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University of South FloridaScholar Commons
Graduate Theses and Dissertations Graduate School
2006
Electrically charged sol-gel coatings for on-linepreconcentration and analysis of zwitterionicbiomolecules by capillary electrophoresisWen LiUniversity of South Florida
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Scholar Commons CitationLi, Wen, "Electrically charged sol-gel coatings for on-line preconcentration and analysis of zwitterionic biomolecules by capillaryelectrophoresis" (2006). Graduate Theses and Dissertations.http://scholarcommons.usf.edu/etd/2604
Electrically Charged Sol-Gel Coatings for On-Line Preconcentration and
Analysis of Zwitterionic Biomolecules by Capillary Electrophoresis
by
Wen Li
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy Department of Chemistry
College of Arts and Sciences University of South Florida
Major Professor: Abdul Malik, Ph.D. Kirpal S. Bisht, Ph.D.
Milton D. Johnston, Jr., Ph.D. Dean F. Martin, Ph.D.
Date of Approval: Feburary 3, 2006
Keywords: protein, amino acid, positively charged surface coating, negatively charged surface coating, sulfonated sol-gel column, C18-TMS sol-gel column
Copyright 2006, Wen Li
DEDICATION
To my son.
ACKNOWLEDGMENTS
I would like to express my sincere appreciation to my major professor Dr.
Abdul Malik, for his instruction, patience, and encouragement that he has shown me
during my graduate education at USF. I am also very grateful to the members of the
dissertation committee for their support and assistance. I want to recognize Mr.
Rafael Fonseca for his technical support to the CE system. Many thanks to all my
colleagues in Dr. Malik’s group for their continuous assistance, encouragement,
friendship and many stimulating conversations over the years.
This research has been partially funded by a subcontract from a grant by the
U.S. Naval Office (N00014-98-1-0848). This financial support is greatly appreciated.
The Department of Chemistry is also appreciated for their financial support
throughout my study at USF.
Finally, I would like to express my gratitude to my parents, my sisters, and my
husband. Their continuous support, encouragement and love have made this possible.
i
TABLE OF CONTENTS
LIST OF FIGURES .................................................................................................... vii
LIST OF SCHEMES..................................................................................................... x
LIST OF SYMBOLS AND ABBREVIATIONS ........................................................ xi
ABSTRACT............................................................................................................... xiv
CHAPTER ONE: CAPILLARY ELECTROPHORESIS:
THEORY AND PRACTICE ........................................................................................ 1
1.1 Development of capillary electrophoresis ................................................. 1
1.2 Fundamentals of capillary electrophoresis theory ..................................... 4
1.3 Typical capillary electrophoresis instrument ........................................... 10
1.4 Modes of operation and their applications............................................... 14
1.4.1 Capillary zone electrophoresis.................................................. 14
1.4.2 Micellar electrokinetic capillary chromatography .................... 14
1.4.3 Capillary electrochromatography.............................................. 17
1.4.4 Capillary gel electrophoresis..................................................... 19
1.4.5 Capillary isoelectric focusing ................................................... 20
1.4.6 Capillary isotachophoresis ........................................................ 20
1.5 References for Chapter One..................................................................... 21
CHAPTER TWO: REVIEW OF PRECONCENTRATION STRATEGIES
FOR CAPILLARY ELECTROPHORESIS ............................................................... 27
2.1 Introduction.............................................................................................. 27
2.2 Electrophoresis-based sample preconcentration ...................................... 28
ii
2.2.1 Normal stacking ........................................................................ 30
2.2.2 Electrokinetic injection/field amplified sample injection ......... 37
2.2.3 Sweeping................................................................................... 41
2.2.4 Capillary isotachophoresis (CITP)............................................ 45
2.3 Chromatography-based sample preconcentration techniques.................. 47
2.3.1 Low-specificity chromatographic preconcentration ................. 47
2.3.1.1 Preconcentration device ............................................. 48
2.3.1.2 Application................................................................. 53
2.3.2 High-specificity chromatographic preconcentration................. 56
2.3.2.1 Immunoaffinity chromatography ............................... 56
2.3.2.2 MIP technology.......................................................... 59
2.4 Conclusions.............................................................................................. 61
2.5 References for Chapter Two .................................................................... 61
CHAPTER THREE: SOL-GEL TECHNOLOGY AND ITS APPLICATION
IN CAPILLARY ELECTROPHORESIS ................................................................... 70
3.1 Introduction.............................................................................................. 70
3.2 Fundamentals of sol-gel process.............................................................. 71
3.3 Characterization of sol-gel materials ....................................................... 77
3.3.1 The morphology of sol-gel materials........................................ 77
3.3.2 Study of the chemical bonds within sol-gel structure............... 78
3.4 General procedures involved in the preparation of CE columns
with sol-gel stationary phases .................................................................. 79
3.4.1 Pretreatment of the capillary..................................................... 79
3.4.2 Sol solution ingredients for the fabrication of the sol-gel
stationary phases ........................................................................ 80
3.4.3 Post-gelation treatment of sol-gel stationary phases.................. 80
3.5 The application of sol-gel technology in CE ............................................ 81
3.5.1 Sol-gel technology for packed columns in CE ....................... 81
iii
3.5.2 Sol-gel open tubular CE columns ........................................... 82
3.5.3 Sol-gel monolithic columns .................................................... 84
3.6 Sol-gel technology for sample preconcentration in CE ............................ 85
3.7 References for Chapter Three ................................................................... 86
CHAPTER FOUR: POSITIVELY CHARGED SOL-GEL COATINGS FOR
ONLINE PRECONCENTRATION OF AMINO ACIDS IN CAPILLARY
ELECTROPHORESIS................................................................................................ 92
4.1 Introduction............................................................................................... 92
4.1.1 Column technology................................................................... 93
4.1.1.1 Dynamic coatings....................................................... 93
4.1.1.2 Chemically bonded coatings ...................................... 94
4.1.2 Positively charged coatings for CE columns ............................ 94
4.2 Experimental ............................................................................................ 95
4.2.1 Equipment ................................................................................. 95
4.2.2 Chemicals and materials ........................................................... 97
4.2.3 Preparation of sol-gel open tubular CE columns with
positive surface charge............................................................. 97
4.2.4 Preparation of samples.............................................................. 99
4.2.5 Procedures for extraction and preconcentration ....................... 99
4.3 Results and discussion ........................................................................... 100
4.3.1 Mechanism of extraction on positively charged sol-gel
coatings .................................................................................... 100
4.3.2 Extraction and preconcentration of amino acid
by sol-gel columns ................................................................... 103
4.4 Conclusions............................................................................................ 128
4.5 References for Chapter Four.................................................................. 128
iv
CHAPTER FIVE: NEGATIVELY CHARGED SOL-GEL COLUMN
FOR ONLINE PRECONCENTRATION OF ZWITTERIONIC
BIOMOLECULES IN CAPILLARY ELECTROMIGRATION
SEPARATIONS ....................................................................................................... 133
5.1 Introduction............................................................................................ 133
5.1.1 The production of a stable EOF.............................................. 134
5.1.2 Negatively charged coatings for CE columns......................... 135
5.2 Experimental .......................................................................................... 137
5.2.1 Equipment ............................................................................... 138
5.2.2 Chemicals and materials ......................................................... 138
5.2.3 Preparation of the sol-gel coating solutions............................ 139
5.2.4 Preparation of CE columns with a negatively charged
sol-gel coating......................................................................... 139
5.2.5 Preparation of samples............................................................ 140
5.3 Results and discussion ........................................................................... 140
5.3.1 Sol-gel reactions involved in the coating process................... 140
5.3.2 Characterization of the sol-gel coating ................................... 148
5.3.3 The zeitterionic solutes preconcentration mechanism
operated in the negatively charged sol-gel columns ............... 154
5.3.4 On-line preconcentration of zwitterionic molecules............... 157
5.3.4.1 On-line preconcentration of amino acids
using sulfonated sol-gel columns............................. 157
5.3.4.2 On-line preconcentration of proteins using
sulfonated sol-gel columns ...................................... 167
5.4 Conclusions............................................................................................ 178
5.5 Reference for Chapter Five.................................................................... 178
APPENDICES………………………………………………………………...……183
ABOUT THE AUTHOR……………………………….…………………….End Page
v
LIST OF TABLES
Table 2.1 Summary of approaches available for increasing on-column detection sensitivity in CE ···········································································29 Table 3.1 Common tetraalkoxysilane precursors and their physical properties ······················································································73 Table 4.1 Names and chemical structures of chemical reagents used in the fabrication of positively charged sol-gel columns·······························101 Table 4.2 Chemical structures and some physical properties of analytes used in the current study············································································102 Table 4.3 Sample extraction and preconcentrations on an electrically charged sol-gel column ·············································································113 Table 4.4 Sensitivity enhancement factors for four amino acids achieved on positively charged sol-gel C18-TMS coated columns ··························114 Table 4.5 Sample extraction and preconcentrations on an electrically charged sol-gel column by method 2 ·····································122 Table 4.6 Sensitivity enhancement factors for four amino acids achieved on positively charged sol-gel C18-TMS coated columns by method 2 ····································································································123 Table 4.7 Repeatability data for the preconcentration by C18-sol-gel coated column using amino acids as test solutes ······································127 Table 5.1 Names and chemical structures of the reagents used in the sol solution to fabricate negatively charged sol-gel sulfonated columns······································································································141 Table 5.2 MPTMS – to- total sol-gel precursor molar ratio in the coating sol solutions used in the fabrication of negatively charged sol-gel sulfonated columns·········································149
vi
Table 5.3 The effect of sol-gel coating on the repeatability of migration time of a neutral EOF marker, DMSO······································152 Table 5.4 Chemical structures and some physical properties of the test amino acids in this work···························································159 Table 5.5 Repeatability of sample preconcentration on a sulfonated sol-gel column using amino acids as test solutes·····················163 Table 5.6 Sensitivity enhancement factors obtained on a sulfonated sol-gel column using amino acids as test solutes·····················164 Table 5.7 Sensitivity enhancement factors (SEFs) obtained on sol-gel coated columns using proteins as test solutes···························173 Table 5.8 Illustration of the preconcentration repeatability on a sol-gel sulfonated column using conalbumin as a test sample ·····························175
vii
LIST OF FIGURES Figure 1.1 Classification of the CE techniques······························································3 Figure 1.2 Growth in the number of publication on CE since 1980 till end of 2005···································································································5 Figure 1.3 Representative functional groups on the surface of fused silica capillary······························································································7 Figure 1.4 Representation of the electrical double layer···············································9 Figure 1.5 Schematic representation of a basic capillary electrophoresis instrument··········································································11 Figure 1.6 Schematic representation of a MEKC system············································16 Figure 1.7 Schematic representations of three different types of columns used in CEC·············································································18 Figure 2.1 Family tree of sampling procedure for preconcentration in CE·················31 Figure 2.2 Mechanism of normal sample stacking······················································32 Figure 2.3 Mechanism of sample stacking in which reversed EOF is utilized············35 Figure 2.4 Illustration of capillary isoelectric focusing ··············································36 Figure 2.5 Behavior of micelles and neutral analytes during FESI-MEKC················40 Figure 2.6 Evolution of analyte zones in CSEI-sweep-MEKC···································42 Figure 2.7 High-salt stacking with a sample matrix of ionic strength greater than the separation buffer·······························································44 Figure 2.8 Representation of capillary isotachophoresis of anions·····························46 Figure 2.9 Schematic illustration of the extraction capillary used for in-capillary CE ·············································································49 Figure 2.10 Device of SPME for CE and its operation················································51
viii
Figure 2.11 Microphotograph of the analyte concentrator fabricated with antibody fragments immobilized to glass beads·······························58 Figure 2.12 Schematic depiction of the preparation of molecular imprints················60
Figure 3.1 Overview of a sol-gel process ···································································72 Figure 4.1 Schematic representation of the capillary filling / purging device ············96 Figure 4.2 Preparation of a sol-gel column for preconcentration ·······························98 Figure 4.3 Extraction of tryptophan on the sol-gel column ······································104 Figure 4.4 Illustration of the events during preconcentration and focusing of zwitterionic analytes on a positively charged sol-gel column ·············106 Figure 4.5 Illustration of the effect of a positively charged sol-gel coating on the preconcentration of alanine ··············································108 Figure 4.6 Effect of sol-gel coating on sample (asparagine) preconcentration ········109 Figure 4.7 Effect of sol-gel coating on sample (phenylalanine) preconcentration·······················································································110 Figure 4.8 Effect of sol-gel coating on sample (tryptophan) preconcentration. ·······111 Figure 4.9 Method 2 for the preconcentration of zwitterionic analytes on the positively charged sol-gel column steps········································116 Figure 4.10 Illustration of the effect of sol-gel coating on sample preconcentration (alanine) by method 2·················································118 Figure 4.11 Illustration of the effect of sol-gel coating on sample preconcentration (asparagine) by method 2···········································119 Figure 4.12 Illustration of the effect of sol-gel coating sample preconcentration (phenylalanine) by method 2·······································120 Figure 4.13 Illustration of the effect of sol-gel coating on sample preconcentration (tryptophan) by method 2···········································121 Figure 4.14 Juxtaposition of the effect of sol-gel coating on sample preconcentration and a blank run with the same method ······················125
ix
Figure 4.15 Preconcentration and separation of a mixture of two amino acids on a positively charged sol-gel column using method 2················126 Figure 5.1 Electroosmotic flow vs. buffer pH values ···············································150 Figure 5.2 Migration time repeatability for a sol-gel sulfonated column··················153 Figure 5.3 Illustration of on-line preconcentration using a negatively charged sol-gel column···························································156 Figure 5.4 Illustration of zwitterionic sample preconcentration on a negatively charged sol-gel sulfonated column using arginine as a test sample··········159 Figure 5.5 Illustration of zwitterionic sample preconcentration on a negatively charged sol-gel sulfonated column using lysine as a test sample··························································································161 Figure 5.6 Illustration of zwitterionic sample preconcentration on a negatively charged sol-gel sulfonated column using asparagine as a test sample··························································································162 Figure 5.7 Preconcentration of asparagine on negatively charged sol-gel coated column. ································································166 Figure 5.8 Preconcentration of conalbumin on a negatively charged sol-gel coated column··································································168 Figure 5.9 Preconcentration of myoglobin on a negatively charged sol-gel coated column··································································169 Figure 5.10 Influence of sol solution MPTMS composition on the preconcentration of myoglobin on a negatively charged sol-gel column························································································171 Figure 5.11 Repeatability of myoglobin preconcentration on a negatively charged sol-gel sulfonated column·························································174 Figure 5.12 Effect of matrix removing media on preconcentration effectiveness using a sol-gel coated column ·········································177
x
LIST OF SCHEMES Scheme 3.1 Sol-gel reaction························································································75 Scheme 3.2 Acid-catalyzed and base catalyzed sol-gel reaction mechanisms············76 Scheme 5.1 Illustration of the complete hydrolysis of sol-gel precursors·················142 Scheme 5.2 Condensations of hydrolysis products from TMSO, MPTMS, and C18-TEOS······························································································143 Scheme 5.3 Covalent bonding of the sol-gel coating to fused silica surface·············145 Scheme 5.4 Deactivation of the sol-gel mediated fused-silica coated surface with PheDMS············································································146 Scheme 5.5 Oxidation of mercaptopropyl group into sulfonic acid moiety by H2O2··············································································147
xi
LIST OF SYMBOLS AND ABBREVIATIONS 1,7-DX 1,7-dimethylxanthine 1-MX 1-methylxanthine 4-OHC 4-hydroxycoumarin 7-OHC 7-hydroxycoumarin AFM atomic force microscopy (AFM) AIBN α,α`-azobis(isobutyronitrile) Arg arginine Asp aspartic acid BGE background electrolyte C18-TEOS n-octadecyltriethoxysilane
C18-TMS N-Octadecyldimethyl[3-(trimethoxysilyl)propyl] ammonium chloride
CGE capillary gel electrophoresis CIEF capillary isoelectric focusing CITP capillary isotachophoresis CSEI cation-selective exhaustive injection CZE capillary zone electrophoresis dc inner diameter of the column DMSO dimethylsulfoxide ∆P pressure difference across the column DSC differential scanning calorimetry (DSC) ε dielectric constant of the buffer E applied electric field ECD electrochemical detectioin EDTA ethylenediaminetetraacetic acid EOF electroosmotic flow ES-MS electrospray mass spectrometry FASI field amplified sample injection FASS field-amplified sample stacking FSCE free solution capillary electrophoresis FTIR Fourier transform infrared spectroscopy
xii
Glu glutamic acid η viscosity of the buffer HCB high-conductivity buffer HEPES N-(hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) His histidine HPAA hydroxyphenylacetic acid HPLC high performance liquid chromatography L length of the column. Linj average length of the sample, LLE Liquid-liquid extraction LOC limit of detection LPME liquid-phase microextraction MEKC micellar electrokinetic chromatography MIP molecularly imprinted polymer MPA 3-mercaptopropionic acid MPTMS Mercaptopropyltrimethoxysilane MS mass spectrometry NMR nuclear magnetic resonance (NMR) OPA o-phthaldialdehyde PDMS poly(dimethylsiloxane) PEO poly(ethylene oxide) PheDMS phenyldimethylsilane pI isoelectric point PS pseudostationary phase PSG photopolymerized sol-gel PVA poly(vinly acetate) PVC poly(vinyl chloride) PVS poly(vinylsulfonate) q q is the charge of the ionized solute r r is the Stokes’ radius of the solute r-CPA replaceable cross-linked polyacrylamide RSD relative standard deviation SDS sodium dodecyl sulfate SEF sensitivity enhancement factor SEM scanning electron microscopy SPE solid-phase extraction SPME solid-phase microextraction tinj injection time TEM transmission electron microscopy TFA trifluoroacetic acid
xiii
tmc the migration time of a micellar aggregate. TMOS tetramethoxysilane Tris-base tris(hydroxymethyl)aminomethane Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride TTAB tetradecyltrimethyl ammonium bromide VEOF velocity of the electroosmotic flow VEF velocity of the electrophoretic flow VOBS observed migration velocity XPS X-ray photoelectron spectroscopy ζ zeta potential
µEF electrophoretic mobility µEOF electroosmotic mobility, ,
xiv
Electrically Charged Sol-Gel Coatings for On-Line Preconcentration and
Analysis of Zwitterionic Biomolecules by Capillary Electrophoresis
Wen Li
ABSTRACT
Novel on-line methods are presented for the extraction, preconcentration and
analysis of zwitterionic biomolecules using sol-gel-coated columns coupled to a
conventional UV/visible detector. The presented approaches do not require any
additional modification of the commercially available standard CE instrument.
Extraction, stacking, and focusing techniques were used in the preconcentration
procedures. The positively charged sol-gel coatings were created using N-
octadecyldimethyl[3-(trimethoxysilyl) proply]ammonium chloride (C18-TMS) in the
coating sol solutions. Due to the presence of a positively charged quaternary
ammonium moiety in C18-TMS, the resulting sol-gel coating carried a positive charge.
The negatively charged sol-gel coatings were due to the presence of sulfonate groups,
which was formed from the oxidation of thiol groups in precursor
mercaptopropyltrimethoxysilane (MPTMS) by hydrogen peroxide. Besides MPTMS,
tetramethoxysilane (TMOS) and n-octadecyltriethoxysilane (C18-TEOS) were also
used to prepare the sol solution for the creation of the negatively charged coatings.
For extraction, the pH of the samples was properly adjusted to impart a net charge
xv
opposite to the sol-gel coatings. When a long plug of the sample was passed through
the sol-gel-coated capillary, extraction was achieved via electrostatic interaction
between the charged sol-gel coating and the charged sample molecules. The extracted
analytes were then desorbed and focused via local pH change and stacking. The local
pH change was accomplished by passing buffer solutions with proper pH values,
while a dynamic pH junction between the sample solution and the background
electrolyte was utilized to facilitate solute focusing. The developed methods showed
excellent extraction and preconcentration effects on both positively and negatively
charged sol-gel-coated columns. On-line preconcentration and analysis results
obtained on the sol-gel coated columns were compared with those obtained on an
uncoated fused silica capillary of identical dimensions using conventional sample
injections. The described procedure provided a 150 000-fold enrichment effect for
alanine on the positively charged sol-gel-coated column. On the negatively charged
sol-gel-coated column, the presented sample preconcentration technique provided a
sensitivity enhancement factor (SEF) on the order of 3 x 103 for myoglobin, and 7 x
103 for asparagines. The developed methods provided acceptable repeatability in
terms of both peak height and migration time.
1
CHAPTER ONE CAPILLARY ELECTROPHORESIS:
THEORY AND PRACTICE
1.1 Development of capillary electrophoresis
As a rapid, high-efficiency analytical technique, capillary electrophoresis (CE)
techniques have been successfully used in chemical 1-3, biochemical 4-6, biomedical 7-9,
pharmaceutical 10-12, environmental13-15, and a host of other areas 16-18. Electrophoresis is
defined as “the movement of electrically charged particles or molecules in a conductive
liquid medium, usually aqueous, under the influence of an electric field” 19. The
separation based on electrophoresis can be traced back to 1930’, when Tiselius conducted
research on the electrophoretic analysis of colloidal mixtures in 1937 20. In 1948, he was
awarded the Nobel Prize in Chemistry due his research in electrophoresis and adsorption
analysis as well as breakthrough discoveries concerning the complex nature of the serum
proteins. In 1940s and early 1950s, anti-convective media such as paper and gels were
introduced to minimize thermal effects caused by application of an electric field to an
electrolyte solution 21.
Capillary electrophoresis emerged in 1960s. Scientists started performing
electrophoresis in narrow diameter tubes to reduce and eliminate problems associated
with the using of larger tubes, which include diffusion caused by convection, and Joule
heat due to poor heat dissipation, etc. Hjerten used capillary of 300 µm internal diameter
to carry out electrophoretic separations in 1967 22. In 1974, Virtanen 23 performed
electrophoresis in small diameter Pyrex tubing with internal diameter 200 to 500 µm. His
research is regarded as one of the earliest demonstrations of the advantages of capillary
electrophoresis 17. Five years later, Mikkers and co-workers used 200 µm i.d. Teflon
capillaries for capillary zone electrophoresis. Their research results showed that when
electrophoresis was performed in capillary the convection problems had been reduced 24.
In 1980s, the inner diameter of the capillary used for electrophoresis was further reduced
to 75 µm in the work by Jorgenson and Lukacs 25. Commercial instruments for capillary
electrophoresis separation technique appeared in 1980s and numerous research groups
2
were attracted to this research field. By the end of 1990s, capillary electrophoresis
separation techniques had reached to some what content of maturity level. Capillary
electrophoresis on microchips is an emerging new technology, which has made great
strides since late 1990s. This miniaturized analytical technology has the potential to assay
hundreds of samples in a very short time (minutes or less). And samples can be prepared
on-board for a completed integration of sample preparation and analysis. These features
make CE on microchips an attractive technology for the next generation of CE
instrumentation26.
Based on the column chemistry and operation theory, CE technique perform by
different operation modes, including capillary zone electrophoresis (CZE), micellar
electrokinetic chromatography (MEKC), capillary electrochromatography 27, capillary
gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), and capillary
isotachophoresis (CITP). Figure 1.1 shows the classification of electrophoresis
techniques.
Compared to other separation techniques, such as gas chromatography and high
performance liquid chromatography, CE has its unique features. The most significant
advantage of CE over other separation techniques is smaller volume of samples and
reagents required. With a typical commercial CE instrument, only a few nanoliters of
sample are sufficient for the repetitive analysis 18. This small consumption of reagents
enables reduced cost. In addition, CE uses buffer solution as the mobile phase, which
imposes much less adverse effect on the environment as hazardous organic solvents
which are widely used as mobile phase in HPLC. Secondly, solutes analyzed in CE move
through a capillary column as a relatively flat profile, rather than the parabolic flow in
HPLC. The sample zones in CE do not spread as much as in HPLC and GC. As a result,
greatly enhanced sepration efficiencies can be obtained. Other advantages of CE include
faster separation times, ease of automation, and availability for analysis of both polar and
nonpolar analytes.
