt
dEdtdc
λ
λ
⋅⋅= equation 5-2
dε2
1dt
dEvλ
λ
⋅⋅⋅= equation 5-3
with
Eλ = extinction at a certain wavelength λ I = intensity of the transmitted light through the sample cuvette in W·m-2 I0 = intensity of the transmitted light through a blank cuvette in W·m-2 c = dye concentration in mmol·L-1
ελ = extinction coefficient at a certain wavelength λ in L·mmol-1·cm-1 d = distance through sample in centimeters t = time in seconds v = conversion rate in mmol·L-1·s-1
Based the chosen assay procedure and on these calculations we will have a look on the
enzyme behavior when immobilized to our polymer coated, functionalized glass beads.
5.1.2 Stability against Leaching
To get the full benefit from an enzyme immobilization, the enzyme has to stay on the
support and must be stable against leaching into the surrounding medium. On the other
hand one has to know how many washing steps have to be performed to wash away non-
bound enzymes. Therefore after the immobilization procedure (10.3.2) several portions of
enzyme-modified glass beads with the identical amounts of glass support have been washed
with buffer media once, twice, three or four times with equal amounts of buffer and
afterwards the activity has been monitored via measuring the extinction as a function of time
as it is shown in Figure 5-4. The washing procedure was kept simple by flushing, shaking
with buffer and decanting the liquid after settlement of the beads.
5 Applications for Functional Copolymers on Silica Substrates 117
0 20.0
0.5
1.0
1.5
2.0
2.5
3.0
1st wash 2nd wash 3rd wash 4th wash
extin
ctio
n (a
bs)
Figure 5-4. Extinction as a function of tim
405 nm (extinction maximum). The leachin
rate between the succeeding washing steps
There is a strong decrease of the con
decrease between steps 2 and 3 and alm
3 and 4. This first strong decrease is du
glass beads and in the pores that give
more effective ways of washing suppo
this was the most practicable one. It sh
caused by leaching of GOD from the
washing step. For the following enzy
comparable results.
v = 18.6 nmol·L-1·s-1
v = 3.8 nmol·L-1·s-1
v = 2.1 nmol·L-1·s-1
v = 2.0 nmol·L-1·s-1
4 6 8 10 12 14time (min)
e monitored by a UV/Vis spectrometer at a wavelength of
g experiments show a declining decrease of the conversion
version rate between washing step 1 and 2, a slight
ost no decrease in enzyme activity between the steps
e to residual enzyme containing solution between the
s an additional conversion of glucose. There may be
rt material but for the small samples of 10 mg each
ows that there is no reduction of glucose conversion
glass beads. The activity stays constant after the 3rd
me experiments 3 washing cycles were used to get
118 5 Applications for Functional Copolymers on Silica Substrates
5.1.3 Influence of the Immobilization Time on the Conversion
Rate of the Enzyme Modified Glass Beads
It is important to know what the appropriate span of time for the enzyme immobilization
step is to get optimal loading. Several bead samples were exposed to the enzyme solution for
varying periods of time. After washing, the activity of the samples were measured. For an
immobilization time span variation from 10 minutes to 4 hours no significant change in
activity nor any trend in conversion rate change was observed (Figure 5-5). Under the given
conditions the conversion rate of the immobilized enzyme just varies between 2 and
3 nmol·L-1·s-1.
0 2 4 6 8 10 12 140.0
0.2
0.4
0.6
0.8
1.0
10 min 30 min 60 min 120 min 240 min
extin
ctio
n (a
bs)
time (min)
v = 2.3 nmol·L-1·s-1 v = 1.9 nmol·L-1·s-1 v = 3.0 nmol·L-1·s-1 v = 2.5 nmol·L-1·s-1 v = 2.0 nmol·L-1·s-1
Figure 5-5. Extinction as a function of time monitored by a UV/Vis spectrometer at a wavelength of
405 nm. The variation of the immobilization time at equal enzyme concentrations in the
immobilization solution shows no significant change or trend regarding the conversion rate.
5 Applications for Functional Copolymers on Silica Substrates 119
5.1.4 Influence of the Immobilization Concentration on the
Conversion Rate of the Enzyme Modified Glass Beads
Here we deal with the variation of the enzyme concentration of the solution and its
influence on the enzyme immobilization. In the initial step the activated ester sites on the
glass beads are exposed to different concentrations of enzymes in solution. With a higher
enzyme concentration the probability of binding an enzyme successfully to the surface is
higher than with lower concentration. It is observed that the activity increases with increase
of the GOD concentration. With our standard GOD concentration of 10-6 mol·L-1 we get an
activity of around 2 nmol·L-1·s-1 as it was observed in the previous experiments.
0 2 4 6 8 10 12 140.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
extin
ctio
n (a
bs)
time (min)
2 *10-6 M 10 *10-6 M 100*10-6 M
v = 1.8 nmol·L-1·s-1 v = 15.1 nmol·L-1·s-1 v =100.9 nmol·L-1·s-1
Figure 5-6. Different GOD concentrations in the immobilization solution have an influence on the
resulting conversion rate of the enzyme-support-system
With two tenfold increases in GOD concentration of 10-5 mol·L-1 and 10-4 mol·L-1 we get
according to Figure 5-6 conversion rates of 15 nmol·L-1·s-1 respectively 101 nmol·L-1·s-1. This
means an increase in glucose conversion rate by a factor of 50 while increasing the enzyme
concentration in solution by a factor of 100.
Combined with the results from the immobilization time variation where no influence was
observed, we get the picture that the enzyme immobilization in the aqueous buffer system is
not diffusion controlled. Under given conditions many of the active ester moieties hydrolyze
120 5 Applications for Functional Copolymers on Silica Substrates
before they can react with a possible enzyme site. The probability of getting hydrolyzed is in
an aqueous solution much higher than catching an enzyme because they are highly diluted.
Therefore a higher enzyme concentration in aqueous solution increases this reaction
probability and leads to higher enzyme loadings on the glass support and finally to
increased conversion rates using the material in the assay procedure. To get an idea how
much GOD actually has been immobilized on the glass beads in the case of using a 10-
4 mol⋅L-1 GOD solution the GOD treated glass beads (51 mg and 150 mg) were heated to
reflux in hydrochloric acid. The enzyme decomposed to its single amino acids which, after
filtering off the remaining solids, can be quantified by liquid chromatography[210] (cf.
10.3.6). The measured values give the concentration of each amino acid in the analyte
solution which was for both samples 1 mL of buffer solution so that the amount of the amino
acids for each sample was calculated by dividing by 1000. As we know the amount of each
amino acid in one GOD molecule[211] and the mass of the modified glass beads the amino
acid were cleaved off from, we are able to calculate the specific GOD loading according to
equation 5-4:
1000mN
cLGOD ⋅⋅= equation 5-4
with
LGOD = specific loading of GOD on the glass beads in nmol⋅g-1
c = amount of the amino acid in the analyte solution in nmol, determined by LC N = number of the amino acid units in 1 mol of GOD m = mass of the analyzed, GOD modified glass beads in g
5 Applications for Functional Copolymers on Silica Substrates 121
Because of the low concentrations of the single amino acids in the analyzed solution only
the three most frequent amino acids are listed and used for calculation of the immobilized
GOD amount on the glass beads (Table 5-1).
Table 5-1. Listed results from amino acid analysis of GOD hydrolysates.
amino acid concentration amino acid sample 1
(51 mg GB) (µmol⋅L-1)
concentration amino acid sample 2
(150 mg GB) (µmol⋅L-1)
# of amino acid units
in one GOD
molecule[211]
calc. GOD amount sample 1 (nmol⋅g-1)
calc. GOD amount sample 2 (nmol⋅g-1)
glycine 21.4 57.9 114 3.7 3.4
serine 17.3 42.8 74 4.6 3.9
glutamic acid 11.4 39.3 60 3.7 4.4
Both samples give the same picture so that an overall average amount of 4.0±0.5 nmol
GOD per gram glass beads is determined as specific loading of GOD on GB80-AA-P(DMAA-
co-MAC2ae(10%)) using an immobilization solution with 100 µmol⋅L-1 GOD. Regarding the
absolute amounts of GOD we used for our immobilization procedure (cf. 10.3.6) in solution
(10-6 mol GOD in the whole solution volume) and the amounts we finally immobilized on
sample 1 (0.2·10-9 mol GOD on 50 mg solid substrate) and on sample 2 (0.6·10-9 mol GOD on
150 mg solid substrate) show that the immobilization quota is below 1%. With 6.022·1023
GOD molecules per mole, an amount of 4.0 nmol GOD per gram glass beads and their
specific surface of 80 m2·g-1 a density of 5·1012 GOD molecules per m2 or 5 GOD molecules per
µm2 is calculated. It is also shown, that an increase of GB80-AA-P(DMAA-co-MAC2ae(10%))
in equal volumes of GOD solution does not influence the final specific GOD loading. The
action to take to improve the immobilization quota is to put as many glass beads into the
immobilization solution as possible to offer the largest number of activated ester moieties per
volume that is possible. That makes immobilization of GOD on the functionalized glass
beads more likely as we already mentioned the competition with the hydrolysis reaction of
the MAC2ae moieties in aqueous solutions.
122 5 Applications for Functional Copolymers on Silica Substrates
5.2 Biomolecule Immobilization The use of immobilized biomolecules is important especially in the design of analytical
methods. Therefore we have tested if solids that were equipped with a functional copolymer
monolayer are capable to bind molecules like biotin or deoxyribonucleic acid fragments
(oligonucleotides) for further use in analytical assays.
5.2.1 Biotin Labeling
Biotin together with avidin is a well known and widely used reagent for many different
analytical purposes. Main field of use is in enzymatic immunoassays[212-214] for detection
of a wide variety of substances, e.g. nucleic acids[215], proteins[216] or food additives[217].
There are also methods for immobilization of nucleotides using a biotin system[218] which
generates the possibility of designing DNA/RNA-chips. Because of the high potential of
biotin systems a short straight forward experimental study shall give an answer to the
question if the active ester sites in a copolymer monolayer as we use it are capable of binding
the biotin and if a more complex reaction system with subsequent reaction steps can be
applied to our copolymer monolayer equipped substrates.
In a first step the functionalized glass beads (Figure 5-7a) are biotinylated by reacting
N-hydroxysuccinimido-biotin in solution with the amino units in the copolymer layer
(Figure 5-7b) on the support surface. Afterwards the solids are treated with an avidin
solution to get glass beads where bound avidin units offer unoccupied binding sites on the
copolymer surface (Figure 5-7c). In a final step from solution biotin-fluorescein binds to the
avidin on the glass beads (Figure 5-7d). If all binding steps are successfully completed
visualization by excitation with a fluorescence reader will show fluorescence of the bound
dye molecules (Figure 5-8).
5 Applications for Functional Copolymers on Silica Substrates 123
Figure 5-7. Steps of a fluorescence labeled biotin assay on glass beads: a) Preparation of the support
material, b) immobilization of N-hydroxysuccinimido-biotin onto the active ester functions, c) linking
the biotin to streptavidin and d) labeling the streptavidin with fluorescence (fluorescein) labeled biotin.
The biotin assay procedure was done on glass beads with varying specific surface area and
with a polystyrene monolayer containing different amounts of amino groups (~ 80 and
~ 150 µmol⋅g-1). The two basic polymer systems (PS & P(DMAA)) containing amino groups
were also compared. In the first comparison it can been seen that with higher content of
functional groups and a higher specific surface area the glass beads are brighter when the
dye bound to the copolymer is fluorescing (Figure 5-8b). A direct quantification via
fluorescence intensity is not possible because of the non-uniform geometry of the beads. In a
second experiment beads with hydrophilic and hydrophobic surface layer are compared.
Interestingly the beads equipped with the more hydrophobic P(S-co-AC3amine) layer show
higher fluorescence intensity after treatment with the biotin-fluorescein assay (Figure 5-9a)
than the beads with a more hydrophilic P(DMAA-co-AC3amine) layer (Figure 5-9b) though
the determined amino contents are nearly the same.
124 5 Applications for Functional Copolymers on Silica Substrates
Figure 5-8. GB80-AA-P(S-co-AC3amine(10%)) (a), GB250-AA-P(S-co-AC3amine(20%)) (b) as glass
bead substrates treated with the aforesaid assay and visualized by fluorescence reading.
Figure 5-9. Fluorescence reading of a substrate with different amino-functionalized polymer layers
treated with the biotin assay: GB80-AA-P(DMAA-co-AC3amine(10%)) (a), GB80-AA-P(S-co-
AC3amine(10%)) (b)
The expected behavior would have been that using polar solvents for the assay procedure
would result in better swelling of the more polar P(DMAA co-AC3amine) monolayer while
carrying out the reactions. Hence the functional groups of the copolymer layer should be
better accessible and the fluorescence would be more intense than for the fluorescein treated
glass beads with a P(S-co-AC3amine) monolayer. This unusual behavior we found may be
due dye-polymer interaction causing fluorescence quenching with a reduced fluorescence
intensity as result like it is suggested by Haugland et al. [219]. Interactions of dyes with
polymers are described by Neumann et al. [220] and it is shown that these interactions
significantly influence the fluorescence behavior of dyes absorbed on or bound to polymers
in terms of a wavelength shift of the adsorped and emitted light.
5 Applications for Functional Copolymers on Silica Substrates 125
5.2.2 DNA Immobilization & Hybridization
An important molecule for biological and medical purposes is deoxyribonucleic acid
(DNA), particular single-stranded oligo nucleotides. DNA molecules attached to planar or
particular SiO2 surfaces are important for DNA analysis. We realize in a setup with glass
beads with an active ester copolymer layer (Figure 5-10a) an oligo nucleotide immobilization
(Figure 5-10b) and subsequent hybridization with an antisense oligonucleotide which
contains a fluorescence label (Figure 5-10c).
For the immobilization step GB80-AA-P(DMAA-co-MAC2ae(10%)) or LC700-ME-
P(DMAA-co-MAC2ae(10%)) as modified supports are used. An amino functionalized oligo
nucleotide with 23 specific bases and 18 thymine units (NH2-DNA) is bound to the active
ester units in the surface-attached copolymer monolayer. To ensure that the immobilization
step is successful, parallel experiments under same conditions with coupling control DNA
(CC-DNA) were carried out and the fluorescence was checked. CC-DNA is a bifunctional,
single-stranded oligo nucleotide with an amino group at the 3’-end and a Cy5 label at the
5’-end.
Figure 5-10. Steps from a solid substrate with a surface-attached copolymer layer with activated ester
groups (a) via immobilized oligo nucleotides (o.n.) (b) to supports with hybridized oligo nucleotides
(h.o.n.) indicated by Cy5 fluorescence (c)
126 5 Applications for Functional Copolymers on Silica Substrates
Upon completing the coupling control experiments solids with a homopolymer P(DMAA)
layer exhibit no fluorescence whereas those equipped with a functional P(DMAA-co-
MAC2ae) layer show fluorescence (Figure 5-11a, b). The beads where the CC-DNA has the
chance to bind to the copolymer layer show bright fluorescence whereas the beads with the
inert homopolymer layer just show minimal reflectance. For the solids with surface
copolymer bound sense DNA treatment with antisense-DNA with 23 bases (AS-DNA) is the
next step. The detailed sequences of the used oligonucleotides are listed in Table 10-1 in the
experimental section. Within this step the AS-DNA hybridizes with the covalently bound
NH2-DNA and binds the Cy5 label to the copolymer monolayer on the support. This leads to
blue colored solids, as Cy5 is a blue dye,. indicating a successful hybridization. Also
examination with the fluorescence reading device reveals bright fluorescence on the beads
(Figure 5-11c, Figure 5-12).
Figure 5-11. Fluorescence detection on glass beads: GB80-AA-P(DMAA) treated with CC-DNA (a),
GB80-AA-P(DMAA-co-MAC2ae(10%)) treated with CC-DNA (b) GB80-AA-P(DMAA-co-
MAC2ae(10%)) treated with NH2-DNA and AS-DNA.
Figure 5-12. False color picture of glass beads with successfully hybridized oligo nucleotides attached
to the copolymer monolayer show fluorescence from Cy5 labeled anti-sense oligo nucleotides.
5 Applications for Functional Copolymers on Silica Substrates 127
Again quantification of the surface fluorescence is difficult because of interfering geometry
of the beads and slight intensity differences between single beads. For glass beads and silica
similar results were observed. Because the silica particles are very small they can hardly be
detected even with a fluorescing surface (Figure 5-13, right bottom).
Figure 5-13. Fluorescence image of glass beads GB80-AA-P(DMAA-co-MAC2ae(10%)) and silica
LC700-ME-P(DMAA-co-MAC2ae(10%)) after successful DNA immobilization & hybridization.
5.3 Catalyst Immobilization Another huge application field for functionalized solids besides biomolecule
immobilization is the immobilization of organometallic species as catalysts for synthesis. All
experiments described here have been carried out in close collaboration with the group of
Prof. Dr. W. Bannwarth. After preparation of the amino modified glass beads further
reaction steps have been carried out by H. Glatz and F. Michalek.
Figure 5-14. Steps for the immobilization of a Ruthenium-metathesis catalyst onto a silica support:
Immobilization of a Hoveyda-type ligand (1) onto the support (b). Ligand exchange of Grubbs catalyst
(2 & 3) binds the species onto the support (c & d).
128 5 Applications for Functional Copolymers on Silica Substrates
Starting point for the catalyst immobilization are glass beads (GB80 & GB250) with a
surface-attached layer of P(S-co-AC3amine) (Figure 5-14a). The amines form an amide link
with the carboxylic acid groups of a Hoveyda-type ligand (Figure 5-14, 1) using N,Nʹ-
dicyclohexyl-carbodiimide (DCC), tert-butanol and Huenig’s base. The ligand is covalently
bound to the support copolymer surface (Figure 5-14b)[221]. Then free Grubbs catalysts of 1st
(Figure 5-14, 2) and 2nd generation (Figure 5-14, 3) in solution is immobilized onto the
support using copper(I)chloride for activation and dichloromethane as solvent giving bound
Hoveyda-type catalyst of 1st (Figure 5-14c, Figure 5-15a) and 2nd generation (Figure 5-14d,
Figure 5-15b)[188].
Figure 5-15. Glass beads with immobilized Ruthenium catalyst species within the functional
copolymer monolayer give a specific color. GB80-AA-P(S-co-AC3amine) as carrier material for Grubbs
catalyst 1st generation (a) and Grubbs catalyst 2nd generation (b) is shown.
To check the performance of this new catalyst hybrid material an exemplary ring closing
metathesis (RCM) reactions were run. This benchmark reaction is displayed in Figure 5-16
where the RCM of N,N-diallyl tosylamide is done by a Ruthenium-metathesis catalyst.
Investigations on olefin metathesis catalysts immobilized on silica have shown that in
organic solvents the immobilized catalyst can be released to perform the reaction as catalytic
carbene species and afterwards may be recaptured by the immobilized ligand[222].
Figure 5-16. RCM reaction of N,N-diallyl tosylamide (a) to N-tosyl-3-pyrrolin (b) catalyzed by an
active Ru-metathesis species.
5 Applications for Functional Copolymers on Silica Substrates 129
In the first RCM experiments the solid phase-bound catalysts are used in dichloromethane
as organic solvent under reflux conditions for 3 h with an catalyst amount of 5 mol% related
to the amount of N,N-diallyl tosylamide. The first two RCM cycles show quantitative
conversion for both catalyst generations. The third cycle reveals for both, immobilized
Grubbs catalyst of 1st and 2nd generation, less activity due leaching of small amounts of the
Ru-metathesis species. But the conversion is for both cases still above 95% conversion. After
each cycle the immobilized catalyst can be easily filtered off the reaction solution and used
for another reaction cycle. To reload the leached solid phase it has to be placed into a
solution of free Grubbs catalyst. There it regains the original loading with surface copolymer-
bound Ru-metathesis species.
After these first promising experiments in organic solvents further experiments where
supercritical carbon dioxide is used as solvent for the RCM reaction were carried out. In
these experiments our functionalized silica polymer hybrids with immobilized Grubbs
catalyst species (Hoveyda type) show even better results concerning conversion and
leaching. The results of these experiments are published and discussed in detail in [188].
130 5 Applications for Functional Copolymers on Silica Substrates
5.4 Discussion & Conclusions Applications The first part of the applications section is concerned with enzyme immobilization on solid
supports. Our investigations are based on the well-known enzyme glucose oxidase (GOD)
and we like to investigate its immobilization behavior on glass beads modified with a
reactive, hydrophilic copolymer layer. In a first set of experiments where the substrates with
the immobilized GOD are washed to test the stability against leaching, it is recognized that
after several washing cycles there is no loss in rate of the conversion of glucose. This
confirms the picture of covalently bound enzymes that are not easily leached out by flushing
the enzyme substrate. The behavior encountered upon variation of the immobilization time
suggests that the final amount of enzyme on the surface is reached fast within a few minutes.
Allowing for additional immobilization time does not add to the activity of the enzyme
modified support as it would be expected for a clearly diffusion based process where the
reactant molecules have enough time to approach the reacting sites. The binding of the
enzyme towards the N-succinimide ester competes with the hydrolysis reaction of the active
ester in the aqueous immobilization solution. Because the hydrolysis step is irreversible
enzymes can only attach as long as the active ester moieties are intact. For the enzyme the
probability of finding a still activated ester site decreases with time. The surrounding
aqueous buffered solution (pH = 7.4) quenches active ester groups quickly before they can
bind any enzyme. The observed conversion rate increase while increasing the initial enzyme
concentration in the immobilization solution is due to the fact that in the initial state more
enzyme entities encounter more intact active ester groups. The probability of binding GOD
covalently to an active ester site increases with GOD concentration while the competitive
hydrolysis reaction of the N-succinimide esters is still quick. This corresponds with the
finding that the increase the glucose conversion rate is not proportional to the increase of
GOD in the immobilization solutions. Caused by the fact that the amount of enzyme
molecules is low (≤ 10-4 mol·L-1) and the active ester moieties are quite prone to hydrolysis
under the given conditions only few enzyme molecules get immobilized. This result is
underlined by the experiments where the immobilized GOD is hydrolyzed to quantify its
amount via liquid chromatography. The immobilization quota is below 1% and because the
aqueous system and the pH-value are necessary to retain the structure and activity of the
enzyme this immobilization quota could only be increased by increasing the amount of
functionalized glass beads in the GOD solution. It is clearly shown that immobilization of
enzymes with our covalently bound, reactive copolymer system on glass beads is feasible.
5 Applications for Functional Copolymers on Silica Substrates 131
We get stable systems with firmly fixed enzymatic entities showing significant activity and
we are able to control the amount of loaded enzyme. To get detail information about the
interaction between enzymes and our copolymer monolayer system it will be necessary to
carry out further experiments where the specific activity of free enzymes and enzymes
bound to the polymer-substrate system will be compared. It is probable that retention of the
enzyme structure is enhanced by a polymer monolayer acting like a cushion on the
immobilization substrate. Also an interesting point for further investigations in this field is to
look if there is a dependence between the active ester loading/density and possible enzyme
loading or specific enzyme activity.
