i
MIXED-MODE SILICA MONOLITH AS AN EFFICIENT SEPARATION
MEDIUM FOR CAPILLARY ELECTROCHROMATOGRAPHY
ALIA SOFIE BINTI JEMALE
Universiti Teknologi Malaysia
v
MIXED-MODE SILICA MONOLITH AS AN EFFICIENT SEPARATION
MEDIUM FOR CAPILLARY ELECTROCHROMATOGRAPHY
ALIA SOFIE BINTI JEMALE
A thesis submitted in fulfillment of the
requirement for the award of the degree of
Master of Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
AUGUST 2010
iii
To my beloved mother, Hajjah Jamelah, and father, Haji Jemale.
and
To dearest husband, Nor Azlan Mohd Aris
iv
ACKNOWLEDGEMENT
First and foremost, I would like to express my countless gratitude to my
project supervisor, Assoc. Prof. Dr. Jafariah Jaafar who made a multitude of useful
comments and perceptive suggestions for this project. Her encouragement, patience
and willingness to motivate me also contribute tremendously in the completion of
this thesis
This project would not have been accomplished without the unconditional
love and support of my family members. My highest appreciation goes to my
beloved parents, Hajjah Jamelah and Haji Jemale. Their understanding and prayer
make the flow of this thesis a lot easier and enjoyable. An honorable mention also
goes to my dearest husband, Nor Azlan for the support and understanding throughout
the completion of this project.
Special thanks to the Department of Chemistry and Faculty of Science for
providing me with a good environment and project facilities. Also, I am thankful to
all the lab assistants in the Department of Chemistry for being co-operative and
helpful in assisting me on technical matters.
Finally, I would like to thank all my friends, especially Nurul Asyikin,
Yuhanees and Noor Fairuzah, as well as other individuals who are not listed here for
giving me endless support and valuable insight.
v
ABSTRACT
Silica monolith column having mixed-mode i.e., hydrophobic (C18) and anion
exchange interaction was successfully synthesized by on-column modification of the
hybrid tetramethoxysilanes (TMOS) and methyltrimethoxysilanes (MTMS) using
octadecyldimethyl-(N-N-diethylamino)silane (ODS) and ion exchange monomer of
N-[3-(dimethylamino)propyl]acrylamide (DMAPAA-Q) methyl chloride-quaternary
salt, for the surface modification. The preparation of the ODS/DMAPAA-Q silica
monolith column was carried out in a fused silica capillary column of 75 µm internal
diameter. At each modification stage, the ODS/DMAPAA-Q silica monolith column
was chromatographically characterized with micro liquid chromatography (µLC) and
produced separation efficiencies of up to 43000 plates/min at the optimum velocity
using the test mixture of alkylbenzenes, and 41800 plates/min for selected
nucleotides. The ODS/DMAPAA-Q silica monolith column was also characterized
physically with scanning electron microscope (SEM), and showed to have an average
through-pore size and skeleton size of 2.2 µm and 2.0 µm, respectively. The
ODS/DMAPAA-Q silica monolith column was then characterized in capillary
electrochromatography (CEC) for the separation of inorganic and organic ions using
a 50 mM phosphate pH 6.9 background electrolyte. The inorganic anions and organic
ions were separated within 10 mins with column efficiency up to 114900 plates/min.
The results suggest that modifying a silica monolith capillary column through in-situ
polymerization of a monomer carrying a functional group can yield high efficiency
columns for the ion-exchange-mode separation as well as for reversed-phased
separations. The analysis of the porous silica monolith columns with CEC yields
higher performance than CZE analysis for the separation of anions.
vi
ABSTRAK
Turus silika monolit yang mempunyai mod saling tindakan campuran iaitu
hidrofobik (C18) dan penukar anion telah disintesis dengan jayanya melalui
pengubahsuaian langsung ke atas permukaan silika di dalam turus hibrid
tetrametoksisilana (TMOS) dan metiltrimetoksisilana (MTMS) menggunakan
oktadekilmetil-(N-N-dietilamino)silana (ODS) dan monomer penukar ion iaitu garam
metil
klorida
kuartenar
N-3[-(dimetilamino)propil]akrilamida
(DMAPAA-Q).
Penyediaan turus silika monolit dilakukan di dalam turus rerambut silika terlakur 75
µm diameter dalaman. Di setiap peringkat pengubahsuaian, turus silika monolit
ODS/DMAPAA-Q dicirikan secara kromatografi dengan kromatografi cecair mikro,
(µLC) dan menghasilkan kecekapan yang tinggi sehingga 43000 plat/min pada
kelajuan optimum menggunakan campuran uji alkilbenzena, dan 41800 plat/min
untuk nukleotida terpilih. Turus silika monolit ODS/DMAPAA-Q juga dicirikan
secara fizikal dengan mikroskop pengimbas elektron (SEM), dan menunjukkan
purata saiz lubang terus dan saiz partikel masing-masing adalah 2.2 µm dan 2.0 µm.
Turus silika monolit ODS/DMAPAA-Q kemudiannya dicirikan secara kapilari
elektrokromatografi, (CEC) untuk pemisahan ion tak organik dan organik
menggunakan 50 mM fosfat pH 6.9 sebagai elektrolit latarbelakang. Ion tak organik
dan organik telah dipisahkan dalam masa 10 min dengan kecekapan turus sehingga
114900 plat/min. Keputusan ini menunjukkan pengubahsuaian turus silika monolit
secara pempolimeran in-situ dengan monomer pembawa kumpulan berfungsi boleh
menghasilkan turus berprestasi tinggi bagi pemisahan dalam mod penukar ion dan
juga pemisahan fasa terbalik. Analisis dengan teknik CEC juga menghasilkan
prestasi yang lebih tinggi berbanding analisis CZE bagi pemisahan anion.
vii
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
x
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xiv
LIST OF SYMBOLS
xvi
INTRODUCTION
1
1.1
General Introduction
1
1.2
Problem Statement
4
1.3
Significance of Study
4
1.4
Objectives of Research
5
1.5
Scope of Research
5
LITERATURE REVIEW
7
2.1
Research Background
7
2.2
CEC
8
2.3
Electroosmosis
12
2.4
Separation in CEC
14
2.5
Column Technology in CEC
16
viii
2.6
Monolith Column
18
2.7
Monolith Column in CEC
19
2.7.1 Polymer-Based Monolith in CEC
23
2.7.2 Silica-Based Monolith in CEC
24
Mixed-Mode Silica Monolith as an
Electrochromatographic Separation Medium
Determination of Corrected Retention Factor
26
2.8
2.9
3
28
EXPERIMENTAL
32
3.1
Chemicals and Reagents
32
3.2
Synthesis of the ODS/DMAPA-Q Silica
Monolith Capillary Column
Instrumentation
33
35
3.3.1
CEC and CZE
35
3.3.2
µLC
36
3.3.3
SEM
36
3.3
3.4
3.5
Preparation of Chemicals
37
3.4.1
Buffer
37
3.4.2
Stock Solution
37
Experimental Procedures
37
3.5.1
CEC Procedures
37
3.5.2
CZE Procedures
38
3.5.3
µLC Procedures
38
3.6
Qualitative Analysis
39
3.7
Data Analysis
39
3.7.1
Mobility of Analyte
39
3.7.2
Separation Efficiency
40
3.7.3
Corrected Retention Factor
41
ix
4
RESULTS AND DISCUSSION
4.1
4.2
4.3
4.4
4.5
5
Synthesis of the ODS/DMAPAAQ Silica
Monolith Capillary Column
SEM Physical Characterization of
ODS/DMAPAAQ Silica Monolith Capillary
Column
µLC Chromatographic Characterization of
ODS/DMAPAAQ Silica Monolith Capillary
Column
ODS/DMAPAA-Q Silica Monolith
Capillary Column as a Separation Medium
in Capillary Electrochromatograph
4.4.1 Anions Separation on
ODS/DMAPAA-Q Silica Monolith
Capillary Column
4.4.2 Determination of Corrected
Retention Factor of the Anions on
ODS/DMAPAA-Q Silica Monolith
Capillary Column
4.2.3 Stability of the ODS.DMAPAA-Q
Silica Monolith Capillary Column
Comparison of Anion Separation with CZE
4.5.1 Reproducibility of Anion
Separation with CZE
43
43
45
46
54
54
56
59
60
62
CONCLUSION AND SUGGESTION
64
5.1
Conclusion
64
5.2
Suggestion
67
REFERENCES
69
x
LIST OF TABLES
TABLE
TITLE
PAGE
NO.
2.1
Comparison of electrodriven separation method
9
2.2
Landmarks in CEC
11
2.3
Features, roles and limitation of monolithic column
19
4.1
Pore properties of ODS/DMAPAA-Q silica
monolith capillary column
46
Theoretical plates, Neff, plate height, H, retention
factor, k and separation factor,  of the ODS silica
monolith column for alkylbenzenes separation
49
Theoretical plates, Neff, plate height, H, retention
factor, k and separation factor,  of the
ODS/DMAPAA-Q silica monolith capillary column
for alkylbenzenes separation
51
Theoretical plates, Neff, plate height, H, retention
factor, k and separation factor,  of the
ODS/DMAPAA-Q silica monolith capillary column
for nucleotides separation
53
The applied mobility, µapp, theoretical plate, Neff and
plate height, H for the separation of ions and anions
on the ODS/DMAPAA-Q silica monolith capillary
column
Data calculated from CZE experiment
55
57
Data calculated from CEC experiment with the
ODS/DMAPAA-Q silica monolith capillary column
57
Retention factor, k, of inorganic anions separated on
DMAPAA-Q silica monolith capillary column with
µLC and corrected retention factor, kCEC,, on
DMAPAA-Q silica monolith capillary column with
CEC (Jaafar et al., 2008)
58
Repeatibility of the ODS/DMAPAA-Q silica
monolith capillary column for short-term stability
59
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
xi
4.10
Reproducibility of ODS/DMAPAA-Q silica
monolith capillary column for long-term stability
60
4.11
Efficiency of anion separation on CZE
62
4.12
Repeatibility of anions separation with CZE for
short-term stability
63
Reproducibility of anions separation with CZE for
long-term stability
63
4.13
xii
LIST OF FIGURES
FIGURE NO.
2.1
2.2
2.3
2.4
2.5
2.6
TITLE
Flow profiles in (A) Pressure driven; (B)
electroosmotic driven
(A) Representation of the surface of fused silica
tubing. (B) Formation of an electrical double layer
near the surface of fused silica tubing
Differential of a solute migration in a column
under voltage gradient
PAGE
10
12
14
CEC of neutral and charged solute. Teof denotes
retention of EOF marker
15
Schematic preparation of direct synthesis for CEC
monolith capillary
21
Schematic preparation of post modification
synthesis for CEC monolith capillary
22
2.7
A generalized reaction scheme for a sol-gel
reaction
3.1
Monomers charged onto the MAS bonded column:
A) octadecyldimethyl-(N-N-diethylamino)silane,
ODS B) N-[3-(dimethylamino)propyl]acrylamide
methyl chloride-quaternary salt, DMAPAA-Q
34
3.2
CEC / CZE schematic diagram
35
4.1Proposed st
Proposed structure of the ODS/DMAPAA-Q silica
monolith packing
44
SEM micrograph of 200 µm I.D. ODS/DMAPAAQ silica monolith capillary column. A: 850x
magnification B: 3500x magnification
45
Chromatogram of uracil at MAS modification
stage. Mobile phase: 80% aqueous methanol.
Column temperature: 23˚C. Pressure: 52 bar.
Linear velocity: 1.0 mm/s
47
4.2
4.3
25
xiii
4.4
4.5
4.6
4.7
4.8
Chromatogram for mixtures of uracil and
alkylbenzenes, C6H5(CH2)nH, (n=0-6) at ODS
modified stage. Mobile phase: 80% aqueous
methanol. Column temperature: 23˚C. Pressure: 52
bar. Linear velocity: 1.0 mm/s
48
Chromatogram for mixtures of uracil and
alkylbenzenes, C6H5(CH2)nH, (n=0-6) at
ODS/DMAPAA-Q modified stage. Mobile phase:
80% aqueous methanol. Column temperature:
23˚C. Pressure: 52 bar. Linear velocity: 1.0 mm/s
50
Chromatogram obtained for mixtures of
nucleotides at ODS/DMAPAA-Q modified stage:
Mobile phase 50 mM phosphate (pH 2.8). Column
temperature: 25˚C. Pressure: 34.3 bar. Linear
velocity: 1.0 mm/s
52
Electropherogram obtained for mixtures of four
inorganic anions and one organic acid. Mobile
phase: 50 mM phosphate pH 6.9. Column
temperature 25°C. Applied voltage: -15 kV.
Separation voltage: -3 kV for 3 secs.
54
Electropherogram obtained for mixtures of four
inorganic anions and one organic acid. Mobile
phase: 50 mM phosphate pH 6.9. Column
temperature 25°C. Applied voltage: -15 kV.
Separation voltage: -3 kV for 3 secs.
61
xiv
LIST OF ABBREVIATION
AX
-
Anion exchange
APS
-
Ammonium persulfate
CE
-
Capillary Electrophoresis
CZE
-
Capillary Zone Electrophoresis
CEC
-
Capillary Electrochromatography
CX
-
Cation exchange
DMAPAA-Q
-
N-3[-(dimethylamino)propyl]acrylamidemethyl]
EOF
-
Electroosmotic flow
EDMA
-
Ethylene dimethacrylate
HPLC
-
High Performance Liquid Chromatography
MAS
-
3-methacrylamidopropyltriethoxysilane
MEAMS
-
2-(methacryloyloxy)ethyltrimethylammonium methyl sulfate
MEKC
-
Micellar Electrokinetic Chromatography
MTMS
-
Methyltrimethoxysilanes
ODS
-
Octadecyldimethyl-(N-N-diethylamino)silane
OT
-
Open tubular
PEG
-
Poly(ethylene glycol)
RP
-
Reversed phased
RP/AX
-
Reversed phased / anion exchange
SAX
-
Strong anion exchange
SCX
-
Strong cation exchange
SEM
-
Scanning Electron Microscope
SEMA
-
2-(sulfooxy)ethyl methacrylate
SWNT
-
Single-wall carbon nanotubes
TEOS
-
Tetraethoxysilane
TCM
-
Traditional Chinese medicine
xv
TMOS
-
Tetramethoxysilanes
VBC
-
Vinylbenzyl chloride
µLC
-
Micro Liquid Chromatography
xvi
LIST OF SYMBOLS
 eo
Electroosmotic mobility
ζ,
Zeta potential
r
Permittivity of the mobile phase

Viscosity of the mobile phase
E
Electric field strength
dp
Diameter of the stationary phase particle
L
Column length
v
Pressure-driven flow velocity

Porosity
p
Pressure drop
kc
Chromatographic retention factor
kapp
Apparent retention factor
t0
Hold up time
tr
Retention time
t’0
Corrected hold up time
µep
Electrophoretic mobility
Leff
Effective length of capillary
Ltot
Total length of capillary
V
Applied voltage
tM
Migration time
w1/2
Width of peak at half height
Veo-0
Electroosmotic velocity
Vep
Electrophoretic velocity
Veff
Effective velocity
Veff-CZE
Effective velocity in CZE
xvii
ECZE
Energy in CZE
Vep-CZE
Electrophoretic velocity in CZE
Veo
Eletcroosmotic velocity in CZE
Neff
Theoretical plates
H
Plate height
k
Retention factor

Separation factor
CHAPTER 1
INTRODUCTION
1.1
General Introduction
Being the youngest member in the family of separation technique, capillary
electrochromatography (CEC), is a micro column separation technique that combines
the features of both capillary zone electrophoresis (CZE) and micro liquid
chromatography (µLC) (Bartle and Mayers, 2001, Wu et al., 2008). In CEC, the
electroosmotic flow (EOF) generated on the surface of the stationary phase, is used
to drive the mobile phase instead of hydrodynamic flow. Due to the plug-flow
profile of the EOF, CEC exhibits separation of higher efficiency than in the pressuredriven µLC.