3
Electrophoresis
Slab-GelElectrophoresis
PaperElectrophoresis
CapillaryElectrophoresis
Electrophoresis on Microchip
CZE MEKC CEC CGE CIEF CITP
CZE: capillary zone electrophoresis; MEKC: micellar electrokinetic capillary chromatography
CEC: capillary electrochromatography; CGE: capillary gel electrophoresis
CIEF: capillary isoelectric focusing; CITP: capillary isotachophoresis
Figure 1.1 Classification of the CE techniques
4
As a relatively new family of techniques for the separation and analysis of
chemical compounds, capillary electrophoresis has attracted more and more attentions of
scientists from various areas since 1990. During the past 25 years, the publications
associated with capillary electrophoresis have been increasing dramatically. Based on the
searching results with database SciFinder Scholar, there are 26771 references containing
“capillary electrophoresis” as entered published during the year from 1981 to the end of
2005. Figure 1.2 shows the distribution of the references based on publication years.
1.2 Fundamentals of capillary electrophoresis theory
Separation in CE depends on the difference in solute migration velocity in an
electric field 16-19, 21. Electrophoretic mobility and electroosmotic mobility are two
important factors affecting the migration velocities of charged solutes under the influence
of an electric field. The observed migration velocity ( OBSν )of molecules is the sum of
the velocities due to electrophoretic flow and electroosmotic flow, and can be shown as
EOFEPOBSννν += (1.1)
where,
EPν is the velocity of the electrophoretic flow
EOFν is the velocity of the electroosmotic flow, both are in cm/s.
Application of voltage across a capillary makes charged solutes migrate through
the conductive media toward the oppositely charged electrode, which is termed as
electrophoretic flow. It depends on the strength of the applied electric field and
electrophoretic mobility of the molecules. Therefore, it is given by
EEPEP
µ=ν (1.2)
where,
E is the applied electric field
µEP is the electrophoretic mobility.
5
0
500
1000
1500
2000
2500
3000
1981 1984 1987 1990 1993 1996 1999 2002
year
num
ber
of r
efer
ence
s
Figure 1.2 Growth in the number of publication on CE since 1980 till end of 2005 (based
on personal search of SciFinder Scholar 2004, up to end of 2005)
6
The strength of applied electric field is the ratio of the applied voltage and the
length of the separation capillary. The electrophoretic mobilities (EF
µ ) of charged
analytes are determined by the properties of the analytes and the buffer, and can be
described by this equation,
ηπ=µ r6/qEF (1.3)
where,
q is the charge of the ionized solute
η is the buffer viscosity
r is the Stokes’ radius of the solute.
The unit of electrophoretic mobility is cm2/V⋅s. From equation (1.3), it follows
that under a certain applied electric field and buffer, the greater the charge-to-size ratio
(q/r), the higher the electrophoretic mobility.
Electroosmotic flow (EOF), the motion of an electrolyte solution, is another
important driving force in CE separation. Analogous to the velocity of electrophoretic
flow, the velocity of electroosmotic flow is given by
EEOF
µEOF =ν (1.4)
Where,
E is the applied electric field
µEOF is the electroosmotic mobility.
In CE, EOF is generated when the following three conditions are met 16, 18, 19, 21: (a)
an electric field is applied across the separation column; (b) a conductive buffer solution
is placed inside the column; and (c) the inner surface of the column acquires charges due
to the existing of the buffer solution. In the case of fused silica capillary used as the
column, the capillary wall is irregularly distributed with silanol groups (-SiOH). Figure
1.3 schematically shows surface structure of a fused silica capillary. The silica surface
acquires a charge when in contact with an electrolyte solution, as expressed by the
following equation 28.
SiOH SiO- + H+ (1.5)
7
O
Si
H
OOSi
OSi
OH
O
OH
SiO
O
SiO
Si
O
OSi
O
O
H
Si
OH
O
O
H
H
Figure 1.3 Representative functional groups on the surface of fused silica capillary.
Reproduced from Reference29.
8
The dissociation constant of silanol groups on the fused silica capillary surface is
estimated to be pKa ≈ 7.5 30. However, it is generally recommended that the point of zero
net charge of fused silica is around pH = 2 18, 19, 21. Consequently, the silica surface is
negatively charged when an electrolyte solution with pH above 2 is used as the running
buffer. The negatively charged silanoate groups attract positively charged cations present
in the buffer solution and an electrical double layer is formed. This double layer is
composed of a fixed layer which is tightly held by the silanoate groups and a diffuse layer,
which is further away from the silanoate groups 19, 21, 29. Under the influence of an
electric field, the diffuse layer of cations then migrates toward the cathode. Since the
cations are believed to be hydrated, a bulk flow of the whole solution within the capillary
is generated 19, 21, 29, 31-33, as shown in Figure 1.4.
The electroosmotic mobility, EOFµ , is given by
πηεζ=µ 4/EOF (1.6)
where,
ε is the dielectric constant of the buffer ζ is the zeta potential
η is the viscosity of the buffer.
Electroosmotic mobility is analogous to electrophoretic mobility, and has the
same units, cm2/V⋅s. For neutral compounds, the electroosmotic flow is the general
transport mechanism in electrophorectic experiment. For charged compounds, the
apparent electrophoretic mobility depends on both electrophoretic and electroosmotic
flow. An important feature EOF possesses is its relatively flat flow profile, which
contributes to its high separation efficiency compared to parabolic flow profile generated
in high-performance liquid chromatography (HPLC) via mechanical pumping. Under
typical experimental conditions, electroosmotic flow is generally greater than
electrophoretic flow 19, 21. A stable EOF is desired to produce consistent analytical results
in CE. If the EOF changes with time, the migration times of the solutes will change,
which may result in misleading the identification and quantitation problems 19.
9
Fu
sed
silic
a ca
pilla
ry w
all
N
NN
N
N
N
N
Fixed layer Diffuse layerPlane of shear
Figure 1.4 Representation of the electrical double layer. Reproduced from Reference34.
10
1.3 Typical capillary electrophoresis instrument
A typical capillary electrophoresis system consists of a high-voltage power supply,
a piece of capillary tube, a detector, sample vials, buffer vials, and a data processing
device. Figure 1.5 is a schematic representation of a capillary electrophoresis system.
A high-voltage power supply is a prerequisite for electrophoresis to occur. The
application of the high-voltage power supply provides high separation efficiency, short
separation time, and improved resolution between peaks 19, 35-37.
In CE, fused silica capillary is the most widely used as the separation column,
although the use of Teflon and Pyrex capillaries have also been reported 38. In many
commercial instruments, the capillary is retained in a cartridge-like device, which is
conjunct with cooling systems. Capillaries with 25 – 100 µm inner diameter and 30 – 100
cm in length are normally used. To make an optical window for on-column detection, the
outer polyimide layer is removed either by scraping or burning. The advantages of the
capillary format include effective heat dissipation and small amount of sample and
mobile phase required. On the other hand, since the inner diameter of the capillary is also
the optical pathlength when UV/vis absorbance or fluoroscence detector is used, the
concentration sensitivity is reduced 18, 19, which is an often-cited drawback of the
technique.
In capillary electrophoresis, only a small volume of sample, usually a few
nanoliters, is required. It is suggested that the sample plug length should be no more
about 1% of the total capillary length to achieve high column efficiency and resolution 21,
39, 40. Either hydrodynamic injection or electrokinetic injection can be used to introduce
samples into CE system. Hydrodynamic injection can be performed by either pressure or
gravity. Compared to electrokinetic injection, this method is usually more precise and
robust but may not be suitable for the use of packed columns. The average length of the
sample, injL , introduced by pressure is given by
L32/PtdLinjinj
2c η∆= (1.7)
11
Run Inject
Autosampler tray
Thermostatted compartment
Power supply
buffer sample
Detector
CE column
Collector bufferAnode Cathode
Figure 1.5 Schematic representation of a basic capillary electrophoresis instrument.
Reproduced from Reference17.
12
where,
cd is the inner diameter of the column
∆P is the pressure difference across the column
injt is the injection time
η is the viscosity of the background buffer
L is the length of the column.
Electrokinetic injection is accomplished by application of a voltage while the inlet
of the separation column is placed in the sample vial. The sample zone generated by
electrokinetic injection is plug-like, but this method is normally associated with sampling
bias 41. The length of the injected zone, injL by electrokinetic method is given by
L/tv)(L injinjEPEOFinjµ+µ= (1.8)
Where,
injV is the voltage applied for the sample injection
EOFµ is electroosmotic mobility
EPµ is electrophoretic mobility
injt is the injection time
L is the length of the column.
Temperature is an important parameter which should be maintained constant
throughout the CE operation to ensure precise analysis. For example, when temperature is
increased, the migration time decreases due to the reduced viscosity of the running buffer.
Since the viscosity of the solution changes with the temperature, the volume of the
injected sample will be different at various temperatures. Thermostatted compartment
allows active temperature control for the column, which is important in obtaining
reproducible migration times 42-44. Although, absolute temperature has little effect on the
separation efficiency, temperature does affect the resolution in some specific cases. It has
been reported recently by Nevado and coworkers 45, during the separation and
13
determination of omeprazole enantiomers in pharmaceutical preparations, resolution
decreased with increase of the temperature due to limited solute-cyclodextrin interaction.
A proper choice of background electrolyte provides stable conditions for CE
separation. Aqueous buffer solutions are the most commonly used as background
electrolytes. An ideal buffer used in CE should possess the following properties: (1)
reasonable water solubility, (2) good buffering capacity in the pH range of choice, (3)
low absorbance at the wavelength of detection 21. Properties of the buffer solution which
affect CE separation include its composition, pH, and concentration. Under constant
capillary temperature, the EOF will decrease with the increase in buffer concentration 46.
The pH of the buffer will influence the ionization of the solutes as well as the silanol
groups on the surface of fused silica capillary 38, the latter being directly related with the
magnitude of EOF.
Once a sample has been resolved into various compartments, it is necessary to
detect the separated zones passing through the column. Most commercial CE instruments
are equipped with a UV/visible absorption detector providing wavelength selection from
190 to 700 nm 17, 19, 21. UV/visible detectors are easy to use and applicable to most
analytes. For UV/Visible detectors, they are usually used on-column to avoid band
broadening 21. However, in this case, effective optical path length of the light equals the
inner diameter of the capillary. And as a result, the absorption signal is relatively low.
Some molecules lack chromophores and cannot be detected by UV/Visible detectors. In
order to visualize these molecules, there are some alternative methods, such as
fluorescence 47, 48, electrochemical detectors 48-50 and mass spectrometry 51-53. Laser-
induced fluorescence 54 is an important detection option for the analysis of trace level
analytes in CE. Since it uses fluorescence of the solutes, derivatization is often needed to
chemically label the solute molecules with a flurophore. Electrochemical detection is
based on the measurements of the current generated from an oxidation or reduction
reaction of the analyte at an electrode surface. This method has high selectivity and
sensitivity for some compounds but is used to only a limited extent in CE 21. The use of
mass spectrometric detection for CE can provide more important information about the
compounds. For example, it enables the identification of chemical structures. However, it
is relatively difficult to couple CE with an MS detector due to the use of buffer.
14
1.4 Modes of operation and their applications
1.4.1 Capillary zone electrophoresis
Capillary zone electrophoresis (CZE) is the most widely used and the simplest
operation mode among various CE separation modes. When molecules are charged, they
can be separated by CZE. In CZE, the separation column is filled with a homogeneous
running buffer, and separation occurs in a free solution. Therefore, CZE is also referred
to as free solution capillary electrophoresis (FSCE) 19. CZE separation is based on
differences in migration velocities of the analytes. Since the electrophoretic mobilities of
cations and anions have opposite directions, they can be separated in the same run. And
cations and anions are separated based on their charge-to-size ratios. On the other hand,
since neutral analytes do not have electrophoretic mobilities, they are carried through the
capillary by EOF at the same velocity. As a result, they cannot be separated in CZE.
Under the influence of a normal electric field (e.g. the polarity of the electric field is from
positive to negative), the order of elution in CZE is: cations, neutrals, and finally anions.
In principle, analytes with different charge-to-size ratios can be separated by CZE.
Many inorganic ions as well as organic ions have been separated. In order to obtain the
best separation, optimization of operation conditions has been investigated. Zare and co-
workers 55used tetradecyltrimethylammonium bromide (TTAB) as flow modifier and
quantitatively analyzed some low molecular weight carboxylic acids in the form of
anions (fomate, acetate, propanoate, butanoate, pentanoate and hexanoate). Macka et al. 56 separated several metallochromic ligands by CZE and MEKC.
1.4.2 Micellar electrokinetic capillary chromatography
Unlike in CZE where separation takes place in free solution, micellar
electrokinetic capillary chromatography (MEKC) utilizes a micellar pseudostationary
phase created from a surfactant or mixed surfactants dissolved in the running buffer
above its critical micelle concentration 21. Surfactants contain a hydrophobic and a
hydrophilic part in their structure. When the concentration of a surfactant in an aqueous
solution exceeds its critical micelle concentration, aggregated structures known as
micelles are formed. Due to the presence of the micelles, the analytes partition between
15
the running buffer and the pseudo-stationary phase, and become separated. Surfactants
are normally charged and move inside the capillary due to their electrophoretic motility
under the influence of an electric field. If an analyte is insoluble in the formed micelles, it
spends all its time with the running buffer and migrates inside the capillary at the same
speed as EOF. If an analyte is totally soluble in the micelles, it migrates at the same speed
as the micelles. For those analytes which partially soluble in micelles, the migration
speed will be different from both EOF and the speed of micelles. Thus, it is important
that the chosen surfactants are charged in some cases, otherwise, MEKC will not take
place. Both chromatographic and electrophoretic principles are represented in MEKC.
The advantage of MEKC over CZE is that it provides a mechanism for the separation of
neutral as well as ionized solutes 57-60. Figure 1.6 schematically shows the separation
principle of a micellar electrokinetic chromatography system. With enhanced selectivity
due to the addition of a surfactant into the background electrolyte, recently MEKC has
been successfully employed by Ewing and co-workers 61 for the separation of biogenic
amines and metabolites in the fruit fly, Drosophila melanogaster. Separations of 14
analytes of biological relevance extracted from the fruit fly were performed in 25 mM
borate buffer containing 50 mM sodium dodecyl sulfate (SDS) and 2% 1-propanol.
Electrochemcial detection (ECD) was utilized. Their results show that the developed
borate- MEKC-ECD system provides a valuable tool to distinguish and monitor
fluctuations in metabolites in the studied subjects and possible application to other
animals.
Trapp et al. 62 reported efficient MEKC methods with laser induced fluorescence
detection for the separation and quantitation of asymmetric dimethyl-L-arginine and L-
arginine in human plasma samples. The influence of buffer and surfactant concentrations
had been investigated and the optimum performance was obtained when 193 mM borate
solution with 100 mM deoxycholic acid as the surfactant was utilized. The strong
influence of buffer and surfactant solution concentration on the peak resolution
demonstrates that both electrophoretic and electrokinetic separation mechanism play
important roles into separation process.
16
Figure 1.6 Schematic representation of a MEKC system. Here, t0 is the migration time of
a neutral ‘unretained’ solute, tR is ‘retention’ time in MEKC, tmc is the migration time of a
micellar aggregate. Reproduced from Reference 63.
17
1.4.3 Capillary electrochromatography
Capillary electrochromatography 27 effectively combines two major separation
techniques: capillary zone electrophoresis (CZE) and high-performance liquid
chromatography (HPLC). Consequently, it possesses their inherent advantages: high
separation efficiency, tunable selectivity and remarkable versatility in separation and may
potentially become a viable alternative to HPLC, micro HPLC and CZE 64. CEC uses
similar instrumention as CZE, and similar columns as microcolumn liquid
chromatography. But unlike CZE, it provides a separation mechanism of both neutral and
charged analytes. In a CEC system, the mobile phase flow through the column is
maintained by an electric field instead of a high-pressure pumping system used in HPLC.
Since the electro-driven flow profile is essentially flat, increased separation efficiency is
achieved in CEC. Compared with MEKC, the surfactant-free mobile phases used in CEC
make it ideally suited for hyphenation with mass spectrometry 65.
In CEC, like in all other chromatographic techniques, stationary phase is a
critically important element directly affecting the separation of analytes. Based on the
format of the stationary phases, the columns used in CEC are classified into three
categories: packed columns 66, 67, monolithic columns68 and open-tubular columns 69, 70.
In CEC, the stationary phase has two-fold roles: (1) it provides chromatographic
interactions with various types of solute molecules, and (2) the CEC stationary also
facilitates the generation of electroosmotic flow through the column. Figure 1.7 is a
schematic representation of different types of columns in CEC. The CEC separation of
neutral compounds is an analogue to reversed-phase liquid chromatography, while CEC
separation of charged analytes is based on a mechanism combining both electrophoresis
and reversed-phase liquid chromatography. During the last decades, CEC, with its unique
separation methodology, has achieved great accomplishment in the separation of a wide
variety of analytes, such as proteins and peptides 71-75, nucleoside and nucleic acid
recognition 76-80, carbohydrates 67, 77, 81-84, and inorganic analytes 1, 85-87.
CEC continues to play an important role among analytical techniques, because a
wide range of stationary phases and thus operation modes are available. Lin et al. 78, for
18
packed bed
open segment
A
frits detection window
B
detection windowopen-tubular coating
C
open segment
detection windowmonolith
polyimide coating capillary wall
open-tubular
monolith
polyimide coating capillary wall
polyimide coating capillary wall
Figure 1.7 Schematic representation of three different types of columns used in CEC: (A)
a typical packed capillary column (adapted from Reference88); (B) an open-tubular
capillary column; and (C) a monolithic capillary column.
19
example, utilized an open-tubular wall-coated macrocyclic polyamine capillary column
separated and determined uridine 5’-monophosphate, guanosine 5’-monophosphate,
adenosine 5’-monophosphate, and cytidine 5’-monophosphate in the tested sample.
Recently, Ohyama et al.89 has developed a CEC method using 3-(1, 8-naphthalimido)
propyl-modified silyl silica gel (NAIP) as the stationary phase to separate caffeine and its
two metabolites 1-methylxanthine (1-MX) and 1, 7-dimethylxanthine (1, 7-DX). This
method also has been successfully coupled with microdialysis and employed to monitor
the caffeine concentration in rat brain.
1.4.4 Capillary gel electrophoresis
Capillary gel electrophoresis (CGE) is adaptation of traditional gel electrophoresis
using capillary columns filled with a polymer solution acting as a molecular sieve. It is
usually used for the separation of biopolymers such as proteins and nucleic acids. When
the solutes migrate through the sieving medium, larger molecules become hindered more
than smaller ones leading their separation. CGE is most suitable for the separations of
molecules with similar mass/charge ratio but different sizes and shapes. Chemical gels
and physical gels are two typical sieving mediums used in CGE. With small mesh
spacings (controlled by adjusting the ratio of acrylamide and the crosslinking reagent),
poly(acrylamide) is almost exclusively used as a chemical gel. Recently, an alternative
sieving matrix, a replaceable cross-linked polyacrylamide (rCPA) was developed for the
separation of a wide range of proteins (~ 4 kD to ~300 kD) by sodium dodecyl sulfate
(SDS)-CGE 90. When compared with most frequently used sieving matrixes, this
developed rCPA was found to be able to permit highest resolutions and faster separation
speed for protein separations.
A methodology for the separation of some oligonucleotide mixtures by CGE has
been developed by Bayer’s research group91. Their method utilized fused silica capillary
(100 µm i.d.x 50 cm), which was permanently coated with poly(vinyl acetate) (PVA).
The gel they used was prepared by dissolving PEG 35000 20% (w/v), 20 mM
bis(hydroxyethyl)amino-tris(hydroxymethyl) (Bis-Tris), 20 mM boric acid and 20% (v/v)
acetonitrile in high-purity water and shaken overnight. Since the PVA coating suppressed
the EOF, analytes migrated inside the capillary exclusively by their electrophoresis. This
20
CGE system was coupled with electrospray mass spectrometry (ES-MS) as detection
unit. Oligonucleotides up to 20 bases in length were successfully separated by the
develop CGE/ES-MS system.
1.4.5 Capillary isoelectric focusing
Capillary isoelectric focusing (CIEF) separates amphoteric analytes based on their
isoelectric points (pI) in a pH gradient covering the range of pI’s of the sample 19, 21. In
CIEF, the electroosmotic flow is eliminated. The outlet buffer vial contains a catholyte
with a high-pH solution, while the inlet buffer vial contains an anolyte with a low-pH
solution. Before separation occurs, the capillary is filled with ampholytes with different
pI values to create a pH gradient along the capillary when a voltage is applied. The
analytes migrate through the capillary and rest at their own isolelectric points, at which it
moves in neither direction due to a net charge of zero, and accumulate into distinct bands.
After this, the focused sample zones are forced through the detector by adding a salt to
either the anolyte or catholyte 92, 93.
The usefulness of CIEF for protein analysis in biological fluids such as human
serum, cerebral spinal fluid, whole cells and cell is well demonstrated 94-98. Crowley and
Hayes 99 utilized off-line coupling of CIEF with matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-TOF MS) for the analysis of human
blood serum. Both pI and mass information were obtained from the collected biological
sample. The newly developed platform demonstrates advanced features such faster (hours
versus days), more automatable, and simpler compared with traditional techniques.
1.4.6 Capillary isotachophoresis
Capillary isotachophoresis (CITP) 100, is an important operation mode in CE. This
method is also employed for sample stacking. CITP is performed in a capillary by
injecting the sample between two discrete buffer plugs: a leading buffer with a higher
mobility ion and a terminating buffer with a lower mobility compared to the analytes of
interest 19, 21. Anions and cations cannot be separated in the same run in CITP mode.
Under the influence of an electric field with a constant current, the anions or cations in
the buffers and the sample migrate toward the anode or cathode and arrange themselves
in order of mobility. Since electroosmotic flow is usually suppressed, all the buffer and
21
analyte ions migrate at the same velocity with the leading ions. CITP has long been used
as a separation as well as a very effective preconcentration method for inorganic ions 101-
104. Inorganic ions build a group of compounds with different nutritional value found food
and feed samples. Their determination in these samples is a specific area of CITP
application. Phytic acid in cereal grains, legumes, and feed was determined by CITP 102.
The electrolyte system consists of 0.01 M hydrochloric acid and 0.0056 M bis-tris-
propane (pH 6.1) as the leading electrolyte, and 0.005 M 2-morpholinoethanesulfonic
acid as the terminating electrolyte. The described method shows quite good
reproducibility, expressed by the relative standard deviation of 3.8%.
In addition, CITP has found application in the analysis of pharmaceuticals and
drug product formulation 105-108. CITP with conductometric detection was used for the
assay determination of tolfenamic, flufenamic, mefenamic, and niflumic acids in drug
product formulations 107. The method used 10 mM HCl/20 mM imidazole aqueous
solution (pH = 7.1) as the leading electrolyte and 10mM 5,5-diethylbarbituric acid
aqueous solution with pH 7.5 as the terminating electrolyte. Analysis can be
accomplished in 20 minutes.
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27
CHAPTER TWO REVIEW OF PRECONCENTRATION STRATEGIES
FOR CAPILLARY ELECTROPHORESIS
2.1 Introduction
Capillary electrophoresis (CE) is a highly efficient separation technique and
possesses a number of advantageous features including high overall separation efficiency,
short analysis times, and small amounts of reagents or samples required. The application
range of CE is possibly the most diverse of all analytical techniques 1-3. With its several
operation modes (free zone, micellar, gel, isotacophoresis, and isoelectric focusing), CE
can be used to analyze a wide range of compounds from large, complex macromolecules,
such as proteins, peptides, nucleotides and nucleic acids, to small solutes, such as organic
drugs and inorganic anions and cations.
Commercial instruments have been available since 1988. Separations are
normally performed employing voltages in the region of 5-30 kV over a piece of fused
silica capillary, typically 25-100 µm i.d. and 25-100 cm in length. Commercial
instruments are commonly equipped with a UV absorbance detector or a diode array
detector. Some instruments also offer the capability of fluorescence, laser-induced
fluorescence, or electrochemical detection. Most CE separations are performed using an
on-column detection mode to prevent loss of separation efficiency due to extracolumn
band broadening that usually takes place if an off-column detection cell is used.
Consequently, the choice of capillary internal diameter largely governs the sensitivity of
the method when UV detector is used as the detection method. However, the necessity of
effective dissipation of Joule heating in the CE column, and to preserve high separation
efficiency, one is forced to use capillaries with small inner diameter (25 ~ 75 µm). Due to
the short path length (equal to the inner diameter of the column), on-column UV
detection on such small-diameter columns is characterized by low concentration
sensitivity, which is the main limitation of C in trace-level analysis and detection of
compounds such as toxins, drug residues, or metabolites in biological samples. In order to
get the maximum sensitivity, the inner diameter of the capillary may be extended to 100
28
µm if necessary, but at the expense of longer analysis and reduced separation efficiency
caused by the necessity to use reduced voltage and/or electrolyte concentration because
of the internal heating problems arising from the increased capillary diameter. In order to
maximize the detection sensitivity in CE, a range of strategies have been used. Table 2.1
shows some of these strategies including the use of low UV wavelengths, increased
capillary diameter, and optimized sampling procedure. Over the past decades, a series of
studies have been undertaken to improve the on-column detection sensitivity in CE. The
developed methods (e.g., increasing the bore of the capillary, changing the operation
voltage or modifying the capillary) have their own strengths and drawbacks as shown in
Table 2.1. Optimizing the sampling procedure to preconcentrate the target analytes is
especially attractive since it involves no additional modification of the commercially
available standard CE instrument, and it can be easily accomplished by carefully
controlling the operation conditions.