The experiments with the amino functionalized polymer layers on glass beads and the
biotin/avidin assay have shown that more complex labeling reactions with several steps can
be successfully carried out with both basic systems, the PS and the P(DMAA) system. In
direct comparison with the P(DMAA)-amine system, the PS-amine system shows the
brighter fluorescence. This is a surprising behavior because the used solvents in the assay
pathway and the affinity of biotin towards hydrophilic environments suggest a better
swellability of the P(DMAA) layer, a better access to the amino reaction sites and a better
penetration of the layer by the biotin and streptavidin. These factors should finally result in a
higher dye density within the copolymer layer. A possible factor that influences the
fluorescence is the interaction between polymer layer and dye. As we have seen in dye
labeling experiments before (Figure 4-8 & Figure 4-12) this interaction provokes shift of
extinction maxima and hence the fluorescence intensity in the observed range. Also polymer-
dye interaction quenching the fluorescence and reducing the observed intensity might be
possible. For a detailed understanding of functional group distribution and accessibility
within polymer monolayers of different polarity further efforts are necessary. Overall, the
feasibility of our functional modified beads for biotin based systems is proven and the
system may be adapted to the particular application.
The complementary pairing of an oligo nucleotide via hydrogen bonds with the
complement forms a reversible link between two DNA strands. Using elevated temperatures
the double-strand can be molten and is split into two complementary species again. In our
experiments oligonucleotides are efficiently immobilized on glass beads and silica gel
equipped with a functional copolymer layer with activated ester moieties. It is shown that
oligonucleotides can be bound specifically to the support and undergo hybridization
reactions in the polymeric environment built by the copolymer monolayer. This possibility
132 5 Applications for Functional Copolymers on Silica Substrates
offers the use of our hybrid system for the whole range of applications in chromatography
and analytics based on the specificity and reversibility as outstanding features of nucleotide
sequences. In addition the surface bound polymer monolayer system is not restricted to
spherical and porous systems but can also be applied to flat substrates as we will show in the
next chapter. This allows one to create DNA-chips based on covalently bound copolymer
monolayers.
For the last application we leave the field of biomolecules and life science and get into
inorganic catalysts for organic synthesis. By covalent attachment of ligands onto the
functional copolymer monolayer it is possible to immobilize transition metal complexes with
catalytic behavior. In our case the immobilized Grubbs catalysts showed quantitative
conversion of the substrate and little leaching during ring closing metathesis reactions in
organic solvents. Also simple reloading of the support with catalyst species can be observed.
This example shows that we have here a potent system offering a large number of
possibilities. It has to be explored which ligands, catalytic species, catalyzed reactions and
reaction conditions can be used with our system. In most cases the polymerization step in the
hybrid material buildup allows tailoring the properties of the interacting layer. This is also
true for the other application fields. For all applications in common are the following
advantages: First to have an inert, non-swellable, easy to handle SiO2 core, second a tailor-
made copolymer monolayer which covers the core and allows to control the interaction with
the environment, third high loading of functional groups within the copolymer and forth
improved mobility of functional groups and immobilized species on a swellable “polymer
cushion” attached to a rigid substrate surface. The advantage last mentioned is important
because many chromatographic and catalytic reactions take place in packed bed reactors
where flowing fluids cause significant shear forces on the surface of the solids. These shear
forces may rip off any immobilized functionality or species when just being surface-attached
by a short, more or less rigid spacer molecule. Having a semi-mobile copolymer layer could
absorb the shear forces by polymer segment movement[223] and could prevent losing active
functionalities due to shear induced rupture.
6 Polymer Systems on Silicon Wafers 133
6 Polymer Systems on Silicon Wafers
6.1 Grafting of Poly(N,N-dimethyl acrylamide) Layers onto
Silicon Wafers through Surface-attached Monomers In previous chapters the surface modification of silica substrates, silica gel and glass beads
by using a “grafting-through” approach was used. Bulk polymerization of styrene on silicon
wafers modified with methacrylate groups was investigated by M. Bialk and O.
Prucker[153]. To close the gap we present results dealing with the radical polymerization of
N,N-dimethyl acrylamide in the presence of monomers attached to silicon wafer surfaces.
The monomers were immobilized using trialkoxy silane anchors. The experiments were
carried out in DMF as solvent because polymerization in bulk DMAA leads into problems
caused by gelation. This effect is for DMAA much more pronounced and occurs already at
much lower conversion than it is the case for styrene. The parameters that were kept
constant are polymerization temperature (60 °C), polymerization time (16 h), AIBN
concentration (3 mmol·L-1) and monomer concentration (3.2 mol·L-1 DMAA). The
experiments are supplemented with similar ones but using styrene as monomer. There we
have as polymerization conditions a temperature of 60 °C, a polymerization time of 24 h and
an initiator concentration of 3 mmol·L-1 AIBN. Variation of single parameters is described in
the following paragraphs. For each set parameter two different wafers are measured with at
least three surface points to get representative values and to calculate the standard deviation.
6.1.1 Influence of Monomer Concentration
The first parameter to investigate is the monomer concentration because it has the
strongest influence on grafting P(DMAA) on silica (3.7.3). As it is known from the
experiments with silica the polymerization solution tends to get viscous and gel at early
stages when conversion has not advanced very far. Using wafers we do not have the
necessity to stir or shake the polymerization vessel in order to prevent concentration or
temperature gradients. After 16 h polymerization time the solutions that started with a
DMAA concentration below 2 mol·L-1 are viscous, but still o.k. Above that concentration the
solution gels. A careful and extensive Soxhlet hot extraction frees the wafer from any non-
covalently bound P(DMAA). This set of experiments gives a clear picture: the higher the
monomer concentration the thicker the P(DMAA) monolayer (Figure 6-1).
134 6 Polymer Systems on Silicon Wafers
0 2 4 6 8 100
10
20
30
40
laye
r thi
ckne
ss (n
m)
c(M) (mol/L)0 2 4 6 8 10
0
10
20
30
40
Figure 6-1. Increase of the P(DMAA) layer thickness with increasing DMAA concentration compared
to similar experiments with styrene (grey values).
At low monomer concentration where the polymerization stays fluid throughout the
polymerization only very thin layers below 3 nm are generated whereas polymerization in
bulk DMAA leads to 30 nm layers. However, at high DMAA concentrations the samples
show a much larger variance in layer thickness. This is an indication for inhomogeneities due
to local gelation at any stage of the polymerization. P(DMAA) builds thicker layers in
comparison with PS, because at comparable monomer concentrations up to bulk
polymerization the PS layer is always thinner.
6 Polymer Systems on Silicon Wafers 135
0 2 4 6 8 100
50100150200250300350400450
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
c(M) (mol/L)
Mn P(DMAA) Mw P(DMAA)
0 2 4 6 8 100
50100150200250300350400450
Mn PS Mw PS
Figure 6-2. Molecular weight averages for the polymerization of DMAA at different monomer
concentrations in comparison with values for PS (grey values).
The measured molecular weights match those of the experiments with DMAA
concentration variation using silica as substrate. The molecular weights increase with higher
initial monomer concentrations for P(DMAA) as well as for PS (Figure 6-2). The number
averages of the molecular weight for both polymers rise linearly with increasing monomer
concentration. The molecular weight distribution for P(DMAA) is much broader than for PS
and the P(DMAA) from bulk polymerization could not be measured properly by our GPC
system because the molecular weight of the polymer was beyond the exclusion volume.
136 6 Polymer Systems on Silicon Wafers
0 2 4 6 8 100.00
0.05
0.10
0.15
graf
t den
sity
(µm
ol/m
2 )
c(M) (mol/L)0 2 4 6 8 10
0.00
0.05
0.10
0.15
Figure 6-3. Graft densities for P(DMAA) on silicon wafers after polymerization with varying
monomer concentrations compared to those of PS on wafers (grey values).
In contrast to graft polymerization on porous substrates P(DMAA) and PS show on MPS-
modified silicon wafers that the increased loading/layer thickness is not altered by higher
molecular weights. For both polymer systems graft densities increase on polymerization
with higher monomer concentrations. For P(DMAA) this effect is more pronounced than for
PS (Figure 6-3). This behavior is strong evidence for a difference between our polymerization
system with surface attached monomers and pure “grafting-to” systems and will be
discussed in paragraph 6.1.4.
6.1.2 Polymerization Time Influence
For the development of the P(DMAA) layer thickness on silicon wafers which is equivalent
with grafted polymer amount on porous substrates we get a bit different result compared
with the latter ones. On glass beads and silica gel the plateau of the final polymer load was
reached quite fast in 30 minutes. After 2 hours of polymerization a level of around 5 nm layer
thickness is reached (Figure 6-4). Apparently for polymerization times above 15 hours the
layer thickness increases and reaches a maximum of 7 nm. With the chosen monomer
concentration of 1/2 (v/v) DMAA/DMF in order to get significant layer thicknesses the
polymerization already suffers from gelation which renders the values obtained not reliable
because of inhomogeneous covering of the wafer.
6 Polymer Systems on Silicon Wafers 137
0 4 8 12 16 20 240123456789
10
laye
r thi
ckne
ss (n
m)
polymerization time (h)
Figure 6-4. Layer thicknesses of P(DMAA) in the course of polymerization.
The development of the molecular weight distribution with polymerization time shows a
similar behavior as observed during the experiments on the porous substrates: In the
beginning we have a higher molecular weights which reach their final level after 4 hours
with a number average of around 60000 g·mol-1 and a weight average of the molecular
weight distribution of around 210000 g·mol-1 (Figure 6-5).
0 4 8 12 16 20 240
50
100
150
200
250
300
350 Mn Mw
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
time (h)
Figure 6-5. Molecular weight averages of P(DMAA) for different periods of polymerization time.
138 6 Polymer Systems on Silicon Wafers
From the layer thickness and the molecular weight the graft density can be calculated. In
the case of P(DMAA) on silicon wafers the graft density increases steadily with
polymerization time (Figure 6-6). Although the differences in film thickness between the
individual samples is small (± 1 nm) the trend appears to be quite stable.
0 4 8 12 16 20 240.00
0.05
0.10
0.15
0.20
graf
t den
sity
(µm
ol/m
2 )
polymerization time (h)
Figure 6-6. Graft densities of P(DMAA) on MPS-modified silicon wafers in the course of
polymerization.
6.1.3 Variation of Initiator Concentration
A change in P(DMAA) layer thickness is observed upon initiator concentration variation.
Using AIBN concentrations in the range from 0.1 to 100 mmol·L-1 the layer thickness slightly
decreases with higher concentrations from 13 nm to 7 nm (Figure 6-7). For P(DMAA) on
porous substrates and for PS on wafers we did not see any influence of the initiator
concentration on the polymer layer thickness.
6 Polymer Systems on Silicon Wafers 139
0.1 1 10 1000
4
8
12
16
20
laye
r thi
ckne
ss (n
m)
c(I) (mmol/L)
Figure 6-7. With increasing AIBN concentration in the polymerization solution the layer thickness of
P(DMAA) on wafers slightly decreases.
As observed during the initiator variation experiments on silica the number average
molecular weight stays constant around 60000 g·mol-1 whereas the weight average has a
trend to higher values with higher initiator concentration thus leading to a broader
molecular weight distribution (Figure 6-8). Again this behavior can be explained by fast
conversion and viscosity effects leading to auto-acceleration of the polymerization reaction.
1 10 1000
50
100
150
200
250
300
mol
ecul
ar w
eigh
t (10
3 g/m
ol)
c(I) (mmol/L)
Mn Mw
Figure 6-8. Molecular weight averages for P(DMAA) after polymerization at different AIBN
concentrations.
140 6 Polymer Systems on Silicon Wafers
The factors described above lead to a decrease in apparent graft density while increasing
the initiator concentration for P(DMAA) on silicon wafers (Figure 6-9). For P(DMAA) on
porous SiO2 there was no influence of initiator concentration on graft density and for PS on
silicon wafers an opposite effect is observed. The different polymerization behavior of
DMAA with its tendency to form highly viscous polymerization solutions may lead to a high
number of unattached but blocking polymer coils for high initiator concentrations.
0.1 1 10 1000.0
0.1
0.2
0.3
0.4
graf
t den
sity
(µm
ol/m
2 )
c(I) (mmol/L)
Figure 6-9. The graft density of P(DMAA) decreases for higher AIBN concentrations.
6.1.4 Discussion & Conclusion P(DMAA) on Wafers
The impact of the varied polymerization parameters such as monomer concentration,
polymerization time and initiator concentration on the polymer loading was differently
pronounced for the single parameters. The monomer concentration variation has the
strongest influence on the polymer loading respective layer thickness like it was the case for
the graft polymerization on porous substrates. Going into more detail there are differences
between the two basic surface geometries: Porous substrates show decreasing graft density
with increasing monomer concentration whereas flat substrates have an increasing graft
density. These trends are the same as investigated with experiments on polystyrene. So this
seems to be a general behavior of systems using surface attached monomers. For a pure
“grafting-to” system one expects decreasing graft densities with higher monomer
concentration. Generated larger polymer coils attached to the surface are more effective in
6 Polymer Systems on Silicon Wafers 141
blocking the way to the surface for further polymer molecules that try approach the
surface[224]. This unusual behavior leads to the conclusion that we deal here with a system
where the “grafting-to” step is most important but the “grafting-from” component is not
negligible. For the latter step it is necessary to provide monomer to the site where the
growing chain is. The diffusion of whole polymer coils through the surface attached polymer
layer is not needed. Higher monomer concentrations supply on the one hand a better
probability of growing the already attached polymer chain further and on the other hand
represses polymer to solvent transfer reactions of the radical that terminate polymer growth.
Another factor is the surface geometry: Monomer concentration in the vicinity of flat
substrates is easier balanced with the surrounding solution than porous substrates because
they get easily clogged pores if a system like P(DMAA) in DMF shows up with high
viscosities. In polymer blocked pores fluidity is more effectively hindered and concentration
may be less balanced with the surrounding media. That is why we observe for porous
substrates for our system indeed decreasing graft densities with increasing monomer
concentrations but this influence is not as strong as for “grafting-to” systems[225]. On flat
substrates the classic “grafting-to” effect is altered in a way that the trend is reversed and we
get increasing graft densities with increasing monomer concentrations. Therefore sometimes
we call systems using surface attached monomers jokingly as “grafting-in-between” because
in fact these systems got components of both “grafting-to” and “grafting-from”. The reason
why for P(DMAA) the final layer thickness on wafers is reached later than the final polymer
load on silica is a combination of monomer balance and diffusion through the polymer layer
and stuck pores due to high viscosity. The latter cause an early final stage for P(DMAA) on
porous substrates. For the polystyrene system we get a stronger diffusion influence as the
viscosity and the propagation rate is not as high as for styrene/polystyrene. This is proven by
the fact that for PS the final layer thickness on wafers is reached faster than the final polymer
loading on porous substrates[153]. A similar picture we can draw from the results of the
initiator concentration variation although the influence is not as strong as for the monomer
variation. With higher initiator concentrations the layer thickness is reduced because the
more initiator molecules start polymer chains the earlier a certain viscosity is reached and
slows down monomer diffusion, polymer mobility and the probability of binding polymer to
the surface (Figure 6-10). For PS the viscosity under similar polymerizing conditions is less
and enables the system to level the layer thickness by diffusion resulting in equal layer
thicknesses over a range of different initiator concentrations[153].
142 6 Polymer Systems on Silicon Wafers
Figure 6-10. Variation of the initiator concentration for the polymerization of DMAA in presence of
surface attached monomers leads to different layer thicknesses/graft densities. With a lower initiator
concentration the growing chains stay mobile for a longer time and their radical and can diffuse
through the monolayer to attach to the surface (a). With a higher initiator concentration the polymer
chains lose their mobility to diffuse through the polymer monolayer earlier because of faster conversion
(b). After extraction all non-attached polymer chains that got stuck in the polymer layer but did not
attach to the surface are removed (lower pictures) and in the case of higher initiator concentration a
lower film thickness/graft density is observed.
6 Polymer Systems on Silicon Wafers 143
6.2 Microstructured Surfaces Target of the experiments described in this chapter is to explore the possibilities of forming
microstructures and micropatterns based on polymer systems using surface attached
monomers. Two different approaches with MPS-modified silicon wafers as base material are
introduced. Both are photolithographic structuring methods that use UV light in the
patterning step. The first one creates a pattern by destroying the MPS units at the surface.
The pattern image is created directly on the flat substrate as first step. Method two creates a
pattern by building a polymer network after a polymer film has been cast onto the wafer
substrate.
6.2.1 Photoablation of Surface-attached Monomers Followed by
Polymerization
For direct patterning of the surface-attached MPS high energetic UV light with
wavelengths λ < 200 nm is used. It is known that deep UV light destroys carbon-carbon
bonds[167] such as those present in the used surface-attached monomers as well as surface-
attached polymer monolayers. In simple contact irradiation experiments the mask is in direct
contact to the substrate while photodecomposition takes place (Figure 6-11a). The MPS in the
covered areas stays intact whereas the irradiated areas lose their monomer functionality
(Figure 6-11b). This method can be performed quickly and requires only short illumination
times below 30 min because the MPS layer is very thin (~1 nm). On the silicon wafer the
residual intact MPS units give a positive image of the mask. The so patterned MPS layer is a
latent image that develops when exposed to a radical polymerization solution creating a
polymer monolayer in the non-irradiated areas (Figure 6-11c). Thicknesses of the built
polymer monolayers are according to our results in this thesis and may be varied up to a
maximum of 10-20 nm for PS and 20-30 nm for P(DMAA) choosing appropriate
polymerization conditions.
144 6 Polymer Systems on Silicon Wafers
Figure 6-11. Direct patterning of MPS-functionalized silicon wafers (a & b) and subsequent
copolymerization of the remaining surface attached double bonds in solution polymerization.
The chosen example for the method depicted in Figure 6-11 is a polymerization of styrene
on silicon wafers with a structured MPS layer. For structure formation different TEM metal
masks are used. The PS monolayer is analyzed by imaging ellipsometry. As result we get
complementary images where on the one hand the intensity of the reflecting light from the
polymer areas (Figure 6-12a, c) and on the other hand from the substrate areas (Figure 6-12b,
d) is minimized. As we use grids as masks the grown polymer monolayer forms also thin
lines to a grid. These polymeric grid lines have a width of a few micrometers as obtained by
ellipsometry. The observed dry PS layer thickness is ~10 nm. These ultrathin layers can be
measured by imaging ellipsometry but the contrast between polymer layer and substrate is
lower than for example polymer brushes with a much higher layer thickness[168].
6 Polymer Systems on Silicon Wafers 145
Figure 6-12. Complementary imaging ellipsometry micrographs of structured polystyrene monolayers
with a dry thickness of ~10 nm on silicon wafers. The grid size of the upper example (a, b) is 128 µm
and of the lower example (c, d) it is 64 µm.
6.2.2 Patterning through Polymer Network Formation
The first step is here the already described immobilization procedure of MPS on silicon to
get the surface-attached monolayers (Figure 6-14a). Then a copolymer film containing
methacryloyl-4-oxy-benzophenone (MABP) units is cast onto the modified substrate. This is
transferred into a polymer network by UV-cross-linking (Figure 6-14b). Different
concentrations of the polymer solution and different coating techniques such as dip coating
and spin casting have been applied in order to get a thick layer on the modified wafers (cf.
Table 6-1). It is also important to get homogeneous polymer layers in order to develop the
intact polymer microstructures with light-guiding masks. By UV-illumination of the
unmasked copolymer areas a network is formed by carbon-carbon linkage of the
benzophenone unit within the polymer. In addition the formed polymer network is fixed to
146 6 Polymer Systems on Silicon Wafers
the wafer by carbon-carbon linkage between the benzophenone in the copolymer and the
MPS molecules on the surface. Network formation through mask guided UV illumination
takes place by using wavelengths of 350 nm to activate the benzophenone units within the
polymer. Copolymerized MABP monomers have been used in the past to form polymer
networks as well as benzophenone with silane anchor group has been immobilized to
surfaces in order to attach polymers covalently to the surface[60, 226]. Here a MPS layer on
the surface is serving as attachment point for the benzophenone units in the copolymer
(Figure 6-13).
Diligent extraction with a good solvent for the copolymer in a Soxhlet extractor leaves the
cross-linked fraction of copolymer in the UV illuminated areas as a network structure. In the
non-illuminated areas the copolymer is washed away down to the wafer surface where still
the attached MPS units are (Figure 6-14c). These surface monomers are then used to bind an
ultrathin copolymer monolayer in another radical copolymerization to the surface (Figure
6-14d). This copolymer monolayer contains functional units for further use.
Figure 6-13. Detailed view on the attachment of a benzophenone copolymer to a surface-attached MPS
group by UV exposure. In the coated copolymer some benzophenone units rest in close proximity to
the MPS-covered surface and form a C-C-bond upon activation with UV light (λ = 350 nm).
6 Polymer Systems on Silicon Wafers 147
Figure 6-14. Four steps leading to a micropatterned wafer with a functional copolymer within: a)
Immobilization of MPS onto the silicon surface, b) casting a benzophenone copolymer on the MPS-
modified wafer and immediately UV illumination through a mask, c) the latent pattern evolves by
extraction of free copolymer chains from the crosslinked & surface bound copolymer network, partially
uncovering the MPS-modified surface, d) another copolymerization with these surface monomers is
possible.
148 6 Polymer Systems on Silicon Wafers
To apply cross-linkable MABP-copolymers to MPS-modified silicon wafers common
coating techniques such as spin casting and dip coating are used. Different coating
conditions are checked and the resulting dry layer thicknesses are listed in Table 6-1 and
Table 6-2:
Table 6-1. Average layer thickness and standard deviation attained by different coating techniques
with P(S-co-MABP) with different amounts of cross linker.
P(S-co-MABP) concentration
(mg⋅mL-1)
benzophenone content (mol%)
coating technique layer thickness average
(nm)
layer thickness deviation
(nm)
20 1 dip coating 64 5
50 1 dip coating 292 8
70 1 dip coating 341 18
100 1 dip coating 315 20
50 1 spin casting 314 8
20 5 spin casting 80 10
20 10 spin casting 94 10
20 5 dip coating 132 126
20 10 dip coating 172 105
Table 6-2. Average layer thickness and standard deviation attained by different coating techniques
with P(DMAA-co-MABP) with different amounts of cross linker.