Separations of ionic solutes in CEC are commonly achieved with ionexchange column packed with particles (Gu et al., 2006; Norton and Shamsi, 2008,
Tanaka and Kobayashi, 2003). However, there are several limitations that need to be
solved on packed-CEC. One of the limitations of the conventional packed column is
the necessity to fabricate frits, which is required for retention of the packed particles
within the column. In addition, packed column also have the tendency to form
bubbles around the packing materials or at the frits. Such problems often results in
an unstable baseline, non-reproducible migration time and even, current breakdown.
Moreover, in packed-CEC, the packing procedure is often more difficult compared to
the normal High Performance Liquid Chromatography (HPLC) column due to the
narrow inner diameter of the capillary (Bartle and Mayers; 2001). It is also observed
that the solute band in this type of chromatographic column is broadened due to the
2
presence of multiple flow paths of different length and velocity, and slow
equilibration of the solute between the mobile phase and the stationary phase,
especially in nm-sized pores of µm-sized particles (Guiochon, 2007, Tanaka and
Kobayashi, 2003). As to obtain separation with high efficiency, the use of small
sized particles helps to reduce the contribution of the above mentioned factors,
therefore enables faster equilibration and narrower bands to be achievable
(Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004; Jaafar et al., 2008,
Klampfl et al., 2000, Lin et al.,2006; Minakuchi et al., 1996; Motokawa et al., 2002;
Tanaka and Kobayashi, 2003; Tang et al., 2000).
The introduction of monolith stationary phases in the field of column
technology is due to the problematic issues associated with packed column. The
major chromatographic features of these monolith columns are providing high
permeability based on its large through-pores, as well as high efficient separation
with large number of theoretical plates per unit pressure drop based on small-sized
skeleton (Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004; Jaafar et al.,
2008, Klampfl et al., 2000, Lin et al.,2006; Minakuchi et al., 1996; Motokawa et al.,
2002; Pesek et al., 2008; Roux et al., 2008; Tanaka and Kobayashi, 2003; Tang et
al., 2000). Guichon (2007) defined monolith column as made up of one piece of
continuous and porous material that is sealed against the wall of the tube. Due to this
characteristic, the mobile phase will tends to percolate through it, avoiding bypassing
any significant length of the bed. Other chromatographic advantages offer by this
monolith material also include low backpressure and fast analytes mass transfer. In
the aspect of monolith column preparation, the simplified preparation procedures
which can be directly performed in-situ within capillaries has brought the monolith
material to be gradually fabricated for application in microscale chromatographic
separation such as in the pressure-driven µLC (Jaafar et al., 2008; Pesek et al., 2008;
Roux et al., 2008; Wu et al., 2008) and the electro-driven CEC (Breadmore et al.,
2001; Ding et al., 2006; Fu et al., 2004; Jaafar et al., 2008; Klampfl et al., 2000; Lin
et al.,2006; Wu et al., 2008). Technically, the preparation of monolith columns in
electro-driven microscale capillaries is similar to that in pressure-driven µLC, except
for the consideration of the generation of EOF for driving the mobile phase, which
requires the incorporation of charged moieties onto the surface of the monolith.
3
Monolith stationary phase can be divided into two main groups which are
silica-based monolith (Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004;
Jaafar et al., 2008, Klampfl et al., 2000; Lin et al.,2006; Minakuchi et al., 1996;
Motokawa et al., 2002) and organic polymer-based monolith (Uysal et al., 2009,
Wang et al., 2009). Organic polymer-based monoliths are often prepared via a onestep polymerization of an organic monomer in the presence of a cross-linker, initiator
and porogen. The results can be classified as either rigid or soft monoliths. As for
silica-based monolith, it can be prepared by a sol-gel process whereby, alkoxysilanes
will undergo a hydrolysis and polycondensation reaction catalyzed by acetic acid in
the presence of porogens. The macroporous structure of the silica monolith prepared
by sol-gel process is controlled by the composition of the starting materials.
Therefore the size of silica skeletons and through-pore can be varied independently.
Silica-based monolith column usually provides greater mechanical stability
compared to the organic polymer-based monolith as the material show less swelling
in solvents.
One drawback of silica is its degradation above pH 7.5, whereas
polymer-based stationary phase can withstand higher pH values.
However, to
circumvent this problem, weak acid eluents can be used instead of highly-alkaline
eluents.
The improvement of selectivity in CEC is powered by the utilization of
various packing materials as its stationary phase. Besides commercially available
capillaries, novel stationary phase are also synthesized to provide sites of required
interaction. Not only to widen the range of selectivity, novel aspect of the material is
their ability to promote strong and constant EOF. Since stationary phase is the heart
of separation technique, improvement of column technology is foremost important
for the continued growth in CEC.
Here, we prepared a mixed-mode silica monolith column synthesized by
modifying the hybrid silica monolith with C18 (ODS) and quaternary ammonium
functional group (DMAPAA-Q), producing a mixed-mode of reversed-phase (RP)
and a strong anion exchange stationary (SAX) phase for CEC. As a mixed-mode
type of column, two different interactions of analytes towards the stationary phase is
offered, thus enabling hydrophobic and positively charge compounds to be
4
separated. Physical characterization of the column exhibits skeleton and throughpore size within the range of that reported by Nakanishi et al. (1996), and it is
expected that this column will produce good or similar performance in terms of its
separation efficiency compared to other silica monolith column reported previously.
A comparison study of the anions separation was also carried out on the CZE mode
with a fused silica capillary.
1.2
Problem Statement
Reversed phase column with ODS functionality is widely applied as the stationary
phase in chromatography due to its proven capability of separating neutral and
hydrophobic analytes. Recently, strong anion exchange (SAX) column of
DMAPAA-Q moiety has been proven successful in separating nucleotides and
inorganic ions (Jaafar et al., 2008). Incorporating the SAX functionality to an ODS
column could afford a dual separation mode which can be applied to a wider range of
analytes of organic species having hydrophobic properties; anion species, organic
and inorganic; and hydrophilic substances, particularly amines. Better column
performance could be expected from this mixed-mode column due to the additional
interaction yield from both the moieties.
1.3
Significance of Study
In obtaining high efficient separation, silica monolith material is an ideal
chromatographic separation medium to be applied in CEC, as the use of monolith
helps to reduce the contribution of the band-broadening factors, therefore enables
faster equilibration and narrower bands to be achievable. It is an added value in this
research as not only physical characterization is carried out at the final stage,
chromatographic characterization of the ODS/DMAPAA-Q silica monolith column
at each modification stage is also conducted using µLC at certain chromatographic
conditions. Analytes separation on normal CZE is also conducted as to distinguish
5
the separation influences on CEC that generate higher efficiency compared to the
conventional CZE method.
1.4
Objectives of Research
The objectives of this research are to:
(i)
synthesize a silica monolith column having mixed-mode interaction of
reversed-phased and ion-exchange using ODS and DMAPAA-Q monomer.
(ii)
conduct a chromatographic evaluation using µLC at each modification stage
of silica monolith column. Those stages consist of (i) MAS-modified stage,
(ii) ODS modified stage and (iii) ODS/DMAPAA-Q modified stage.
(iii)
conduct a physical characterization on the ODS/DMAPAA-Q silica monolith
capillary using Scanning Electron Microscope (SEM) with the aid of Image
Pro-Plus Analyzer.
(iv)
evaluate the performance of the ODS/DMAPAA-Q silica monolith column as
an electrochromatographic separation medium in CEC using selected anionic
compounds.
(v)
compare the efficiency and separation mode between the normal CZE and
CEC for the separation of selected inorganic anions and ionic compounds.
1.5
Scope of Research
This research is carried out to explore the capability of mixed-mode
monolithic silica as an electrochromatographic separation medium on selected
anionic compounds (bromide, bromated, iodate, nitrate and benzoic acid) with CEC.
6
The ODS/DMAPAA-Q silica monolith is synthesized with the collaboration of
Tanaka’s Laboratory, Kyoto Institute of Technology, Japan.
The ODS/DMAPAA-Q silica monolith prepared in the fused-silica capillary
of 75 m I.D. from the mixture of TMOS and MTMS first evaluated under µLC
mode with specific chromatographic condition proposed by Motokawa et al. (2002).
Evaluation based on column separation efficiency, are at the unmodified stage,
modified ODS stage and lastly at the modification stage with ODS/DMAPAA-Q.
The synthesized capillary is then tested for the separation of some common
nucleotides. Since the mixed-mode monolithic silica is obtained in the form of
capillary rod, physical characterization is limited to using SEM. The morphology
study is conducted with the aid of Image Pro-Plus analyzer under cross-sectional
view obtained from SEM.
The ODS/DMAPAA-Q silica monolith capillary with the mixed-mode
functionalities is then applied as an electrochromatographic separation medium in
CEC. The column is tested for the separation of selected anionic compounds and its
performance is evaluated based on the calculation of theoretical plates, N and plate
height, H. In this part of research, an optimum separation conditions are applied,
adopted from the previous study (Jaafar et al., 2008). Lastly, a comparison study of
the ion analysis with the normal CZE mode is conducted and results obtained are
discussed based on its separation elution and efficiency.
7
CHAPTER 2
LITERATURE REVIEW
2.1
Research Background
Taking the advantage offered by CEC, this study is focused on the ability of
silica monolith material with dual functionality for the separation of anionic
compounds. Monolithic column when coupled with CEC exhibit sharp, narrow and
compact band of analyte due to the plug flow profile generated across the column
(Bartle and Mayers, 2001; Wu et al., 2008). The characteristic of the monolith
material itself that consist of small skeleton and large through-pore size also
contributed to the high efficiency separation, producing the lowest plate height
(Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004; Jaafar et al., 2008,
Klampfl et al., 2000, Lin et al.,2006; Minakuchi et al., 1996; Motokawa et al., 2002;
Pesek et al., 2008; Roux et al., 2008; Tanaka and Kobayashi, 2003; Tang et al.,
2000).
The chromatographic capillary investigated in this work is a silica-based
monolith created with sol-gel method patented by Nakanishi and Soga (1997). This
method is developed over the course of five years documented in a series of papers
published by two groups at Kyoto University and Kyoto Institute of Technology,
Japan. A key feature of this synthesis is product riddled with pores in two size
ranges: tiny ones that provide high surface area and access to the silica surfaces on
which separations occur and the large ones that facilitate easy flow of material
through the monolith.
8
The simplicity of derivatization process of the functional group on monolithic
silica has captivated researchers’ attention to synthesize column of mixed-mode
characteristic which is normally a combination of two different functionalities. The
mixed-mode column of reversed-phased/anion exchange (RP/AX) (Ding et al., 2006;
Fu et al., 2004; Klampfl et al., 2000) were synthesized and its column performance
was tested for numerous anionic compounds, as well as inorganic anions. This type
of mixed-mode monolithic silica column, besides widening the range of analytes
selectivity, also promotes strong and constant EOF. In CEC of the mixed-mode type
of column, the elution trend of the analytes could be under the influence of either
electrophoresis or chromatographic mechanisms. This phenomenon is clarified
through the migration of EOF marker (Bartle and Mayers, 2001; Ding et al., 2006;
Fu et al., 2004; Klampfl et al., 2000).
In this research, a hybrid-typed monolithic silica column is prepared from the
mixture of TMOS and MTMS before surface modification with ODS and
DMAPAA-Q monomer to exhibit mixed-mode characteristic of reversed-phased and
strong anion exchange functionalities. The synthesized ODS/DMAPAA-Q
monolithic silica column is then applied as the electrochromatographic separation
medium on CEC, an intermediate technique bridging the gap between µLC and CZE,
for the separation of anionic analytes.
2.2
CEC
Separation techniques such as CE and HPLC are particularly useful in the
analysis of complex mixtures because of their high resolving power. Continued
refinement of these techniques has led to a novel separation method known as CEC,
also referred to as capillary electrokinetic chromatography, which is a combination
of HPLC and conventional CZE (Bartle and Mayers, 2001).
As in CZE and micellar electrokinetic chromatography (MEKC), small
diameter of typically 50-100 µm capillary column with favourable surface area-to-
9
volume ratio are employed to minimize thermal gradients from ohmic heating, which
can have an adverse effect on band widths. CEC differs crucially from CE and
MEKC. However, in terms of separation principle, CEC, CZE and MEKC are
identical as these separation techniques are based on partition between the liquid and
solid phases. In summary, the comparison of these three electro-driven separation
methods are tabulated in Table 2.1 (Bartle and Mayers, 2001).
Table 2.1:
Comparison of electrodriven separation method
CE
MEKC
CEC
Separation
principle
Different
mobilities of ions
in electric field
Partition between
bulk solution and
micelle moving in
opposite direction
of analyte
Partition between
solid stationary
phase and mobile
phase
Typical column
diameter (µm)
50-100
50-100
50-100
Stationary phase
None
None
Particle packed,
open-tubular, or
monolithic
Mobile phase
Sample type
Electrolyte solution Electrolyte solution Electrolyte solution
Charged species
Neutral and
charged species
Neutral and
charged species
In CEC, the flow of mobile phase is driven through the column by an electric
field, a phenomenon known as electroosmosis, rather than by applied pressure in
normal HPLC. This electroosmotic flow (EOF) is generated by applying a large
voltage across the column. The positive ions of the added electrolyte will accumulate
in the electrical double layer of the column packing, move towards the cathode, and
drag the liquid mobile phase with them (Bartle and Mayers, 2001). This resulted in a
plug flow profile characteristic of the EOF, in comparison to the parabolic flow
profile in HPLC. Figure 2.1 illustrates the differences in flow profile of CEC and
HPLC.