Commonly used sampling techniques for preconcentration in CE can be divided
into two different categories 4: (a) electrophoresis-based sample preparation; and (b)
chromatography-based sample preparation. These categories can be further divided into
several sub-categories. Figure 2.1 shows the family tree of sampling procedures for
preconcentration, and was constructed based on several reviews published during last
decade 4-10.
2.2 Electrophoresis-based sample preconcentration
The preconcentration method related to electrophoretic processes is based on the
differences in mobility and conductivity of sample and running buffer systems. Normal
stacking, field amplified sample injection, sweeping and isotachophoresis and dynamic
pH junctions are electrophoresis-based sample preconcentration techniques that are
commonly used in CE.
29
Table 2.1 Summary of approaches available for increasing on-column detection
sensitivity in CE. Reproduced from Reference1
Action to improved sensitivity Drawbacks to consider
Employ low-UV wavelength Increased background noise----determine
wavelength to give optimum signal-to-
noise ratio
Increase capillary bore Increased current, reduced EOF giving
possible alternation in selectivity
Increase injection time Reduction in separation efficiency;
excessive time will result in run failure
Appropriately use electrokinetic injection Sampling bias for more mobile ions,
sample matrix effects on injection amount
Increase electrolyte strength Increased current, increased Joule heating,
and associated noise
Optimize electrolyte composition Effects on selectivity and current
Decrease operating voltage Increase in analysis time
Decrease temperature Increase in analysis time
Modify the capillary Reduction in separation efficiency and
resolution, increased cost
Derivatize the sample Additional sampling handling
Perform indirect detection Extra method development considerations
Use wide-bore capillaries EOF profile disturbed, adjustments to rinse
and injection times, siphoning effects more
pronounced
Increase detector slit width Reduction in separation efficiency and
resolution
30
2.2.1 Normal stacking
Sample stacking was first used by Ornstein in disc electrophoresis in 1964. It is
generally used to describe zone-sharpening effects resulting in sample concentration
phenomenon 11. In CE, stacking is one of the most widely used sample preconcentration
techniques, in which the velocity of analyte ions is changed by using discontinuous
buffers 12-14. The sample is usually dissolved in an electrolyte solution that has a lower
conductivity than the running buffer, and the injection is made hydrodynamically. When
a high voltage is applied, a higher electric field is developed in the injected sample
characterized by lower conductivity than in the more concentrated running buffer because
of the higher resistance of the sample zone. As known, electrophoretic velocity is
proportional to electric field. Therefore, the analyte ions migrate more rapidly in the
dilute sample solution than in the running buffer. This feature makes the ionic analysts
stack at the boundary between the sample plug and the buffer and forms a narrow
“stacked” zone 12, 13, 15 1, 16-20. Under a normal voltage polarity, cations should be stacked
at the front of the sample plug, while anions are stacked at the rear of the sample plug.
Neutral solutes are not stacked since they are forced through the capillary only by the
influence of electroosmotic flow. Figure 2.2 schematically illustrates sample stacking. By
using this method, Chien and Burgi 12 preconcentrated phenyl-thiohydantoin-aspartic acid
(PTH-Asp), PTH-glutamic acid (PTH-Glu), PTH-arginine (PTH-Arg) and PTH-histidine
(PTH-His). Their study showed the possibility of preconcentration and baseline
separation of PTH-Asp and PTH-Glu. In clinical application, sample stacking technique
was used in capillary electrophoresis-mass spectrometry system by Clench and co-
workers 21. The sample they tested was urine, obtained from five male smokers. Their
analytes of interest, nicotine and its metabolites in urine were extracted by solid-phase
extraction. The solutes then obtained were analyzed using sample stacking capillary
electrophoresis-mass spectrometry. The electrolyte they used for CE analysis was 10 mM
ammonium formate dissolved in a mixture of 75% acetonitrile and 25% deionized water.
The pH of the buffer was adjusted to 2.5 using HCl or formic acid. Results shows sample
stacking procedure with MS detection achieved the LODs (limit of detection) of nicotine
and cotinine as 0.11 and 2.25 µg/mL respectively.
31
Figure 2.1 Family tree of sampling procedure for preconcentration in CE (based on several reviews published during last decade 4-10).
32
+
+
+
EOF
EOF
EOF
Inject sample
Apply voltage
Electrophoresis proceeds
Anode
Anode
Anode
Cathode
Cathode
Cathode
Figure 2.2 Sample stacking with a sample dissolved in a solution, such as deionized water,
that has a lower conductivity than the electrophoresis run buffer. The circles represent a
cationic solute. Top: the sample plug is injected. Middle: voltage is applied and since the
electric field in the sample solution is higher than in the rest of the capillary, the cations
rapidly migrate through the sample solution until they reach the low electric field in
buffer, where they slow down and become stacked at the boundary between the solutions.
Anions are stacked at the back of the sample plug. Bottom: the stacked ions migrate
through the capillary as a zone that is narrower than the sample plug. Reproduced from
Reference20.
33
As shown in Figure 2.2, the migration speed of the analytes is changed by the
discontinuous buffer system and the concentration ratio of the sample buffer and support
buffer play important roles in achieving optimum separation 14, the preparation and
composition of discontinuous buffers greatly affect the preconcentration effect. Studies
have shown that discontinuous buffers can be prepared simply by addition of salts into
buffer, by dissolving the sample in a low ionic strength buffer, or by adjusting their pH
values 19, 20, 22. Aebersold and Morrison 23 reported a method to achieve stacking by pH
gradient within the capillary. The sample plug with a high pH was introduced inside the
capillary surrounded by buffer at a low pH. This results in the migration of anionic
peptides toward the anode when the high voltage is applied. When the analytes reach the
acidic (low pH buffer zone), the charge reverses and the migration direction is reversed.
The sample is then stacked. A novel strategy for the formation of a sharp pH gradient was
developed by Wei and Yeung 22. Their method is based on electrochemical reactions
occurring at the ends of a short platinum wire which was inserted into the separation
capillary. After the application of a high voltage, OH- and H+ were generated at the ends
of Pt wire. A pH gradient was then formed along the capillary, while those created ions
began to titrate the buffer ions and distribute down the column.
It was found that large volume injection accompanying sample stacking normally
cause some problems such as broadened peaks and increased temperature in the diluted
sample zone 24, 25. Meantime, when a large volume of sample is introduced into the
separation column for stacking, the solute zone is as wide as the length of the sample
plug. In order to minimize the problems caused by the large volume sample injection,
several techniques had been developed to achieve a narrow stacked sample band for
further analysis. Chien and Burgi 12 introduced a method of stacking, in which a reverse
EOF was used to back out the sample matrix after injection into the capillary. The
reversed EOF can be generated by switching the polarity of the applied voltage. For
instance, if the separation voltage is from anode to cathode, the sample matrix should be
removed out of the capillary by an applied voltage from cathode to anode. This procedure
is illustrated in Figure 2.3. First, a large volume of dilute sample solution (e.g., an
injection length of 65 cm in a 100 cm long capillary column) was injected into the
column. A negative voltage (from cathode to anode) was then applied to the column ends
34
to obtain an electroosmotic flow (EOF) toward to the capillary inlet. Consequently, the
sample matrix was gradually pushed out of the capillary by the EOF and the anions were
stacked at the boundary between sample solution and the background electrolyte. The
resulting sample zone was narrow. When the separation voltage with polarity was
opposite to the back-out voltage polarity was applied, CE separation of the sample
components was achieved in the capillary under the normal EOF.
Chien and Burgi 12 compared the separation efficiency in CE using sample
stacking procedure with and without sample matrix removal. The results showed that the
peaks obtained by stacking with sample removal were narrower than those without
sample removal. The large volume sample injection stacking method with sample matrix
removal greatly enhanced the separation efficiency. Using this strategy, Kim and
coworkers 17 successfully preconcentrated some protains and an enhancement factor of
more than 100 was achieved. A mixture of chlorophenols and chlorophenoxy acids was
preconcentrated and separated by Marriott and co-workers 26 using sample stacking
strategy. Compared to standard CE analysis, 40- and 10- fold improvement of sensitivity
was obtained by using sample stacking with and without the removal of sample matrix,
respectively. Recently, this sample stacking technique was reported to have been used in
the determination for metallothioneins in rabbit and eel livers 27. This same research
group 28 also used this technique to determine some metal element, such as Cd, Cu, and
Zn speciation in fish liver metallothioneins.
In addition to the method invented by Chien and Burgi 12 to focus the sample by
reversed EOF, capillary isoelectric focusing (CIEF) 29-35 is another widely used focusing
technique in CE. In this approach, differences in the isoelectric points (pIs) of analytes
are utilized to achieve the focusing effect. First, the separation capillary is filled with a
solution of ampholytes. Upon application of an electric field across the capillary, a pH
gradient is formed along the length of the ampholyte plug. When zwitterionic analytes are
injected into such a capillary, they electrophoretically migrate through the pH gradient of
the ampholyte solution. When a charged analyte reaches a location in the ampholyte plug
where pH equals the pI value of the analyte, it becomes electrically neutral, and ceases to
move via electrophoretic migration. Without the influence of EOF, focused discrete
neutral analyte zones line up along the capillary based on their pI values. Figure 2.4
35
+
+
+
EOF
EOF
Inject sample hydrodynamically
Reverse polarity and apply voltage
Reverse polarity
Anode
Anode
Anode
Cathode
Cathode
Cathode
+ EOF
Electrophoresis proceeds
Anode Cathode
(A)
(B)
(C)
(D)
Figure 2.3 Sample stacking of a large sample volume. The circles represent a mixture of
two anionic solute. (A) A large volume of sample is hydrodynamically injected. (B) A
negative voltage is applied at the capillary inlet, causing the anions to stack and the
sample solution to be forced out of the capillary. Cations and neutrals, not shown, are
also forced out of the capillary at the inlet. (C) When most of the sample solution has
been removed, the polarity is reversed. (D) The stacked anions are separated by CZE.
Reproduced from Reference20.
36
+ -Anolyte Catholyte
Anode Cathode
pH GradientLow pH High pH
Capillary
pI
Solutes
Figure 2.4 Illustration of capillary isoelectric focusing (CIEF). The capillary is filled with
a mixture of ampholytes and the sample containing zwitterionic solutes. When an electric
field is applied, the solutes migrate to the locations in the capillary where the pH = pI for
individual solutes, and are focused into narrow zones. Solutes and ampholytes are
mobilized to force them through the detector. The anolyte is an acidic solution with a pH
lower than the pI of the most acidic ampholyte, and the catholyte is a basic solution with
a pH higher than the pI of the most basic ampholyte. Reproduced from Reference36.
37
illustrates the steps of CIEF. CIEF is widely used for analytes with different pI values 30-
35. This technique is reported to be used with mixtures with very small differences in
isoelectric points (as low as 0.01 pH unit). And an enhancement factor of 500 was
achieved for polypeptide mixtures resulting from digestion of proteins 34. There are
several review papers published recently on the theory and application of CIEF 37-40.
2.2.2 Electrokinetic injection/field amplified sample injection
In CE, the sample is introduced into the capillary by different techniques, in
which the most commonly used methods are hydrodynamic and electrokinetic injections.
Hydrodynamic injection is done either by pressure or siphoning. Siphon injection is also
known as gravity injection, which is performed by raising the sample vial above the
capillary making the sample to be introduced into the capillary. Pressure injection is
carried out by pressurizing or vacuuming the sample into the capillary.
In electrokinetic injection mode, an electric field is applied over the capillary
length which causes sample to migrate into the capillary by electroosmotic flow and
electrophoretic mobility 2, 3, 20, 41, 42. The amount of analytes injected depends on the
electrophoretic mobility of the analytes in the sample, electroosmotic flow rate, applied
voltage, capillary dimensions, and sample concentrations.
Field-amplified sample injection (FASI) is a sample stacking technique operated
by electrokinetic injection. The principle of FASI is the same as normal stacking. The
main difference between FASI and normal stacking technique is that in normal stacking
the focusing process occurs when the separation electric field is applied, while in FASI,
the process occurs during injection. In order to produce an amplified electric field on the
sample zone, the sample is prepared in a solution of smaller ionic strength than the
running buffer. When a high voltage is applied, the electric field over the sample zone is
much higher than that in the running buffer, which causes the solutes migrate rapidly into
the capillary. When the solutes reach the running buffer zone with lower electric field,
they slow down and get concentrated. It was found when the capillary was switched
directly from the running buffer to the dilute sample buffer, the boundary was disturbed
and the electric field at the inject point might not be amplified properly, which leads to a
lower concentration enhancement than expected. This problem was successfully solved
38
by Chien and Burgi 43 with introduction of a water plug prior the injection of the dilute
sample. With this modification, a high electric field was produced at the injection point
and several hundred-fold enhancement was obtained. The advantage of FASI over
normal stacking method is that a larger quantity of solute ions and just a small volume of
dilute buffer are injected. Consequently, the problems associated with injection of a large
volume of dilute buffer solution, such as the overall electroosmotic velocity changes,
production of laminar flow due to mismatched velocities, are limited.
FASI technique has been frequently used in various fields. Its application include
the determination of derivatized amino acids 44, small inorganic 45-47 and organic 48, 49
ions in water. In biological applications, pH-mediated sample stacking was used to the
analysis of coumarin metabolites in microsomal incubations 50. The metabolites studied
included 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC) and 2-
hydroxyphenylacetic acid (HPAA). Low limits of detection at the range of 0.1-0.5 µM
was achieved for those analytes when UV absorbance detection was employed. Yin 45
and Zhang et al. 46 applied this method to concentrate different arsenic species. Velocity
difference-induced focusing based on the pH changes in sample and run buffer zones was
used in their studies. In Yin’s study, detection of limits (LOD) obtained was as low as
5.0-9.3 µg. L-1 when atomic fluorescence spectrometry was used for detection. Finally,
both researchers showed the applicability of their methods to real environmental samples.
Sample stacking has been used not only in aqueous CE but also in nonaqueous CE.
It was reported by Kim and Chung 51 that methanol was used as the running liquid in
nonaqueous CE to preconcentrate and separate ten organic acids in the concentration
range of 10-100 nM.. After CE separation they were detected using conventional UV
absorption detection. Excellent linear responses and reproducibility in the migration
times, and corrected peak areas were obtained.
Electrokinetic injection depends on the electrophoretic mobilities of the solutes as
well as the EOF. Solutes with high mobilities are injected in a larger amounts than those
with lower mobilites. This phenomenon is called sampling bias, which is one of the
drawbacks of FASI. However, sampling bias is not regarded as a serious problem if the
samples are all dissolved in the same solutions and the concentrations are similar 20. In
39
order to determine and concentrate neutral compounds, field-enhanced sample injection
micellar electrokinetic chromatography (FESI-MEKC) was used to get sample
preconcentrated 52. Figure 2.5 shows the mechanisms and steps contributing to FESI-
MEKC. As Chien and Burgi 43 described, the introduction of a water plug prior to the
sample plug provided a higher electric field. In FESI-MEKC, this water plug also
provided a fast pathway for the micelles to carry the neutral analytes. In this process, an
anionic surfactant butylacrylate-butyl methacrylate-methacrylic acid copolymers, sodium
salt (BBMA), served as the pseudostationary phase, and was used in both sample and
buffer solutions. Due to the enhanced electric field in the water plug, the electrophoretic
velocities were higher than the bulk electroosmotic flow (EOF). During the electrokinetic
injection, micelles carrying the neutral analytes entered the capillary and got stacked in
the front portion of the water plug. At the meantime, the water plug was pumped out by
the EOF. Once the water was forced out the capillary, the inlet of the capillary was put
inside a vial containing the running buffer and the polarity of the electric voltage is
switched. This step is followed by the separation and detection. Using this FESI-MEKC
procedure, more than 20- and 100- fold improvements were obtained for estradiol. Using
normal stacking, the same research group also successfully preconcentrated and analyzed
neutral compounds (e.g., resorcinol, 1-naphthol, and 2-naphthol) in MEKC 53. For
resorcinol, the limit of detection (S/N = 3) was in the order of 10-7 M with UV detection.
Sample stacking was first used in CE with low-concentration electrolyte by Chien
and Helmer in 199124. Since then, it has been extensively studied over last decade.
Besides “field amplified sample injection (FASI)”, it is also called as “head column
sample stacking” and “field-amplified sample stacking” (FASS) 11. Overall, sample
stacking is an important technique to increase the detection sensitivity in many
electrophoresis systems.
With the development in microchip-based capillary electrophoresis 54-56, sample
stacking techniques have also been used in the microfluidic devices. A few orders of
magnitude in sample enrichment have been reported 57.
40
B1detector
water plug BGS
y z
xxx
xy
y
yy
zz
zz
xxx
yyy
zzz
yy
z z
x
x
xB1
detector
detectorB1
detector
x
A
B
C
D
Figure 2.5 Behavior of micelles and neutral analytes during FESI-MEKC. (A) initial
situation (water plug, unshaded; micellar separation solution shown as BGS , shaded); (B)
micelles enter the capillary and carry neutral analytes emanating from the cathodic vial.
The migration order of neutral analytes is dependent on their retention factors to micelles;
(C) micelles and neutral analytes stacked at the concentration boundary (shown as B1),
voltage is cut and the sample vial is replaced by another BGS vial when the measured
current is approximately 97-99% of the predetermined current, voltage is then applied at
positive polarity; (D) separation of zones develops. Reproduced from Reference52.
41
2.2.3 Sweeping
Sweeping analytes in MEKC was introduced by Quirino and Terabe 58-60. It
involves the interactions between a pseudostationary phase (PS) in the separation solution
and a sample. The theoretical model of this technique is that the analytes are swept by the
pesudostationary phase (micelles, polymers, dendrimers, etc.) which migrates through the
sample zone and gets the sample focused. In this technique, the sample is prepared in a
solution having a lower, similar, or higher conductivity than the background solution
(running buffer). Analytes are preconcentrated because of chromatographic partitioning,
complexation, or any interaction between analytes and PS. It is noticed that the length of
the sample zone is dependent on the retention factor 58. For both neutral and charged
compounds, the length of the sample zone is shorter for analytes with higher retention
factors. Sweeping combined with field-enhanced sample injection (FESI), which was
named as cation-selective exhaustive injection and sweeping (CSEI-sweep), was used to
concentrate naphthylamine. Outstanding improvement in sensitivity as high as million-
fold was achieved 60. The procedure of CSEI-sweep is shown in Figure 2.6. At first, the
capillary was filled with a high-conductivity buffer devoid of organic solvent (HCB)
followed by a short zone of water. The cationic sample prepared in a low-conductivity
solution was then electrokinetically injected into the capillary for a long time (about 10
min). After that, the sample zone was focused by means of sweeping by placing a low-pH
buffer solution containing anionic micelles or micellar BGS in the inlet reservoir upon
the application of a negative voltage. Separation was achieved by MEKC. The effect of
preconcentration is dictated by the strength of the interactions involved. Sensitivity
enhancements from tens to several thousand-fold have been achieved.
In sweeping, both neutral and charged PS can be used based on the properties of
the analytes. For neutral analytes, only charged PS are available for their separation. On
the other hand, uncharged PS makes it possible to separate many important charged
molecules. In this case, the sample penetrates the neutral PS zone to start the sweeping
procedure other than the penetration of PS into the sample zone.
Additionally, Landers and coworkers have made significant contribution to
sample preconcentration via sweeping, especially for the samples from high salt matrixes 61-63. Contrary to conventional sample stacking procedure, the samples were prepared in
42
Figure 2.6 Evolution of analyte zones in CSEI-sweep-MEKC: (A) starting situation,
conditioning of the capillary with a nonmicellar background buffer, injection of a high-
conductivity buffer void of organic solvent, and injection of a short water plug; (B)
electrokinetic injection at positive polarity (FESI) of cationic analytes prepared in a low-
conductivity matrix or water, nonmicellar background buffer found in the outlet end;
cationic analytes focus or stack at the interface between the water zone and high-
conductivity buffer void of organic solvent zone; (C) injection is stopped and the micellar
background solutions are placed at both ends of the capillary, shows the profile of the
analytes after FESI; (D) application of voltage at negative polarity that will permit entry
of micelles from the cathodic vial into the capillary and sweep the stacked analytes; (E)
separation of zones based on MEKC. Reproduced from Reference60 .
43
high-conductivity matrixes, which is the key to their technique in micellar capillary
electrophoresis (MCE), more commonly known as MEKC. Since the separation buffer
has a lower conductivity compared with that in sample matrix, a filed amplification
occurs within the separation buffer zone, which leads to stacking of the charged micelles
at the detector side of the sample matrix and separation buffer interface. The stacked
micelles then sweep the neutral compounds through the capillary to the detector. The
proposed mechanism of the technique is illustrated in Figure 2.7. The advantage of this
method is it allows free optimization of separation buffer parameters including
concentration, pH, ionic strength, and organic modifier without affecting the sample
stacking method. This novel, robust, and widely applicable technique does not only solve
the low sensitivity problem in CE but also the problems associated with the sample
preconcentration from high-salt matrixes, which is frequently met in real-life situations.
As discussed above, the sweeping techniques used by Quirino used both
continuous 58 and discontinuous 60 buffer systems. In contrast, Landers and coworkers 61-
63 utilized the advantages of discontinuous buffer systems. Since high micelle
concentration was used in the sample matrix, a stacking of micelles at the
sample/separation buffer interface occurred. Therefore, this technique is also called high-
salt stacking. Following the stacking of micelles, the stacked micelles entered the sample
zone and carried or swept the sample through the capillary and got detected. Recently, the
principles of high-salt stacking and sweeping has been compared and clarified in a paper
by Palmer 64.
No matter what kind of buffer system and PS phase is used, the purpose of
sweeping is to preconcentrate the analytes and analyze them. Britz-McKibbin 65, 66
developed a preconcentration method involving dynamic pH junction and sweeping
modes of focusing. Dilute flavins in biological samples with as low as picomolar
concentrations have been examined by capillary electrophoresis with laser-induced
fluorescence detection. Gavenda and coworkers 67 added sodium dodecyl sulfate (150
mM) into the background buffer as the PS phase. Trace amounts of biologically active
anthracyclines were analyzed with UV absorption detection. Some basic drugs and their
metabolites have been preconcentrated and analyzed by sweeping technique in capillary
44
Figure 2.7 High-salt stacking with a sample matrix of ionic strength greater than the
separation buffer. High-conductivity sample matrix induces a stacking of the cholate
micelles at the interface (t = 1). Analytes (e.g. corticosteroid) experience a reduction in
velocity upon encountering the stacked micelle / sample zone interface (t = 2). The
chloride in sample matrix has diffused and results in a neat interface of the sample matrix
with the stacking micelles (t = 3). Reproduced from Reference61.
45
zone electrophoresis using field-amplified sample injection (FASI) by Taylor and co-
workers 68.
Since sweeping is applicable to both neutral and charged analytes, it is regarded
as a highly versatile and effective on-line preconcentration technique in CE 69.
2.2.4 Capillary isotachophoresis (CITP)
Capillary isotachophoresis (CITP) 70 can also be employed for sample
preconcentration. CITP is accomplished in a capillary where the sample is sandwiched
between two discrete buffer plugs: a leading buffer with a higher mobility ion and a
terminating buffer having a lower mobility ion than the charged analytes. Upon the
application of an electric field with constant current, the ions existing in the sample are
distributed into narrow and concentrated zones based on the differences in their
mobilities. Figure 2.8 schematically shows the procedure of CITP, in which solute 1 has
the highest mobility and solute 3 has the lowest mobility. Transient ITP is a technique in
which sample is concentrated and separated by CZE in the same capillary 71, 72. During
the separation process, the buffer is changed and ITP does no longer occur. Therefore, the
term “transient” is used to name the technique 71. Similar to the principle of CITP, in
transient ITP sample is injected between a leading and a tailing electrolyte. After the
solutes are concentrated in ITP, separation of the analyte zones occurs.