P(DMAA-co-MABP) concentration
(mg⋅mL-1)
benzophenone content (mol%)
coating technique layer thickness average
(nm)
layer thickness deviation
(nm)
50 1 dip coating 675 34
70 1 dip coating 742 16
25 5 dip coating 347 1
50 5 dip coating 770 3
25 10 dip coating 325 1
6 Polymer Systems on Silicon Wafers 149
Goal of the diverse coating experiments is to determine conditions to get homogeneous
and relatively thick (> 200 nm) copolymer layers. Dip coated P(S-co-MABP) layers show in
most cases dewetting on silicon wafers whereas spin cast P(S-co-MABP) layers are stable.
MABP-copolymer solutions are influenced by even traces of daylight and so crosslinking and
viscosity changes may occur. The higher the MABP content the more difficult the handling of
the solution and the application of the coating. Based on these insights 50 mg⋅mL-1 of
P(S-co-MABP(1%)) are spin cast and 70 mg⋅mL-1 of P(DMAA-co-MABP(1%)) are dip coated
onto MPS-modified silicon wafers for use in the following photolithographic experiments. It
is important to mention that by 1H-NMR measurements the benzophenone content of
P(S-co-MABP(1%)) was determined as 9 mol% and for P(DMAA-co-MABP(1%)) the actual
value was 1 mol%.
Different masks, self-made printouts and commercially available transmission electron
microscopy (TEM) grids, have been applied to P(S-co-MABP)- and P(DMAA-co-MABP)-
coated silicon wafers prior to UV exposure to create polymer network microstructures.
Details on used masks and patterns are given in paragraph 11.3 of the experimental section.
From this great variety some examples are chosen to illustrate the possibilities and limits of
the described photolithographic technique.
Figure 6-15 shows images of polymer network structures evolved from UV illumination
through self-made masks. These masks are printouts of a laser printer using common
transparencies for overhead projectors. The line width and space between two lines are the
same. The top view with a CCD camera shows that structures down to a feature size of
200 µm are clearly separated and only the printer resolution limits the structure dimensions.
The color gradient of the polymer structures reveals different thicknesses within one section.
150 6 Polymer Systems on Silicon Wafers
Figure 6-15. CCD image of a cross-linked P(S-co-MABP) network. The colored areas show the
polymer. A self-made mask with a line width of 400 µm (a) and 200 µm was used. The black line
marks the taken profile of Figure 6-16.
Profilometric measurements allow to have a more detailed look on a cross section of these
structures. Figure 6-16 shows polymer network walls with a maximum height of nearly
400 nm. The structures have no sharp edge between areas where the copolymer has been
irradiated (wall) and where not (ground level). This is due to the fact that the transparencies
as carrier of the printed masks do not totally overlie on the coated copolymer and the prints
neither have a sharp contour nor they are completely black and impermeable for light. These
factors have the effect that the polymer network structure got a rounded silhouette on the
top edge. Upon inspection of the Figure 6-16 should be noted that the y-axis is given in
nanometer, whereas the x-axis is in µm, so that all structures look much more rounded then
they actually are.
6 Polymer Systems on Silicon Wafers 151
0 200 400 600 800 1000 12000
100
200
300
400
500
heig
ht (n
m)
width (µm)
Figure 6-16. Profilometric cross section of a PS network created by using a mask with 400 µm line
width (cf. Figure 6-15a).
The effects of the mask design on the resulting polymer network with its less pronounced
contours is also tangible with imaging ellipsometry. When alternately minimizing the
intensity of the reflecting light from the top of the polymer structure(Figure 6-17a) and from
the substrate (Figure 6-17b) there is a small area in between that is not dark on both images.
This is the transition area where the polymer network structure has a slope.
Figure 6-17. Complementary imaging ellipsometry micrographs of a PS network: reflectance
minimized on polymer (a) and on substrate(b). The line width of the mask was 200 µm.
152 6 Polymer Systems on Silicon Wafers
Mask patterns printed on flexible plastic have the drawback that they become distorted
upon exposure to a UV lamp as they get significantly hot. If the mask support or material
does not fit to the copolymer layer closely there are penumbra regions where the copolymer
layer is only partially irradiated by the UV light. This results in rounded profiles instead of
sharp edged ones. A comparison between a microstructure built with a closely fit mask and a
distorted mask is given in Figure 6-18. For image a) the metal mask fitted nicely to the PS
copolymer layer surface and the resulting structures have sharp edges. On image b) the
metal was slightly twisted and did not lie tight on the PS copolymer surface which leads to
structures that mingle with each other.
Figure 6-18. TEM grids with different mesh sizes (mesh 200 = 128 µm (a), mesh 400 = 64 µm (b)) are
used to pattern PS networks. The profiles of Figure 6-19 and Figure 6-20 are marked as black lines.
The impressions from the top view of Figure 6-18 are confirmed by profilometry. A cross
section taken from the mesh 200 polymer microstructure reveals a relatively flat top with a
height of around 414 nm and a width of about 80 µm and a groove down to the base
substrate (Figure 6-19). Another picture gives the cross section of the mesh 400 polymer
microstructures (Figure 6-20). The top of the structures is more rounded and the grooves
between the polymer microstructures do not reach down to the substrate.
6 Polymer Systems on Silicon Wafers 153
0 25 50 75 100 1250
100
200
300
400
500
heig
ht (n
m)
width (µm)
Figure 6-19. Profilometric cross section of a PS network created by using a metal grid with 128 µm
grid size (cf. Figure 6-18a).
0 25 50 75 100 1250
100
200
300
400
500
heig
ht (n
m)
width (µm)
Figure 6-20. Profilometric cross section of a PS network created by using a TEM grid with a mesh size
of 64 µm (mesh 400) (cf. Figure 6-18b).
154 6 Polymer Systems on Silicon Wafers
Figure 6-21. Square ‘pillars’ are constructed from PS network using mesh 200-type TEM grids as
shown in the complementary ellipsometry images.
Figure 6-22. From ellipsometric data computed 3-dimensional view of the structured PS network. The
z-axis (height) is exaggerated to pronounce the polymer network structures and reveals that single
structure elements look like pillars.
The ellipsometric data from mesh 200 PS microstructures shown in Figure 6-21 is used to
calculate a three-dimensional computer image where the axis perpendicular to the substrate
base is stretched for a better visualization of the polymer structures (Figure 6-22). This three-
dimensional view shows, as already seen in the graphs and images before, that with even an
6 Polymer Systems on Silicon Wafers 155
apparently sharp edged microstructure there is a slope from top level down to the base. In
the 3D view the polymeric pattern looks like single pillars.
Polymer micropatterns built by cross-linked P(DMAA-co-MABP) copolymers are
investigated as well. P(DMAA) networks are very attractive for applications where good
swellability in polar solvents is needed. With the chosen dip coating conditions relatively
thick copolymer layers are deposited on the wafer surface. These layers can be structured in
an exact way with a clear cut between the single pattern elements as it is depicted in the
following images (Figure 6-23 to Figure 6-25).
Figure 6-23. CCD image of an evolved P(DMAA) network after UV irradiation with a mesh 200
TEM grid.
Figure 6-24. Complementary imaging ellipsometry micrographs of the P(DMAA) network shown in
Figure 6-23.
156 6 Polymer Systems on Silicon Wafers
The evaluation of the ellipsometric data (Figure 6-24) by calculating a 3D image (Figure
6-25) corroborates the assumption that the P(DMAA) networks are structured more clearly
with definite contours than PS networks with their rounded profiles.
Figure 6-25. This image shows a 3D relief computed from the ellipsometric data shown in Figure 6-24.
The pattern elements have rectangular shape and well defined edges.
Figure 6-26. CCD image of a P(DMAA) network built by photolithography using a TEM grid with
line bars having a spacing of 64 µm.
That cross-linked P(DMAA-co-MABP) copolymers have a much sharper confined
structure after the photolithography process is confirmed in the next example where a TEM
grid with line bars was used. For the sample shown in Figure 6-26 profilometric
6 Polymer Systems on Silicon Wafers 157
measurements give the following dimensions: The height of the polymer structures is
~670 nm and the lateral dimensions are 38 µm for the polymer network and 26 µm for the
base substrate crossing perpendicular to the orientation of the polymer walls.
In this case the profilometric findings are supported by data from imaging ellipsometry
(Figure 6-27). Steeper slopes at the edges of the polymeric structures provide better contours
(Figure 6-28) than it is the case for the PS network structures.
Figure 6-27. Complementary ellipsometry images focused on P(DMAA) network structures built by
photo cross-linking with a TEM grid with line bars.
158 6 Polymer Systems on Silicon Wafers
Figure 6-28. A three-dimensional image has been calculated from the ellipsometric data shown in
Figure 6-27. The polymer walls are well confined.
With the P(DMAA-co-MABP) copolymers we are in the position to create small, separated
and surface-attached microstructures with a simple photolithographic process as the last
example in Figure 6-29 and Figure 6-30 shows.
Figure 6-29. Microstructures formed by UV cross-linking through a mesh 400 TEM grid are well
applied to P(DMAA) copolymer. Each square has a side length of nearly 40 µm.
6 Polymer Systems on Silicon Wafers 159
Figure 6-30. Complementary imaging ellipsometry pictures mapping the P(DMAA) network
structures shown in Figure 6-29.
The last step of our proposed method is the application of a ultrathin functional copolymer
layer onto the substrate copolymerizing with the surface-attached MPS. This step has been
chosen because this functionalization is not accessible by simple printing of a copolymer
onto a substrate and subsequent fixation with any technique. Several problems were
encountered during realization of this reaction step. Preparation of the polymerization
solution for the final polymerization step and degassing using freeze & thaw cycles leads to
fissured and detached polymer networks. The microstructures could still be identified but
they were not fully intact anymore. This problem is successfully solved by putting the wafer
samples into the polymerization solution after the degassing step. The functional groups of
the second ultrathin copolymer layer between the first copolymer networks are tested by
reaction with appropriate fluorescence dyes. Upon analysis with the fluorescence reader it
turned out that fluorescence has the highest intensity in the areas of the network structures
and not between the patterned network where only the functional copolymer is attached to
the surface. This leads to the assumption that the copolymer network structures do not
prevent attachment of the functional copolymer in the final polymerization step in their
areas. That is plausible because photo attachment of the first copolymer via benzophenone
groups onto the MPS-modified silicon wafer is strong enough to fix the polymer network
layer to the surface but the most MPS units are still active underneath the polymer network
after the photolithographic step. The other way round the attached polymer network even
160 6 Polymer Systems on Silicon Wafers
enhances the probability of entanglements of functional free copolymer coils within the
network besides the surface attached polymer chains. This leads to higher fluorescence
intensities in areas where the polymer network is attached.
6.2.3 Discussion & Conclusion Micropatterning
In the first set of experiments it is shown that surface attached monomers can be actively
used for building polymer microstructures. Upon deep UV irradiation through a mask we
get fast ablation of the MPS layer. This is not surprising because this layer is according to the
ellipsometric measurements just around 1 nm thin. The subsequent radical polymerization
creates a polymer monolayer in the non-illuminated areas. The thickness of the resulting
polymer monolayer is according to the polymerization conditions as it has been determined
in [153] and paragraph 6.1. The size of single pattern elements can be varied down to a few
micrometers as we have shown with imaging ellipsometry. This patterning technique may
be interesting when using a combination of monomers introducing functionalities which
lead to applications we already have shown as examples. Especially bioanalytical purposes
using fluorescence are applicable to this micropatterned system. The advantage of this
system is the simplicity of generating micropatterns with a broad range of different (co-
)polymers and the stability of the polymer monolayer as it is covalently bound to the
substrate surface. Drawback of this method is on the one hand that immobilization of MPS is
limited to oxidic surfaces and in lab analytics glass is more and more substituted by plastic
substrates. On the other hand there are significant amounts of polymer generated which are
not bound to the substrate surface even when the substrates are densely packed inside the
polymerization solution through appropriate holders.
With the combination of a benzophenone containing copolymer and a methacryloyl propyl
trimethoxysilane one is able to form micropatterned polymer networks and attach them
covalently to the surface. The advantage of MPS layers is that they are very stable compared
to the so far often for surface attachment used benzophenone silane monolayers. MPS-
modified silicon wafers stay active for copolymerization with other monomers or attach by
benzophenone after weeks of storage in air under normal conditions in the presence of
oxygen and humidity and exposure to daylight. In the past it has been shown that a
benzophenone content of 1 mol% (and below) in the copolymer is sufficient to build stable
polymer networks[60]. But here we discover that such a benzophenone content is also
sufficient to fix a polymer network via covalent bonds effectively to surface that offers
6 Polymer Systems on Silicon Wafers 161
aliphatic carbon-carbon bonds. In principle also alkyl silanes would do the trick but if as the
surface attached silane layer a monomer is used, it can be employed for another
polymerization step.
The evaluation of the micrographs reveals that the quality of the mask is important for the
quality of the polymer network’s shape. That means a mask for the patterning of
benzophenone copolymer layers has to be strictly planar and must lie closely on the layer in
order to get detailed and sharp edged pattern elements. We have observed a persistent
difference between PS and P(DMAA) networks regarding the slope between polymer
network surface and substrate surface. Other factors that effect the section profile are the
molecular weight in combination with the benzophenone content of the copolymer. The used
P(S-co-MABP) got higher molecular weight and higher benzophenone content by nearly a
factor of 10 in comparison with the used P(DMAA-co-MABP). These two copolymer inherent
factors provide for an extension of the polymer network into the threshold between
illuminated and non-illuminated areas. This accounts for the previously mentioned
differences documented and proven by profilometry and imaging ellipsometry. In general
well defined structures in the micrometer range are possible to build by polymer cross-
linking.
The second polymerization step was not successful the way it was intended. Blocking an
area with surface attached polymer networks for functionalization with a second copolymer
is not possible with the chosen network density and polymer mesh size respectively. But
these findings show the way to go: Application of patterned polymer networks onto a
surface where the apparent density of functional/reactive groups can be increased by using
three dimensional structures. With irradiation of functional terpolymers (base unit +
benzophenone unit + reactive unit) the combined advantages of patterns and polymer
networks on solid substrates can be used as it is similarly filed by Johnson et al.
(Motorola)[227] for immobilizing biomolecules and cross-linking copolymers in a single step.
However for simple deposition of functional copolymers patterns printing is faster and more
effective concerning resources, e.g. monomers, polymers and cross-linking agent and
photoresist techniques for patterning[228] are widely acknowledged as well. For future
research this has to be taken in account and it should come into focus if substrates modified
with immobilized monomers bring improvements for applicable systems based on
imprinting.
162 7 Outlook
7 Outlook
This work has described the mechanism of the buildup of polymer monolayers using
surface-attached monomers. Having explored these details for two basic polymer (PS and
P(DMAA)) on various porous and flat substrates it has turned out that the influence of the
polymerization parameters on the resulting polymer monolayers is strongly dependent from
the used monomer-solvent system. Therefore we suggest also to have more detailed looks on
further monomers building polymer monolayers and if possible allocate them to certain
polymerization groups dependent of their polymerization behavior (e.g. “styrene type”, or
“acrylic type”). Although efforts to reduce the amount of generated free polymer during the
grafting polymerization process have been successful, there is still some room left for
improvements. In this regard one may speculate/dream about performing a polymerization
with monomer modified substrates where the monomer is nearly completely consumed in
the generation of the surface-attached chains.
If one wants to study copolymer monolayer systems in more detail it has to be checked
which other monomer units bearing desired functional groups can be incorporated into the
polymer layer and to overcome solubility or copolymerization problems or other restrictions,
in order to drive the specific loading of the surface-attached functional groups to even higher
values.
Especially in the field of applications for functional hybrid system many possible
applications have been tested in experiments and completed successfully. A promising case,
immobilization of Grubbs catalyst and subsequent ring closing metathesis, is investigated in
great detail by our cooperation partners at the Institute for Organic Chemistry, University of
Freiburg. As life science and bio analysis are future markets similar endeavors should be
attempted for the suggested biomolecule immobilizations. When combined with spatial
patterning through photolithographic techniques the “grafting-through” system might
evolve as a good toolbox for various chip applications, e.g. “lab on a chip”.
8 Experimental I – Materials & Methods 163
8 Experimental I – Materials & Methods
8.1 Materials
8.1.1 Material List Acetone .................................................................................................................................................................. (p.a., Fluka)
Acryloyl chloride ................................................................................................................................................. (96%, Fluka)
Aerosil 300 ......................................................................................................................................................... (Degussa AG)
Aluminum oxide, basic ............................................................................................................................................... (Fluka)
2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt................................. (99.9%, Sigma-Aldrich)
2,2’-Azobisisobutyronitrile ................................................................................................................................(p.a., Merck)
Benzene ............................................................................................................................................................... (>96%, Fluka)
Fluorescein biotin ............................................................................................................................................... (90%, Sigma)
Calciumhydride .................................................................................................................................................... (p.a., Fluka)
Chloroform ........................................................................................................................................... (p.a., Riedel de Haën)
Chloroform-d3 ................................................................................................................................................ (>99.8%, Fluka)
Copper-I-Chloride ................................................................................................................................................ (p.a., Fluka)
Dichloromethane .................................................................................................................................................. (p.a., Fluka)
N,N’-Dicyclohexylcarbodiimide .................................................................................................................. (puriss., Fluka)
Diethylether ......................................................................................................................................(technical grade, Fluka)
N,N’-Dimethylacrylamide .................................................................................................................................. (p.a., Fluka)
Dimethylsulfoxide ............................................................................................................................................ (puriss., Roth)
Dimethylsulfoxide-d6 .................................................................................................................................... (>99.8%, Fluka)
DY-635-NH2 .................................................................................................................................................(Dyomics GmbH)
Ethanol ...............................................................................................................................................(technical grade, Fluka)
Ethanol ..................................................................................................................................................................(p.a., Merck)
Fluorescein ........................................................................................................................................................ (p.a., Aldrich)
Fluorescein-5-isothiocyanate .......................................................................................................................(Sigma-Aldrich)
Glass beads (details see below) ..................................................................................... (research samples, Grace GmbH)
3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluorodecylacrylat ............................................................(purum, Aldrich)
Hydrazine hydrate .........................................................................................................................................(50-60%, Fluka)
4-Hydroxybenzophenone ............................................................................................................................. (purum, Fluka)
N-Hydroxysuccinimido biotin .......................................................................................................................... (98%, Sigma)
Isopropanol .......................................................................................................................................... (p.a., Riedel de Haën)
N-Isopropyl acrylamide ...................................................................................................................................... (97%, Fluka)
LiChrospher Si60.......................................................................................................................................................... (Merck)
Methacrylic acid ................................................................................................................................................... (98%, Fluka)
Methanol ......................................................................................................................... (technical grade, Riedel-de-Haën)
Methanol ..............................................................................................................................................................(p.a., Merck)
Methyl methacrylate............................................................................................................................................ (99%, Fluka)
164 8 Experimental I – Materials & Methods
Methylamine ................................................................................................................................................... (puriss., Fluka)
Oligonucleotides (details see below)....................................................................... (purified by HPLC, Thermo Hybaid)
Potassium peroxodisulfate ................................................................................................................................(>98%, Fluka)
Sodium ..........................................................................................................................................................................(Merck)
Sodium carbonate ............................................................................................................................................... (p.a., Merck)
Sodium hydroxide ........................................................................................................................................ (purum, Merck)
Sodium sulfate .................................................................................................................................................... (p.a., Merck)
Streptavidin ................................................................................................................. (from Streptomyces avidinii, Sigma)
Styrene ..................................................................................................................................................................(99%, Fluka)
Toluene ................................................................................................................................................(p.a., Riedel-de-Haën)
1,1,2-Trichloro trifluoro ethane .................................................................................................(Freon© 113, 99.5 %, Fluka)
Triethylamine .................................................................................................................................................... (>99.5., Fluka)
8.1.2 Material Preparation
The silica gels LiChrospher Si60 (LC700, Merck) and Aerosil300 (AE300, Degussa AG) were
dried at elevated temperature and reduced pressure for 3 hours (120 °C, 10-2 mbar). The
mesoporous glass beads of different sizes and specific surfaces (GB40, GB80, GB250, Grace
GmbH & Co. KG) were dried under the same conditions for 1 hour. The specifications of the
different solids are listed in Table 8-1:
Table 8-1. Specifications of the different silica gels and glass beads used for grafting polymer
monolayers
solid LiChrospher Si60
LC700
Aerosil300
AR300
glass bead
GB250
glass bead
GB80
glass bead
GB40
specific surface
(m2·g-1)
700 300 250 80 40
particle size
(µm)
~25 10-20 70-100 100-300 90-130
pore size
(nm)
< 2 - 20 50 100
3-methacryloylpropyl trimethoxysilane (MPS, Fluka), 3-acryloylpropyl trimethoxysilane
(APS, Fluka), octyl trimethoxysilane (OS, Fluka), and azobis(isobutyronitrile) (AIBN, Fluka)
were used as received. Styrene (Fluka) was chromatographically purified over basic
8 Experimental I – Materials & Methods 165
aluminum oxide, distilled under reduced pressure from copper(I)chloride (60 °C, 50 mbar)
and stored under dry nitrogen at -20 °C until used. N,N’-Dimethylacrylamide was also
chromatographically purified and destabilized over basic aluminum oxide, distilled under
reduced pressure from copper(I)chloride (80 °C, 20 mbar) and stored under dry nitrogen at
-20 °C until used. Toluene (Fluka) was dried and distilled using molten sodium and
benzophenone as an indicator. After distillation it was stored over molecular sieve under dry
nitrogen. Triethyl amine (Fluka) was dried and distilled over calcium hydride prior to use.
N-methacryloyl-N-methyl-propyl trimethoxysilane (MNPS) and N-acryloyl-N-methyl-
propyl trimethoxysilane (ANPS) were synthesized from 3-amino-N-methyl-propyl
trimethoxysilane (ABCR) and methacryloyl chloride (Fluka), or acryloyl chloride (Fluka).
Coupling of the methoxysilane and the acrylic unit was performed in dichloromethane at
0 °C under nitrogen. Triethylamine was used to capture hydrochloric acid. After completion
of the reaction the solvent is removed and residuals resolved in diethylether. Solids were
filtered off. After removal of ether the product was obtained as a brown oil. Further
purification was done with a small silica gel column and leaves a yellow oil. The
characterization was done in the analytical labs of the Department for Organic Chemistry,
Albert-Ludwigs-University, Freiburg.
1H-NMR (500 MHz, CDCl3, TMS):
δ = 5.51 (bs, 1H, vinyl), 5.00-5.03 (m, 1H, vinyl) 3.55-3.60 (m, 9H, -OCH3), 3.27-3.45 (m, 2H, -CH2N), 2.90-3.03 (m, 3H, -NCH3), 1.96 (s, 3H, -CH3), 1.53-1.75 (m, 2H, -CH2-), 0.50-0.68 (m, 2H, -CH2Si).
13C-NMR (100 MHz, CDCl3, TMS):
δ = 168.8; 141.3; 115.0; 53.2; 50.6; 36.5; 21.8; 20.7; 6.3.
