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“‘3“ LIBRARY

. Michigan State
I 7 UI IIVGI’SIty

o w

 

 

 

This is to certify that the
dissertation entitled

ENCAPSULATION OF POLYMERIC
COMPLEXANTS IN MESOSTRUCTURED
SILICA AND THE ENRICHMENT OF
PHOSPHORYLATED PEPTIDES ON GRID
SUPPORTED MESOPOROUS METAL OXIDE
THIN FILMS

presented by

Christian Paule Canlas

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Chemistry

 

 

 

 

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ENCAPSULATION OF POLYMERIC COMPLEXANTS IN
MESOSTRUCTURED SILICA AND THE ENRICHMENT OF
PHOSPHORYLATED PEPTIDES ON GRID SUPPORTED
MESOPOROUS METAL OXIDE THIN FILMS

By

Christian Paule Canlas

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Chemistry

2010

ABSTRACT

ENCAPSULATION OF POLYMERIC COMPLEXANTS IN
MESOSTRUCTURED SILICA AND THE ENRICHMENT OF
PHOSPHORYLATED PEPTIDES ON GRID SUPPORTED
MESOPOROUS METAL OXIDE THIN FILMS

By

Christian Paule Canlas

The advent of mesoporous silicas has opened new fields of research. The
inherent high surface area, chemical and mechanical stability, and tailorable pore
size have made mesoporous silica as an ideal substrate in many applications
such as catalysis, enzyme immobilization, drug delivery, chemical sensing and
separation chemistry. Mesoporous silica by itself is not very useful even with the
above mentioned properties. Functionalities must be introduced to make them
suitable for each preferred application. Two of the most common techniques of
introducing functionality onto mesoporous silica surface are thru post-synthesis
grafting and direct-synthesis. In the first half of this study, an alternate approach
was explored, that is the utilization of the surfactant template as an organic
functionality. Oleyl amine surfactants were used to assemble worrnhole silica
products. The surfactant template was crosslinked using divinylbezene to prevent
Ieeching out of the framework pores. The cross-linked template in turn acts as a
polymeric complexant which was later tested for trapping Pb(|l). Moreover, the
mesoporous silicas assembled using oleyl amine surfactants under varying
solvent polarity and temperature were explored. Results revealed that it is

possible to control pore size down to the supermicroporous range (1-2nm) using

oleyl amine surfactants with the proper solvent polarity and assembly
temperature.

The second part of the study is the use of mesoporous metal oxide thin
films for the enrichment of model phosphopeptides. Phosphorylation is one of
several post-translational modifications and is very important as it is involved in
the regulation of many cellular processes. In order to understand cellular
regulation on a molecular level, post-translational modifications need to be
identified and characterized. Despite recent advancements in mass spectrometry
techniques, the characterization of protein phosphorylation sites is challenging
due to the low abundance of phosphorylated proteins. Enrichment prior to mass
spectrometry analysis will greatly facilitate the analysis of phosphorylated
proteins. In this study, an alternative method to immobilized metal-ion affinity
chromatography (IMAC) and metal oxide columns is introduced. Instead of
columns packed with IMAC or metal oxide particles, transmission electron
' microscopy (TEM) grid supported mesoporous metal oxide thin films were used.
These mesoporous metal oxide thin films were synthesized and characterized.
These were tested by enriching B-casein and ovalbumin digests. Results
revealed a successful enrichment of the phosphopeptides up to 50 fmoI

concentration.

Copyright by
Christian Paule Canlas

2010

for Pam...for inspiring me to pursue my dreams and for teaching me things that
the four corners of a classroom, and in this case, a lab could not

ACKNOWLEDGMENTS

First of all, I want to thank God Almighty, for giving me the gift of intellect
and as my source of strength. For always guiding me in every endeavor I take.

Prof. Tom Pinnavaia for his unending patience and for taking me under his
wing. I really appreciate how he stimulates me to think creatively and how he
molded me into what I am today as chemist. I

Dr. Fan for all his expertise in TEM.

Prof. Tepe for his expertise in the proteomics project and Prof. Reid for his
mass spec expertise and for letting us use his MALDl-MS instrument.

I would also like to thank my parents and my siblings for their support and
inspiration and for always believing in me.

The Pinnavaia group members, for their friendship, with special mentions
to Kim, Joel, Susan, Noemi and Xin.

To my friends who kept me sane all through these years. Especially to
Janir and Armando for all the help they have extended to me.

The MSU Filipino Club for serving as an avenue to do things other than
chemistry, special mention to Ann, Maricris, Gizelle, and Charrise.

Jerry...for not giving up on our friendship, for always believing in me...for
being a brother.

Matt for teaching me how to be a kid again and appreciate things I’ve
forgotten because I grew up too fast. For being a best friend.

And lastly, Pam, for teaching me so much about life and for inspiring me to
pursue my doctorate degree. You will always have a special place in my heart.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................. ix
LIST OF FIGURES ............................................................................................... x
ABBREVIATIONS .............................................................................................. xvii
CHAPTER 1
SYNTHESIS AND APPLICATIONS OF MESOPOROUS SILICA ......................... 1
1.1 Introduction ..................................................................................................... 1
1.1.1 Synthesis of Mesoporous Silica ........................................................ 1
1.2 Mesoporous Silica Applications ...................................................................... 6
1.2.1 As Stationary Phase for High Performance Liquid Chromatography]
1.2.2 Immobilization of Biomolecules ....................................................... 10
1.2.3 Catalysis .......................................................................................... 15
1.2.3.1 Preparation of Mesoporous Catalysts ................................ 15
1.2.3.2 Applications of Mesoporous Silica Catalyst ....................... 19
1.2.4 Mesoporous Silica As Adsorbents .................................................. 21
1.2.5 Polymer Reinforcement .................................................................. 23
1.3 Objectives .................................................................................................... 24
1.4 References ................................................................................................... 26
CHAPTER 2
SUPRAMOLECULAR ASSEMBLY OF MESOSTRUCTURED SILICA FROM
OLEYLAMINE SURFACTANTS .......................................................................... 38
2.1 Introduction .................................................................................................. 38
2.1.1 Background on Supramolecular Assembly ...................................... 38
2.1.2 Pore Size and Morphological Control of Mesostructured Silica ....... 43
2.1.3 Tailoring the Pore Size Distribution Between the Microporous and
Mesoporous Region ................................................................................. 47
2.1.4 Objectives ....................................................................................... 48
2.2 Experimental ................................................................................................. 50
2.2.1 Reagents ......................................................................................... 50
2.2.2 Material Synthesis ........................................................................... 50
2.2.2.1 Mesostructure Synthesis via the Electrostatic S"l+
Pathway ......................................................................................... 51
2.2.2.2 Mesostructure Synthesis via the Electrically Neutral S°I0
Pathway ......................................................................................... 52

2.2.2.3 Effect of Solvent Polarity and Assembly Temperature
on Mesostructured Silica Assembled Using Oleyl Amine
Surfactants .................................................................................... 54

vii

2.3 Physical Measurements ................................................................................ 57

2.4 Results and Discussion ................................................................................. 58
2.5 Conclusions ................................................................................................ 112
2.6 References .................................................................................................. 116
CHAPTER 3
ENCAPSULATION OF POLYMERIC NEUTRAL AMINE SURFACTANT ON
MESOPOROUS SILICA ................................................................................... 119
3.1 Introduction to Mesoporous Silica Functionalization ................................... 119
3.1.1 Post-Synthesis Grafting Technique ............................................... 120
3.1.2 Direct Assembly / Co-Condensation .............................................. 121
3.1.3 Polymerization of Surfactants ........................................................ 125
3.1.4 Polymerization In Mesoporous Silica ............................................. 128
3.1.5 Heavy Metal Trapping Using Mesoporous Silica ........................... 129
3.2 Objectives ................................................................................................... 130
3.3 Experimental .............................................................................................. 130
3.3.1 Co-Polymerization of the Templating Agent Inside HMS Wormhole
Mesostructures with Divinylbenzene as the Cross-linker ....................... 130
3.3.2 Leaching Studies ........................................................................... 134
3.3.3 Lead (ll) Trapping by Batch Studies .............................................. 135
3.3.4 Characterization ............................................................................ 135
3.4 Results and Discussions ............................................................................. 137
3.5 Conclusions ................................................................................................ 149
3.6 References .................................................................................................. 154
CHAPTER 4
PHOSPHOPROTEIN ENRICHMENT USING GRID SUPPORTED
MESOPOROUS METAL OXIDE THIN FILMS .................................................. 158
4.1 Introduction to Post-translational Modification ............................................ 158
4.1.1 Background on Protein Phosphorylation ....................................... 161
4.1.2 Enrichment of Phosphoproteins .................................................... 165
4.2 Objectives ................................................................................................... 175
4.3 Experimental ............................................................................................... 176
4.3.1 Materials ........................................................................................ 176
4.3.2 Precursor Solution Preparation ..................................................... 176
4.3.3 Preparation of Thin Films .............................................................. 177
4.3.4 Characterization of Mesoporous Metal Oxide Thin Films .............. 178
4.3.5 Protein Digestion ........................................................................... 179
4.3.6 Phosphoprotein Enrichment .......................................................... 179
4.4 Results and Discussions ............................................................................. 184
4.4.1 Fabrication of Grid Supported Mesoporous Metal Oxide Thin Films190
4.4.2 Phosphoprotein Enrichment ......................................................... 196
4.5 Conclusions ................................................................................................ 213
4.6 References ......................................................................... ' ......................... 215

viii

LIST OF TABLES

Table 1.1 Classification of mesoporous silica based on the interaction of
inorganic species and organic template. ................... 3

Table 1.2 Mesoporous silica used as stationary phases for HPLC. ..................... 9

Table 1.3 Examples of fine chemical reactions catalyzed over mesoporous
catalysts .................................. 20

Table 2.1 The different packing parameter range corresponding to different
micellar aggregates. ........................................................................................... 42

Table 2.2 Structures and quantities of SO surfactants used for the synthesis of
mesoporous silica. .............................................................................................. 55

Table 2.3 Reaction Temperature and Solvent Compositions Used for the
Mesostructure Templating by Anneen OLD (oleyl-NHZ) and Duomeen O (oleyl-

NH(CH2)3NH2) surfactants ................................................................................ 56

Table 2.4 Structural properties of calcined wormhole silica materials ................. 67

Table 2.5 Summary of the pore properties of mesoporous silica products
assembled using Duomeen O as a templating agent under varying assembly
temperature and varying water composition. .................................................... 102

Table 2.6 Summary of the pore properties of mesoporous silica products
assembled using Arrneen OLD as a templating agent under varying assembly
temperature and varying water composition. .................................................... 103

Table 3.1. Wormhole Silica Products Synthesized in 60 volume % Ethanol Media.
.......................................................................................................................... 133

Table 3.2 Summary of pore properties of calcined and extracted silica products
assembled with crosslinked surfactant templates. ............................................ 144

Table 3.3 Pb(ll) uptake of mesoporous materials by crosslinked oleylamine
surfactant encapsulated in mesoporous silica .................................................. 152

Table 4.1. Phosphorylated peptides from tryptic digests of B-casein and
ovalbumin ......................................................................................................... 198

LIST OF FIGURES

Figure 1.1 Functionalization techniques of mesoporous silica for catalytic

applications ........................................................................................................ 17
Figure 2.1 Schematic representation of MOM-41 synthesis ............................... 39
Figure 2.2 Structure of CTAB and Ethoquad ..................................................... 51

Figure 2.3 Powder x-ray diffraction of calcined silica products synthesized using
(A) Ethoquad (oleyl-N+(CH3)(CH2CH20H)2 CI') and (B) cetyl trimethyl
ammonium bromide (CTAB) as the templating surfactants ................................. 62

Figure 2.4 Low angle powder x-ray diffraction of calcined wormhole silicas
synthesized from different electrically neutral oleyl amine surfactants (60/40
ethanol water by volume). The structure of the oleylamine surfactants are given
in Table 2.1. The pattern for the wormhole mesostructure made from
dodecylamin is included for comparison. Each product was synthesized at a
reaction temperature of 25°C. ............................................................................. 63

Figure 2.5 Low angle powder x-ray diffraction calcined wormhole silicas
synthesized from oleyl amine surfactants in water rich media (30/70
ethanol/water by volume).The structure of the oleyl surfactants are given in Table
2.1. The pattern for the silica template by DDA is included for comparison. The
reaction temperature was 25°C. ......................................................................... 64

Figure 2.6 Nitrogen isotherms for the wormhole silicas synthesized at 25°C under
ethanol rich media (60 vol %) in the presence of oleyl amine surfactants. The
isotherm for the silica template by dodecylamine (DDA) is included for
comparison. ........................................................................................................ 65

Figure 2.7 Nitrogen isotherms for the wormhole silicas synthesized at 25°C under
water rich media (70 vol %) in the presence of oleyl amine surfactants. The
isotherm for the silica template by dodecylamine (DDA) is included for
comparison. ........................................................................................................ 66

Figure 2.8 Powder x-ray diffraction of mesostructured silica products synthesized
from Duomeen O (oleyl-NH(CH2)3NH2) at 25, 45, and 60°C. The labeling
scheme is described in the text. .......................................................................... 71

Figure 2.9 Powder x-ray diffraction of mesostructured silica products synthesized
from Anneen OLD (oleyl-NH2) in reaction medium containing 10% to 100% H20

at 25, 45, and 60 0C. The labeling scheme for each sample is described in the
text. ..................................................................................................................... 72

Figure 2.10 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Duomeen O at 25 °C with varying water fraction (Top) 10-50%, (Bottom)
60-100%. ............................................................................................................ 74

Figure 2.11 BJH pore size distribution of mesostructured silica synthesized from
Duomeen O at 25 0C with varying water fraction (Top) 10-50% (Bottom) 60-

100% ................................................................................................................... 75
Figure 2.12 TEM images of mesostructured silica synthesized from

Duomeen O at 25 °C with varying solvent polarity a liquid. ............................... 76
Figure 2.12 (cont’d) ............................................................................................. 77

Figure 2.13 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Duomeen O at 45 °C with varying water fraction (Top) 10-50% (Bottom) 60-
100% ................................................................................................................... 80

Figure 2.14 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Duomeen O at 60 0C with varying water fraction (Top) 10-50% (Bottom) 60-
100% .................................................................................................................. .81

Figure 2.15 BJH pore size distribution of mesostructured silica synthesized from

Duomeen O at 45 °C with varying water fraction (Top) 10-50% (Bottom) 60-
100% ................................................................................................................... 82

Figure 2.16 BJH pore size distribution of mesostructured silica synthesized from
Duomeen O at 60 0C with varying water fractions of (Top) 10-50% and (Bottom)

60-100°/o. ............................................................................................................ 83
Figure 2.17 TEM images of mesostructured silica synthesized from Duomeen O
at 45 °C with varying solvent polarity. ................................................................. 84
Figure 2.17 (cont’d) ............................................................................................. 85
Figure 2.18 TEM images of mesostructured materials synthesized from
Duomeen O at 60 °C with varying solvent polarity .............................................. 86
Figure 2.18 (cont’d) ............................................................................................. 87

xi

Figure 2.19 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Armeen OLD at 25 0C with varying water fraction (Top) 10-50% (Bottom)
60-100%. ............................................................................................................ 89

Figure 2.20 BJH pore size distribution of mesostructured silica synthesized from
Armeen OLD at 25°C with varying water fraction (Top) 10-50% (Bottom) 60-

100% ................................................................................................................... 90
Figure 2.21 TEM images of mesostructured materials synthesized from Armeen
OLD at 25 °C with varying solvent polarity. ........................................................ 91
Figure 2.21 (cont’d) ............................................................................................. 92

Figure 2.22 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Armeen OLD at 25 0C with varying water fraction (Top) 10-50% (Bottom)
60-100%. ............................................................................................................ 93

Figure 2.23 BJH pore size distribution of mesostructured silica synthesized from
Duomeen O at 25 0C with varying water fraction (Top) 10—50% (Bottom) 60-

100% ................................................................................................................... 94
Figure 2.24 TEM images of mesostructured materials synthesized from Armeen
OLD at 45 0C with varying solvent polarity. ........................................................ 95
Figure 2.24 (cont’d) ............................................................................................. 96

Figure 2.25 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Armeen OLD at 25 °C with varying water fraction (Top) 10-50% (Bottom)
60-100%. ............................................................................................................ 97

Figure 2.26 BJH pore size distribution of mesostructured silica synthesized from
Duomeen O at 25 °C with varying water fraction (Top) 10-50% (Bottom) 60-

100% ................................................................................................................... 98
Figure 2.27 TEM images of mesostructured materials synthesized from Armeen
OLD at 60 0C with varying solvent polarity. ........................................................ 99
Figure 2.27 (cont’d) ........................................................................................... 100

Figure 2.28 Schematic illustration on how the oleyl surfactant decreases their
hydrophobe chain length ................................................................................... 108

Figure 2.29 Nitrogen isotherms (top) and BJH pore size distributions (bottom) of

mesostructured silicas synthesized from tallow amine (TA) at 25 °C under
varying water fractions (10-100%). ................................................................... 109

xii

Figure 2.30 Pore size variations of mesoporous silicas assembled under varying
water fractions using Armeen OLD (oleyl-NHZ) with respect to temperature

variations. (Note: A10-60 (10 volume % water) was not included in the plot since
it did not produced a mesostructured silica.) ..................................................... 110

Figure 2.31 Pore size variations of mesoporous silicas assembled under varying
water fractions using Duomeen O (oleyl-NH(CH2)3NH2) with respect to

temperature variations. .................................................................................... 111

Figure 3.1 Schematic representation of the post-synthesis grafting technique for
introducing organic functionality in the pore walls of mesoporous silica. .......... 122

Figure 3.2 Schematic representation of direct assembly or co-condensation
technique of introducing organic functionality in mesoporous silica. ................. 123

Figure 3.3 Two dimensional, highly idealized polymeric micelle with
polymerizable group on the hydrophobic tail (A) and on the hydrophilic head (B);
CD: Sherrington's concept of a polymerized micelle with polymerizable group on
the hydrophobic tail (C) and on the hydrophilic head (D). ................................. 127

Figure 3.4 Two possible schemes on incorporating a polymeric complexant
inside mesoporous silica materials. .................................................................. 132

Figure 3.5 Low angle powder x-ray diffraction pattern of crosslinked oleyl
surfactant in mesostructured silicas made by pre-assembly and post-assembly
crosslinking of the surfactant porogen. ............................................................ 138

Figure 3.6 Representative TEM images mesostructured silica assembled in the
presence of oleyl surfactant cross-linked by divinylbenzene prior to the addition
of TEOS (pre-assembly crosslinking) ................................................................ 139

Figure 3.7 Representative TEM images calcined mesostructured silica
assembled in the presence of oleylamine surfactant followed by crosslinking of
the surfactant by divinylbenzene. ...................................................................... 140

Figure 3.8 (Top) Nitrogen adsorption isotherms and (Bottom) BJH pore size
distributions for non-crosslinked, calcined and ethanol extracted silicas made by
pre-assembly DVB crosslinking of the surfactant. ............................................. 142

Figure 3.9 (Top) Nitrogen adsorption isotherms and (Bottom) BJH pore

distributions for non-crosslinked, calcined and ethanol extracted silicas made by
post-assembly DVB crosslinking of the surfactant. ........................................... 143

xiii

Figure 3.10 Thermal gravimetric analysis plots for: Left panel: A. As-made
mesostructured silica templated with unpolymerized oleyl amine surfactant, B.
Mesostructured silica from A after water extraction; Central Panel: C.
Mesostructured silica whose surfactant template was crosslinked with DVB
before supramolecular assembly(post—assembly crosslinking), D. Mesostructured
silica C after water extraction; Right panel: E. Water extracted mesostructured
silica material whose surfactant template was crosslinked after supramolecular
assembly, F. Material E after water extraction. ................................................. 147

Figure 3.11 Distribution of Pb(ll) species as a function of pH based on the
equilibrium constants. ....................................................................................... 151

Figure 3.12 Pb(ll) uptake by mesoporous silica with encapsulated crosslinked
oleylamine surfactant as a complexant. The amount of Pb(ll) added varied from
0.22 to 4.73 mmole Pb(ll) per gram as-made material. The ratio of N : Pb for pre-
and post-assembly polymerization are 3.63 and 3.73 respectively. .................. 153

Figure 4.1 The two general schemes for post-translational modification: covalent
modification and covalent cleavage. ................................................................. 159

Figure 4.2 The five major types of covalent additions to protein side chains 160
Figure 4.3 The phosphorylated amine residues ................................................ 162

Figure 4.4 General scheme for the modification for phosphoserine by B-
elimination ......................................................................................................... 167

Figure 4.5 Enrichment of a modified phosphopeptide coupled with a biotin affinity
tag. .................................................................................................................... 168

Figure 4.6 Modified phosphopeptide enrichment via direct attachment to a solid
suppon. ............................................................................................................. 169

Figure 4.7 Structures of A. Fe(Ill) iminodiacetic acid complex B. Fe(lII)
nitrilotriacetic acid complex C. phos-tag ............................................................ 171

Figure 4.8 Schematic illustration of the phosphopeptide enrichment process
using grid supported mesoporous metal oxide thin films. Washing solution 1 is
2,5-dihydroxybenzoic acid in acetonitrile. Washing solution 2 is trifluoroacetic
acid in acetonitrile. ............................................................................................ 182

Figure 4.9 Schematic representation of grid-supported mesoporous metal oxide
thin film containing bound phosphopeptide being mounted on a MALDI plate and
the subsequent release of the bound phosphopeptide for MALDI mass spec
analysis. ............................................................................................................ 183

xiv

Figure 4.10 Different types of grids. A square mesh B. hexagonal mesh C. slots
D. parallel E. combination of parallel and mesh F. folding and G. tabbed. ...... 184

Figure 4.11 A. Bar B. Hole C. Pitch ................................................................... 185

Figure 4.12 Wide angle x-ray diffraction of calcined alumina, titania and zirconia
powders. ........................................................................................................... 187

Figure 4.13 Electron diffraction of grid supported mesoporous zirconia thin films
taken from TEM selected area diffraction mode showing several rings indicating
a polycrystalline structure. ................................................................................ 188

Figure 4.14 Electron diffraction of grid supported mesoporous titania thin film
taken from TEM selected area diffraction mode showing several rings indicating
a polycrystalline structure. ................................................................................ 189

Figure 4.15 TEM images of mesoporous titania thin film supported on a gold
TEM grid (400 mesh). (Left) The lighter shades on the image represent the
mesopores. (Right) Variable orientations of lattice fringes indicate the
polycrystalline nature of the film ........................................................................ 190

Figure 4.16 TEM images of mesoporous zirconia thin film supported on a gold
TEM grid (400 mesh). (Left) The lighter shades on the image represent the
pores. (Right) Lattice fringes on different directions indicate the polycrystalline
nature of the material. ....................................................................................... 191

Figure 4.17 TEM image of mesoporous alumina thin film supported on a gold
grid. The image shows the mesopores that arises from the stacking of lath-like
nanocrystals of alumina. ................................................................................... 192

Figure 4.18 SEM images of bare gold grid. The left image was taken from the
matted side of the grid and the right image was taken from the glossy side of the
grid. ................................................................................................................... 193

Figure 4.19 SEM image of a calcined mesoporous titania thin film supported on a
gold grid. The crystalline anatase coats the bars of the grid and extends into the
holes of the grid. The crystallization of the anatase phase during calclnations
causes shrinkage which fragments the films ..................................................... 194

Figure 4.20 SEM image of a calcined mesoporous alumina thin film showing
completely filled holes on the TEM grid as well as the coated bars of the grid. 195

Figure 4.21 Schematic illustration of phosphopeptide binding to a metal oxide
surface and the release of the protein by reaction with ammonium hydroxide..196

XV

Figure 4.22 MALDI spectrum of un—enriched B-casein. The specimen was
supported on a stainless steel MALDI plate ..................................................... .199

Figure 4.23 Incubation studies of 100 fmol B-casein on grid supported
mesoporous titania (TiOz) thin film. .................................................................. 200

Figure 4.24 Incubation studies of 100 fmol B-casein on grid supported
mesoporous alumina (AI203) thin film ............................................................... 201

Figure 4.25 Enrichment of the monophosphorylated and tetraphosphorylated
peptides in a tryptic digest of 50 fmol B-casein on TEM grid supported
mesoporous zirconia (top), alumina (middle), and titania (bottom). A three-hour
incubation time was used in obtaining each spectrum. ..................................... 204

Figure 4.26 Enrichment of 50 fmol B-casein on gold TEM grid showing high
selectivity towards tetraphosphorylated peptides — 4P2956 and 4P3122. ........ 205

Figure 4.27 Enrichment of 50 fmol of B-casein tryptic digest on bare gold (bottom)
and bare titanium (top) TEM grids ..................................................................... 207

Figure 4.28 Enrichment of a 50 fmol tryptic digest ovalbumin on grid supported
mesoporous zirconia, alumina, and titania. ....................................................... 210

Figure 4.29 MALDI spectrum of 100 fmol ovalbumin tryptic digest enriched on

grid supported mesoporous zirconia, alumina and titania thin films. Note the
weak or absence of enrichment for 1P2511 and 1P2901 fragments. ................. 211

Figure 4.30 MALDI spectrum of the 50 fmol ovalbumin phosphorylated
peptide enriched using titanium metal grid ........................................................ 212

xvi

AIBN

BET

BJH

DDA

DVD
EtOH

fmol
H-bonding
HK

HMS

lo
IUPAC
MCM—41
mmol
nm

No

P/Po

PEO

ABBREVIATIONS

Azobisisobutyronitrile

Brunauer-Emmett-Teller

Barrett-Joyner-Halenda

Dodecylamine

Divinylbenzene

Ethanol

Femtomoles

Hydrogen bonding

Horvath and Kawazoe

wormhole mesostructured silica synthesize with a neutral amine
surfactant using hydrogen bonding interactions

Anionic inorganic precursor

Cationic inorganic precursor

Neutral inorganic precursor

International Union of Pure and Applied Chemistry

Mobil Composition of Matter No. 41

Millimoles

Nanometer (10’9 m)

Non-ionic amphiphilic PEO based surfactant

Relative Pressure P = pressure, P0 = saturation pressure

Polyethylene oxide

xvii

pmol

S-I-

S+|‘

S+X'l"’

SBA-15

SO

S°I°

TEOS

XRD

Picomoles

Anionic amphiphilic surfactant

Cationic amphiphilic surfactant

Electrostatic Assembly between cationic surfactant and anionic
silica precursor

Electrostatic assembly between cationic surfactant and cationic
silica precursors with halogen ions as mediating counter ions
Mesostructure silica assembled under high acid conditions with
TEOS and triblock copolymer PEO based surfactant

Neutral amphiphilic amine surfactant

Neutral assembly pathway between neutral amine surfactant
and TEOS

Tetraethylorthosilicate

X-ray Diffracttion

xviii

Chapter 1 Synthesis and Applications of Mesoporous Silica

1. Introduction

The International Union of Pure and Applied Chemistry (IUPAC) has
classified porous materials into three classes: microporous (pore size <2 nm),
mesoporous (2-50 nm) and macroporous (>50 nm) materials.1 Other porous
materials such as pillared clays, anodic alumina, carbon nanotubes and related
porous carbons also have been extensively described in literature.2 Among the
best known microporous materials are the zeolites. Zeolites have narrow and
uniform pore size distributions due to their crystallographically defined structures.
The ability of zeolites to act as solid acids and in some cases as bases, has
attracted strong attention. However, zeolites applications are severely limited for
applications involving large molecules. This limitation is most evident in the
synthesis of fine chemicals where mass transfer is very difficult to achieve in
zeolite micropores. Efforts have been made to improve the diffusion of reactants
to the catalytic sites of zeolites by increasing framework pore sizes,3 decreasing
the zeolites crystal size,4 or providing an additional mesopore system within the

microporous crystals.5’6 A very important line of research has focused on the

enlargement of the pore sizes.