10
Figure 2.1:
Flow profiles in (A) Pressure driven; (B) Electroosmotic driven
Avoiding the use of pressure results in a number of important advantages for
CEC over HPLC (Bartle and Mayers, 2001). Firstly, in the pressure-driven
separation mode, the flow rate through the packing inside the column depends
directly on the particle diameter and inversely on the column length. For such case,
the particle diameters of the column packing are seldom less than 3 µm, with column
length restricted to approximately 25 cm long. By contrast, in CEC, the electrically
driven flow rate is independent of particle diameter and column length, therefore in
principle, smaller particles and longer columns can be used and thus, higher
efficiency is generated in CEC than that offered in HPLC. A second consequence of
employing the electrodriven separation mode is that increase in column efficiency is
expected since the plug-like flow velocity profile in the EOF reduces dispersion of
the solute band as it passes through the column. Thus, these combined effects of
reduced particle diameter, increased in column length and the plug flow
characteristic leads to CEC separation of higher efficiency and substantially
improved resolution.
Strain (1939) first reported the use of EOF in chromatography. He recognized
the difference between electrophoresis and electrochromatography on the one hand,
11
and partitioning of analytes between the mobile and stationary phase on the other.
The importance of the EOF in electrochromatography was then recognized, since the
aid of electrophoretic mobility and electroosmosis in this electrodriven separation
mode enabled the movement of analytes through the separation medium. The pioneer
of CEC was however by the Pretorius group (1974). They reported that the EOF used
to drive the mobile phase, that generated a plug flow profile in contrast to the
hydrodynamic flow in HPLC, yield separation with the lowest plate height. They
also pointed out the absence of pressure drop across the column if the EOF was used.
Significant progress in CEC began in 1980 when Joergenson and Lukacs
(1981) demonstrated the use of electroosmosis in capillaries and showed the
possibilities for low reduced plate heights. Tsuda (1987) then showed that CEC was
possible in coated open tubular (OT-CEC) and recognized the factors that control the
EOF as well as the importance of practical effects, such as bubble formation in
packed columns. The detail theoretical analysis of CEC and its technique
development was published by Knox and Grant (1987), followed by practical
demonstrations by the same group in 1991. The important observation made was that
column driven electrically showed higher efficiencies than the same column with
pressure drive. Some landmarks in the history of CEC are listed in Table 2.2.
Table 2.2:
Landmarks in CEC
Landmarks in CEC
Year
Reference
First report of use of EOF in
chromatography
1939
Strain, 1939
Use of EOF in column chromatography
1974
Pretorius et al., 1974
Electroosmosis in capillaries
1981
Jorgenson and Lukacs,
1981
CEC in open tubular column
1987
Tsuda, 1987
Theory of CEC and technique development
1987, 1991
Knox and Grant, 1987;
Knox and Grant, 1991
12
Since the re-emergence of CEC, there has been a very rapid increase in the
number of publications and reviews relating to CEC. To this date, due to the
attractive potential of CEC, it is widely applied as the separations technique for the
analysis of drugs (Aturki et al., 2009), food (Uysal et al., 2009) and pharmaceutical
analysis (Adu et al., 2008), enantiomeric separation (Fanali et al., 2008), as well as
for the determination of organic acids (Klampfl, 2007) and inorganic metals
(Breadmore et al., 2001; Kuban et al., 2008; Wang et al., 2009).
2.3
Electroosmosis
Electroosmosis is described as the movement of liquid relative to a stationary
charged surface under an applied field (Bartle and Mayers, 2001). Substances tend to
acquire a surface charge as a result of ionization of the surface and/or by the
interaction of ionic species. In a fused silica capillary, the ionization of silanol groups
give rise to a negatively charged surface, which affects the distribution of nearby
ions in solution. Ions of opposite charge (counter-ions) are attracted to the surface to
maintain the charged balance whilst ions of like charge (co-ions) are repelled. The
resultant is the formation of electrical double layer, illustrated in Figure 2.2.
Figure 2.2:
(A) Representation of the surface of fused silica tubing. (B) Formation
of an electrical double layer near the surface of fused silica tubing.
13
The counter-ions are arranged in two layers, which are the fixed and diffuse
layer, with a surface of shear beyond the interface. The voltage drop between the
wall and the surface of shear is known as the zeta potential, ζ. In the diffuse layer, the
potential falls exponentially to zero, and the distance over which it falls is known as
the double layer thickness,  . When voltage is applied, the solvated cations in the
diffuse layer migrate towards the cathode, dragging the solvent molecules along with
them.
The linear velocity of the EOF,  eo , is described by Smoluchowski (Knox,
1988) in Equation 2.1. This shows how the EOF is governed by ζ, the permittivity
and viscosity of the mobile phase,  r and  respectively, and the electric field
strength, E.
 eo 
 0  r E

(2.1)
The flow profile in CEC is assumed to be near-plug-like since it originates
from the capillary wall, but in reality it depends on the capillary internal diameter, d
and  . The use of microscope optic to image flow profiles in narrow capillaries has
produced conflicting results. While the plug flow profile predicted from theory has
been observed by Taylor and Yeung (1993), Tsuda et al. (1993) found a higher EOF
at the capillary wall than at the centre. Therefore, the importance of the EOF profile
in CEC will necessitates further research.
From Equation 2.1, it can be seen that neither the diameter of the stationary
phase particle (dp) nor the column length (L) affect the mobile phase velocity. This is
in contrast with pressure-driven flow velocity, v , which was described by KonzenyCarman in Equation 2.2, where  is the porosity and p is the pressure drop.
2
d 2 p p
v
180(1   ) 2  L
_
(2.2)
14
In CEC, both the capillary wall and the column packing carry surface charges
that are capable of supporting the EOF. To date, most of the separation analysis
carried out on CEC (Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004; Jaafar
et al., 2008; Klampfl et al., 2000; Lin et al.,2006; Motokawa et al., 2002; Pesek et
al., 2008; Roux et al., 2008; Uysal et al., 2009; Wang et al., 2009) suggests that the
column packing is responsible for the generation of EOF. This is because, there is a
greater number of free silanol groups presents since the solid packing has a far larger
surface area compared to that provide by the internal silica wall.
2.4
Separation in CEC
Separation in CZE is a consequence of differential migration of charged and
neutral species, as depicted in Figure 2.3. When a negative separation voltage is
applied, depending on the charge of analytes, cations will be attracted to the cathode,
followed by neutral solute and lastly the anions.
Figure 2.3:
Differential of a solute migration in a column under voltage gradient
CEC may be compared with CZE and classified in the hierarchy of separation
methods employing liquids by unified description proposed by Rathore and Horvath
(1996). The differential migration of solute bands can be divided into components
that are separative (selective interaction with the stationary phase, or differences in
electrophoretic migration velocities), and components that are non-separative
(migration not contributing to the separation.
15
In CEC of neutral solute (Bartle and Mayers, 2001), depicted as in Figure 2.4
(A), the solute and the mobile phase move in the same direction, and the sample
components emerge in the order of retardation by the stationary phase. However, if
the solute is charged, there are three operational modes depending on the direction
and magnitude of electrophoretic migration with respect to the direction of EOF
(Figure 2.4) (Bartle and Mayers, 2001).
Figure 2.4:
CEC of neutral and charged solute. T eof denotes retention of EOF
marker
16
Referring to Figure 2.4 (B), a negative voltage is applied for the separation.
The positive charged species emerged before the EOF marker due to the strong
attraction at the cathodic end. Therefore, the EOF is of co-directional, whereby the
migration velocities of charged species are always greater than that of the EOF
marker. Similarly, a negative separation voltage was also applied as in the case of 2.4
(C). However, since the solute exhibit negative charged, it is strongly attracted to the
anodic end, resulting it to emerge after the EOF marker. A counter-directional EOF
is generated because the EOF velocity is greater than the electrophoretic velocity of a
charged component. Unlike in the three other cases, in 2.4 (D) a positive separation
voltage was applied. In this case, the EOF marker is not detected. A counterdirectional EOF is generated where the EOF velocity is less than the electrophoretic
velocity. The detection of a charged component is only possible with instrument of
reversed polarity.
2.5
Column Technology in CEC
In the development of CEC, three types of column configuration had been
reported (Gu et al., 2007; Tang and Lee, 2000; Yuan et al., 2006). These include
packed (Norton and Shamsi, 2008), open-tubular (Zaidi et al., 2009) and monolith
stationary phase (Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004;
Guiochon, 2007; Jaafar et al., 2008; Klampfl et al., 2000; Lin et al., 2006; Minakuchi
et al., Motokawa et al., 2002; 1996; Tanaka and Kobayashi, 2003; Tang et al., 2000).
Generally, a packed column is a capillary packed with packing materials that are
confined between two end-frits, while an open-tubular column is a capillary bonded
with a wall-supported stationary phase that can be a coated polymer, bonded
molecule monolayer, or a synthesized porous layer network. As for the monolith
stationary phase, it is usually made up of a porous continuous bed that is formed in
situ inside the capillary.
Normally in CEC, it uses fused silica capillary with inner diameter measured
from 50 m to 100 m is used (Bartle and Mayers, 2001; Tang and Lee, 2000).
Small diameter capillaries are employed as to minimize thermal gradient from ohmic
17
heating, which will cause an adverse effect on band widths. Fused silica capillary is
the preferred choice for CEC due to its superior properties such as high electrical and
pressure resistance, good UV transparency, high thermal conductivity, good
chemical inertness, good flexibility as well as high mechanical strength.
In the early beginning of CEC, the capillary is packed with typical HPLC
stationary phase such as octadecyl silica (ODS) (Gu et al., 2007; Norton and Shamsi,
2008; Tang and Lee, 2000; Yuan et al., 2006). However, there are several problems
that need to be solved on packed-CEC. One of the limitations of the conventional
CEC is the necessity to fabricate frits, which are required for retention of the packed
particles within the column. In addition, packed capillaries also have the tendency to
form bubbles around the packing materials or at the frits. Such problems often results
in an unstable baseline, non-reproducible migration time and even, current
breakdown. In packed-CEC, the packing procedures are more difficult compared to
HPLC due to the narrow inner diameter of the capillary.
Since problems encountered using packed-CEC, as an alternative approach, is
the introduction of open-tubular capillary electrochromatography (OT-CEC) (Gu et
al., 2007; Tang and Lee, 2000; Yuan et al., 2006; Zaidi et al., 2009). In OT-CEC, the
stationary phase is coated to the capillary wall. This helps to increase the separation
efficiency than in packed-CEC as the eddy diffusion contribution is eliminated. In
OT-CEC, inner diameter of capillary smaller than 25 m is normally being used.
Still, there are some drawbacks of such alternative as the loadability is very low, and
lower retention factors are obtained than on packed-CEC.
Interest regarding monolith porous materials as chromatographic stationary
phase in capillary electrochromatography (CEC) is the result of many advantages
over packed-CEC and OT-CEC (Gu et al., 2007; Norton and Shamsi, 2008; Tang
and Lee, 2000; Yuan et al., 2006; Zaidi et al., 2009). In the past few years, the
monolith types of stationary phase, with wall supported porous continuous bed
columns have been developed for CEC. The monolith technology is widely made
into application as to create separation media that fulfill the requirement needed for
specific application.
18
2.6
Monolith Column
Separation of solute in liquid chromatography is commonly achieved by
using a column packed with particles (Gu et al., 2006; Norton and Shamsi, 2008;
Tanaka and Kobayashi, 2003). The particles are to provide large surface areas to
adsorb solutes onto the stationary phase from the mobile phase. Chromatography was
first carried out in this format at the beginning of 20 th century. Although most
analytical columns still consist of a packed bed, monolith materials has been recently
proposed as a chromatographic separation medium of higher performance
(Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004; Guiochon, 2007; Jaafar et
al., 2008; Klampfl et al., 2000; Lin et al., 2006; Minakuchi et al., Motokawa et al.,
2002; 1996; Tanaka and Kobayashi, 2003; Tang et al., 2000).
Previously, with particles packed stationary phase, the solute band is
broadened, either in a pressurized flow of electrodriven flow of solvent by the
presence of multiple flow paths of different length and velocity, slow equilibrium of
a solute between mobile phase and stationary phase and the diffusion of solute in the
mobile phase (Guiochon, 2007; Minakuchi et al., 1996; Tanaka and Kobayashi,
2003). The higher efficiency separation is obtainable with the use of smaller particles
sizes, as it reduces the above-mentioned factors.
Monolith column can be defined as a column consisting of one piece of solid
that possesses interconnected skeletons and interconnected flow paths (throughpores) (Guiochon, 2007; Tanaka and Kobayashi, 2003). A monolith column with
small-sized skeletons and large through-pores, having a large ratio (throughpore/skeleton size), commonly 1-4 can simultaneously provide high permeability and
high column efficiency. In contrast to the particle-packed column, monolith column
provide higher porosities, which is 65-70% for monolith prepared in a mold and
higher than 80% for those prepared in a capillary, compared to 40% for a particlepacked column. The features, roles and limitation of monolithic column are
summarized as in Table 2.3 (Tanaka and Kobayashi, 2003).
19
Table 2.3:
Features, roles and limitation of monolithic column
Monolith Column
Features
1) Continuous one-piece structure, fritless (mechanical stability)
2) High porosity (high permeability)
3) Small skeleton, large through-pores (high efficiency at high
speed)
4) Facile design and surfaces (selectivity)
Roles
1) High-efficiency and high speed separations beyond the limit of
particle-packed column
2) Stable and high-performance miniaturized columns for LC and
chip channels
3) Column for hyphenation with MS
4) Material for sample preparation by SPME and SPE
Limitation
1) Limited availability in size (50-100 µm, ca. 4-8 mm diameter)
and in chemical structure at present
2) Limited availability of skeleton size and through-pore size
3) Relatively low sample loading capacity (1/2-1/10 of a particlepacked column
Monolithic columns offer faster analysis by bypassing the limitations
imposed by pressure via through-pores, which allow higher flow rates than
particulate columns at reasonable column backpressures just like other continuous
media. Monolithic columns consist of a single, rigid or semi-rigid, porous rod.