MS (EI, 200 °C, 70 eV):
m/z (%): 261 (22) [M+], 229 (37) [M+-MeOH], 160 (37), 121 (77) [MeO)3Si+], 112 (100).
166 8 Experimental I – Materials & Methods
8.2 Methods
8.2.1 Elemental Analysis
All elemental analysis measurements were carried out on a Vario EL
(Elementaranalysensysteme GmbH, Germany) at the Institute for Inorganic and Analytical
Chemistry, University of Freiburg.
8.2.2 Infrared Spectroscopy
The Fourier transform infrared (FT-IR) spectra were recorded on a BioRad Excalibur FTS
3000 spectrometer collecting 128 scans within a wavenumber range of 4000 to 400 cm-1 and a
resolution of 4 cm-1. For measurements on modified silica the device was equipped with a
DRIFT compartment and purged with nitrogen to reduce carbon dioxide and water bands
from the gas phase. The incidence angle was 45° and dry potassium bromide was used for
sample preparation. The spectra evaluation was done with the ʺWin-IR Proʺ (v2.95) software.
8.2.3 Gel Permeation Chromatography (GPC)
For GPC measurements an Agilent 1100 system with ʺWinGPC scientific (v6.20)ʺ software
from PSS (Polymer Standards Service, Mainz) was used. In the case of polystyrene samples the
SDV oligo column system was run with tetrahydrofurane as eluent and calibrated with
polystyrene standard samples with polystyrene with a narrow molecular weight
distribution. For the poly(N,N-dimethyl acrylamide) samples the GRAM column system was
used with DMF as eluent and calibrated with PMMA standard samples. The separations
were carried out at a flow rate of 1 mL·min-1. All calibration polymers were bought from PSS.
8.2.4 Ellipsometry & Imaging Ellipsometry
An ELX-2 ellipsometer (Riss, Germany) was used for null ellipsometry. With an incident
angle of 70° the planar substrates were exposed to a Helium-Neon-laser with a wavelength
of 632.8 nm. The refractive indices used for the layer models for the software to perform
correct calculations were as follows: a) air nD = 1.000, b) silicon nD = 3.882, c) SiO2 nD = 1.457,
d) MPS nD = 1.431, e) PS nD = 1.450.
8 Experimental I – Materials & Methods 167
For imaging ellipsometry a Nanofilm I-elli 2000 device was used. With an incidence angle of
50° a He-Ne-laser with a wavelength of 632.8 nm was led onto the patterned, planar
substrates.
8.2.5 X-ray Photoelectron Spectroscopy
X-ray photoelectron spectra (XPS) were recorded by using Mg Kα radiation using a Perkin
Elmer PHI 5600 spectrometer. The analyzer was positioned at an angle of 45° relative to the
substrate holder. For survey scans the step width while collecting the spectra was 0.8 eV with
a pass energy of 187.85 eV and a step time of 100 ms. For detail scans a step width of 0.06 eV
and a pass energy of 23.50 eV were used.
8.2.6 Nuclear Magnetic Resonance Spectroscopy 1H and 13C nuclear magnetic resonance (NMR) spectra were obtained from a Bruker Avance
250 MHz spectrometer. All substances and polymers were dissolved in deuterated solvents
(CDCl3, D2O or (CD3)2SO) and the solutions were measured at room temperature.
8.2.7 UV/vis Spectroscopy
For the UV/visible absorption measurements a Varian Cary 50 Bio spectrometer was used at
a wavelength range from 800 down to 200 nm with variable scan rates. In most cases the
“medium” speed program with 1 nm interval at 10 nm per second was used. In some cases
the “slow” speed program with 0.5 nm interval and 5 nm per second was used for better
resolution.
8.2.8 Fluorescence Microscopy & Reading
An Olympus Fluoview confocal scanning laser microscope including the Fluoview software
(v1.26) was used for microscopical fluorescence investigations. It is equipped with an
Krypton-Argon laser unit that emits light at 488 nm and 568 nm. For the FITC-stained
sampled the 488 nm excitation wavelength is used. FITC has its emission peak at 518 nm and
is exclusively detected by the microscope optics using colored filters. The laser intensity was
set to 20% output to minimize bleaching of the dye and not to overload the detector. To have
a high vertical resolution an aperture size of 60 µm (Aperture 1) was used. Result of the
measurements are digital pictures.
168 8 Experimental I – Materials & Methods
For determination of fluorescence intensities on glass beads (and silica) the biochip
analyzer BioDetect 645/4 (BioChip Technologies GmbH, Freiburg) was used. This multicolor
(4 colors) reading device can be used to excite different dyes. For our measurements FITC,
Cy5 and DY-635 were used as chromophores. For FITC the biochip analyzer uses an
excitation range from 485 to 495 nm and a detection range from 515 to 545 nm. Using Cy5 or
DY-635 the ranges are 635 to 645 nm for excitation and 671 to 693 nm for detection. The
emitted light intensities are detected by a CCD array which gives a gray scale picture. For
better distinction of the different intensities the gray scale pictured are calculated into false
color pictures (Figure 8-1).
Figure 8-1. Chart for transfer from a CCD detector gray scale image to a false color image.
8.2.9 Profilometry
Profilometric line scans on patterned surfaces were done on a Tencor Alpha-Step IQ
profilometer. The survey scan width was 1000 µm with a vertical resolution of 0.8 nm and
the detail scan width was 130 µm with a vertical resolution of 0.08 nm.
8.2.10 Titration
The amount of amino groups on copolymer modified silica was determined by titration
with a TitroLine 96 (Merck Eurolab, Germany) pH titration device using a Blueline pH11
(Schott, Germany) pH-electrode which was calibrated with buffer solutions at pH = 4 and
pH = 7.
9 Experimental II – Polymerizations 169
9 Experimental II – Polymerizations
In order to obtain a more detailed understanding of the reaction during the polymerization
reactions always one parameter was varied while the other were kept constant. Therefore the
preparation of the individual samples differs and is described for each varied parameter
separately. Degassing, isolation and drying procedures were carried out as described in
paragraph 9.3. Ingredients and reaction vessels were all strictly kept under dry nitrogen.
Larger scale polymerizations (> 50 mL reaction volume) were carried out in appropriate
Schlenk flasks with reflux condenser. 50 mL-Schlenk tubes were used as reaction vessel for
smaller scale polymerization reactions. The silica gel with the attached polymer monolayer
was separated through centrifugation and repeated washing with a good solvent for the
polymer. The free polymer was precipitated from the reaction mixture through a slow
addition of the solution to 10x excess methanol, filtered off and dried. The molecular weights
were determined by GPC. After several washings the modified silica gel was dried from
benzene and analyzed qualitively by FT-infrared spectroscopy and quantitively by elemental
analysis.
9.1 Immobilization of Silanes
9.1.1 Immobilization of Trimethoxy Silanes onto Silica Gel
In an atmosphere of dry nitrogen 6 g silica gel were suspended in 150 mL toluene. A
solution of 2.08 g MPS (8.4 mmol) in 50 mL toluene and 10 mL triethyl amine (~74 mmol)
were added to the mixture. The reaction vessel was heated until the solution started to reflux
(120°C) and stirred for 3 h. To avoid grinding of the silica beads with magnetic stir bars the
reaction mixture was agitated with a circular shaking device (IKA KS 260 control) at 160
rounds per minute (rpm).
The modified silica gels were centrifuged (Sorvall Super T21, 12500 rpm, 16.750 x g) for 15
minutes in the case of LiChrospher and 30 minutes for Aerosil300. Then washed with toluene,
ethanol, ethanol/water (1/1 v/v, acidified with HCl), ethanol/water (1/1 v/v), ethanol and
diethyl ether. The remaining colorless solid was dried for 18 h at 10-2 mbar. The reaction
conditions for immobilization of all monomers are summarized in Table 9-1.
170 9 Experimental II – Polymerizations
Table 9-1 Reaction conditions for the immobilization of different silanes on varying solid supports.
substrate n (silane)
(mmol)
m (silica)
(g)
V (toluene)
(mL)
n (Et3N)
(mmol)
LC700 + MPS 8.4 6.0 200 54
LC700 + MNPS 2.4 2.0 66 18
LC700 + APS 9.0 6.0 200 50
LC700 + ANPS 12.5 9.0 300 50
LC700 + OS† 1.1 0.5 20 6
AR300 + MPS 8.4 6.0 200 54
GB250 + MPS 2.5 5.0 60 21
GB80 + MPS 4.2 10.0 125 36
GB80 + MNPS 1.0 2.0 25 7
GB80 + APS 2.1 4.0 50 14
GB80 + ANPS 4.2 10.0 80 28
GB40 + MPS 1.0 4.0 25 7
GB40 + MNPS 0.5 2.0 13 4
† reference sample with surface-attached octyl silane monolayer
SiO2-MPS. IR (DRIFT) ν [cm-1]: 3112, 2986, 2961, 2899, 2857, 1724, 1704, 1637, 1250-
1000 cm-1. elemental analysis: LC700-ME: C 7.30 %, H 1.15 %, AR300-ME: C 6.55%, H 0.92%,
GB250-ME: C 4.76 %, H 0.84%, GB80-ME: C 1.95 %, H 0.30 %, GB40-ME: C 1.33 %, H 0.26 %
SiO2-APS. IR (DRIFT) ν [cm-1]: 3113, 2959, 2901, 2851, 1733, 1714, 1627, 1250-1000 cm-1.
elemental analysis: LC700-AE: C 6.02 %, H 1.05 %, GB250-AE: C 4.76 %, H 0.84%, GB80-AE:
C 1.71 %, H 0.43 %
SiO2-MNPS. IR (DRIFT) ν [cm-1]: 3091, 2981, 2944, 2892, 1647, 1603, 1250-1000 cm-1.
elemental analysis: LC700-MA: C 7.94 %, H 1.38 %, N 1,31 %, GB80-MA: C 2.22 %, H 0.24%,
N 0.24%, GB40-MA: C 1.89 %, H 0.28 %, N 0.31 %
9 Experimental II – Polymerizations 171
SiO2-ANPS. IR (DRIFT) ν [cm-1]: 3108, 2947, 2896, 2851, 1645, 1590, 1250-1000 cm-1.
elemental analysis: LC700-AA: C 6.45 %, H 1.00 %, N 0.84 %, GB250-AA: C 5.18 %, H 0.96%,
N 0.70%, GB80-AA: C 1.92 %, H 0.47 %, N 0.24 %
SiO2-OS. IR (DRIFT) ν [cm-1]: 2986, 2961, 2932, 2899, 2857 cm-1. elemental analysis:
LC700-OS C 6.50 %, H 1.42 %
9.1.2 Immobilization of Trimethoxy Silanes onto Glass Beads
Under dry nitrogen glass beads were suspended in toluene and a toluene solution of the
silane monomer unit and triethyl amine were added to the mixture. The flask with the
reaction mixture was heated until the solution started to reflux (120°C). Agitation for 3 h was
done with a circular shaking device at 160 rpm. The immobilization recipes are summarized
in Table 9-2. In case of the glass beads sedimentation was fast enough to skip centrifugation.
Washing with toluene, ethanol, ethanol/water (1/1 v/v, acidified with HCl), ethanol/water
(1/1 v/v), ethanol and diethyl ether removes excess triethyl amine. The remaining colorless
solids were dried for 18 h at 10-2 mbar.
Table 9-2. Mixtures for the immobilization of different silanes onto glass beads.
substrate & surface
modification*
n (silane)
(mmol)
m (silica)
(g)
V (toluene)
(mL)
n (Et3N)
(mmol)
GB250-ME 2.5 5 60 21
GB250-AA 2.5 7 60 21
GB80-ME 4.2 10 125 36
GB80-MA 3.9 10 100 35
GB80-AE 2.1 4 50 14
GB80-AA 4.2 10 80 28
GB40-ME 1.0 4 25 7
GB40-MA 0.5 2 13 4
172 9 Experimental II – Polymerizations
9.1.3 Co-Immobilization of MPS and OS onto Silica Gel
MPS and OS were added to the reaction solution in certain molar ratios (4/1, 1/1, 1/4, 1/19,
pure OS). The composition of each batch is given in Table 9-3. The mixtures, heated to reflux
by an oil bath (120 °C), were agitated by circular shaking for 3 h at 160 rpm. After the
reaction was completed and between each washing step during work up, the modified silica
gels were centrifuged (15 minutes at 12500 rpm). They were washed with toluene, ethanol,
an mixture of ethanol/water (1/1 v/v, acidified with HCl), ethanol/water (1/1 v/v), ethanol
and diethyl ether. Remaining colorless solids were dried for 18 h (overnight) at <10-2 mbar.
Table 9-3. Reaction mixtures for the co-immobilization of MPS and OS.
batch (MPS/OS)
(n/n)
V (MPS)
(µL)
V (OS)
(µL)
m (LiChrospher)
(mg)
V (toluene)
(mL)
n (Et3N)
(mL)
1/0 2000 - 6000 200 10.0
4/1 190 50 715 28 1.0
1/1 120 130 715 28 1.0
1/4 50 200 715 28 1.0
1/19 15 240 715 28 1.0
0/1 - 300 509 20 0.7
The actual ratio between immobilized “active” silane (with double bond) and immobilized
“inactive” silane (without double bond) was examined by FT-infrared spectroscopy To get
an idea of the real silane composition on the surface, as it that may differ from the
composition of the initial immobilization mixture, the DRIFT data were compared to FT-IR
data of liquid mixtures with know ratios of MPS and OS. The quotient of the integral of the
C=O-valence vibration band divided by the integral of the C-H-valence vibration band of
methylene gave the extinction ratio. From the transmission-IR measurements of the liquid
silanes a calibration curve was generated. The initial DRIFT data points were aligned to the
calibration curve in order to get the surface composition of the silanes. For details
cf. paragraph 3.3.7.1.
9 Experimental II – Polymerizations 173
9.2 Homopolymerizations
9.2.1 Synthesis of Poly(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-
heptadecafluorodecyl acrylate)
1.0 g of the solid phase with surface attached monomers is suspended in 7.5 mL Freon®-113
(1,1,2-trichloro-trifluoro ethane) and 2.5 mL distilled HDFDA are given to the suspension.
Then 24.6 mg AIBN as initiator are added and dissolved in the polymerization solution. The
whole mixture then is degassed in vacuum by six freeze & thaw cycles and finally heated to
50.0±0.1 °C with a water thermostat. After 24 h the polymerization was stopped by cooling
down. The suspensions with LiChrospher were centrifuged at 12500 rpm for 20 min for
separation of the silica gel and the glass beads were isolated by sedimentation. The solids
were put into a filter inside of an extraction case and extraction at 48 °C with Freon®-113 was
carried out over night (~ 18 h). The substrates with the perfluoro polymer monolayer then
were qualitively analyzed by x-ray photoelectron spectroscopy (cf. Table 3-3) and DRIFT and
quantitively by elemental analysis.
SiO2-P(HDFDA). IR (DRIFT) ν [cm-1]: 2955, 2851, 1738, 1193 cm-1, elemental analysis:
LC700-AE-P(HDFDA) C 15.48 %, H 1.19 %, GB80-AA-P(HDFDA) C 11.13 %, H 0.76 %,
N 0.17 %.
9.2.2 Synthesis of Polystyrene & Poly(methyl methacrylate)
First the solid phase is suspended in toluene and the monomer is added. Afterwards AIBN
is dissolved in the polymerization solution. The whole mixture is then degassed in vacuum
through five freeze & thaw cycles and finally heated with a water thermostat (60.0±0.1 °C).
After 25 h of polymerization for styrene and 16 h for MMA the polymerizations were
stopped and cooled down. Then the polymerization solutions with LiChrospher were
centrifuged at 12500 rpm for 20 minutes in order to separate the silica gel. In the mixtures
with glass beads sedimentation was fast. After decanting, the remaining solids were put into
a filter inside of an extraction case and hot extraction with toluene was carried out over night
(~ 18 h). The substrates modified with polystyrene and poly(methyl methacrylate)
monolayers were freeze-dried from benzene. All samples were analyzed by DRIFT and
elemental analysis. The different free polymers were precipitated from the reaction solution
through a slow addition to 10x excess of methanol. The precipitates were filtered off and
dried. The molecular weights were determined by gel permeation chromatography (GPC).
174 9 Experimental II – Polymerizations
A glass bead sample with surface-attached polystyrene monolayer was also characterized by
XPS (cf. Table 3-3).
SiO2-PS. IR (DRIFT) ν [cm-1]: 3080, 3061, 3028, 2920, 2852, 1724, 1602, 1493, 1450, 1200-1000,
762, 697 cm-1, elemental analysis: LC700-ME-PS C 32.85 %, H 3.45 %, GB80-AA-PS C 11.13 %,
H 0.76 %, N 0.17 %.
SiO2-P(MMA). IR (DRIFT) ν [cm-1]: 2993, 2951, 2846, 1730, 1481, 1450, 1200-1000 cm-1,
elemental analysis: LC700-ME-P(MMA) C 27.51 %, H 4.04 %, GB80-ME-P(MMA) C 16.46 %,
H 2.38 %.
9.2.3 Synthesis of Poly(N-isopropyl acrylamide)
In 12 mL of MilliQ purified water 3.0 g NIPAAm and as water-soluble initiator 29 mg
(108 µmol) potassium peroxodisulfate were dissolved within 30 minutes under rapid
stirring. Then 300 mg of either LC700-ME, or GB80-ME are suspended in the solutions for the
polymerization reaction. By ultrasonification under reduced pressure the mixtures were
degassed. For polymerization the reaction vessels were heated at 60.0±0.1 °C with a water
thermostat. After a time period of 16 hours gelation of the solutions was observed and the
polymerization reactions were terminated by cooling down. The gelatinous mixture
including the solids was dissolved in methanol to reveal the substrates with the surface-
attached P(NIPAAm) monolayers. The solids were filtered off, extracted with hot methanol
for 2 days to remove free adsorbed P(NIPAAm) and freeze-dried from water. The resulting
solids were characterized by elemental analysis and DRIFT. The free polymer was
precipitated from the filtrates through slow addition to a 10fold excess of a mixture of
diethyl ether and acetone (2/1 v/v), filtered off and dried. GPC measurements of the free
P(NIPAAm) with the SUPREMA column system were not possible, because even very dilute
solutions in water could not be filtered with a 0.2 µm filter.
SiO2-P(NIPAAm). IR (DRIFT) ν [cm-1]: 2972, 2938, 2881, 1650, 1530, 1460, 1200-1000 cm-1,
elemental analysis: LC700-ME-P(NIPAAm) C % 20.60, H % 3.57, N % 3.75,
GB80-ME-P(NIPAAm) C % 16.51, H % 3.15, N % 2.34.
9 Experimental II – Polymerizations 175
9.2.4 Synthesis Poly(N,N-dimethyl acrylamide)
300 mg LiChrospher or 400 mg glass beads, both modified with surface-attached
monomers, were given into a solution of DMAA in DMF. The ratio of the two liquid
components was in most cases 1 part DMAA in 9 parts DMF. The initiator AIBN was added
to the polymerization solution (10 mmol·L-1 for most experiments). The solution then was
degassed in vacuum by five freeze & thaw cycles. A water thermostat kept the
polymerization at 60.0±0.1 °C. After 24 to 25 h polymerization time the reactions were
stopped by cooling down and air exposure. For separating the silica gel the polymerization
solutions with LiChrospher inside were centrifuged at 12500 rpm for 20 minutes. The
sedimentation of the glass beads was fast although the reaction solutions were quite viscous.
After decanting, the remaining solids were washed once with DMF and the supernatant
solution was discarded. The solids were collected into a filter paper inside of an extraction
case and extracted with DMF inside of a Soxhlet apparatus overnight (~ 18 h). Last step was
freeze-drying from water. Elemental analysis and DRIFT were used to characterize the
substrates with the P(DMAA) monolayer. The decanted polymerization solution was slowly
poured under fast stirring into 10fold excess of diethyl ether to precipitate the free
P(DMAA). The free polymers were filtered off, dried under vacuum and characterized by
molecular weight determination via GPC. If any amount of substrate, monomer, solvent,
initiator or the polymerization conditions themselves is different from this description it will
be described separately in the paragraph 9.4.
SiO2-P(DMAA). IR (DRIFT) ν [cm-1]: 2928, 1641, 1496, 1399, 1353, 1250-1000 cm-1. elemental
analysis: LC700-ME-P(DMAA) C % 11.34, H % 2.24, N % 1.33; GB80-AE-P(DMAA) C % 6.43,
H % 1.17, N % 1.11.
9.2.5 Synthesis of Poly(methacrylic acid)
300 mg of a solid compound, with monomers attached to the surface, were suspended in a
solution of 3 mL methacrylic acid and 6 mL pure water (MilliQ). Before degassing via
ultrasonification under reduced pressure for 30 minutes, 22 mg (81 µmol) potassium
peroxodisulfate were added. The temperature of 60.0±0.1 °C during the polymerization
period of 16 hours is controlled by a water thermostat. In the end gelation occurred and the
substrates with P(MAA) layer were recovered by washing with methanol and filtration.
Then the modified substrates were extracted with hot methanol in a Soxhlet apparatus for
176 9 Experimental II – Polymerizations
2 days to remove all free P(MAA) and finally freeze-dried from water. DRIFT and elemental
analysis characterized the surface-attached polymer monolayers. The filtrate of the reaction
mixture was slowly poured into a well agitated mixture of acetone and diethyl ether (1/1 v/v)
to precipitate the free P(MAA). The polymer was filtered off, dried but GPC measurements
were not possible because the aqueous solution of the free P(MAA) went not through a
0.2 µm filter.
SiO2-P(MAA). IR (DRIFT) ν [cm-1]: 2992, 2955, 2630, 1710, 1480, 1451, 1391, 1200-1000 cm-1,
elemental analysis: LC700-ME-P(MAA) C 16.58 %, H 2.81 %; GB80-ME-P(MAA) C 19.20 %,
H 2.87 %.
9.3 Polystyrene – Polymerization Parameters
9.3.1 Influence of Reaction Time
Under dry nitrogen a monomer-modified silica substrate was suspended in a mixture of
styrene and toluene and AIBN as initiator was added (Table 9-4). The solution was degassed
in vacuum through four freeze & thaw cycles. The mixture was heated with an oil bath at
60±1 °C. After chosen time periods samples were withdrawn from the reaction under
nitrogen (for silica gels 20 mL, for glass beads 10 mL). The silica gel samples were
centrifuged at 12500 rpm (15 min LiChrospher Si60, 30 min Aerosil300) to separate the silica
gel. For all types of glass beads centrifugation was not necessary because they settle fast at
the bottom. After decanting, the remaining solids were washed with toluene (~ 40 mL)
resuspended and again centrifuged. These steps were repeated (usually five times) until no
precipitate formed when the decanted washing solution was added dropwise to excess
methanol. Finally the silica gel with the polystyrene monolayer was filtered off and freeze-
dried from benzene (7 wt.% silica gel ≈ 100 mg silica gel in 1.63 mL benzene) The
supernatant reaction mixture containing the nonattached polymer chains was slowly poured
in excess methanol (>10x volume) under rapid stirring. The white precipitate was filtered off
and dried in vacuum (<10-2 mbar) overnight.