On-column transient ITP technique has been used to preconcentrate and separate
various analytes including inorganic 73, 74 75, organic 76, 77 and biomolecules 78. Tu and
Lee 73 used chloride ion as the leading ion and dihydrogenphosphate as the terminating
ion in ITP to determine the nitrate in seawater. Since the mobility difference for chloride
and nitrate ions is small, a zwitterionic surfactant [3-(N, N-
dimethyldodecylammonio)propane sulfonate] was added to the background buffer and
the selectivity was successfully increased 73. Iodide and iodate in seawater was
determined by this technique 75. Several metal cations including La3+, Ce3+, Pr3+, Nd3+,
Sm3+, and Eu3+ in low-ppb range have been determined by transient ITP with α-
hydroxyisobutyric acid as the complexing agent to enable the separation of analyte ions
that have similar mobilities 74. Shihabi 77 has developed a method in which some water
miscible organic solvents such as acetonitrile, acetone and small alcohols were used as a
46
Inject sample and buffers
Terminating
Buffer
Leading
Buffer
Terminating
Buffer
Leading
Buffer3 2 1
Solute Mixture
Apply electric field
Separated Solutes
Mobility
+
+
Cathode
Cathode
Anode
Anode
Figure 2.8 Representation of capillary isotachophoresis of anions. A leading buffer is
selected which has anions with higher mobility than any solute anions. A terminating
buffer is selected with anions that have lower mobility than any solute anions. When an
electric field is applied, the solutes migrate to form individual zones on the basis of their
mobilities with solute 1 having the highest mobility and 3 the lowest. The length of each
zone is proportional to the amount of solute in the zone. After the solutes migrate into
their zones, an equilibrium is established and the buffers and solutes migrate through the
capillary. Reproduced from Reference20.
47
terminating ion in transient isotachophoresis, while salts existing in samples function as
leading ions. This technique has been suggested to be termed as transient “pseudo-
isotachophoresis” (pseudo-ITP). Compared to traditional ITP, pseudo-ITP is much easier
to perform. Tyrosine and theophylline were preconcentrated and separated using the
developed method. Transient isotachophoresis was also used to perform on-line
preconcentration of enantiomers (clenbuterol). Prior to the enantioseparation, ITP was
used to concentrate the sample, in which a counterflow was used to increase the sample
loadability of the system and the CZE separation path length. The counterflow was
achieved by a counterpressure that counterbalanced the electrophoretic migration of the
compounds. The chiral selector used in this research was dimethyl-β-cyclodextrin 76. In
the area of biology, human insulin, a polypeptide was concentrated by the use of transient
ITP 78.
2.3 Chromatography-based sample preconcentration techniques
Chromatography-based methods represent an important category of
preconcentration techniques. Compared to electrophoresis-based preconcentration
methods, chromatography-based techniques are more selective. Based on the adsorbent, a
wide range of analytes can be preconcentrated by these technoliques. As shown in Figure
2.1, it is further divided into two sub-categories as low-specificity and high-specificity
chromatographic methods. In this section, principles, mechanisms, applications and the
practical aspects of chromatographic-based preconcentration techniques are discussed.
2.3.1 Low-specificity chromatographic preconcentration
Solid-phase extraction (SPE) is the most widely used low-specificity
chromatographic preconcentration method. SPE is used to isolate and preconcentrate
target analytes from a gas, fluid or liquid sample by passing it through a sorbent bed,
thereby allowing their transfer to and retention on the solid sorbent. The sorbent with the
extracted analytes on it is then isolated from the sample and the analytes recovered by
elution with a liquid or fluid, or by thermal desorption 79. In SPE-CE approach, samples
are purified and extracted from a liquid mixture onto the solid adsorbent where it is
concentrated, and then, the concentrated sample is released from the adsorbent and
48
analyzed by CE. Various SPE matrices including C2-, C8-, and C18-bonded silica have
been reported 80-82.
2.3.1.1 Preconcentration device
In-line SPE-CE technique allows the completion of sample purification and
preconcentration in a single step. Viberg et al. 80 have developed a miniaturized device to
analyze and detect heterocyclic aromatic amines 83 by micro solid-phase extraction
coupled to CE (µSPE-CE) with nanospray ionization (nESI) mass spectrometric (MS)
detection. The schematic illustration of the extraction capillary for this process was
shown in Figure 2.9. As shown in this Figure, it was made of an inlet capillary, an
extractor and separation capillary column. The capillary was first conditioned by
methanol and water. Sample solution was then introduced. Salt and hydrophilic
substances were eluted by water and electrolyte, while HAs were extracted by the packed
bed. Methanol was then used to elute the HAs out of the capillary for further analysis.
Compared to the limit of detection (LODs) obtained from HPLC-atmospheric-pressure
chemical-ionisation MS analyses, µSPE-CE-nESI-MS technique provided 10-100-fold
improvement in LODs for HAs.
Because of the use of packing material and the frits, the electroosmotic flow (EOF)
usually is disturbed in such a µSPE-CE device by the increased back pressure. In addition,
in-line SPE methods are often associated with some disadvantages such as the loss of
resolution, peak broadening, and peak tailing. In membrane preconcentration (mPC-CE) 84, 85, these drawbacks can be overcome, in which the bed of solid phase are replaced with
membranes with conventional LC stationary phases. The mPC-CE cartridge was
constructed with membrane adsorptive phase inserted inside a piece of Telfon tubing84.
The fused silica capillary for CE separation was connected to the ends of the Telfon
tubing.
Visser et al. 86 developed an interface, through which an SPE and CE parts are
coupled together. First, sample was transferred from the sampling loop to the SPE
cartridge by using the loading solvent. After desalting, the extracted sample was desorbed
by the elution solvent and transferred to the interface. A hydrodynamic injection was
performed when the sample passed the micro-injection vial inside the interface. The
49
Figure 2.9 Schematic illustration of the extraction capillary used for in-capillary µSPE,
CE and nESI. A: The inlet capillary serves as a transfer line to the extractor. B: The
extractor is a short packed bed of particles for extraction and purification of the sample. C:
In the CE-nESI capillary the sample molecules are separated by CE and electrosprayed
into the mass spectrometer. Reproduced from Ref. 80.
50
interface was then flushed with the CE electrolyte, after which, the CE separation was
started.
Solid-phase microextraction (SPME) is a solvent-free sample preconcentration
technique developed by Belardi and Pawliszyn 87. In SPME the sample solution is
exposed to a sorbent coating applied to a fiber surface and the analytes are extracted by
this coating. SPME and SPE are similar. Compared to SPE, no clean-up step is necessary
for SPME. With many advantages including simplicity, rapid extraction, solvent-free,
SPME has been used with gas chromatography with automation 88-93. SPME-HPLC
makes it possible to analyze less volatile or thermally labile compounds 94, 95. Trace
impurities such as PAHs, ketones, and alkylbenzenes in aqueous samples have been
preconcentrated and analyzed by SPME-HPLC with titania-based material functioning
extraction 96. The applications of SPME-CE have been reported by several research
groups during last decade 97-100.
Li and Weber 99 developed a preconcentration device for CE, which is very
simple, inexpensive and solvent waste free. The device was made of a stainless steel
extraction rod coated with poly (vinyl chloride) (PVC), a Teflon tube and a microsyringe.
The extraction device and operation was illustrated in Figure 2.10. First, the analytes
were exposed to the PVC coated rod. Secondly, the extracted analyte was transferred into
an aqueous back-extraction solution through the Teflon tube. Finally, the back-extraction
solvent containing interested analytes was collected and injected into CE system to be
separated. With this device, it took less than 30 min to complete extraction, back-
extraction, and separation of 10 barbiturates. Due to the small sample volume in CE, this
device is difficult to couple the CE on-line. Another device developed by Nguyen and
Luong 98 utilizes an optical fiber with poly(dimethylsiloxane) (PDMS) coating. Analytes
were absorbed in the PDMS coating, and then released into CE system. A piece of 2 cm
long heat shrinkable tubing and a piece of 1.5 cm long capillary segment was used to
connect the extraction rod and the separation capillary with zero dead volume at the
interface. A normal CE separation can be carried out after the releasing the extracted
analytes by methanol. The technique described by Nguyen and Luong 98 realized the
direct connection between the extraction fiber and the inlet end of separation capillary in
CE system, compared with Weber’s off-line method 99. Besides, Whang and Pawliszyn 97
51
Figure 2.10 SPME device and operation in hyphenation with CE . (1) Put the extraction
rod in the sample solution for a specific time. (2) Inject 5 µL of back-extraction buffer
solution into the Teflon tube. (3) Put the extraction rod into the Teflon tube. The droplet
of the back-extraction buffer solution can be forced to cover the whole surface of the
PVC coating by adjusting the position of droplet with the syringe handle before or after
placing the rod. (4) Take out the rod after letting it stand horizontally for a specific time.
The majority of back-extraction solution will form a droplet near the end of the tube. (5)
Collect the solution by moving the droplet spanning the diameter as a piston and transfer
the drop to an injection vial. Reproduced from Reference 99.
52
designed an on-column interface for the coupling of the SPME into CE. Since their
device facilitated the direct insertion of the SPME fiber inside the injection end of the CE
capillary, the extracted analytes can be completely desorbed into the separation capillary.
The fiber-in-tube SPME device developed by Jinno et al. 101 is composed of a fiber-
packed capillary, which possesses the dual feature of being an extraction medium as well
as a separation medium.
Liquid-liquid extraction (LLE) and liquid-phase microextraction (LPME), two
other techniques are also used for the sample preconcentration in GC, HPLC, and CE as
low-specificity chromatographic preconcentration methods102-111. Liquid-liquid extraction
normally uses a supported liquid membrane (SLM). An organic liquid phase is
immobilized in the pores of the support, for instance, a membrane. Generally, a water-
immiscible organic solvent including pentane, hexane, diethyl ether, ethyl acetate,
chloroform and methylene chloride is used to extract hydrophorbic components from
aqueous samples. The basic theory of LLE, its compatibility with CZE, and applications
have been discussed and reviewed 109. LLE provides excellent clean-up for sample
preparation. On the other hand, the loss of analytes in the evaporation step greatly limits
its attractiveness for CZE.
The miniaturized verstion of LLE is called liquid-phase microextraction (LPME).
Compared with LLE, LPME can achieve higher preconcentration effects using greatly
reduced volume of the solvent. A disposable LPME device was developed by Pedersen-
Bjergaard and co-workers 106, which consisted of a vial containing the sample solution
(donor solution), a porous hollow fiber impregnated with an organic solvent, a screw top
with silicone septum, two needles connected to the ends of the hollow fiber. During the
extraction, the acceptor solution was injected into the hollow fiber with one of the
needles the extracted analytes in the acceptor solution were collected by another needle.
Chung and coworkers 111 developed a simple and efficient sample preconcentration
method for CE using liquid-phase microextraction (LPME). The extraction is completed
by transferring the analytes twice between liquid-liquid phases. First, the aqueous sample
solution was exposed to a droplet of acceptor (e.g., a thin layer of octanol on the surface).
Analytes were extracted from the original sample solution (so-called donor phase) into
octanol layer. The pH value of the sample was previously adjusted so that the sample had
53
higher distribution in the organic solvent. After the equilibrium was achieved, the
extracted analytes were back-extracted into the separation buffer (so-called acceptor
phase). For this, the pH of the acceptor phase was carefully adjusted so that the analytes
had higher solubility than in octanol. After copletion of the LPME procedure, the
acceptor phase containing concentrated sample was injected into the CE column for the
separation and analysis of the extracted solutes.
2.3.1.2 Application
SPE-CE, SPME-CE and LLE-CE and LPME-CE are becoming increasingly
popular in analytical chemistry. Their applications include preconcentrating and
analyzing a wide range of analytes, generally applied to biological and environmental
samples.
In order to monitor therapeutic drug, recently Fonge and co-workers 112 used off-
line SPE to extract tobramycin from human serum. A carboxypropyl bonded phase (CBA)
cartridge was used. The extracted analyte was then eluted from the CBA by a mixture of
NH3 (25%, w/v in water)-methanol (30:70, vol./vol. in water). After evaporation of the
solvent, the analyte was derivatized with o-phthaldialdehyde (OPA)/3-mercaptopropionic
acid (MPA). The tobramycin-OPA/MPA derivative was analyzed by CZE using UV
detector at 230 nm. A limit of detection (LOD) of 0.1 µg/mL was achieved, which
indicates the sensitivity of this method. Lingeman et al. have published several papers
based on the applications of SPE-CE, such as the determination of amphoteric
compounds in biological samples, including determination of sulfonamides in blood,
serum, and urine 113, 114, online dialysis-SPE-CE of acidic drugs in urine and serum 115,
the determination of the anticoagulant phenprocoumon in plasma and urine 116. Peptides
in the low nanograms per mL range has been extracted and concentrated by method
developed by Landers et al. 117 118. In the mean time, peptide mapping was accomplished
using SPE-CE system 119. A review by Martinez et al. summarized the application of
SPE-CE in the environmental analysis 120. Other applications of SPE-CE in biological
area include the determination of insulin derivatives in urine, serum and plasma by on-
line SPE-CE developed by Visser and co-workers 86, preconcentration, separation and
54
quantitation of nonsteroidal anti-inflammatory drugs (NSAIDS) in human urine and
serum 121, measurement of urinary free cortisol (UFC) by Rao and co-workers 122.
Due to its attractive features such as simplicity, speed, and solvent-free operation,
SPME has few applications in CE. The first application of off-line SPME with CE was in
biomedical area presented by Li and Weber 99. Barbiturates were extracted and analyzed.
Their extraction device was introduced in section 2.3.1.1. The extraction effect was
quantified by the preconcentration factor, which was defined as the ratio of the
concentration of analyte in back-extraction solvent to the concentration in the sample.
The extracted samples were separated in a CE system equipped with a UV detector. The
results showed that barbiturate concentrations in the range of 0.1 – 0.3 ppm in urine and
about 1 ppm in serum could be determined. In the work by Jinno et al. 101, an on-line
fiber-in-tube SPME-CE system has been developed for the separation of four tricyclic
antidepressant (TCA) drugs: amitriptyline, imipramine, nortriptyline, and desipramine.
After extraction on the fiber, the analytes were desorbed with acetonitrile and then
injected into the CE separation capillary. The running buffer for the separation consisted
Na2HPO4, β-cyclodextrin, and acetonitrile. UV detector was employed. Research results
showed that the on-line fiber-in-tube SPME-CE system is a powerful tool to determine
analytes in biological matrices. Cifuentes and co-workers123 developed a highly sensitive
procedure to detect multiple pesticides including pyrimethanil, pyrifenox, cyprodinil,
cyromazine, and pirimicarb at trace levels spiked in different food samples such as grapes
and orange juice. In this SPME procedure, commercial extraction fibers were used.
Samples were mixed with NaCl solution and the pH was adjusted to 6.0. The extraction
process was followed by desorption of the pesticides in methanol. The optimum
background electrolyte was determined as 0.4 M acetic acid at pH 4. Mass spectrometry
was used as the detector. Their approach achieved the LODs down to 15 ng/mL for the
pesticides studied. Another method based on SPME/CE/MS has been developed by Pico
et al. 124 to determine some acidic pesticides in fruits. The studied pesticides included o-
phenylphenol, ioxynil, haloxyfop, acifluorfen, picloram. Commercial SPME fibers were
used to extract pesticides from the fruits and mthanol was used to desorb extracted
pesticides. The optimum running buffer was 32 mM ammonium formate/formic acid
buffer at pH 3.1. The limits of quantification were found in the range of 0.02 - 5 mg/kg.
55
In environmental science, SPME and CE hyphenated system was used for the analysis of
polycyclic aromatic hydrocarbons 98, phenol 97, and explosives125.
The combination of liquid-liquid extraction (LLE) and CZE has been applied to
the analysis of drugs from biological fluids, such as plasma, serum, and urine126-128. The
advantages of using LLE in sample preparation include: (a) removing inorganic salts
from the sample matrix, which interfere the optimal operation conditions for sample
injection with field-enhanced sample stacking method in CE; (b) effective removing of
plasma and serum proteins which usually deteriorate the peak shapes because of their
adsorption to capillary surface. Thormann and co-workers 127 determined an
antihelminthic drug albendazole (ABZ) in human plasma using CE with on-line UV
detector. The sample preparation was conducted via liquid/liquid extraction using
dichloromethane. Extract reconstitution was performed in N-methylformamide. During
CE separation, borate buffer with apparent pH 9.9 in a mixture of methanol and N-
methylformamide (1:3) was used. The limits of detection (LODs) for ABZ, ABZSO
(ABZ’s major metabolite albendazole sulfoxide) and ABZSO2 (ABZSO’s further
oxidization product albendazole sulfone) were in the order of 10-7 M. An overview of
published methods based on LLE-CZE applications of drugs from biological fluids has
been summarized in a review by Pedersen-Bjergaard et al.109, in which the analytes,
sample matrix, extraction solvent, reconstitution solvent, enrichment detection, detection
mode as well as limit of quantification were clearly listed. Pedersen-Bjergaard and
coworkers also validated and successfully used the combination of LPME and CE to
determine chiral drugs in biological matrices 102, 106, 129. For example, the chiral
antidepressant drug mianserin in plasma was determined using LPME-CE technique.
First, the analytes were extracted with di-n-hexyl ether and further transferred into an
acidic solution. The limit of detection (S /N = 3) was 4 ng / mL when a UV detector was
employed 106.
Low-specificity chromatographic preconcentration, as shown in its name, allow
enhancement of the sensitivity but with low selectivity. Sometimes, other componenets in
the matrix may be preconcentrated in addition to the analytes of interested, which may
hinder determination of the target analytes. To avoid this interference, high-specificity
chromatographic preconcentration can be used.
56
2.3.2 High-specificity chromatographic preconcentration
When very selective isolations need to be made, high-specificity chromatography
is more appropriate than methods based on low-specificity chromatography. The most
frequently used preconcentration techniques based on high-specificity chromatography
include immunoaffinity chromatography and molecularly imprinted polymer technology.
2.3.2.1 Immunoaffinity chromatography
Immunoaffinity chromatography, sometimes called immunoadsorption
chromatography, is based on highly specific interaction of a ligand with its counterpart.
Antigen-antibodies and lectin-carbohydroattes are the best representative examples of
this kind of interaction. Because an antibody recognizes only a very small part of the
antigen, this method is highly specific. Normally immunoaffinity chromatography
utilizes an antibody or antibody fragment as a ligand which is immobilized onto a solid
support matrix in such a manner that it retains its binding capacity for the analytes. Due
to the ability to provide powerful clean-up and selective enrichment, the potential of
immunoaffinity chromatography 130 combined with CE has been demonstrated for the
determination of analytes at low levels in complex biological matrices 131, 132.
The immunoaffinity preconcentration device or enrichment chamber is composed
of a solid support with immobilized the antibody. A good solid support should have a
maximum surface to immobilize the antibodies and a minimum of hydrostatic resistance.
The simplest enrichment chamber utilizes a portion of a capillary containing an
immobilized selector on the wall to capture a specific analyte 133. The remaining portion
of the capillary is used as the actual electrophoretic separation column. A second type of
enrichment chamber uses a membrane placed in a Teflon cartridge, an antibody or
receptor is impregnated on the membrane. A third type of on-line immunoaffinity
preconcentration system contains HPLC-pakcing beads. In this case, frits are used to
retain the packing beads. Because of the high specificity of immunoaffinity
chromatographic technique, a substantial increase in concentration of analytes can be
achieved, generally in the range of 100-500 folds 134. Kennedy and Cole 135 used
capillaries packed with a perfused protein G as the chromatographic support. Antibody
was loaded on it for create an immunoaffinity stationary phase. This immunoaffinity
57
preconcentrator could be coupled with CZE either online or off-line. For on-line
combination, a flow-gated interface was needed to connect the preconcentration and
separation columns, which enabled the direct injection of the desorbed sample plug into
the electrophoresis system. For off-line coupling, the desorbed fractions were collected
and then injected into the CE system. Using this system, insulin samples was
preconcentrated and analyzed.
Immunoaffinity chromatography coupled with CE has been widely used to
identify and characterize bioanalytes. Here is an example from Guzman 9, 131, 136, 137, who
has published several papers since 1990. Immunoreactive gonadotropin-releasing
hormone (GnRH) in serum and urine specimens was determined. The controlled-porous
glass beads were incubated with FAb peptide, an antibody fragment. A piece of fused-
silica capillary was partially filled with the glass beads covalently attached with FAb
fragments (e.g., antigen binding fragment of an immunoglobulin G containing the hinge-
region cysteine); the beads were retained by two porous frits. This piece of capillary was
then fused to two longer capillary columns using a Teflon sleeve and glued with an epoxy
resin. Figure 2.11 illustrates the microphotograph of the concentrator. The enriched
GnRH was desorbed using a solution containing glycine-HCl buffer pH 2.5. For the
separation, a solution composed of sodium tetraborate (80 mM, pH 9.1) and 1% (v/v)
acetonitrile was used as the running buffer. When a UV detector was employed, 1 ng/mL
was determined as the LOD for the analysis of GnRH 136. Immunoaffinity
chromatography was also used by Dalluge and Sander 138 to prepare clinical disease-state
marker protein samples prior to capillary electrophoresis separations. In their protocol,
the receptor monoclonal anti-cTnI antibodies were covalently immobilized on the pakced
bed of porous silica. This procedure was accomplished by putting the pretreated silica
particles into phosphate buffer solution containing monoclonal anti-cTnI antibodies and
allowing the system to stand for 1 h at room temperature. After this, the nonspecifically
bound proteins were removed by washing the silica with Tris-Cl, NaCl, and KCl,
containing Tween-20 nonionic detergent, pH 7.2. This was followed by the packing of
antibody-loaded silica particles into a piece of capillary and the preconcentration column
is ready. When the sample containing cardiac troponin I (cTnI) was passed through the
preconcentration column, anti-cTnI selectively extracted cTnI due to their
58
Figure 2.11 Microphotograph of the analyte concentrator fabricated with FAb antibody
fragments immobilized to glass beads. The glass beads were retained between two frits.
The analyte concentrator device was connected to two separation capillaries by a Teflon
sleeve. The plastic connector was glued to the separation capillaries by an epoxy resin.
The entire fabrication process was monitored by a stereo microscope. Reproduced from
Reference136.
59
complementary structure features. After the sample injection, the column was rinsed
extensively with running buffer to remove any nonspecifically bound serum proteins,
salts, and other impurities. The target analyte cTnI was then released from the column
with the elution buffer consisting of Tris-acetate, β-mercaptoethanol,
ethylenediaminetetraacetic acid (EDTA), and urea, pH 3.5. The desorbed cTnI was
analyzed directly by CE coupled to a UV detector. The limit of detection (LOD) was 2
nmol/L. Selectivity, sensitivity, efficiency, and sample recovery of this method were
remarkable.
2.3.2.2 MIP technology
MIP is the abbreviation for molecularly imprinted polymer. The synthetic
materials made by MIP technology are capable of selectively recognizing specific
molecules because of the binding sites created by the template molecules complementing
molecules in size, shape and chemical functionality 139. The procedure to make MIPs
includes several steps 140. Figure 2.12 schematically shows the preparation of an MIPs.
First, an appropriate amount of a functional monomer is mixed with a cross-linker in an
organic solvent containing an initiator and a template molecule. After polymerization, the
template molecules are removed. The synthesized material has both steric (size and shape)
and chemical (spatial arrangement of complementary functionality) memory for the
template and is able to selectively rebind imprint molecules from sample mixtures. Either
the analyte of interest or a structural analogue of it can be the template molecule. For
instance, Ratner and co-workers 141 developed a method for imprinting surfaces with
protein-recognition sites. With the powerful molecular recognition feature, MIPs have
been used in several analytical tools such as liquid chromatography (LC) 142, 143, CE 144-147,
solid phase extraction 148, 149, ligand binding assay 150, and sensor technology 151.
Since MIPs display characteristics similar to biological receptor, they have been
used as selective chiral stationary phases. De Boer et al. 144 prepared spherical MIP
particles for the chiral separation of ephedrine and salbutamol enantiomers. Owing to its
high selectivity, this method might be used for the concentration of samples prior to the
CE separation,. However, extensive investigation is needed to reveal the potential
application of MIPs in preconcentration methods (online or offline) coupled with CE.
60
Figure 2.12 Schematic depiction of the preparation of molecular imprints. Reproduced
from Reference151.
61
2.4 Conclusions
Capillary electrophoresis has been drawing increasing attention in a wide range of
research fields with its inherent virtues. Its low concentration sensitivity, however, has
induced a new research subject in the fields related to CE aimed at accomplishing trace-
level analysis by CE. As shown in this chapter, enrichment of sample has been achieved
to reduce the detection limit using various practical methods in CE.
Both electrophoresis-based and chromatography-based preconcentration methods
have been developed to achieve this goal. Each technology has its own advantages and
priorities. Obviously, there is no universal method which is suitable to all situations. In
the view of practice, the identification of the most suitable method should be made based
on the requirement in specific analytes, the magnitude of sensitivity enhancement,
modification of the existing devices, convenience for operation, as well as the costs.