1.1 Synthesis of Mesoporous Silica
The first synthesis of an ordered mesoporous silica was described in the
patent literature of 1969. However, due in part to the lack of analysis and

analytical knowhow, this product was not recognized].8 In 1992, a similar

material was reported by Mobil Oil Corporation scientists.9 This material was
denoted Mobil Composition of Matter No. 41 (MGM-41). MCM-41 opened up a
whole field of research. This mesoporous silica has highly ordered hexagonal
array of unidimensional pores with a very narrow pore size distribution“).11 The
walls of MGM-41 resembles that of amorphous silica. Along with MGM-41, MCM-
48 and MGM-50 having cubic and lamellar structures, respectively, also were
reported. Depending on the assembly conditions, the silica source and the
surfactant template used in the assembly process, mesoporous silica can be
synthesized following co-operative assembly based on electrostatic interactions
or electrically neutral hydrogen bonding.12'14 In addition, ordered mesoporous
silica can also be synthesized using liquid crystalline templates15 and by
nanocasting.15.17 Table 1.1 presents the classification of mesoporous forms of
silica based on the interaction of the inorganic species and organic template
molecule. The first ordered mesoporous silicas were prepared from ionic
surfactants (quaternary ammonium salts) as the structure directing agent. The

formation of the inorganic-organic composite is based on the electrostatic
interactions between the positively charged surfactants (denoted S“) and the

negatively charged silicate species in solution (denoted l').

Table 1.1 Classification of mesoporous silica based on the interaction of

inorganic species and organic template a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. Synthesis
Template Interaction Conditions Examples
MCM-41, MGM-48,
s+I- Basic MOM-50,941 FSM-
Direct 1618‘19
Interaction
(Electrostatic) (Aluminum, iron, lead
Ionic S'l“ Neutral-Basic oxides, etc.13:14)
surfactant AM820-21
- _ _ 22
lntermediated S”X'l+ Acidic SBA 1’ SBA 21' 583A 3
Interaction TLCT
Electrostatic
( ) _ +_ B . (Aluminum, zinc oxide
SX l asrc etc‘I4i15)
Non—Ionic solo HM323.24
Surfactant/ H-bonding . . MSU25IZ6, SBA-
Co-polymer Nolo AC'd'C 1527,28, TLCT15
(Ligand Covalent 34 Nb-TM829v30, Ta-
Assisted) Bonding TMS31
Nanocasting CMK-n16i32
33+ and So denote cationic and electrically neutral surfactant templates,
respectively; I', l+ and I0 denote anionic and electrically neutral inorganic

precursor; X' and X+ denote inorganic ions participating in triple ion formation.

The prediction of surfactant aggregates (micelles) and the corresponding
mesophases in surfactant-water systems (liquid crystals) was made possible by
Israelachavili and co-workers33v34 through the development of a model that
describes the packing parameter 9. Briefly, 9 is the ratio of the molecular volume
of the surfactant molecule to the volume the molecule would occupy if it were
enclosed in a cylinder. It is important to note that the value the packing

parameter, which is typically between 0.3 and 1.0, is not constant as it is affected

by solution conditions such as ionic strength, pH, co-surfactant concentration or
temperature.34

The synthesis-space diagram for a ternary system composed of NaOH,
cetyltrimethylammonium bromide (CTAB) as a surfactant porogen, and
tetraethylorthosilicate (TEOS) as the inorganic reagent has been described
previously.35’35 It was shown that the surfactant to silica ratio has a substantial
effect on the composite structure obtained.37 Also, the mesopore size was found
to be controlled by the length of the hydrophilic segment of the surfactant and the
corresponding diameter of the surfactant micelle. However, the addition of
auxiliary organic molecules such as aromatics,11’38 n-alkanes,39 or fatty acid40
can act as micelle swelling agents and lead to expansion of the mesopore size.

Pinnavaia et al. developed non-electrostatic pathways for the synthesis of
mesoporous silica, denoted the solo pathway (see Table 1.1). This approach
utilizes the H-bonding interactions between the silica precursor (silicic acid
formed by tetraethylorthosilicate hydrolysis) and the electrically neutral amine
surfactant or poly(ethylene oxides) to prepare wormhole HMS and MSU silicas.
This approach is advantageous in both economical and ecological points of view
for as much as 90% of the surfactant template can be recovered by simple
extraction with ethanol23 or aqueous acids.41 Compared to MOM-41, HMS and
MSU silicas have very similar surface areas and pore volumes but the pore size
distributions are broader. Also, many of the modifications that can be applied to

MOM-41 are applicable to HMS wormhole silicas such as the tailoring of the pore

size by changing the alkyl chain length of the surfactant or by the addition of
mesitylene to the reaction mixture in order to swell the templating micelles.42:43

In the ligand assisted assembly approach (denoted S-l in Table 1.1),
covalent bonds are formed between the organic precursors and the organic
surfactant molecule prior to assembly. The approach was first developed by
Antonelli and Ying for the preparation of mesoporous niobium oxide29 and
tantalum oxide.31 The assembly of Nb-TMS (Transition Metal oxides
mesoporous molecular Sieve) and Ta-TMS involves the hydrolysis of long-chain
amine complexes of niobium or tantalum. The removal of the template by
extraction afforded open porous structures with surface areas of up to 500 m2/g.
Mesostructured niobia with 3D hexagonal symmetry, as well as a cubic phase
and a layered structure, was obtained.30

The true liquid crystal templating (TLCT) had already been suggested as
one possible mechanism for the formation of MGM-41. The first example of this
approach was reported by Attard and co-workers15 for the hydrolysis of
tetramethylorthosilicate (TMOS) in a liquid crystalline phase. The liquid crystalline
phase is destroyed upon the formation of methanol as the reaction product but

reforms when methanol evaporates. This method was later found to be useful in
the synthesis of mesoporous metals‘t‘t'47 and mesostructured assemblies of
chalcogenide nanoparticles.48

Another distinct method for producing mesostructure phases is the
nanocasting method that was first reported by Ryoo and co-workers in 1999.35 In

this approach, no surfactant was utilized as a porogen. Instead an ordered

mesoporous silica was used as a hard template. The method involves infiltrating
the mesoporous silica (eg. MGM-41) with a carbon precursor such as sucrose or
furfuryl alcohol which is subsequently converted to carbon at high temperature
under inert atmosphere. The mesoporous silica is removed by treatment with HF
or NaOH leaving the carbon negative of the original mold (denoted as CMK-n).
While this method is already established to produce carbon base materials,4950
the use of silca as hard template was only recently reported for some examples
such as CeOz,51 0203,52 NiO,53 0504,54 In203,55.56 and C002.56 The limited
applicability of the method most likely is due to the incompatibility of many oxide

materials with the conditions needed for leaching away the hard silica template.

1.2 Mesoporous Silica Applications

Although there are many reviews regarding the synthesis, formation
mechanism, modification and control of their characteristics57'54, there are
relatively few reviews of the material applications of mesoporous metal oxides.
This introduction will provide a summary of recent studies and applications of
mesoporous silica in liquid chromatography, the immobilization of biomolecules,

catalysts, trapping agents and agents for polymer reinforcement.

1.2.1 As Stationary Phase for High Performance Liquid Chromatography
One major area of application for mesoporous silica is in liquid
chromatography as a column packing material for the stationary phase.

Mesoporous silicas are of interest as stationary phase materials for HPLC

 

 

 

because of their inherent high surface area and their organized pore structure.
The silanol group density and the mechanical stability of the mesophase under
chromatographic operating conditions are similar to those of the precipitated
silica. Table 1.2 identifies several studies of mesoporous silica for use as
stationary phases for HPLC.
In liquid chromatography, the efficiency (N, number of theoretical plates)

of a column (length L) is given by the equation:

N = L2/0'2 = 16tR / tw 1

H = 02/L = UN 2
where H is the plate height and o?- is a variance of a Gaussian-shaped band the
solute acquire during migration through the column. N is calculated from
retention time of a solute (tR) and the base peak width (tw = 40', 0' is the standard
deviation)

H is dependent on the particle size (dp) and mobile phase linear velocity
(u) as shown in equation 3 where CE, CD, CS are the coefficients indicating the

contributions of the eddy diffusion, molecular diffusion and slow mass transfer to

band broadening, respectively. The smaller clp gives a higher efficiency by the

faster mass transfer based on the shorter diffusion path length inside and outside

the particles, whereas a small dp causes a large pressure drop due to small
interstitial flow paths (n, solvent viscosity), where I» (a flow resistance factor) is

commonly 700-1000 for a particle packed column.77

H = CEdp + CD/u + Csdpu 3

AP = (anL I dp2 4
For high resolution and high speed it is desirable to have high efficiency
(large N) and low pressure (small AP). Separation time is dictated by L, and the
smaller dp allows the use of shorter column. Actually, H is approximately 2dp at
optimum linear velocity. Recently, 1.7-2 pm particles, have started to provide
greatly enhanced N per unit time73i79 compared to 3-5 urn particles, although
accompanied by high pressure in proportion to 1/dp2. Their optimum

performance can be obtained with a specially designed instrument. Common
HPLC equipment possesses a 35-40 MPa pressure limit. Monolithic silica
columns having continuous structures can simultaneously provide higher
efficiency and lower pressure that a particle packed column.30'82 Prouzet and
co—workers summarized the chromatographic application of MSU-X silica

particles with controlled morphology.83

Table 1.2 Mesoporous silica used as stationary phases for HPLC

 

 

 

 

 

 

 

 

 

 

 

 

 

pm) with trans-(1R,2R)-
diaminocyclohexane

 

 

chiral HPLC

 

Chromatographic
Mesoporous Material Applications Ref
MGM-41 constituted by loose NP-HPLC (aromatic polycyclic 64
particles agglomerates hydrocarbons)
Solid obtained in acidic medium by
using cationic surfactant and NP-HPLC (phthalic esters and 65
TMOS. It was grinded and aromatic compounds)
separated in aggregates of 15pm
NP-HPLC (ferrocene and
SBA-3 type spherical particles acetylferrocene) y RP HPLC 66
(aromatic hydrocarbon and
racemic mixtures)
MSU-X type spherical particles NP-HPLC and RP-HPLC 67
(diameter ~7.6 um) (aromatic hydrocarbon)
SBA-15 or SBA-16 type spherical
o polyhedral particles (diamter 3— NP'HP'TC and RP'HPLC 68-70
10 um) (aromatic hydrocarbon)
MSU-X type snlica agglomarates of NP-HPLC (hydrocarbon) 71
nanometnc particles
MGM-41 type spherical particles
(d'ameter 5 mm) °bta'”“?d by RP-HPLC (alkylbenzenes) 72
pseudomorphlc synthesrs from
macroporous silica gel
SBA-15 type spheroidal particles Capillary RP-HPLC 73
(diameter ~1 pm) (biomolecules)
MOM-41 type silica with pore Exclusion size HPLC (low
diameter ~3.38 nm molecular weight 74
polystyrene compounds)
MGM—41 type silica with pore Separation of three aluminun
diameter ~2.5 nm species (cationic, tridecameric 75
and polynuclear)
Chiral stationary phases supported a-methylbenzylamine 66
on MGM-48 or SBA-3 type silica enantiomers
Bifunctionalized mesoporous
organosilica spheres (diameter 5 Resolution of DL-valine by 76

 

 

1.2.2 Immobilization of Biomolecules

Biomolecules are frequently immobilized on silica supports through a sol
gel process. Mesoporous silica molecular sieves and related materials have
received much attention as inorganic supports for biomolecules. Aside from the
fact that they have very high surface area and large pore volume, they are
chemically and mechanically stable and resistant to microbial attack. The
mesostructured materials can be reliable in the accommodation or immobilization
of biomolecules.

One of the current trends in material research is innovation in the fields of
intelligent or smart materials. Smart materials are materials that display
functionalities capable of detecting changes in the near environment and store,
transfer or convert a signal in way analogous to a living cell.34'86 The design of
smart materials focuses on the concept of biomimetic materials in order to
reproduce specific and selective properties not deliverable by standard chemical
or physical processes. The engineering of biohybrid enzyme based materials and
their applications is limited by the intrinsic fragility of enzymes toward various
environmental stresses and their narrow range of working conditions. A suitable
inorganic host should allow high levels of enzyme uptake with minimum enzyme
degradation or inactivation. In general, the support must have a high capacity to
bind enzyme. The available surface area, chemical inertness and mechanical
stability are usually the decisive factors. Moreover, other factors that must be
considered are charge of the surface and hydrophilicity as well as diffusion ability

in bulk. There are several immobilization approaches available but the most

10

common of which are sol-gel encapsulation and covalent grafting. The sol-gel
encapsulation method has attracted much attention due to its potential for
creating a high concentration of encapsulated enzyme. This approach is used
mainly for immobilizing an enzyme in a silica matrix. In comparison with other
methods, the sol-gel method generally exhibits simplicity, and versatility in
composition and physio-chemical properties. However, since enzymes are
weakly bound, leakage of entrapped enzyme is often observed through diffusion
out of the matrix. Another hurdle is that the matrix acts as a mass transfer barrier
that can limit or hamper the diffusion of large substrate molecules to the
embedded enzyme. The covalent binding of the enzyme to a solid support
appears to be the most appropriate treatment when enzymes must be used in
drastic environments where high stability is required or leakage must be
prevented. Proteins that display side chains with reactive groups able to undergo
condensation reaction with various organic functions or complexation reactions
with metallic cations can be used to fix biomolecules onto inorganic supports.

The immobilization of proteins in mesoporous MGM-41 was first attempted
by Diaz and Balkus.87 Several research groups then investigated the influence of
various physical factors such as pore size, ambient pH, and ionic strength on the
adsorption efficiency of proteins.33'104 Vinu and co-workers did an extensive
study on the immobilization of hen egg white Iysozyme into MCM-41. Base on
their results they tried to explain the adsorption behavior of Iysozyme inside the
pores of mesopore.105 The effect of pH changes and incorporated heteroatoms

into a mesoporous framework (Al-substituted MGM-41) were also studied.

11

Immobilization of proteins also can be done through direct addition of the
protein during the mesoporous silica assembly. Blin and co-workers105 also
demonstrated that hexagonally arranged pore structures can be synthesized
using a fluorinated surfactant, CF3(CF2)7C2H4(OCZH4)90H in the presence of
glucose oxidase as long as the protein concentration was below 3.2 mg/mL.
Galarneau and co-workers107 proposed the use of lecithin and cationic dodecyl
amine as the structure directing agent in the presence of lactose for direct protein
immobilization. In this study, the lecithin is thought to protect the enzyme and
prevent the direct interaction between the silica and the enzyme. Ying and co-
workers108 reported a pressure driven method for entrapping lipase into siliceous
mesocellular foam. A high pressure (3000-5000 psi) was used to achieve a high
enzyme loading within the silica support. The immobilized lipase demonstrated
high activity and enhanced thermal stability and the enzyme leaching was not
significant.

Post treatment of adsorbed proteins in mesoporous silica is often
beneficial for the stability of the immobilize protein. The use of gluteraldehyde for
the immobilization of a-chymotrypsin in hierarchically ordered mesocellular
mesoporous silica materials (HMMS) enables the crosslinking of the enzyme that
resulted to an impressive stability with very high loadings with 10 times the
activity.109 Polymers have also been used for the stable entrapment of proteins
inside mesoporous silica. Poly(methacrylate)-type polymers has been used to

reduce the pore openings SBA-15 support that is loaded with porcine pancreatic

lipase.110 This prevents the lipase from leaching out of the support. Wang and

12

Caruso111 proposed a unique method to prepare a porous protein assembly
using mesoporous silica as a hard template. In this approach, proteins such as
Iysozyme, cytochrome c and catalase were adsorbed in the channels of a
mesoporous silica sphere. Next, a polyelectrolyte was infiltrated into the pore
channels to bridge the proteins. The silica template is then removed leaving a
nanoporous protein particle. Such materials are seen to find applications in the
field of biosensing, catalysis, separations and controlled drug delivery.

Hudson and co-workers112 discussed the driving forces for protein
adsorption on mesoporous materials using SBA-15 silica and MSE, a periodic
mesoporous organosilica. Cytochrome c and xylanase were adsorbed onto the
two mesoporous materials. Electrostatic attraction dominated the protein
interactions with SBA-15 silica, while weaker hydrophobic interactions were more
prominent with MSE for both proteins. Moreover, their study also revealed that
cytochrome c immobilized on SBA-15 through electrostatic interaction showed
resistance to leaching with an enhance activity compared to the free protein.

Nucleic acids, RNA and DNA are attractive biopolymers that can be used

for biomedical applications,113v114 nanostructure application,115v116

computingII7»118 and materials for electron concluction.119'120 The
immobilization of DNA and RNA in a well defined structure would be one of the
most unique subjects in current nanotechnology. Unfortunately, the silica surface
cannot adsorb duplex DNA in aqueous solution due to electrostatic repulsions

between the silica surface and polyanionic DNA. Recently a method was devised

for adsorption of duplex DNA in protonated phosphoric acid form.121

13

Mesoporous silica alone adsorbs a negligible amount of DNA but the exchange
of divalent cations such as Mg2+ and 032+ into the pores of mesoporous silica
prior to DNA uptake was found to cause a significant amount of DNA to be
adsorbed.122 Post synthetic treatment with aminopropyl groups further increased
the adsorption capacity that 2-3 times higher in comparison to that observed for
metal ion-exchanged mesoporous silica.123

There are several potential applications for the immobilization of
biomolecules in mesoporous materials. One is the production of microscopic
reactors. Galarneau et al. have shown that lipase immobilized in mesoporous
silica produce higher activity.124 Cytochrome c immobilized on mesoporous silica
has high specific activity toward the oxidation of polycyclic aromatic
hydrocarbons. Immobilized aldolase antibody 8463 in mesoporous silica with 15-
25 nm pores exhibit highly accelerated aldol reactions and stability as compared
to a free antibody.125 The enhanced activity is attributed to a favorable
microenvironment that results in higher efficiency due to size exclusion. A
mesoporous nanospace can also provide an environment where molecular
motions and orientations are highly constrained. This situation is similar to that in
the confined spaces found in enzyme pockets.

Another application is selective binding and separation of biomolecules.
Brennan and co-workers126 reported the application of protein doped monolithic
silica columns for immobilized enzyme reactor chromatography. This allowed the
screening of enzyme inhibitors in mixtures using mass spectrometry for

detection. The mesoporous materials also possess highly reliable and

14

controllable pore geometries that regulate the release of drugs from mesopores.
Photo controlled regulation of drug storage and release using functionalized
mesoporous silica has been reported as well as colloid capped mesoporous

materials for controlled release and delivery. Biomolecules and mesoporous

silica hybrids are very promising for biosensor applications.127'128

1.2.3 Catalysis

1.2.3.1 7 Preparation of Mesoporous Catalysts

The advantages of using mesoporous silica in catalysis include the
relatively large pores which facilitate mass transfer and the very high surface
areas which allows a high concentration of active sites per mass of material.
There are several ways to modify mesoporous silica to provide the desired
catalytic function. These include the introduction of highly dispersed heteroatoms
in the framework walls, occlusion of metal, metal oxide or metal sulfide
nanoparticles in the pores, and anchoring of molecular catalyst to the framework
walls. Figure 1.1 shows these different approaches.

Incorporation of Heteroatoms

Much effort has been made on the introduction of heteroatoms such as B, Fe,
Ga, Ti, V, Sn, and Al in siliceous frameworks to modify the composition of the
inorganic walls. It should be noted that the walls of mesoporous silica resembles
that of amorphous silica. The incorporation of heteroatoms allows the fine tuning
of the acidity or redox properties, as observed in amorphous alumino silicates or

zeolites. The incorporation of Al into the walls affords enhanced hydrothermal

15

stability129 and broader pore size distributions than the pure silica
analoguesIO-130. This also creates Bronsted acidity and ion exchange sites. The
effect of the aluminum source such as AI(OH)3, aluminum isopropoxide or
NaAl02 on the degree of aluminum incorporation has also been studied.131 AI-
MGM-41 prepared using NaA|02 as the aluminum source gave the strongest
Bronsted acidity. A comparison of AI-, 63- and Fe- functionalized silicas,
showed that the Bronsted acidity decreased in the order of Al>Ga>Fe but for
Lewis acidity the sequence was Ga z Al >> Fe."32

Zeolitization of the walls of mesoporous silica is another interesting
development that can be achieved either through the conversion of the silica

walls into zeolitic units or through the use of precursors that originally contain
zeolitic structural elements.138‘140 The cations can also be exchanged with 03“,
Zn”, or Fe3+ and to achieve catalytic activity as in the DieIs-Alder reaction of
cyclopentadiene and methyl methacrylate141 or in the selective catalytic
reduction of No.142 Not only inorganic cations but also cationic organometallic
compounds such as [Fe(phen)3Cl2],143 [Fe(8-quinolinol)3]CI2,144 [Mn(2,2-
bipyr)2]N03,145 [Mn(porphyrin)]CI,146 or [Mn(salen)]C|1”"”148 are also
exchangeable. Titanium is incorporated by either grafting‘49'154 or direct

assembly.1552156

16

Figure 1.1 Functionalization techniques of mesoporous silica for catalytic
applications

 

H Direct Assembly or Post-Synthesis Grafting II

 

 

Substitution Enzyme
grafting encapsulation
Immobilization Non-silica
material
Surface Organic-
coating inorganic
. hybrid
Mesoporous silica h'gh surzface area materials
(MOM-41, HMS, SBA-15) (1000"1 /9)
narrow pore size
distribution

thermal stability

Incorporation of Metal, Metal Oxide and Metal Sulfide Nanoparticles

The deposition of catalytically active nanoparticles on support materials
with high dispersion is an important and effective strategy for the design of
mesoporous silica catalysts with very high surface areas. Mesoporous silicas
have the advantage of confining nanoparticles and preventing their growth to a
size larger than the framework pore size. Metal nanoparticles have been used in

catalysis for many decades. These materials have great potential as catalyst

because of the large surface area and high fraction of atoms present at the
surface.157

Nanoparticles can be incorporated into mesoporous silica by different
methods such as direct inclusion of metal particles during the mesoporous
assembly process and the impregnation and immobilization of polynuclear
molecular transition metal complexes. The direct inclusion approach has been
reported for the Pt and Rh.158.159 Conventional methods such as the
impregnation technique yield supported nanoparticles of various sizes in the
mesopore and on the external surface of the support material. Immobilization of
polynuclear molecular transition metal complexes is another promising method
for the preparation of supported metal catalyst. Typically, metal carbonyl
complexes are used to react with the surface silanol groups at elevated
temperature. This approach has been used for immobilization of platinum and

ruthenium nanoparticles.150-162

Anchoring of Molecular Catalyst to the Surface

The immobilization of homogeneous catalysts on solid supports is of high
commercial interest since the heterogenized catalyst becomes easier to handle,
retrieve and recycle. However, the immobilized catalyst often suffers from
reduced activity upon immobilization. But occasionally, the catalytic performance
can be improved as compared to the homogeneous counterpart.153'164

Immobilization of Mn(bipy)22+, for example exhibits a higher catalytic activity in

terms of peroxide oxidation and selectivity for styrene oxidation than the

18

corresponding homogeneous catalyst.”5 This is explained by the fact the
Mn(bipy)?_2+ easily forms a dimer and in this state preferentially decomposes

peroxide rather than oxidize any substrate. The isolation of the catalytic metal

centers prevents this dimerization.

1.2.3.2 Applications of Mesoporous Silica Catalyst

Among the most common uses for mesoporous silica catalysts are
hydrocarbon cracking and other petrochemical conversions, acid/base catalyst
for reactions in fine chemical synthesis and redox catalysis.

The patent literature contains many examples of the use of mesoporous

silica catalyst for cracking and petrochemical conversions.165 The low acid
strength and low hydrothermal stability is an unfortunate disadvantage for MCM-
41 but this is countered by the advantage of having a material with accessible
pores even for larger molecules. Nevertheless, MGM-41 catalyst show
substantial cracking activity for bulky substances such as palm oil and
asphaltene.155'167 For small molecules, such as 1-butene, n-hexane, n-heptane
or small cyclic hydrocarbons such as tetralin and decalin, the cracking activity of
MOM-41 is much lower than that of USY or Beta Zeolites, and similar to that
amorphous silica-alumina.158'172

Although the inherent weakness of the acid sites in mesoporous silica
limits its applicability in many petrochemical reactions, is still can use in reactions
requiring weaker acidity such as reactions for synthesizing fine chemicals. Table

1.3 summarizes some of the reactions catalyzed by mesoporous catalyst.

19

Table 1.3 Examples of fine chemical reactions catalyzed over mesoporous

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

catalysts
Reaction Mesoporous catalysts Substrates Ref
AI(OPri)3 grafted Phenol + methanol 220
Friedel_Crafls Gin 21:33:53 ’ Benzene + benzyl chloride 221
alkylation Al-substituted 2,4-Di-tert-butylphenol + 209
cinnamyl alcohol
Fe-impregnated Benzene + benzyl chloride 210
Heptanal, 2-
.. . hen l ro anal,
Siliceous/AI-substrtuted diphFenylgcgtaIIdehyde + 211
Acetalization trimethyl orthoformate
AI-substituted Heptanal + methanol 212
siliceous Cyclohexanone + methanol 213
AIIOPTIIB grafted Cyclopentadiene + 214
. crotonaldehyde
Duels-Alder , ,
Zn2+-ron exchanged Cyclopentadiene + 141
methylacrylate
Beckmann Al-substituted Cyclohexanone oxime 215
rearrangement
Aldol Al- substituted Heptanal + benzaldehyde 212
condensation (Ijasminaldehyde)
Prins Sn-substituted B-Pinene + 216
condensation paraforrnaldehyde
MPV-reduction ZTIOPF). AI(OPI‘I)3- 4-tert-butylcyclohexanone 219
grafted + 2-propanol
Metathesis Siliceous Propene 217
(Esterification) Cst-ion exchanged Triolein + glycerol 218
(Etherification) Cs-impregnated Glycerol 222

 

 

 

Redox reactions also can be catalyzed using of mesoporous metal oxide.
Titanium containing mesoporous materials as well as those modified with early
transition metals, such as Zr, V, Cr, Mn, or Fe, are promising oxidation catalysts.
Titanium modified MGM-41 allows relatively bulky peroxides to be used as

oxidants for sterically demanding substrates. It also widens the range of catalysts

20

available for selective oxidation reactions with hydrogen peroxide or organic

peroxides as oxidants.173

1.2.4 Mesoporous Silica As Adsorbents

The high surface area and large pore of mesoporous silica makes them
very suitable for adsorbing or trapping metal ions, acidic gases and organics.
Heavy metals, particularly lead and mercury are important environmental
pollutants, threatening the health of human populations and natural ecosystems.
There are several methods for metal ion removal which include precipitation,
coagulation/flocculation, ion exchange, reverse osmosis, complexation
lsequestration, electrochemical operation and biological treatment. Some
limitations of these are high operating and energy cost and relatively low and
variable loading capacities.174»175 Functionalization of the framework walls with
thiol groups tunes the mesoporous silica for mercury trapping while the
introduction of amino group makes it suitable for Pb and As trapping. The
functionalization of with thiol groups can be achieved either by post synthesis
grafting or by direct assembly. Various mesostructures with thiol groups have
been synthesized through direct assembly including MOM-41.176478 MCM-
48,178 SBA-15,179'181 SBA-16, and KIT-6,132"183 HMS,183 MSU,184'185 SBA-
1135'187. The aminopropyl functionalization has been used on trapping metal

ions such as Ni,188 Cr,188 Mn188 , Fe133, A3189, Cu,190 00,190 and Zn190.
Amine modified mesoporous silica has also been used to trap acidic gases such

as 00;; and H28. The chemistry of amine and 002 has been employed for

21

selectively removing C02 from gas streams. The idea of combining amines with
solid supports to adsorb C02 has been examined by several groups.191'198 The
most common problems encountered when developing amine loaded solids
supports are low capacity for 002 due to the limited quantity of amine retained
on the support, operation in a narrow temperature range and poor thermal
stability. Yang and co-workers199 investigated the selective adsorption of 002
and H28 from natural gases using silica xerogel and MOM-48. The trapping of
organic molecules on mesoporous silica has also been studied. It has been
reported that calcined and phenyl modified MGM—41 show great affinity for
benzene200 and cyclohexane in the gas phase.201 Both calcined pure silica and
aluminosilicate MGM—41 have a high sorption capacity for cyclohexane (>04 9 lg
dry solid).201 Vance and co-workers202 reported the use of as-made MGM-41

for the removal of trichloroethylene and tetrachloroethylene in waste water.