9 Experimental II – Polymerizations 177
Table 9-4. Mixtures for the investigations on the influence of polymerization time for the
polymerization of styrene onto different modified silica substrates.
substrate & surface modification*
m (solid X)
(g)
V (styrene)
(mL)
V (toluene)
(mL)
c (AIBN)
(mmol·L-1)
LC700-ME 2.0 66 133 9
AR300-ME low Mn 2.0 66 133 9
AR300-ME high Mn 1.2 60 60 3
GB80-ME 2.1 23 46 9
GB80-AE 1.8 30 60 3
GB80-AA 1.0 16 32 3
GB40-ME 0.9 10 20 3
9.3.2 Variation of the Overall Concentration of the Solid
The investigations were divided into two sets of experiments where one was made with
high specific surface LiChrospher silica gel and a second with porous glass beads with a lower
specific surface.
In 50 mL Schlenk tubes five different amounts of MPS-modified LiChrospher (LC700-ME =
0.15 g; 0.6 g; 1.2 g; 2.4 g and 4.8 g) were suspended under dry nitrogen in 15 mL aliquots of a
mixture consisting of 30 mL styrene, 60 mL toluene and 132 mg AIBN (0.8 mmol). The
Schlenk tubes with the degassed suspensions were mounted on a shaking device and heated
in a thermostat to 60°C for 24 hours. Afterwards polymer modified solids were separated
from the polymer solution.
Under dry nitrogen, in 50 mL Schlenk tubes, four different amounts of MPS-modified glass
beads (GB80-ME = 0.6 g, 1.2 g, 2.4 g and 4.8 g) were suspended each in a reaction mixture of
12 mL. These aliquots were taken from a mixture of 16 mL styrene, 32 mL toluene and 24 mg
AIBN (3 mM). The vessels were degassed, kept in motion by a shaking device (160 RPM) and
heated in a thermostat to 60°C for 24 hours. Then the solids were separated from the free
polymer solution.
178 9 Experimental II – Polymerizations
9.3.3 Monomer Concentration Variation
In each of the five experiments with silica gel the total volume of solution was 40 mL with
400 mg MPS-modified LiChrospher and 20 mg (3 mM) AIBN. The input volumes of monomer
and solvent (styrene/toluene, v/v) were 1/0, 1/1, 1/2, 1/4 and 1/8. All mixtures were heated at
60±1 °C for 38.5 hours. Three experiments with styrene concentration variation and glass
beads were carried out. The total volume of solution was 20 mL with 200 mg MPS-modified
GB80 and 10 mg (3 mM) AIBN. The input volumes of styrene and solvent (v/v) were 1/0, 1/1
and 1/2. All mixtures were heated at 60±1 °C for 24 hours. Separation, isolation and analysis
follow the usual procedure.
9.3.4 Initiator Concentration Variation
The polymerizations were carried out in 40 mL styrene/toluene mixtures (1/2 v/v) with
400 mg MPS-modified silica gel each. Five different initiator (AIBN) concentrations were
delivered to the solutions: 10-4 mol⋅L-1 (0.7 mg), 10-3 mol⋅L-1 (6.6 mg), 3⋅10-3 mol⋅L-1 (19.7 mg),
10-2 mol⋅L-1 (65.7 mg), 10-1 mol⋅L-1 (656.9 mg). Then all batches were heated at 60 °C for a
duration of 26.2 hours.
The reaction mixture compositions for the investigations on the initiator influence are
summarized in Table 9-5. To the solutions five (LC700-ME, GB80-ME), four (GB80-AE) and
two (GB250-ME) different initiator (AIBN) concentrations were added and after degassing all
batches were heated to 60 °C and agitated via shaking for a duration of at least 24 hours.
Separating and isolating PS-modified substrates as well as the free monomer lead to the
results of elemental analysis and GPC.
9 Experimental II – Polymerizations 179
Table 9-5. Ingredients list for the experiments on initiator concentration influence on polymerization
of styrene onto different solids.
m (solid X)
(mg)
V (styrene)
(mL)
V (toluene)
(mL)
substrate &
surface
modification* per concentration
c (AIBN)
(mmol)
LC700-ME 400 13.3 26.4 0.1 1 3 10 100
AR300-ME low Mn 2000 66 133 - - - 9 -
GB250-ME 600 8 4 - - 3 10 -
GB80-ME 600 8 4 0.1 1 3 10 100
GB80-AE 600 8 4 0.1 1 - 10 100
9.3.5 Variation of the Polymerization Temperature
In each reaction vessel 300 mg of MPS-modified silica gel were suspended in 20 mL
toluene, 10 mL styrene and 43 mg (0.26 mmol) AIBN under dry nitrogen. The mixtures were
heated to chosen temperatures in a range from 60 to 90 °C. The heating bath temperature
was in all experiments controlled by a contact sensor inside the Schlenk tubes. For one set of
polymerization reactions at different temperatures the polymerization time was varied so
that it amounted to exactly one half life time of the initiator. For another set of experiments
the polymerizations were performed for a constant period of 21.5 hours. For details see
Table 9-6.
180 9 Experimental II – Polymerizations
Table 9-6 Parameters used for the investigation of the temperature dependence of the grafting process.
temperature
(°C)
decomposition rate
(10-6⋅s-1)
polymerization time
(h)
constant initiator conversion (50%)
60 9 21.50
62 12 16.05
65 19 10.85
70 40 4.00
80 150 1.05
90 490 0.40
constant polymerization time
60 9 21.50
62 12 21.50
65 19 21.50
70 40 21.50
80 150 21.50
90 490 21.50
9.3.6 Influence of MPS Surface Concentration on the Polystyrene
Graft Density
The essential step is the co-immobilization of MPS and OS as described above (9.1.3.). The
resulting different modified silica gels were suspended in a polymerization solution under
equal reaction conditions. Each 300 mg MPS/OS-modified LiChrospher were placed in 30 mL
styrene/toluene (1/2 v/v) with 43 mg (9 mM) AIBN and heated at a temperature of 60 °C for
25 hours.
9 Experimental II – Polymerizations 181
9.3.7 Variation of the Overall Concentration of Glass Beads
Under dry nitrogen, in 50 mL Schlenk tubes, four different amounts of MPS-modified glass
beads (GB80-ME: 0.6 g, 1.2 g, 2.4 g and 4.8 g) were suspended in 12 mL aliquots of a mixture
consisting of 16 mL styrene, 32 mL toluene and 24 mg AIBN (3 mM). The vessels were
degassed, kept in motion by a shaking device (160 RPM) and heated in a thermostat to 60°C
for 24 hours. Then the solids were separated from the free polymer solution.
9.3.8 Two-step Grafting
LiChrospher samples with different polystyrene monolayers that were previously attached
to the surface with this immobilized monomer approach, were used in this set of
experiments (Table 9-7). The polystyrene monolayers, covalently bound to the surface,
consist of polymer coils of different molecular weight.
Table 9-7. Molecular weights of the PS monolayers on LiChrospher used for the two-step experiments
sample PS1 PS2 PS3 PS4 PS5
nM PS (1st) (g/mol) 230000 100000 60000 32000 6500
Five different polystyrene/silica samples (300 mg/batch) were placed into a polymerization
solution consisting of 20 mL toluene, 10 mL styrene and 246 mg (50 mM) AIBN as initiator.
Polymerization took place at 60 °C for 24 hours. After separation and freeze-drying, the
solids were again characterized by elemental analysis to evaluate the changes in polymer
load in comparison to the silica after the first polymerization step.
9.3.9 Polymerization Reactions without Surface-attached
Monomers (blind tests)
300 mg LiChrospher (used as received) and 300 mg LiChrospher with immobilized octyl
trimethoxysilane were each placed into a polymerization mixture consisting of 10 mL styrene
20 mL toluene and 43 mg (0.26 mmol) AIBN and were separately heated at 60°C for 24 hours.
The workup was carried out in the same way as described before. The results of the
experiments are listed in Table 3-4.
182 9 Experimental II – Polymerizations
9.4 Poly(N,N-dimethyl acrylamide) – Polymerization
parameters
9.4.1 Influence of Reaction Time
Under dry nitrogen 1.2 g GB250-ME was suspended in a solution of 20 mL DMAA, 40 mL
DMF and 98.4 mg AIBN as initiator were added in a first series. For a second series 984 mg
AIBN were used in the polymerization mixture. The mixture was degassed in vacuum by
five freeze & thaw cycles. Then the Schlenk flask was heated with an oil bath at 60±1 °C and
agitated with a shaking device at 180 rpm. After chosen time periods (0.5, 1, 2, 4, 8 and 24 h)
samples of 10 mL were withdrawn from the reaction under nitrogen. The glass beads settled
at the bottom and after decanting, the solids were washed with THF and put into an
extraction thimble. After Soxhlet extraction overnight with dichloromethane the P(DMAA)-
modified glass beads were freeze-dried from water (10 wt.% glass beads) and analyzed by
DRIFT and elemental analysis. The decanted polymerization solution was added dropwise
to excess methanol under rapid stirring to precipitate the nonattached polymer chains. The
white precipitate was filtered off, dried in vacuum (<10-2 mbar) overnight and analyzed by
GPC.
GB250-ME-PDMAA. IR (DRIFT) ν [cm-1]: 2930, 1643, 1499, 1400, 1355, 1250-1000 cm-1.
elemental analysis: cf. Table 9-8.
Table 9-8. Elemental analysis and GPC measurements after polymerization for time dependence of
P(DMAA) formation on glass beads.
carbon
(%)
Γ0
(µmol·m-2)
substrate & surface
modification
c (AIBN)
(mmol·L-1)
monomer P(DMAA) monomer P(DMAA)
nM
(g·mol-1)
GB250-ME 10 4.8 20.3 2.0 0.07 9600
GB250-ME 100 4.8 20.7 2.0 0.19 25700
9 Experimental II – Polymerizations 183
9.4.2 Monomer Concentration Variation
For each experiment the total volume of solution was 10 mL with partial input volumes of
monomer and solvent (DMAA/DMF, v/v) of 1/2, 1/4 and 1/9. 300 mg MPS-modified
LiChrospher and 16.4 mg (10 mM) AIBN were added under an atmosphere of dry nitrogen
into the Schlenk tube. All mixtures were degassed by five freeze & thaw cycles and then
heated in a thermostat at 60±1 °C and agitated by a shaker at 160 rpm for 24 hours. After
stopping the polymerization by adding air and cooling down the P(DMAA) layered silica
and the free polymer were separated, isolated and analyzed as described in 9.4.1.
LC700-ME-PDMAA. IR (DRIFT) ν [cm-1]: 2928, 1635, 1497, 1403, 1357, 1250-1000 cm-1.
elemental analysis: see Table 9-9.
Table 9-9. Elemental analysis and GPC measurements after the monomer concentration dependent
polymerization experiments for P(DMAA) on glass beads.
carbon
(%)
Γ0
(µmol·m-2)
substrate & surface
modification
DMAA/DMF
(v/v)
monomer P(DMAA) monomer P(DMAA)
nM
(g·mol-1)
LC700-ME 1/9 5.7 11.3 0.85 0.011 18300
LC700-ME 1/4 5.7 14.1 0.85 0.010 30400
LC700-ME 1/2 5.7 19.3 0.85 0.009 59400
9.4.3 Initiator Concentration Variation
The polymerizations were carried out in 40 mL-Schlenk tubes under an atmosphere of dry
nitrogen. Five different initiator concentrations were used in the experiments: 10-4 mol⋅L-1,
10-3 mol⋅L-1, 10-2 mol⋅L-1 and 10-1 mol⋅L-1 For experimental details see Table 9-10. All batches
were heated at 60 °C for a duration of 24 hours and again the P(DMAA) layered silica and
the free polymer were separated, isolated and analyzed as described in 9.4.1.
184 9 Experimental II – Polymerizations
Table 9-10. Recipes and investigated initiator concentrations for the initiator concentration variation
experiments.
m (solid)
(mg)
V (DMAA)
(mL)
V (DMF)
(mL)
substrate &
surface
modification per AIBN concentration
c (AIBN)
(mmol)
LC700-ME 300 1 9 0.1 1 10 100
GB250-ME* 1200 20 40 - - 10 100
GB80-AE 600 1 9 0.1 1 10 100
* larger amounts because time dependence was examined in parallel
DRIFT results were shown exemplarily for glass beads and silica in 9.4.1 and 9.4.2. The
results of the elemental analysis and GPC measurements were summarized in Table 9-11.
Table 9-11. Measured carbon contents from elemental analysis, calculated graft densities and
molecular weights from GPC for the initiator concentration variation experiments.
carbon
(%)
Γ0
(µmol·m-2)
substrate & surface
modification
AIBN
(mmol·L-1)
monomer P(DMAA) monomer P(DMAA)
nM
(g·mol-1)
LC700-ME 0.1 5.7 11.1 0.9 0.011 16800
LC700-ME 1.0 5.7 11.3 0.9 0.011 18300
LC700-ME 10.0 5.7 12.0 0.9 0.011 20700
LC700-ME 100.0 5.7 11.6 0.9 0.011 18800
GB250-ME 10.0 4.8 20.3 2.0 0.069 9600
GB250-ME 100.0 4.8 20.7 2.0 0.186 25700
GB80-AE 0.1 1.7 6.4 2.3 0.068 17250
GB80-AE 1.0 1.7 6.4 2.3 0.065 18320
GB80-AE 10.0 1.7 6.4 2.3 0.055 21281
GB80-AE 100.0 1.7 6.4 2.3 0.059 20125
9 Experimental II – Polymerizations 185
9.5 Copolymerizations
9.5.1 Synthesis of Poly(styrene-co-N-acryloyl-N-methyl-propyl
phthalimide), P(S-co-AC3pht)
The substrate with surface-attached monomers was put into a Schlenk tube and toluene
and styrene in given volumes were added. Then the desired amount of N-acryloyl-N-
methyl-propyl phthalimide (AC3pht) and AIBN as initiator were dissolved in the liquid
phase by circular shaking. The solution was degassed in vacuum by five freeze and thaw
cycles and the mixture was heated by an thermostat to 60.0±0.1 °C. After a certain period of
time the polymerization reaction was stopped by air exposure and cooling down.
Sedimentation of glass beads and centrifugation of silica gel to separate the polymer
monolayer modified solid product from the polymer solution. The supernatant solution
containing the non-attached copolymer was added dropwise to 10x excess methanol to
precipitate the free copolymer, filtered off and dried in vacuum. The solids were extracted
overnight in a Soxhlet apparatus using toluene and freeze-dried from benzene. All
polymerization conditions are listed in Table 9-12. Solids were characterized by elemental
analysis (Table 9-13) and DRIFT, free copolymers by 1H-NMR, FT-IR spectroscopy and GPC
(SDV oligo system).
Table 9-12 Ingredients and reaction times for the copolymerization of styrene as main monomer (M)
and N-acryloyl-N-methyl-propyl phthalimide as functional monomer (fM) in toluene as solvent (S)
onto different silica substrates.
substrate & surface
modification
m (solid)
(mg)
m(fM)
(mg)
c(fM)
(mol%)
V (M)
(mL)
V (S)
(mL)
m (I)
(mg)
c (I)
(mmol·L-1)
t
(h)
LC700-ME 240 946 5 8.0 16 36 9 24
LC700-ME 5000 1320 10 5.0 10# 22 9 30
GB80-ME 2000 1426 5 12.0 24 18 3 24
GB80-ME 2000 2836 10 10.8 24 17 3 25
GB80-ME 2000 5673 20 9.6 24 17 3 25
GB80-AA 13500 3160 10 12.0 100 80 5 30
GB250-AA 5000 2370 20 4.0 8# 18 9 26
186 9 Experimental II – Polymerizations
Table 9-13. Elemental analysis results of the styrene-co-N-acryloyl-N-methyl-propyl phthalimide
monolayer modified substrates.
product C*
(%)
H*
(%)
N*
(%)
LC700-ME-PS-AC3pht(5%) 29.27 2.83 1.32
LC700-ME-PS-AC3pht(10%) 27.01 2.94 0.64
GB80-ME-PS-AC3pht(5%) 11.63 0.92 0.15
GB80-ME-PS-AC3pht(10%) 12.99 1.05 0.30
GB80-ME-PS-AC3pht(20%) 12.76 0.92 0.52
GB80-AA-PS-AC3pht(10%) 8.39 0.96 0.50
GB250-AA-PS-AC3pht(20%) 22.29 2.25 1.33
* obtained from elemental analysis
1H-NMR (250 MHz, CDCl3, TMS) δ [ppm]:
l), m) (4H, d) 7.6 – 8.0
c), d), e) (5H, m) 6.3 – 7.4
i), k) (4H, b) 3.3 – 3.7
h) (3H, b) 3.0 – 3.3
g) (1H, b) 3.0 – 2.8
j) (2H, b) 2.5 – 2.8
b) (1H, m) 1.8 – 2.5
N
NO
O
O
co
co
a)b)
c) c)
d) d)e)
f)g)
h)
j)
k)
m)m)
i)
l)
l)
a), f) (2H + 2H, m) 0.8 – 1.8
9 Experimental II – Polymerizations 187
LC700-ME-P(S-co-AC3pht). IR (DRIFT) ν [cm-1]: 3083, 3062, 3027, 2926, 2850, 1771, 1715, 1602, 1493, 1450, 1395, 1250-1000,
759, 723, 698 cm-1.
elemental analysis: cf. Table 9-13.
GB80-AA-P(S-co-AC3pht).
IR (DRIFT) ν [cm-1]: 3084, 3065, 3029, 2929, 2854, 1773, 1716, 1642, 1609, 1491, 1450, 1400, 1364,
1250-1000, 721, 699 cm-1.
elemental analysis: cf. Table 9-13.
GB250-AA-P(S-co-AC3pht).
IR (DRIFT) ν [cm-1]: 3085, 3063, 3029, 2927, 2853, 1772, 1715, 1643, 1604, 1491, 1450, 1399, 1370,
1250-1000, 721, 699 cm-1.
elemental analysis: cf. Table 9-13.
9.5.2 Synthesis of Poly(styrene-co-N-acryloyl-N-methyl-propyl
amine), P(S-co-AC3amine)
Silica substrates with a poly(styrene-co-N-acryloyl-N-methyl-propyl phthalimide)
monolayer were covered with THF and a sufficient amount (up to 50fold excess) of
hydrazine monohydrate (N2H4·1.5H2O) was added. The mixture was then shaken with
180 rpm in a one-neck flask with reflux condenser for 18 h at 60 °C. Afterwards the reaction
solution is filtered with a Büchner funnel and the remaining solids were washed with
toluene, dichloro methane and again with toluene. Last step was freeze-drying from
benzene. Then the substrates were analyzed by DRIFT and elemental analysis (cf.
Table 9-14). To quantify the number of amino groups on the solids a [1,1-bis(4-
methoxyphenyl)-1-phenyl]methyl (DMT) containing dye[229] (λmax = 495 nm, ε = 71700[230])
with disulfide linker was coupled to the amino function[190] for further characterization of
the solids. The glass beads with the amino functions in the copolymer monolayer were also
treated with 1 mM HClaq. and the filtrate then was titrated against 1 mM NaOHaq. to calculate
the concentration of converted amino groups. 500 mg of the phthalimide copolymer were
dissolved in a emulsion of 1 mL (17.6 mmol) hydrazine hydrate in 5 mL THF and well stirred
for 18 h at 60 °C to deprotect the amine functions. The whole emulsion is evaporated until
188 9 Experimental II – Polymerizations
there is a dry residue. This residue was dissolved in toluene and precipitation of the free
amino copolymer took place by dropwise addition of the copolymer solution in excess (10x)
methanol. The colorless polymer is filtered off and dried carefully in vacuum for
examination with 1H-NMR.
The first phthalimide conversions in the case of GB80-ME-P(S-co-AC3pht(10%)) and GB80-
ME-P(S-co-AC3pht(20%)) were carried out with methyl amine. A suspension of 1.9 g solid
substrate and 20 mL of a 2 M MeNH2 solution in THF was heated to 60 °C and shaken
(160 rpm) for 18 hours. During cooling down the glass beads settled and the supernatant
solution was decanted. Six washing steps with 40 mL THF each and two washing steps with
40 mL diethyl ether purified the glass beads before they were dried in vacuum for 8 hours.
To deprotect the amino groups in the free phthalimide copolymer 125 mg of the copolymer
were dissolved in 5 mL of a 2 M MeN2 solution in THF and stirred for 18 h at 60 °C. Then the
solution is evaporated until dry. The residues were dissolved again in toluene and the free
amino copolymer was precipitated by dropwise addition of the polymer solution in excess
(10x) methanol. The copolymer is filtered off, dried in vacuum and finally characterized with 1H-NMR.
1H-NMR (250 MHz, CDCl3, TMS) δ [ppm]:
c), d), e) (5H, m) 6.8 – 7.3
i) (2H, b) 3.1 – 3.3
h) (3H, b) 2.8 – 3.1
k) (2H, b) 2.7 – 2.8
g) (1H, b) 2.6 – 2.7
j) (2H, b) 2.4 – 2.6
b) (1H, m) 1.7 – 2.4
NH2
NO
co
co
a)b)
c) c)
d) d)e)
f)g)
h)
j)
k)
i)
a), f) (2H + 2H, m) 1.0 – 1.7
9 Experimental II – Polymerizations 189
LC700-ME-P(S-co-AC3NH2).
IR (DRIFT) ν [cm-1]: 3083, 3063, 3027, 2925, 2850, 1724, 1602, 1493, 1450, 1250-1000, 758,
699 cm-1.
elemental analysis: cf. Table 9-14.
GB80-AA-P(S-co- AC3NH2).
IR (DRIFT) ν [cm-1]: 3086, 3066, 3030, 2929, 1632, 1494, 1450, 1250-1000, 699 cm-1. elemental
analysis: cf. Table 9-14.
GB250-AA-P(S-co- AC3NH2).
IR (DRIFT) ν [cm-1]: 3085, 3065, 3029, 2926, (2855) 1638, 1491, 1450, 1250-1000, (758), 699 cm-1.
elemental analysis: cf. Table 9-14.