With the increasing demands on sensitive detection of analytes in biological
matrices, preconcentration technique with high selectivity has shown great potential
applications in the future. In this dissertation, on-line electrophoresis-based
preconcentration methods were developed for amino acids and proteins with sol-gel
materials coated fused silica capillaries which were coupled with commercial CE systems.
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70
CHAPTER THREE SOL-GEL TECHNOLOGY AND ITS APPLICATION
IN CAPILLARY ELECTROPHORESIS
3.1 Introduction
The sol-gel process1, 2 generally involves the transition of a system from a liquid
"sol" phase, into a solid "gel" phase. Sol-gel process provides a versatile approach to the
synthesis of inorganic polymers and organic-inorganic hybrid materials 1. The
advantageous features of sol-gel technology include (1) mild reaction conditions, (2)
possibility to create products of various shapes, sizes, and formats (e.g., monoliths, films,
fibers, and monosized particles), (3) ease in the design and fine-tuning of material
structure and property through proper selection of sol-gel ingredients.
Sol-gel technology’s appearance can be traced back to mid 1800s 1. In early 1960s,
contemporary sol-gel processing emerged as a result of specialized requirements for
ceramic nuclear fuels 3. Due to its numerous inherent advantages, sol-gel technique has
found ever increasing application in a diverse range of fields, such as ceramic industry 4-6,
nuclear-fuel industry 7, electronics 8, 9, and chemistry 10, 11 etc..
The application of sol-gel technology for the creation of chromatographic
stationary phase started only two decades ago. In 1987, Cortes et al. 12 reported the use
of sol-gel technology to create monolithic ceramic beds within small-diameter capillaries
and applied such capillaries as separation columns in liquid chromatography (LC). Crego
et al. 13developed a method to prepare a sol-gel stationary phase in situ for open tubular
liquid chromatography. Using a similar method, Guo and Colon 14, 15 prepared sol-gel
based open tubular columns for CEC. The sol-gel technology provided an effective
means to chemically bind chromatographic stationary phases to the capillary inner
surface and brought new promise to produce stationary phases with high stability and
column efficiency in liquid-phase separations. These pioneering works stimulated further
developments in the area of sol-gel stationary phases in chromatographic 16-22 and
electrophoretic separation 23-29 and sample preparation technologies 30-36.
71
3.2 Fundamentals of sol-gel process
Figure 3.1 illustrates a typical sol-gel process. Generally, sol-gel process consists
of hydrolysis and polycondensation of metal alkoxide precursors. During the process, a
liquid like colloidal suspension is formed, which is known as the sol 37. The sol is then
transformed into an interconnected network with submicrometer size pores and polymeric
chains greater than a micrometer, which is called a gel 38. When the liquid is extracted
from the wet gel under supercritical conditions, a low density aerogel is produced. If the
liquid is removed by thermal evaporation, a material termed xerogel is generated 2. Under
elevated temperatures (above 1000 ˚C), the number of pores inside the xerogel is reduced
significantly and the density of the material increased substantially. As a result, the
porous gel is transformed into a dense ceramic material.
In order to properly control the whole sol-gel process and fine-tune the properties
of the target product, it is important to understand the chemical reactions involved in the
sol-gel process. A typical sol solution generally contains the following chemical
components: (1) at least one sol-gel precursor, which is usually a metal alkoxide M(OR)x 39; (2) a solvent to disperse the precursor(s) and a catalyst, which can be an acid 1, 40, 41, a
base 42 or a fluoride 43, 44 based on the type of desired end products; and (3) water.
Precursors that commonly used in sol-gel process include silica-based and non-silica-base
alkoxides (e.g., metal alkoxides) 39. Many metal elements, such as titanium, aluminum,
vanadium, zirconium, and germanium, can be used to prepare metal alkoxides. However,
silica based alkoxides are the most widely used precursors due to their well known
chemistry, pH stability of Si-C bond, well documented sol-gel methodology, facility of
characterizations and commercially available starting materials 45. Table 3.1 lists some
common silica based sol-gel precursors.
The sol-gel process is a relatively straightforward procedure for the preparation of
inorganic or organic-inorganic hybrid materials through hydrolysis of the precursor(s)
and alcohol- or water condensation of the sol-gel-active species present in the sol solution.
The sol-gel-active species may include the alkoxysilane-based precursors as well as any
other chemical species reactive to alkoxysilane, silanol and analogous silica species. A
polycondensation occurs with the linkage of additional ≡ Si-OH tetrahedral to the
condensation products, and eventually materials with three-dimensional network
72
Figure 3.1 Overview of a sol-gel process. Reproduced from Reference 1.
73
Table 3.1 Common tetraalkoxysilane precursors and their physical properties*
Name MW bp
(˚C)
nD
(20˚C)
d
(20˚C)
η
(ctsks)
Dipole
Moment
Solubility
SiMeO
MeO OMe
OMe
Si(OCH3)4 Tetramethoxysilane
152.2
121
1.3688
1.02
5.46
1.71
Alcohols
SiEtO
EtO OEt
OEt
Si(OC2H5)4 Tetraethoxysilane
208.3
169
1.3838
0.93
-
1.63
Alcohols
SiC3H7O
C3H7O OC3H7
OC3H7
Si(OC3H7)4 Tetra-n-propoxysilane
264.4
224
1.401
0.916
1.66
1.48
Alcohols
SiC4H9O
C4H9O OC4H9
OC4H9
Si(OC4H9)4 Tetra-n-butoxysilane
320.5
115
1.4126
0.899
2.00
1.61
Alcohols
Tetrakis(2-
methoxyethoxy)silane
328.4
179
1.4219
1.079
4.9
-
Alcohols
*Reproduced from Reference 1
74
structures are produced2. Scheme 3.1 illustrates hydrolysis, condensation and
polycondensation of tetramethoxysilane (TMOS) as an example.
The catalysts used in sol gel process play an important role. They not only change
the reaction speed, the type of the catalyst also affects the structure of the resulting sol gel
materials. A generally accepted notion is that acid-catalyzed sol-gel processes are more
likely to produce linear polymers because under acidic condition, the hydrolysis of
alkoxide precursors undergo faster than the condensation process 46. The mechanism of
hydrolysis reaction under acidic conditions involves protonation of the alkoxide group,
which is followed by nucleophilic attack by water to form a pentacoordinate intermediate 1, 47. On the other hand, under basic condition, condensation reaction is faster and the rate
of the overall sol-gel process is determined by the relatively slow hydrolysis step. In this
case, highly condensed particulate structure is more likely to be generated 48. The
hydrolysis reaction under basic condition is believed to start with the nucleophilic attack
on the silicon atom by the hydroxide anion and form a penta-coordinated intermediate.
This step is followed by the substitution of a alkoxide group by a hydroxyl group 1, 47, 49,
50. This is an important feature which enables researchers to manipulate experimental
conditions to fine-tune the formation of the end products with desired characteristics.
Scheme 3.2 illustrates both acid-catalyzed sol-gel reaction and base-catalyzed sol-gel
reaction mechanisms. In addition to the use of catalysts, sol-gel processes can also be
initiated by irradiation. It has been reported by Zare and co-workers that the precursor
methacryloxypropyltrimethoxysilane is initiated by the application of UV light at 350 nm
wavelength 29, 51, 52.
Solvents in the sol solution play an important role in the formation of a
homogeneous sol solution and progression of the gelation procedure1. Artaki et al.
systematically investigated the solvent effects on the condensation reaction of the sol-gel
process 53. With the aids of Raman spectroscopy, molybdenum chemical reaction, and
electron microscopy, the authors concluded that the mechanism of particle aggregation as
well as the extent of condensation of the polymeric network is dramatically affected by
the presence of organic additives, such as fomamide, dimethyl formamide, acetonitrile
and dioxane due their influence on hydrogen bonding and electrostatic interactions,
75
hydrolysis
Si OCH3OCH3
H3COOCH3
4H2O Si OHOH
HOOH
4CH3OH
Si OCH3OCH3
H3COOCH3
Si OCH3OCH3
HOOCH3 alcohol condensation
Si OOCH3
H3COOCH3
Si OCH3OCH3
OCH3CH3OH
Si OHOCH3
H3COOCH3
Si OCH3OCH3
HOOCH3 water condensation Si O
OCH3
H3COOCH3
Si OCH3OCH3
OCH3H2O
Si OOH
HOOH
Si OHOH
OH water polycondensation6Si(OH)4
Si OO
OO
Si OO
O
Si OHHOOH
Si OHHOOH
Si OHOH
HO Si OHOH
HO
Si OHOH
OHSiOH
HOOH
6H2O
Scheme 3.1 Sol-gel reaction 48
76
Acid-catalyzed sol-gel reaction mechanism.
a. Hydrolysis mechanism
Si ORRO
RO
ORH+
SiHO
RO
RO
OR
R SiHO
RO
RO
ORH
R
SiRO
RO
OR
OH
H3O+
OH
H
SiRO
RO
OR
O
HORH2O
H
HH2O
b. Condensation mechanism
R-Si(OH)3 H+k1
k-1
R-Si(OH)2O
H H
R-Si(OH)2O
H HR-Si(OH)3
k2
k-2
Si
OH
OH
R O Si
OH
OH
R H3O+
A. Base-catalyzed sol-gel reaction mechanisms
a. Hydrolysis mechanism
Si OR
RO
RORO
HO- Si
OR
RO OR
HO ORδ− δ−
SiOR
OR
HO
OR
RO-
b. Condensation mechanism
R-Si(OH)3 OH-k1
k-1
R-Si(OH)2O- H2O
R-Si(OH)2O- R-Si(OH)3
k2
k-2
R-Si(OH)2-O-Si(OH)2R OH-
Scheme 3.2 Acid-catalyzed and base catalyzed sol-gel reaction mechanisms
77
which modulate the nucleophilic substitution mechanism associated with the sol-gel
process.
In addition to the catalyst type, several other factors, such as water-to-silane ratio,
nature of solvent system and the alkoxide precursors, can affect the physical properties of
the obtained sol-gel materials 47. In order to get a fine-tuned porosity of the monoliths,
the amount of water in sol system should be carefully controlled. Constantin and Freitag
reported that there is an optimum content of water (approximately 200%) that facilitates
the formation of uniform porous monoliths 54. These authors observed no significant
microstructure developments (pores) in the monoliths when the content of water was
much less than 200%. On the other hand, with water content larger than 300%, sol-gel
beads with broad distribution and blocks of non-macrophorous structures were formed.
Gelation is followed by the drying step. During this step, the liquid is removed
from the interconnected pore network either through extraction to produce an aerogel,
ambient conditions to evaporate liquid and generate a xerogel, or extreme thermal
condition to obtain ceramic material 2 (Figure 3.1).
3.3 Characterization of sol-gel materials
With the development of new analytical and computational techniques for
investigation on a nanometer scale, it is possible to get more information associated with
sol-gel process (e.g., hydrolysis, polycondensation, and drying), which enable researchers
to have a deeper insight into the fundamental aspects of sol-gel chemistry providing
increased scientific understanding. Various techniques have been used to investigate the
sol-gel process as well as the properties of the organic-inorganic hybrid materials created
though sol-gel process. These techniques include nuclear magnetic resonance (NMR), X-
ray small-angle scattering (XSAS), Raman spectroscopy, X-ray photoelectron
spectroscopy (XPS), differential scanning calorimetry (DSC), dielectric relaxation
spectroscopy (DRS), etc.
3.3.1. The morphology of sol-gel materials
To study the surface characteristics and fine structural details of the sol-gel
materials, scanning electron microscopy (SEM) is a powerful tool. With SEM, sample
78
surface is scanned by a fine electron incident beam which produces an image with great
depth of field and an almost three-dimensional appearance. With this feature, SEM is the
most widely used technique to evaluate the morphology of sol-gel materials 24, 25, 28, 29, 51,
52, 54-61. Hernandez-Padron et al. 62 reported an SEM morphological study and
transparency properties of hybrid SiO2-phenolic resin materials. In the case of sol-gel
surface-coated open tubular columns, SEM can reveal the uniformity of sol-gel coating
thickness and structural defects therein. It is also used to study the effects of various
experimental parameters on the properties of produced sol-gel materials 52. In addition to
SEM, atomic force microscopy (AFM) 63, X-ray absorption 64 and transmission electron
microscopy 65, 66 are also used to investigate the morphology of sol-gel materials. For
example, Almeida et al. 64 used extended x-ray absorption to study fine structure and
near-edge structure of silic-titania sol-gel film. Yan et al. 66 used TEM to characterize the
magnesium silicate thin films obtained through sol-gel technique.
3.3.2. Study of the chemical bonds within sol-gel structure
Techniques like SEM, AFM, etc. provide the picture of sol-gel materials depicting
heterogeneous or homogeneous morphologies, while spectroscopic results confirm the
existence of chemical bonds within sol-gel structure. To study the chemical bonds in sol-
gel structure, various spectroscopic techniques including Fourier transform infrared
spectroscopy (FTIR) 66-68, fluorescence spectroscopy 67 and nuclear magnetic resonance
(NMR) 69-72 have been used.
Since spectra can be scanned, recorded, and transformed in an extremely rapid
pace, FTIR enables the study of sol-gel process in its progression with time. Toyo’oka
and co-workers 73 monitored the content of residual silanol groups in sol-gel material as
the gelation process progressed using attenuated total reflectance (ATR) FTIR hybrid
technique. The encapsulation of bovine serum albumin 74 in the sol-gel matrix was
confirmed by FTIR by Zuo et al.63. IR was used to characterize capillaries modified with
macrocylic dioxopolyamine 75. The typical IR absorptions of NH, C=O, and CH obtained
provided the evidence of successful surface modification in capillaries for open-tubular
capillary electrochromatography.
79
The esterification reaction between stearic acid and the epoxy groups of
glycidoxypropyltrimethoxysilane was investigated by X-ray photoelectron spectroscopy
(XPS) by Zhao and coworkers76. The XPS results provided evidence on the existence of
carbon in the reaction product indicating the success in the octadecyl silylation reaction.
Another powerful analytical technique, nuclear magnetic resonance (NMR) was
used by Rodriguez and Colon 72 to investigate the species present in the sol-gel solution
used to modify the inner surface of an open tubular CEC column. It is established that in
a sol-gel solution containing more than one precursor, a homogenous hybrid system is
usually formed if the monomeric precursors undergo hydrolysis reactions at similar rates.
However, if one of the precursors has much faster rate for the hydrolysis reaction leading
to pronounced self-condensation 77, a heterogeneous composition will be produced. Since
the properties of the final sol-gel columns can be indicated by the species present in the
sol-gel solutions prior to the coating process, it is very important to understand the
characteristics of the sol-gel solution in details.
3.4 General procedures involved in the preparation of CE columns with sol-gel stationary phases
Several steps are involved in the preparation of CE columns with sol-gel
stationary phase. The preparation procedures vary depending on the types of the columns
and the intended applications. They include pretreatment of the capillary, fabrication of
the sol-gel stationary phases, and the post-gelation treatment of the CE stationary phases.
3.4.1 Pretreatment of the capillary
The purpose of capillary pretreatment is to increase the concentration of surface
silanol groups. Since sinanol groups on the capillary surface represent the principal
binding sites for in situ created sol-gel stationary phases, higher concentration of these
binding sties on the capillary surface would facilitate the formation of highly secured sol-
gel stationary phases through chemical bonding with the capillary inner walls. Alkali
solutions are used to clean the capillary surface in addition of some organic solvents 14, 15.
In the reported one-step synthesis of monolithic silica column by Constantin and Freitag 54, the pretreatment of the bare fused silica was accomplished by flushing with NaOH,
then, with HCl, and followed by rinsing with purified water. The similar pretreatment
80
method was used by other researchers to prepare sol-gel open-tubular 76 and monolithic
columns 55-57. Hayes and Malik 24, 25 reported the use of hydrothermal treatment of the
inner surface of the fused silica capillary for the preparation of both sol-gel monolithic
and sol-gel open tubular columns. The purpose of hydrothermal pretreatment was
explained as cleaning the capillary inner surface and increasing the surface concentration
of silanol groups to effectively anchor the in situ created sol-gel stationary phases, and it
is also being used by other research groups78, 79.
3.4.2 Sol solution ingredients for the fabrication of the sol-gel stationary phases In addition to the typical components in the sol solution (e.g., precursor(s), a
solvent system, a catalyst and water), the actual operations for creating sol-gel stationary
phases often involve the use of various additives to provide the desired end products. A
porogen is often used in the sol solution, especially in creating a porous monolithic bed.
Toluene was found to be a suitable porogen for photopolymerized sol-gel monolithis for
CE 29, 52. Poly(ethylene oxide)(PEO) and polyethylene glycol 80 were also used as
porogens by different researcher 58, 60, 81. Porogens generally play a dual role: they serve
as a thorough-pore template and as a solubilizer of silane reagent.
Deactivation reagents represent another important type of sol solution additives
used to derivative residual silanol groups on the stationary phase, and thereby reduce
harmful adsorptive effects of the latter on CE separation. Hayes and Malik reported the
use of phenyldimethylsilane (PheDMS) as a deactivation reagent for both open-tubular
and monolithic sol-gel columns 24, 25. The deactivation reagent reacts with the residual
silanol groups on the stationary phase resulting in the reduction of chromatographically
harmful adsorption sites on the stationary phase. The effect of the deactivation was
evaluated by the comparing the column performance obtained on columns prepared with
and without the addition of deactivation agent.
3.4.3 Post-gelation treatment of sol-gel stationary phases
In order to minimize or eliminate the volume shrinkage during the fabrication of
sol-gel stationary phase, especially for sol-gel monoliths, post-gelation treatment is
needed. Various techniques have been developed to accomplish post-gelation treatment.
81
In the case of open-tubular columns, organic solvents were used to flush the sol-gel
stationary, followed by equilibrating the columns with running buffers 14, 15, 76. It was
found by Zuo and co-workers 63 that aging under moist conditions at lower temperatures
benefits the encapsulation of biological macromolecules. While an accelerated rate of
aging may lead to cracks on the dried gel.
3.5 The application of sol-gel technology in CE
Like all other chromatographic systems, in CE the column is a fundamentally
important component. Column technology, therefore, can be regarded as the key to the
success of CE. A careful review of the published papers devoted to the fabrication of CE
columns over the last decade shows that sol-gel technology shows a promising direction
in CE column technology. It is applicable to the preparation of CE columns in three
different formats: open-tubular columns with sol-gel surface coatings, capillary columns
packed with micro/submicro particles, and capillary columns with monolithic beds.
3.5.1 Sol-gel technology for packed columns in CE
Sol-gel technique has been used in three distinct areas in packed columns for CE.
They are: (1) preparation of micrometer and submicrometer size sol-gel particles used to
pack the capillary, (2) creation of sol-gel frit in packed columns, and (3) preparation of
sol-gel entrapped chromatographic packing material.
Retaining end frits are commonly used in CE packed columns to keep the
particulate packing material inside the capillary. Traditionally, on-column frits are
produced by sintering of silica based packing materials by heating a short segment of the
packed bed with a flame or applying low- voltage resistive heating for a short period of
time. Consequently, the particles of the packing material in this segment become
connected with neighboring particles and the capillary wall at their contact points to form
a permeable barrier and retain the stationary phase. These methods to produce on-column
frits put a high thermal stress on the protective polyimide coating of the fused silica
capillary and may lead to fragility, variable permeability, and destruction of the chemical
bonds in the frit region 82-85. Since sol-gel process can occur under mild conditions, it is a
good alternative method to prepare frits for packed columns 22, 86, 87. The frits prepared
82
by sol-gel technology have been proved to possess good mechanical strength, and high
pH and solvent stabilities 88. Piraino and Dorsey studied the performance of several types
of frits, including sintered frits, photopolymerized frits, and frits made by sol-gel process.
Their research results suggested that capillaries with sol-gel frits showed the greatest
electroosmotic mobility 89.
It is believed that the use of frits is associated with a number of problems, such as
bubble formation during CE operation 90, column fragility 91, variable permeability and
related shortcomings 84, 85. To avoid the use of frits, sol-gel technology has been used to
entrap the bonded stationary particles inside the capillary 86. The methodology involved
the preparation of a sol-gel solution containing tetraethoxysilane, ethanol, and
hydrochloric acid followed by addition of packing particles to create a suspension. This
suspension containing packing particles was then introduced into the capillary to generate
a packed column. Another approach to fabricate packed columns with sol-gel process
was developed by Tang et al. 92. Based on their method, the capillary was first packed
with the chromatographic particles prior to the application of sol solution. A sol solution
was then introduced into the particle packed capillary. After the conversion of the sol to a
gel, the sol-gel bonded packed column was dried using supercritical CO2.
3.5.2 Sol-gel open tubular CE columns
In open tubular columns, the stationary phase is bonded to or spread as a coating
on the inner surface of the capillary, which constitute an important category of CE
columns. They represent an alternative to packed columns and free from the problems
caused by the use of frits in traditional packed columns. In order to provide sufficient
retentive properties and sample capacity, open tubular columns should have thick
stationary phase coatings. However, with conventional fabrication methods, it is usually
difficult to achieve thick and stable coatings. Research works devoted to solving these
problems include four main directions 93. They are (1) using thick, immobilized organic
polymer coatings to improve the column phase ratio, (2) creating etched inner surface of
the capillary with bonded organic ligands to provide higher surface area and enhanced
solute/stationary phase interactions, (3) using dynamic nanoparticles as pseudostationary
phase, and (4) coating the capillary with sol-gel technology. Based on experimental
83
results, researchers 14, 15 found that columns prepared by sol-gel technology had enhanced
surface area, improved hydrolytic stability, and enhanced retentive characteristics.
Compared with traditional methods, sol-gel approach is much simpler.
Using sol-gel precursors containing an alkyl chain (such as C6, C8, C16, and C18),
sol-gel technology allowed the preparation of stationary phase coatings within fused
silica capillaries, where these chemically bonded alkyl chains served as retentive moieties
for the reversed-phase separation of uncharged polycyclic aromatic hydrocarbons,
aromatic ketones and alcohols 14, 15, 24, 94, 95.
Wu et al. 96 developed a reversed-phase open-tubular column coated with a sol-
gel stationary phase containing an amine moiety. An enhanced EOF in an acidic buffer
and reduced adsorption of peptides on the capillary wall were achieved. These columns
provided fast separation fro peptide samples: six peptides were baseline resolved within 3
min.
Hayes and Malik 24 developed a positively charged sol-gel ODS stationary phase
for open-tubular column providing reversed electroosmotic flow. The key precursor used
by was (N-octadecldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride). The
methoxysilyl groups enabled the creation of sol-gel network structure and attachedment
of the created sol-gel stationary phase onto the capillary wall. The presence of the
octadecyl group reinforced the chromatographic interactions between organic analytes
and the sol-gel stationary phase. The quaternary amine group chemically incorporated in
the stationary phase structure provided a positively charged capillary surface which is in
contrast with the electrical properties of bare fused silica capillary surface that carries a
negative charge.
Constantin and Freitag 94 introduced ion exchange groups into open-tubular
column by sol-gel process. The ion exchange groups were the results from the addition of
(pentafluorophenyl)dimethylchlorosilane into sol solution. The obtained open-tubular
column was successfully used for the separation of amino acids.
The use of fluorinated stationary phase for the separation of fluorinated organic
compounds and halogenated organic compounds was demonstrated by Narang and Colon 97. The open-tubular column bearing sol-gel-deived fluorinated stationary phase were
84
prepared by using a sol solution containing tridecafluoro-1,1,2,2-tetrahydrooctyl-
triethoxysilane (F13-TEOS), TEOS, ethanol, hydrochloric acid and water.
Wang and Zeng 75, 98 used 3-(2-cyclooxypropoxyl)propyl-tri-methoxy silane as a
bridge to connect 1,4,7,10-tetraazacyclotridecane-11,13-dione and 2,6-dibutyl-β-
cyclodextrin to TEOS. By doing this, macrocyclic polyamine derived-and β-cyclodextrin
derived stationary phases were attached onto the sol-gel matrix. The obtained open-
tubular columns were used to separate isomeric nitrophenols and benzenediols, isomeric
aminophenols, diaminobenzenes, dihydroxybenzenes, and biogenic monoamine
neurotransmitters.
3.5.3 Sol-gel monolithic columns
In chromatographic science, monolithic columns are referred to as continuous bed
columns, fritless columns or rod columns 99. Compared to the traditional packed columns,
monolithic columns have many advantages such as ease in construction, higher surface
area and porosity, improved mass transfer, absence of retaining end frits, and elimination
or significant reduction of certain operation problems inherent in packed columns due to
the presence of the retaining end frits 12. Compared to open-tubular columns, the solute
molecules do not have to diffuse a long distance through the liquid mobile phase to reach
the stationary phase in monolithic columns.