1.2.5 Polymer Reinforcement

Inorganic particles with domain size less than 100 nm in at least one
dimension can enhance the tensile properties of the polymer through beneficial
interaction at the polymer-particle interface.202 Layered silicate clays in
exfoliated form has been widely investigated as reinforcing agents as well as
barrier and flame retardation agents for a variety of organic polymer systems.203
Mesostructure silica and other metal oxides were also considered for polymer

composite formation. MGM-41 has been used to direct polymerization of

conjugated polymers such as polydiacetylene,204 polyvinylene,205‘3a206 and

22

polythiophene.207 The alignment of the polymers within the pores resulted in
unusual chromatic changes, polarized luminescence and stability. Pinnavaia and
co-workers203 have improved the tensile modulus, strength, and toughness of
mesocomposites formed from as-made and calcined MSU-J silica in comparison
to pure rubbery epoxy polymer. These reinforcement benefits were achieved
without the need for organic modification of the silica surface. Mark et al.208
reported the preparation of poly(ethylene oxide)/MCM-41 mesoporous silica
composites and they found that after pressing the physical blend at 100 0C the
polymer melting transition vanished. That effect was more clearly seen for

polymers with low molecular weight.

1.3 Objectives

This thesis research effort has two general objectives. The main objective
of the first half of the study was to synthesize mesoporous silica from biologically
derived oleyl amine surfactants. All of the applications described in the foregoing
introduction involve the use of mesophases for which the structure-directing
porogens has been removed by ion exchange, solvent extraction or calcinations.
There are no examples of the use of as-made surfactant-intercalated
mesophases for a materials application. This is understandable because the
organic surfactant occupies the pores of the inorganic framework. Nevertheless,
the intercalated surfactant bears organic functionality that could be useful for the

adsorption of organic and inorganic species from aqueous solution. The

23

limitation, however, is the leaching of the surfactant phase to solution and the
eventual loss of the functionality provided by the surfactant.

Being renewable plant derived molecules, oleyl amines are attractive
templating agents in their own right for the synthesis of “green” mesophases.
Oleyl surfactants have not yet been reported as structure-directing agents for
silica mesophases. Additionally, owing to the presence of the polar amine head
group and the olefinic bond in the hydrophobic segment, these surfactants offer
unique opportunities for the design of functional as-made mesophases
adsorbents. For instance, the amine group may serve as a complexing agent for
removing metal ions from aqueous solution. This approach may make it possible
to circumvent the need for cost-intensive use of organosilanes as grafting agents
for the introduction of ligand functionality in the framework walls. Also, the
presence of the cis double at the 9-position of the hydrophobic group offers the
possibility of cross linking of the surfactant, thus avoiding, or at least, minimizing
surfactant leaching. The plausibility of these concepts is demonstrated through
synthesis and oleyl amine surfactant/silica mesostructures and their ability to
adsorb Pb(ll) from aqueous solution.

The second general objective concerns the development of thin-film forms
of mesoporous alumina, titania, and zirconia for the enrichment of
phosphorylated protein fragments from protein digests. These oxides are
recognized as selective media for the phosphoproteins enrichment by a
chemisorption mechanisms. But the use of the oxides in packed column form

requires the use undesirably large quantities of protein digest and cumbersome

24

procedures for the release of the enriched protein for identification by mass
spectroscopy. Thin film forms of the mesophases represent a promising
approach to circumventing these unattractive aspects of this chemistry. More
specifically, we show that by supporting the oxide films on metal grids normally
used to host specimens for transmission electron microscopy it is possible to
reduce the amount of digest for the enrichment and MALDI mass spectroscopic
analysis for two model phosphoproteins (namely, B-casein and ovalbumin) to 50
femtomoles or less. Moreover, the processing of the specimens can be achieved
using 10-microliter volumes of reagents and this greatly facilitates through put of

samples for analysis.

25

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37

Chapter 2. Supramolecular Assembly of Mesostructured Silica from
Oleylamine Surfactants

2.1 Introduction
2.1.1 Background on Supramolecular Assembly
The supramolecular synthesis of mesoporous silica generally follows two

pathways - the electrostatic assembly pathway and the electrically neutral
pathway. The electrostatic S+I' pathway utilizes charge matching interactions

between the cationic surfactant (8*) and the anionic inorganic silica precursor (l'
). This type of interaction was used by Mobil Corporation researchers in the
synthesis of MGM-41 (hexagonal), MCM-48 (cubic) and MGM-50 (lamellar)1.
Figure 2.1 shows the synthetic scheme proposed for the assembly of hexagonal
MGM-41 silica in the presence of surfactant micelle formed from quaternary
ammonium ion. For the synthesis of MGM-41, a surfactant with long hydrophobic
alkyl chain and a hydrophilic group in the form of a quaternary ammonium ion
was used. When dispersed in solution, these surfactant molecules associate
themselves into micelles. These micelles then serve as templates from which the
silica precursor can condense. One such phase consists of cylindrical rod-like
surfactant micelles. The addition of basic silicate anions to the micelle dispersion,
under suitable hydrothermal conditions results in the formation of a surfactant
silica composite1 .2.

Moreover, surfactants can form different mesophases depending on
certain factors such as concentration, temperature and packing parameter.
Concentration is an important parameter because for mesophase tailoring, the

surfactant concentration should be at the critical micelle concentration (CMC).

38

  

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The CMC is the minimum concentration for surfactant molecules to aggregate to
form micelles. This process happens to minimize energy and is entropy driven. It
has also been noted that a surfactant with low CMC value is an important
criterion toward increasing the regularity of a mesostructure3. Temperature is
also an important factor as it affects the solubility of the surfactant in the reaction
media thus affecting the CMC. The packing parameter (Q) is also an important
parameter in predicting and explaining the final mesostructure. The packing
parameter, 9 = V/ (aol) where V is the total volume of the surfactant hydrophobic
chains plus any co-solvent between chains, 30 is the effective hydrophilic head
group area at the aqueous-micelle surface and I is the kinetic surfactant tail

length. For a 9 value that is <1/3, the surfactant forms spherical micelles. For g

39

values of 1/3 to <1/2 would give rise to cylindrical micelles, values1/2 to < 2/3
would give rise to 3D cylindrical micelles, a value of 1 would mean a lamellar
structure and >1 would give rise to a reverse micelle. Table 2.1 provides the
approximate ranges for the packing parameter and the type of micellar
aggregates formed4.

MGM-41 and related silicas are characterized by long range ordered
mesopores with uniform pore size distributions that can be tailored through
variation in surfactant chain length, addition of auxiliary co-surfactant agents and
by post-synthesis ' treatment. Powder x-ray diffraction of MGM-41
characteristically show 3 peaks that can be indexed to the hexagonal unit cell

with the unit cell parameter a0 = 2d100 / w/3.1 The nitrogen adsorption isotherm

of MCM 41 is characterized by a sharp step that is due to capillary condensation.
Capillary condensation is a phenomenon on porous materials when multilayer
adsorption from the vapor phase proceeds to a point where the pores are filled
with the condensed vapor. This usually occurs below the saturation pressure of

the liquid phase. Brunauer-Emmett-Teller (BET) surface areas of MGM-41

materials are estimated to be 1000 m2/g for MGM-41 and pore sizes can be

tuned to a range of 3.0 to ~10nm by adding trimethyl benzene (T MB) as a co-
surfactant or by varying the alkyl chain length of the surfactant.5

The electrostatic S“X'l+ pathway on the other hand involves assembly
using the interactions between a cationic surfactant (8") and a cationic silica
precursor 0") as mediated by a counter ion (X'). Stucky and co-workers

produced SBA-15 through this pathways]. By balancing the charge interactions

40

with charge matching interactions in aqueous synthesis enhances the long range
periodic order of the mesoporous silica. The formation of the mesostmctures
occurs below the isoelectric point of silica (pH ~ 2). The solubilization of nonionic
poly(alkylene oxide) surfactants and block copolymers in aqueous media is due
to the association of water molecules with the alkylene oxide moieties through
hydrogen bonding and is enhanced in strongly acidic media through association
with hydronium ions instead of water molecules.8 SBA-15 also has hexagonal
pores like MGM-41 but these mesopores are interconnected by micropores.
SBA-15 has much thicker walls than MGM-41 giving it greater hydrothermal

stability but as a result of the thicker walls, the surface area is decreased. A
typical SBA-15 surface area is ~800 m2/g. Moreover, the pore diameter of SBA-
15 can be tailored to range from 5-30 nm.6

The neutral assembly pathway, denoted S°I°, utilizes hydrogen bonding
interactions between a neutral surfactant (SO) and an uncharged silica precursor

(l0). Tanev and Pinnavaia9 synthesized wormhole silica materials using this
pathway. As opposed to MGM-41 and SBA-15 that utilize electrostatic
interactions which results to long range order of hexagonal pores of MGM-41 and

SBA-15, wormhole silica materials make use of hydrogen bonding interaction

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42

that is relatively weaker thus resulting to short disordered channels. The pore
motif of HMS materials has been termed “wormhole-like”. The surface area of a
wormhole silica is typically ~1000 m2/g and pore diameters range from 3-5nm.10
Moreover, the synthesis of wormhole silica materials afford milder conditions and
does not require the addition of acids. The surfactant template is also easily
recovered through solvent extraction and can be reused instead of the
destructive calcination used to remove the quaternary ammonium template.
Another electrically neutral pathway is the NOIo pathway. Like the SOI0
pathways used for synthesizing HMS wormhole type materials, N°I° pathway
utilizes amphiphilic PEO-based surfactants that have hydrophilic polyethylene

oxide (-(CHZCH20)n-) segments connected to hydrophobic R groups of varying

lengths and functionality. The R group can be an alkyl group as in the case of Brij
and Tergitol surfactants an alkylated aromatic group as in the case of IGEPAL-
RC and TRITON-X surfactants or a polypropylene oxide segment as in the
Pluronic family of block co-polymer surfactants. The resulting materials have
disordered pore wormhole structure with BET surface area of up to 1200 m2/g

with framework pore diameters of up to 5.8 nm.11,12

2.1.2 Pore Size and Morphological Control of Mesostructured Silica
The quest for mesostructured silica with larger pores has pushed
researchers to develop methods of increasing the framework pore diameter.

There are several known approaches on controlling the size of the framework

pores, including the nature of the hydrophobic tail of the surfactant templatel, the

43

incorporation of an auxiliary organic solvent or swelling agent7, varying the
assembly temperature13'18 and various post-synthesis treatment.3

Whether synthesized through the electrostatic pathway using quaternary
ammonium surfactants or the neutral templating pathways using neutral amine
surfactants, the mesopore framework diameter can be controlled by varying the
hydrophobic chain length of the surfactant template. The original work of the
Mobil group who developed MGM-41 has demonstrated that the pore size is
dependent on the length of the hydrophobic tail of the quaternary ammonium
surfactant.1 Also, Tanev and Pinnavaia achieved pore size control for HMS
wormhole by using a family of surfactants with different hydrophobic chain
lengths.9

The addition of auxiliary organic solvent or swelling agents is also a
significant way to expand mesopore diameter. The principle behind this method
is that hydrophobic organic species can be solubilized inside the hydrophobic
regions of the surfactant micelles which lead to the swelling of the micelles. The
solubility of the auxiliary solvent in aqueous media should also be considered for
their swelling roles. Examples of successful swelling agents include dodecane,
trimethyl benzene (TMB), triisopropylbenzene, tertiary amines and
poly(propylene glycol).19'23 With the aid of TMB, pore sizes can be expanded to
as much as 40nm for acidic triblock copolymer systems or to 10 nm in basic
CTAB surfactant systems though the resulting materials are disordered. The pore
size of ordered mesostructures can only be increased to 13 nm and 6 nm for

SBA-15 and MGM-41, respectively, by the addition of TMB. The addition of large

44

amounts of TMB to a synthesis system of SBA-15 results in the formation of

mesocellular foams (MCF).20 Hanrahan and co-workers24 also reported the use

of super critical 002 in tailoring the pore size of hexagonal mesoporous silica.

Assembly temperature also plays a role in tailoring the mesopore
diameter. Studies have shown that increase in the assembly temperature can be
used to increase the framework pore size. It has been suggested that increases
in assembly temperature can lead to the decomposition of the quaternary
ammonium ion surfactant through reaction with surface —OH groups to yield the
formation of neutral amines or by Hoffman degradation to yield alkenes.13 Either
way both products are efficient swelling agents and can act as auxiliary co-
surfactants for increasing framework pore size. For the case of MSU-X silicas
formed under electrically neutral assembly conditions, pore size expansions up to
2.4 nm can be achieved using a single PEO surfactant simply by increasing the
assembly temperature over the range of 25-65°C.25 In this case, the mechanism
leading to the increase in framework pore size with increasing assembly
temperature has been attributed to temperature-dependent changes in the
polarity of the S°I° interface and consequently, a decrease in H-bonding at the
interface. As the assembly temperature is increase, the framework cross-linking
is enhanced through further condensation of the silanol groups, leading to the
reduction in the framework polarity. The degree of H-bonding, along with the
hydration of the amine headgroup, decreases as the polarity of the interface

decreases. This leads to an increase in the surfactant packing parameter (9) with

45

decreasing effective head group area (a0) and therefore an increase in the

framework diameter as the interfacial curvature decreases.26

Post-synthesis treatment of as-made assembly products is used to
improve not only the pore diameter of the mesostructure but also to improve the
thermal stability of the mesoporous structure. For example, hydrothermal
treatment of as—made mesostructures is one of the most efficient methods to
improve the mesoscopic regularity of the products}:27 After the initial assembly
reaction, the mesostructure undergoes reorganization, growth, and crystallization
during hydrothermal treatment. The post-synthesis treatment temperature is
usually low, between 80 and 150°C, but more typically the range of 95-100°C
because higher temperatures usually leads to the degradation of the surfactant
template.28 The post-synthesis treatment of wormhole silica provides an example
of the effectiveness of post-synthesis hydrothermal treatment in mediating the

pore structure of the mesophase. As-made HMS wormhole materials assembled
at 25°C with l°/S° = 4 was washed free of the mother liquor and then was

digested in distilled water at 65°C for 1-5 days. A substantial increase in pore-
pore correlation distance (d-spacing) from 3.8 to 4.6 nm was observed after 1
day of digestion, followed by a more gradual increase to 5.3 nm after 5 days. It
was also observed that this method afforded a d-spacing increase of 1.5 nm,

which is somewhat larger than the 1.2 nm expansion achieved through direct

assembly at 65°C after a reaction time of one day.29

46

2.1.3 Tailoring the Pore Size Distribution Between the Microporous and
Mesoporous Region

Over the past decade a considerable number of studies have focused on
the synthesis of mesoporous materials with pore diameters >2 nm and on making
the framework pores even larger.30 However, much less attention has been
devoted in the synthesis of materials with pores in the super-microporous region.
Super-microporous silicas have framework pore diameters between 1 and 2 nm.
It is said that these materials bridge the gap between the microporous zeolites
(pore diameters s 1.2 nm) and the mesoporous silica (2-50 nm).31'35 It has also
been suggested that microporous materials are important materials for catalytic
applications since they exhibit the potential for size and shape selectivity for
organic molecules that are too large for to have access to the pores of the
microporous zeolites.36 Moreover, size-selectivity seldom is seldom observed for
mesoporous materials because the sizes of the reagent and products involved in
the catalytic reaction is smaller than the mesopores of the catalyst. There have
been very few reports on the synthesis of super-microporous silicas. Most reports
utilize specialized surfactants such as bolafonn amines37i33,
adamantanamine39, short double chain alkylammonium halides‘IO and ionic
liquids41 as templating agents. Moreover, these surfactant templates produce
materials with only one super-microporous diameter. Changing the diameter of
the super-micropores requires a change in the size of the templating surfactant.
As the hydrophobic chain length is decreased, the pore diameter also decreases
but only to an extent. As the hydrophobe becomes shorter, the amphiphilic

property of the molecule also decreases and at a certain chain length, the

47

molecule loses its amphiphilic property and no longer forms a micelle. Instead, it
becomes soluble in the reaction media. The challenge for synthesizing silicas
with super-micropores with diameters less than 2 nm diameter is in finding a

suitable temperature dependent surfactant template.

2.1.4 Objectives
This chapter describes the exploratory studies of the use of five oleyl

amine surfactants as template for the supramolecular assembly of mesoporous

assembly of mesoporous silica via the S°I° pathway. These oleyl surfactant were
first tested for their ability to template a mesostructure. The best surfactant
templates then were chosen for further study. To the best of our knowledge,
these oleyl surfactants have not been reported as templating agents for
mesoporous silica. Thus, a profile of mesoporous silica synthesized under
different reaction conditions was carried out. The effect of varying the polarity of
the reaction medium was investigated as well as the effect of assembly
temperature.

Our interest in using oleyl surfactants as mesostructure templates also is
centered on the possible use of the as-made mesostructure as complexants for
the trapping of heavy metal ions from aqueous solution. The amine head groups
of the templating amine surfactant have been recognized as potential trapping
agents for mercury, arsenic, lead and even carbon dioxide. However, a serious
drawback prevents the use of as-made amine template mesostructures as

trapping agents is the tendency of the intercalated surfactant to desorb in

48

aqueous suspension. One possible solution to this limitation is to cross-link the
surfactant in-situ. The introduction of the organic functionality needed for the
templating of this approach will require a great deal from the surfactant. First, the
surfactant must be able to form a mesostructure. For the synthesis of a
mesostructured silica, it is preferred that the surfactant be either cationic or
electrically neutral. It is also important to consider the chain length of the
hydrophobe and hydrophilic head because these two factors determine the
packing parameter from which the types of micellar aggregates arise. Second,
the surfactant must have functional groups that can act as a ligand for metal
binding. Eventually, the synthesized materials will be used for metal trapping.
Third, the surfactant must have a polymerizable group preferably a double bond
so that it can form a polymer network inside the mesostructure. Fourth, the
polymerized template should not be soluble in solvents like water or ethanol. And
lastly, it would be preferred if the surfactant to be used are commercially
available.

Finding the surfactant that fits all these requirements can be a daunting
task. After reviewing the fundamentals of the synthesis pathways for
mesostructured silica, I concluded that the oleylamine surfactants are the best
candidates for the preparation of functional as-made mesostructures. The
presence of a double bond in the hydrophobic tails should make it possible to

immobilize the structure directing template in the pores of the mesostructure.

49

2.2 Experimental
2.2.1 Reagents

Oleylmethylbis(2-hydroxyethyl)ammonium chloride (Ethoquad), bis(2-
hydroxyethyl)oleylamine (Ethomeen), oleylamine (Armeen OLD),
oleyldimethylamine (Armeen DMOD), N-oleyl-1,3-diaminopropane (Duomeen 0)
were provided by Akzo Nobel (www.surface.akzonobel.com) and were used
without further purification. Tetraethyl orthosilicate (T EOS), dodecylamine (DDA),
and cetyl trimethylammonium bromide (CTAB) were purchased from Aldrich.
Absolute ethanol was purchased in-house. The water used in the experiments
was double exchanged using a Millipore filter apparatus to remove cations and

anions.

2.2.2 Material Synthesis
To the best of our knowledge, there have not been reports describing the
synthesis of mesoporous silica using oleylamine surfactants. In this regard, the

five surfactants were evaluated based on their ability to template mesostructure
silica via the electrostatic S”I+ assembly pathway as well as the electrically

neutral S°I° pathway

50

2.2.2.1 Mesostructure Synthesis via the Electrostatic S+l+ Pathway

The cationic surfactant, oleylmethylbis(2-hydroxyethyl)ammonium chloride
(Ethoquad) was used as the template for the attempted S+l' synthesis of a
mesostructured silica by the S+l' pathway according to the method of Edler and
White.42 A control experiment for the S+l' synthesis of MGM-41 also was carried

out using CTAB as the S+ surfactant. The structures of the two surfactants used

for this synthesis are shown below in Figure 2.2.

Ethoquad

Figure 2.2 Structure of CTAB and Ethoquad

For the synthesis of the MCM-41 reference compound, CTAB (6.08g
0.0167mol) was dissolved in H20 (459, 2.5mol), then 70149, 0.0315mol of

sodium silicate (14 wt% NaOH, 27 wt% Si02) was added and the mixture was

stirred for 10 minutes. The pH was then adjusted to around pH 11 by adding 1M

51

sulfuric acid solution and was heated to 100°C for 3 days. Over the 3-day
reaction period the pH was periodically checked and maintained at pH 11
through the addition of sulfuric acid. The product was filtered and was washed 3
times with deionized water. The product was allowed to air dry. The surfactant
template was removed by calcination at 600°C for 4 hours with a heating rate of
2°C per minute.

In place of CTAB, the same procedures were used in an attempt to
synthesize MGM-41 type material from oleylmethylbis(2-hydroxyethyl)ammonium
chloride (12.799, 0.0315mol) as structure director. At the end of the three day
synthesis the product was recovered by filtration and was washed with deionized

water 3 times. The material was air-dried and the surfactant template was

removed by calcination at 600°C for 4 hours at a heating rate of 2°C per minute.

2.2.2.2 Mesostructure Synthesis via the Electrically Neutral S°I° Pathway
The method used for the synthesis of mesoporous silica via the S°I°

pathway was based on the paper of Pauly and Pinnavaia.43 Table 2.1 provides
the structures and quantities of surfactant used for the assembly of mesoporous
silica based on the recipe that uses 10 g of TEOS as the silica source. Included
for comparison purposes is a reaction mixture in which a non oleyl surfactant was
used as structure director, namely dodecyl amine (DDA). As described below,
the assembly reactions were carried out in an ethanol-rich and water rich solvent

medium corresponding to 60% and 30% ethanol volume, respectively.

52

Mesostructures Prepared in 60 volume % Ethanol

The desired electrically neutral amine surfactant (0.013mol) was added to
a mixture of ethanol (33.6mL) and water (22.4mL) in polypropylene bottles and
the mixture was stirred for 30 minutes at room temperature. TEOS (109,
0.048mol) was then added to the reaction mixture and the resulting mixture was
stirred for 20 hours. The products were recovered by filtration and were air dried.
The surfactant template was removed by calcination at 600°C for 4 hours at a

heating rate of 2°C per minute

Mesostructures Prepared in 30 volume % Ethanol Rich Media

The desired electrically neutral (S0) amine surfactant (0.013mol) was
added into a mixture of ethanol (16.8mL) and water (39.2mL) in polypropylene
bottles and stirred for 30 minutes at room temperature. TEOS (10g, 0.048mol)
was then added to the reaction mixture and was stirred for 20 hours at room

temperature. The products were recovered by filtration and were washed at least
3 times. The surfactant template was removed by calcination at 600°C for four
hours with a heating rate of 2°C per minute. The surfactant template was

removed by calcination at 600°C for 4 hours at a heating rate of 2°C per minute.

53

2.2.2.3 Effect of Solvent Polarity and Assembly Temperature on
Mesostructured Silica Assembled Using Oleyl Amine Surfactants
Mesostructures were synthesized under different conditions of solvent

polarity through variation in the volume ratio of ethanol and water over the range
of 10:90 to 100:0 v/v. Assembly reactions were done at room temperature, 45°C

and 60°C. The details of the syntheses conditions are shown in Table 2.2.

In all of the syntheses, the ratio of silica to surfactant was maintained at
1.0 : 0.27, in accordance with the optimum ratio found in previous studies of
amine template silicas by Pauly43. For a typical reaction 0.013 mol surfactant
template was weighed into a 250 mL polypropylene bottle and was dispersed in
an appropriate amount of water/ethanol solution. The mixture was then either
stirred (for room temperature synthesis) or shaken in a hot water bath. TEOS
was added after 30 mins of stirring or shaking. The reaction was then aged for 20
hours with continuous stirring or shaking. The products were recovered by

centrifugation and were washed with copious amount of water. The surfactant
was removed by calcination at 600 °C for 4 hours in air with a heating rate of

2°C/min.

54

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56

2.3 Physical Measurements

The physical properties of the mesostructured silica reaction products
were determined by nitrogen adsorption isotherms, powder x-ray diffraction,
transmission electron microscopy and scanning electron microscopy.

Nitrogen adsorption and desorption isotherms were obtained at -196 0C
on a Micromeritics Tristar 3000 sorptometer. The calcined samples were
outgased overnight at 150 0C and 10'3 torr.

Powder x-ray diffraction patterns were obtained using Cu Kat radiation (l =
1.542 A) and a Rigaku Rotaflex diffractometer equipped with a rotating anode
operated at 45 kV and 100 mA. Counts were accumulated every 0.02 degree two
theta at a scan speed of 0.5 degree per minute.

Transmission electron microscopy (TEM) images were obtained on a
JEOL 2200FS with an accelerating voltage of 200kV. Samples were prepared by
sonicating powdered samples in ethanol for 15 minutes and evaporation two
drops of the suspension onto a carbon coated film supported on a 3 mm, 300
mesh copper grid.

Scanning electron microscopy (SEM) images were obtained on a 6400
SEM with an accelerating voltage of 220 kV. Samples were coated with gold prior

to taking images.

57

2.4 Results and Discussion

According to "er“, the condensation-polymerization of silicate ions in
aqueous solution to form silica occur in three general stages: polymerization of
monomers to form particles, growth of particles by Ostwald ripening and the
linking of the particles into chains that form networks. The initial polymerization
can be described by the following reaction equilibria starting with a silica ester as

the silica source:

Hydrolysis

—-—————¥

Esterification
Ester Silicate

I | Condensation I
—Si-OH + —Si-OH = —Si-O-Si— + HOH

I I Hydrolysis I I

Alcohol
I I Condensation I

—Si-0R + —Si-OH -————= —sr-o-sr— + ROH

Solvolysis I I

This incorporation of a polymeric complexant inside the framework pores
of a mesostructured silica formed by condensation polymerization requires a
suitably functionalized surfactant. As noted above the choice of an appropriate
surfactant is critical.

First of all, the surfactant must be capable of templating a mesostmcture.