Table 9-14. Elemental analysis results for styrene-co-N-acryloyl-N-methyl-propyl amine monolayer
modified substrates after polymer analogue reaction of the phthalimide containing substrates with
hydrazine.
product C*
(%)
H*
(%)
N*
(%)
LC700-ME-PS-AC3amine(10%) 25.60 3.43 0.53
GB80-ME-PS-AC3amine(5%) 11.00 0.86 0.22
GB80-ME-PS-AC3amine(10%) 11.17 0.86 0.33
GB80-ME-PS-AC3amine(20%) 11.84 1.14 0.66
GB80-AA-PS-AC3amine(10%) 7.83 1.19 0.60
GB250-AA-PS-AC3amine(20%) 19.46 2.51 1.62
* obtained from elemental analysis
9.5.3 Synthesis of Poly(styrene-co-N-methacryloyl-β-alanine
succinimide ester), P(S-co-MAC2ae)
884 mg (3.5 mmol, 5 mol%) N-methacryloyl-β-alanine succinimide ester (MAC2ae) were
dissolved in a solution of 8 mL styrene (66 mmol, 95 mol%) in 16 mL DMF. Then 240 mg
190 9 Experimental II – Polymerizations
LC700-ME substrate and 35 mg (0.2 mmol, 9 mM) AIBN were added. Degassing in vacuum
by five freeze & thaw cycles was followed by a heating period of 24 h at 60 °C and shaking
with 160 rpm. The polymerization reaction was terminated by air exposure and cooling. The
mixture was centrifuged to separate the polymer modified silica gel The solid product was
extracted in a Soxhlet apparatus with DMF and toluene, 8 hours each and in the end freeze-
dried from benzene. The free copolymer precipitated on dropwise addition of the
polymerization solution to excess (10fold) methanol.
Into a solution of 1,397 mg MAC2ae (5.5 mmol, 5 mol%), 12 mL styrene (10.4 mmol,
95 mol%), 24 mL DMF 2 g GB80-ME were added. 18 mg (0.1 mmol, 3 mM) AIBN were added
as initiator. The mixture was degassed in vacuum by five freeze & thaw cycles. The
polymerization reaction was started by heating to 60 °C and the temperature was kept
constant for 24 h by an oil bath. Afterwards the polymerization reaction was stopped by air
exposure and cooling down. After the glass beads had settled, the supernatant solution was
added dropwise to an excess (10fold) of cold methanol to precipitate the free copolymer. The
beads were extracted with DMF an toluene (8 h each) in a Soxhlet apparatus and finally
freeze-dried with benzene.
1H-NMR (250 MHz, CDCl3, TMS) δ [ppm]:
c), d), e) (5H, m) 6.3 – 7.3
h) (2H, b) 3.1 – 3.4
j), i) (6H, b) 2.6 – 2.9
b) (1H, m) 1.7 – 2.4
a), f), g) (2H + 5H, m) 0.9 – 1.7
NHO
co
co
O O
NO
O
h)i)
a)b)
c) c)
d) d)e)
f)g)
j)j)
SiO2-ME-P(S-co-MAC2ae).
IR (DRIFT) ν [cm-1]: 3083, 3063, 3028, 2925, 2851, 1826, 1788, 1732, 1603, 1493, 1450, 1250-1000,
699 cm-1.
elemental analysis: LC700-ME-P(S-co-MAC2ae) C 27.37 %, H 2.68 %, N 1.61 %
GB80-ME-P(S-co-MAC2ae) C 11.21 %, H 0.87 %, N 0.50 %.
9 Experimental II – Polymerizations 191
9.5.4 Synthesis of Poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-
methyl-propyl phthalimide), P(DMAA-co-AC3pht)
First 1.47 g AC3pht (4.8 mmol, 10 mol%) were dissolved in a solution of 5 mL DMAA
(48.5 mmol, 90 mol%) in 10 mL DMF. Then 5 g of GB80-AA and 22 mg (0.13 mmol, 9 mM)
were added and the mixture was degassed in vacuum by five freeze & thaw cycles. The
polymerization reaction was run for 6 h at 60 °C (oil bath). After that time period the
polymerization solution was highly viscous and dichloro methane was added to make
filtration of the glass particles possible. The organic solvents of the filtrate were removed by
rotational evaporation. The residual substance was dissolved again in THF and dropwise
addition to cold diethyl ether precipitates the free copolymer. The filtered glass beads were
extracted with dichloro methane in a Soxhlet apparatus over night and finally dried in high
vacuum (< 10-3 mbar) to give the product.
1H-NMR (250 MHz, CDCl3, TMS) δ [ppm]:
j), k) (4H, b) 7.5 – 7.8
g), i) (4H, b) 3.4 – 3.7
c), f) (3H + 3H, m) 2.6 – 3.4
b), e), h) (1H + 3H, b) 2.1 – 2.7
a), d) (2H + 2H, b) 1.0 – 2.1
N
O NNO
O
O
co
co
a)b)
c)
c)
d)e)
g)h)
j)
j)k)k)
i)
f)
GB80-AA-P(DMAA-co-AC3pht).
IR (DRIFT) ν [cm-1]: 2928, 1643, 1772, 1715, 1495, 1398, 1356, 1250-1000, 724 cm-1.
elemental analysis: C 16.90 %, H 2.87 %, N 3.51 %.
192 9 Experimental II – Polymerizations
9.5.5 Synthesis of Poly(N,N-dimethyl acrylamide-co-N-acryloyl-N-
methyl-propyl amine), P(DMAA-co-AC3amine)
6.4 g GB80-AA-P(DMAA-co-AC3pht(10%)) were covered with 15 mL dry ethanol. After
addition of 2 mL of hydrazine monohydrate (N2H4·1.5H2O) the reaction suspension was kept
for 18 h at 60 °C. At the end of the reaction the glass beads were separated from the reaction
solution by sedimentation and decanting. Several washing steps were done analogous. The
washing steps were in detail: 4x ethanol/water (v/v 1/1), methanol and 2x dichloro methane.
The particles were filtered off and dried in high vacuum (< 10-3 mbar) overnight. For a
quantitative determination of the amino groups a DMT-dye with disulfide linker was
coupled to them, followed by elemental analysis and cleavage of the dye for quantitative
UV/vis spectroscopy (λ = 499 nm, ε = 71900). The glass beads with the amino functions in the
copolymer monolayer were also treated with 1 mM HClaq. and the filtrate then was titrated
against 1 mM NaOHaq. to obtain the amount of amino groups.
1H-NMR (250 MHz, CDCl3, TMS) δ [ppm]:
g) (2H, b) 3.1 – 3.3
c), f), i) (6H + 5H, m) 2.6 – 3.1
b), e), h) (1H + 3H, b) 2.1 – 2.7
a), d) (2H + 2H, b) 1.0 – 2.1
GB80-AA-P(DMAA-co-AC3NH2).
IR (DRIFT) ν [cm-1]: 2928, 1641, 1496, 1399, 1353, 1250-1000 cm-1.
elemental analysis: C 15.42 %, H 3.01 %, N 3.68 %.
9 Experimental II – Polymerizations 193
9.5.6 Synthesis of Poly(DMAA-co-N-methacryloyl-β-
alanine succinimide ester), P(DMAA-co-MAC2ae)
A solution of 1.36 g MAC2ae (5.4 mmol, 5 mol%) and 5 mL DMAA (48.5 mmol, 95 mol%)
as monomers in 10 mL DMF was filled under dry nitrogen in a Schlenk flask. Then
5 g GB80-AA and 25 mg (10 mM) AIBN were added before degassing in vacuum by five
freeze & thaw cycles. The mixture was heated for 25 h at 60 °C and rapid circular agitation at
200 rpm. All solids were filtered off. The filtrate solution was freed from DMF by rotational
evaporation and the residues were dissolved in THF. The free copolymer was obtained by
dropwise addition of the solution to cold diethyl ether where a white substance precipitates.
After an extraction with dichloro methane in a Soxhlet apparatus for 24 hours the copolymer
modified glass beads were dried in high vacuum (< 10-3 mbar).
1.36 g MAC2ae (5.4 mmol, 5 mol%) were dissolved in 5 mL DMAA (48.5 mmol,
95 mol%)and 10 mL DMF. 5 g LC700-ME and 22 mg (0.13 mmol, 9 mM) AIBN were added to
this solution. After that the suspension was degassed in vacuum by five freeze & thaw cycles
and the polymerization reaction kept running for 30 h at 60 °C. The reaction leaves a highly
viscous solution and in order to make a filtration of the silica gel possible dichloro methane
was added. The CH2Cl2 and DMF of the filtrate solution were removed by rotational
evaporation. The polymeric residues were dissolved in THF and dropwise addition to cold
diethyl ether precipitates the free copolymer. The filtered silica particles were extracted with
dichloro methane in a Soxhlet apparatus for 25 hours and as last step dried in high vacuum
(< 10-3 mbar).
194 9 Experimental II – Polymerizations
1H-NMR (250 MHz, CDCl3, TMS) δ [ppm]:
f) (2H, b) 3.3 – 3.5
g), h) (6H, b) 2.8 – 3.1
c) (6H, m) 2.6 – 3.0
b) (2H, b) 2.2 – 2.6
a), d) (2H + 2H, b) 1.3 – 1.9
NNHO
co
co
O
O ON
O
O
a)b)
c)
c)
d)e)
g)
h)h)
f)
e) (3H, b) 0.9 – 1.3
SiO2-P(DMAA-co-MAC2ae).
IR (DRIFT) ν [cm-1]: 2938, 1817, 1786, 1740, 1644, 1496, 1399, 1356, 1250-1000 cm-1.
elemental analysis: GB80-AA-P(DMAA-co-MAC2ae) C 14.07 %, H 2.36 %, N 3.07 %
LC700-ME-P(DMAA-co-MAC2ae) C 18.51 %, H 3.49 %, N 3.51 %.
10 Experimental III – Reactions with Functional Groups 195
10 Experimental III – Reactions with Functional Groups
10.1 Two Step Grafting on Functionalities In this section two methods of grafting polystyrene onto functional copolymer layers on
glass beads are described. In both cases the starting substrate is GB80-AA-P(S-co-AC3amine)
with 10 mol% of amino groups within the copolymer layer.
10.1.1 Immobilization of Acryloyl Chloride as Monomer
1 g of GB80-AA-PS-AC3amine were suspended in 3 mL dichloromethane (distilled and
dried) and brought to a temperature of 0 °C with an ice-bath. According to the estimated
amount of amino groups 14 µL (139 µmol) acryloyl chloride and 24 µL (172 µmol) Et3N were
added drop wise to the suspension that was agitated by a shaking device. After 20 minutes
the ice-bath was removed and the suspension let shaken for 4 hours at room temperature.
Afterwards the reaction solution is removed by a porous glass filter. Glass beads a washed
with acidified ethanol/water (pH = 3, v/v 1/1) to remove excess base, then washed neutral
with ethanol/water. Basic ethanol/water treatment (pH = 11) to recovered amino groups and
again washed neutral with ethanol/water. Two washing cycles with pure ethanol and diethyl
ether followed for easy removable volatiles. Drying overnight under reduced pressure yields
colorless glass beads (GB80-AA-P(S-co-AC3acrylamide)) that were characterized by DRIFT
and elemental analysis:
GB80-AA-P(S-co-AC3acrylamide).
IR (DRIFT) ν [cm-1]: 3086, 3066, 3030, 2928, 1624, 1609, 1491, 1450, 1408, 1250-1000, 799,
700 cm-1.
elemental analysis: C 7.27 %, H 1.03 %, N 0.57 %.
10.1.2 Immobilization of 2-Brom-Propionic Acid Bromine as Starter
for ATRP
In a 25 mL-Schlenk flask with a rubber septum a suspension of 500 mg GB80-AA-P(S-co-
AC3amine) and 3 mL acetone (p.a.) was agitated at 160 rpm in a dry nitrogen atmosphere.
Via syringe a few drops triethyl amine and 2-brom-propionic acid bromine were added to
196 10 Experimental III – Reactions with Functional Groups
the suspension. While shaking at room temperature the mixture turns yellow. After a
reaction time of six hours the glass beads were filtered off and washed with acetone. Then
the modified glass beads are subsequently washed with acidified (pH = 3) ethanol/water (v/v
1/1), ethanol/water, ethanol (3x) and diethyl ether (3x). Drying under high vacuum for 6
hours gives GB80-AA-P(S-co-AC3(2-brom-propionic amide)) what was immediately used for
the polymerization (10.1.4).
10.1.3 Polymerization of Styrene with Polymer-attached Monomers
In a 40 mL-Schlenk tube 200 mg GB80-AA-P(S-co-AC3acrylamide) were suspended in 8 mL
dry toluene. 4 mL styrene and 18.6 mg (9 mM) AIBN were added. The mixture was degassed
by five freeze & thaw cycles and finally heated to 60 °C and shaken at a speed of 160 rpm for
26 hours. After stopping the polymerization by cooling down the supernatant liquid was
decanted and added dropwise to 120 mL methanol to precipitate the free polystyrene. The
glass bead were flushed into a filter and extracted with toluene overnight in a Soxhlet
apparatus. Then the glass beads were freeze dried in 3 mL benzene to get GB80-AA-P(S-co-
AC3AA-PS) as colorless product that was characterized per elemental analysis and DRIFT.
GB80-AA-P(S-co-AC3AA-PS).
IR (DRIFT) ν [cm-1]: 3086, 3066, 3030, 2929, 1630, 1494, 1450, 1250-1000, 699 cm-1.
elemental analysis: C 9.53 %, H 1.08 %, N 0.56 %.
As blind test without polymer attached monomers the same polymerization procedure as
described above was carried out with the precursor support GB80-AA-P(S-co-AC3amine) to
get GB80-AA-P(S-co-AC3amine)-PS as product and afterwards characterized in the same
manner:
GB80-AA-P(S-co-AC3amine)-PS.
IR (DRIFT) ν [cm-1]: 3085, 3065, 3029, 2928, 1629, 1495, 1450, 1250-1000, 699 cm-1.
elemental analysis: C 8.33 %, H 1.06 %, N 0.65 %.
10 Experimental III – Reactions with Functional Groups 197
10.1.4 Atom Transfer Radical Polymerization of Styrene with
Polymer-attached Initiators
In a 40 mL-Schlenk tube 48 mg (0.217 mmol) copper(II)bromide were suspended in 25 mL
(0.217 mol) styrene and 376 µL (2.17 mmol) PMDETA were added and agitated until a slight
green color of the copper complex appears. Then 230 mg GB80-AA-P(S-co-AC3(2-brom-
propion amide)) were mixed into the solution followed by two freeze & thaw cycles for
degassing. Under an atmosphere of dry nitrogen 311 mg (2.17 mmol) copper(I)bromide was
added and again two degassing cycles took place. The polymerization reaction was started
by raising the temperature to 90 °C. For 21 hours the mixture was shaken at 190 rpm at
constant temperature. The viscosity of the suspension increased slightly during the
polymerization. The reaction was stopped by cooling down and flooding the vessel with air.
Parallel a degassed mixture of 25 mL (0.217 mol) styrene with 376 µL (2.17 mmol) PMDETA,
230 mg GB80-AA-P(S-co-AC3(2-brom-propion amide)), 296 µL (2.17 mmol) PEBr (as free
initiator) and 311 mg (2.17 mmol) copper(I)bromide was polymerized for 2 hours in order to
get free polystyrene under same polymerization conditions for GPC analysis. For this case
viscosity of the reaction mixture increased much faster during polymerization. The glass
beads were filtered off and washed with toluene. Then the solids were extracted with THF
for 22 hours. The glass beads remained light green in color even after treatment with solvents
of different polarity. Finally the polystyrene grafted glass beads were freeze-dried in benzene
and characterized by elemental analysis. Free polystyrene was isolated by first reducing the
volume of filtrate with a rotary evaporator and adding drop wise to excess (10fold) methanol
to precipitate the polymer. Because of slight blue color of the polystyrene it was dissolved in
THF and again precipitated in methanol. After drying under high vacuum overnight the free
PS is characterized by GPC.
GB80-AA-P(S-co-AC3(propionic amide)PS).
elemental analysis: C 50.95 %, H 4.46 %, N 0.30 %.
198 10 Experimental III – Reactions with Functional Groups
10.2 Coupling of Dyes to the Functionalities of the Copolymer
10.2.1 Coupling of Fluorescein Isothiocyanate
A solution of 10 mg FITC (fluorescein isothiocyanate) in 10 mL distilled and dried DMF
was prepared and glass beads with different surface-attached polymer monolayers as well as
the corresponding free polymers were mixed or dissolved at room temperature. The amount
of dye molecules was always calculated so that an excess compared to all functionalities
within the polymer (monolayer) was added. 20 mg of GB80-ME-P(S-co-AC3amine(10%))
were put into 1 mL of FITC solution and shaken for 2 hours. Afterwards the supernatant
FITC solution was discarded and the glass beads were washed repeatedly with methanol
until no further coloring of the solution takes place. Then the now yellow glass beads were
washed with toluene and diethyl ether and dried in a high vacuum overnight. The stained
glass beads remained yellow and the fluorescence of the dye was examined a confocal
scanning laser microscope to have many slices in z-direction. As blind test experiments
20 mg GB80-ME-P(S-co-MAC2ae) and 20 mg GB80-ME-PS were also put into 1 mL of FITC
solution and received the same treatment as the amino-functionalized glass beads. During
the washing steps the color faded away and after drying both substrates are again colorless.
100 mg P(S-co-AC3amine(5%)), P(S-co-AC3amine(10%)) and P(S-co-AC3amine(20%)) were
each dissolved in a solution of 60 mg FITC in 20 mL DMF and stirred for one hour at room
temperature. Then the DMF of each solution was nearly removed and the remaining solution
was added dropwise to excess (10x) cold methanol to precipitate the colored copolymers.
Filtration and washing with methanol removed unbound dye. The colored copolymers were
dried in vacuum overnight. It was not possible to dissolve the colored P(S-co-AC3thiourea-
fluorescein(10%)) and P(S-co-AC3thiourea-fluorescein(20%)) again for UV/vis spectroscopy
characterization in any solvent or mixture of solvents. Due to this insolubility only the
P(S-co-AC3thiourea-fluorescein(5%)) was examined with the photospectrometer at a
concentration of 1 mg·mL-1in DMF. For the other two polymer species only a description of
the optical properties was done.
10 Experimental III – Reactions with Functional Groups 199
10.2.2 Coupling of DY-635-NH2
A solution of 1 mg (1.356 mmol) DY-635-NH2 (8-(3-(1-(5-(2-amino-ethylcarbamoyl)-
pentyl)-3,3-demethyl-5-sulfonato-1,3-dihydro-indol-2-ylidene)-10-tbutyl-2,3,5,6,11b,11c-hexa-
hydro-1H,4H-11-oxonia-3a-aza-benzo(de)anthracene) in 0.5 mL EtOHabs. was prepared and
100 µL of this solution were diluted with 170 µL ethanol to a 1 mmol·L-1 solution. Then some
milligram of silica solids with a monolayer of P(DMAA-co-MAC2ae) or P(S-co-MAC2ae) were
suspended in a few milliliters of the dye solution. After 20 minutes shaking at room
temperature the solids were filtered off, washed three times with methanol and hot extracted
in THF for 18 h to remove unbound dye molecules. Finally all particles were dried in
vacuum.
A solution of 50 mg P(S-co-MAC2ae(5%)) in 5 mL dry toluene was mixed well with 50 µL
of a 1 mM DY-635-NH2 ethanol solution. After a reaction time of 10 minutes at room
temperature the mixture was added dropwise to cold methanol to precipitate the labeled
copolymer. It was filtered off, washed with methanol and dried in vacuum. The product
again was dissolved in toluene and analyzed with UV/vis spectroscopy at a concentration of
1 mg·mL-1.
10.3 Enzyme Immobilization
10.3.1 Activity Assay
The activity assay for glucose oxidase (GOD) is performed using four different solutions:
The glucose solution contains the substrate, the GOD solution delivers the enzyme to the
immobilization site on the glass beads, the horseradish peroxidase solution transforms the
GOD product hydrogen peroxide into electrons that reduce the dye 2,2‘-Azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) for a color change from colorless to deep
green in the fourth solution.
1 liter of phosphate buffered saline stock solution (10x PBS) at pH = 7.4 consists of 0.19 mol
NaH2PO4, 0.81 mol Na2HPO4, 1.38 mol NaCl and 0.027 mol KCl in distilled water and was
used in all described handling steps with 10 mmol·L-1 phosphate concentration (1x PBS).
To prepare the glucose solution 301.4 mg D-(+)-glucose were dissolved in 3 mL phosphate
buffered saline (PBS, pH=7.4). To get the 1 µmol·L-1 GOD solution 16.1 mg (0.1 µmol) GOD
200 10 Experimental III – Reactions with Functional Groups
were dissolved in 10 mL PBS and 1 mL of this solution was diluted with 9 mL PBS and kept
cool (~4 °C). 15 mg horseradish peroxidase were dissolved in 5 mL PBS and kept cool. The
dye solution consisted of 274.5 mg ABTS in 10 mL PBS and was kept cool and stored in the
dark.
To measure the GOD activity the freshly prepared solutions were mixed in a cuvette with
the following dosages: 1 mL glucose solution, 0.2 mL peroxidase solution, 0.2 mL dye
solution and 1.6 mL PBS (pH=7.4). Upon addition of GOD loaded active ester polymer layers
on glass beads the assay turned green with time and a UV spectrometer measured the
extinction change at a wavelength of λ=405 nm. The cuvette was equipped with a magnetic
stirring bar and stirred for equal color distribution. For comparison also experiments with
30 µL of 1 µmol·L-1 GOD solution and experiments with inert solids with a polystyrene layer
were carried out.
For each experiment 10 mg of dry solid support (GB80-AA-P(DMAA-co-MAC2ae(10%)))
were put into 1 mL GOD solution for a certain time period. Then the glass beads were
decanted and washed several cycles with PBS. After filtering off the beads were immediately
used in the GOD activity assay. Specific details of the enzyme experiments are explained in
the following paragraphs.
10.3.2 Washing Cycles
After 30 minutes of enzyme immobilization in 1 µmol·L-1 GOD solution four samples of
GB80-AA-P(DMAA-co-MAC2ae(10%)), 10 mg each, were washed with PBS for a different
number of cycles. For one washing cycle the filtered solids from the prior solution were
taken, put into 1 mL PBS, agitated for approximately 5 minutes and allow to settle. The
supernatant liquid was discarded. The washed, enzyme containing supports were then used
in the GOD activity assay.
10.3.3 Varied Immobilization Time
Five samples of 10 mg GB80-AA-P(DMAA-co-MAC2ae(10%)) were put into 1 µmol·L-1
GOD solution for immobilization and kept in motion for different periods of time. The
examined time periods were 10, 30, 60, 120 and 240 minutes. After the immobilization
procedure all samples were washed with three PBS washing cycles to get rid of free GOD
and finally measured with the GOD activity assay.