Preparation of monolithic columns using sol-gel technology provides a variety of
important advantages over other methods. Cortes et al. 12 reported the pioneering research
work using sol-gel technology to create silica-based monolithic beds inside fused
capillary in 1987. Fujimoto published a detailed procedure for the preparation of sol-gel
monolithic columns for CE 99. First, the capillary was filled with the sol solution for 20
hours at 40 °C. The formed material was then washed with water and treated with
ammonium hydroxide solution for 24 hours at 40 °C. After thermal conditioning, the
chromatographic moiety C18 was bonded to the sol-gel matrix with a 10% solution of
dimethylocadecylchlorosilane.
Tanaka and co-workers 60, 100 also fabricated sol-gel monolithic columns suibable
for both HPLC and CE. These researchers used a two-step procedure: (1)a sol-gel silica
monolithic bed was created inside the capillary, (2) a surface-derivatization reaction was
85
used to chemically bind the desired chromatographic ligand to the surface of the porous
silica monolith. The sol solution they used included TMSO as the precursor, PEO as the
porogenic agent and acetic acid as the catalyst.
Hayes and Malik 25 developed a method to prepare sol-gel monolithic column for
CEC in a single-step procedure. These researchers used octadecyl group as the organic
component of the stationary phase facilitating the solute/stationary interactions during CE
separation. In this method, all the process including the formation of sol-gel monolithic
matrix, the introduction of the organic moiety C18 and the deactivation of the unreacted
silanol groups were accomplished in one step, which provided a much simpler and faster
method for the preparation of sol-gel ODS monolithic CE column.
3.6 Sol-gel technology for sample preconcentration in CE
The organic-inorganic hybrid materials synthesized by sol-gel technology have
been explored applications in sample preparation, such as sample preconcentration. Malik
and co-workers introduced sol-gel coated fibers and capillaries for solid-phase
microextraction (SPME) and capillary microextraction (CME or in-tube SPME) 30, 34, 35.
The sol-gel coated capillary was also used for the sample preconcentration in high
performance liquid chromatography (HPLC) 33 and CE 101, 102.
Quirino and co-workers 102, 103 developed a photopolymerized sol-gel (PSG)
material for sample preconcentration in CE. The porous PSG monolith has a high mass-
transfer rate, which promotes preconcentration of dilute samples. In this method,
(methacryloxypropyl)trimethoxysilane was used as the precursor, toluene as the
porogenic agent. The photopolymerization reaction was initiated by exposing the fused
silica capillary filled with sol-gel solution to 365 nm light. The obtained sol-gel monolith
acted as a solid-phase extractor as well as a separation stationary phase. The
preconcentration effect was calculated in terms of peak heights, up to 100-fold increase
for the PAH mixture, 30-fold for the alkyl phenyl ketone mixture, and 20-fold for the
peptide mixture when UV detection was used.
Oguri and Toyo’koa et al. 101 developed a method of on-line preconcentration
prior to on-column derivatization CEC. A sol-gel octadecasiloxane capillary column was
created using thermal sol-gel reaction of tetraethyl orthosilicate to capture ODS particles
86
in a piece of capillary. A standard biogenic amine solution consisting of histamine,
methylhistamine, and serotonin were effectively concentrated at the inlet site of the
capillary column by filed-amplified sample stacking, a gradient effect, or both. An
increased sensitivity by a factor of 1000-fold greater than that of normal on-column
derivatization CEC was obtained when a fluorescence detector was equipped.
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92
CHAPTER FOUR POSITIVELY CHARGED SOL-GEL COATINGS FOR
ONLINE PRECONCENTRATION OF AMINO ACIDS IN CAPILLARY ELECTROPHORESIS
4.1 Introduction Capillary electrophoresis (CE) represents a class of highly efficient electro-driven
liquid phase separation techniques that are often complementary to HPLC 1. Compared to
HPLC, CE provides good mass detection limits, especially when the sample volume is
limited. But in some cases, samples are not volume limited and concentration detection
limits are more important 2, 3. CE separation techniques provide poor concentration
sensitivity compared with liquid chromatography, especially when on-column UV
detection technique is used to prevent loss of separation efficiency due to extracolumn
band broadening that usually takes place in the detection cell when an off-column
detection scheme is used. Generally, low concentration sensitivity of CE techniques is a
consequence of the small sample injection volume and the short optical path length of the
capillary 4. Several preconcentration techniques, including electrophoresis-based 5-19 and
chromatography based 20-26 methods, have been developed to enhance the concentration
sensitivity of CE separations.
In this chapter, a positively charged sol-gel coating for on-line preconcentration of
amino acids zwitterionic biomolecules in general is presented. The preconcentration
methods based on the utilization of capillary with such a sol-gel coating do not require
any additional modification of the commercially available standard CE instrument.
Extraction, stacking, and focusing techniques were used in the preconcentration
procedures. With zwitterionic properties, amino acids can carry either a positive or a
negative charge when the pH of the samples is properly adjusted. Therefore, the
electrostatic interaction between the analytes and CE column can be used to facilitate the
extraction procedure.
93
4.1.1 Column technology Unlike HPLC, CE does not require high-pressure pumping systems for mobile
phase delivery. The typical high-voltage operation in CE is associated with
electroosmotic pumping mechanism that serves as the driving force for mobile phase
flow through the CE column. The pressure-free operations in CE provide the technique
with some significant advantages over HPLC, and makes CE operation practically free
from particle size, column length and available maximum pressure limitations inherent in
HPLC. Thus, CE opens up real possibilities to achieve extremely high separation
efficiencies and column performances in liquid-phase separation. However,
materialization of this great potential of CE will require effective solution of a number of
problems − mostly in the area of column technology development.
The silanol groups on the fused silica capillary surface are responsible for the
generation of electroosmotic flow (EOF) and the adsorption of sample ions. In some
cases, surface coatings are used to screen these active sites to suppress or reduce EOF, or
to reverse the direction of EOF 27-29. Capillary wall coating techniques normally can be
classified into two categories: dynamic coating methods and chemical bonding methods 1-
3.
4.1.1.1 Dynamic coatings
Dynamic coatings are produced by sequential adsorption of various kinds of
coating reagents on the surface of a fused silica capillary 1, 3, 30. The advantages of this
type of coating technique made simplicity in column preparation by using the rinse
function of standard CE instrumentation, and the dynamically modified capillaries exhibit
improved EOF reproducibility compared to an unmodified fused silica capillary.
Therefore, this method is widely used to coat capillaries for the separation of various
analytes 30-34. Various types of coating agents have been used 1, including alkylamines
and diamines 27, 28, 35, 36, neutral and ionic polymers 29, 37, 38, cationic surfactants 28, 39, and
polyelectrolyte multilayers 40, 41. For instance, Bendahl and colleagues 41 developed a
simple procedure for the generation of dynamic coatings by noncovalent adsorption of
ionic polymer Polybrene and poly(vinylsulfonate) (PVS). The dynamic coating produced
by their method was proved to be stable over a wide pH range of 2-10. Fast separation of
94
basic test compounds was achieved at low pH. Recently, Chang and co-workers 42 used
commercially available coating reagent EOTrolTM to dynamically modify the capillary
for CE. The coating procedure involved pretreatment of the capillary with 1 M NaOH
and water under pressure (20 psi) for 10 min and 1 min, respectively. And this
pretreatment process was followed by the dynamic coating process with EOTrolTM in
running buffer at a concentration of 0.15% w/v. The coatings allowed reproducible EOF
modulation in the cathodal direction and rapid separation of isofoms of three model
proteins (bovine serum albumin, transferrin, a1-antitrypsin).
4.1.1.2 Chemically bonded coatings
A number of procedures have been employed to prepare chemically bonded
coatings, which are not usually destroyed due to rinsing of the capillary with buffer
solution. The coating techniques include surface derivatization using silane chemistry 43-
45. By this technique, both halo and alkoxy silanes can be coupled to fused silica capillary
surface through one or more siloxane bonds 46-48. Poly(ether) chains with different length
or different substituent groups can also be covalently bonded to a sublayer generated by
the silanization reaction 1. In addition, organic polymerization-based techniques 49-52
represent another important method to generate chemically bonded coatings. Chemically
bonded coatings can be obtained by cross-linking of prepolymers or by polymerization of
monomers on the capillary surface. Sol-gel approach 53-59 providing organic-inorganic
hybrid materials have been attracting more and more interests in the column technology
due to its various advantageous features.
4.1.2 Positively charged coatings for CE columns In a CE column with a positively charged inner surface, the diffuse component of
the electrical double layer contains anions and generates an anodic electroosmotic flow
instead of the cathodic EOF produced by untreated fused silica capillary characterized by
a negatively charged surface. Both dynamic coating techniques and chemical bonding
methods have been used in order to obtain positively charged coatings on the capillary
surface. Positively charged sites have been generated using a cryptand-containing
polysiloxane 60 or quaternary ammonium group 61-65. In Lee and co-workers 60 prepared
95
positively charged quaternary amine surface coating using iodopropyltrimethoxylsilane
and poly(4-vinylpyridine-co-butylmethacrylate). Iodopropyltrimethoxylsilane was first
used to bond onto the capillary surface through a siloxane bond, and poly(4-
vinylpyridine-co-butylmethacrylate) was immobilized through a reaction between the
carbon-iodo bonds and the pyridine groups in the polymer, producing quaternary amine
moieties. The positively charged quaternary amine effectively provided a reversed EOF
compared to an untreated fused silica capillary. Columns with positive surface charges
obtained in this way were successfully used to separate proteins.
As mentioned in Chapter Three, sol-gel columns offer many advantages over
conventional columns for the separation in GC 66, 67, HPLC 53, 54, 56, 68, and CE 53-55, 69. In
addition, sol-gel coated capillaries have been employed for the isolation and
preconcentration of a wide variety of polar and nonpolar analytes by solid-phase
microextraction analysis 70, 71. This chapter, describes positively charged sol-gel coatings
created by using N-octadecyldimethyl[3-(trimethoxysilyl)propyl] ammonium chloride as
a sol-gel precursor. The obtained positively charged sol-gel columns were used for on-
line preconcentration and CE analysis of amino acids.
4.2 Experimental
4.2.1 Equipment All sample preconcentration and CE experiments were performed on a Bio-Rad
BioFocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules, CA)
equipped with programmable, multiwavelength UV/visible detector. The BioFocus 3000
operating software system (version 6.0) was used to collect and process the CE data. A
Barnstead model 04741 Nanopure deionized water system (Barnstead/Thermodyne,
Dubuque, IA) was used to prepare deionized water, ~ 17 MΩ/cm. A homemade gas
pressure-operated capillary filling / purging device53 was used for coating the fused-silica
capillary. Figure 4.1 schematically illustrates the filling / purging device. A Microcentaur
model APO 5760 centrifuge (Accurate Chemical and Scientific Corp., Westbury, NY)
was used for centrifugation of the sol solutions. A Fisher model G-560 Vortex Genie 2
system (Fisher Scientific, Pittsburgh, PA) was used for thorough mixing of various
chemical ingredients in the sol-gel coating solution. A Chemcadet model 5984-50 pH
96
Fused silica capillary
Flow control valveFlow control valve
Pressurization chamber
Sol solution vial
Gas flow outlet
Threaded detachable cap
inert gas supply
Figure 4.1 Schematic representation of the home made capillary filling / purging device, adapted from Reference 53
97
meter (Cole-Palmer Instrument Co., Chicago, IL) equipped with a TRIS-specific pH
electrode (Sigma-Aldrich, St. Louis, MO) was used to measure the buffer and sample pH.
4.2.2 Chemicals and materials
Fused-silica tubing of 50-µm i.d. was purchased from Polymicro Technologies
(Phoenix, AZ) for the preparation of sol-gel coated columns. Sample vials (600µL),
HPLC grade methylene chloride, methanol, and acetonitrile were purchased from Fisher
Scientific (Pittsburgh, PA). Tetramethyl orthosilicate (99+%) and trifluoroacetic acid
(99%) were purchased from Aldrich (Milwaukee, WI). Tris
(hydroxymethyl)aminomethane hydrochloride (reagent grade) and amino acids (DL-
alanine, DL-asparagine, DL-phenylanlaine, and DL-tryptophan) were purchased from
Sigma (St. Louis, MO). N-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium
chloride and phenyldimethylsilane were purchased from United Chemical Technologies,
Inc. (Bristol, PA).
4.2.3 Preparation of sol-gel open tubular CE columns with positive surface charge
Sol-gel open tubular C18-TMS columns with a positive surface charge were
prepared according to the procedure described by Hayes and Malik 54 Briefly, the sol
solution was prepared by thoroughly mixing appropriate amounts of the following
ingredients: (a) two sol-gel precursors tetramethoxysilane (TMOS) and N-
Octadecyldimethyl[3-(trimethoxysilyl)propyl] ammonium chloride (C18-TMS), (b) a
deactivation reagent [phenyldimethylsilane (PheDMS)], and (c) a sol-gel catalyst
[trifluoroacetic acid (TFA) (containing 5% water)]. A piece of previously cleaned and
hydrothermally treated fused-silica capillary was first sealed at one end using an
oxyacetylene flame. The prepared sol solution was then introduced into the capillary
from its open end, creating a pressurized gas pocket at the sealed end. This filling process
was carried out under 40 psi of helium pressure using a homemade filling device 53.
Because of the presence of a pressurized gas pocket at the sealed end of the capillary, the
inner surface of the gas-containing part of the capillary remained untouched by the sol
solution. Figure 4.2 illustrates the process. After a 20-min in-capillary residence time, the
98
polyimide coating capillary wall
pressure
sol solution
sol-gel coatingoptical window
A
B
C
D
E
Figure 4.2 Preparation of a sol-gel column for preconcentration adapted from Ref.55
A: a piece of hydrothermally pretreated fused silica capillary.
B: sealing one of the capillary ends via an oxyacetylene torch.
C: filling part of the capillary with the sol solution by helium pressure.
D: depressurization of the capillary followed by opening of the sealed end of the capillary
through the open end.
E: preparation of a UV detection window after condition.
99
filling gas pressure was released to allow the pressurized gas pocket to expel the sol
solution from the capillary through its open end. The sealed end was cut open and the
capillary was further purged with helium from the previously sealed end, allowing a
segment of the capillary at this end to remain untouched by the sol solution. Thus, a
segment of the capillary at the open end was coated, leaving at the initially sealed end an
uncoated segment (~25 cm). Once the remaining steps of the column preparation are
complete, this uncoated segment can be used to create an optical window for on-column
UV detection. After coating and purging, both ends of the capillary were sealed with an
oxy-acetylene torch and the capillary was further conditioned in a GC oven at 150°C for
2 hours. Following thermal treatment, the sealed capillary ends were cut open. The
column was purged under 40 psi helium pressure for an additional 30 min and then
sequentially rinsed with 100% acetonitrile, deionized water and desired running buffer.
The UV detection window was created by burning the extenal polyimide coating on the
uncoated segment of the capillary.
4.2.4 Preparation of samples DL-alanine, DL-asparagine, DL-phenylalanine and DL-tryptophan purchased
from Sigma (St. Louis, MO) were dissolved in deionized water to make the test samples.
4.2.5 Procedures for extraction and preconcentration After the sol-gel coated column was installed on the CE system, it was first filled
with the running buffer using gas pressure. The inlet end of the column was then inserted
into the sample vial. Samples were hydrodynamically injected for 3 min at 100 psi. Under
these conditions, the pH of the sample solution was kept above the isoelectric point (pI)
of the test amino acid to impart a net negative charge to the solute amino acids (Table
4.2). Electrostatic interaction between the positively charged sol-gel coating and
negatively charged amino acid molecules led to their extraction on the sol-gel column. To
show the extraction effect of the sol-gel coating, the sample matrix was removed from the
column either by purging with deionized water or by reversed electroosmotic flow. After
this, the inlet end of the capillary was returned back to the buffer reservoir, which
100
contained an acidic running buffer. The extracted analytes were then desorbed by this
acidic running buffer and analyzed under the effect of a high applied electric field.
4.3 Results and discussion Table 4.1 lists the names and chemical structures of sol-gel solution ingredients
used in this research to create a positively charged sol-gel coating. The sol-gel co-
precursor, C18-TMS, possesses a number of important structural features. The
methoxysilyl groups in the C18-TMS and TMOS are sol-gel active, and they participate in
the formation of the sol-gel polymeric network through hydrolysis and polycondensation
reaction 53-55, 72. Its quaternary amine moiety is responsible for the positive charge on the
sol-gel coating, which not only provides the basis for electrostatic interaction between
sol-gel coating and analytes in samples but also supports reversed electroosmotic flow in
the CE column. In addition, the octadecyl chain like a pendant group is capable of
providing the chromatographic interactions. Although a number of other chemicals
including poly(diallyldimethylammonium chloride) 64, chitosan 73 and cyrptand-
containing polysiloxane 60 have been used by different research groups to generate a
positively charged coating surface, in the present study, C18-TMS was used. This sol-gel
approach to achieve this goal was introduced by our group and is characterized by its
simplicity and effectiveness.
4.3.1 Mechanism of extraction on positively charged sol-gel coatings The mechanism of extraction in a CE column with a positively charged sol-gel
C18-TMS coating is based on the electrostatic interaction. The structures of amino acid
test solutes used in the current study together with their dissociation constants are shown
in Table 4.2. Due to their zwitterionic properties, amino acids can bear a net positive
charge, a net negative charge, or be electrically neutral in different pH environments. At
pH values above its isoelectric point, an amino acid will possess a net negative charge.
Because of the electrostatic interactions, the negatively charged species will get extracted
by the positively charged C18-TMS coating on the inner surface of the CE column.
101
Table 4.1 Names and chemical structures of chemical reagents used in the fabrication of positively charged sol-gel columns
Regent Function and Reagent Name Reagent Structure
Sol-gel precursors:
Tetramethoxysilane (TMOS)
OCH3
Si
OCH3
OCH3H3CO
N-Octadecyl-dimethyl[3-(trimethoxysilyl)propyl] ammonium chloride (C18-TMS)
N+H3C-(H2C)17
CH3
CH3
(CH2)3 Si
OCH3
OCH3
OCH3 Deactivation reagent:
Phenyldimethylsilane (PheDMS) Si CH3CH3
H
Catalyst:
Trifluoroacetic acid (TFA) F 3 C
COH
O
102
Table 4.2 Chemical structures and some physical properties of analytes used in the current study
Name Structure pKa of
α-COOH Group 60a
pKa of
α-NH3+ Group60a
Isoelectric point*
Alanine O
NH2
CH3OH
2.3 9.7 6.0
Asparagine O
O
NH 2
NH 2
O H
2.0 8.8 5.4
Phenylalnine O
NH2
OH
1.8 9.1 5.5
Tryptophan O
NH2NH
OH
2.4 9.4 5.9
* Data were calculated according to the procedure described in Reference74.
103
Unlike Quirino and Terabe 13, 75-78, who used MEKC-based sweeping of the
analytes from dilute sample solutions by employing surfactants, we used an acidic buffer
to elute the extracted amino acid analytes from the sol-gel surface coating and collect
them in the form of a compressed zone. This was accomplished in a simple step during
CE operation without using any micellar solution. The use of surfactants in MEKC
mobile phase often makes the technique difficult to interface with different detection
systems, most notably with mass spectrometry. The presented sol-gel approach to sample
preconcentration does not use any surfactants, and thereby elegantly overcomes a serious
shortcoming inherent in MEKC approach.
4.3.2 Extraction and preconcentration of amino acid by sol-gel columns Based on reported stacking and sweeping methods8, 9, 13, 75-82, the maximum
volume of the sample that could be injected into the CE system is the volume of the
column itself. In the case of very dilute samples, even such a sample volume may not be
enough to enrich a detectable amount of the analyte after preconcentration. In the sample
preconcentration technique described in this chapter, the sample volume that can be used
for analyte enrichment is not limited to one column volume. It allows for the injection of
multiple column volumes of sample. By continuously passing the sample solution for an
extended period through the sol-gel coated capillary possessing enhanced surface area
and appropriate surface charge, the analytes can be extracted from a large volume of the
sample. The pH of the sample solution should be carefully chosen, so that the
zwitterionic analyte of interest assume a net electric charge which is opposite to the
surface charge on the sol-gel coating. The extracted analytes can be further focused into a
narrow band by manipulating the buffer pH.
Figure 4.3 illustrates the enrichment of the tryptophan sample (pI = 5.9) using
extraction on a capillary with positively charged sol-gel coating followed by focusing of
the extracted analytes. At the end of an extended period of sample injection (3 min), the
sample matrix was pushed out of the column by a flow of deionzied water (A) or the
running buffer (B).
After this, a high voltage was applied and CZE was performed using an acidic
buffer (50 mM Tris-HCl, pH 2.2). No sample peak could be detected, if a voltage was
104
-3
-2
-1
0
1
2
3
4
5
6
7
0 10 20min.
mA
U.
A
B
Figure 4.3 Extraction of tryptophan on the sol-gel column. Experimental conditions: sol-
gel coated column 75 cm x 50 µm; the effective length of the column is 70.4 cm; mobile
hase 50 mM Tris-HCl (pH 2.2); injection 3 min at 100 psi; running voltage V = -15 kV;
wavelength of UV detector 200 nm; sample 10 µM tryptophan (pH 7.67). After injection,
the sample matrix was removed by (A) deionized water 3 min at 100 psi, (B) mobile
phase 3 min at 100 psi.
105
applied after rinsing the column with the low-pH buffer (Figure 4.3B) capable of washing
the extracted analytes off the sol-gel coating. On the other hand, when the sample matrix
was removed by deionized water (pH 7.0) (Figure 4.3 A), the extracted amino acid
molecules remained attached to the positively charged capillary surface due to
electrostatic attractive forces between negatively charged solute ions and the positively
charged capillary surface. Desorption of the extracted amino acid and its focusing into a
narrow zone was accomplished by using a high electric field (V = - 15 kV) and a low-pH
buffer, as illustrated in Figure 4.4. The whole procedure consisted of three steps: (a)
extraction, (b) removal of the sample matrix and (c) desorption and enrichment of the
extracted analyte using a low-pH running buffer and a high electric field. In the first step,
the column was filled with sample solution. Negatively charged analytes were extracted
on the positively charged inner surface of the sol-gel column. This process was followed
by the removal of sample matrix with deionized water. In the third step, a high electric
voltage (V = -15 kV) was applied between the ends of the sol-gel capillary, using pH 2.2
Tris-HCl (50 mM) as the running background electrolyte. The cathode was on the inlet
side and a node on the outlet side of the capillary. Under the applied electric field, an
anodic EOF was generated in the CE capillary with positively charged sol-gel coating.
Once the acidic running buffer (50 mM Tris-HCl pH 2.2) came in contact with the front
of the extracted solute zone, it reversed the net charge of the amino acid molecules,
providing a repulsive mechanism for their desorption from the capillary surface. EOF
moved the desorbed analyte molecules forward, gradually desorbing more and more
amino acid molecules and focusing them into a narrow zone.
106
Cathode Anode
1. Filling the capillary with the sample
2. Sample matrix is removed by water
3. Applying a high electric voltage V= -15kV
4. Analytes are focused by an acidic buffer
EOF
Cathode AnodeEOFEOF
cation
anion
neutral analyte
Figure 4.4 Illustration of the events during preconcentration and focusing of zwitterionic
analytes on a positively charged sol-gel column: (1) sample was passed through the
column under helium pressure (e.g., 100 psi for 3 min); (2) sample matrix was removed
from the column by water under helium pressure (e.g., 100 psi for 3 min); (3) application
of high electric voltage (e.g., V = -15 kV); (4) the acidic running buffer desorbed the
extracted analytes and carried them to the detector.