For the synthesis of a well-ordered mesoporous structure, it is preferred that the

58

surfactant be either cationic or neutral. Anionic surfactants have not afforded
derivatives of comparable pore size regularity. It is also important to consider the
chain length of the hydrophobic tail and the size of the hydrophilic head group
because these two factors determine the packing parameter Which determines
the type of micellar aggregate and the final structure of the pore network. Most
importantly, the surfactant must have functional groups that can act as a ligand
for metal binding. This second requirement favors neutral surfactant over a
cationic quaternary ammonium ions because the latter cannot function as a
ligand. Third, the surfactant must have a polymerizable group, preferably a
double bond, so that it can form a polymer network inside the mesostructure. And
lastly, the polymerized template should not be soluble in solvents like water or
ethanol. For these reasons, commercially available oleyl amine surfactants were

investigated as templating agents for mesoporous silica.

Attempted Synthesis of Silica Using a Cationic Oleyl Quaternary Ammonium Ion
Surfactant
It was of interest for comparison purpose to attempt the synthesis of silica

mesostructure using a cationic oleyl surfactant. However, the commercial
cationic surfactant oleyl-N+(CH3)(CH2CH20H)2 Cl' (Ethoquad) failed to template
a mesostructure form of silica under the reaction conditions where CTAB is
known to template MGM-41 silica. Based on the x-ray patterns obtained, no
mesostructure was formed in the presence of the ethoxylated oleyl surfactant.

Figure 2.3 compares the powder x-ray diffraction patterns for the reaction

59

products obtained from oleyl-N+(CH3)(CH2CH20H)2 Cl' and CTAB under

equivalent reaction conditions. CTAB forms a well ordered structure as evidence

by the presence of four Bragg reflections indexed as d100, d110, 9200 and d210.
However, oleyl-N+(CH3)(CH2CH20H)2 Cl‘ exhibit no Bragg reflections indicating
the absence of an ordered framework. A possible explanation for the lack of a

template structure in the presence of oleyl-N+(CH3)(CH2CH20H)2 Cl' is the

presence of the hydroxyethyl groups. In the S+l‘ pathway, the electrostatic

interaction between the cationic surfactant and the anionic silica precursor is very

important. In the case of oleyl-N+(CH3)(CHZCH20H)2 Cl’, the cationic N center is

shielded by the relatively bulky hydroxyethyl groups. The large head group may
have favor ion pairing of the onium ion center with the chloride rather than the

larger silicate anions.

Synthesis of Mesostructured Silica with an Electrically Neutral Oleyl Amine
Surfactant

The Sol0 assembly of mesoporous silicas with wormhole framework
structures was accomplished using different commercially available oleyl amine
surfactants. All the polymerizable surfactants have a double bond in the 09
position of the hydrophobic tail. For initial survey experiments, reactions were
carried out both in ethanol rich media (60 volume %) and in water rich reaction
media (70 volume %). Varying the solvent polarity makes it possible to form
amine template products with the same wormhole framework structure but with

different hierarchical textural porosity. The importance of solvent polarity was

60

previously demonstrated for silica templated by DDA where a high textural
porosity was obtained at low ethanol fractions and low textural porosity formed at
low water fractions.43

Figures 2.4 and 2.5 provide the powder x-ray diffraction patterns for the
products in ethanol-rich and water-rich media respectively. Wormhole structures
are known to exhibit a single low angle reflection that indicates an average pore-
pore correlation distance. The wormhole product made using DDA as a structure—
directing template serves as reference structure. Based on the x-ray diffraction
patterns, we see that for products synthesized under ethanol-rich conditions (60

volume % ethanol), only Ethomeen (oleyl-N(CH2CH20H)2) produced a poorly

ordered material. 0n the other hand, for the silicas synthesized under water-rich
conditions (70 volume % water), Ethomeen again provided the most poorly
ordered wormhole structure. As judged on the basis of the half widths of the

diffraction peaks, Armeen OLD (oleyl-NHZ) and Duomeen 0 (oleyl-
NH(CH2)3NH2), afforded the most ordered wormhole structure in both reaction

media.
Nitrogen adsorption isotherms were obtained to confirm the
mesostructured assignments based on x-ray diffraction. Figure 2.6 and 2.7 show

the nitrogen adsorption isotherms for each of the reaction products.

61

 

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Figure 2.3 Powder x-ray diffraction of calcined silica products synthesized using
(A) Ethoquad (oleyl-N+(CH3)(CH2CH20H)2 Cl') and (B) cetyl trimethyl
ammonium bromide (CTAB) as the templating surfactants.

62

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The nitrogen isotherms revealed more information regarding the pore structure,
morphology and most importantly, the mesoporosity of the materials. From the
isotherms, it can be seen clearly that among the tested surfactants, oleyl-

N(CHZCH20H)2 does not provide products with well expressed mesostructured

pores in either reaction medium as evidenced by the absence of the adsorption

step in the region of P/Po = ~O.35. Also, oleyl-N(CH3)2 does not template well

ordered mesopore in water rich medium but it performs better in ethanol rich
conditions. This adsorption step found in the nitrogen isotherm is due to the filling
of ordered mesopores template by the surfactant.

Table 2.3 summarizes the structural properties of the materials
synthesized in the presence of oleyl surfactants. Note that the ordered products
made from the polymerizable surfactants have larger d-spacings and pore
diameters in comparison to the silica template by DDA. This observation was
readily explained by the longer chain length of the polymerizable surfactant. On
the basis of the surface area and framework pore volumes, two surfactants

namely oley-NH2 (Armeen OLD) and oleyl-NH(CH2)3NH2 (Duomeen O),

standout as being the best oleyl surfactants for templating a wormhole
mesostructure under ethanol-rich and water-rich reaction conditions. In ethanol
rich media, however, neither surfactant is as effective as dodecylamine (DDA) in
templating a mesostructure, but in water-rich conditions, these two surfactants

are superior to DDA.

68

Detailed Study of the Effect of Solvent Polarity and Assembly Temperature on
the Fidelity of the Oleylamine Templated Mesostructures

In order to better elucidate the effect of solvent polarity and assembly
temperature on the pore structures of mesoporous silica template by oleylamine

surfactants, oleyl-NH2 and oleyl-NH(CH2)3NH2 were selected as templating

agents and the reaction medium was varied over the composition range 90/10 to

100/0 EtOH/HZO v/v. Also, the assembly reactions were carried out at three

temperatures, namely 25°, 45° and 60°C. The polarity of the reaction medium
and the reaction temperature both mediate the H-bonding interactions between
the amine head group and the walls of the mesostructure and thereby affect the
properties of the mesostructures.

The following labeling scheme was adopted in order to identify the 60
reaction products. X-Y-Z where X identifies the templating surfactant (“A" for
Armeen OLD, “D” for Duomeen O), Y provides the volume % of water in the
reaction medium and Z is the reaction temperature in oC.

The XRD powder patterns for the products obtained in the presence of
Duomeen O and Armeen OLD are provided in Figure 2.8 and 2.9, respectively.
The products template by Armeen OLD over the temperature range 250-600C all
with the exception of the product made in 10% water at 60°C, are
mesostructured as evidenced by the presence of at least one low angle
diffraction peak. The room temperature products assembled in 20-70% water

exhibit a very weak second order peak, suggesting thepresence of a major

69

lamellar phase. The presence of a single low angle reflection is characteristic of a
wormhole framework structure.

With the exception of the silica template in 100% water at 25°C, all of the
products made with Duomeen 0 over the range of 25-60 0C are mesostructured.
Moreover, the products assembled in the 10-80 vol % water exhibit a clear
second order diffraction peak indicative of the presence of a lamellar structure.
This structural feature is retained for products made at 450 and 60 oC when the
reaction medium contains 30-80 vol % water. The products made outside the
range of solvent composition exhibit a single diffraction peak characteristic of a
wormhole framework. The dependence of mesostructure formation as assembly
temperature and the polarity of the solvent medium attest to the importance of
salvation effects in determining the pore structure of the template products. The
salvation of both the polar head groups and the apolar tail of the surfactant is
temperature and solvent dependent. Consequently, the packing parameter and

templating properties of the surfactant also depend on temperature and solvent

polarity.

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The nitrogen adsorption isotherms confirm mesoporosity and usually,
reveal more information on pore size than the power x-ray diffraction. The

nitrogen adsorption isotherms and BJH pore distributions for the materials

synthesized at 250C using Duomeen O are presented on Figures 2.10 and 2.11,
respectively. At first glance, the isotherms confirm the presence of mesopores. It
is normally expected for interparticle textural porosity to appear when the water
fraction during the synthesis becomes high, because high solvent polarity
normally favors small particles. However as can be seen from the isotherms,
there is little or no nitrogen uptake of nitrogen at high partial pressure (P/Po > 90)
indicating the absence of significant textural porosity and the presence of large
particles. As shown in the TEM images in Figure 2.12, most solvent compositions
give large particles. Moreover, there is a decrease in the volume of adsorbed
nitrogen at very low solvent polarity. This can be explained by the amount of
surfactant in micellar form. As the solvent polarity decreases, the surfactant
template becomes more soluble in monomeric form and fewer go to form
micelles. This results in a decrease in porosity.

Another feature of the isotherm is the shifting of the adsorption step
corresponding to framework mesopore filling. In the process of nitrogen
adsorption, nitrogen first forms a monolayer of molecules on the surface of the
pores. From this information the surface area is calculated. As the partial
pressure of nitrogen is increased, the gaseous nitrogen that is adsorbed begins
to form multi-layers and these multi-layers of nitrogen begin to condense and

behaves like

73

on

O

O
I

—l— D10—25
—0— 020-25
—A— D30-25
-—-v— D40-25
—<>— D50—25

 

 

 

Volume of Adsorbed Nitrogen (cm3/g, STP)

 

0.0 0.2 0.4 0.6 0.8 1.0

Relative Pressure (P/Po)

 

 

 

 

1000-
E —-— D60-25
l7, —._ 070-25
03 300‘ —A— 080-25 .
0‘ -v— 09025 fig: : 1’.
E —<>— 0100-25 3*!" v v/
v ’— Iv/ v
QC, 600‘ ',v V' ’ 'v Q
a ,, -
:0: ’. v «900
z 0/0/
8 4004 / f 000/
‘9 <>
0
(I)
'U
E 200-
O
0
E r
2 o .
o l i‘ I l I ' I v I I j
> 0.0 0.2 0.4 0.6 0.8 1,0

Relative Pressure (P/P°)

Figure 2.10 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Duomeen O at 25 °C with varying water fraction (Top) 10-50%, (Bottom)
60-100%

74

  
 

 

1.2 —
1.0 —
’5
C
@08 -
0')
g —I—— D10-25
50.6 — + 020-25
g —A— D30-25
'o 0.4 — + 040-25
0.2 —
0.0 - - “
1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm
1.4 -
1.2 -
- o
1.0 —
E .
9 0.8 - '
2’
"E —-— 060-25
30.6 - —o— D70-25
% —A— 080-25
% Q4 _ —v— 090-25
. —<>— D100-25
0.2 —
f’.”?3».».°-z.s
0. 0 _ —1\\\\.A,‘ A‘ .

 

 

 

I ' l ' l ' I 7 I ‘ l

. . , .
1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

Figure 2.11 BJH pore size distribution of mesostructured silica synthesized from
Duomeen O at 25 0C with varying water fraction (Top) 10-50% (Bottom) 60-100%

75

 

Figure 2.12 TEM images of mesostructured silica synthesized from
Duomeen O at 25 °C with varying solvent polarity a liquid.

76

 

Figure 2.12 (Cont'd)

77

This has been described as capillary condensation which is represented by a
step in the nitrogen adsorption isotherm when the mesopore are uniform in size.
During this stage, the mesopores are being filled. For the templated products
made at 25°C, there is a shift in the partial pressure for framework adsorption as

the water content is increased over the range of 10 to 50 % H20. This shift

signals a change in the pore diameter. The changes in pore diameters with
respect to increasing solvent polarity are best seen by the BJH pore distribution
plot shown in Figure 2.11. From these plots, it can be seen clearly that the pore
diameters increases from ~2.0 nm for sample D10-25 to ~35 nm for sample
D50-25 and remains more or less constant there after. To the best of our
knowledge, this ability to change framework pore structure from 2.0 nm to 3.5 nm
by changing solvent polarity is unprecedented.

The TEM images shown in Figure 2.12 confirm the suspected presence of
layered structures, particularly as shown by products 030-25 to product D90-25.
Fiber-like morphologies in conjunction with the wormhole structure also are
observed. The presence of lamellar structures is in agreement with the XRD

results.

Mesostructure synthesis at 45° and 60°C was also done to observe the
temperature behavior of the templating agent. At higher temperature the
surfactant becomes more soluble in water but the H-bonding interaction also is
weakened. Figure 2.13 and 2.14 shows the nitrogen isotherms of products
synthesized at 45°C and 60°C respectively, and Figure 2.15 and 2.16 and Figure

2.17 and 2.18 provide the respective pore size distributions and TEM images for

78

the products. As was observed for the materials synthesized at room
temperature, there is a shift to higher partial pressures for the mesopore
adsorption step as the solvent polarity was increased under both temperature
conditions. The shift to higher partial pressure continues until the water fraction is
50% (D50-x). Moreover, little or no texture porosity is observed up to 50% water
media but products D80-45 and D80-60 exhibit substantial textural porosity.
From these results, only at 80% water and at elevated temperature provide
products with high textural porosity.

The mesopore adsorption step shift to higher partial pressure represents
an increase in pore diameter which is better seen on the BJH pore distribution
plots on Figure 2.15 and 2.16. Figure 2.17 and 2.18 shows the TEM images of
the materials. The materials still exhibited wormhole pore structure but there are
still parts where fiber like morphologies (e.g. products D40—45, D60-45, D50-60,
D70-60) and lamellar vesicle-like particles are observed (D100-45, D20-60 and
D60-60).

Figure 2.19 shows the nitrogen adsorption isotherms for materials
synthesized at 25°C with Armeen OLD. These templated products exhibit
properties similar to those synthesized with Duomeen O. The mesopore
adsorption step shifted to higher partial pressure as solvent polarity is increased.

There is little or no texture porosity for the products made at room temperature.

79

800 -

     
 

E —l - 010-45
la; —O— 020-45
03 —A— 030-45
00‘ 600 - —v— 040-45 _

_<>_ 050.45 6W

- , - w

_, M110...“

. 400 ‘ "1 ”—ooooo—o-o—“M

j

N

O

O
1

 

 

Volume of Adsorbed Nitrogen (cm

0.0 0.2 0.4 0.6 0.8 1.0

Relative Pressure (P/Po)

1400-
--— 060-45
—0— 070-45
120° ‘ —A— D80-45 .0
‘ —v— 090-45 Age
1°°°~ —<>— 0100-45 fl

4
/
800 -
V
"" /
(A ‘. ‘
I ' -‘ . . R? f" .7. I
7‘ KB I" ‘ I I" "

 

Volume of Adsorbed Nitrogen (cm3/g, STP)

 

 

0 I ' I . I ' I . I . I
0.0 0.2 0.4 0.6 0.8 1.0

Relative Pressure (P/PO)
Figure 2.13 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Duomeen O at 45 °C with varying water fraction (Top) 10-50°/o (Bottom) 60-
100%.

80

800 1
—I — 010-60

—0— 020-60
—A- 030-60

——v— 040-60 W
——o— 050-60 0’83“

O)
O
O
4

  

Volume of Adsorbed Nitrogen
(cm3/g, STP)
h
0
O

 

 

200-
0 I ' I ' l ' r I l 1 fl
0.0 0.2 0.4 0.6 0.8 1.0
Relative Pressure (P/Po)
16007 --l- 060-60
- —0— 070-60
1400 - —A- D80-60
A - —v— D90-60
,0: 1200 - —<>— 0100-60 A

Volume of Adsorbed Nitrogen

 

 

 

0.0 0.2 0.4 0.6 0.8 1.0

Relative Pressure (P/Po)

Figure 2.14 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Duomeen O at 60 °C with varying water fraction (Top) 10-50% (Bottom) 60-
100%.

81

1.4-

   
     
 

 

i

1.2-

01.0-

E .

‘F
90.8-
0")

{5, ‘ —-— 01045
50-6 ‘ + 02045
2 1 —4— 03045
% 0-4‘ + 04045
‘ —<>— 050-45

1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

   
   
 
 

 

 

1.4 7
1.2 -
1.0 -
E -
C
00° -
(0
g + 06045
30.6 - + 07045
2 —A— 08045
5 0,4 - + 09045
—<>— 010045
0.2J
0.0 - H ‘

 

1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

Figure 2.15 BJH pore size distribution of mesostructured silica synthesized from

Duomeen O at 45 °C with varying water fraction (Top) 10-50% (Bottom) 60-
100%.

82

   
 
 
    
   

 
   

       

 

1.8 -
1.6 -
-1-4 -
E u!
3,12 -
m\
g 1-0 - + D10-60
50.8 _ + 020—60
2 —A— D30-60
5 0.6 — —v— 040-60
- —<>— 050-60
0.4 -
0.2 - .-
A
0.0; "'5285‘g1o293' ‘ ,1-

 

I I l 5 l I l l
1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

2.0 -
1.8 -
1.6 -
1.4 —
E _
g 1.2 -
m\
g 1-0 -_ + 060-60
0 0.8 _‘ + D70'60
g . —e— D80-60
% 0.6 4 —v— 090-60
- —<>— D100-60

 

 

1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

Figure 2.16 BJH pore size distribution of mesostructured silica synthesized from

Duomeen O at 60 °C with varying water fractions of (Top) 10-50% and (Bottom)
60-100%.

83

 

ized from Duomeen 0

images of mesostructured Silica synthes

Figure 2.17 TEM
at 45 °C with vary

ing solvent polar

84

 

Figure 2.17 (cont'd)

85

 

Figure 2.18 TEM images of mesostructured materials synthesized from
Duomeen O at 60 0C with varying solvent polarity.

86

 

Figure 2.18 (cont’d)

87

Figure 2.20 and 2.21 provide the corresponding BJH pore distributions
and TEM images for these template products. The BJH pore size for products
A10-25 and A20-25 are in the supermicroporous range. As the water fraction ( >
30 volume % water) is increased, the pore size increases to about 3.1 nm where
it remain more or less constant for all silica products template for Armeen OLD.
The TEM images in shows the dominant wormhole framework morphology.

At 45°C, the increase in temperature is enough to disrupt the hydrogen
bonding interactions between the template and the silica precursor that the
mesostructure that is form is not as well defined as the others, resulting in a
product with high textural porosity as revealed by the uptake of nitrogen at high
P/Po for the products made in media containing 60-100 volume % water. This
observation is confirmed by the isotherm in Figure 2.22, the pore size distribution
in Figure 2.23 and the TEM images on Figure 2.24. As with the other templated
products, increasing the water fraction from 20-50% results in increased pore
diameter (Figure 2.26) with the formation of high texture porosity materials above
50% water fraction. The nitrogen isotherms of the products A20-45 to A50-45
showed a steady increase in the volume of adsorbed nitrogen. This is indicative
of how much surfactant is in micellar form. As the percentage of water is

increased, more surfactant aggregates to template the mesostructure.

At 60 0C, Armeen OLD a non structured product is formed in 10% water
medium. As the water fraction is increased to 40%, the products produced are

without textural porosity and from 50% to 100% water, the isotherms and pore

88

800 -

 

 

       
  

         
 

 

 

5 -—I—A10-25
8’ —0—A20-25 H
1%” 4430-25 .. - - 1 1 ~-
$600~ —v—A40-25 - prim"
2 (.7) _O_ A50'25 /’/' /A/:.£WM
o — 10
w 2’
2 mg I/ ooooo-o—o—oououl
g .81.. I-lalll-I—I—nlfl
2 <30
g 93.12.
200 ‘97“.. -'V .r .__ ’-
0 I W I v I v I u T f I
0.0 0.2 0.4 0.6 0.8 1.0
Relative Pressure (P/Po)
1400-
g ‘ —I—A60-25
8’ A —o— A70—25
.12 1200 —A— A80-25
2 E —v-A90- 25 2:. 11 11.-”11’”
'0 29
.8 '0 100°— —<>—A100-25 :3”
8 23 ,,
2 ”E 800 - / :01 «1741- .1"-
“5 3 (v4; I g -3, .1L.-.+_e‘w
g 600- "J
2 .
o
> 400-
,.
ZOO-f ~
0 I, . I ' I ' I ' I . i
0.0 0.2 0.4 0.6 0.8 1.0

Relative Pressure (P/Po)

Figure 2.19 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Armeen OLD at 25 °C with varying water fraction (Top) 10-50% (Bottom)
60-100%.

89

 

 

  
 
 
 
   

+ A10-25
—O—— A20-25
—A— A30-25
—v— A40-25
—<>— A50-25

 

1 2 3 4 5 6 7 8 9 10
Average Pore Diameter. nm

   
      

 

2.0 -
1.8 —
1.6 —
1.4 -
C
£31.21
(0
E 1-0 ‘ —-— A60-25
008 _ + A70-25
2 —A— A80-25
%06 - + A90-25
- —<>— A100-25
0.4 -
0.2 -
0.0 —

1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

Figure 2.20 BJH pore size distribution of mesostructured silica synthesized from
Armeen OLD at 25°C with varying water fraction (Top) 10-50% (Bottom) 60-

100%.

90

 

Figure 2.21 TEM images of mesostructured materials synthesized from Armeen
OLD at 25 0C with varying solvent polarity.

91

 

Figure 2.21 (cont'd)

92

800 —
—I— A10-45

—o— A20-45

' —A— A30-45 I
500 - —v— A40-45
—<>— A50-45 O?8<>

Volume of Adsorbed Nitrogen
(cm3/g, STP)
14'
‘ l
i
\

 

 

 

2
I
O | I l ' l

0.0 0.2 0.4 0.6 0.8 1.0
Relative Pressure (P/Po)
1400 -
— I — AGO-45
—o — A70-45
1200 ' -A— A8045 "
6? —v—- A90-45 / ’
{7,1000 - —-<>-— A100-45 f9,

(‘0

Volume of Adsorbed Nitrogen
(cm lg

 

 

 

0.0 0.2 0.4 0.6 ' 0.8 ' 1.0

Relative Pressure (P/PO)

Figure 2.22 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Armeen OLD at 25 °C with varying water fraction (Top) 10-50% (Bottom)
60-100%.

93

1.2 -
1.0 -
g .
C
90.8 -
0")
g + A10-45
50.6 — —o— A2045
2 . ~A— A3045
% Q4 _ + A4045
- . -—<>— A5045

 

 

1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

  
  
  

 

1.4 -
1.2 -
1.0 -
g .
C
L508 -
0')
g + A6045
50.6 - —o— A70-45
g —4-— A80-45
1>a 0_4 —v— A90-45
7 —<>— A100-45
0.2 4
0.0 -

 

" I I I I I I I-
1 2 3 4 5 6 7 8 9 10
Average Pore Diameter, nm

Figure 2.23 BJH pore size distribution of mesostructured silica synthesized from

Duomeen O at 25 °C with varying water fraction (Top) 10-50°/o (Bottom) 60-
100%.

94

 

Figure 2.24 TEM images of mesostructured materials synthesized from Armeen
OLD at 45 °C with varying solvent polarity.

95

 

Figure 2.24 (cont’d)

96

1000-

     

 

 

— I — A10—60
5 —.— A2060
5’ 800 _ —A— A30-60
5 A —v— A40—60 :-
8 g I —<>—— A50-60 g
E ‘0 .
8 23 600 0
'O m
< E J 0
‘8 8
0 400 4
S w—v—W
g I . , - . 0 —A-AM—A—A-MMAM
200 - .- - 1 ' oun—ouuw
O I l l l l l
0.0 0 2 0.4 0 6 0 8 1 0
Relative Pressure (P/PO)
1400 -
‘ —-— A6060
—0— A70—60 q
1200 “ —-A—— A80-60
' —v— A90-60
1°°° - —<>— A100—60

Volume of Adsorbed Nitrogen
(cm3/g, STP)

 

 

 

0 I I I I I I I I I I I

0.0 0.2 0.4 0.6 0.8 1.0

Relative Pressure (P/Po)

Figure 2.25 Nitrogen adsorption isotherms of mesostructured silica synthesized

from Armeen OLD at 25 °C with varying water fraction (Top) 10-50% (Bottom)
60-100%.

97

1.0—

  
 
 
  
 

0.8 -
*
E
30.6 -
0‘
g 1 +A1o-60
D 0.4 _ + A20'60
2 +A30-6o
% —v—A40-60
—<>— A50-60

 

  

 

Average Pore Diameter, nm

  
  
 

1'01
0.8 -
E0 6
9'» .
(’0
,E, 1 —-— A60—60
50 4 _ —o— A70-60
32 ' + A80-60
% + A9060
—<>— A100-60
0.2 -

 

 

Average Pore Diameter, nm

Figure 2.26 BJH pore size distribution of mesostructured silica synthesized from
Duomeen O at 25 °C with varying water fraction (Top) 10-50% (Bottom) 60-
100%.

98

 

Figure 2.27 TEM images of mesostructured materials synthesized from Armeen
OLD at 60 °C with varying solvent polarity.

99

 

Figure 2.27 (cont’d)

100

distributions for the products are similar as shown in Figures 2.25 and 2.26. The
TEM images (Figure 2.27) confirm the formation of high texture porosity products
with wormhole framework structure.

Table 2.4-2.5 summarizes the pore properties for the products. Overall the
most remarkable result of the study was the ability to control the pore diameter
through simple mediation of solvent polarity and assembly temperature. For
assembly reactions carried out at very low solvent polarity, the mesostructured
silica products are super-microporous. Super-microporous silicas have pore
diameter between 1-2 nm and bridge the gap between the microporous zeolites
and the mesoporous silica. Relatively few studies have been reported for the
synthesis of supermicroporous silicas. It has been suggested that they offer more
selectivity towards size and shape-selective separation or catalysis. These
materials are not easily synthesized. Even though their synthesis is based on
supra-molecular assembly using surfactant templates, finding a suitable
surfactant to produce the desired 1-2 nm diameter pores is not easy because
solvated surfactant micelle typically are larger than 2 nm in diameter. Making the
hydrophobe of the surfactant template shorter compromises the amphiphilic
property of the surfactant. Typical surfactants, such as alkylammonium ions and
block co-polymers used for mesostructured silica synthesis produce micelles that
are >2 nm in diameter. When the surfactant hydrophobe becomes too short, it
does not form a micelle anymore, instead, it becomes soluble in monomer form.