10 Experimental III – Reactions with Functional Groups 201
10.3.4 Varied Initial Immobilization Concentration
Immobilization of the enzyme on GB80-AA-P(DMAA-co-MAC2ae(10%))took place for 30
minutes in GOD solution with three different GOD concentrations. In each of the solutions,
1·10-6, 10·10-6, 100·10-6 mol·L-1, 10 mg of the solid support were put and after immobilization
washed with PBS three times. The activity of the immobilized GOD enzyme was measured
by UV spectroscopy of the activity assay.
10.3.5 Immobilization of Dyed Enzymes
In order to get colored GOD enzymes, a DY-635-NH2 solution was added to a GOD
solution where reaction with free accessible carboxyl groups at room temperature on the
enzyme took place. This deep blue colored enzyme solution (6.2 10-5 mol·L-1) was used as
stock solution for the following enzyme immobilization steps. For 10-6 mol·L-1 GOD solution
161 µL of the stock solution were diluted with 839 µL PBS and for 10-6 mol·L-1 GOD solution
16 µL of the stock solution were diluted with 9846 µL PBS. Each immobilization
concentration was used with either GB80-AA-P(DMAA-co-MAC2ae(10%)) or LC700-ME-
P(DMAA-co-MAC2ae(10%)). To immobilize the colored enzyme 50 mg of solid support were
suspended in 500 µL GOD solution and agitated for 30 minutes at room temperature. Four
subsequent washing cycles with 1 mL deionized water and vivid agitation for 15 minutes
were used to remove non-covalently bound GOD. The enzyme modified solids were
investigated with optical methods (CCD camera and fluorescence reading device
(Biodetect®).
10.3.6 Hydrolysis of Immobilized Enzymes
16.2 mg GOD were dissolved twice in 10 mL PBS (pH = 7.4) to get two 10-4 mol·L-1
solutions. 51 mg and 150 mg GB80-AA-P(DMAA-co-MAC2ae(10%)) as separate samples
were suspended in these solutions and agitated for 30 minutes in order to immobilize the
enzyme on the support surface. After the immobilization procedure the solids were
separated by decanting the supernatant solution and washed three times with distilled water
for 10 minutes.
In a 25 mL flask equipped with a reflux condenser the GOD-covered glass beads were
suspended into 5 mL of 6 mol·L-1 hydrochloric acid, stirred and heated to reflux (115 °C oil
bath) for 24 hours. The solids were filtered off and the filtrate was concentrated in a rotary
202 10 Experimental III – Reactions with Functional Groups
evaporator until dry. Then the residue was incorporated in 1 mL of a buffer (pH = 2.0)
provided by the Pediatric hospital Freiburg where the analysis via liquid chromatography of
the single amino acid units took place[210].
10.4 Oligo Nucleotide Immobilization For the DNA experiments different oligo nucleotides with functional groups and
fluorescent dyes were used as supplied by Thermo Electron GmbH, Ulm (Table 10-1). The
oligo-nucleotide reacted with the active ester surface groups on the solid supports got at the
5’-end an amino group with a C6-spacer (“aminolink”) and the following nucleotide
sequence: 5’-TTT-TTT-TTT-TTT-TTT-TTT-TTA-TGG-TGT-AAA-CTT-GTA-CCA-GT-3’. This
oligo nucleotide is called NH2-DNA. To check if the immobilization procedures were
successful an oligo-nucleotide that bears an amino group as linker and a fluorescent dye for
detection was used. This connection control DNA has a Cy5 dye as 5’-modification, an amino
group with C7-spacer as 3’-modification and a nucleotide sequence as follows: 5’-CAA-
GAA-CTA-TGC-TTC-AAG-CAG-ACA-TCA-ACC-GTT-TTT-TTT-TTT-3’. This oligo-
nucleotide is called CC-DNA (coupling control). The complementary oligo-nucleotide for
hybridization on the NH2-DNA got a Cy5 fluorescent dye as 5’-modification and 5’-ACT-
GGT-ACA-AGT-TTA-CAC-CAT-AA-3’ as nucleotide sequence. This oligo-nucleotide is
called AS-DNA (anti-sense). All Cy5-labelled oligo-nucleotides have a deep blue color.
Table 10-1. List of functionalized oligonucleotides used in the DNA-immobilization experiments.
abbreviation structure
NH2-DNA aminolink-C6-(5’-TTT-TTT-TTT-TTT-TTT-TTT-TTA-TGG-TGT-AAA-CTT-
GTA-CCA-GT-3’)
CC-DNA Cy5-(5’-CAA-GAA-CTA-TGC-TTC-AAG-CAG-ACA-TCA-ACC-GTT-
TTT-TTT-TTT-3’)-C7-aminolink
AS-DNA Cy5-(5’-ACT-GGT-ACA-AGT-TTA-CAC-CAT-AA-3’)
For the handling of DNA oligo-nucleotides special salt solutions were used. NaPi is an
sodium phosphate buffer. For pH = 8.0 with an overall salt concentration of 300 mmol·L-1 it
10 Experimental III – Reactions with Functional Groups 203
consists of 285 mmol Na2HPO4 and 15 mmol NaH2PO4. A 10-fold sodium salt citrate (SSC)
stock solution consists of 1.5 mol NaCl and 0.15 mol sodium citrate in 1 liter distilled water,
has a pH-value of 7.0 and. The aqueous sodium dodecyl sulfate stock solution had 10 wt.%
of the surfactant.
10.4.1 Immobilization Reaction
To handle the oligo-nucleotides 100 µmol·L-1 solutions were made from 0.05 µmol DNA
and 500 µL NaPi buffer at a pH-value of 8. The solids used for the oligo-nucleotide
immobilization were GB80-AA-P(DMAA-co-MAC2ae(10%)) and LC700-ME-P(DMAA-co-
MAC2ae(10%)). For every immobilization reaction 40 mg solid support were suspended in
400 µL SSC (10x)inside of an Eppendorf vial and 100 µL of DNA-solution (= 10 nmol DNA)
were added. For each support, glass beads and silica gel, one immobilization with NH2-DNA
and one immobilization with CC-DNA were carried out. The immobilization lasted two
hours at a temperature of 50 °C in a water bath and the vials were agitated with a shaking
device at 230 rpm. After reaction the solids were let settle and the supernatant solution was
decanted. The supports reacted with the coupling control oligo-nucleotide were colored blue.
All remaining solids were washed three times with SSC buffer (2x) including 0.1 wt.% SDS
using a vortex apparatus for 10 minutes each cycle. Then three washing cycles with
deionized water (MilliQ, 18.2 MΩ) followed. After the washing procedure the glass beads
and the silica gel with the immobilized CC-DNA stayed blue. They were dried for detection
of the fluorescent DNA-label with a DNA-fluorescence-reading device. Solids with the
immobilized NH2-DNA (GB80-AA-P(DMAA-co-MAC2amido-DNA) and LC700-ME-
P(DMAA-co-MAC2amido-DNA)) were prepared for the hybridization step.
10.4.2 Hybridization
Each sample with immobilized NH2-DNA, 50 mg of GB80-AA-P(DMAA-co-MAC2amido-
DNA) and 40 mg of LC700-ME-P(DMAA-co-MAC2amido-DNA), was suspended in 900 µL
SSC (6x, 0.5 wt.% SDS). Then 100 µL of AS-DNA solution were added to each solution. Both
reaction mixtures were agitated at room temperature for 30 minutes on a vortex apparatus.
Subsequent washing cycles (same agitation, 15 minutes each) were carried out three times
with 1 mL SSC buffer (2x, 0.1 wt.% SDS) and finally with 1 mL SSC buffer (0.5x). Filtration
and drying led to blue colored solids.
204 10 Experimental III – Reactions with Functional Groups
10.5 Biotin assay GB80-AA-P(S-co-AC3amine(10%)), GB250-AA-P(S-co-AC3amine(20%)) and GB80-AA-
P(DMAA-co-AC3amine(10%)) as solid supports equipped with a covalently bound
N-acryloyl-N-methyl-propyl amine containing copolymer were used for the following biotin
assay procedure:
15.4 mg (45 mmol) N-hydroxysuccinimido-biotin were dissolved in 3 mL dry DMF. 1 mL
of this solution was given to 100 mg of each solid. Reaction took place at room temperature
overnight. Then the supernatant was removed from the settled solids with filter paper. The
residuals were suspended in methanol and washed two times (30 minutes vortex agitation).
After drying in air overnight the remaining solids were put each into 1 mL of 1 µmol·L-1
solution of streptavidin in PBS (1x) for 30 minutes at room temperature and 6 hours at 4 °C.
The supernatant was discarded. 0.2 mg of fluorescein labeled biotin (M = 644.7 g·mol-1) was
dissolved in 10 mL PBS (1x) giving 30 µmol·L-1 solution. Every solid was suspended in 1 mL
of the biotin-fluorescein solution and kept at 4 °C overnight. Again the supernatant was
discarded and the solid were washed two times with distilled water and in a final step with
acetone. After drying the solids were analyzed by a fluorescence reading device (Biodetect®).
10.6 Catalyst Immobilization Basic support material for the catalyst immobilization was GB80-AA-P(S-co-
AC3amine(10%)). The covalent attachment of the Ruthenium catalyst species onto the
support was realized by immobilization of a styrene based Hoveyda type ligand[221] via
reaction of its carboxyl group with the amino groups of the support material[188] using
DCC, tBuOH and Huenig’s base. Subsequently free Grubbs catalyst complexes were fixed to
the immobilized ligands in CH2Cl2 under reflux conditions with Cu(I)Cl as activator. The
ring closing metathesis of N,N-bis(allyl-tosyl amide) was used as benchmark test for the
reactivity respectively the effectivity of the whole catalyst system. The reaction was carried
out in dried dichloromethane under reflux conditions with an amount of 5 mol% of
immobilized Grubbs catalyst species related to the amount of N,N-bis(allyl-tosyl amide). The
substrate with the catalyst was filtered off after each cycle and used for at least three reaction
cycles. The product of the ring closing metathesis was isolated by distillation to determine
the conversion.
11 Experimental IV – Silicon Wafers 205
11 Experimental IV – Silicon Wafers
11.1 Silane Immobilization Standard silicon wafers with a 2.5 nm silicon oxide layer (polished face) were used as
substrates. For silane immobilization and polymer deposition the silicon wafers were cut into
pieces between 2 x 2 and 2 x 4 cm. The pieces were cleansed of organic traces by subsequent
ultrasound exposure in methanol, toluene and acetone. Silicon dust particles were then
removed by SnowJet treatment using a carbon dioxide snow plume.
For the silane immobilization the wafer pieces were placed in a Schlenk tube equipped
with a reflux condenser in an inert atmosphere of dry nitrogen. Under nitrogen counter flow
dried toluene was added until the silicon was covered. Some drops of freshly distilled
triethylamine were added as catalyst. 1 mL of methacryloyl propyl trimethoxysilane MPS
was added in excess amount to react most of the surface groups. The immobilization reaction
was carried out at 120 °C for 3.5 hours heated by an oil bath and the solution was stirred
with a magnetic stir bar. After cooling down the silicon wafer pieces were removed and
rinsed with toluene and methanol. Finally they were dried with dry nitrogen and
characterized by ellipsometric measurements to determine the silane layer thickness. Until
the use for polymerization experiments the surface modified silicon was stored under dry
nitrogen.
11.2 P(DMAA) Layers Generally the MPS-modified silicon wafers were put into a Schlenk tube under an
atmosphere of dry nitrogen, containing the polymerization mixture consisting of the
monomer N,N-dimethyl acrylamide, the solvent DMF and the initiator AIBN. Five freeze-
thaw cycles degassed the polymerization system carefully. The polymerization was started
by placing the reaction vessel into a thermostat at 60±0.1 °C. After the chosen polymerization
time the silicon sample was withdrawn from the polymerization solution, rinsed with DMF
and extracted in a Soxhlet apparatus for at least 18 h with dichloromethane. Finally the
samples were dried under vacuum and measured with ellipsometry to determine the
polymer film thicknesses. At least two silicon wafer samples were prepared for every
polymerization condition variation.
206 11 Experimental IV – Silicon Wafers
The polymerization in the solution was stopped by cooling down to ambient temperature
and air exposure. The produced free P(DMAA) was precipitated by dropwise adding the
polymerization solution to cold excess (10x) diethyl ether and filtrated. If the precipitate was
still viscous due to embedded DMF, the polymer was dissolved in THF and again
precipitated in cold diethyl ether. After filtration the remaining colorless polymer is dried
under high vacuum and characterized by GPC measurements.
11.2.1 Influence of the Polymerization Time
For the time dependent polymerization the polymerization mixture was prepared of 3 mL
N,N-dimethyl acrylamide in 6 mL DMF and 4.5 mg AIBN (3 mmol·L-1). The chosen periods
of time were 10 min, 30 min, 1 h, 2 h, 4 h, 14 h, 16 h and 21.5 h. Silicon wafer preparation,
polymerization and isolation of the wafers and free polymers were carried out as described
above.
11.2.2 Influence of the Monomer Concentration
The overall volume of the polymerization solution was in every case 10 mL. Chosen ratios
between monomer and solvent were 1:0, 1:1, 1:2, 1:4 and 1:9 to vary the monomer
concentration. 5 mg (3 mmol·L-1) AIBN was used as initiator. For the polymerization the
reaction vessels were kept at 60 °C for 16 hours.
11.2.3 Influence of the Initiator Concentration
For the variation of the initiator concentration the polymerization mixture was prepared of
3 mL N,N-dimethyl acrylamide in 6 mL DMF. 0.15 mg (0.1 mmol·L-1), 1.5 mg (1 mmol·L-1),
4.5 mg (3 mmol·L-1), 15 mg (10 mmol·L-1) and 150 mg (100 mmol·L-1) were the chosen
amounts of initiator. For 16 hours the polymerization was kept at 60 °C.
11.3 Microstructured Layers For all microstructure experiments MPS-modified silicon wafers were used. There were
different methods to microstructure the silicon surfaces with polymer layers and two
different kind of masks have been used. The first type of masks were double printed laser
images on common overhead projector transparencies. Here the patterns were designed with
a vector oriented graphics program (CorelDRAW 9.0) and they include squares, lines and
grids with feature widths of 100, 200 and 400 µm (Figure 11-1).
11 Experimental IV – Silicon Wafers 207
Figure 11-1. Template used for laser printed masks on plastic with different widths and structures.
Squares give a polymer grid with dimples, lines give polymer walls and grids give polymer pillars.
For sizes of 100 µm and below the printing procedure by printing twice on the same plastic
sheet to get a deep black pattern is too inaccurate and the resolution of the printer (300 dpi)
was insufficient. The second type of masks were small metal nets with different patterns
normally used for transmission electron microscopy (TEM) These patterns were lines and
grids with mesh sizes of 128 µm (mesh 200 = 200 grid bars per inch) and 64 µm (mesh 400 =
400 grid bars per inch).
11.3.1 UV Photoablation of Surface-attached Monomers
Metal electron microscopy nets were put on MPS-modified silicon wafers. The UV light
source was a pen ray lamp (high pressure Hg, 500 W, L.O.T. Oriel) that emits in the far
(deep) UV range (<200nm). Exposure to the emitted light for 20 minutes decomposed the
MPS in the illuminated areas. Afterwards the silicon samples were rinsed with 1 M KOHaq.,
distilled water and methanol to remove the molecular debris of the ablation.
11.3.2 Grafting of Polystyrene
Silicon samples with an ablation patterned MPS-layer were put into a Schlenk tube under
an atmosphere of dry nitrogen. The polymerization solution, consisting of 20 mL styrene,
20 mL toluene and 19.6 mg (3 mmol·L-1) AIBN was added. Four freeze-thaw cycles removed
trace oxygen from the polymerization system. By placing the reaction vessel into a
thermostat at 60±0.1 °C the polymerization was started. After 24 hours the silicon samples
were withdrawn from the polymerization solution, rinsed with toluene and extracted in a
Soxhlet apparatus for 24 h with toluene. Finally the samples were dried under vacuum.
Ellipsometric measurements determined film thickness and the lateral extension of the
208 11 Experimental IV – Silicon Wafers
structures (imaging ellipsometry). The free PS was characterized by GPC measurements with
the SDV poly columns in THF:
Mn = 154000 g·mol-1 , Mw = 301000 g·mol-1.
11.4 Combined Functional Layers & Networks To prepare a combined system of a polymer network and a covalently bound, thin
polymer layer with chemical functionalities based on a planar MPS-modified silicon wafer a
sequence of several steps was necessary. First the modified wafers were coated with a UV-
cross-linkable copolymer with benzophenone units with layer thicknesses ranging from
60 nm to several hundred nanometers. Then the coated wafers were equipped with a mask.
Exposition to UV light cross-linked the polymer layer and bound it to the MPS-layered
surface in the illuminated areas. The patterned polymer layer evolved during extraction with
a good solvent for the copolymer. Finally the prepared wafers were used in a
copolymerization reaction. A covalently bound, functional copolymer layer of P(S-co-
AC3pht) was formed on the wafer and by treatment with hydrazine solution transferred to
P(S-co-AC3amine). Thickness could not be measured because the scale towards the polymer
network was too small for proper detection but a detection of fluorescence after a subsequent
reaction of the amino groups with FITC was carried out.
11.4.1 Preparation of Copolymers for Network Formation
All polymerization solutions were prepared in Schlenk tubes under an atmosphere of dry
nitrogen and degassed by 3 freeze & thaw cycles to remove residual oxygen traces. For
polymerization the reaction vessels were heated in a thermostat to 60 °C for the given
amount of time. The recipes are listed in Table 11-1:
11 Experimental IV – Silicon Wafers 209
Table -1. Ingredients and reaction times for the copolymerization of styrene and N,N’-dimethyl
acrylamide with benzophenone-methacrylic ester.
11
MABP styrene toluene AIBN t
(mol%)
m (mg)
n (mmol)
V (mL)
n (mmol)
V (mL)
m (mg)
c (mmol·L-1)
(h)
1 540 2.0 22.76 198.0 0.0 11.2 3.00 19
5 666 2.5 4.48 47.5 15.5 0.5 0.15 16
10 1332 5.0 4.25 45.0 15.7 0.5 0.15 16
MABP DMAA DMF AIBN t
(mol%)
m (mg)
n (mmol)
V (mL)
n (mmol)
V (mL)
m (mg)
c (mmol·L-1)
(h)
1 107 0.4 4.11 39.6 25.0 13.1 3 16
5 666 2.5 4.53 47.5 31.5 5.9 1 21
10 1332 5.0 4.29 45.0 30.0 5.6 1 21
All polymerization solutions were prepared in Schlenk tubes under an atmosphere of dry
nitrogen and degassed by 3 freeze & thaw cycles to remove residual oxygen traces. For
polymerization the reaction vessels were heated in a thermostat to 60 °C for the given
amount of time. To precipitate the polymers the reaction mixture of P(S-co-MABP) was
slowly poured into cold methanol while stirring and for the P(DMAA-co-MABP) polymers
the precipitation solvent was cold diethyl ether. In both cases the colorless polymer was
filtered off and vacuum dried overnight. All copolymers were analyzed by 1H-NMR to verify
the incorporation of benzophenone units (Table 11-2). Molecular weights were determined
by GPC measurements:
P(S-co-MABP(1%)) GPC: Mn = 220000 g·mol-1 , Mw = 415000 g·mol-1.
P(DMAA-co-MABP(1%)) GPC: Mn = 28400 g·mol-1 , Mw = 95300 g·mol-1.
210 11 Experimental IV – Silicon Wafers
1Table 11-2. H-NMR measurements of the copolymers reveal the amount of incorporated
benzophenone after polymerization.
Copolymer 1H-NMR integrals Benzophenone content*
5H arom. styrene 9H arom. MABP (mol%)
P(S-co-MABP(1%)) 82.7 13.9 9.3
P(S-co-MABP(5%)) 102.1 31.1 16.9
P(S-co-MABP(10%)) 44.2 24.1 30.3
6H+1H methyl+methin
DMAA
9H arom. MABP
P(DMAA-co-MABP(1%)) 145.0 2.0 1.0
P(DMAA-co-MABP(5%)) 43.4 3.0 5.3
P(DMAA-co-MABP(10%)) 40.8 5.1 9.8
* the P(S-co-MABP) copolymers show a preferred MABP incorporation
11.4.2 Coating & UV-cross-linking
The use of the following copolymers and following coating methods worked out best (cf.
Table 6-1 & Table 6-2): 0.5 g P(S-co-MABP(1%)) were dissolved in 10 mL toluene and applied
to the MPS-modified silicon surface via spin casting with a speed of 2000 rpm (B.L.E.
Delta10). The spin casting procedure was chosen because the PS-co-BP layers tend to dewet
upon dip casting. 0.7 g P(DMAA-co-MABP(1%)) were dissolved in 10 mL isopropanol and
MPS-modified wafer pieces were dipped into the solution and slowly retracted with a speed
of 1 mm·s-1 (Zwick Z2.5). The copolymers with 5 mol% and 10 mol% benzophenone were not
used for further experiments because they had a strong tendency to crosslink early and give
inhomogeneous polymer layers. The air dried copolymer layer thickness was
elipsometrically measured for each sample (cf. Table 6-1 & Table 6-2). Then masks as
described in 11.3 were put onto the coated wafer pieces and illuminated for 100 minutes with
light of a wavelength of 365 nm (Stratalinker, Stratagene). The masks were removed and the
wafers were extracted in either toluene or isopropanol for at least 8 hours. The originated
copolymer network patterns were measured by imaging ellipsometry, profilometry and
visually magnified with a CCD camera.
11 Experimental IV – Silicon Wafers 211
11.4.3 Functional layers
In a Schlenk tube under dry nitrogen a polymerization solution consisting of 72 mg AC3pht
(1% based on the styrene amount) and 4.5 mg AIBN dissolved in 6 mL DMF and 3 mL
styrene was prepared. The solution was degassed by 5 freeze & thaw cycles. Then the silicon
wafers with an attached and patterned P(DMAA) polymer network were given under dry
nitrogen into this polymerization mixture. The polymerization reaction was run for 16 h at
60 °C in a water thermostat. After the chosen time period the silicon wafers were taken out of
the polymer solution and extracted with toluene in a Soxhlet apparatus for 16 h. The patterns
were observed with a CCD camera. The generated free copolymer was analyzed with 1H-
NMR and GPC. The patterned surfaces were treated with hydrazine solution for 3 hours in
order to convert the phthalimide function into an amine. The samples were well rinsed with
acetone and methanol. Finally one prepared wafer sample was brought to reaction overnight
in a solution of 5 mg FITC in 5 mL CH2Cl2 and another wafer sample was used for biotin
assay treatment (same reactant solutions used as in 5.2.1). These samples were analyzed with
the biochip fluorescence reader.