107
Figure 4.5, 4.6, 4.7 and 4.8 show the electropherograms of four amino acids
alanine, asparagine, phenylalanine and tryptophan preconcentrated from 10 µM aqueous
on a positively charged sol-gel column using an extended injection time (3 min). To show
the enrichment effect, two samples of the same amino acid at two different concentration
levels were analyzed on an uncoated fused silica capillary column with the identical
dimensions using conventional hydrodynamic injections. One of the samples had the
same concentration as the one used in the preconcentration experiment, and the other
sample had at least 1000-fold higher concentrations of the amino acids. From these
figures, it is evident that the sample is greatly preconcentrated when analyzed on a sol-gel
column. For example, a 10 µM alanine sample solution was preconcentrated on the sol-
gel column and a peak of more than 6 mAU was obtained (Figure 4.5A). With
conventional mode of injection on an uncoated capillary column, an alanine solution (100
mM) only gave a peak height of less than 2 mAU (Figure 4.5B). Figure 4.5 C shows that
with an uncoated capillary column and conventional injection, no peak was detected for
10 µM alanine sample solution. Similarly, the sol-gel column preconcentrated a 10 µM
tryptophan sample and gave a peak height of more than 6 mAU in Figure 4.8(A). While
with the uncoated fused silica capillary and with conventional mode of hydrodynamic
injection, no peak was obtained for 10 µM tryptophan in Figure 4.8(C). Using an
uncoated capillary, we also ran a tryptophan sample of 1000 times higher concentration
(10 mM). As a result, a peak with a little more than 10 mAU in height was obtained
shown in Figure 4.8(B).
Based on these results, the limit of detection values (LOD, S / N = 3) were
calculated and the results are presented in Table 4.3. We can see that with the positively
charged sol-gel C18-TMS coated column, the presented preconcentration method
provided significantly lower LODs of these amino acids. The most effective
preconcentration result was obtained for alanine. LOD of alanine was reduced from 10.2
mM on an uncoated column to 139 nM on the sol-gel coated column, which corresponds
to an enrichment factor of more than 73,000 times.
108
Alanine
-4
-2
0
2
4
6
8
10
12
0 10 20 30min.
mAU
A
BC
Figure 4.5 Illustration of the effect of a positively charged sol-gel coating on the
preconcentration of alanine. The sample concentrations of A, B, and C was 10 µM, 100
mM and 10 µM. pH of the prepared samples: 7.7. Electropherogram A was obtained
using the sol-gel coated column (75 cm x 50 µm). The effective length of the column was
70.4 cm; mobile phase, 50 mM Tris-HCl (pH 2.2). Hydrodynamic injection for 3 min at
100 psi. UV detection at 200 nm. Sample matrix was removed by deionized water for 3
min at 100 psi, running voltage V = -15 kV. Electropherograms B and C were obtained
on an uncoated column (75 cm x 50 µm) with the same mobile phase. The effective
length of the column is 70.4 cm. Hydrodynamic injection at 10 psi ⋅ sec., running voltage
V = +15 kV. UV detection at 200 nm.
109
Asparagine
-4
-2
0
2
4
6
8
10
12
0 10 20 30min.
mAU
A B
C
Figure 4.6 Effect of sol-gel coating on sample (asparagine) preconcentration. The sample
concentrations of A, B, and C was 10 µM, 50 mM, and 10 µM. Operation conditions are
the same as shown in Figure 4.5.
110
Phenylalanine
-4
-2
0
2
4
6
8
10
12
0 10 20 30min.
mAU
AB
C
Figure 4.7 Effect of sol-gel coating on sample (phenylalanine) preconcentration. The
sample concentrations of A, B, and C was 10 µM, 10 mM and 10 µM. Operation
conditions are the same as shown in Figure 4.5.
111
Tryptophan
-4
-2
0
2
4
6
8
10
12
0 10 20 30min.
mAU
A
B
C
Figure 4.8 Effect of sol-gel coating on sample (tryptophan) preconcentration. The sample
concentrations of A, B, and C was 10 µM, 10 mM and 10 µM. Operation conditions are
the same as shown in Figure 4.5.
112
In order to calculate the sensitivity enhancement factor (SEF), we employed peak
areas as well as the corrected peak areas (the ratio between peak area and the migration
time) using the following equation 75.
factordilution rationpreconcent without obtainedparameter peak
rationpreconcent with obtainedparameter peak SEF ×=
The sensitivity enhancement factors (SEF) for the studied amino acids are
presented in Table 4.4. The SEF values were different for different samples.
In addition, the observation that the migration time of the sample running in an
uncoated column was much longer than that of obtained in sol-gel coated column is due
to the different electroosmotic flow in coated and uncoated columns. When a low-pH
acidic buffer is used as the mobile phase in an uncoated column, a significant portion of
the silanol groups on the fused silica surface remain protonated by the acidic mobile
phase, resulting in a decreased surface charge, and hence reduced EOF. Experiments with
a neutral marker, DMSO, showed that when the pH of the running buffer was 2.22, the
electroosmotic mobility in the untreated fused silica column was 1.02 x 10-4 cm2/V.s. On
the other hand, an acidic running buffer practically did not influence the positive charge
on the sol-gel surface of the column. This is explained by the fact that dissociation of the
quaternary amine group anchored to the surface coating practically remains unaffected by
this pH change. The direction of electroosmotic mobility obtained on the sol-gel coated
column using the same buffer and DMSO as an EOF marker had a value of 4.02 x 10-4
cm2/V.s., being reversed in direction and about four times greater in magnitude than the
EOF in the uncoated column under identical operating conditions.
113
Table 4.3 Sample extraction and preconcentrations on an electrically charged sol-gel column
Aa Bb Cb
Sample
Concentration
µM
LOD
nM (S/N=3)
Concentration
mM
LOD
µM (S/N=3)
Concentration
µM
LOD
(S/N=3)
Alanine 10 139 100 10,170 10 N/Ac
Asparagine 10 98 50 864 10 N/A
Phenylalanine 10 141 10 195 10 N/A
Tryptophan 10 115 10 203 10 N/A
aA: column (75 cm x 50 µm) with a positively charged sol-gel coating, the effective length of the column is 70.4 cm; mobile
phase 50 mM Tris-HCl (pH=2.2). Samples were injected hydronamically for 3 min at 100 psi. Running voltage V= -15 kV.
Wavelength of UV detector: 200 nm. bB and C: uncoated column (75 cm x 50 µm), the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl
(pH=2.2). Samples were injected hydronamically for 10 sec⋅psi. Running voltage V= +15 kV. Wavelength of UV detector:
200nm. c N/A, not applicable.
114
Table 4.4. Sensitivity enhancement factors for four amino acids achieved on positively
charged sol-gel C18-TMS coated columns
SEF by Method 1
Sample By Height By Area
Alanine 55,374 61,048
Asparagine 3,596 1,817
Phenylalanine 995 1,730
Tryptophan 928 1,496
115
4.3.3 Preconcentration of amino acids with the removal of sample solutions by reversed EOF
Remarkably high sample-enrichment factors were achieved using the sample-
preconcentration method mentioned above. However, it might be possible to further
improve this SEF, if we consider the following. Deionized water was used during the
sample matrix removal step after extraction for an extended period of time. Undoubtedly,
this led to the elution, and therefore loss of portion of the analytes extracted on the sol-gel
column. To prevent this loss, the following experiment was designed. The procedure is
illustrated schematically in Figure 4.9.
The sample solution was passed through the sol-gel column for an extended
injection period (3 min at 100 psi). The positively charged sol-gel coating extracted the
anions in the sample solution. Next, a high voltage (+15 kV) was applied with anode in
the inlet side and cathode in outlet end. In this stage, a number of processes occurred.
One of them is that the anions in sample solution migrated to anode while cations
migrated toward the cathode side by electrophoretic flow. In addition, the electroosmotic
flow was generated towards the capillary inlet. EOF, being stronger than the
electrophoreitc flow of the ions, forced the sample matrix to move toward the capillary
inlet and leave the capillary from the inlet end. At the same time, because the running
buffer was acidic, on contact it reversed the electric charge of the extracted analytes and
provided an effective mechanism for their desorption from the positively charged surface
coating in the column via mutual repulsion. During this process, the analyte was focused
at the boundary of the sample solution and the running buffer. The current was observed
carefully to decide the time when the voltage polarity needed to be reversed. While the
column was filled with sample solution, the current was low due to the low conductivity
of the dilute sample solution. With more and more sample matrix being pushed out of the
column, more and more running buffer filled in the column, the current increased because
of the higher conductivity of the media filling the column. Just before the current soared
up quickly, the polarity of the voltage was reversed. The focused sample zone was carried
by the resulting electroosmotic flow towards to the outlet of the capillary and detected by
the UV/vis detector. It can be noticed that instead of mechanically rinsing the column
with deionized water, a reversed electroosmotic flow was applied in conjunction with a
low-pH buffer to remove the sample matrix. Unlike the water-rinsing procedure
116
1. Filling the capillary with the sample
2. Applying a high electric voltage V=+15kV
Anode Cathode
3. After a high electric voltage V=+15kV has been applied for a certain time. Anode Cathode
EOF
4. Switching the polarity of the high electric voltage to V=-15kV. Cathode Anode
EOF
EOF
cation
anion
neutral analyte
Figure 4.9 Method 2 for the preconcentration of zwitterionic analytes on the positively
charged sol-gel column steps. 1. Sample was passed through the column under helium
pressure (e.g. 100 psi for 3 min). 2. High voltage was applied (V= +15 kV), acidic
running buffer came into the column, where extracted analyte desorption occurred via the
local pH change, which caused the redistribution of ions inside the column. 3. Under the
reversed EOF, the sample matrix was removed from the capillary, and the positively
charged analytes were focused at its anodic end. 4. The polarity of the electric field was
switched to V=-15 kV, the focused analytes were carried to the detector.
117
described in the previous section, this method prevented the loss of analytes that have
already been extracted on the sol-gel column.
The sample preconcentration results obtained by this method are shown in Figure
4.10, 4.11, 4.12 and 4.13. The results show that with this method, (hereafter referred to as
Method 2), the sample preconcentration effect is more significant even compared with
the results obtained by the method we described in the previous section (hereafter
referred to as Method 1). This indicates that in Method 1, when the sample matrix was
removed by water, some analytes loss took place. However, with Method 2, the sample
matrix was pushed out of the capillary by reversed electroosmotic flow, and the amount
of analyte loss was greatly reduced.
The LOD (S / N = 3) and SEF by Method 2 were calculated and are listed in
Table 4.5 and Table 4.6. Comparing these data with those listed in Table 4.3, we can see
that for samples of equal concentration the LODs were greatly reduced with Method 2.
For example, with tryptophan as the test sample, Method 2 allowed to lower the LOD to
24.5 nM from 115 nM that was achieved by Method 1. The enhancement in sensitivity is
more than five times. Comparing these data with the results obtained from a bare fused
silica column, the sensitivity enhancement factor were calculated and shown in Table 4.6.
It can be observed that the best results for both preconcentration methods belong to the
same amino acid, alanine. This can be explained from its smaller size compared with
other amino acid samples. According to Beer-Lambert Law, the amount of absorbed light
is proportional to the product of sample concentration and its molar absorptivity
coefficient. Since the inner surface area of the column is constant, the smaller the analyte,
the more amino acids can be extracted on the same area of the sol-gel column. After they
are desorbed from the column, the smaller molecule possesses a higher concentration.
From Table 4.4, we note two important points: (a) both methods greatly increased the
detection sensitivity, and (b) Method 2 is more effective compared with Method 1
because it reduced the sample loss during the sample matrix removal step.
118
Alanine
-5
0
5
10
15
20
25
0 10 20 30
min.
mAU
A
BC
Figure 4.10 Illustration of the effect of sol-gel coating on sample preconcentration
(alanine) by Method 2. The sample concentrations of A, B, and C were 10 µM, 100 mM
and 10 µM. pH values for all samples were 7.7. Electrophoregrams marked with A were
obtained using sol-gel coated column (75 cm x 50 µm), the effective length of the column
was 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.2). Hydrodynamic injection for 3
min at 100 psi. Sample matrix was removed by reversed EOF. Running voltage V=-15
kV. UV detection at 200 nm. Electrophoregrams marked with B and C were obtained on
an uncoated column (75 cm x 50 µm) with the same mobile phase, the effective length of
the column was 70.4 cm. Hydrodynamic injection for 10 sec * psi. Running voltage
V=+15 kV. UV detection at 200 nm.
119
Asparagine
-5
0
5
10
15
20
25
30
35
0 10 20 30min.
mAU
A
B
C
Figure 4.11 Illustration of the effect of sol-gel coating on sample preconcentration
(asparagine) by Method 2. The sample concentrations of A, B, and C were 10 µM, 50
mM and 10 µM. Operation conditions are the same as described in Figure 4.10.
120
Phenylalanine
-5
5
15
25
35
45
55
65
75
85
95
0 10 20 30min.
mAU
A
B
C
Figure 4.12 Illustration of the effect of sol-gel coating sample preconcentration
(phenylalanine) by Method 2. The sample concentrations of A, B, and C were 10 µM, 10
mM and 10 µM. Operation conditions are the same as described in Figure 4.10.
121
Tryptophan
-5
5
15
25
35
45
55
65
75
0 10 20 30min.
mAU
A
B
C
Figure 4.13 Illustration of the effect of sol-gel coating on sample preconcentration
(tryptophan) by Method 2. The sample concentrations of A, B, and C were 10 µM, 10
mM and 10 µM. Operation conditions are the same as described in Figure 4.10.
122
Table 4.5 Sample extraction and preconcentrations on an electrically charged sol-gel column by Method 2.
Aa Bb Cb
Sample
Concentration
µM
LOD, nM
(S/N=3)
Concentration
mM
LOD, µM
(S/N=3)
Concentration
µM
LOD
(S/N=3)
Alanine 10 60.7 100 10,170 10 N/Ac
Asparagine 10 47.3 50 864 10 N/A
Phenylalanine 10 23.3 10 195 10 N/A
Tryptophan 10 24.5 10 203 10 N/A
aA: column (75 cm x 50 µm) with a positively charged sol-gel coating, the effective length of the column is 70.4 cm; mobile phase 50
mM Tris-HCl (pH=2.22). Samples were injected hydronamically for 180 seconds at 100 psi. Running voltage
V= -15 kV. Wavelength of UV detector: 200 nm. bB and C: uncoated column (75 cm x 50 µm), the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.22).
Samples were injected hydronamically for 10 sec*psi. Running voltage V= +15 kV. Wavelength of UV detector: 200 nm. cN/A: not applicable
123
Table 4.6 Sensitivity enhancement factors for four amino acids achieved on positively
charged sol-gel C18-TMS coated columns by Method 2a
SEF by Method 2
Sample By Height By Area
Alanine 153,770 66,782
Asparagine 16,773 21,427
Phenylalanine 11,248 63,469
Tryptophan 6,326 10,754
a Operation conditions are as same as shown in Figure 4.10.
124
Figure 4.14 represents experimental data showing the preconcentration of alanine
by Method 2 (trace A), which was compared with a blank run (trace B). This experiment
was designed to verify whether the peaks obtained by the described preconcentration
methods were artifacts of system peaks. The absence of such a peak in the blank run
clearly indicates that the peak in (A) is not a system peak and confirms the real possibility
of performing sample preconcentration using the described methods.
Unlike Method 1 (which includes extraction and focusing operations only),
Method 2 includes an additional step allowing electrophoretic migration of the extracted
charged analytes. This provides a real opportunity to achieve separation of the extracted
analytes by Method 2. Figure 4.15 highlights this point and illustrates the practical utility
of Method 2 by providing an example of online preconcentration and separation of two
amino acids: tryptophan and asparagine. As can be seen in Figure 4.15, the two
preconcentrated amino acids are more than baseline separated with a wide gap between
them.
The reproducibility of the sample preconcentration methods was examined by a
series of experiments and shown in the terms of the relative standard deviation (RSD) of
migration time and peak height. Table 4.7 shows the experimental and calculation results.
Quite good repeatability in migration times was obtained with both preconcentration
methods for all test analytes. The RSD values in terms of migration time are no more
than 3.7%. The RSD values in the range of 3.8% to 28%were obtained for peak height
repeatability. The presented data reveals that in both cases, Method 2 provided
significantly better repeatability than Method 1. The sample matrix removal procedure in
Method 1 probably caused the inferior reproducibility for some solutes in Method 1.
125
Alanine
-5
0
5
10
15
20
25
0 10 20 30min.
mAU
A
B
Figure 4.14 Juxtaposition of the effect of sol-gel coating on sample preconcentration by
Method 2 and a blank run with the same method. Trace (A) is 10 µM alanine. Sample pH
value was 7.7. Trace (B) is a blank run. Sol-gel coated column (75 cm x 50 µm), the
effective length of the column was 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.2).
Hydrodynamic injection for 3 min at 100 psi. Sample matrix was removed by reversed
EOF. Running voltage V=-15 kV. UV detection at 200 nm.
126
-5
-3
-1
1
3
5
7
9
0 10 20 30min.
mAU.
A
B
a
b
Figure 4.15 Illustration of the preconcentration and separation of a mixture of two amino
acids on a positively charged sol-gel column using Method 2. pH value of the sample was
7.7. Electropherograms were obtained using sol-gel coated column (75 cm x 50 µm), the
effective length of the column was 70.4 cm; mobile phase 50 mM Tris-HCl
(pH=2.2)/ACN 50/50. Hydrodynamic injection for 3 min at 100 psi. Sample matrix was
removed by reversed EOF. Running voltage V=-15 kV. UV detection at 200 nm.
Electropherogram marked with A was obtained by preconcentrating and separating an
amino acid mixture, in which peak a is tryptophan and peak b is asparagine.
Electropherogram B was obtained by running a blank.
127
Table 4.7 Repeatability data for the preconcentration by C18-sol-gel coated column using amino acids as test solutes
Method 1a Method 2b
Analytes av. tR
(min)
RSD (%)
(n=4)
av.height
(mAU)
RSD(%)
(n=4)
av. tR
(min)
RSD (%)
(n=4)
av.height
(mAU)
RSD(%)
(n=4)
Alanine 4.37 2.77 5.80 27.70 17.67 1.62 20.80 10.10
Asparagine 4.36 1.32 5.73 13.81 12.08 0.95 26.53 9.45
Phenylalanine 4.44 1.11 7.57 3.83 14.43 1.73 94.04 5.91
tryptophan 4.45 2.27 6.61 9.18 9.56 3.70 86.26 15.93
a Experimental conditions: same as in Figure 4.5. bExperimental conditions: same as in Figure 4.10.
128
4.4 Conclusions For the first time, we have shown on-column extraction and preconcentration
effect offered by positively charged sol-gel column in capillary zone electrophoresis.
Using a positively charged sol-gel coating, a 150,000 fold enrichment effect was obtained
for alanine. The newly developed methods do not limit the volume of the injected sample
and they do not require modification of a standard CE system to achieve the
preconcentration effect. They allow large-volume injection of the sample for an extended
period of time, and are very effective in enriching trace concentrations of zwitterionic
solutes. Large sensitivity enhancement factors (SEF) on the order of 105 were obtained in
the study. Further sensitivity enhancement should be possible in a number of ways by (a)
performing the preconcentration step without the removal of sample buffer; (b) using
thicker sol-gel coatings or monolithic beds; and (c) derivatizating the amino acids with
proper derivatization reagents.
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133
CHAPTER FIVE NEGATIVELY CHARGED SOL-GEL COLUMN FOR
ONLINE PRECONCENTRATION OF ZWITTERIONIC BIOMOLECULES IN CAPILLARY
ELECTROMIGRATION SEPARATIONS
5.1 Introduction
Capillary electrophoresis (CE), characterized by high column efficiency and
separation speed, has been successfully used in a wide range of areas 1-15. While CE
provides a number of significant advantages, its use in conjunction with on-column UV
detection results in low concentration sensitivity due to short optical path length. To
improve the concentration sensitivity in capillary electrophoresis, a number of strategies
have been developed such as sample preconcentration, use of alternative capillary
geometry, and using other detection modes 16. The first strategy has attracted a great
degree of interest since it neither involves any modification of commercially available CE
instrument nor does it increase the cost associated with using alternative detection modes.
In Chapter Four, we have shown the possibility of using a positively charged sol-
gel coating for on-column preconcentration and analysis of amino acids using UV
detection. In this approach, the sample pH values were initially maintained above the
amino acid isoelectric points. Under these conditions, amino acids (zwitterionic analytes,
in general) carried a net negative charge. When such a sample was passed through a
capillary column with a positively charged surface, electrostatic interaction between the
solutes and the coating led to effective extraction of the amino acids. The extracted
analytes were further desorbed by a local pH change and analyzed by CE.
This chapter describes a method for the creation of a negatively charged sol-gel
coating in a fused silica capillary and demonstrates how such a sol-gel column can
effectively combine principles of capillary microextraction 17 with those of stacking and
focusing techniques 18, 19 to increase the sample concentration sensibility in CE.
Another important issue discussed in this chapter is providing a stable EOF in CE.
It is well known that among CE operation modes, such as capillary zone electrophoresis
134
(CZE), capillary electrochromatography (CEC) , and micellar electrokinetic
chromatography (MEKC) 20, 21, electroosmotic flow (EOF) plays an important role by
serving as the driving force for the mobile phase flow through the column. In CE
separation, if EOF changes with time, the migration times of the solutes will change. This
may lead to inaccurate results in identification and quantitation 21. Therefore, a stable
EOF is highly desired for consistent analytical results in CE separations 21.
5.1.1 The production of a stable EOF
As mentioned in previous section, the origin of EOF lies on the electrical double
layer formed on the capillary inner wall. Under high electric field, the counter ions on the
diffuse layer of the double layer begin to move, and drag the solvated water molecules
with them, which give rise to the bulk movement of the liquid in the columns. In fused
silica capillary columns, commonly used in CE separations, the electrical double layer
forms as a result of the deprotonation of the silanol groups present on the inner surface of
the capillary wall 21, 22. The concentration of deprotonated silanol groups residing on the
inner surface of capillary is a major factor that determines the magnitude of EOF 23. In
addition, various operational parameters such as the concentration, pH, viscosity of the
buffer solution, type of electrolyte used to prepare the buffer solution, and operation
voltage also affect the the magnitude of EOF 21, 24.
For silanol groups on the fused silica capillary surface, the pKa value is estimated
at around 7.5 25 . However, in contact with buffer solutions with pH above 2, some of
these silanol groups start dissociating into negatively charged silanate. The dissociation
of the silanol groups increases with an increase in buffer pH resulting in an increased
EOF, which may cause poor migration time reproducibility in CE when fused silica
capillary severs as the separation column. A second cause for the migration time
fluctuation is the absorptive interaction between the column and solutes like proteins and
peptides. Adsorption of solutes on the capillary surface not only affects consistency in
their migration times, but also produces poor separation efficiencies. In fact, not a single
mode of binding contributes to protein adsorption 26-29. It is commonly assumed that
adsorption is associated with the electrostatic interactions between the net charge on the
135
molecule and the silica surface 23, hydrogen bonding with OH, NH or CO groups, as well
as hydrophobic interaction 30.
To minimize the migration time fluctuation problem, several strategies have been
developed to prepare columns with stable EOF in CE. One of the most commonly used
approaches is to create an appropriate coating on the fused silica capillary inner surface to
derivatize or shield the silanol groups. These coatings can be either electrically neutral or
carry net charges. In the case of neutral coating, the separation will only depend on the
electrophoresis due to the suppression of EOF. With a charged coating, a relatively
stable EOF may be generated if the coating material is carefully selected.
Poly(viny1 alcohols) (PVA) with a molecular weight about 50 000 were applied
by Gilges et al. to modify the fused silica surface dynamically and permanently for the
separation of charged molecules such as proteins 31. The dynamic thin polymeric
coatings were formed by adsorption of PVA on the capillary surface when a PVA
solution was passed through the capillary for a short time before actual separations. The
generation of PVA coatings is based on the fact that poly(vinyl alcohol) becomes water
insoluble by thermal treatment at temperatures of up to 160 ˚C. These PVA coatings
successfully suppressed the EOF. Other neutral polymers used as coating materials
include methylcellulose 32, poly(methylglutamate) 33, polyethylene glycols 34, 35 and Ucon 36, 37.
Angulo and co-workers 38 developed a CE analysis method to study kin17
protein-DNA affinity by using a nonpermanent poly(ethylene oxide) (PEO) based coating
to avoid adsorption of kin17. Their coating procedure was as follows: the capillary was
first pretreated with NaOH, HCl and Milli-Q water. Then A 0.2% w/v PEO solution in
0.1 M HCl was used to flush through the capillary to achieve reprotonation of the
capillary with water and HCl. After each run, the coating was regenerated by a series of
rinses with water, HCl as well as a solution of PEO. It is reported that this coating
procedure was optimized to provide a residual and stable EOF.
5.1.2 Negatively charged coatings for CE columns
Depending on the desired direction of EOF and the properties of analytes, either
positively or negatively charged coatings have been created 39-45. In a CE column with a
136
positively charged inner surface, the diffuse component of the electrical double layer
contains anions and generates an anodic electroosmotic flow instead of the cathodic EOF
produced by untreated fused silica capillary characterized by a negatively charged surface.
To generate positively charged sites on the inner surface of column, cryptand-containing
polysiloxane 40 and quaternary ammonium group 39, 41-46 have been used.