The oleyl surfactants which have 18 carbons on its hydrophobic tail are expected

101

Table 2.5 Summary of the pore properties of mesoporous silica products
assembled using Duomeen O as a templating agent under varying assembly

temperature and varying water composition.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Framework
. Avera e BET Pore
M . Calcmed Port:J .Wall Surface Volume,
aterial d- D' t Thickness, A Ll
spacing, iame er, rim rtzea, m 9
nm nm m lg (taken @
P/Po = 0.75)
25°C
D10-25 3.9 2.0 1.9 1263.5 0.691
D20-25 4.4 2.7 2.0 923.7 0.888
D30-25 4.7 3.0 1.7 871.9 0.861
D40-25 4.5 3.2 1.3 855.0 0.766
D50-25 4.6 3.4 1.2 786.2 0.720
D60-25 4.7 3.7 1.0 892.1 1.124
D70-25 4.9 3.8 1.1 928.6 1.237
D80-25 4.6 4.2 0.4 941.3 1.200
D90-25 5.4 3.7 1.7 872.2 0.874
D100-25 - 3.4 - 801.6 0.698
45°C
D10—45 3.9 1.6 1.4 990.9 0.519 (0.75)
D20-45 4.2 1.9 2.3 1098.1 0.609(0.75)
D30-45 4.6 2.5 2.1 862.9 0.670 (0.8)
D40-45 4.6 2.7 1.9 865.4 0.760 (0.8)
D50-45 4.8 2.8 2.0 883.6 0.880 (0.75)
D60-45 4.7 3.1 1.6 884.0 0.942 (0.75)
D70-45 4.8 3.3 1.5 986.1 1.176 (0.75)
D80-45 4.3 3.0 1.5 1106.1 1.146 (0.8)
D90-45 4.5 3.1 1.4 1114.2 1.094 (0.8)
D100-45 4.5 3.0 1.5 1048.0 0.931(08)
60°C
D10-60 4.2 1.4 2.8 738.4 0.387 (0.75)
D20-60 4.2 1.9 2.3 909.8 0.510 (0.75)
D30-60 4.6 2.4 2.2 880.0 0.658 (0.8)
D40-60 4.9 2.8 2.1 826.4 0.822 (0.8)
D50-60 5.0 3.1 1.9 834.3 0.932 (0.8)
D60-60 5.0 3.3 1.7 785.3 0.854 (0.75)
D70-60 5.1 3.4 1.7 876.5 1.141 (0.75)
D80-60 4.9 3.8 1.1 884.3 1.210 (0.8)
D90-60 5.1 3.5 1.6 967.7 1.486 (1.0)
D100-60 5.1 3.7 1.4 969.9 1.189 (0.8

 

102

 

Table 2.6 Summary of the pore properties of mesoporous silica products
assembled using Armeen OLD as a templating agent under varying assembly
temperature and varying water composition.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Framework
Calgined Ail/garage Wall SBr'fET VFiore
. - ore . u ace oume,
Material spacing, Diameter, Thicknes, Area, mL/g
nm nm nm m2/g (taken @
P/Po = 0.75)
-2539... MW...
A10-25 3.4 1.)_2__ 2.2 1003.8 0.533
A20-25 3.8 2.1 1.7 1179.4 0.693
A30-25 6.7 3.8 2.9 666.4 0.845
A40-25 5.2 3.0 2.2 868.4 0.980
A50-25 4.7 2.9 1.8 683.1 1.485
A60-25 4.7 3.4 1.3 910.4 1.132
”Am-25 .. W4] 30 1.7 1050.9 1.115
A80-25 4.6 2.9 1.7 1183.5 1.057
A90-25 -.. 4.5 3.2 1.3 1175.2 1.272
A100-25 5.0 3.2 1.8 1506.8 1.637
45°C
A10-45 4.6 1.9 (broad) 2.7 794.8 0.676 (0.8)
A20-45 3.7 1.6 2.1 859.7 0.458
L A30-45 5.2 2.1 3.0 902.1 0.587
64045 5.3- 21,-M 2.6 820.4 0.725 (0.8)
. A50—45 - 5.0 3. 1.9 820.2 0.910(0.8)
A60-45 5.3 3.5 1.8 853.3 1.117 (0.8)
A70-45 5.3 3.5 1.8 900.5 1.192(0.8) -
A80-45 6.1 3.6 2.5 832.1 1.099 (0.8)
- A90-45 f 6.1 3.5 2.6 908.0 1.124(0.8)
A100-45 6.0 3.4 2.6 896.3 1.132 (0.8)
60°C '_
A10-6O - 6.0 (broad) __ - 1.191 1.191 (1.0)
A20-60 4.0 1.0 3.0 586.8 0.316
A30-60 4.7 1.8 2.9 698.4 0.413
A40-60 5.1 2.3 2.8 784.3 0.524
A50-60 5.1 3.3 1.8 730.2 0.873 (0.8)
A60-60 5.7 3.5 2.2 767.8 1.192 (0.9)
.. A70-60 6.0 3.6 2.4 761.6 1.041 (0.8)
A80-60 . 6.2 3.7 2.5 770.3 --.-1__-__0.§-9_.(Q-§L-.
A90-60 - 3.9 - 803.2 1.052 (0.8)
A100-60 5.8 3.9 1.9 804.5 1.026 (0.8)

 

103

 

to produce micelles with diameter about 3-3.5 nm but instead at the proper
solvent polarity it is able to produce micelles with diameters within the super-
microporous range. This flexibility arises is due to the double bond present on the
oleyl chain. This double bond provides a kink that gives the hydrophobe the
propensity to fold in a hair pin conformation thus decreasing the chain length and
consequently the micelle diameter. This is thought to happen at low solvent
polarity (i.e. high EtOH fraction). As illustrated in Figure 2.28, when there is
inadequate water to solvate the head groups of a micelle with fully extended
hydrophobic chains. This results to a more fully hydrated but smaller micelle. As
the water concentration is increase, there is enough water to hydrate the micelle
being formed thus the hydrophobe is able to extend completely to its maximum
size. This interpretation is supported by the observed increase in pore diameters
as the water fraction is increased up to a volume fraction where the hydrophobe
segment is fully extended. Beyond that point, further increase in the volume
fraction of water does not result in an increase in templating micelle size.

To further explore this hypothesis, a parallel experiment was done using a
saturated amine surfactant template, namely, hydrogenated tallow amine
(abbreviated TA). The hydrophobic tails of this surfactant contains primarily 18
carbon atoms with a minority of the tails containing 14 or 16 carbon atoms. The
template silica products were synthesized the same way as the oleyl-templated
silicas. The nitrogen isotherms and BJH pore size distribution of the products
synthesized at room temperature are presented in Figure 2.29. The isotherms

exhibit qualitatively similar step shaped (Type IV) isotherms, indicating the

104

presence of mesopores with diameters >2 nm, regardless of the solvent polarity.
The BJH pore size distributions indicate the absence of pores below the
mesopore region of (< 2 nm). The smallest average pore size was obtained at
10% water (TA10-25) which is about 2.5 nm. Thus, the effect of solvent polarity
on the average pore size of TA templated silica is much more limited and
confined entirely to smaller variations in the small mesopore range. The lack of
double bond in the hydrophobic tail limits the hairpin folding thus limiting the
effect of solvent polarity on the template mesopore size of the silica.

Figure 2.30 presents the plot of pore sizes of mesoporous silica products
assembled using Armeen OLD. In the studies conducted by Pauly26 regarding
the effect of assembly temperature on the BJH pore size distribution of wormhole
silica, an increase in assembly temperature corresponds to an increase in pore
diameter. In their experiment, they maintained the water to ethanol volume ratio
to 90/10 while varying the assembly temperature. In the experiments done,
mesoporous silica products assembled at 90 volume °/o water showed a similar
behavior where an increase in assembly temperature results in the increase in
pore diameter. However, since the synthesis using oleylamine surfactants
covered most of the possible water/ethanol ratios, the results gave a bigger
picture on how the surfactant aggregates at different temperatures and
water/ethanol ratios. For silica products assembled using Armeen OLD, the
expected increase in pore diameter is observed from 50 — 100 °/o water fractions.
When the water fraction decreases to 40 to 10 percent, an increase in

temperature has an opposite effect where the smaller pore diameters are

105

observed at higher temperature. This result can be attributed to the greater
solubility of the surfactant at elevated temperature. The high ethanol fractions
already make the surfactant more soluble in the dispersing medium thus less
surfactant molecules aggregates to form micelles. Moreover, the increase in
temperature also increases the movement of molecules thus disrupting the H-

bonding interaction of the H20 molecules solvating the micelle. The presence of

water solvating the micelle increases the diameter of the micelle but at elevated
temperatures, some of these water molecules are lost.

Figure 2.31 presents the variation of pore sizes with respect to
temperature as a function of water to ethanol ratio. The plot shows a different

trend as opposed to oleyl-NHZ. In all cases except for A100-60 (100 volume %

water at 60°C using Armeen OLD), silica products obtained at room temperature
have bigger pores than the products obtained at 45° and 60°C. For materials
assembled at 60°C, all have bigger pore sizes except for A10-60 (10 volume %
water at 60°C using Armeen OLD) as opposed to silica products assembled at
45°C. Compared to oleyl-NH2, the results did not show a shift in the trend for
pore sizes. This suggests that more factors come into play for the assembled
mesoporous silica using Duomeen O. The diamino functionality of the surfactant
gives it greater stability with regards to the micelle that is produced. The
presence of the diamino functionality gives more sites for H-bonding thus making
the micelle less prone H-bonding disruptions due to an increase in temperature.

Moreover, the headgroup of the surfactant also increases the amount of H20

hydrating the micelle thus increasing its apparent size. This also might explain

106

why at elevated temperature, the templated silica products have smaller pore
sizes since it will be expected that there will be less water of hydration around the

micelle.

107

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g 1200 - —v—- TA70-25
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1 .44 —A- TA80-25
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; 1.2 - —<>— TA60-25
”2’ 1.0: —< — TA50-25
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% . —*— TA20-25
'2 0-6j r _._ TA10-25
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1 2 3 4 5 6 7 8 9 10
Pore Diameter, nm

Figure 2.29 Nitrogen isotherms (top) and BJH pore size distributions (bottom) of

mesostructured silicas synthesized from tallow amine (TA) at 25 0C under
varying water fractions (10-100%).

109

 

Effect of Assembly Temperature on the
Framework Pore Size of Mesoporous Silica
Templated with Armeen OLD
4 -l
I
A
. ‘ Agni—c
A I
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f 1 fl 1 ' l ' l ' l ' l ' l i l ' l '
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Water Fraction During Assembly

Figure 2.30 Pore size variations of mesoporous silicas assembled under varying
water fractions using Armeen OLD (oleyl-NHZ) with respect to temperature

variations. (Note: A10-6O (10 volume % water) was not included in the plot since
it did not produced a mesostructured silica.)

110

 

Effect of Assembly Temperature on the
Framework Pore Size of Mesoporous Silica
Templated with Duomeen O
I
4 _
I . A I
E
r: I A A
(T.; I A '
‘53 3-1 I . I .
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I I ' I fl I ' I ' I ' I ' I I ' T T
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Water Fraction During Assembly

Figure 2.31 Pore size variations of mesoporous silicas assembled under varying
water fractions using Duomeen O (oleyl-NH(CH2)3NH2) with respect to

temperature variations.

111

2.5 Conclusions
Among the oleylamine surfactants that were investigated on their ability to

template a mesostructured silica, Ethomeen (oleyl-N(CHZCHZOH)2) did not

produce a mesoporous silica with well defined pores on both water-rich and
ethanol-rich media. This conclusion is based on the results obtained from the
powder x-ray diffraction where the product obtained using Ethomeen showed no
low angle diffraction peak typical of mesostructured wormhole structures and the
lack of mesoporous adsorption step in nitrogen adsorption isotherms.

The attempted synthesis of well ordered hexagonal mesoporous silica

similar to MCM 41 using Ethoquad (oleyl-N+(CH2CHZOH)2CH3) was not

successful as evidenced by the lack of the characteristic peaks in the low angle
powder x-ray diffraction.

Ethomeen (oleyl-(CHZCHZOH)2) and Ethoquad (oleyl-(CHZCH20H)2CH3)
did not produce mesostructures but instead produce high surface area silica gels

at 25°C regardless of the polarity of the reaction medium. This result was
attributed to the hydroxyl ethyl group present on the head group of the surfactant.
The relatively bulky hydroxyl ethyl group poses steric hindrance on the H-
bonding interactions for the case of Ethomeen and electrostatic interaction for the
case Ethoquad.

Armeen DMOD (oleyl-N(CH3)2) did not produce a good mesostructured

silica. This is evidenced by the weak diffraction peak on the powder XRD and
very weak mesopore adsorption step on its nitrogen adsorption isotherm. This

result is attributed to the weakened H-bonding interaction due to the presence of

112

the methyl groups on the nitrogen. Replacing the H atoms of Armeen OLD (oleyl-

NH2) with methyl groups reduces the sites for possible H-bonding interactions

between the surfactant and oxygen atoms of the silica framework.
Among the five surfactants surveyed for the assembly of mesoporous

wormhole silicas, oleyl-NH2 and oleyl-NH(CH2)3NH2 are judged to be the best

among the oleylamine surfactant template. Both surfactants produced better
mesostructured wormhole silica as compared to dodecylamine (DDA) template
products when the synthesis is done under water-rich (70 volume %) media as
evidenced by the volume of adsorbed nitrogen and the framework pore volume.
The powder x-ray diffraction peaks also suggest the presence of lamellar
structures on the products obtained. Although mesoporous silica products of both
surfactants are superior to DDA in water-rich media, under ethanol-rich media,
both produced silica with lower framework pore volume as opposed to DDA. The
templating properties of the latter two surfactants was used on the succeeding
steps of the study in an effort to determine the effects of solvent polarity and
assembly temperature on framework structure and pore size.

Mesostructured silica templated by Duomeen O and Armeen OLD under
varying solvent polarities and temperatures were successfully synthesized. TEM
images revealed that the silica products have a wormhole pore network with
some fractions having a lamellar morphology. The mesostructured mesoporous
silicas synthesized from these two templates have pore structures that extend
into the super—microporous range. Also, the two surfactants are able to produce

mesostructured silica over the entire super-microporous range of 1-2 nm by

113

merely modifying the solvent polarity. This capability of the surfactant is attributed
to the double bond present on the hydrophobe. This double bond provides a kink
for the surfactant tail to fold into a hairpin structure that assembles into micelles
of smaller diameters. At higher solvent polarity the surfactant favor the elongated
conformation that results to larger pore diameters. Parallel studies of
mesoporous silica products assembled using tallow amine template showed that
the double bond on the hydrophobic tail plays a key role in the supramolecular
assembly of supermicroporous silica. At high ethanol volume fractions (10-20
volume % ethanol), the tallow amine surfactant did not template silica products
with pore sizes below 2 nm.

The effect of temperature on the products also was observed. The effect
of temperature on silica products templated using Armeen OLD and Duomeen 0

showed very different trends. For Armeen OLD (oleyl-NH2). high water fraction

(50-100 volume % water) synthesis along with elevated temperature results to
increased pore size while low fractions (< 30 volume % EtOH) results in a
decrease in pore size. As for silicas assembled using Duomeen O, it is observed
that the pore size of the materials assembled at room temperature are bigger
than those silicas assembled at elevated temperature. It is hypothesized, that
there are more water hydrating the Duomeen O micelle because of the two
amino groups on the hydrophilic tail thus results to a bigger micelle diameter. At
elevated temperature, the H-bonding interactions get disrupted and the amount

of water molecules solvating the micelles are less thus the smaller micelles.

114

Overall, the assembly of mesostructured silica material using Armeen
OLD for all water/ethanol ratios is best carried out under a 45°C temperature.
This temperature provides more control over the morphology and pore
dimensions of the mesoporous silica products because at this temperature where
one can assemble silicas with low and high textural porosities with similar pore
size distribution. As for Duomeen O, the best assembly temperature would be at
60°C. The diamino functionality of surfactant template gives it more sites for H-
bonding thus giving it the flexibility for elevated temperature assembly. The 60°C,
the silica products have better pore size fidelity as evidenced by the nitrogen

adsorption isotherms and the BJH pore size distribution.

115

2.6 References

1.

10.
11.
12.

13.
14.

15.

16.
17.

18.
19.

Beck, J. S. Vartuli, J. C., Roth, W. J., Leonowicz, M.E., Kresge, C.T.
Schmitt, K. D., Chu, C.T-W., Olsom, D.H., Sheppard, E.W., McCullen, B.,
Higgins, J.B., Schlenker, J.L. J. Am. Chem. Soc. 1992, 114, 10834

Kresge, C.T., Leonowicz, M.E., Roth, W. J., Vartuli, J.C., Beck, J.C.
Nature 1992, 359, 710

Hou, Q.S., Margolese, D.|., Stucky, G.D. Chem Mater. 1996, 8, 1147
Wan, Y. and Zhao, D. Chemical Reviews 2007 107, 7 2821-2860

Beck, J.S., Vartuli, J.C., Kennedy, G.J., Kresge, C.T., Roth,
W.J.,Schramm, S.E. Chem. Mater.1994 6,1816-1821

Zhao, D.Y., Feng, J.L., Huo, Q.S. Melosh, N. Fredrickson, G.H., Chmelka,
B.F., Stucky, 6D. Science 1998, 279, 548-552

Zhao, X.S., Lu, 6.0. J. Phys. Chem. B 1998, 102, 1556-1561

Zhao, D.Y.Q., Huo, Feng, J.L. Chmelka, B.F., Stucky, G.D. J. Am. Chem.
Soc. 1998. 120, 6024-6036

Tanev, P.T.; Pinnavaia, T.J. Science 1995, 267, 865-867
Pauly, T.R.; Pinnavaia, T.J. Chem. Mater. 2001, 13, 987
Bagshaw, S.A., Prouzet, E. Pinnavaia, T.J. Science 1995, 269, 1242-1244

Bagshaw, SA. and Pinnavaia, T.J. Angew. Chem-Int. Edit. Engl. 1996, 35,
1 102-1 105

Cheng, C.F., Zhou, W., Klinowski, J. Chem. Phys. Lett. 1996, 263, 247

Corma, A., Kan, Q., Navarro, M.T.,Perez-Pariente, J.,Rey, F. Chem.
Mater. 1997, 9, 2123

Khushalani, D.; Kuperman, A., Ozin, G.A., Tanaka, K.; Garces, J.; Olken,
M.M.; Coombs, N. Adv. Mater. 1995, 7, 842

Sayari, A., Liu, P. Kruk, M. Jaroniec, M. Chem Mater. 1997, 9, 2499

Sayari, A., Kruk, M. Jaroniec, M. Moudrakovski, l. Adv. Mater. 1998, 10,
16, 1376

Sayari, A., Liu, P., Kruk, M., Jaroniec, M. J. Phys. Chem B 1999 103, 4590
Sayari, A. Angew. Chem. Int. Ed. 200039, 2920

116

20.

21.
22.

23.

24.

25.
26.
27.

28.
29.

30.

31.

32.

33.

34.

35.

36.
37.
38.

Lettow, J.S., Han, Y.J., Schmidt-Winkel, P., Yang, P.D., Zhao, D.Y.,
Stucky, G.D.,Ying, J.Y. Langmuir, 2000, 16, 8291

Blin, J.L., Su, B.L. Langmuir, 2002, 18, 5303

Jana, S.K., Nishida, R., Shindo, K., Kugita, T.,Namba, S. Microporous
Mesoporous Mater. 2004, 68, 133

Park, 8.6., Guo, W.P., Cui, X.G., Park, J.Y., Ha, C.S. Microporous
Mesoporous Mater, 2004, 68, 133

Hanrahan, J.P. Copley, M.P., Ryan, K.M., Spalding, T.R., Morris, M.A.,
Holmes, J.D. Chem. Mater. 2004 16, 424

Prouzet, E., Pinnavaia, T.J. Angew. Chem. Int. Ed. Engl. 1997, 35, 5, 516
Pauly, T.R., Pinnavaia, T.J. Chem Mater. 2001, 13, 987-993

Patarin, J., Lebeau, B. Zana, R. Curr. Opin. Colloid Interface Sci. 2002, 7,
107

Chen, X.Y., Huang, L.M. Li, 0.2. J. Phys. Chem. B. 1997, 101, 8460

Di Renzo, R., Testa, F., Chen, J.D., Cambon, H., Galarneau, A. Plee, D.,
Fajula, F. Microporous Mesoporous Mater. 1999, 121, 8835

Nanoparticles and Nanostructured Films (Ed: J.H. Fendler), Wiley-VCH,
Weinheim, 1998

Park, M., Harrison, 0., Chaikin, P.M. Register, R.A., Adamson, D.H.
Science, 1997, 276, 1401

Metal Nanoparticles (Eds: Feldheim, P.M., Foss, C.A. Jr. Marcel Dekker,
New York 2002

Cheng, J.Y., Ross, C.A., Chan, V.Z.H., Thomas, E.L. Lammertink, R.G.H.
Vancso, G.J. Adv. Mater. 2001, 13, 1147

Spatz, J.P., Chan, V.Z.H., Mobmer, S. Moller, M. Herzog, T., Krieger, M.,
Boyen, H.G., Ziemann, P. Langmuir, 2000, 14, 407

Spatz, J.P., Chan, V.H.Z., Mobmer, S., Kamm, F.M., Plettl, A., Ziemann,
P., Moller, M. Adv. Mater. 2002, 14, 1827

Zhou, Y., Antonietti, M. Adv. Mater. 2003, 15, 17, 1452-1455
Bagshaw, S.A., Hayman, A.R. Chem. Commun. 2000 533
Bagshaw, S.A., Hayman, A.R. Adv. Mater. 2001, 13, 1011-1013

117

39.
40.
41.
42.
43.

44.

Sun, T. Wong, M.S., Ying, J.T. Chem Commun. 2000, 2057
Yuan, Z.Y., Zhou, W., Peng, L.M. Chem. Lett. 2000, 1150
Zhou, Y., Antonietti, M. Chem. Mater. 2004, 16, 544-550
White, J., Edler Chem. Mater. 1997, 9, 1226

Pauly, T.R., Liu, Y., Pinnavaia, T.J., Billinge, S.J.L., Rieker, T.P. J. Am.
Chem. Soc. 1999, 121, 8835-8842

ller, R.K. The Chemistry of Silica. 1979. John Wiley & Sons. New York

118

Chapter 3. Encapsulation of Polymeric Neutral Amine Surfactant on
Mesoporous Silica

3.1 Introduction to Mesoporous Silica Functionalization

The advent of mesoporous materials opened new possibilities for the
catalysis of compounds that large for processing over traditional zeolite catalysts.
In order to expand the usefulness of high surface area mesoporous silica,
organic functionality must be introduced. Catalysis and environmental
remediation are two areas where functionalized mesostructures are employed.
The incorporation of the appropriate functional group on the pore walls tunes the
mesoporous silica for a particular application. For instance, the introduction of
amine functionality into the mesoporous framework pores can allow for arsenate
adsorption1:2 or as a basic catalyst.3.4 Thiol functionalization makes the
mesoporous silica suitable for mercury adsorption.515 In addition, the fields of
protein immobilization}9 drug delivery systems‘o'12 and chiral catalysis13‘14
also are probable applications for functionalized mesoporous silica materials.
Aside from their inherently large pores, mesoporous silicas have the advantage
of greater catalytic activity and stability than modified silica gels.15 The two most
common methods of introducing functionality into mesoporous silica are post-

synthesis grafting and direct assembly (co-condensation)

119

3.1.1 Post-Synthesis Grafting Technique

Post-synthesis grafting is a method of functionalizing silica where in the
organic moieties are anchored through coupling to the hydroxyl groups on the
surface of the incompletely condensed silica. Figure 3.1 illustrates the post-
synthesis grafting technique. Beck and co-workers were the first to employ this
technique using chloromethylsilane as a coupling agent.16 Since then, this
approach has been used to add a variety of organic functionalities to MCM-41,6
SBA-1517 and HMS2 wormhole silicas. Mesostructural cellular foams (MCF) are
attractive for the immobilization of bulky molecules such as enzymes and the
separation of proteins. The large pore size in the 30 nm range enables the
accommodation of bulky functional groups for separation and reactions of large
molecules. MCF’s have been functionalized by a two-step grafting approach for
protein separation and the immobilization enzymes like chloroperoxidase.18-19

Post-synthesis grafting method has several drawbacks. The lack of control
over the distribution of silanol groups on the surface of the silica can yield islands
of functional groups that lead to limited loading.6 Also for calcined silicas, the
silica has to be rehydrated to regenerate the surface silanol groups that have
been lost during the calcinations process. This adds another step to the process.
This grafting method also is limited by the possibility of the organofunctionalized
silica condensing onto itself which can lead to pore blockage.20 Furthermore, the
surface area and the framework pore volume can be compromised upon organic
modification by post-synthesis grafting.1’21'23 Concomitantly, as the organic

loading is increased, the more pronounced is the decrease in surface area and

120

framework pore volume. Also, the accessibility of the grafted derivatives typically

is less than that of derivatives made by directly assembled material.

3.1.2 Direct Assembly I Co-Condensation

As shown in Figure 3.2, direct Assembly is a method of functionalizing
mesoporous silica wherein the organosilane co-condense with the inorganic
silica precursor in the initial reaction mixture. The resulting products have the

anhydrous formula (Si02)1_X (LSiO1.5)x , where L is the organic moiety and x is

the fraction of the framework silicon centers that is functionalized. Thus the
organic moiety is covalently bonded within the structure through the non-
hydrolyzable Si-C bond. The advantage of this approach in comparison to the
grafting method is that it affords greater loading for the organic functional group,
fewer steps in the overall synthesis and a more homogeneous distribution of the
organic functional groups. Until recently, direct-assembly method has been
limited by the miscibility of the organosilanes and TEOS. This process has been
extended to the use of sodium silicate as the inorganic silica source.“-25
Functionalities such as mercaptopropyl, aminopropyl, cyanopropyl and

calyx[8]arene moiety have been incorporated into mesoporous silica using direct

assembly approach.25.27

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The most commonly seen challenge in using the direct-assembly
approach is the incorporation of the organic functional group into the wall so that
the organogroup is oriented toward the pore surface and not on the pore wall
making it inaccessible. Overall, direct assembly is the preferred method for
organic incorporation, except for amines. The difficulty of incorporating amines
using this approach is its reactivity. Under acidic conditions, the amine gets
protonated and can interact with the silanol groups thus disrupting the interaction
of the silane with the surfactant. Another problem with the basic nature of amines
is that they can contribute to local hydrolysis that can result to the collapse of the
mesostructure.

Stein and co-workers21 examined the stability and reactivity of vinyl group
incorporated by direct assembly as opposed to grafting onto MOM-41. The use of
the grafting technique afforded products with greater hydrothermal stability but

the distribution of vinyl groups was not uniform. The grafting process allowed the
hexagonal long range order of the material to be retained but directly assembled
materials generally lose their long range order above 20% loading of the
functionalized silane.

Mercier and Pinnavaia28 also compared various organo-functionalized
mesoporous wormhole silica prepare by direct assembly and grafting.
Mesoporous wormhole silicas prepared by direct assembly method exhibited
higher surface area and pore volumes. It was also observe that the directly
assembled products displayed a more uniform distribution of functional groups in

the pore channels as opposed to grafted mesoporous wormhole silica, as judged

124

by the narrower pore distributions from the Horvath-Kawazoe method of pore

distribution.

3.1 .3 Polymerization of Surfactants

Although Freedman and co-workers29 synthesized potassium styryl
undecanoate and studied the ‘poly soap’ properties of its copolymers in 1958, the
first premeditated and attempted micellar polymerization of an amphiphilic
monomer reported in open literature appears to be the work of Hyde and Robb.30
In their work, they synthesized undecenyltrimethylammonium bromide and
undecenyl sulfate and polymerized these and sodium oleate above their critical
micelle concentration (CMC) in order to produce stabilized micelles. Larrabee
and Sprague31 in 1979 reported micelle polymerization using y-irradiated
aqueous micelles of sodium 10-undecanoate. They confirmed their observation
by the disappearance of the vinyl signals in the 1l-l-NMR spectrum. Further

studies on the polymerization of sodium 10-undecenoate were done by Paleos
and co-workers,32 wherein they demonstrated using electrical conductivity
measurements that the “polymerized micelle’ exhibited a CMC equal to zero.

The polymerization of allyldimethyldodecylammonium bromide under bulk
and micellar conditions has been reported by Paleos and co-workers.33'34
Photochemical and thermal polymerization in quantitative yield was reported, but
this result was not reproduced by the groups of Rodriguez35 and McGrath.35 In

all of these reports, the polymerization has been carried out above the CMC.