Within the monolithic structure, analyte capacity is usually provided by smaller
mesopores.
2.7
Monolith Column in CEC
Monolith stationary phases have been widely used in fast efficiency
separation system. The preparation and application of monolith capillary in CEC
have gained increasing interest due to the distinct features of the porous monolith
material, such as the facile preparation, versatile surface chemistry, fritless design,
good permeability and fast mass transfer. The preparation of porous monolith
capillary is actually similar to that in HPLC. However, charged groups should be
20
incorporated to the surface of the monolith to generate EOF since EOF is the driving
force for the mobile phase.
In general, the derivatization of the functional groups onto the polymer or
silica monolith is fabricated using single step (direct synthesis) or multiple steps
(post-modification) procedures (Wu et al., 2008). Technically, the preparation of
CEC monolith capillaries is similar to that in µLC. This is demonstrated by Ishizuka
and coworkers (1998) for the preparation of ODS modified column, containing a
layer of octadecyl ligand on a silica monolith. Although their earlier work was
focused on µLC, their preparation process was then adapted by most researchers to
synthesize column for the utilization as stationary phase in CEC.
Direct synthesis of polymer based and silica based monolith column was
practiced by some researchers as this technique offers advantages such as timesaving, simplicity and also produces good reproducibility of column (Ding et al.,
2006; Lin et al., 2006). Nevertheless, obtaining the reliable silica monolith support
with desired chromatographic properties is not an easy task, due to the complexity of
the reaction that consist of a mixture containing monomers, cross-linker and initiator
in the presence of porogenic solvent. While this kind of silica monolith still needs to
go further investigation as to obtain support with the expected chromatographic
properties, based on the original method introduced by Minakuchi et al. (1996), the
study on the post-modification of the silica monolith column has been developing to
fit the various needs for the wide separation of compounds in µLC and CEC (Fu et
al., 2004; Jaafar et al., 2008). Through the post-modification step, monolithic
columns allow completely independent control of the porous and the chemical
properties. The schematic preparation steps for the monolith in CEC using direct and
post-modification method are depicted in Figure 2.5 and 2.6 respectively.
Monolith stationary phases applied in CEC are of two types, which are the
polymer-based monolith and the silica-based monolith. Examples of these types of
monolith will be discusses briefly in the following sub-topics.
21
Bare capillary
Polymerization
mixture
a) Silanization
b) Filling
Partially filled capillary
c) Polymerization
Thermal initiation
UV initiation
d) Washing
Mechanical pump
Electroosmotic flow
Monolithic column ready for use
Figure 2.5:
Schematic preparation of direct synthesis for CEC monolith capillary
22
Bare capillary
a) Silanization
Silanized capillary
Polymerization
mixture
b) Silanization
Partially filled capillary
c) Polymerization
UV initiation
Thermal initiation
d) Washing
Mechanical pump
Electroosmotic flow
Capillary filled with monolithic matrix
e) Coupling of chromatographic ligand
f) Coupling of charged group
Functionalized monolithic CEC column
g) Creation of on-column detection
window
Monolithic column ready for use
Figure 2.6:
Schematic preparation of post modification synthesis for CEC
monolith capillary
23
2.7.1 Polymer-Based Monoliths in CEC
Reversed-phase (RP) stationary phases is most widely applied in CEC. As the
most common polymer monolith column in capillary, poly(methacrylate),
poly(acrylamide), poly(PS-DVB)-based monolith column were the good candidate to
RP stationary phases in CEC. Li et al. (2005) incorporated single-wall carbon
nanotubes (SWNTs) into the preparation of polymer monolith column via in situ
copolymerization of vinylbenzyl chloride (VBC) and EDMA for CEC. The prepared
poly(VBC-EDMA-SWNT) monolith column was evaluated by separating a mixture
of small organic molecules. It was observed that the incorporation of SWNTs indeed
enhanced the retention of small neutral molecules on the monolith column. Another
type of RP polymer monolith was prepared by Ding et al. (2006), of a lysine
immobilized poly(GMA-co-EDMA). On this post-modified monolith column, the
hydrophobic interaction was responsible for separation of neutral analytes, and the
separation of ionic or ionizable analytes in CEC involved electrostatic interaction and
electrophoretic migration, in addition to the hydrophobic interaction.
Ion-exchange is also an important separation mode in CEC. Thus, ionexchange stationary phase with high degree of ionic interaction for CEC is always
desirable. Wu et al., (2002) developed SCX polymer monolith stationary phase by in
situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene
dimethacrylate (EDMA). The sulfate group provided by the monomer SEMA on the
monolith bed was used for the generation of electroosmotic flow from anode to
cathode, and these negatively charged groups also served as a SCX stationary phase.
But, because of the hydrophobicity of the polymeric skeleton, the CEC separation of
peptides was explained under both electrostatic interaction of SCX and hydrophobic
interaction of RP.
An anion-exchange stationary phase was prepared by Bisjak et al. (2005) by
grafting the ionizable amino groups onto the poly(GMA-co-DVB) monolith by the
post modification with deithylamine via the ring opening of the epoxy groups.
Wieder et al. (2006) further extended this approach to prepare the SAX stationary
phase by a two-step derivatization of poly(GMA-co-DVB) monolith first with
24
diethylamine and then with diethyl sulfate to form the designated quaternary
ammonium group via the ring opening epoxy groups from GMA.
The coating of latex-nanoparticles onto the polymer-based monoliths has also
been applied for the functionalization of monolith columns. Hilder et al. (2004)
prepared an anion-exchange polymer monolith column for the separation of
polysaccharides by coating 60 nm quaternary ammonium latex-nanoparticles on a
poly(BMA-co-EDMA-co-AMPS) monolith matrix via the electrostatic interaction.
The SEM images before and after coating of latex particles on the monolith
demonstrated that the macroporous structure of the monolith did not change but the
microglobules of coated nanoparticles played as the anion-exchange stationary phase
were found on the surface of the monolith. By applying this method, Hutchinson et
al. (2005) and Zakaria et al. (2005) realized the separation of inorganic anions over a
short separation period, and the elution order indicated the contribution of both ionexchange and electrophoresis mechanisms on the latex-nanoparticles coated
polymer-based monolith column. However, because the electrostatic adsorption of
quaternary ammonium latex onto the monolithic matrix was restricted by the amount
of negatively charged groups on monolith, the increase of the density of surface
active groups will accordingly improve the adsorption of latex-nanoparticles and
consequently increase the ion-exchange capacity of the latex-coated monolith
stationary phase.
2.7.2 Silica-Based Monoliths in CEC
Silica-based monolith columns provide good mechanical stability and specific
mesoporous and macroporous structure as well as the variety of chemical
modification for CEC. The aspirations of exploring the performance of silica
monolith column in CEC have driven researchers to develop a variety of new
methods for the preparation of silica-based monolith column. Silica-based monolith
columns can be prepared by hydrolysis and polycondensation of alkoxysilanes
catalyzed by acetic acid in the presence of porogenic agent using the sol-gel method.
25
Figure 2.7 shows a generalized reaction scheme for a sol-gel reaction. After drying
and heating, the sol-gel network is derivatized by on-column silylation reaction.
Figure 2.7:
A generalized reaction scheme for a sol-gel reaction
For modifying the surface feature for chromatographic separation, the functional
groups can be introduced onto the silica monolith matrix either by post-modification
or direct incorporation of desired functional monomers during the fabrication
process.
Hayes and Malik (2000) prepared porous silica monolith inside the fused
silica capillaries for the use as a separation bed in CEC. In this method, a sol-gel
precursor, N-octadecyldimethyl [3-(trimethoxysilyl)propyl] ammonium chloride was
incorporated into the sol solution for the preparation of the silica-based monolith
column. The prepared silica-based monolith with C18 moiety could be directly
applied in CEC, and therefore, the post-modification of the silica-based monolith is
avoided. Constantin and Fretag (2003) developed a similar one step sol-gel process
for the preparation of a C8 silica monolith column inside the fused silica capillary,
which also does not require the additional derivatization. Different from the previous
26
method, Dulay et al. (2002) reported the chemical modification approach of sol-gel
monoliths
by
silanizing
the
sol-gel
surface
with
organochlorosilane
or
organoalkoxysilane coupling reagents to form the stationary phases with the moieties
of pentafluorophenylpropyldimethyl, pentafluorophenyl, 3, 3, 3-trifluoropropyl, noctadimethyl,
perfluorohexyl,
and
aminopropyl,
respectively.
After
the
derivatization, the modified monoliths had higher stability at pH values below 4
compared to the underivatized monolith matrix. The separations of different mixtures
containing nucleosides, positively charged peptides and taxol derivatives were
observed on these phase-bonded PSG columns in CEC.
Using mercapto silane reagent, Xie et al. (2005) prepared the SCX silicabased monolith column for SCX separation in CEC. In this method, the silica
monolith matrix from a sol-gel process was first chemically modified by (3mercaptopropyl)-trimethoxysilane), and then followed by in situ oxidation of thiol
groups to sulfonic groups to produce the SCX stationary phase. The SCX stationary
phase was characterized by its substantial and stable electroosmotic flow (EOF), and
it was observed that the EOF value of the prepared column remained unchanged at
different buffer pH values and slowly decreased with increasing phosphate
concentration in the mobile phase. The silica monolith column with SCX stationary
phase was then employed in CEC for the separation of β-blockers and alkaloids,
which were extracted from the traditional Chinese medicines (TCMs).
2.8
Mixed-Mode Silica Monolith as Electrochromatographic Separation
Medium
Initially, the stationary phases that had been applied in CEC are the cation
exchange (CX) (Xie et al., 2005), RP (Motokawa et al., 2002; Pesek et al., 2008;
Roux et al., 2008) and anion exchange (AX) (Jaafar et al., 2008; Lin et al.,2006)
functionalities modified on silica monolith. Minakuchi and co-workers (1996)
prepared an ODS modified column, containing a layer of octadecyl ligand on a silica
monolith. Although their earlier work was focused on µLC, their preparation process
was then adapted by most researchers to synthesize column for the utilization as
27
stationary phase in CEC. An AX column was synthesized and evaluated by Jaafar et
al. (2008) for the separation of some common nucleotides and inorganic anions by
CEC. Polymerization of the DMAPAA-Q moities on the column exhibit a strong
anion exchange functionality, produced up to 90,000 theoretical plates with a BGS of
50 mM phosphate pH 6.9. Column short term stability was reported to be moderate
with RSD's values ranging from 4.7-8.8%. Another CEC AX column containing a
propyl-N,N,N-trimethylammonium group was also reported by Lin et al. (2006), and
its performance was examined using mixtures of inorganic anions. A plate height of
7.5 µm was obtained from HCrO4 - ion with 40 mM phosphate buffer pH 3.5, while
its short term and long term stability were reasonably good with RSD's values of 1.73.9% and 5.5-8.2% respectively.
In the separation of anions, mixed-mode phases offer more optimization
potential that RP or AX chromatography. Mixed-mode retention mechanism results
in which both electrophoretic mobility and hydrophobic interactions with the
stationary phase contribute to the overall retention of the analytes. Scherer and
Steiner (2001) synthesize a porous encapsulated silica monolith with a mixed-mode
of SAX and RP. The column exhibit high efficiency, H=7 µm for neutral solutes in 5
mmol/L phosphate, pH 4.0-water-acetonitrile eluent, while slightly higher H values
were obtained for some carboxylate ions. A mixed-mode column of RP/SAX with
the introduction of C6-alkyl chain and quarternary ammonium group modified on the
surface of the silica monolith was reported by Klampfl et al. (2000). A BGE of 5
mM Bis-Tris (pH 6.5 with HCl)-45% acetonitrile and addition of 2.5 mM chloride
was used to separate the inorganic anions, organic acid, organic bases and neutral
compounds. This column exhibited a high efficiency, N=197,000 for phenol but
slightly lower theoretical plates of 7000 for salicylic acid.
Fu et al. (2004), prepared a mixed-mode of RP/SAX type of column on a
silica
monolith
modified
with
charged
monomer
of
2-
(methacryloyloxy)ethyltrimethylammonium methyl sulfate (MEAMS) and ethylene
dimethacrylate (EDMA) for the separation of neutral and ionic compounds. The
column yields plate height of 5.2 µm and 5.5 µm for thiourea and benzyl alcohol
respectively, using a 10 mM phosphate buffer containing 40% (v/v) acetonitrile at
28
pH 2.0. The separation of analytes in the column is predominantly under the
influence of electrophoresis, due to the late elution of thiourea; an EOF marker. A
RP/WAX silica monolith column synthesized by Ding et al. (2006) used a mixture of
three precursors, tetraethoxysilane (TEOS), aminopropyltriethoxysilane (APTES)
and octytriethoxysislane (C8-TEOS). Fast and efficient separation of six aromatic
acid was obtained using a 10 mM phosphate buffer (pH 4.7) and 20% acetonitrile
with column efficiency up to 160,000 theoretical plates by CEC. The early elution of
its EOF marker, thiourea, at approximately 3 mins with a plate height of 5.7 µm,
suggested that separation is determined under chromatographic mechanisms. For
both of the above-mentioned cases, separations of the analytes were carried out with
a electrokinetic injection and a positive applied voltage.
2.9
Determination of Corrected Retention Factor
In column chromatography, retention factor, k, is a measure of the time the
sample component resides in the stationary phase relative to the time it resides in the
mobile phase (Kucera, 1985; Swadesh, 2001; Dong, 2006). k also expresses how
much longer a sample component is retarded by the stationary phase than it would
take to travel through the column with the velocity of the mobile phase.
Retention in CEC of charged solute depends on both chromatographic
retention and electrophoresis. Electrophoresis drives the charged solute through the
capillary at a rate which depends on it mass-to-charge ratio while the
chromatographic interaction is based on its chemical constituents towards the
stationary phase.
Several researchers (Al-Rimawi and Pyell; 2007; Knox and Grant, 1996;
Rathore and Horvath; 1999; Tsuda, 1988) have proposed different CEC retention
models. Tsuda (1988) introduced the following relationship as in Equation 2.3.
29
L
tR 
u pre
(2.3)
(1  k ' LC )  u ep
In Equation 2.3, upre and uep are corresponding migration velocities under pressure
and electric field mode. The variables, tR and L are solute retention and capillary
length, while k’LC is the chromatographic retention factor. Here CEC retention is
separated into two components, one being the chromatographic factor and another,
the electrophoretic factor. According to Equation 2.3, when upre and uep have the
same mathematical sign, the CEC retention will decrease; otherwise, it will increase.
However, the CEC retention factor, k’ is not included in Equation 2.3, and therefore
cannot be further evaluated.