The silicon wafers with an attached and patterned PS polymer network were placed in a
degassed polymerization solution of 75 mg MAC2ae (1% based on DMAA amount) and
4.5 mg AIBN dissolved in 3 mL DMAA and 6 mL DMF. The polymerization mixture was
kept at 60 °C for 6 h. GPC measurements on PSS GRAM columns calibrated with PMMA
standards of the free P(DMAA-co-MAC2ae(1%)) GPC gave the following molecular weight
averages: Mn = 64000 g·mol-1 , Mw = 279000 g·mol-1. After copolymerization the wafers were
retracted from the polymer solution and extracted with dichloromethane in a Soxhlet
apparatus for 16 h. Afterwards prepared wafer samples were put overnight into a 1 mmol·L-1
solution of DY-635-NH2 in EtOHabs. to bind the dye to the functional layer. These samples
were again analyzed with the biochip fluorescence reader.
212 12 Summary
12 Summary
In this thesis the surface modification of various solid substrates with monomer-bearing
silanes and subsequent use in radical polymerization is described. The surface-attached
monomers provide during the polymerization process the buildup of a covalently bound
polymer monolayer. A variety of homopolymers with different polarities from a strongly
hydrophobic perfluorinated polymer to a very hydrophilic polymer like poly(methacrylic
acid) have been successfully grafted onto solids. Styrene and N,N-dimethyl acrylamide have
been investigated in detail describing the influence of different polymerization parameters
on polymer loading and graft density. In order to determine these values the hybrid
materials were characterized by elemental analysis and GPC. The achieved polymer load,
layer thickness and graft densities are above of those for “grafting-to” systems and below of
those for “grafting-from” systems. As a result of our investigations we find in most cases the
“grafting-to” step dominating. For characterization of the chemical identity of the silanes and
polymer layers DRIFT and XPS measurements were carried out as well as 1H-NMR
spectroscopy of the free polymer. Generally the polymer formation and the graft process
follow the expected behavior for simple radical polymerization as there is much more free
monomer in solution compared to the number of surface-attached monomers on the
substrates. The investigated polymerization parameters are polymerization time, initiator
concentration, monomer concentration in solution and on the substrate and polymerization
temperature. It turns out that this polymerization system is very robust in respect to run-to-
run variations, e.g. monomer density on the surface, residual oxygen in solution, and in
respect to certain polymerization parameter variations depending on the polymerization
behavior of the used free monomer. An influence which cannot be neglected is that of the
surface topology of the substrate onto the amount of polymer attached in the immobilization
reaction. The substrate topology was varied from microporous to mesoporous spherical
substrates and also silicon wafers as typical flat substrates were used. It is shown that the
size of the pores influences the surface accessibility during the graft polymerization process.
Combining the results with substrates of different pore size and with silicon wafers we find
evidence that though the “grafting-to” step has superior influence on the whole grafting
process there is a significant portion of grafted polymer created by the “grafting-from” step.
Also the fact that at each stage of the polymerization new polymer chains are generated that
are small enough to diffuse through the existing polymer monolayer contributes significantly
to the deposition of surface-attached polymer. Therefore we like to call this process using
12 Summary 213
surface-attached monomers in radical solution polymerization as a “grafting-through”
technique. Starting from the theoretical considerations of Cohen et al.[149] and Chakrabarti
et al.[152] and the experiments of Hamann et al.[147] we got an detailed insight in the
mechanism of such systems where radical polymerization is carried out in the presence of
surface-attached monomers.
Furthermore with this technique we are able to create copolymer monolayers including
units with functional groups. The buildup of these copolymer layers follows the rules
determined by the polymerization conditions and the used base polymer. The amount of
integrated functional units can be controlled just by using an adequate amount of functional
monomer in solution. We exemplified the high potential of the “grafting-through” process
by combining a base monomer with a functional monomer. As base monomer served mostly
two monomers in our study for which we looked into polymerization behavior in some
detail: styrene and N,N-dimethyl acrylamide. This delivers a functionalized hydrophobic or
hydrophilic polymer monolayer attached to the substrate surface and gives the possibility to
use the hybrid material in different solvent environments later on. The monomers bearing a
functional group are N-acryloyl-N-methylpropyl phthalimide as an amine group precursor
and N-methacryloyl-β-alanine succinimide ester as an activated ester. These two functional
groups are reactive towards a large number of reagents and thus give us a whole palette of
possibilities for further polymeranalogous reactions. This approach can be used as a toolbox
system for a broad range of applications based on the “grafting-through” approach. The
copolymer coated materials were analyzed by elemental analysis and GPC in order to
determine the copolymer loading on the substrates and the corresponding graft densities.
The chemical nature of the surface layers is investigated using DRIFT and XPS
measurements on the solids and 1H-NMR spectroscopy on the free copolymers. Accessibility
and reactivity of the functional groups is tested by labeling and fluorescence experiments. In
the case of N-acryloyl-N-methylpropyl phthalimide where the amino groups are created
after film formation by a deprotection step, additional tests and reaction have been
performed to determine the actual number of amino groups within the copolymer layer on
the hybrid material.
As we are able to generate significant amounts of hybrid material consisting of a SiO2 core
and a functional copolymer monolayer as shell, we use this material in different applications.
In the field of biooriented applications we use glucose oxidase as an example for an enzyme,
biotin for assay procedures and oligonucleotides for DNA analysis. All these compounds are
214 12 Summary
immobilized on the functional copolymer layer of the hybrid material with good success. For
the enzyme immobilization the results of the study suggest a quick immobilization reaction.
A stable covalent linkage prevents leaching of the enzyme and the amount of immobilized
enzyme can be controlled. A very promising use of this new functional hybrid material is the
use for catalyst immobilization. Via ligand linkage to a system with polystyrene-amino layer
one is able to immobilize catalytic species. In our case we used different Grubbs catalyst for
immobilization and found very good conversions for exemplary ring closing metathesis
reactions. Despite that leaching was not a problem, the solid material can be easily reloaded
with free Grubbs species in solution.
Based on the studies with silicon wafers where the “grafting-through” approach has been
applied to using styrene or N-N-dimethyl acrylamide the system is expanded to polymer
network formation and fixation. To form polymer networks copolymers including
benzophenone moieties are irradiated. Here the monomers attached to the surface of silicon
wafers are used as attachment points for benzophenone during irradiation. The layer of
surface-attached monomers is much more robust than a layer of benzophenone silane so far
used for such purposes. Not just an alternate attachment for polymer networks onto silicon
wafers has been studied but also photolithographic methods to form patterned polymer
monolayers and networks. The microstructures are characterized by profilometric,
ellipsometric and microscopic measurements. The spatial dimensions of the generated
patterns are in the micrometer range and the layer thicknesses in the nanometer range. For
such systems an application in analytic chip design is possible especially when combined
with functional monomers to terpolymer network layers.
The bottom line is: The “grafting-through” process is a very simple way to attach
functional molecules to a surface. It allows to attach a wide spectrum of polymers to
surfaces, irrespective of the details of the topography of the sample. The recognition of
details of the mechanism is worthwhile as it enables one to built polymer monolayers in a
desired manner within the physically and chemically given boundaries. The docking step of
growing polymer chains (“grafting-to”) in combination with the buildup of a kinetic barrier
by polymer monolayer formation mostly dominates, but partly polymer growth from the
surface (“grafting-from”) comes into play when shorter, growing polymer chains penetrate
the polymer monolayer and successfully bind to the surface, before growing further. Precise
tailoring of chemical and physical properties of the surface of the substrates is possible by
careful choice of the monomers used in solution (co-)polymerization. It is also possible to
12 Summary 215
build surface microstructures using surface-attached monomers combined with different
irradiation techniques.
The investigated graft technique is simple and robust and gives nicely reproducible
results. It can be hoped that the “grafting-through” technique becomes an equal partner for
surface decoration with polymers next to the attachment of preformed polymers (“grafting-
to”) and growth of polymers through surface-initiated polymerization reactions (“grafting-
from”).
216 13 Zusammenfassung
13 Zusammenfassung
In dieser Arbeit wird die Oberflächenmodifizierung verschiedener fester Substrate mit
Silanen, die eine Monomergruppe tragen, beschrieben. Die so modifizierten Substrate
werden anschließend in einer radikalischen Polymerisation verwendet, wodurch die
oberflächengebundenen Monomere dafür sorgen, dass sich während der Polymerisation eine
kovalent an das Substrat gebundene Polymermonolage aufbaut. Eine Vielfalt von
Homopolymeren mit unterschiedlichen Polaritäten wird erfolgreich auf die Feststoffe
gepfropft. Dabei reicht die Bandbreite vom stark hydrophoben Perfluoropolymer bis hin zu
sehr hydrophilen Polymeren wie Poly(Methylacrylsäure). Styrol und N,N-
Dimethylacrylamid werden im Detail untersucht und der Einfluss verschiedener
Polymerisationsparameter auf Polymerbeladung und Propfdichte der Substrate beschrieben.
Um diese Größen zu bestimmen werden diese Hybridmaterialien mittels Elementaranalyse
und Gelpermeationschromatographie charakterisiert. Die erzielten Polymerbeladungen,
Schichtdicken und Pfropfdichten liegen über denen, die durch Aufpfropfen reaktiver
Polymere („grafting-to“) auf vergleichbare Substrate erreicht werden und unter denen, die
durch oberflächeninitiiertes Polymerwachstum („grafting-from“) erzielt werden. Ein
Ergebnis unserer Untersuchungen ist es, dass in den meisten Fällen der „grafting-to“-Schritt
dominierend ist. Um die Silan- und Polymermonolagen chemisch zu charakterisieren
werden sowohl Diffuse-Reflexion-IR-Spektroskopie (DRIFT) und Röntgen-Photoelektronen-
Spektroskopie (XPS) an den modifizierten Substraten, als auch Protonen-Kernresonanz-
Spektroskopie (1H-NMR) am freien Polymer durchgeführt. Generell folgen die
Polymerbildung und der Pfropfprozess dem erwarteten Verhalten einer einfachen
radikalischen Polymerisation, da im System wesentlich mehr freies Monomer in Lösung als
oberflächengebundenes Monomer auf den Substraten existiert. Die untersuchten
Polymerisationsparameter sind Polymerisationszeit, Initiatorkonzentration,
Monomerkonzentration sowohl in Lösung, als auch auf der Substratoberfläche sowie
Polymerisationstemperatur. Es zeigt sich, dass das untersuchte Polymerisationssystem sehr
unempfindlich gegenüber Schwankungen einzelner Versuchsdurchläufe ist, wie z.B.
Monomerdichte auf der Substratoberfläche, oder Restsauerstoff in der
Polymerisationslösung. Der Einfluss der Oberflächentopologie auf die Menge des während
der Polymerisation gepfropften Polymers kann nicht vernachlässigt werden. Die
Oberflächentopologie der Substrate wird von mikroporösen zu makroporösen kugeligen
Substraten variiert, aber auch planare Substrate, wie z.B. Siliziumscheiben, kommen zum
13 Zusammenfassung 217
Einsatz. Es wird gezeigt, dass die Porengröße die Verfügbarkeit der Oberfläche für den
Pfropfprozess beeinflusst. Kombiniert man die Resultate der Substrate mit verschiedenen
Porengrößen und die der Siliziumscheiben miteinander, so finden sich Belege dafür, dass
trotz des überlegenen „grafting-to“-Einflusses, eine signifikante Menge des gepfropften
Polymers durch den „grafting-from“-Schritt erzielt wird. Da in jeder Phase der
Polymerisation neue Polymerketten generiert werden, die klein genug sind, um durch die
bereits existierende Polymermonolage hindurchzudiffundieren, trägt dieser Anteil
maßgeblich mit zur Pfropfmenge bei. Daher schlagen wir vor, den Prozess, der
oberflächengebundene Monomere in einer radikalischen Polymerisation verwendet, als
„grafting-through“-Technik zu bezeichnen. Ausgehend von den theoretischen
Betrachtungen von Cohen et al.[149] und Chakrabarti et al.[152] sowie den Experimenten
von Hamann et al.[147] haben wir einen detaillierten Einblick in die Mechanismen eines
System erlangt, bei dem eine radikalische Polymerisation in der Gegenwart von
oberflächengebundene Monomeren durchgeführt wird.
Weiterhin ist man in der Lage mit dieser Technik oberflächengebundene
Copolymermonolagen herzustellen, die funktionelle Gruppen beinhalten. Der Aufbau dieser
Copolymerschichten erfolgt nach den Regeln für die Polymerisation des verwendeten
Basismonomers. Der Anteil an eingebauten funktionellen Gruppen kann über eine
entsprechende Menge an funktionellem Monomer in Lösung gesteuert werden. Das hohe
Potential des „grafting-through“-Prozesses durch Kombination funktioneller Monomere mit
Basismonomeren wird beispielhaft erläutert. Als Grundmonomere dienen meist zwei
Monomere in unserer Studie, für die das Polymerisationsverhalten im Detail untersucht
wurde: Styrol und N,N-Dimethylacrylamid. Somit wird eine hydrophobe oder hydrophile
funktionelle Copolymermonolage auf die Substratoberfläche gebracht. Dies ermöglicht es,
das Hybridmaterial später in unterschiedlichen Lösungsmitteln zu verwenden. Bei den
verwendeten funktionellen Monomeren handelt es sich um N-Acryloyl-N-Methylpropyl-
Phthalimid als eine Vorstufe für Aminogruppen und N-Methacryloyl-β-Alanin-
Succinimidester als einen aktivierten Ester. Die genannten funktionellen Gruppen sind
gegenüber einer Vielzahl von Reagenzien reaktiv und eröffnen uns eine breite Palette an
polymeranalogen Umsetzungen. Dieser Ansatz, basierend auf der „grafting-through“-
Technik kann somit als Baukastensystem für einen breiten Bereich an Anwendungen genutzt
werden. Die copolymerbeschichteten Materialien werden per Elementaranalyse untersucht,
um die Polymerbeladung zu bestimmen und GPC des freien Copolymers zur
Molekulargewichtsbestimmung ermöglicht eine Berechnung der entsprechenden
218 13 Zusammenfassung
Pfropfdichten. Chemisch charakterisiert werden die copolymeren Oberflächenschichten
mittels DRIFT und XPS Messungen. Die freien Copolymere werden mit 1H-NMR-
Spektroskopie untersucht. Zugänglichkeit und Reaktivität der copolymerisierten
funktionellen Gruppen werden durch Markierung mit Farbstoffen und
Fluoreszenzuntersuchungen getestet. Im Falle des N-Acryloyl-N-Methylpropyl-Phthalimids,
bei dem nach der Copolymerisation die Aminogruppen entschützt werden, werden
zusätzliche Tests durchgeführt, um die tatsächliche Anzahl der Aminogruppen in der
oberflächengebundenen Copolymerschicht zu bestimmen.
Da wir in der Lage sind einiges an Hybridmaterialien herzustellen, die aus einem SiO2-
Kern und einer Schale, gebildet durch eine Copolymermonoschicht, bestehen, verwenden
wir diese Materialen für unterschiedliche Anwendungen. Im Bereich der biologisch
orientierten Anwendungen verwenden wir folgende Beispiele: Glucoseoxidase für ein
Enzym, Biotin für einen Assay und ein Oligonukleotid für die DNA-Analyse. All diese
Moleküle werden erfolgreich durch die funktionellen Copolymerschichten auf den
Hybridmaterialien gebunden bzw. immobilisiert. Für die Enzymimmobilisierung lässt sich
aus den Ergebnissen eine schnelle und stabile Bindung an die Oberfläche folgern. Die Menge
des Enzyms auf der Oberfläche kann gesteuert werden und es verrichtet weiterhin seine
enzymatisch Arbeit. Ein sehr vielversprechendes Einsatzgebiet für die neuen
funktionalisierten Hybridmaterialien ist die Katalysatorimmobilisierung. Die katalytische
Spezies bindet auf die Oberfläche per Ligandenaustausch an einen Liganden, der an die
vorhandenen Aminogruppen geknüpft wurde. In unserem Fall verwenden wir für die
Immobilisierung verschiedene Grubbs-Katalysatoren. Die entstandenen
katalysatorbeladenen Materialien zeigen bei exemplarisch durchgeführten Ringschluss-
Metathese-Reaktionen sehr gute Umsätze. Obwohl Auswaschen des Katalysators bei diesem
System kein Problem ist, kann das Feststoffmaterial durch Zugabe in eine Lösung des
entsprechenden Katalysators leicht wieder beladen werden.
Basierend auf den Studien mit Siliziumscheiben als Substrat, werden
oberflächengebundene Monomere durch Belichtung (Ablation) mikrostrukturiert, um in der
Folge mikrostrukturierte Polymerschichten durch den „grafting-through“-Ansatz zu
generieren. Weiterhin können oberflächengebundene dazu eingesetzt werden, um polymere
Netzwerke auf der Oberfläche anzubinden, die durch Bestrahlung von einpolymerisierten
Benzophenon-Einheiten gebildet werden. Es stellt sich heraus, das die Schicht
oberflächengebundener Monomere sich robuster handhaben lässt als die für gleiche Zwecke
13 Zusammenfassung 219
eingesetzten Benzophenon-Silan-Schichten. Es werden nicht nur polymere Netzwerke
gebildet, sondern auch photolithographische Methoden angewendet, um mikrostrukturierte
Netzwerkarchitekturen aufzubauen. Die räumlichen Abmessungen der hergestellten
Polymerstrukturen werden durch Profilometrie, Ellipsometrie und Mikroskopie
charakterisiert. Die Ausdehnung der Strukturen in der Ebene liegt im Mikrometerbereich
und die Schichtdicken im Nanometerbereich. Ausgestattet mit funktionellen Co- oder
Terpolymeren ist für solche Systeme eine Anwendung im Bereich analytischer Chips
denkbar.
Die Quintessenz ist, dass der „grafting-through“-Prozess ein sehr einfacher Weg ist, um
funktionale Moleküle an eine Oberfläche zu binden. Er erlaubt das Anknüpfen eines weiten
Spektrums an Polymeren auf Oberflächen, unabhängig von Topologiedetails der
verwendeten Substrate. Die Erkenntnis der Details des Mechanismus ist lohnenswert und
hilfreich insofern dies es ermöglicht polymere Monoschichten in gewünschter Form,
innerhalb der physikalisch und chemisch gegebenen Grenzen, aufzubauen. Der
Andockschritt wachsender Polymerketten („grafting-to“), der den Aufbau einer kinetischen
Barriere in Form einer polymeren Monoschicht mit sich bringt, ist meist dominierend. Aber
teilweise wird Polymerwachstum von der Oberfläche („grafting-from“) mit ins Spiel
gebracht, dadurch dass kürzere, wachsende Polymerketten die Polymermonoschicht
durchdringen und erfolgreich an die oberflächengebundenen Monomere anbinden, um dann
von der Oberfläche wegzuwachsen. Präzises Maßschneidern von chemischen und
physikalischen Eigenschaften der Oberflächen der Substrate ist durch eine durchdachte
Wahl der eingesetzten Monomere für die Copolymerisation in Lösung möglich. Es ist auch
möglich Oberflächenmikrostrukturen mittels oberflächengebundener Monomere durch
verschiedene Belichtungsmethoden zu erzeugen.
Die untersuchte Pfropftechnik ist einfach, robust und ergibt gut reproduzierbare Resultate.
Es bleibt zu hoffen, dass die „grafting-through“-Technik es schafft, ebenbürtig neben der
Anbindung von reaktiven, fertigen Polymeren („grafting-to“) und dem Polymerwachstum
ausgehend von oberflächengebundenen Initiatoren („grafting-from“), für die Ausstattung
von Oberflächen mit Polymeren eingesetzt zu werden.
220 14 References
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Acknowledgements
In first place I like to thank my supervisor, Prof. Dr. Jürgen Rühe, for the chance to work
on an interesting research topic that evolved many different facettes during my
investigations. The helpful discussions with him and his support were the foundation of this
thesis. Further I want to thank him for the excellent working conditions regarding
infrastructure as well as the great team spirit within the whole working group.
PD Dr.-Ing. Thomas Hanemann for kindly act as co-referee for this thesis.
Dr. Oswald “Ossi” Prucker for his introduction to the “grafting-in-between” topic and his
continuous support on all questions I had, including long or latenight discussions.
Of great importance were my cooperation partners at the department for organic
chemistry: I want to thank Prof. Dr. Willi Bannwarth for many fruitful discussions in a
pleasant atmosphere, how to drive my research results to application. This especially
concerns Heiko Glatz, who synthesized some of the special monomers and started using
“G.I.B.” material for solid phase supports. Thanks to Florian Michalek the work on catalyst
immobilization was continued and evolved to a real success story. Last but not least
cooperation member Tobias Cramer did fluorous phase stuff while grafting somewhere in
between.
Bernd “Barney the butcher” Berchtold, Daniel “das kommt auf den Hut” Freidank,
Rupert “there can be only one Rupilander” Konradi, Ulrike “sÜrface design” Mock,
Jörg “phosphonate anchor-man” Pahnke for helping me in many different issues of my work
and of course for their friendship exceeding working time.
Sidar Loschonsky and Marc Loschonsky for their great support during “the final
countdown” to the thesis submission.
Bong Jun Chang, Doungporn Sirikittikul, Dr. “JP” J. D. Jeyaprakash S. Samuel, Kamlesh
Shroff and Haining Zhang for great international colleagueship spirit and fantastic culinary
cooking events.
In the end I would like to thank all the people who spent work and time together with me,
supported, encouraged and enlightened me and therefore share an essential part of this
thesis with me.
Natalia Schatz for her great work on “G.I.B.” in the lab and spending many hours with the
self-developped shaking-device; “Shaken not stirred!”
Dorothea Freidank for the good experiments with enzymes and their UV measurements.
Daniela Mössner and Martin Schönstein for the XPS measurements on porous and flat
substrates.
Kirstin Seidel for her help on enzyme assay development and Dr. Holger Klapproth for his
valuable hints on DNA preparations.
Dr. J. D. Jeyaprakash S. Samuel for his introduction and help on ATRP experiments.
Dr. Jörn O. Saß for amino acid analysis of immobilized enzymes on “G.I.B.” supports and
Dipl.-Ing. (FH) Michael Reichel for introducing profilometry to me using my micropatterned
samples.
Waltraud Hanser and Petra Hettich for their help “at the office desk” handling all that
bureaucratic stuff.
Gerhard Baaken, Dr. Markus Biesalski, Petra Böhringer, Julien Couet, Franziska
Degenhardt, Alexey Kopyshev, Meike Moschallski, Thorsten Neumann, Dr. Svetlana Santer,
Christian Schlemmer, Falko Stenzel, Julia Viertel, Anke Wörz and Hyun-Kwan Yang for their
good colleagueship and conversations at the coffeebreak.
Life at CPI was fun, I had a good time I’ll always have in good remembrance and so I
wanna thank again those I maybe forgot to list here and the whole funky bunch at CPI. Keep
up the fine spirit!
Finally I am deeply grateful for the continual support I got from my friend Karen, from my
parents and my grandparents, who always believed that “I’ll make it”, throughout my
studies and PhD work.