125

Emulsion polymerization is an area where polymerizable surfactants or
surfmers (surfactant monomers) are used. Studies have shown that incorporating
a polar group such as a surfactant molecule on the surface of the latex particle
adds stability to the emulsion and helps prevent the coagulation of the emulsion
upon aging. Polymerization of alkyl methacrylate,41'43 acrylate“3 and maleate44'
45 surfactants has also been studied. Surfmers have been used to make
polymeric47 and barium sulfate‘f8 nanoparticles and as a possible drug delivery
system.49

Sherrington published several papers describing the synthesis and
characterization of mono and divalent quaternary cationic surfmers.37‘40 He
questioned the report of ‘polymerized micelles’ by Paleos, saying that the term
insinuates topochemical polymerization, that is, the linking the monomers in such
manner that the final polymer mirrors the parent micelle. He argued that micellar
systems are dynamic systems. There exists a rapid exchange of surfactant

molecules between micelles that occurs in the time scale range of 10'5-10'95 and
the complete breaking and reforming of the micelle occurs on the average every
10'3-10'23. Because the average rate of chain propagation during polymerization
is 10'5-10'23, the rate of surfactant exchange between micelles should be faster
than polymerization, leading to the belief that topochemical polymerization is
extremely unlikely.33v 5052 Instead, he proposed a more accurate model based

on the formation of oligomeric species that exhibit micellar properties,38 as

shown in Figure 3.3.

126

The influence of monomer reactivity and the position of the polymerizable
group within the surfactant on the nature of the polymerized species has been
emphasized. Surfmers can be classified as T-type if the polymerizable group is

located at the hydrophobic tail or H-type if the polymerizable group is located on

the hydrophilic end of the surfactant. 51

I
..
.A‘ . I
O. .

 

Figure 3.3 Two dimensional, highly idealized polymeric micelle with
polymerizable group on the hydrophobic tail (A) and on the hydrophilic head (B);
C-D: Sherrington's concept of a polymerized micelle with polymerizable group on
the hydrophobic tail (C) and on the hydrophilic head (D).

It also was observed that polymerization for T-type surfmers was faster than for
H-type surfmers. This observation was readily explained by the higher
concentration of the methacrylate groups in the interior of the micelle for a T-type

surfmer and by the electrostatic repulsions between adjacent headgroups which

retards the propagation rate of the H-type surfmer.

127

3.1.4 Polymerization In Mesoporous Silica

Several studies have been reported regarding the encapsulation of
organic polymers inside mesoporous silica materials. Polyaniline polymerization
inside MCM-41 has been reported by Bein and co-workers.17 Such systems,
wherein conjugated polymers with mobile charge carriers are confined within
nanometer-size galleries, can be a considered a significant step toward
designing nanoscale electronic devices.

Moler and co-workers studied the polymerization of methyl methacrylate
inside microporous and mesoporous silica53. It was observed that the resulting
polymer has distinct properties compared with the bulk polymer. Tolbert and co-
workers54 showed that semiconducting polymers aligned inside mesopores
exhibit unique energy transfer and photophysical properties that can be use for
developing electronic and optoelectronic devices. Dendrimers55 and polystyrene
nanofibersfl28 have also been synthesized in the pores of mesoporous silica. In
all these reports, the polymer completely fills the mesopores, resulting in a non-

porous material.

In a recent paper by Ryoo and co—workers56 a new way a new way was
shown for functionalizing mesoporous silica. This involved polymerizing
poly(chloromethylstyrene) in SBA-15 while keeping the mesopores open. It was
noted that the micropores of SBA-15 play a crucial role in restricting the polymer
layer to the mesopore surface and in stabilizing the entire polymer network. The

poly(chloromethylstyrene) polymer inside the mesoporous is a good starting

128

material for transformation into other functional groups suited for ion exchange,

catalysis and protein immobilization.

3.1.5 Heavy Metal Trapping Using Mesoporous Silica
Heavy metals in water can be harmful to ones health. This has been
enough for researchers to engage in extensive research for trapping metals from

water. Traditional methods of trapping metals include the use of activated

carbon, zeolites, silica gel, sulfide precipitates, ion exchange resins and clays.58
The low loading capacity and poor selectivity have impeded the use of these
materials for remediation purposes. Mesoporous materials overcome these
factors, which makes them a very good candidates for trapping metals. The

metals that were previously studied were arsenic,58 copper,59 zinc,59

mercury,5:53 palladium,50v61 platinum,50:61 cadmium51 and nickel.51 The amine
functional groups were the best for trapping copper ions. The thiol functional
group has a high affinity for trapping mercury in aqueous media. Radioactive
element trapping has also been demonstrated for mesoporous silica. One ligand
that is known to bind with an actinide cation is carbamoylphosphine oxide

(CMPO). The CMPO MCM-41 have been shown by Fryxell et al to be highly

efficient and selective in binding plutonium62

129

3.2 Objectives

In Chapter 2, two oleyl surfactants, namely oleyl-NH2 (Armeen OLD) and
oleyl-NH(CH2)3NH2 (Duomeen O) were identified as potential candidates for

trapping lead. But since Duomeen O is capable of chelating metal ions, the
efforts were concentrated on using this templating agent for the synthesis of
mesoporous silica for lead trapping. The extra amino group suggests greater
trapping capacity and the formation of a more stable coordination compound due
to chelate effect.

For this part of the study, mesoporous silica derivatives are prepared
using the Duomeen O surfactant and divinyl benzene (DVB) as a cross-linking
agent. An effort was made to incorporate the maximum amount of polymeric
complexant inside the framework structure in order to optimize the trapping
capacity. The amount of the encapsulated complexant was determined
quantitatively, as well as its resistance to leaching. Finally, the cross-linked

products were evaluated for their ability to trap lead ions in solution.

3.3 Experimental

3.3.1 Co-Polymerization of the Templating Agent Inside HMS Wormhole
Mesostructures with Divinylbenzene as the Cross-linker

Initial attempts to polymerize Duomeen O using azobisisobutyronitrile
(AIBN) as initiator showed no success. The internal double bond in the

oleylamine chain is sterically hindered and its polymerization by a radical

130

pathway is limited. This was circumvented by adding a crosslinking agent, divinyl
benzene to facilitate the polymerization process.

Initial polymerization attempts were done by varying the mole ratio of
oleylamine surfactant to divinylbenzene, DVB over the range of 1.0201 to 1.0-
0.5). The highest yield of copolymer was obtained at the ratio of 1205. Thus, this
ratio was used for polymerization within the framework walls of the
mesostructure. The addition of DVB also raised the question if the surfactant will
still be capable of templating mesostructured silica. This was addressed by the
hypothesis that DVB will behave like trimethylbenzene (TMB) as a co-surfactant
when added to the reaction mixture. TMB still permit the formation of
mesostructures although with expanded pores.

The incorporation of a polymeric templating agent inside the framework
pores of mesostructured silica can be approached in two ways. As can be seen
on Figure 3.4, the template can be polymerized prior to the formation of
mesostructure (pre—assembly polymerization) or the mesostructure can first be

assembled and the template polymerized later (post-assembly polymerization).

131

Polymerizable surfactant

Co—Polymerize Surfactant in Make a Mesostructure
solution 1 1
Make a Mesostructure Polymerize Surfactant

in mesopores

l 1

Metal Trapping Metal Trapping
Scheme 1 Scheme 2
Pre-Assembly Polymerization Post-Assembly Polymerization

Figure 3.4 Two possible schemes on incorporating a polymeric complexant
inside mesoporous silica materials.
Scheme 1 Pre-Assembly Polymerization

The oleylamine surfactant (0.013mol) was added the desired mixture of
ethanol and water (see to Table 3.1) in a round bottom flask. Then the divinyl
benzene (DVB, 0.87g, 0.007mol) was added followed by the addition of the
initiator. This reaction mixture was stirred until all the AIBN (0.0349, 0.0002mol)
was completely dispersed. The mixture was then heated to 90°C for 3 hours.
After the polymerization, the resulting product was cooled to room temperature.
TEOS was then added and the reaction mixture was stirred for 15 hours. The
reaction products were separated from the mother liquor by filtration followed by

washing with copious amount of deionized water. The mesoporous silica

132

products were then air dried. Some of the sample was calcined at 620°C for 4

hours for TEM analysis.

Scheme 2 Post-Assembly Polymerization

The oleylamine surfactant (0.013mol) was added into a mixture of ethanol
and water (see to Table 3.1) in a polypropylene bottle. Then the divinyl benzene
(DVB, 0.87g, 0.007mol) was added, followed by the addition of the initiator. This
reaction mixture was stirred until all the AIBN (0.034g, 0.0002mol) was
completely dispersed. TEOS was then added and the reaction mixture was
stirred for 15 hours at room temperature. The reaction products were separated
from the mother liquor by filtration. The DVB was expected to reside inside the
micelle template of the as-made mesostructure. Copolymerization with the
surfactant was achieved by placing the freshly filtered product in a 90°C oven for

2 hours.

Table 3.1. Wormhole Silica Products Synthesized in 60 volume % Ethanol Media.

 

Mass of Mass of Mass of Volume of Volume of
Surfactant DVB Initiator H20 Ethanol
Oleyl-

NH(CH2)3NH2 4.369 0.879 0.0349 22.4mL 33.6mL

Mole Ratio: 1 SiOz : 0.27 Surfactant: 0.135 DVB
Initiator: AIBN @ 1mo| °/o of Surfactant + DVB

133

3.3.2 Leaching Studies

One of the goals of this study is to synthesize a mesoporous silica
material with an entrapped polymeric complexant that also serve as a structure-
directing agent for mesoporous silica. The polymeric complexant has to be
resistant to Ieeching from the framework pores. The use of the interconnected
framework pores of a wormhole structure is considered to be a good approach to
preventing the polymeric complexant from Ieeching since a polymer can get
entangled within the 3D framework pore network.

To examine whether the polymer inside the mesoporous wormhole resists
leaching, the material was subjected to two types of extraction experiments using
ethanol and water as solvents for extraction. For the water extraction experiment,
0.4 g of the as-made mesoporous silica-copolymerized surfactant composite was
added to 200 mL of deionized water was added. This mixture then was stirred
overnight. The composite was separated from water through filtration and air-
dried. The pore volumes of the products before and after water extraction were
compared by thermal gravimetric analysis.

For the ethanol extraction experiments, 1.0 g of the mesoporous silica was
measured into a 350 mL round bottom flask. Absolute ethanol (150 mL) was
added and this mixture was refluxed for 15 hours. The silica was separated from
ethanol by filtration and air-dried. The pore volumes of the products before and

after ethanol extractions were compared by nitrogen adsorption isotherms.

134

3.3.3 Lead (ll) Trapping by Batch Studies
Preparation of Standards
A 500 ppm, 1 L lead stock solution was prepared by dissolving 0.64 g of

Pb(N03)2 (99.999 %) in water. From this solution, aliquots of Pb(ll) were taken to

prepare the standards for the calibration curve for the analysis through atomic
absorption spectroscopy. Standard solutions were prepared with lead

concentrations in the range of 1-100 ppm Pb(|l).

Trapping Pb(ll) in Water using Mesoporous Silica with Encapsulated Polymeric
Complexant

Approximately 0.1g mesoporous silica composite was measured into
250mL polypropylene bottles. Then, a 100mL of a 100 ppm aliquot of Pb(ll)
solution was added. This mixture was then allowed to equilibrate. with stirring at
room temperature for 15 hours. The mesoporous silica was separated from
solution by filtration and the filtrate was analyzed by atomic absorption

spectroscopy.

3.3.4 Characterization

The products were characterized using powder X-ray diffraction (XRD)
and nitrogen adsorption-desorption isotherms. The powder X-ray diffraction
(XRD) patterns were obtained using a Rigaku Rotaflex Diffractometer with CuKo
radiation (A = 0.154nm). Counts were accumulated every 0.02 degree (20) at a

scan speed 0.5 degree per minute.

135

N2 adsorption—desorption isotherms were obtained at -196°C on a
Micromeritics Tristar 3000 sorptometer. Samples were outgassed under vacuum
at 100°C for 16 hours. BET surface areas were calculated from the linear part of
the BET plot according to IUPAC recommendations.

Transmission electron microscopy (TEM) images were obtained on a
JEOL 2200FS with an accelerating voltage of 200kV. Samples were prepared by
sonicating powdered samples in ethanol for 15 minutes and evaporating two
drops of the suspension onto a carbon coated film supported on a 3 mm, 300
mesh copper grid.

Atomic absorption spectroscopy was employed to determine the lead
trapped in mesoporous silica by determining the concentration of Pb(ll) left in the
filtrate. The difference in the initial and final concentration of Pb(ll) in the filtrate
provided the amount of Pb(ll) trapped. Pb(ll) was analyzed using a Varian
Spectra AA 200 Model in flame absorption mode at 283.3 nm wavelength.

Thermal gravimetric analysis was done using a therrnobalance (l' GA 7, ‘
Perkin-Elmer) coupled with a thermal analysis controller (TAC 7/DX, Perkin-
Elmer) was employed. The instrument was calibrated with “alumel” alloy and
nickel for temperature settings and with a 100-mg standard for weight accuracy.
Approximately 5 mg of each sample were added to a tared aluminum balance

pan. The pan was then placed in the furnace at room temperature, and the exact
sample weight was determined. The temperature was then increased to 100 °C
at the rate of 10 °C min-1, and the sample was held at this temperature for 15

min to remove any residual water. Thereafter, the sample was heated to 800 °C

136

at the rate of 10 °C min-1. Air was chosen as the sample purge gas (flow of 50

cm3 min-1), whereas N2 was used as balance purge gas at the flow rate of 75

cm3 min-1.

3.4 Results and Discussions
Synthesis and Leeching Studies

As mentioned earlier, the neat oleylamine surfactant does not polymerize
due to steric limitations. The addition DVB as a crosslinker solved this problem,
but it also raised the issue of whether the surfactant will still be able to template a
mesostructured silica. The initial goal was to evaluate the mesoporous silica
templated by the crosslinked surfactant. Another goal was to attempt the
crosslinking of the surfactant in the as-made mesostructure.

Figure 3.5 shows the low angle powder x-ray diffraction of a
mesostructured silica obtained by pre- and post-assembly crosslinking of the
oleyl surfactant. The diffraction peaks show the pore-pore correlation typically
seen for a mesostructure with a wormhole framework structure. The product
obtained by post-assembly crosslinking shows a diffraction peak typical of
wormhole pore structure. On the otherhand, the material obtained through pre-
assembly surfactant cross-linking suggests the presence of layered structure
based on the sharpness of the diffraction peak. TEM images (Figure 3.6 and 3.7)
show the pore structures of the two products. The TEM images also revealed the

suspected layered structure of the materials. The structure of the silica products

137

synthesized is a combination of wormhole and layered structure similar to the

previously synthesized silica without DVB.

Low Angle Powder X-ray Diffraction

OlGYI-NH(CH2)3NH2

  
   

Pre-Assembly
d=53nm

Relative Intensity

 

Post-Assembly
d=49nm

  

 

 

 

28

Figure 3.5 Low angle powder x-ray diffraction pattern of crosslinked oleyl
surfactant in mesostructured silicas made by pre-assembly and post-assembly
crosslinking of the surfactant porogen.

138

 

Figure 3.6 Representative TEM images mesostructured silica assembled in the
presence of oleyl surfactant cross-linked by divinylbenzene prior to the addition
of TEOS (pre-assembly crosslinking).

139

 

Figure 3.7 Representative TEM images calcined mesostructured silica aeembled
in the presence of oleylamine surfactant followed by crosslinklng of the surfactant
by divinylbenzene.

140

The formation of mesostructured silica from a divinylbenzene crosslinked
oleyl surfactant is a bit counterintuitive. Sherrington described a polymerized
micelle as being irregularly shaped. For a surfactant template to give rise to a
narrow and uniform pore distribution, the surfactant micelle should be regularly
shaped. The representative nitrogen adsorption isotherms and BJH pore
distributions for the mesoporous silica assembled by polymerizing the surfactant
template prior to the addition of TEOS are shown on Figure 3.8. Based on the
BJH pore distribution plots, the narrow pore size distribution suggest a template
that is uniform in size and shape. This suggests that the DVB crosslinked micelle
must have preserved the topology of the micelle before polymerization. The
kinetics of radical polymerization and micelle formation suggests that the
polymerized micelle could not have preserved the spherical shape or its initial
micellar shape. But the presence of the hydrating solvent may forced the micelle
to adapt a shape other than the irregularly shape polymer that will minimize
energy and maximize entropy in the mixture. It is plausible that the crosslinked
micelle re-adapts a spherical or its original micellar shape to minimize its energy
and maximize the entropy. This in turn permits the crosslinked surfactant to
template a mesostructured silica with a very uniform pore distribution.

After evaluating the fidelity of the mesostructure silica produced, we step
evaluated whether the crosslinked template could be leached out of the
framework pores by ethanol extraction. This is best evaluated by examining the

nitrogen adsorption isotherms in Figure 3.8 and 3.9.

141

 

 

(O
E
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2
2 600 ~
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J

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0.6-
D
g A = Calcined product
g ()4. B = Ethanol extracted

product

  

 

 

2 4 ' 6 8 1o
Framework Pore Size (BJH), nm

Figure 3.8 (Top) Nitrogen adsorption isotherms and (Bottom) BJH pore size
distributions for non-crosslinked, calcined and ethanol extracted silicas made by
pre-assembly DVB crosslinking of the surfactant.

142

1000-

800 -

    
   
 

600 -

Ethanol etracted
product

 

 

0.0 ' 0:2 T 04 ' 0:6 ' 0:8 ' 1:0
Relative Pressure, P/Po

Volume of Adsorbed Nitrogen, cm3

1 .0 1
, Ethanol extracted

product
0.8 - non-crosslinked

0'6“ Calcined product

 

dV/dD
O
1‘

 

\\\\

 

2 ' 4 6 8 .- 10
Framework Pore Size (BJH), nm

Figure 3.9 (Top) Nitrogen adsorption isotherms and (Bottom) BJH pore
distributions for non-crosslinked, calcined and ethanol extracted silicas made by
post-assembly DVB crosslinking of the surfactant.

143

Table 3.2 Summary of pore properties of calcined and extracted silica products
assembled with crosslinked surfactant templates

 

 

 

 

 

 

Framework
. Average Wall BET Pore Volume,
. Calcrned Pore .
Material . . Thickness, Surface mL/g
d-spacrng, Diameter, 2
nm Area, In lg (taken @ P/Po
nm nm
= 0.75)
Non-
polymerized
Duomeen O 4.7 3.7 1.0 892.1 1.12
template
Calcined
Pre-
Assembly 5.3 3.1 2.2 642.3 0.58
Crosslinked
Extracted
Pre-
Assembly 5.3 3.0 1.6 198.5 0.31
Crosslinked
Calcined
Post-
Assembly 4.9 3.3 1.6 948.1 1.20
Crosslinked
Extracted
Post
Assembly 4.9 3.2 1.7 671.2 0.70
Crosslinked

 

 

 

 

 

 

 

 

Ideally, non-porous materials are desired after ethanol extraction of the
as-made products because this would mean that the polymerized surfactant
resisted the Ieeching process. The nitrogen adsorption isotherms show that the
ethanol extracted products are porous which means that a substantial fraction of
the crosslinked surfactant leeched out. On the basis of comparison of the
nitrogen adsorption isotherms for the calcined and ethanol extracted materials
synthesized by post-assembly crosslinking (Figure 3.8), there is a significant
decrease in the volume of adsorbed nitrogen for the extracted sample compared
to the calcined sample. Moreover, the partial pressure for mesopore adsorption
occurs is about the same partial pressure for both products. This suggests that

both products have about the same framework pore diameters. This also means

144

that the leaching process does not ocCur uniformly throughout the mesostructure.
The crosslinked surfactant near the periphery of the mesoporous silica particle is
extracted first and the crosslinked surfactant within the inner parts of the particle
is retained. This explains why the partial pressure for the mesopore filling is not
shifted after ethanol extraction.

Figure 3.9 also showed the same trend. Typically, an ethanol extracted
mesophase would exhibit the same adsorption isotherm as with the calcined
material. A single ethanol extraction is sufficient enough to remove most of the
templating agent. The fact that the synthesized material with the polymerized
template partially resisted complete extraction proves that the crosslinking of the
surfactant template was successful. The partial extraction in ethanol more likely
is due to the solubility of the polymerized surfactant in ethanol. The pore and
surface area analysis are summarized in Table 3.2.

Comparing the nitrogen isotherms for the silicas with non-crosslinked
surfactant template with the silicas with crosslinked templates, the silicas whose
surfactant template was cross-linked after assembly (post-assembly

crosslinking), the N2 isotherm resembles that of the non-crosslinked product with

a slightly higher framework pore volume as evidenced by the slightly volume of
adsorbed nitrogen. This is explained by the expansion of the pores when DVB is
added. The silicas whose surfactant template was crosslinked prior to the
addition of TEOS (pre-assembly crosslinking) presented a different adsorption
curve. This tells us that the micellar aggregates that templated the mesostructure

is different from the non-crosslinked aggregates.

145

The extent of surfactant extraction using water was evaluated using TGA.
The results for this experiment are shown in Figure 3.10. From the TGA plots, the
as—made silica assembled from unpolymerized surfactant (A) contains about 55%
organic surfactant. The extent of extraction by water is shown by curve B where
the organic content of the water extracted product is about 10%, indicating that
more than 80% of the surfactant template is extracted by water. C and D in
Figure 10 represent the TGA plots for silica made by the pre-assembly
crosslinking of the surfactant. The plots show that the material did not lose a
significant amount of the weight and the TGA results reveal that there is about
10% increase in the organic composition of the material after water extraction.
This slight increase in the percentage of organic component can be explained by
the slight dissolution of some of the silica when the water extraction was
conducted. This also demonstrates that the polymerized surfactant is not soluble

in water.

Batch Studies of Pb(ll) Uptake from Water

Lead exposure is hazardous to health. The EPA has set the levels for
drinking water at zero mg/L though the maximum contaminant level for treatment
technique was set at 0.015 mg/L (15ppb). Prolonged exposure to lead among
infants can cause delays in physical or mental development and among children

it can cause deficits in attention span and learning disabilities. Among adults,

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Pb2+ can cause kidney problems and high blood pressure. Figure 3.11 shows
the pH dependence of lead species in water at a concentration of 0.01M. From
the species distribution, it is inferred that lead adsorption experiments should be
done at pH below 8 where most of the lead is in cationic form andsuitable for
complexation.

Table 3.3 presents representative results for Pb(ll) trapping by crosslinked
oleyl amine surfactants encapsulated in mesoporous silicas. As-made and
ethanol extracted mesoporous silica products with pre- and post-assembly
crosslinked surfactant reduced the concentration of 100 ppm Pb(ll) in water. The
silica with post-assembly crosslinked template trapped the most Pb(ll) with
capacity of 0.4 mmol Pb(|l)/g of as-made material while the silica with pre-
assembly crosslinked template gave a lower amount of Pb(ll) trapping capacity of
0.36 mmol Pb(|l)/g of as-made product. The equilibrium N:Pb ratio revealed that
the post-assembly crosslinked product was more efficient in trapping lead with
ratio of N:Pb ratio of about 6.00 while the pre-assembly polymerized product has
a N:Pb ratio of 7.54. The extracted counterparts of the as-made silica products
gave lower trapping capacity which is expected since both have less surfactant
content.

Figure 3.12 presents the curves for the uptake of Pb(ll) under batch
equilibrium conditions. The uptakes curves show that the as-made silica product
containing pre-assembly crosslinked template has a capacity of trapped Pb(ll)
about 0.75 mmol per gram as-made silica with a N:Pb ratio of 3.63. The post-

assembly polymerized silica has a lower uptake of about 0.55 mmol Pb(ll) per

148

gram as-made silica with a N:Pb ratio of 3.73. Overall the difference in terms of
the stoichiometry of the N:Pb is not significant for both material. The reason why
there is a difference in the uptake capacity toward Pb(ll) is due to the amount of
encapsulated polymerized template. Pre-assembly crosslinking is slightly more
effective in maximizing the polymeric complexant loading of the mesoporous
silica. A possible explanation of this result lies in the assembly process. For the
pre-assembly crosslinking approach, the template is already crosslinked after the
condensation of the silica. Thus, washing the as-made sample with water after
the removal of the mother liquor does not extract the surfactant template. On the
other hand, in the post-assembly polymerization approach, the as-made silica
products are washed with water after separating them from the mother liquor and
in this step, there is a chance that some of the non-crosslinked surfactant
template is extracted thus decreasing the amount of surfactant template available

for the succeeding crosslinking step.

3.5 Conclusions

The synthesis of mesoporous silica with crosslinked oleyl diamine
template was accomplished. The surfactant template is crosslinked with the use
of DVB. Because internal double bond in the hydrophobic tail of the oleyl
surfactant is sterically hindered, it is difficult for the surfactant to polymerize in
neat. The use of DVB as a cross-linking agent circumvents this limitation. A 120.5
mole ratio of surfactant to DVB was found to be best for the synthesis of

mesoporous silica with encapsulated polymeric complexant. The incorporation of

149

DVB in the as-made mesophase under post assembly crosslinking conditions
occurs through adsorption inside the hydrophobic interior of the micelle.

Two approaches were used for the encapsulation of the surfactant
complexant inside mesostructured silica, namely pre-assembly crosslinking by
DVB and post-assembly DVB crosslinking. The low angle powder x-ray
diffraction showed a single diffraction peak typical of wormhole to lamellar
framework pores for the products obtained using both approaches. The nitrogen
isotherms and the BJH pore size distributions showed well defined framework
pores of ~3.0 nm. TEM images also revealed the wormhole and lamellar
morphology of the mesoporous silica is similar to the silica products assembled
without DVB. Of the two approaches both are effective in preventing the
surfactant template from leaching from of the framework pores during the metal
absorption process. The unpolymerized template of an as-made silica product is
readily leached out into solution as shown by the TGA. The silica product
obtained by pre-assembly polymerization has a slightly higher loading of
polymeric complexant than the other silica product but it provides a much lower
mesopore volume. This slight difference is attributed to the loss of surfactant
during the washing of the as-made product before polymerization. The Pb(ll)
trapping studies have shown that the capacity of the silica product is dependent
amount of complexant present as the ratio of N:Pb(ll) for both silica products is

about the same.

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152

O Post-Assembly Crosslinking of Surfactant

5 _ El Pre—Assembly Crosslinking of Surfactant
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mmol Pb(ll) adsorbed
Figure 3.12 Pb(ll) uptake by mesoporous silica with encapsulated crosslinked
oleylamine surfactant as a complexant. The amount of Pb(ll) added varied from

0.22 to 4.73 mmole Pb(ll) per gram as-made material. The ratio of N : Pb for pre-
and post-assembly polymerization are 3.63 and 3.73 respectively.

153

3.6 References

10.
11.

12.

13.

14.

15.

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Yoshitake, H., Yokoi, T., Tatsumi, T., Chem. Mater. 2002, 14, 4603-4610
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Rao, Y.V.S., De Vos, D.E., Jacobs, P.A. Angew. Chem, Int. Ed. Engl
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Mercier, L., Pinnavaia, T.J. Adv. Mater. 1997, 9, 500

Feng, X., Fryxell, G.E., Wang, L.Q., Kim, A.Y., Liu, J., Kemmer, K.M.
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Takahashi, H., lnagaki, S., Kajino, T., Usuki, A., Li, B. In Eur. Pat. Appl.
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Kulak, A., Hall, S.R., Mann, S. Chem. Commun. 2004, 576-577

Lai, C.Y., Trewyn, B.G., Jeftinja, D.M., Jeftinja, K., Xu, 8., Jeftina, 3., Lin,
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Lin, V.S.Y., Lai, C.Y., Trewyn, B.G., Abstracts of Papers, 225'" ACS
National Meeting, New Orleans, LA, United States, March 23-27, 2003
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Bellocq, N., Abramson, S., Lasperas, M., Brunel, D., Moreau, P.
Tetrahedron-Asymmetry1999, 10 3229-3241

Johnson, B.F.G., Raynor, S.A., Shephard, D.S., Mashmeyer, T., Thomas,
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26.