Knox and Grant (1996) proposed that the overall migration velocity (ux) of
any charged solute in CEC could be expressed as in Equation 2.4, where k’LC is the
chromatographic retention factor of a solute and ueo is the electroosmotic flow
velocity.
ux 
u eo  u ep
1 k ' LC
(2.4)
Knox and Grant (1996) equation also describes the CEC retention in terms of
migration velocity. According to Equation 2.2, the CEC retention consists of two
independent factors, which are chromatographic and electrophoretic. However,
similarly as in Equation 2.3, CEC retention factor, k’ is also not expressed in the
Equation 2.4. The omission of the k’ thus restricted the physical understanding of the
CEC retention factor.
Rathore and Horvath (1999) introduced the concept of virtual migration
distances into CEC. Based on this theory, the migration process in CEC can be
divided into separative and non-separative components. The separative components
describe selective interaction with the stationary phase and differences in the
30
electroporetic migration velocity while the non-separative component describes
migration by convection that does not contribute directly to separation. Rathore and
Hovath (1999) defined CEC retention factor, k’ as in Equations 2.5 and 2.6, where
k’e is the electrophoretic velocity factor.
k '  k ' LC  k ' e  k ' e
k 'e 
u ep
u eo
(2.5)
(2.6)
All features of CZE and liquid chromatography (LC) are combined in Equation 2.5.
In principle, this model should be applicable to evaluate the CEC retention factor.
Recently, Al-Rimawi and Pyell (2007) proposed the corrected retention
factor, kc in CEC. The determination of corrected retention factor for charged analyte
in CEC is different from the normal apparent value, kapp, as the kc reflect both
chromatographic interaction and electrophoretic migration. Apparent retention factor,
kapp can be calculated as in Equation 2.7, where tr is the retention time of analyte and
t0 is the hold-up time.
k app 
tr  t0
t0
(2.7)
For the determination of corrected retention factor, kc, Al-Rimawi and Pyell (2007)
modified Equation 2.5 (Rathore and Horvath, 1999) into Equation 2.8, where tr is the
migration time of analyte ant t’0 is the corrected hold up time.
kc 
t r  t '0
t '0
(2.8)
31
In calculating the kc, it is necessary to take into consideration the
electrophoretic mobility, µep of the analyte. The electrophoretic mobility, µep can be
determined from CZE experiment with an empty fused silica capillary. The
electrophoretic mobility, µep is expressed as Equation 2.9, where Leff and Ltot is the
effective length and the total length of capillary, V is the applied voltage, tM is the
migration time of analyte and t0 is the hold up time.
 ep 
Leff Ltot  1 1 

 
V  t M t 0 
(2.9)
32
CHAPTER 3
EXPERIMENTAL
3.1
Chemicals and Reagents
All aqueous solutions were prepared with deionized water of 18 M.cm
purified by a Mili-Q A10 system from Millipore (Massachusetts, USA). All
chemicals were used without further purification, unless otherwise stated. Toluene
purchased from Nacalai Tesque (Kyoto, Japan) was distilled from calcium hydride
after refluxing the mixture for 2 h.
Salts
for
dihydrogenphosphate;
background
electrolyte
NaH2PO4.2H2O
and
(BGE)
consist
disodium
of
sodium
hydrogenphosphate;
Na2HPO4.12H2O were supplied by GCE Laboratory Chemicals (Eppelheim,
Germany), while analysis grade phosphoric acid; H3PO4 was from QRёC (Auckland,
New Zealand). 1 N of sodium hydroxide; (NaOH) and methanol; MeOH of HPLC
grade were both purchased from J. T. Baker (New Jersey, USA).
Salts for sample solutions were of potassium bromide; KBr, potassium
iodate; KBrO3 and potassium iodide; KI, purchased from GCE Laboratory Chemicals
(Eppelheim, Germany). Potassium nitrate; KNO3, and salt for EOF marker, thiourea;
(NH2)2CS were supplied by QRёC (Auckland, New Zealand). Standards of
adenosine-5’-monophosphate; (5’-AMP), cytidine-5’-monophosphate; (5’-CMP),
guanosine-5’-monophosphate; (5’-GMP) and uridine-5’-monophosphate; (5’-UMP)
33
were purchased from Wako (Osaka, Japan), while the sample solution of
alkylbenzenes were from Sigma-Aldrich (Wisconsin, USA).
Tetramethoxysilane, TMOS and methyltrimethoxysilane, MTMS were
purchased from Shin-Etsu Chemical (Tokyo, Japan). Solution of poly(ethylene
glycol), PEG, urea, acetic acid; CH3COOH were from Nacalai, Tesque (Kyoto,
Japan). N,N-dimethylaminopropylacrylamide methyl chloride quaternary salt,
(DMAPAA-Q) was from Kohjin (Tokyo, Japan), and ammonium persulfate (APS)
was from Wako (Osaka, Japan).
3.2
Synthesis of the ODS/DMAPAA-Q Silica Monolith Capillary Column
The preparation conditions of the ODS/DMAPAA-Q silica monolith capillary
column were similar to the procedures reported earlier (Motokawa et al., 2002).
Typical conditions were as follows: Mixture of TMOS and MTMOS (9 mL) was
added to a solution of PEG (1.05 g) and urea (2.03 g) in 0.01 M acetic acid (20 mL)
and stirred at 40°C for 45 mins. The resultant homogeneous solution was charged
into a fused-silica capillary tube from Polymicro Technologies (Arizona, USA),
which had been treated in advanced with 1 N NaOH solution at 40°C for 3 h, and
allowed to react at 40°C. Gelation occurred within 2 h and the gel was subsequently
aged in the capillary overnight at the same temperature. Then, the temperature was
raised, and the monolithic silica was treated for 3 h at 120°C to complete the
formation of the mesopores with ammonia generated by the hydrolysis of urea,
followed by water and methanol washes. After drying, heat-treatment was carried out
at 330°C for 25 h, resulting in the decomposition of organic moieties in the capillary.
Surface modification of the silica monolith was carried out on-column with
an anchor group, 3-methacrylamidopropyltriethoxysilane (MAS). Then each
monomer solution (Figure 3.1) was charged into the MAS bonded column. Firstly,
solution of octadecyldimethyl-N,N-diethylaminosilane monomer, ODS (2 mL) in 8
mL of toluene was continuously fed under a pressure of 50 mbar at 60°C for 3 h.
34
This was followed by continuous feeding of DMAPAA-Q monomer (0.2 mL) under
similar conditions as mentioned above.
CH
A)
3
N
+
N
Si(CH2CH3)
H3)
CH
3
B)
CH3
Figure 3.1:
C
N
O
H
(CH2)3
N+
CH3
Cl-
CH3
Monomers charged onto the MAS bonded column: A)
octadecyldimethyl-(N-N-diethylamino)silane,
ODS
B)
N-[3(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt,
DMAPAA-Q
Then both ends of the column were dipped in the reaction mixture in a 1 mL
vial and allowed to react at 68°C in a water bath for 2 h. After the reaction, the
column was washed quickly using water with a HPLC pump at 15 MPa for 6 h. The
washing procedure was repeated twice to obtain the mixed-mode ODS/DMAPAA-Q
silica monolith capillary column.
35
3.3
Instrumentation
3.3.1 CEC and CZE
CEC and CZE experiments were carried out on a HP3D CE Instrument from
Agilent Technologies (Waldron, Germany), equipped with a diode array detector and
ChemStation software. The schematic diagram of both the CEC and CZE mode is
depicted as in Figure 3.2.
Figure 3.2:
CEC / CZE schematic diagram
Separations in CEC were carried out on a packed column, 34 cm x 75 μm
I.D. x 360 μm O.D. capillary. Off-column detection was performed for the CEC
measurement by connecting the ODS/DMAPAA-Q silica monolith capillary column
to an empty fused-silica capillary column from Polymicro Technologies (Arizona,
USA), 10 cm x 75 μm I.D. x 360 μm O.D. using a 1 cm union, Teflon tubing of 33
mm I.D., part number 6010 from GL Science (California, USA). A detection window
about 2 mm in length, 1.5 cm from the union joint was made by burning out the
polyamide coating by a capillary burner. The capillary was then inserted into the
alignment of the CE cassette holder. UV detection wavelength was measured at 200
nm.
36
Separations on CZE were carried on a fused silica column from Polymicro
Technologies (Arizona, USA), 50 cm x 75μm I.D. x 360 μm O.D.. A detection
window about 2 mm in length, 8.5 cm from the end was made by burning out the
polyamide coating by a capillary burner. The capillary was then inserted into the
alignment of the CE cassette holder. UV detection wavelength was also measured at
200 nm.
3.3.2 µLC
The µLC evaluation of the ODS/DMAPAA-Q silica monolith capillary
column was carried out using a LC-10AD VP pump from Shimadzu (Kyoto, Japan)
and a UV detector, model CE1575 from JASCO (Tokyo, Japan) operated at 254 nm,
a data processor model C-R6A from Shimadzu (Kyoto, Japan) and a injector valve
model 7125 from Rheodyne (California, USA) fitted with a T-union which serves as
a splitter, with one end connected to the ODS/DMAPAA-Q silica monolith capillary
column, and the other end to a flow restrictor of an empty fused silica capillary
tubing.
3.3.3 SEM
Scanning Electron Microscopy from JEOL (Montana, USA), was used for the
assessment of the structural parameters in the monolithic column. The size of
skeleton and through-pore were calculated manually using the Image Pro-Plus
software from MediaCybernatic Inc (Montana, USA).
The ODS/DMAPAA-Q column was cut into duplicate fragments of 0.50 cm
respectively before mounted perpendicularly onto a 12 mm pin-type aluminum stub
using carbon paste. High resolution images were obtained by coating the capillary
with platinum (~40 nm thick), operated at high vacuum mode using an accelerating
low voltage of 2 kV.
37
3.4
Preparation of Chemicals
3.4.1 Buffer
The BGE of 50 mM phosphate buffer at pH 6.9 was prepared using the
following procedures. 0.1950 g of NaH2PO4.2H2O was mixed with 0.4477 g of
Na2HPO4.12H2O in a 50 mL volumetric flask and top-up with deionized water.
Before being used, the pH was measured using pH/Ion 5.5 Eutech Instrument pHmeter (Singapore). Prior to use, buffer solutions were filtered through a 0.22 μm
Millipore syringe (Massachusetts, USA), sonicated for 10 min, and 30 min degassing
using a Branson Ultrasonic cleaner (Connecticut, USA).
3.4.2 Stock Solution
A stock solution of 1000 ppm for each studied ion was prepared by weighing
appropriate amount of salt before made up to mark with deionized water. Each of the
stock solution was stored at 4˚C in a refrigerator until needed. Prior to analysis, the
standard solution mixture was prepared by diluting certain amount of stock solution
with deionized water.
3.5
Experimental Procedures
3.5.1 CEC Procedures
Prior to CEC measurement, the ODS/DMAPAA-Q silica monolith capillary
column was preconditioned by a syringe pump from KD Scientific (Massachusetts,
USA), with a pump flow of 10 μL/h for 3 h using methanol. The capillary was then
further conditioned via the CE instrument with water under 8 bar of nitrogen
pressure, applied to the inlet vial for 1 hr and followed by a BGE for another 1 h. The
38
column was further conditioned electrokinetically at -5 kV applied voltage with the
background electrolyte until a stable baseline was achieved.
Standard solutions mixture was introduced into the ODS/DMAPAA-Q
column through electrokinetic injection mode, -3 kV for 3 s from the cathodic end
(Jaafar et al. 2008). The EOF was determined by using thiourea as a marker.
Between each injection, the capillary was flushed with running electrolyte for 2 min
to ensure repeatibility between runs.
3.5.2 CZE Procedures
The fused silica capillary was preconditioned with 1 N NaOH (30 min),
methanol (30 min), deionized water (30 min) and finally BGE (30 min). Standard
solutions mixture was introduced into the fused-silica capillary through electrokinetic
injection mode, at -3 kV for 3 s from the cathodic end. The EOF was determined by
using thiourea as a marker. Between each injection, the capillary was flushed with
0.1 N sodium hydroxide (3 min), methanol (3 min), deionized water (3 min) and
BGE (3 min) to ensure repeatibility between runs.
3.5.3 µLC Procedures
µLC experiments were performed on a LC-10AD VP pump and a UV
detector, operated at 210 nm for the separations of the alkylbenzenes (n=0-6).
Separations were carried out on ODS/DMAPAA-Q monolithic silica column, and
samples were injected in a split-flow injection mode at a linear velocity, u = 1.00
mm/s. Prior to being used, the ODS/DMAPAA-Q monolithic silica column was
preconditioned with the mobile phase consisting of 80% methanol until a stable baseline was achieved. Temperature of the column was kept constant at 23°C with
pressure, P of 52.0 bar.
39
µLC experiments for the separations of nucleotides (5’-CMP, 5’-AMP, 5’UMP and 5’-GMP) were also carried on the same instruments under similar injection
mode at u=1.00 mm/s. Detection was determined at 254 nm. Prior to being used, the
ODS/DMAPAA-Q monolithic silica column was pre-conditioned with methanol (1
h), degassed deionized water (1 h) and finally with the mobile phase of 50 mM
phosphate buffer (pH 2.8) until a stable baseline was achieved. Temperature of the
column was kept constant at 24°C with P of 34.3 bar.
3.6
Qualitative Analysis
In qualitative analysis, each of the analyte (100 ppm) was injected into CEC
or CZE individually. The migration time of each analyte was noted from the
electropherogram. A mixture of standard solution was also prepared prior to the
determination via CEC or CZE. Each peak in the standard solution mixture found in
the chromatogram was analyzed by comparing their retention time with the retention
time of the individual standard injected under identical CEC or CZE condition.
3.7
Data Analysis
3.7.1 Mobility of Analyte
In CEC and CZE, the mobility of analytes, defined as applied mobility, µapp
and the electrophoretic mobility, µ eo of EOF marker were calculated using Equations
3.1 and 3.2 respectively (Camilleri, 1998; Henry, 2006). The effective mobility was
calculated using Equation 3.3(Camilleri, 1998; Henry, 2006).
µapp =
Leff  Ltot
kV  t m
(3.1)
40
µeo =
Leff  Ltot
(3.2)
kV  t 0
µeff = µapp - µeo
Where
(3.3)
Leff
: Effective length of capillary (m)
Ltot
: Total length of capillary (m)
kV
: Applied voltage (V)
tm
: Migration time of analyte (s)
t0
: Migration time of EOF marker
3.7.2 Separation Efficiency
Separation efficiency is evaluated with regard to the value of theoretical
plates, N and plate heights, H. For both parameters, the calculations were as in
Equations 3.4 and 3.5 respectively.