27.
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31.

32.

33.

34.

Beck, J.S., Vartuli, J.C., Roth, W.J., Leonowicz, M.E., Kresge, C.T.,
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Higgins, J.B., Schlenker, J.L., J. Am. Chem. Soc. 1992, 114, 10834-10843

Morey, M.S., O’Brien, 8., Schwarz, S., Stucky, G.D. Chem. Mater. 2000,
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Han, Y.J., Watson, J.T., Stucky, G.D., Butler, A. J. Mol. Catal. B: Enzym.
2002, 17, 1-8

Schmidt-Winkel, P. Lukens, W.W., Zhao, D.Y., Yang, P.D., Chmelka, B.F.,
Stucky, G.D., J. Am. Chem. Soc. 1999, 121, 254-255

Walcarius, A., Etienne, M., Lebeau, 8., Chem. Mater. 2003, 15, 261-2173

Pauly, T.R., Liu, Y., Pinnavaia, T.J., Billinge, S.J.L., Rieker, T.P. J. Am.
Chem. Soc. 1999, 121, 8835-8842

Lim, M.H., Stein, A. Chem. Mater. 1999, 11 , 3285-3295
Yokoi, T., Yoshitake, H., Tatsumi, T. J. Mater. Chem. 2004, 14, 951-957
Shah, J., Kim, S.S., Pinnavaia, T.J., Chem. Commun. 2004, 572-573

Yu, N. Gong, Y., Wu, D., Sun, Y., Luo, 0., Liu, W., Deng, F. Microporous
and Mesoporous Mater. 2004, 72, 25-32

Liu, C., Naismith, N., Fu, L., Economy, J. Chem. Commun. 2003, 2472-
2473

Liu, C., Lambert, J.B. Polym. Mater. Sci. Eng. 2003, 88, 433-434
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Freedman, H.H.; Mason, J.P.; Medalia, A.|. J. Org. Chem. 1958, 23, 76
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Larrabee, C.E. Jr.; Sprague, E.D. J. Polym. Sci. Polym. Lett. Ed. 1979, 17,
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Paleos, C.M.; Stassinopoulou, C.l.; Milliaris, A. J. Phys. Chem. 1983, 87,
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Paleos, C.M.; Dais, P. Rec. Adv. Liq. Cryst. Polym, 1985, 6, 1985

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155

35.

36.
37.
38.
39.
40.
41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

Rodriguez, J.L.; Soltero, J.F.A.; Puig, J.E.; Schulz, P.C.; Espinoza-
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McGrath, K.M. Drummond, C.J. Colloid Polym. Sci. 1996, 274, 316
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Joynes, D.; Sherrington, D.C. Polymer, 1996, 8, 1453

Joynes, D.; Sherrington, D.C. Polymer, 1997, 38, 1427-1438
Eastoe, J.; Summers, M. Chem. Mater. 2000, 12, 3533-3537

Summers, M.; Eastoe, J.; Davis, S.; Du, Z. Richardson, R.M.; Heenan,
R.K.; Steytler, D.; Grillo, l. Langmuir 2001, 17, 5388-5397

Eastoe, J.; Summers, M. Langmuir 2003, 19, 6357-6362
Guyot, A.; Goux, A. J. Appl. Polym. Sci. 1997, 65, 2289-2296

Zicmanis, A.; Hamaide, T.; Graillat, C. Monnet, C.; Abele, S.; Guyot, A.
Colloid Polym. Sci. 1997, 275, 1-8

Montoya, A.; Sherrington, D.C. Polymer 1999, 40 1067-1079

Herold, M.; Brunner, H.; Tovar, G.E.M. Macromol. Chem. Phys. 2004,
204, 770-778

Summers, M.; Eastoe, J.; Davis, S. Langmuir, 2002, 18, 5023-5026

Scholz, C.’ Iijima, M.; Nagasaki, Y.; Kataoka, K. Polym. Adv. Technol.
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Eastoe, J.; Summers, M. Adv. Coll. Interf. Sci. 2003, 100-102, 137-152
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56.

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McKimmy, Emily Jane. Supramolecular Assembly of Organofunctional
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1043-1049

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Commun. 2002, 1374-1375

157

 

Chapter 4. Phosphoprotein Enrichment Using Grid Supported Mesoporous
Metal Oxide Thin Films

4.1 Introduction to Post-translational Modification

The proteome is the inventory of all the proteins in a cell or organism.
There are two major mechanisms in which the proteome is expanded beyond
that what can be predicted by the genes in eukaryotic organisms. The first route
for diversifying proteins occurs at the transcriptional level through mRNA splicing

including the tissue specific alternate splicing which is the central topic in RNA

metabolism in eukaryotic bilology.1’2 The second route for proteome

diversification and expansion is through covalent post-translational modification

(PTM). These covalent modifications occur after the DNA has been transcribed

into RNA and translated into proteins.3 Hundreds of enzymes are dedicated to

such protein modifications. In general, post-translational modification can occur
either through the covalent addition of a chemical group or by the cleavage of a
protein backbone. (See Figure 4.1)

PTM achieved through covalent addition reaction includes
phosphorylation, acylation, alkylation, glycosylation and oxidation. The resulting
modified proteins comprise the phosphoproteome, acyl proteome, alkyl

proteome, glycoproteome and the oxidized proteome subsets. (See Figure 4.2)

158

x X—YR
+ Y-R —>
.NIW. .NIY
H o H o

Covalent modification

__. “N o + 3
H o

Rn+1

R‘n H O
%N \‘(Nflt‘
H o

Rn+1

Cleavage of Protein Backbone

Figure 4.1 The two general schemes for post-translational modification: covalent
modification and covalent cleavage.

159

 

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Directed post-translational modifications enable the expansion of distinct
covalent forms of proteins beyond that is predicated by DNA encoding. Presently,
the phosphorylation of proteins is the most studied and best understood pathway

among PTMs. It is also the most ubiquitous among the PTMs.

4.1.1 Background on Protein Phosphorylation

Beginning with the isolation of phosphoserine of Levene and Lipmann,
protein phosphorylation has turned out to be one of the most biologically relevant
and ubiquitous post translational protein modifications.5:5 Phosphorylation is a
reversible modification process effecting both the folding and function of proteins
and regulating essential functions such as cell division, signal transduction,
enzymatic activity, cell cycle, cell proliferation and differentiation.5- 7 It has been
estimated that about 30-50% of proteins are phosphorylated at some point 3 and
about 2-3% of the genes in the entire eukaryotic genome are involved in the
phosphorylation process.9 It is further estimated that one-third of all cellular
functions are directly or indirectly affected by protein phosphorylation.11 Among
the amino acids that can be phosphorylated, the ortho-phosphates are the most
abundant, mostly attached to serine, threonine and tyrosine residues.
Phosphorylation of serine and threonine residues is more frequent than on
tyrosine with an approximate ratio of 1800:200:1 for pSer/pThr/pTyr respectively.
Phosphoramidates of arginine, histidine and lysine also occur, as do acyl
derivatives of aspartic and glutamic acid, but they are less abundant”,13 (See

Figure 4.3)

161

l 0'
- : — l
0=P- 0' — - A u
l 0: F: O O N\ N-F:-O’ O
o o- o
%N 1.” an
H o o o
phosphoSer phosphoThr phosphoTyr phosphoHis phosphoAsp
(pS) (M) <le (pHis) (pAsm

Figure 4.3 The phosphorylated amine residues.

The disruption of the phosphorylation and dephosphorylation events involved in
signal transduction pathways has been implicated in diseases such as
cancer,14v15 type II diabetes,1‘5:17 cystic fibrosis18 and Alzheimer’s disease.19v20

Given the importance of protein phosphorylation in cell function, one must
conclude that the characterization of the phosphoproteome will be the key to
understanding the mechanism of cellular processes and in identifying targets for
therapeutic intervention.10 The great interest in acquiring a complete knowledge
of phosphorylation mechanisms also is due to the fact that more than a hundred
different protein kinases have been implicated in human cancer.21 It is therefore
not surprising that protein kinases have emerged as the most studied class of
new drug targets in oncology and other disease areas.22 Approximately 100,000
potential phosphorylation sites are present in the human proteome and currently
very little is known about them. This emphasizes the need for sensitive, high-

throughput methods to identify, characterize and monitor new sites of protein

phosphorylation.13

162

Traditional methods have been long employed in studying and detecting
phosphopeptides such as gel electrophoresis and radiolabeling of
phosphopeptides or proteins-23242549 The use of these methods is well
documented but they do require a large initial amount of protein samples. The
advancement of technology in mass spectrometry has greatly improved the study
of the phosphoproteome. Mass spectrometry when combined with
phosphoprotein enrichment methods is the analytical tool of choice given its
speed and sensitivity.5»23:25

Even with the development of mass spectrometry, proteomic techniques
relevant to the elucidation of signal transduction have lagged in deveIOpment
when compared to genomic technologies. The challenge is due to the complexity
of cellular samples and the low stoichiometry of the phosphoproteins when
compared to the non-phosphorylated counterparts. This makes it necessary to
develop efficient enrichment technologies for studying the phosphoproteome at
the cellular level. Through enrichment technologies, the complexity of cellular
samples is reduced.

Phosphoproteomics focuses on the analysis of protein phosphorylation

and makes use of mass spectrometry (MS) and specific methods to purify

phosphorylated proteins and peptides.30“"3 However, the use of mass
spectrometry in analyzing the phosphoproteome is far from becoming a routine.
Aside from the inherent problem of substoichiometric phosphorylation, three
other reasons as to why phosphorylation analysis is so problematic are often

quoted, including increased hydrophilicity of the phosphopeptide with a

163

concomitant loss during the loading onto reversed-phase columns, selective
suppression of their ionization/detection efficiencies in the presence of large
amounts of unphosphorylated peptides, and lower detection efficiencies of
phosphopeptides as compared with their unphosphorylated cognates.
Phosphopeptides are often lost during loading onto the reversed-phase
columns because the addition of anionic/acidic phosphate groups increase the

hydrophilicity resulting in reduce retention. This is one reason why LC/MS failed.

Steen and co-workers36 hypothesized that phosphorylation indeed increases
hydrophilicity. However if the peptide contains basic amino acid residues that are
positively charged under standard LC/MS conditions, the increase in
hydrophilicity can be overcompensated by charge neutralization. The presence of
a phosphate group decreases hydrophilicity thus increasing the LC retention
time as long as the positive net charge decreases but once the phosphate
groups exceeds the number of basic residues, the hydrophilicity increases again.
The same group also tested the notion of phosphorylated species being
selectively suppressed in the presence of unmodified peptides. They found that
there is no significant change in the signal intensity of the phosphorylated peptide
in the presence of a 100-fold excess of non-phosphorylated peptides. To test
further peptide/phosphopeptide pairs, samples were spiked with 1000-fold
excess of tryptic bovine serum albumin (BSA) digest. As expected, the signal
intensity of the peptides and phosphopeptides were significantly reduced but no
selective suppression was observed. The common belief that phosphopeptides

showed lower ionization/detection efficiencies that their non-phosphorylated

164

cognates could not be substantiated as a general fact. When testing the
peptide/phosphopeptide pairs using liquid chromatography in combination with
electro spray ionization — mass spectrometry (LC/ESl-MS), the majority of the
phosphopeptides tested actually showed better ionization/detection than their
unmodified cognates. This indicates that LC combined with ESl-MS is a good
choice for routine phosphorylation analyses. F

Despite the recent advancements in mass spectrometry, lability of the

phosphate group under mass spectrometry conditions can lead to dominant

 

neutral losses of H3PO4 (98 Da) and HPO3 (80 Da) in MS and MS/MS analyses

makes peptide sequencing and phosphorylation site assignment more

difficult.40""1

4.1.2 Enrichment of Phosphoproteins

The major challenge for the application of phosphoproteomics lies within
successfully isolating phosphoproteins from the whole cell lysate with a focus on
identification of low abundant phosphoproteins. To date, several strategies have
been developed to enrich samples for phosphopeptides and to remove non-

phosphorylated acidic peptides.

Immunoprecipitation
Immunoprecipitation utilizes antibodies specific to phosphorylated
residues."'4s45 Antibodies specific to phosphorylated residues are used to

immunoprecipitate total protein rather than phosphopeptides.46 The

165

immunoprecipitation of phosphotyrosine containing proteins is more frequent
than immunoprecipitation using phosphoserine or phosphothreonine specific
antibodies as the former is more reliable than the antibodies of the latter. This is
due to the fact that at present, antibodies specific for phosphoserine and
phosphothreonine suitable for immunoprecipitation are not known.45'49 The
success of this approach is due to the high-quality antiphosphotyrosine
antibodies that have little crossreactivity with other phosphorylated or non-
phosphorylated residues. However, in practice, the immunoprecipitation of
phosphoproteins usually requires an expensive mixture of different antibodies.
Therefore, there have only been a limited number of reports that used this

approach.

Chemical Modification of Phosphate Groups
In chemical modification approach, the phosphorylated peptides are
chemically altered to an affinity tag to selectively fish them out of the pool of non-

modified peptides. Most of these approaches exploit the B—elimination chemistry

of the phosphate esters of serine and threonine under basic conditions.50v51 This
B-elimination is accompanied by the formation of a,B-unsaturated double bonds

as illustrated in Figure 4.4.

166

 

OH Nu

O=P-OH -OH nucleophile j\Ӣ
| 1A _’ In
0 < |- N N
H O H

O
1" N H3PO4
H dehydroalanine

O
phosphosefine

Figure 4.4 General scheme for the modification for phosphoserine by [3-
elimination.

This interrnediate species reacts readily with a thiol reagent thus providing a way
to introduce an affinity group. Oda and co-workers have demonstrated that it is
possible to isolate phosphorylated peptide or protein on an Avidin matrix via the
Michael addition of ethanedithiol, followed by the attachment of biotin (See
Figure 4.5).52 Mclachlin and co-workers introduced a thiol tag as the ligand for
affinity purification via disulfide linkage exchange with an activated thiol resin
(See Figure 4.6).53 Apart from minor degree of side reaction in which the non-
phosphorylated peptides were also modified, this strategy has shown to be

effective.

167

Ski

 

_(')H HSNSH l\l
O-P-OH -OH H s
1. ——>
C) <§r' N
H
“N H3PO4 H
H O dehydroalanine O
phosphosenne
Biotin
5
7
COOH O

H
BiotinV‘Nw/fi BiotinU‘N l
3 H20 5

 

Figure 4.5 Enrichment of a modified phosphopeptide coupled with a biotin
affinity tag.

168

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169

Moreover, phosphotyrosine does not B-eliminate, making this approach specific
for phosphorylated serine and threonine residues. Also, deglycosylation of the
sample appears to be a prerequisite since o-glycosylated peptides also 8-
eliminates producing a modified peptide indistinguishable from the modified
phosphorylated peptides.

Zhou and co-workers employed carbodiimide catalyzed cysteamine
addition of free phosphate groups as a phosphospecific chemical modification
strategy. The cysteamine is first reduced then the phosphorylated peptides are
immobilized onto an iodoacetyl matrix. The procedure requires several chemical
reactions and purification steps that lead to sample Ioss.54

Recently, Tao and co-workers introduced another approach of enriching
phosphoprotein through single step covalent coupling to a synthetic polyamine
dendrimer.55 The phosphopeptides are recovered via acid hydrolysis and can be

subsequently analyzed by mass spectrometry.

Immobilized Metal-Ion Affinity Chromatography (IMAC)

The isolation of phosphoproteins through their natural affinity to metal
cations has been explored over the years. In 1975, Porath and co-workers
originally proposed this approach in purifying histidine-tagged proteins.56 Back
then, it was called ‘metal chelating affinity chromatography. Subsequent studies
revealed that phosphorylated amino acids also bind to the iron(lll) charged
species coordinated to agarose bound iminodiacetic acid that later became what

is known as immobilized metal affinity chromatography (IMAC).57

170

IMAC is currently the most popular method for phosphopeptide
enrichment for MS-based phosphoproteome studies. It is based on the formation
of reversible coordination complexes between the amino acid side chains and the
positively charged metals bound to chelating groups such as nitrilotriacetic acid
(NTA) or iminodiacetic acid (IDA) immobilized on a support. Immobilized metal

ions of Fe3+, Ga3+, Al3+ and recently Zr4+ have shown very high selectivity

toward phosphopeptides.53'60 IMAC materials are synthesize by the addition of
metal ions to chelating agents such as iminodiacetic acid (IDA) or nitrilotriacetic
acid that are immobilized on a resin, bead, glass chip or membrane. The metal-
lDA and the NTA complex result to coordinatively unsaturated metal centers with
three and two sites available for coordination, respectively (See Figure 4.7).
These coordination sites are apparently only available to the phosphate at a pH
range between 2.0-3.5. Thus, in order to develop an IMAC material that can be

use at a physiological pH, phos-tag, an alkoxide-bridged di-nuclear Zn (II) was

developed.61

O

V.

 

ll _ ‘,N
0% 5—0‘0 Ox, :x3+
-.-Fe§* Fe‘
0" ‘~o- .\
A B o- c

Figure 4.7 Structures of A. Fe(l|l) iminodiacetic acid complex B. Fe(l|l)
nitrilotriacetic acid complex C. phos-tag.

Enrichment using IMAC is typically carried out by the introduction of a
protein digest or cellular extract onto a column or surface that contain the

immobilized cations. The phosphate functional group forms a coordination

171

complex with the metal thereby allowing the removal of unbound non-
phosphorylated peptides. The bound phosphopeptides are released from the
affinity material through washing the column with an alkaline solution or by the
addition of excess ligand such as a phosphate buffer. The eluted
phosphopeptides are typically analyzed directly either by matrix assisted laser
desorption/ionization mass spectrometry (MALDI-MS) or electrospray ionization
mass spectrometry (ESl-MS) to obtain the peptide sequence and to determine to
phosphorylation site. IMAC procedures have become very popular due to its
good compatibility with subsequent separation and detection techniques such as
ESl-MS and MALDI-MS. Moreover, the main advantage of this technique is that it
has been proven to be useful in large scale analysis. IMAC based techniques

have been reported to recover up to 70-90% of the total phosphoprotein samples

for whole cell extracts based on 32F or 33F radioactivity.

However, there are several drawbacks with this technique. The first
challenge is the recovery of multiply phosphorylated peptides that has stronger
affinity to the immobilized support, resulting in poor elution and requiring harsher
conditions to liberate the phosphopeptides.39v62 Another challenge is the non-
specific binding of phosphorylated peptides though the carboxylate groups of
aspartic and glutamic acid residues and the strong binding of the multiply
phosphorylated peptides.39 The non-specific binding of the acidic peptides is
diminished by esterification of the carboxylic acids to methyl esters using HCl-
saturated in dried methanol. Reaction conditions have to be chosen carefully to

avoid both the incomplete esterification of carboxylate group and side reactions

172

because they increase sample complexity. Despite following an apparently
simple common scheme (binding-washing-eluting), IMAC experiment conditions
are very variable and care should be taken as small variations in the experiment
conditions such as pH, ionic strength and organic composition of the solvents

could drastically affect the selectivity of the IMAC stationary phase.

Strong Ion Exchange Chromatography

In addition to IMAC, strong cation exchange chromatography (SCX)‘53»64
has been used for the enrichment of phosphorylated peptides. This procedure is
based on the fact that under acidic conditions (pH 2.7), tryptic phosphorylated
peptides are singly positively charged and amenable to further separation from
non-phosphorylated tryptic peptides that usually have a net charge of 2+ at same
pH. Although this technique does not have high specificity and the fractions
enriched in phosphopeptides also contain a high percentage of contaminant,
SCX enrichment have been used for massive phosphoprotein proofing mainly

due to the fact that the tryptic mixtures can be analyzed directly.

Metal Oxide Affinity Chromatography

Metal oxides have emerged as alternatives to the use of IMAC.65'67
Pinkse and co-workers59 first described the use of titania as a possible
alternative for IMAC for the enrichment of phosphorylated peptides. This
approach is based on the selective interaction of water soluble phosphates with

porous titania microspheres via a bidentate binding on the titania surface. In this

173

technique the phosphopeptides are trapped on the surface of titania precolumn
under acidic conditions and are desorbed under alkaline conditions. As with
IMAC, non-specific binding of acidic non-phosphorylated peptides also is
encountered with titania but this problem also can be minimized by methyl
esterification. Larsen and co-workers also have demonstrated that the inclusion
of 2,5-dihydroxybenzoic acid (DHB) in the loading buffer enhances the selectivity
towards phosphopeptides while retaining high binding affinity.68 The exact
mechanism on how DHB improves selectivity is not clearly understood but it
appears that DHB competes with the acidic nonphosphorylated proteins for
binding sites on the titania surface. This hypothesis was confirmed by studies of
the coordination of substituted aromatic carboxylic acids with the surface of
titania. Spectroscopic studies have revealed that substituted aromatic carboxylic
acids (such as DHB) coordinate more strongly with titania as compared to
monofunctional carboxylic acids (such as benzoic acid).59

Metal oxides of zirconium,66 aluminum,"O gallium,71 tin72 and niobium73
also have been reported to be very selective for phosphopeptide enrichment.
Overall, the metal oxide affinity chromatography technique offers fast,
straightforward enrichment since it can easily be coupled with ESl-MS or MALDI-
MS. Moreover, the metal oxide materials are robust and chemically inert. One
drawback however, is that desorption process can be considerably slower.

The discovery of mesostructured metal oxides such as titania, alumina
and zirconia are of particular interest for applications in catalysis, catalyst

supports and molecular separations because of their large pore size and high

174

 

specific surface area. The high specific surface area of mesoporous metal oxides
together with reports of metal oxides being good materials for enriching
phosphoprotein make mesoporous metal oxides good candidates for
phosphoproteomic applications. The inherent high surface area will give the

material higher enrichment capacity.

4.2 Objectives

There is no single technique that can be considered the ‘holy grail’ for
phosphoprotein enrichment. In this study modified metal oxide affinity
chromatography approach for enriching phosphorylated proteins is introduced.
This approach serves as an alternative to the use of IMAC and metal oxide pre-
columns. The general objective of the study is to further simplify the
phosphopeptide enrichment process with a technique that will allow high
throughput without sacrificing high selectivity and capacity toward
phosphopeptides. This will be accomplished by utilizing mesoporous metal oxide
thin films of titania, alumina and zirconia. These metal oxides have high
selectivity towards phosphorylated proteins and peptides. Thus, it is expected
that the high surface area would translate to high capacity. Specifically, this study
fabricates mesoporous thin films of titania, alumina and zirconia on supports that
permits easier enrichment procedures. Metal TEM grids will be used as support
for the thin films. The metal TEM grids are very well suited for this application

since they have small enough dimensions to carry out the enrichment

175

procedures. Moreover the physical properties of the mesoporous metal oxide thin
films also were elucidated. Ultimately, the end goal would be to successfully
enrich model phosphoproteins at the femtomolar level and to develop a protocol

for phosphoprotein enrichment using grid supported mesoporous metal oxide thin

films.

4.3 Experimental

4.3.1 Materials
‘ Copper, nickel, gold, molybdenum, titanium and aluminum TEM grids
were purchased from Ted Pella. Silver and stainless steel grids were procured
from SPI Supplies. All grids have a 400 mesh size except for stainless steel,
aluminum and titanium where they have 100, 200 and 300 mesh sizes
respectively. All of the grids were used as received. Titanium chloride, zirconium

tetrabutoxide and aluminum isopropoxide were procured from Aldrich.

4.3.2 Precursor Solution Preparation
Preparation of Alumina Precursor Solution
Aluminum tri-sec-butoxide (0.1 mol) was dissolved in 100 mL water. This

initial mixture was then mixed with 10 ml of 0.1 M HNO3 solution. The resulting

solution was allowed to incubate overnight at 100°C. The mixture was then

mixed with 9.0 g of Tergitol 15-S-9 surfactant. Finally, the resulting mixture was

diluted with 100 mL ethanol.74

176

 

Preparation of Titania Precursor Solution

A precursor mixture with a composition of 1 TiCl4 : 22 EtOH : 0.004 F127
was prepared by first adding TiCl4 to absolute ethanol under vigorous stirring.
This was followed by the addition of the Pluronic F127 (EO1OGPO7OEO106)

porogen dissolved in ethanol.

Preparation of Zirconia Precursor Solution

Zirconium propoxide (Zr(PrO)4), 70% wt. solution in n-propanol and non-
ionic triblock copolymer Pluronic F127 (E0106PO70EO106) were used as the
inorganic source and the structure directing agent, respectively. A precursor
solution has a molar composition of 1.0 Zr(PrO)4 : 0.0075 F127 : 2.4 HCI : 35.2
EtOH and 2.4 n-propanol.

The solution was prepared by mixing the Zr(PrO)4 and half the required
volume of ethanol together. In a second vessel, Pluronic F127 was dissolved
using the remaining volume of ethanol together with concentrated HCI. The two

solutions were then mixed together to produce a homogeneous solution. The

solution was kept in the fridge when not in use and was brought back to room

temperature for the casting of grid supported thin films.75

4.3.3 Preparation of Thin Films
Gold grids were dipped into the mesoporous metal oxide precursor

solution were allowed to dry to form the surfactant intercalated metal oxide thin

177

 

film on the grid- The films were allowed to incubate in a 100% humidity chamber
for at least 48 hours. The films were then calcined at the appropriate temperature
to obtain the metal oxide form for the phosphoprotein enrichment. In the case of
mesoporous alumina thin films, the grids were calcined at 500°C for 2 hours at a

heating rate of 2 degrees per minute.

4.3.4 Characterization of Mesoporous Metal Oxide Thin Films

Powder x-ray diffraction patterns were measured using Cu Ka radiation (l
= 1.542 A) and a Rigaku Rotaflex diffractometer equipped with a rotating anode
operated at 45 kV and 100 mA. Counts were accumulated every 0.02 degree 2
theta at a scan speed of 0.5 degree per minute. Thin films specimen for X-ray
diffraction analysis were prepared by dropping the precursor solution onto a
glass slide and allowing it dry. The films were then calcined at the appropriate
temperature before they were analyzed.

Transmission electron microscopy (TEM) images were obtained on a
JEOL 2200FS with an accelerating voltage of 200kV. TEM specimen were
prepared by dip-coating bare grids (3 mm, 400 mesh gold grid) into a metal oxide
precursor solution. These grids were dried and subsequently calcined at the
appropriate temperature to obtain the grid supported mesoporous metal oxide
thin films.

Scanning electron microscopy (SEM) images were obtained on a 6400
SEM with an accelerating voltage of 220 kV. Samples were coated with gold prior

to recording images.

178

 

4.3.5 Protein Digestion

The protein digests were obtained from Bruening's and Reid’s group. The

digestion procedures are described below.

B-casein and chicken ovalbumin were dissolved in GM urea containing 50
mM Tris-HCI to fom solutions containing 50pg protein / pL The disulfide bonds of
the ovalbumin were cleaved by adding 5uL, 10mM 1,4-dithio-DL-threitol (DDT) to
20 pL of protein solution prior to heating in a water bath at 70°C for 1 hour.
Though B-casein does not contain disulfide bonds, the same procedure also was
employed for preparing B-casein digest for consistency. A 160uL aliquot of 50mM
ammonium bicarbonate and a 10 IL aliquot of 100 mM was added to the
solutions at room temperature and then the solutions were place in the dark for 1
hour. Next, trypsin (10uL, 0.5pg/pL) was added and the solutions were incubated
for about 16 hours at 37°C. The digestion was quenched by adding enough

glacial acetic acid to make a 5% solution. The digests were stored in a -70°C

freezer until use.

4.3.6 Phosphoprotein Enrichment
Effects of incubation time

Figure 4.8 schematically illustrates the enrichment process. Initially,
individual TEM grids are placed inside 1.5mL Eppendorf tubes using self closing
tweezers. Then, 10pL of tryptic protein digest is carefully added to the tube to

ensure that the TEM grid was submerged in the protein digest. Special care also

179

is made so that the grid does not fold and the digest has no bubbles or air
pockets. To determine the optimum incubation time, the grids together with the
protein digest are allowed to incubate for 1-5 hours with samples being analyzed
at 1 hour intervals. After incubation, the TEM grid is separated from the protein
digest by simply picking it out using a tweezer. Then, the grid is immediately
placed in a new Eppendorf tube so that the grid did not dry out. Next, 10 uL of
the first washing solution of 2,5-dihydroxybenzoic acid in acetonitrile is added
and allowed to equilibrate for 10 minutes. After 10 minutes, the grid is again
picked out from the washing solution and place in a new Eppendorf tube for the
second washing with 10uL of the washing solution was added and was allowed
to equilibrate for another 10 minutes. The second washing solution this time was
composed of trifluoroacetic acid in acetonitrile solution. Both washing steps were
employed to remove nonphosphorylated peptidess adsorbed onto the grid. After
10 minutes, the grid was again removed and placed in an Eppendorf tube for a
final wash with distilled water. The washed grid was then mounted on a stainless
steel MALDI plate using conducting carbon cement. Once the carbon

cement was dried, the phosphopeptides were desorbed or eluted from the grid by
the addition of 3 pL of ammonium hydroxide solution onto the top of the grid.
(See Figure 4.9). The ammonium hydroxide solution was allowed to dry
completely and them 3 uL of the DHB matrix solution was dropped onto the

gridand dried before the MALDI spectrum was obtained.

180

 

Mass Spectrum Analysis

The MALDI mass spectrum shown were representative of many similar
spectra as each experiment was replicated three times using different TEM grids.
All mass spectra were obtained using a MALDI linear ion trap mass spectrometer

(Thermo vMALDI LTQ XL)

 

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183

4.4 Results and Discussions
4.4.1 Fabrication of Grid Supported Mesoporous Metal Oxide Thin Films

In this study, three mesoporous metal oxides of titania, alumina and
zirconia were used for phosphoprotein enrichment. The approach involved the
fabrication and characterization of TEM grid supported mesoporous metal oxide
thin film. A TEM grid is a circular shaped metal usually 3mm in diameter that is
use to mount samples for Transmission Electron Microscopy (TEM). These grids
can be segmented in many ways like square mesh, hexagonal and diamond

mesh, slots and holes, parallel, folding and tabbed. (See Figure 4.10)

   

A B C D E F G
Figure 4.10 Different types of grids. A square mesh B. hexagonal mesh C. slots
D. parallel E. combination of parallel and mesh F. folding and G. tabbed.

Grids are usually made from copper, nickel, gold but other metals and
materials are also used like beryllium, molybdenum, aluminum, titanium. Grids
are also described by their mesh size and the most popular mesh sizes are 200,
300 and 400 mesh. Aside from mesh sizes, grids can vary based on the size of
the hole and the bar (See Figure 4.11). Sometimes pitch is also used to describe

TEM grids, which is the sum of the bar and the hole.

184

 

 

 

 

 

 

 

 

 

 

 

 

 

0

A

—|l—_ll__ll—

Figure 4.11 A. Bar B. Hole C. Pitch

 

 

 

 

 

 

 

 

The use of TEM grids as supports for mesoporous metal oxide offers the
advantage of very high throughput because more samples can be, processed at
the same time. Moreover, the phosphoprotein can be separated from the digest
by simply pulling out the grid from the digest and washing off the non-
phosphorylated peptide off the grid. The TEM grid can also be use as received
and MS analysis can be done on the grid itself. The only disadvantage seen on
using TEM grids for phosphoprotein enrichment is that a steady hand is needed,
because the grids since the TEM grids are very thin and are easy folded.

Figure 4.12 shows the x-ray diffraction patterns for the mesoporous titania,
zirconia and alumina thin films. Based on the x-ray diffraction patterns, the
alumina pattern is characteristic of the gamma form of alumina. The titania
pattern is characteristic of anatase and the zirconia phase is cubic. Figure 4.13
and 4.14 also shows the electron diffraction patterns of titania and zirconia taken

using selected area diffraction mode of the TEM microscope. The diffuse

185

 

diffraction rings indicate the nanocrystalline nature of the films. The diffuse
reflections of the grid-supported aluminum films could not be resolved.

Figure 4.15 shows high resolution TEM images of a titania thin film. The
image on the left shows contrast where in the lighter shades represents the
pores of the mesoporous film. The image on the right shows a higher
magnification of the image on the left. At this magnification, the fringes due to the
crystalline nature of the material are visible. The different orientations of the
fringes indicate the polycrystalline nature of the material.

Figure 4.16 shows TEM images of mesoporous zirconia thin film. The
image on the left reveals the mesopores present in the film. The image on the
right shows a higher magnification of the material where the crystal fringes are
clearly seen. Figure 4.17 shows the TEM image of the mesoporous alumina film.
The image shows the random stacking of the lathlike alumina nanocrystals that
give rise to the pores of the materials.

Figure 4.18 shows the SEM image of a bare gold grid. The left image was
taken from the matted side of the grid and the image on the right was taken from
the glossy side of the bare gold TEM grid. These images were taken to ascertain
whether a thin film was deposited by knowing the initial morphology of the bare
grid. Figure 4.19 shows SEM image of the mesoporous titania supported by a

gold TEM grid. As the film is calcined to form the anatase structure, the Ti02

rearranged itself and in the process produce the broken glass-like morphology.
On the other hand, the lath-like structure of alumina permitted the formation of a

film that completely fills the holes of the TEM grid as shown in Figure 4.20.

186

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101 . .
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1015 20 25 3O 35 4O 45 50 55 60 65 7O

 

111 Zirconia

Intensity

 

 

 

 

10 15 20 25 30 35 40 45 50 55 60 65 70
2 theta

Figure 4.12 Wide angle x-ray diffraction of calcined alumina, titania and zirconia
powders.

187

 

 

 

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193

 

 

 

Figure 4.19 SEM image of a calcined mesoporous titania thin film supported on a
gold grid. The crystalline anatase coats the bars of the grid and extends into the
holes of the grid. The crystallization of the anatase phase during calcinations
causes shrinkage which fragments the films.

194

 

’9 1‘qu um

 

 

Figure 4.20 SEM image of a calcined mesoporous alumina thin film showing
completely filled holes on the TEM grid as well as the coated bars of the grid.

195

4.4.2 Phosphoprotein Enrichment
The chemistry behind this approach is based on the reported use of metal

oxides such as Ti02, Al203 and Zr02 for phosphoprotein enrichment. The metal

oxide surface provides a coordinatively unsaturated metal center where the
phosphate group of the phosphopeptide can form a covalent bond to the metal
as illustrated in Figure 4.21. The phosphate group of the protein can act either as

a monodentate or a bidentate ligand.

 
       

0H2 phosphopeptide I
,. /OH2
: “a
\0H2
Thin Thin
Fllm Film
16' .' '. 0H2
x“
M\O/ .. M;
I '. .' ° I \OHZ
M = Ti, Al, Zr M = Ti, Al, Zr unbound

phosphopepfides
Figure 4.21 Schematic illustration of phosphopeptide binding to a metal oxide
surface and the release of the protein by reaction with ammonium hydroxide.
B-Casein and ovalbumin were used to demonstrate the ability of TEM grid
supported mesoporous metal oxide thin films to enrich phosphoproteins. B-casein
represents 36% of bovine casein. It has a variety of functional properties which in

some cases are similar to but mostly differ from, those of (131- and K- caseins.

The B-casein molecule is a single chain of known sequences with five
phosphoserine residues and a molecular weight of 20,020 Da. Tryptic digestion

of B-casein yields three phosphorylated peptides fragments — two

196

 

tetraphosphorylated peptides and one monophosphorylated peptide (See Table

4.1). Ovalbumin is the major protein in avian egg-white and was one of the first

protein to be isolated in pure form.1 Its availability in large quantities has led to

the widespread use as a standard preparation studies of the structure and

properties of proteins and in experimental models of allergies.2 Ovalbumin is also
a glycoprotein with a relative molecular mass of 45,000. The amino acid

sequence of hen ovalbumin is comprised of 386 amino acids. This sequence was
deduced from the mRNA sequence by McReynolds and co-workers.3 This result
was also in agreement with the sequence of purified protein4 and the cloned

DNA.5 It has two potential phosphorylation sites at serine 69 and 345.6 Trypsin
cleaves on the carboxyl side of arginine and lysine residues. Tryptic digests of
ovalbumin typically yields three phosphorylated peptides with the third fragment
resulting from a miscleavage.7 The tryptic digest fragments for ovalbumin and [3-

casein are shown in Table 4.1.

197

 

 

Table 4.1. Phosphorylated peptides from tryptic digests of B-casein and
ovalbumin.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Peptide Sequence mlz
_._-A--__._._-_J3_-Casein
,,_.__ ___WMJEg‘ng FQpSEEQQQTEDELQDK 2062
V {92955 ELEELNVPGElVEpSLpSpSpSEESITR 2966
nglgg RELEELNVPGEIVEpSLpSpSpSEESITR 3122
., ------QXQLQtlmin
1P2088 EWGpSAEAGVDAASVSEEFR 2088
1P2511 LPGFGDpSlEAQCGTSVNVHSSLR 2511
1Png1 FDKLPGFGDpSlEAQCGTSVNVHSSLR 2901

 

 

Effect of Incubation Time

The binding of phosphopeptide onto a metal oxide surface will be
governed by equilibrium. In this case, the maximum amount of specific binding
for the phosphopeptides is desired. But the specific binding of the
phosphopeptide is difficult to achieve without the proper protocol, particularly with
regard to the length of incubation time. Tryptic digest of B-casein yields
phosphopeptide fragments composed of a monophosphorylated peptide (m/z =
2062) and two fragments both tetraphosphorylated with m/z values of 2966 and
3122. The non-specific binding of phosphopeptide is usually due to the presence

of basic (i.e. —NH2) and/or acidic (-COOH) donor groups in the peptide

sequence. This problem is usually circumvented by adding trifluoroacetic acid
(TFA) to protonate the amino side chain of basic amino acid residue as well as to
keep the carboxylic acid side chain protonated. For the case of titania, it has
been reported that 2,5-dihydroxybenzoic acid (DHB) was most effective in

achieving this effect thus, DHB was used in the studies.

198

 

Figure 4.22 shows the MALDI spectrum of 100 frnol of B-casein tryptic
digest without enrichment. From the spectrum, phosphorylated peptides are not
visible. Figure 4.23 shows the enrichment studies of 100 frnol B-casein on grid
supported mesoporous titania thin films. The figure shows MALDI spectrum after
different incubation times in the presence of DHB. The results of this experiment
were very interesting. Previously, it was thought that the longer the incubation

time, the better the enrichment becomes until it reaches equilibrium where no

 

100 -
[El-casein without enrichment
8
: 75 -1
(0
'O
C
3
2
0 50 -
.2
‘0'
is
0:

 

 

 

25

 

‘1 ~11:
. ill .

 

     

1 500 2000 2500 3000 3500 4000

m/z

 

Figure 4.22 MALDI spectrum of un-enriched B-casein. The specimen was
supported on a stainless steel MALDI plate.

199

 

 

    
      

 

0 WMWd®m¢uh9md ........... ..u. ...- .. , , ...
1500 2000 2500 3000 3500 4000
1 00

4P
/ 3122

 

 

 

 

 

 

     
 

 

G)
o 1:-‘.LI‘1.II1A‘ . ..1 l .1. l
9 1500 2000 2500 3000 3500 4000
c 10 3
3 0 ‘— P2062 4P
0 . 3122
< 50 — /
g 4P2966—-—> 3 hours
% O — illum.‘ I A I l l A I: .L .-
0; 1500 2000 2500 3000 3500 4000
100
, ‘— P2062 4P3122
50 - 2 hours
. 4P2966—.
O 4.1. L A... I .. I __ ,
1500 2000 2500 3000 3500 4000
100
4P3122
50 1 hour
0
1500 2000 2500 3000 3500 4000

W2

 

Figure 4.23 Incubation studies of 100 fmol B-casein on grid supported
mesoporous titania (TiOZ) thin film.

200

 

  
 
 

100
5 hours

.\\

50 4P3122

 
 
 

 

     

 

   

 

 

 

 

 

 

  

 

    

 

  
 

 

 

 

 

 
  

 

 

 

 
  

 

 

   

 

 

  
   

 

 

    

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1500 2000 2500 3000 3500 4000
100« 13 ‘//,
2 62
. 0 4ng66 4P3122
8 50- \ / 4 hours
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(u . .
3g 0, ' . . , .....-..----H- .
jg 1500 2000 2500 3000 3500 4000
<
100
a) P
{3 50 4p2966 ///
1r \ 3 hours
0 ' ' l I r
1500 2000 2500 3000 3500 4000
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o. .. . - .
1500 2000 2500 3000 3500 4000
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P2062——~0'
50- 4P2966 4P3122
\\\ 1 hour

 

    

  

 

 

0.
1500

2000

2500

rn/z

3000

  

 

3500 4000

Figure 4.24 Incubation studies of 100 frnol B-casein on grid supported
mesoporous alumina (Al203) thin film.

further improvement in enrichment is observed. Results showed that very good

enrichment can be achieved at 1-3 hours of incubation. Good enrichment in this

201

case was defined as obtaining a MALDI spectrum very high signal to noise
intensity and with high selectivity towards the phosphorylated peptides. MALDI
spectra taken after 3 hours started to show non-specific binding and the signal to
noise ratio decreased. It seems that the addition of DHB only delays the non-
specific binding of the non-phosphorylated protein but doesn’t stop it. DHB is said
to compete with the carboxylic acid side chain of the peptide chain thus delaying
non-specific binding. But as the system moves towards equilibrium, the
carboxylic acid group side chain of the non-phosphorylated peptide will
eventually compete with the phosphorylated peptide for binding sites on the
metal oxide surface as shown on the MALDI spectra at 4 and 5 hours incubation.

Figure 4.24 shows the MALDI spectra for 100 fmol B-casein on grid
supported mesoporous alumina thin film at different incubation times. As in the
case of mesoporous titania thin films, highly specific binding was observed from
1-3 hours of DHB incubation time. Non—specific binding starts to appear in the
spectra after 3 hours. From these two studies, it was decided that 3 hours is the
incubation time needed to ensure optimum specific binding. Thus, three-hour
incubation time was used for subsequent studies of both ovalbumin and [3-

casein.

202

. 9'

Enrichment of ,B-casein

The results for this part of the study are shown in Figure 4.25 where 50
frnol of tryptic digest of B-casein was enriched using mesoporous zirconia,
alumina and titania thin film. As previously reported in literature, the three metal
oxides showed highly specific enrichment.

In all of the spectra, the monophosphorylated peak (m/z = 2062) was the
most intense. The monophosphorylated peak was enriched at least 2600 times
as shown by the peak intensities. Also, the tetraphosphorylated peaks were
observed, though at very low intensity. Peaks corresponding to the lost of
phosphate group/s were also observed.

The spectrum for the control sample where a bare gold grid is used for the
enrichment process is shown in Figure 4.26. The results for this spectrum were
remarkable and unprecedented in part because there has not been previous
reports of gold surface being used for phosphopeptide enrichment and because
the surface is highly selective for the enrichment of two tetraphosphorylated
peptide of 1.3-casein. This is a level of enrichment that the mesoporous metal
oxide thin films failed to achieve. The spectrum for bare gold also showed peaks
corresponding to the lost of the phosphate moieties. Another interesting
observation is that the monophosphorylated peak is very weak to almost non-

existent.

203

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100- / 50 frnol B-casein on Zr02 - 2890
(D
0 q .
g 75- - 2167 g:
g - P2062 - 2
d)
g 50- - 1445 E
0 . 4P . _
E. 25- “32966 3122 - 722
0 /
E) -1 \ d
O l I A O
1500 2000 2500 3000 3500 4000
8 100- 50 frnol B-casein on Al203 - 2826
C a d
(U
3 75.. / 9 2120
§ . P2062 . .5;
C
2:, 50‘ 4P2966 4P3122 ‘ 1413 9
'4: d / ' —
g 25- \ _ - 707
c: - .
O I I l I O
1500 2000 2500 3000 3500 4000
100- 50 frnol B-casein on Ti02 - 2679
m . / .
0
C
§ 75- [52062 - 2009 0
f? 50- - 1339 g
g - 4P2965 4P3122 . 5
a; 25- \ / 3 670
E . .
0W 0

 

1 500 2000 2500 3000 3500 4000
m/z

Figure 4.25 Enrichment of the monophosphorylated and tetraphosphorylated
peptides in a tryptic digest of 50 frnol B-casein on TEM grid supported
mesoporous zirconia (top), alumina (middle), and titania (bottom). A three-hour
incubation time was used in obtaining each spectrum.

204

 

 

100 -l q 3325

 

 

1 P2062

4P2966 1 4.53122
% 75- - 2494
C
0 , 4P - 2H PO ,
1.5 3122 3 4 ,«E‘
0 4P -rrPo w
2.; 50* 2966 3 4 _ 1663 0:)
9 E
0 -
a: . .
4P2966 - 2H3PO4
25- 1 .4 831

 

 

 

 

0*. f. o
1500 2000 2500 3000 3500 4000

m/z

 

 

Figure 4.26 Enrichment of 50 frnol B-casein on gold TEM gn’d showing high
selectivity towards tetraphosphorylated peptides - 4P2965 and 4P3122.

The above results prompted an investigation of other metallic TEM grids
for potential use in enriching phosphopeptides. Among the metal grids tested
were tested were molybdenum, aluminum, copper, nickel, titanium, silver and
stainless steel. These metal TEM grids were tested using B-casein as the best
model system, because it contains both mono and tetraphosphorylated peptide.
Test results showed that molybdenum, nickel and copper showed did not show
any evidence of enriching B-casein. On the other hand, silver and stainless steel
showed some evidence of enrichment but they were not as good as the metal
oxides or of gold.

Aluminum was expected to give very good enrichment results since as a

metal, it is easily oxidize to form a thin Al203 layer. This is the same oxide as the

205

 

 

mesoporous alumina where enn'chment can be performed. Unfortunately, the
aluminum grid did not show any enrichment. This result was explained by the fact

that Al203 is amphoteric and the enrichment conditions are highly acidic. Though

the chemical composition of the aluminum surface oxide and the mesoporous
alumina are the same, they have different structure. The aluminum oxide film on
the metal surface is unstable towards acids and dissolves during the process of
enrichment, thus the observed lack of enrichment. The test results for the
titanium metal grid also are shown on Figure 4.27.

For the titanium grid, as in the case of the grid supported mesoporous
titania, all three phosphorylated fragments are visible with the
monophosphorylated fragment having the highest intensity. This was quite

expected since just like other metals, titanium forms a thin layer of Ti02 on its

surface. This oxide is more stable thus it persists in acidic conditions and it is

responsible for the enrichment process.

206

 

 

 

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207

The results for the bare gold and titanium complements each other.
Titanium is very effective in enriching the monophosphorylated peptide of B-
casein while gold is very effective in enriching the tetraphosphorylated peptides.
Moreover, comparing the intensity of the MALDI spectrum for the bare grids and
the mesoporous metal oxides, signal to noise the intensity for the bare metal
grids was higher than for the mesoporous metal oxide thin films. This is
explained by the presence of nooks and crannies on the mesoporous metal oxide
thin film. It has been suggested that a high surface area material is needed for
higher capacity material for enrichment. A high surface area material can have a
very high capacity and will be very useful when enriching relatively large amounts
of proteins but experimental data showed that it can also pose as a disadvantage
when working on very low protein concentration (ie. femtomole concentration). If
the phosphorylated peptide gets bound deep into the mesoporous film, the
desorption of the peptide during MALDI analysis can be difficult because it would
have to go through a more complicated path and it may even result to desorption
failure. The non-porous and relatively flatter morphology of the bare metal grid
does not have this problem. The bound phosphopeptide will be on its surface and
would be easily desorbed during analysis. Though the bare metal grids might not
have the surface area of the mesoporous metal oxide thin films, it seems that
their surface area is enough in enriching phosphoprotein in the femtomole
concentration range. They not only have the advantage of highly specific binding
but they are commercially available and can be used without preliminary

cleaning. This further simplifies the process of enriching phosphoproteins.

208

Enrichment of ovalbumin

Ovalbumin is a phosphoprotein whose tryptic digest yields three
monophosphorylated fragments. Figure 4.28 shows the MALDI spectra of the
enrichment of ovalbumin on grid supported mesoporous metal oxides and on a
bare titanium grid. Gold was also tested but it gave no enrichment thus the
spectrum was not included. This was quite expected from gold since from the
results in B-casein, where it weakly enriched the monophosphorylated fragment
in comparison to the tetraphosphorylated segments.

Mesoporous titania and alumina showed highly selective enrichment
where the m/z = 2088 peak was the strongest. Titania also show some non-
specific binding at m/z = 1774. This has been attributed to the residue that
contains the histidine group. Alumina doesn’t show this non-specific binding but
the intensity of the 2088 peak was lower compared to titania. The absence of
non-specific binding still can’t be explained but the lower intensity of the 2088
peak could be due to the relatively more porous nature of alumina where the
bound peptide have difficulties desorbing dun'ng MALDI analysis. Zirconia on the

other hand

209

 

   
  
 

 

 

 

  
 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

0
g 100 ‘ \ 50 frnol ovalbumin '7 413
<5 ‘ 1P on zirconia ‘
E 75 - 2°83 - 310 %f
3 _ d c
.0 0
<3: 50 - 1p - 207 E
9 q 2511 . —
“g 25 - I / - 103
a -
n: o I . 1 0.....- _.._......_,._______., _- . ._ , 0
1 500 2000 2500 3000 3500 4000
0
g 100 - \ 50 frnol ovalbumin“ 555
‘ 1P on alumina ‘
g 75 - 2°38 - 424 g
2 ‘ ‘ 5
(D 50 - - 283 E
.2 - ~
5 25 - - 141
0 .
0: WM
0 I I I ' I ' I ' I ' 0
1500 2000 2500 3000 3500 4000
0 _
g 100 - \ 50 fmol ovalbumin - 1092
m .. . . u
t t
E 75 _ 1P2088 on name _ 819 .—
3 (D
.0 ‘ q C
< 50 — - 546 .9
g - 1P2511 - 5
E 25 — / — 273
a) . .
CE 0 0%?“ - .1“ ‘ 0
1 500 2000 2500 3000 3500 4000
mlz

Figure 4.28 Enrichment of a 50 frnol tryptic digest ovalbumin on grid supported
mesoporous zirconia, alumina, and titania.

210

 

100 - 100 frnol ovalbumin

75 _ \ Zr02 _ 608

- P2088 ~

 

 

 

 

   

 

 

 

 

  
  
  

 

 

   

 
        

 

   

 

 
   

 

 

 

  
    

 

o , , o

1500 2000 2500 3000 3500 4000
8100 - , - 284
c . \ 100 frnol ovalbumin .
(U

75- —213

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a) ' l' E
.5 25 -71
Lo ‘ , .

SE 0 I I I I ' I .0..-00-.. ‘ 0
1500 2000 2500 3000 3500 4000
100': \ 100 frnol ovalbumin :381
75- T -285

I0
- P2088 2 r
50 - 0190
25 -95
o , , r -...._._ " o
1500 2000 2500 3000 3500 4000

m/z

Figure 4.29 MALDI spectrum of 100 fmol ovalbumin tryptic digest enriched on
grid supported mesoporous zirconia, alumina and titania thin films. Note the
weak or absence of enrichment for 1P2511 and 1P2901 fragments.

211

 

 

 

1000

"““ P2088
754
P2511
E
(I)
g l
E: 50-
P2901
25J 1

 

 

 

  

 

0,1...,.
1500 2000

2500' 1 I 56000. I 5506 ' ' 4000
mlz

 

1

Figure 4.30 MALDI spectrum of the 50 frnol ovalbumin

phosphorylated peptide enriched using titanium metal grid.
showed the monophosphorylated peaks at mlz = 2088 and 2511 but also showed
more non-specific binding. The reason to why this happen on zirconia and not on
alumina and titania is still yet to be investigated further. The results for the bare
titanium grid are more promising. Compared to titania, the spectrum did not show
non-specific binding. Titania showed two phosphorylated peptide peaks with m/z
= 2088 and 2511 while all three monophosphorylated peptides mlz = 2088, 2511

and 2901 are all visible for the case of the bare titanium grid.

212

4.5 Conclusions

Gold grid supported mesoporous metal oxide thin films were successfully
fabricated. Ideally, the holes of the grid should be filled with the metal oxide thin
film. Of the three metal oxides produced, only alumina was able to completely fill
the holes of the TEM grids as revealed by the SEM and TEM images. The lath-
like nature of alumina helped the thin film that was produced to be relative more
flexible than that of the films from titania or zirconia where the film doesn’t
completely fill the hole. In principle, the holes on the TEM grid can be filled by
adjusting the concentration of the precursor solution such that enough material
can be deposited during the dip-coating process. It was reasoned out that at this
point of the study that it is more important first to concentrate efforts on exploring
the properties of the material towards phosphoprotein enrichment. Results also
showed that there seems to be enough metal oxide surface area on the as
supplied grid. On the basis of x-ray diffractions patterns the alumina that as
produced was gamma alumina, titania as anatase and zirconia as the cubic
polymorph. TEM imaging also revealed that the thin films were indeed
mesoporous.

From these results, the feasibility of using grid supported mesoporous
metal oxide thin films for phosphorylated protein enrichment has been
demonstrated. This approach simplifies the enrichment process as well as
increasing sample throughput because multiple grid samples can be processed

simultaneously.

213

 

 

 

DHB incubation studies have shown that for highly selective enrichment,
one hour incubation time is enough and this can be extended to no more than
three hours. Beyond three hours, non-specific binding starts to appear on the
MALDI spectrum.

For the mesoporous metal oxide thin films tested, titania, alumina and
zirconia, all were very effective in enriching the mono- and tetraphosphorylated
segments of B-casein. For the enrichment of ovalbumin, mesoporous alumina
was the best among the mesoporous thin films tested since non-specific binding
was not observed. The next best material was mesoporous titania showing some
non-specific binding. The worst was zirconia because of very large amounts of
non-specific binding.

The results with the bare TEM grids of gold and titanium were the biggest
breakthrough in this study. There have not been reports of using bare gold and
bare titanium metal for enriching phosphoproteins. The capability of gold and
titanium complement each other. Gold is very selective on tetraphosphorylated
peptides. Titanium is capable of enriching both tetra- and monophosphorylated
peptides but it is more selective towards the monophosphorylated peptide
fragments. These results together with the protocol that was develop in enriching
these model proteins will help make the study of phosphoproteomics easier

because of the simplicity of the protocol and the relatively cheap TEM grids.

214

 

 

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