2
 t
N = 5.54 R
 w1 / 2



Where
w1/2 : Width of peak at half height
H=
Leff
N
(3.4)
(3.5)
41
3.7.3 Corrected Retention Factor
The CEC retention factor, kCEC can be calculated using Equation 3.6.
Veo 0  Veff  Vep
kCEC =
Vep
Where
Veo-0
: Electroosmotic velocity
Vep
: Electrophoretic velocity
Veff
: Effective velocity
(3.6)
The Veo-0, Vep and Veff of the EOF marker and charged species in CEC is given by
Equation 3.7, 3.8 and 3.9 respectively.
Veo-0 =
Vep =
Veff =
Leff
t0
Leff
(3.7)
(3.8)
tm
 eff CZE kV
Leff
(3.9)
To calculate the effective mobility in CZE, µeff-CZE, a CZE experiment using fused
silica capillary was carried out under identical chromatographic conditions as applied
in CEC. µeff-CZE is calculated using Equation 3.10.
42
µeff-CZE =
Veff CZE
(3.10)
ECZE
Where
Veff-CZE
: Effective velocity in CZE
ECZE
: Energy in CZE
The ECZE and Veff-CZE value are also obtained from CZE experiment. The equations
for both ECZE and Veff-CZE are shown in Equations 3.11 and 3.12 respectively.
ECZE =
kV
Leff
(3.11)
Veff-CZE = Vep-CZE + Veo-CZE
Where
(3.12)
Vep-CZE
: Electrophoretic velocity in CZE
Veo
: Eletcroosmotic velocity in CZE
The VCZE and Veo-CZE value are from the CZE experiment. The equations for Vep-CZE
and Veo-CZE are as in Equations 3.13 and 3.14 respectively.
Vep-CZE =
Veo-CZE =
Leff
tm
Leff
t0
(3.13)
(3.14)
43
CHAPTER 4
RESULTS AND DISCUSSION
4.1
Synthesis of ODS/DMAPAA-Q Silica Monolith Capillary Column
The ODS/DMAPAA-Q silica monolith capillary column was synthesized
according to previous report (Motokawa et al., 2002), and consisted of two-steps
process. It involved the preparation of the hybrid silica monolith as the backbone for
the attachment of monomers, using the sol-gel method, and followed by surface
modification through co-polymerization process to obtain the mixed-mode
ODS/DMAPAA-Q functionalities.
As a hybrid type of silica monolith, the
preparation involved the addition of TMOS and MTMS with the presence of PEG
and urea to firstly form the silica network. The role of PEG and urea was to set the
monolithic structure during the gelation and ageing process. An advantage offered
by hybrid silica monolith was that it could effectively avoid shrinkage and cracking
of the column packing (Ding et al., 2006; Motokawa et al., 2002).
In this experiment, a sol-gel reaction was employed for the formation of silica
monolith backbone. The network was achieved via hydrolysis of an alkoxy silicate,
followed by condensation and polymerization reaction. The silanol groups at the
inner surface of the capillary took part in this process to produce the monolithic bed
bonded to the capillary wall.
The ODS/DMAPAA-Q silica monolith capillary
column was prepared in a 100 cm fused-silica capillary with 75 µm I. D., since
reported work (Motokawa et al., 2002) and review regarding the column technology
for liquid chromatography (Gu et al., 2007; Tang et al., 2000; Wu et al., 2008; Yuan
44
et al., 2006), showed some difficulty when prepared in a 50 µm I.D. capillary
column. Although it is easier to prepare the monolith in capillaries of 100 µm and
200 µm I.D., it was reported that larger I.D. of capillary will create the effect of
ohmic heating which leads to the current leaking during analysis (Bartle and Mayers,
2001).
After synthesizing the silica network, next is the process of surface
modification. The derivatization of the mesoporous monolithic silica to C 18 with
ODS before the attachment of DMAPAA-Q, both by on-column polymerization
results in a reversed phase and strong anion exchange silica monolith capillary
column. Figure 4.1 shows the proposed structure of the polymer inside the capillary.
The C18 group which is a hydrophobic compound enables separation to be carried out
under the RP-mode while the ammonium groups from the DMAPAA-Q moieties
served to yield negatively charged surface, thus generating anodic EOF.
The
presence of dual functionalities inside the capillary, either with hydrophobic, and
anion (Breadmore et al., 2001; Ding et al., 2006; Fu et al., 2004; Klampfl et al.,
2000) or cation moieties create separation medium that has the ability to generate
strong and constant EOF.
H
O
Si
N
H
O
Hybrid
silica monolith
Figure 4.1:
Si
O
O
N
N-(3-triethoxysilylpropyl) methacrylamide,
MAS
ODS-DEA
MONOMER
DMAPAA-Q
MONOMER
Functional
group
Proposed structure of the ODS/DMAPAA-Q silica monolith packing
45
4.2
SEM Physical Characterization of ODS/DMAPAA-Q Silica Monolith
Capillary Column
The ODS/DMAPAA-Q silica monolith was characterized using SEM to
obtain an estimate of the skeleton and through-pore sizes. The SEM micrographs of
the cross-section of the monolith at 850x and 3500x magnification are shown in
Figure 4.2.
ODS/DMAPAA-Q monolithic silica column showed a continuous
porous network and was successfully prepared without any shrinkage as shown in the
micrograph.
A
B
Figure 4.2
SEM micrograph of 200 µm I. D. ODS/DMAPAA-Q silica monolith
capillary column. A: 850x magnification B: 3500x magnification.
46
The pore properties of the ODS/DMAPAA-Q silica monolith capillary
column are listed in Table 4.1.
The ODS/DMPAA-Q silica monolith capillary
column prepared from the mixture of TMOS/MTMS gave network structure with
larger through-pore and skeleton size, as well as higher domain and smaller ratio
value compared to the reported columns of MS-H(50)-II (Motokawa et al., 2002).
Minakuchi et al. (1996) reported that silica monolith capillary columns showed pore
properties of small-sized skeletons (1-3 µm), large through-pores size (1.5-5 µm) and
large through-pores size/skeleton size ratios (~1.2-1.5), as such properties could
create a column with performance beyond the limit of particle-packed column
especially when operated at high flow velocities. A good attachment of the silica
monolith to the capillary wall was attributed to the small domain size (Ding et al.,
2006; Fu et al., 2004; Jaafar et al., 2008, Lin et al., 2006; Motokawa et al., 2002)
showed by the ODS/DMAPAA-Q silica monolith capillary column, thus reducing
the void volume of the column.
Table 4.1:
a
Pore properties of the ODS/DMAPAA-Q silica monolith capillary
column
Through-pore size Ratioa Domain sizeb
(μm)
(μm)
Capillary
Skeleton size
(μm)
ODS/DMAPAA-Q
2.0
2.2
1.1
4.2
MS(50)-D
(Motokawa et al.,
2002)
MS-H(50) II
(Motokawa et al.,
2002)
1.0
2.0
1.0
3.0
1.5
2.0
1.3
3.5
: through-pore size to skeleton size ratio
: skeleton size + through-pore size
b
4.3
µLC Chromatographic Characterization of ODS/DMAPAA-Q Silica
Monolith Capillary Column
The ODS/DMAPAA-Q silica monolith capillary column was characterized
under chromatographic mode utilizing µLC after each modification step. These steps
consist of modification stage with MAS as the anchoring group, modification stage
47
with the ODS-DEA monomer, and lastly modification stage with the introduction of
DMAPAA-Q monomer to yield silica monolith capillary column with mixed-mode
functionalities of ODS/DMAPAA-Q. The purpose of conducting chromatographic
characterization at each modification stage was to ensure that the monomers were
successfully polymerized inside the capillary column, through the evaluation of the
chromatograms obtained. All chromatographic conditions and the choice of test
compounds applied in this part of experiment were based on method reported by
Motokawa et al. (2002).
The modified silica monolith capillary column with the MAS-anchoring
group was applied for the separation of uracil, an unretain marker, and alkylbenzenes
(n=0-6). Alkylbenzenes were selected as the test compounds due to the existence of
methyl group that provide hydrophobic characteristic. At this stage, as shown in
Figure 4.3, the uracil peak was observed at ca. 6.7 min, showing that the MAS group
was successfully anchored onto the silica monolith backbone.
Figure 4.3:
Chromatogram of uracil at MAS modification stage. Mobile phase:
80% aqueous methanol. Column temperature: 23˚C. Pressure: 52 bar.
Linear velocity: 1.0 mm/s.
The MAS-silica monolith capillary column was then co-polymerized with
ODS-DEA monomer to yield the reversed-phase functionality. The chromatogram,
shown in Figure 4.4 indicated that ODS-silica monolith capillary column yield
48
excellent peak symmetry within 12 min of analysis for the separation of
alkylbenzenes.
From the chromatogram, it was observed that the ODS-silica
monolith capillary column yielded excellent peaks symmetry within 12 min of
analysis for the separation of alkylbenzenes.
Peak Identifications:
(1) Uracil
(2) C6H5(CH2)nH; n = 0
(3) C6H5(CH2)nH; n = 1
(4) C6H5(CH2)nH; n = 2
(5) C6H5(CH2)nH; n = 3
(6) C6H5(CH2)nH; n = 4
(7) C6H5(CH2)nH; n = 5
(8) C6H5(CH2)nH; n = 6
Figure 4.4:
(2)
(3)
(4) (5)
(6)
(1)
(7)
(8)
Chromatogram for mixtures of uracil and alkylbenzenes,
C6H5(CH2)nH, (n=0-6) at ODS modified stage. Mobile phase: 80%
aqueous methanol. Column temperature: 23˚C. Pressure: 52 bar.
Linear velocity: 1.0 mm/s.
Results showed that the ODS-silica monolith capillary column provide
similar selectivity in terms of the alkylbenzenes separation orders as reported
previously (Motokawa et al., 2002) on a silica monolith capillary column.
According to Motokawa et al. (2002), the selectivity of the column was not
significantly affected by the type of chromatographic support such as in monolith or
particle packed column. This was because, as long as a sufficient dense layer of
chromatographic ligand can be grafted to the silica surface, the selectivity of a
column will not be much affected. It was also found out that the compounds were
eluted in the order of increasing hydrophobicity.
The column performance
evaluation in terms of theoretical plate, Neff, plate height, H, retention factor, k and
separation factor, α of the ODS-silica monolith column for alkylbenzenes are
tabulated in Table 4.2.
49
Table 4.2:
Theoretical plates, Neff, plate height, H, retention factor, k and
separation factor,  of the ODS silica monolith capillary column for
alkylbenzenes separation
Alkylbenzenes, C6H5(CH2)nH
Neff
H (m)
k

n=0
30 900
12.9
0.12
-
n=1
36 000
11.1
0.22
1.80
n=2
39 100
10.2
0.26
1.18
n=3
25 700
15.6
0.33
1.27
n=4
29 300
13.7
0.42
1.27
n=5
34 500
11.6
0.54
1.29
n=6
43 000
9.3
0.72
1.33
At the optimum linear velocity, the ODS-silica monolith capillary column
produced 25700–43000 theoretical plates/min with a minimum plate height of 9.3
m. These results were very similar to or greater than the efficiency of a ODS-DEA
MS-H(100)-II hybrid silica monolith capillary column prepared from the mixture of
TMOS/MTMS using similar procedures (Motokawa et al., 2002).
This was
presumably due to the network of silica monolith of the MS-H(100)-II capillary
column when being prepared in a larger I. D. capillary, although was easier in term
of preparation, it however tends to easily shrink and therefore could cause adverse
affect on the column performance. Another reason that contributed to this scenario
was because of the different skeleton and through-pore sizes yield from both
capillaries. Although both types of capillary column were prepared from the same
procedure and feed, the effect of I. D. will somehow cause differences in the column
performance.
The retention time of neutral solutes like alkylbenzenes on the modified
ODS-silica monolith capillary column depends on the carbon content and the density
of ligands of the stationary phase (Motokawa et al., 2002; Roux et al., 2008). This
retention behavior can therefore be used to evaluate the hydrophobicity of the
stationary phase. The hydrophobicity was usually determined by the ratio of the
retention factors of two neutral solutes differing in a single CH2 groups such as
ethylbenzene and toluene. The retention factors of the alkylbenzenes on the ODS-
50
silica monolith capillary column were found to be slightly larger than those obtained
from the column prepared only from TMOS (Motokawa et al., 2002). This was due
to the more hydrophobic nature of the hybrid capillary column that possessed
abundant methyl groups as well as C18 alkyl chains after chemical modification. The
separation factor for hexylbenzene and amybenzene, αCH2, as shown in Table 4.2 was
1.33 for the hybrid ODS modified column. Compared to the value from previous
ODS MS-H(100)II silica monolith capillary column, the value obtained was 1.51
(Motokawa et al., 2002). Therefore, it can be concluded that the new synthesized
column was much better in term of its performance due to the existence of small
skeleton and large through-pore sizes.
Since the column exhibit excellent peak symmetry and separation, further
modification with the co-polymerization of strong anion monomer, DMAPAA-Q
was carried out.
The capillary column was again, evaluated under the same
chromatographic condition as in previous step. The chromatogram obtained for the
mixed-mode ODS/DMAPAA-Q silica monolith capillary column is shown in Figure
4.5.
Peak Identifications:
(1) Uracil
(2) C6H5(CH2)nH; n = 0
(3) C6H5(CH2)nH; n = 1
(4) C6H5(CH2)nH; n = 2
(5) C6H5(CH2)nH; n = 3
(6) C6H5(CH2)nH; n = 4
(7) C6H5(CH2)nH; n = 5
(8) C6H5(CH2)nH; n = 6
Figure 4.5:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(1)
Chromatogram for mixtures of uracil and alkylbenzenes,
C6H5(CH2)nH, (n=0-6) at ODS/DMAPAA-Q modified stage. Mobile
phase: 80% aqueous methanol. Column temperature: 23˚C. Pressure:
52 bar. Linear velocity: 1.0 mm/s.
51
At the optimum linear velocity of ~1.0 mm/s, rapid analysis was obtained for
the separation of the alkylbenzenes on the ODS/DMAPAA-Q silica monolith
capillary column, compared to the ODS-silica monolith capillary column modified at
the second stage. However, in this stage, the drawback of this capillary column was
that, a base line separation for the first three alkylbenzenes was not achieved
probably due to weak interaction of the analytes towards the C 18 sites as the surface
was more shielded with the introduction of the anion exchange functional group.
The performance in terms of theoretical plates, Neff and plate height, H, for the base
line separated peaks at the ODS/DMAPAA-Q column are listed in Table 4.3.
Table 4.3:
Theoretical plates, Neff, plate height, H, retention factor, k and
separation factor,  of the ODS/DMAPAA-Q silica monolith
capillary column for alkylbenzenes separation
Alkylbenzenes, C6H5(CH2)nH
n=0
n=1
n=2
n=3
Neff
39 800
H (m)
10.1
K
0.06
0.08
0.11
0.14

1.33
1.38
1.27
n=4
n=5
n=6
24 300
26 800
30 300
16.5
14.9
13.2
0.18
0.27
0.35
1.29
1.50
1.30
The separation of alkylbenzenes was achieved within 9 min of analysis time
with the ODS/DMAPAA-Q silica monolith capillary column. At this stage, the
ODS/DMAPAA-Q silica monolith capillary column was compared to the ODS-silica
monolith capillary column that was synthesized in the previous stage, and showed to
have a poorer column performance as it produced an average of 30300 theoretical
plate and H=13.7 µm for propylbenzene, butylbenzene, pentylbenzene and
hexylbenzene. Although the separation time of the alkylbenzenes were faster in the
ODS/DMAPAA-Q silica monolith capillary column compared with the ODS-silica
monolith capillary column synthesized in the second stage, the average retention
factor and the separation factor for the alkylbenzenes were of smaller value than
those obtained using ODS silica monolith column. This was due to the relatively
weaker hydrophobic surface of the ODS/DMAPAA-Q silica monolith capillary
52
column, probably due to the existence of the quaternary ammonium functional group.
The anion exchange property of the ODS/DMAPAA-Q silica monolith capillary
column was examined using 50 mM phosphate buffer (pH 2.8) and four nucleotides,
5’-AMP, 5’-CMP, 5’-GMP and 5’-UMP. Figure 4.6 shows the separation of the
nucleotides on the ODS/DMAPAA-Q silica monolith capillary column.
Peak Identifications:
(1) 5’-CMP
(2) 5’-UMP
(3) 5’-AMP
(4) 5’-GMP
*Unknown peak
*
Figure 4.6:
*
Chromatogram obtained for mixtures of nucleotides at
ODS/DMAPAA-Q modified stage: Mobile phase 50 mM phosphate
(pH 2.8). Column temperature: 25˚C. Pressure: 34.3 bar. Linear
velocity: 1.0 mm/s.
Using the 50 mM phosphate buffer at pH 2.8, 5’-CMP and 5’-AMP were
protonated, while 5’-UMP and 5’-GMP were in their neutral state, therefore it was
expected that the former pair is more retained that the latter pair (Watanabe et al.,
2009). However, it was observed that among the protonated and neutral pairs,
nucleotides with purine base (5’-AMP and 5’-GMP) possess greater retention than
those with pyrimidine (5’-CMP and 5’-UMP) bases. This result was explained by
the differences in the hydrophobic nature of these nucleotides. A more hydrophobic
pair was retained longer inside the ODS/DMAPAA-Q silica monolith capillary
column while the less hydrophobic pair was eluted earlier.
All the four nucleotides were base-line separated within 11 min of analysis
time. From the chromatogram in Figure 4.6, it was observed that the order of
53
nucleotides elution were different on a strong anion exchange column, DMAPAA-Q
silica monolith capillary column reported previously (Jaafar et al., 2008) for the
retention time order of 5’-AMP and 5’-GMP. The nucleotides separation on the
DMAPAA-Q silica monolith capillary column was much faster, within 6 min of
analysis time.
The ODS/DMAPAA-Q silica monolith capillary column performance based
on calculated theoretical plates, Neff, plate height, H, retention factor, k and separation
factor, for the nucleotides separation are listed in Table 4.4. As tabulated in Table
4.4, the values of the retention factor were less than 1, resulted in a moderate
retention time for all of the analytes.
Table 4.4:
Theoretical plates, Neff, plate height, H, retention factor, k and
separation factor,  of the ODS/DMAPAA-Q silica monolith capillary
column for nucleotides separation
Nucleotides
Neff
H (m)
k

5’-CMP
5’-UMP
5’-AMP
41800
38000
21900
8.1
8.9
15.5
0.06
0.52
0.53
8.67
1.02
5’-GMP
18300
18.6
0.56
1.06
The separation factor of α5’-UMP/5’-CMP=8.76 was high presumably due to the
strong interaction of 5’-UMP towards the stationary phase, which results in longer
retention time compared to 5’-CMP. Nevertheless, at the optimum linear velocity,
the column was efficient, producing an average 30000 plates/min and H=12.8 µm for
the four nucleotides. These values were lower in comparison with the DMAPAA-Q
silica monolith capillary column reported by Jaafar et al. (2008), which exhibit
higher column efficiency with an average theoretical plates and plate height of 35000
plates/min and 9.5 µm respectively.
54
4.4
ODS/DMAPAAQ Silica Monolith Capillary Column as Separation
Medium in Capillary Electrochromatography
4.4.1 Anions Separation on ODS/DMAPAA-Q Silica Monolith Capillary
Column
The ODS/DMAPAA-Q silica monolith capillary column was tested for the
separation of inorganic and organic ions on CEC. The pH and concentration of
BGE, and separation pressure and temperature of capillary was adopted from
previous study (Jaafar et al., 2008). Phosphate buffer employed in this study as the
BGE has a low UV absorbance, providing a stable baseline compared to either
acetate or ammonium buffers.
Since solutes exhibit a negative charge, an
electrokinetic injection from the cathodic end was applied so that solutes can migrate
towards the anodic end.
As compared to the hydrodynamic injection, the
electrokinetic injection was applied as it produced good repeatability and
reproducibility between runs. Figure 4.7 shows the electropherogram of results on
the separation of a standard mixture of four inorganic anions and one organic ion. It
was observed that the anions were base-line separated within 10 min in the order of
bromate, nitrate, bromide, iodate and benzoic acid. No obvious tailing peaks were
observed for the inorganic anions except for benzoic acid.
mAU
2
Peak Identifications:
3535
(1) Bromate, 20 ppb
(2) Nitrate, 30 ppb
(3) Bromide, 20 ppb
(4) Iodate, 20 ppb
(5) Benzoic acid, 20 ppb
3030
3
2525
2020
1
1515
4
1010
5
5
5
0
0
2
Figure 4.7:
4
6
8
10
min
Electropherogram obtained for mixtures of four inorganic anions and
one organic acid. Mobile phase: 50 mM phosphate pH 6.9. Column
temperature 25°C. Applied voltage: -15 kV. Separation voltage: -3 kV
for 3 secs.
55
Thiourea, a neutral solute, was used to determine the t 0. Functioning as a
neutral marker, the late migration of thiourea at 15 min suggests that the separation
of solute was predominantly under the influence of electrophoresis. The applied
electrochromatographic condition generated a co-directional EOF where the
migration velocities of charged species were always greater than the EOF marker,
thus enabling the anions to emerge before the EOF marker (Bartle and Mayers,
2001). The migration of EOF marker, after the migration of anions was also reported
in several previous studies (Jaafar et al., 2008; Klampfl et al., 2001).
An advantage of a mixed-mode capillary column was its ability to provide
two different mode of interaction of solute towards the stationary phase (Breadmore
et al., 2001; Ding et al., 2006; Fu et al., 2004; Klampfl et al., 2000). In the case of
the ODS/DMAPAA-Q silica monolith capillary column, the separation of the
inorganic anions was due to the interaction with the quaternary ammonium group
that exhibit positive charge surface.
Benzoic acid was retained longest by the
column and resulted in a tailing peak. The retention of benzoic acid was probably
governed by several different retention mechanisms, namely electrophoretic, ionexchange, as well as hydrophobic interaction with the alkyl-chains on the stationary
phase (Klampfl et al., 2001). The applied mobility, µapp, number of theoretical plates,
Neff, and plate heights, H, of each analytes are listed in Table 4.5.
Table 4.5:
The applied mobility, µapp, theoretical plate, Neff and plate height, H
for the separation of ions and anions on the ODS/DMAPAA-Q silica
monolith capillary column
Analyte
µapp (m2Vs-1)
Neff
H (µm)
Bromate
-3.37 x 10-8
98 100
3.5
Nitrate
-3.09 x 10-8
114 900
3.0
Bromide
-2.59 x 10-8
82 600
4.1
Iodate
-2.24 x 10
-8
73 100
4.7
Benzoic acid
-1.37 x 10-8
16 900
20.1
56
In CEC, the movement of solutes through the column was a result of the EOF
generated by the applied field. The plug-flow profile produced an improvement of
separation efficiency in CEC. Good efficiency with low plate height of H=3.0-4.7
µm was obtained from the ODS/DMAPAA-Q silica monolith column, except for
benzoic acid.
Overall, for the separation of inorganic anions, the efficiency of the
ODS/DMAPAA-Q silica monolith capillary column was slightly higher but with
lower ion mobility compared to the DMAPAA-Q strong anion exchange capillary
column (Jaafar et al., 2008).
The ODS/DMAPAA-Q silica monolith capillary
column also provided lower plate height values than other mixed-mode (Klampfl et
al., 2001) and ion exchange (Lin et al., 2006) monolithic columns reported.
However, compared to other mixed-mode capillary columns (Breadmore et al., 2001;
Ding et al., 2006; Fu et al., 2004; Klampfl et al., 2001), limited selectivity of
analytes for ODS/DMAPAA-Q silica monolith capillary column was obtained.
4.4.2 Determination of Corrected Retention Factor of the Anions on
ODS/DMAPAA-Q Silica Monolith Capillary Column
All charged analytes separated on CEC have an electrophoretic mobility, µ ep,
in addition to the retention due to chromatographic interaction (Klampfl et al., 2000).
The studied inorganic anions and organic ions produced an effective electric charge
depending on the pH of the mobile phase and therefore have an µ ep which will affect
the elution time.
In an attempt to describe the retention of charged species, Al-Rimawi and
Pyell (2007) defined the corrected CEC retention factor, kCEC as in the Equation 3.6,
which includes the interaction of analyte towards the ODS/DMAPAA-Q silica
monolith column, and the contribution of the applied voltage generated towards the
mobility of the analytes. The proposed equation was modified from the original
equation defined by Rathore and Horvath (2002). In order to calculate the kCEC, it
was necessary to take into consideration the effective mobility, µ eff of the analyte.
57
The µeff was calculated based on experiment performed on CZE using fused-silica
capillary, with identical background electrolyte and separation conditions as in CEC.
The energy generated from the CZE experiment is -58823 Vm-1, calculated using
Equation 3.11. Other data related to the determination of the µ eff are tabulated in
Table 4.6.
Table 4.6:
Data calculated from CZE experiment
Bromate
tm
(s)
315.6
t0
(s)
-
Vep-CZE
(ms-1)
8.08 x 10-4
Veff-CZE
(ms-1)
2.18 x 10-3
eff
(m2Vs-1)
-3.71 x 10-8
Nitrate
325.8
-
7.83 x 10-4
2.15 x 10-3
-3.66 x 10-8
Bromide
435.0
-
5.86 x 10-4
1.97 x 10-3
-3.35 x1 0-8
Iodate
531.06
-
4.80 x 10-4
1.85 x 10-3
-3.15 x 10-8
Thiourea
-
185.04
1.37 x 10-3
-
-
Analyte
Referring to the calculated µeff from the CZE experiment, it is possible to
calculate the kCEC. To further calculate the kCEC, data were based on CEC experiment,
and the values were calculated using Equation 3.6-3.10. All data calculated are listed
in Table 4.7. The values for kCEC for the other four inorganic anions were between
0.92-1.49, indicating that all analytes have sufficient interaction with the stationary
phase.
Table 4.7:
Analyte
Data calculated from CEC experiment with ODS/DMAPAA-Q silica
monolith capillary column
Bromate
tm
(s)
230.76
t0
(s)
-
Vep-CZE
(ms-1)
1.30 x 10-3
Veo-0
(ms-1)
-
Veff
(Vm-1)
2.18 x 10-3
kcec
0.92
Nitrate
249.48
-
1.20 x 10-3
-
2.15 x 10-3
1.06
Bromide
297.72
-
1.01 x 10-3
-
1.97 x 10-3
1.27
Iodate
343.86
-
0.87 x 10-3
-
1.85 x 10-3
1.49
Thiourea
-
932.22
-
0.32 x 10-3
-
-
58
In comparing the retention factor, k, between CEC and the pressure-driven
separation, µLC, the normal chromatographic k values of bromate, nitrate, bromide
and iodate from DMAPAA-Q silica monolith capillary column (Jaafar et al., 2008)
were calculated manually using the following Equation 5.1. The k values calculated
are listed in Table 4.8
k=
tr  t0
t0
Table 4.8:
(5.1)
Retention factor, k, of inorganic anions separated on DMAPAA-Q
silica monolith capillary column with µLC and corrected retention
factor, kCEC, on DMAPAA-Q silica monolith capillary column with
CEC (Jaafar et al., 2008)
Analyte
K
kCEC
Iodate
0.05
0.87
Bromate
0.24
0.95
Nitrate
0.48
1.18
Bromide
0.52
1.24
Although the DMAPAA-Q silica monolith capillary column (Jaafar et al.,
2008) only possesses anion exchange functionality compared to the dual
functionalities of C18 and anion exchange on the ODS/DMAPAA-Q silica monolith
column, the k values from DMAPAA-Q silica monolith column on µLC are still
comparable to the kCEC of the ODS/DMAPAA-Q silica monolith column on CEC. In
comparison with the kCEC value on CEC, it was observed that the value of k was
found to be lower on µLC. Jaafar et al. (2008), observed that the kCEC values
generated from CEC were higher than the k value produced from µLC, due to the
additional separation based on electrophoretic mobility, µ ep, of the charged analytes.
In comparison, the kCEC values were higher on the ODS/DMAPAA-Q silica monolith
as analytes are retained slightly longer inside the column, allowing the analytes to
have sufficient time for interaction and therefore producing separation of high
efficiency. Also, according to Rathore and Horvath (1999), the value of retention
59
time in CEC was always larger than in µLC when the electroporetic and
electroosmotic velocity are in the same direction.
4.4.3 Stability of the ODS/DMAPAA-Q Silica Monolith Capillary Column
The stability of the ODS/DMAPAA-Q silica monolith capillary column was
examined based on the measurement of relative standard deviation (RSD) of the
migration time of the inorganic anions, organic ions and EOF marker. Repeatability
measurements of solutes migration time on the same day were used to determine the
short-term stability of the column. As shown in Table 4.9, the short-term stability of
the column was excellent with RSDs ranging from 0.6–1.8%. The repeatability was
found to be better than the DMAPAA-Q silica monolith capillary column reported by
Jaafar et al. (2008).
Table 4.9:
Repeatability of the ODS/DMAPAA-Q silica monolith capillary
column for short-term stability
Analyte
Migration time (min) ± SD
RSD; n=2 (%)
Bromate
3.85 ± 0.05
1.3
Nitrate
4.16 ± 0.05
1.2
Bromide
4.96 ± 0.07
1.4
Iodate
5.73 ± 0.08
1.4
Benzoic Acid
9.35 ± 0.09
1.0
Thiourea
15.54 ± 0.09
0.6
Similarly, in evaluating the performance of the ODS/DMAPAA-Q silica
monolith capillary column for its long term stability, migration time data for the
solute for three successive days were examined. Data as tabulated in Table 4.10
indicated that the column showed good long-term stability (RSDs of 3.0-6.7%).
60
Table 4.10:
Reproducibility of the ODS/DMAPAA-Q silica monolith capillary
column for long-term stability
Analyte
Migration time (min) ± SD
RSD; n=2 (%)
Bromate
3.86 ± 0.26
6.7
Nitrate
4.10 ± 0.21
5.1
Bromide
4.82 ± 0.29
6.0
Iodate
5.66 ± 0.25
4.4
Benzoic Acid
9.30 ± 0.28
3.0
Thiourea
15.5 ± 0.21
1.4
Although higher values of reproducibility were obtained for the
ODS/DMAPAA-Q silica monolith capillary column compared to an anion exchange
capillary column containing a propyl-N,N,N-trimethylammonium group on silicon
alkoxide monolith, as reported by Lin et al. (2006), these values were still acceptable
for a CE method (Jaafar et al., 2008).
4.5
Comparison of Anions Separation with CZE
Separation of the anions was also performed on CZE under identical
condition as in CEC. The electropherogam generated is illustrated in Figure 4.8. CZE
with fused-silica capillary and BGS of 50 mM phosphate pH 6.9 was only capable of
separating four inorganic anions. Benzoic acid was unable to be detected within 20
min of analysis time. A baseline separation was achieved for the inorganic anions
within 10 min in the order of bromide, nitrate, bromate and iodate.
61
mAU
Peak Identifications:
20
17.5
(1) Bromate, 20 ppb
(2) Nitrate, 30 ppb
(3) Bromide, 20 ppb
(4) Iodate, 20 ppb
(5) Benzoic acid, 20 ppb (not detected)
15
12.5
10
4
7.5
3
1
5
2
2.5
0
-2.5
2
Figure 4.8:
4
6
8
10 min
Separation of the test mixture of four inorganic anions and one
organic acid. Mobile phase: 50 mM phosphate pH 6.9. Applied
voltage: -15 kV. Separation voltage: -3 kV for 3 s. Capillary column
temperature: 25°C.
In conventional CZE with utilization of acidic buffer, a negatively charged
species was normally separated under the influence of counter-EOF when no EOF
modifier was added into the BGS (Breadmore et al., 2001; Klampfl 2007). As a
result, the determination of EOF marker requires a relatively longer time, and in
some reports it was unable to be determined at all (Breadmore et al., 2001; Bartle
and Mayers, 2001; Klampfl 2007). In this experiment, thiourea gave no signal within
20 min of analysis time. However, when a positive mode of separation potential was
applied, thiourea showed a peak at 3.01 min. This phenomenon supports the evident
that under the previous approach, anions were separated under the influence of
counter-EOF.
The separation of anions under counter-EOF lengthens the analysis time as
the anions tend to migrate in the opposite direction to the EOF. This was observed
through the values of µapp as tabulated in Table 4.11. Evaluation of separation
efficiency in terms of Neff and H yielded lower performance than in CEC which
might be due to the effect of counter-EOF.
62
Table 4.11:
Efficiency of anion separation on CZE
Analyte
µapp (m2Vs-1)
Neff
H (µm)
Bromide
-3.30 x 10-8
46 300
7.7
Nitrate
-3.20 x 10-8
39 600
9.0
Bromate
-2.39 x 10-8
46 500
7.6
-8
43 400
8.2
Iodate
-1.96 x 10
Although baseline separation of the inorganic anions was achievable in CZE
with the same condition as applied in CEC, results clearly highlighted that CEC with
the utilization of the mixed-mode monolithic silica column provided different
selectivity from CZE for bromide and bromate. Separation in CZE was determined
by the electrophoretic mobilities of the solute (Breadmore et al., 2001; Klampfl
2007) while in CEC, chromatographic retention was available to help improve the
separation selectivity (Breadmore et al., 2001; Ding et al., 2006; Fu et al., Jaafar et
al., 2008; 2004; Klampfl et al., 2000; Lin et al., 2006). The characteristic of the
stationary phase which is attached to the quaternary ammonium functional group
provides positive charge surface, thus enabling the separation of solutes under the
direction of EOF. Unlike in CEC, separation in CZE under co-EOF was impossible
without the addition of EOF modifier.
4.5.1 Stability of Anions Separation with CZE
The repeatability and reproducibility of the anions separation with CZE was
examined based on the measurement of relative standard deviation (RSD) of the
migration time of the inorganic anions, organic ions and EOF marker. Repeatability
measurements of solutes migration time on the same day were used to determine the
short-term stability. As shown in Table 4.12, the short-term stability for the anions
separation is excellent with RSDs ranging from 0.2–0.6%.
63
Table 4.12:
Repeatability of anions separation with CZE for short-term stability
Analyte
Migration time (min) ± SD
RSD; n=2 (%)
Bromide
5.26 ± 0.03
0.4
Nitrate
5.43 ± 0.03
0.6
Bromate
7.25 ± 0.02
0.4
Iodate
8.85 ± 0.02
0.2
Thiourea
3.08 ± 0.01
0.3
Similarly, in evaluating the performance of the analytes separation for its
long term stability, migration time data for the solute for three successive days were
examined to determine its reproducibility. Data as tabulated in Table 4.13 indicated
that the CZE separation also showed excellent long-term stability (RSDs of 0.31.1%).
Table 4.13:
Reproducibility of anions separation with CZE for long-term stability
Analyte
Migration time (min) ± SD
RSD; n=2(%)
Bromide
5.25 ± 0.05
0.9
Nitrate
5.42 ± 0.06
1.1
Bromate
7.24 ± 0.04
0.6
Iodate
8.84 ± 0.03
0.3
Thiourea
3.08 ± 0.02
0.6
64
CHAPTER 5
CONCLUSION AND SUGGESTION
5.1
Conclusion
The silica monolith capillary column having mixed-mode hydrophobic (C18)
and anion exchange functionalities was successfully synthesized by on-column
polymerization onto the hybrid TMOS/MTMS silica backbone using ODS and ionexchange monomer, DMAPAA-Q in capillary of 75 µm I. D.. The ODS/DMAPAAQ silica monolith capillary column showed properties of continuous porous silica
with average through-pore size of 2.2 µm and skeleton size of 2.0 µm, while the
domain size of this column was calculated to be 4.2 µm. It was observed that there is
no shrinkage or cracking of the column packing and also, no polymer agglomerate
was formed through the co-polymerization of monomers and the anchor group.
A multiple step synthesis of the ODS/DMAPAA-Q column was adopted as
the technique offers flexibility and allows completely independent control of the
porous and the chemical properties. A hybrid silica monolith backbone was first
synthesized with mixture of TMOS/MTMS using the sol-gel method. The hybrid
silica monolith provides more hydrophobicity of the column nature due to the
abundant methyl group present from the mixture of TMOS/MTMS as well as the C 18
carbon alkyl chains after undergoing the chemical bonding. Therefore, DMAPAAQ, besides serving as the anion exchange monomer, it is also responsible for the
generation of the EOF across the column.
65
The
ODS/DMAPAA-Q
silica
monolith
was
characterized
chromatographically using µLC. The silica monolith was first modified with MASanchoring group and tested for the separation of uracil and alkylbenzene. The uracil
peak was observed at ca. 6.7 min while alkylbenzene peaks was not retained,
showing that there is no interaction of the solutes toward the stationary phase. Copolymerization on the MAS-silica monolith capillary column with ODS-DEA
monomer yield a reversed-phase column with excellent column efficiency of up to
43000 plates/min and H=9.3 µm for alkylbenzenes (n=0-6) separations.
The modified ODS-silica monolith capillary column was then subjected with
the co-polymerization of DMAPAA-Q monomer to afford a mixed-mode capillary
column, ODS/DMAPAA-Q silica monolith. However, the performance between the
ODS/DMAPAA-Q silica monolith and the ODS-silica monolith was poorer as not all
alkylbenzenes were baseline separated, and the column efficiency too decreased with
theoretical plates and plate height of 24300-39800 plates/min and H=10.1-16.5 µm
respectively. Nevertheless, the evaluation on the ODS/DMAPAA-Q silica monolith
capillary column towards the separation of anionic mixture of selected nucleotides
were again excellent with the average value of N=41800 and H=9.6 m. The results
gathered from this part of experiment, showed that the ODS-DEA and DMAPAA-Q
monomer were successfully co-polymerized onto the hybrid silica monolith, while
the last evaluation for the separation of nucleotides suggested that the
ODS/DMAPAA-Q column has the capability of fast separation of anions.
Application of the ODS/DMAPAA-Q column on CEC exhibited good
performance for the inorganic anions; bromate, nitrate, bromide, iodate, with large
number of theoretical plate, although tailing peak was observed for late eluting
anion; benzoic acid. The ODS/DMAPAA-Q column gave an enhanced separation
efficiency of up to N=114900 and H=3.0 µm due to the production of sharp, narrow
and compact band. In the ODS/DMAPAA-Q silica monolith capillary column, a
negative separation mode was applied to avoid the counter-EOF due to the positive
charged surface of the column.
66
The corrected retention factor, kCEC for the separation of charged ions on the
ODS/DMAPAA-Q column was calculated using modified equation proposed by AlRimawi and Pyell (2007). kCEC has to be determine since in CEC, two types of
separation mode are combine, electrophoretic from CZE and chromatographic from
µLC. The calculated kCEC were within 2.02-2.89, were slightly of higher value than
the DMAPAA-Q silica monolith capillary column. The kCEC values yield from CEC
was also found to be higher than in µLC, suggesting that the electrophoretic and
electroosmotic velocities generated across the column are in the same direction. The
kCEC values also showed that the analytes are provided with enough time for
interaction towards the stationary phase, and also interaction based on analyte
electrophoretic mobility, µep.
Overall, the separation of ions and anions of this column was predominantly
under the electrophoresis basis due to the late elution of thiourea, a EOF marker.
However, the interaction of solute towards the stationary phase was a combination of
eletcrophoretic, ion-exchange as well as the hydrophobic interaction with the alkylchains. The short term stability of the ODS/DMAPAA-Q silica monolith capillary
column was excellent with RSDs ranging from 0.2–0.6% while its long term stability
was good (RSDs of 0.3-1.1%) for a period of three successive days.
Although the plug-flow profile was also generated on conventional CZE, the
comparison of separation efficiency for anions obtained from CZE was lower. The
values of theoretical plates and plate height were between 39600-46500 plates/min
and 9.0-7.6 µm respectively. In another aspect, the mobilities of the ions were higher
than in the ODS/DMAPAA-Q column, indicating that the ions were slightly faster
eluted with the CZE. Lower efficiency and ions mobilities observed with the CZE
was due to the counter-EOF generated across the column. Unlike in CEC, separation
in CZE under co-EOF is impossible without the addition of EOF modifier. The
short-term repeatability and long-term reproducibility were excellent with RSDs
values of 0.1-0.4% and 0.8-1.0% respectively.
As a conclusion, the ODS/DMAPAA-Q silica monolith capillary column
besides being used as a stationary phase in µLC, it also showed potential as a
67
electrochromatographic separation medium on CEC.
As a monolith separation
medium, the analytes were separated within the shortest analysis of time, both on
µLC and on CEC. An enhanced in the separation efficiency of the column was
observed on CEC than on µLC and CZE. Highest theoretical plate, along with the
lowest plate height was due to the separation medium itself, and the plug-flow profile
generated across the column that produces sharp, narrow and compact band of
analyte. The evaluation of ODS/DMAPAA-Q silica monolith capillary column with
CEC demonstrated it to be useful separation technique that could offers separation of
high efficient for the inorganic anions as compared to the conventional CZE.
5.2
Suggestion
Although results obtained for the inorganic anions separation on the
ODS/DMAPAA-Q silica monolith capillary column was good, the column should be
further evaluated for its performance for the separation of organic species having
hydrophobic properties, organic and inorganic ions as well as hydrophilic substance
such as amines.
Good examples of separation of these types of analytes, separately and in
mixtures would make this work more significant. The behavior of amines on the
ODS/DMAPAA-Q silica monolith capillary column will be of interest as these
compounds provide problems with the reversed-phase mode such as tailing peak and
longer analysis time.
The advantage of the ODS/DMAPAA-Q silica monolith
capillary column shown by the separation impedance, E, is also useful to describe the
total performance of column based on the required time and column backpressure to
produce one theoretical plate.
Study on the effect of domain size (through-pore size + silica skeleton size)
and the ratio between through-pore size and skeleton size on the performance of the
column is important for the complete characterization of the column. Study on the
factors determining band-broadening should also be carried out for the
68
ODS/DMAPAA-Q silica monolith capillary column. In the silica monolith, the
band-broadening is dominated by A-term, or the contribution of slow mass transfer
in the mobile phase, especially when the silica monolith posses large through-pore
size. A slower mass transfer (C-term of the van Deemter equation) is predicted for
the ODS/DMAPAA-Q silica monolith capillary column when evaluated under
pressure driven compared to electro-driven conditions.
The theoretical and
experimental study on band-broadening will give some fundamental knowledge in
optimizing the synthesis of mixed-mode silica monolith capillary column.
Further work on the fundamental characterization of the ODS/DMAPAA-Q
silica monolith capillary column must be conducted to study the band broadening
effect. The van Deemter plot is necessary to study the contribution of A-, B- and Cterms to band broadening.
A detail study on band broadening in the
ODS/DMAPAA-Q silica monolith capillary column should be compared with
column of ODS and DMAPAA-Q functionality respectively. Further study on the
ODS/DMAPAA-Q silica monolith stationary phase is also necessary in order to
reduce A- and C- terms contributions by increasing homogeneity of silica structure
as well as optimizing bonded polymer structures.
69
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