The commonly used negatively charged coatings are prepared using sulfonate-
containing materials 40, 47. Lee and co-workers 40 created sulfonic acid groups on the
capillary surface by in situ copolymerization. In their method, the capillary surface was
first treated with 7-oct-1-enyltrimethoxysilane. Then, 2-acryloyl-amido-2-2-
methylpropane sulfonic acid and acrylamide mixtures were copolymerized with α,α`-
azobis(isobutyronitrile) (AIBN) as an initiator. The most important advantage to use
sulfonate-containing materials is that sulfonic acid group, unlike the silanol group, can
stay dissociated under low pH conditions. Therefore, a CE column with a surface coating
containing the sulfonic acid moiety is expected to provide a stable cathodic EOF within a
wide pH range. It should be noted that EOF in an untreated fused silica capillary changes
with pH since the silanol groups represent a weak acid whose dissociation is greatly
affected by pH changes.
Recently, Wiedmer and co-workers 48, 49 developed a method to coat fused silica
capillary with anionic liposomes in the presence of N-(hydroxyethyl)piperazine-N’-(2-
ethanesulfonic acid) (HEPES) as background electrolyte solution. The coating was done
by rinsing the pretreated capillary with liposomes in HEPES background electrolyte
solution for 10 min at a pressure of 930-940 mbar. The coating solution was allowed to
stay inside the capillary for 15 min and washed with background electrolyte solution for
10 min to remove unbound liposomes. The capillary modified with the liposomes coating
was successfully used to separate uncharged steroids as model compounds.
Among various methods to control EOF in CE, sol-gel approach is a new
direction in achieving this goal 36, 41-43, 50-52. For example, Hayes and Malik 36 used sol-gel
based technology to prepare a Ucon-coated fused silica capillary column in CE. The
coating procedure involves (1) preparation of sol solution containing proper ingredients,
(2) filling the capillary with the sol solution, and (3) formation of sol-gel network inside
the capillary. The obtained sol-gel coated column was successfully used to separate test
137
samples (e.g. basic proteins, and nucleotides). Allen and El Rassi 50 developed a two-step
preparation method to create positively charged sol-gel monolithic column in CE, in
which the sol-gel silica backbone was first prepared, and followed by the introduction of
organic moieties that contributed to the positive charge on the obtained columns. A
similar pathway was reported by these authors to produce sol-gel monolithic columns
with cyano or cyano/hydroxyl functional groups 53. By sol-gel technology 54, 55, an in situ
created sol-gel layer is chemically bonded to the capillary surface, greatly increasing
columns’ operational stability. In the meantime, sol-gel chemistry makes it possible to
synthesize organic-inorganic hybrid materials with advanced properties. Columns
prepared by sol-gel technology offers many advantages over their conventional
counterparts in GC 56, 57, HPLC 58, 59, and CE 36, 42, 43, 51, 60.
In this chapter, we described the use sol-gel chemistry to create a sulfonic acid-
containing sol-gel coating on the inner surface of a fused silica capillary. The electrostatic
interaction between the analytes and the capillary surface was used to extract analytes
from dilute sample solutions. Extraction techniques including solid-phase extraction (SPE) 61 and liquid-liquid extraction (LLE) 62 have been used for sample preconcentration in CE.
Compared with electrophoresis-based preconcentration methods, extraction-based
approaches are more selective, and a wide range of analytes can be preconcentrated by
these methods. Here, coupling of the preconcentrator with CE usually requires
modification of the instrument, and may be considered as a drawback. In the present
study, microextraction and dynamic pH junction were combined to increase sample
preconcentration sensitivity in CE using a commercial instrument without any
modifications. Sulfonic acid (pKa ~2) 63 is fully ionized over a wide range of pH 40.
Therefore, it is less influenced by the change of pH environment compared with silanol
groups (pKa ~ 7.5) 25 on fused silica capillary. Thus, the created sol-gel coating not only
provided a more stable EOF, but also offered an electrostatic repulsive mechanism to
prevent adsorption of anionic bioanalytes on the capillary surface.
5.2 Experimental
138
5.2.1 Equipment
On-line sample preconcentration and CE experiments were performed on a Bio-
Rad BioFocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules,
CA) equipped with a programmable, multi-wavelength UV/Vis detector. BioFocus 3000
operating software system (version 6.00) was used to collect and process the CE data. A
Barnstead model 04741 Nanopure deionized water system (Barnstead/Thermodyne,
Dubuque, IA) was used to prepare deionized water (~16MΩ cm). A homemade gas
pressure-operated capillary filling/purging device 36 was used for coating the fused-silica
capillary. A Microcentaur model APO 5760 centrifuge (Accurate Chemical and Scientific
Corp., Westbury, NY) was used for centrifugation of the sol solutions. A Fisher model G-
560 Vortex Genie 2 system (Fisher Scientific, Pittsburgh, PA) was used for thorough
mixing of the ingredients in sol solution. A Chemcadet model 5984-50 pH meter (Cole-
Palmer Instrument Co., Chicago, IL) equipped with a TRIS-specific pH electrode
(Sigma-Aldrich, St. Louis, MO) was used to measure the buffer pH.
5.2.2 Chemicals and materials
Fused-silica tubing of 50-µm i.d. was purchased from Polymicro Technologies
(Phoenix, AZ) for the preparation of sol-gel coated columns. Sample vials (600µL),
HPLC grade methylene chloride and methanol were purchased from Fisher Scientific
(Pittsburgh, PA). Mercaptopropyltrimethoxysilane (MPTMS), n-octadecyltriethoxysilane
(C18-TEOS) and phenyldimethylsilane (PheDMS) were purchased from United Chemical
Technologies, Inc. (Bristol, PA). Tetramethoxysilane (TMOS) (99+%), trifluoroacetic
acid (TFA) (99%), dimethylsulfoxide (DMSO), and hydrogen peroxide 30 wt. % in water
were purchased from Aldrich (Milwaukee, WI). Tris (hydroxymethyl)aminomethane
hydrochloride (reagent grade) (Tris-HCl), tris(hydroxymethyl)aminomethane (reagent
grade) (Tris-base) as well as myoglobin, conalbumin, arginine, lysine and asparagine
standards were purchased from Sigma (St. Louis, MO).
The background electrolyte (BGE) solutions were prepared by dissolving Tris-
base and/or Tris-HCl in deionized water. The pH adjustments for the BGE solutions and
samples were carried out by the addition of 1 M HCl, or 0.1 M NaOH.
139
5.2.3 Preparation of the sol-gel coating solutions
The sol-gel solutions were prepared using the following ingredients: (a) three
precursors (mercaptopropyltrimethoxysilane (MPTMS), tetramethoxysilane (TMOS) and
n-octadecyltriethoxysilane (C18-TEOS)), (b) a deactivation reagent (phenyldimethylsilane
(PheDMS)) and (c) a sol-gel catalyst (trifluoroacetic acid (TFA) containing 5% water)).
The sol solution was vortexed to mix thoroughly and then centrifuged to remove the
precipitates (if any). The clear sol solution in the top portion of the centrifuge tube was
transferred to a clean vial and was further used to create a negatively charged sol-gel
coating on the inner surface of a fused silica capillary.
5.2.4 Preparation of CE columns with a negatively charged sol-gel coating
Preparation of the sol-gel open tubular CE columns involved the following two
major steps: (1) hydrothermal treatment of the fused-silica capillary inner surface and (2)
creation of sol-gel coating on the hydrothermally treated surface. The purpose of the
hydrothermal treatment was to clean the capillary inner surface and to enhance the
surface silanol concentrations by hydrolysis of siloxane bridges, thereby increasing the
bonding sites for chemical anchoring the sol-gel coating. Detailed coating procedure was
described elsewhere by Hayes and Malik 42. Briefly, the capillary was coated by using an
in-house built gas pressure-operated filling/purging system 36. Under pressure, the
capillary was filled with the sol-gel coating solution. The sol solution was allowed to stay
inside the capillary for a predetermined period (typically 15-30 min) to facilitate the
hydrolytic polycondensation reactions responsible for the formation and growth of sol-gel
network structures within the sol solution filling the capillary. At this point, the sol
solution in the capillary was still a liquid and contained fragments of sol-gel network. In
the course of sol-gel process, a portion of the sol-gel network fragments evolving in the
vicinity of the fused silica capillary inner wall became chemically bonded to it via
condensation reaction between the silanol groups on the capillary walls and the sol-gel-
active functional groups on the sol-gel network. This bonded part of the sol-gel material
formed the surface coating. The unbonded liquid portion of the partially gelled sol
solution was then removed from the capillary using helium pressure (40 psi), and the
coated capillary was conditioned at 150˚C for 2 hours. To convert the mercaptopropyl
140
groups into sulfonate moieties 64, a solution containing 30 wt % hydrogen peroxide was
subsequently passed through the column at 40 psi for 5 hours. The coated capillary with
negatively charged sulfonate groups in the surface-bonded sol-gel coating was further
conditioned at 100° C for 10 hours. This step was followed by rinsing the sol-gel coated
columns with 1 mL each of deionized water and the desired running buffer at 100 psi
using the filling/purging system. After installation in the CE system, before the first run
the column was further rinsed with deionized water and running buffer for 10 min each at
100 psi. Before each replicate run, the column was rinsed at 100 psi with DI water and
running buffer but for a shorter time depending on the length of the column.
5.2.5 Preparation of samples
Myoglobin, conalbumin, arginine, lysine and asparagines purchased from Sigma
(St. Louis, MO) were dissolved in deionized water to make the test samples.
Concentrations of each sample are shown in section 5.3.
5.3 Results and discussion
5.3.1 Sol-gel reactions involved in the coating process
Table 5.1 lists the names and chemical structures of sol-gel solution ingredients
used in the present study. The ethoxysilyl groups in C18-TEOS and methoxysilyl groups
in TMOS and MPTMS are sol-gel-active. They participate in the formation of the sol-gel
polymeric network through hydrolytic polycondensation reactions. Scheme 5.1 illustrates
complete hydrolysis of MPTMS, TMOS and C18-TEOS.
This is a simplified scheme and illustrates only one possible outcome of the
hydrolysis reactions. It is implied that partial hydrolysis of the alkoxy groups is possible
and the reaction mixture is likely to contain a variety of other products with varying
numbers of hydrolyzed alkoxy groups. The hydrolysis products from sol-gel co-
precursors can further enter into polycondensation reactions, which provide a simple and
effective pathway for the formation of a sol-gel network inside the capillary. A simplified
scheme of polycondensation reactions likely to take place in the sol-gel process is
presented in Scheme 5.2.
141
Table 5.1 Names and chemical structures of the reagents used in the sol solution to
fabricate negatively charged sol-gel sulfonated columns.
Regent Function and Reagent Name Reagent Structure
Sol-gel precursors:
Tetramethoxysilane (TMOS)
OCH3
Si
OCH3
OCH3H3CO
n-octadecyltriethoxysilane (C18-TEOS)
Si (CH2)17H5C2O
OC2H5
OC2H5
CH3
Mercaptopropyltrimethoxysilane (MPTMS)
Si (CH2)3H3CO
OCH3
OCH3
SH
Deactivation reagent:
Phenyldimethylsilane (PheDMS)
Si
H
CH3 CH3
Catalyst:
Trifluoroacetic acid (TFA)
HO
O
CF3
142
Scheme 5.1 Illustration of the complete hydrolysis of sol-gel precursors
a. Hydrolysis of MPTMS:
3H2O 3CH3OHSi (CH2)3
OCH3
H3CO
OCH3
SH Si (CH2)3
OH
HO
OH
SH
b. Hydrolysis of TMOS:
Si OCH3
OCH3
H3CO
OCH3
Si OH
OH
HO
OH
4H2O 4CH3OH
c. Hydrolysis of C18-TEOS:
Si (CH2)17
OC2H5
C2H5O
OC2H5
CH3 3H2O Si (CH2)17
OH
HO
OH
CH3 3C2H5OH
143
Scheme 5.2 Condensations of hydrolysis products from TMSO, MPTMS, and C18-TEOS
x Si (CH2)3HO
OH
OH OH
Si
OH
OHHO Si (CH2)17CH3HO
OH
OH
+ y uSH
-nH2O
Si (CH2)3
O
O
SH
O
Si
O
OO
Si (CH2)17CH3
O
OO
Si
O
OSi
O
OHO
Si
**
+
OH
m
144
The condensation process may further extend to chemically bind a portion of the
evolving sol-gel network to the silanol groups on the capillary inner surface (shown in
Scheme 5.3).
The deactivating reagent, PheDMS, was added to the sol solution to derivatize
residual silanol groups on the sol-gel coating or the capillary surface during thermal
conditioning step that follows the coating process, and thereby provides deactivation of
the sol-gel coated capillary (Scheme 5.4).
MPTMS possesses the mercaptopropyl group, which is further oxidized into
sulfonic acid moiety by passing hydrogen peroxide 64 through the coated capillary.
Scheme 5.5 shows the oxidation of mercaptopropyl group into sulfonic acid.
145
Scheme 5.3 Covalent bonding of the sol-gel coating to fused silica surface
Si (CH2)3
O
O
SH
O
Si
O
OO
Si (CH2)17CH3
O
OO
Si
O
OSi
O
OHO
Si
**Si
O
OH
O
-H2O
Si (CH2)3
O
O
SH
O
Si
O
OO
Si (CH2)17CH3
O
OO
Si
O
OSi
O
O
Si
Si
O
O
O
Silica surface
Sol-gel coating bondedto the silica surface
OH
m
m
OH
Silica surface
146
Scheme 5.4 Deactivation of the sol-gel mediated fused-silica coated surface with
PheDMS
Si (CH2)3
O
O
SH
O
Si
O
OO
Si (CH2)17CH3
O
OO
Si
O
O
OH
Si
O
O
Si
**Si
O
O
O
SiHH3C CH3
-H2
Si (CH2)3
O
O
SH
O
Si
O
OO
Si (CH2)17CH3
O
OO
Si
O
O
O
Si
O
O
Si
Si
O
O
O
SiH3C CH3
Sol-gel coating bonded to silica surface
Deactivated sol-gel coatingbonded to silica surface
Silica surface
m
Silica surface
m
147
Scheme 5.5 Oxidation of mercaptopropyl group into sulfonic acid moiety by H2O2
Si (CH2)3O
OSH
OSiO
OO
Si (CH2)17CH3O
OOSiO
OOSiO
O
Si
**SiO
OO
SiH3C CH3
3H2O2
Si (CH2)3O
OSO3H
OSiO
OO
Si (CH2)17CH3O
OOSiO
OOSiO
O
Si
SiO
OO
SiH3C CH3
-3H2O
Si (CH2)3O
OSO3
OSiO
OO
Si (CH2)17CH3O
OOSiO
OOSiO
O
Si
SiO
OO
SiH3C CH3
Deprotonation
Deactivated sol-gel coating bonded tosilica surface
Deactivated sulfonated sol-gel coating bonded tosilica surface
Negatively charged sol-gel coating bonded tosilica surface
Silica surface
Silica surface
Silica surface
m
m
m
148
5.3.2 Characterization of the sol-gel coating
A series of electroosmotic flow measurements were performed to investigate the
magnitude of the EOF under different pH conditions using dimethylsulfoxide (DMSO) as
an EOF marker. Since the sulfonic acid moiety is primarily responsible for the negative
charge on the prepared sol-gel columns, the relative concentration of MPTMS in the sol
solution used to prepare these columns is important. To show the effect of the sol solution
MPTMS content on the EOF at different pH conditions, sol solutions with three different
MPTMS-to-total precursor molar ratios were used to coat the capillaries. Table 5.2 lists
the relative compositions of precursors in the used sol solutions.
Figure 5.1 depicts the results from EOF evaluations on two sol-gel sulfonated
columns and one uncoated fused silica capillary.
As can be seen in Figure 5.1, the buffer pH has less influence on the EOF of
sulfonated sol-gel columns. Also, the effect of buffer pH on the EOF decreases with the
increase of MPTMS content in the sol solution which translates into an increase of
sulfonate groups on the surface of the capillary. The silanol groups on the fused silica
capillary surface is estimated have a pKa value of about 7.5 25. Unlike the silanol group
on the fused silica, sulfonic acid has a pKa of about 2 63, and can stay dissociated at low
pH conditions. However, the point of zero net charge generally recommended for fused
silica in the field of capillary electrophoresis is pH ≈ 2.0 21, 22, 65. The measured EOF
values for sulfonated sol-gel coated columns under acidic conditions are higher than that
for uncoated fused silica capillary. As shown in Figure 1, when pH was 3.75, the EOF
obtained from the sol-gel coated column (74% MPTMS in the sol solution) was 1.57 x
10-4 cm2/V.s, which increased 162% compared with the value of 5.99 x 10-5 cm2/V.s
obtained from an uncoated fused silica capillary. This significant difference in EOF
between sulfonate coated and uncoated fused silica columns can be explained by the fact
that sulfonic acid represents a much stronger acid than silanol. Under basic buffer
conditions, the measured EOF values for the sol-gel sulfonated columns were lower than
that of bare silica columns. For example, at pH 8.88, the EOF measured from the sol-gel
coated column (74% MPTMS in the sol solution) was 3.84 x 10-5 cm2/V.s, which was
34% lower than the EOF value obtained on an uncoated column at same pH (5.81 x 10-5
cm2/V.s). Under such conditions, both silanol and sulfonic acid groups dissociate. The
149
Table 5.2 MPTMS – to- total sol-gel precursor molar ratio in the coating sol solutions
used in the fabrication of negatively charged sol-gel sulfonated columns
Column I Column II Column III
Sol-gel precursors: mol. % mol. % mol. %
Tetramethoxysilane (TMOS)
46%
26%
14%
n-Octadecyltriethoxysilane
(C18-TEOS)
14%
14%
12%
Mercaptopropyltrimethoxysilane
(MPTMS)
40%
60%
74%
150
0.00E+00
2.00E-04
4.00E-04
6.00E-04
8.00E-04
0 1 2 3 4 5 6 7 8 9 10 11
pH
EO
F (c
m2 /V
.s)
uncoated
40% MPTMS
74% MPTMS
Figure 5.1 Electroosmotic flow vs. buffer pH values. 30 cm x 50 µm i.d. columns, running buffer 50 mM tris-base, DMSO as the
neutral marker, V = + 9 kV, injection 10 psi ⋅ s, UV detection at 214 nm.
151
lower EOF infers that the density of negative charge on the column surface has been
reduced. The overall profile of the curves shows that pH has less prominent influence on
EOF of the sol-gel sulfonated columns. However, the different EOF values obtained
under basic and acidic conditions indicate that some silanols, either on the fused silica
capillary surface or the sol-gel coating, are not shielded or deactivated.
The effect of sol-gel coating on the repeatability of EOF was investigated by
comparing relative standard deviations (RSD) for replicate EOF measurements carried
out on a sulfonated sol-gel columns and an uncoated fused silica capillary of same
dimensions (75 cm x 50 µm i.d.) at pH 2.2 and 8.0, respectively. The sulfonated sol-gel
column provided more consistent EOF than that for the bare fused silica capillary as
reflected by the smaller RSD values for the sol-gel sulfonated column. For example, the
RSD value obtained on a sol-gel sulfonated column (MPTMS 40%) for 5 replicate runs at
pH = 2.2 was 4.8%; while the RSD value obtained on an uncoated fused silica capillary
was 24% under the same pH conditions. Figure 5.2 shows the run-to-run EOF
repeatability data obtained on a sol-gel sulfonated column in four replicate experiments
performed at pH 8.8 (10 mM buffer solution), using DMSO as the EOF marker. The
migration times measured was (4.56 ± 0.03) min.
In order to investigate the reproducibility of the described coating procedure, the
electroosmotic mobility has been measured on 5 columns prepared by the developed
method. Dimethylsulfoxide was used as the EOF marker, and 50 mM Tris buffer (pH =
8.8) as the background electrolyte. An RSD value of 6.8% was obtained, which is
indicative of good column-to-column reproducibility for the described sol-gel coating
procedure.
152
Table 5.3 The effect of sol-gel coating on the repeatability of migration time of a neutral
EOF marker, DMSO
pH = 8.0
pH = 2.2
Uncoated1
tR (min)
Coated2
tR (min)
Uncoated1
tR (min)
Coated2
tR (min)
Run #1 11.18 14.38 31.77 24.86
Run #2 12.08 14.45 46.66 22.05
Run #3 11.04 14.46 41.04 22.83
Run #4 10.97 14.42 51.94 22.49
Run #5 10.88 14.37 56.97 23.67
RSD (%) 4.3 0.3 21 4.8
1: Uncoated fused silica capillary column 75 cm x 50 µm i.d. 2: Fused silica capillary column 75 cm x 50 µm i.d. with a sol-gel sulfonated coating
(MPTMS 40%).
Operation conditions: sample DMSO, buffer 50 mM Tris HCl/Base for pH 8.0, and 50
mM Tris HCl for pH 2.2, applied voltage +15.00 kV, injection 10 psi⋅s, UV detection at
214 nm.
153
0
2
4
6
8
10
12
14
16
18
0 5 10 15 20
min.
mA
U
4.54min
4.52min
4.57min
4.61min
Figure 5.2 Migration time repeatability for a sol-gel sulfonated column. Operation
conditions: column 50 cm x 50 µm i.d., sample DMSO, buffer 10 mM 50/50 Tris HCl /
Base pH 8.8, applied voltage + 15.00 kV, injection 10 psi⋅sec, UV detection at 214 nm.
154
5.3.3 The zeitterionic solutes preconcentration mechanism operated in the negatively charged sol-gel columns
The attractive electrostatic interaction of the zwitterionic solutes (protein or amino
acid) and the negative charge on the inner walls of the sol-gel coated fused silica
capillary forms the basis for solute preconcentration on the described sol-gel columns.
When such a zwitterionic test analyte is passing through the sulfonated sol-gel column
with the opposite charge on the surface coating, the positively charged analytes will
electrostatically interact with the sol-gel coating leading to their extraction. Therefore,
microextraction is accomplished by passing the dilute sample solution through the
column. In the presented method, the sample volume that can be used for analyte
enrichment is not limited by one column volume as imposed by some other
preconcentration techniques 18, 66. The sol-gel approach allows for the injection of
multiple column volumes of the sample. By continuously passing the zwitterionic sample
solution for an extended period through the sol-gel coated capillary with enhanced
surface area and appropriate surface charge, the extraction can be accomplished from a
large volume of the sample. With this described preconcentration method, sample was
introduced into the column at 100 psi for an extended period. For example, sample was
injected at 100 psi for 30 sec into a 30 cm long column. This approximately corresponded
to more than 7 capillary volumes of the sample passed through the column for on-line
extraction. This is an important advantage of the described on-line sample
preconcentration method compared with other methods 18, 19, 66, 67, in which the possible
maximum volume of the sample that could be injected into the CE system is limited by
the volume of the column itself. It should be pointed out that in the case of very dilute
samples, even one column volume of the sample may not be enough to provide detectable
amount of the analyte.
The extracted analytes are then desorbed from the sol-gel coating by using a
running buffer with a pH value above the pI of the extracted analytes. Under such pH
conditions, the extracted zwitterions assume a net negative charge and desorb from the
155
negatively charged sol-gel coating due to mutual repulsion. The desorbed zwitterions can
be further focused and analyzed.
The focusing of desorbed analytes was achieved by taking advantage of the
dynamic pH junction between the sample solution and the background electrolyte zone 68.
A schematic diagram (Figure 5.3) is presented below to illustrate the whole process
including sample injection, extraction, desorption, and focusing of myoglobin or
asparagine used as representative zwitterionic test biomolecules. As shown in Figure 5.3,
after an extended period of sample injection (e.g., ≥ 30 s at 100 psi), facilitates the
extraction of positively charged analytes on the negatively charged sol-gel coating (2a).
A high electric field (e.g., E = +300 V/cm) is then applied using a running buffer (pH =
8.8), whose pH is greater than the pI of extracted analyte (for myoglobin pI ~ 7.0 69, for
asparagine pI ~ 5.4 70) (2b). Under the effect of high electric field, a cathodic EOF is
generated which drives the buffer (e.g. pH = 8.8) towards the cathode. Once the basic
buffer enters the capillary filled with sample solution, a dynamic pH junction is formed
between the running buffer and the sample solution. By coming in contact with the basic
buffer, the extracted zwitterionic analyte molecules in the junction area acquire a net
negative charge, and desorb from the sol-gel coating because of the repulsive forces
between same sign of the charge on the analyte and the sol-gel surface. The direction and
migration rate of the desorbed analytes are under control of two opposite forces: (a) EOF
and (b) electrophoretic mobility of the desorbed analyte molecules that have now turned
into anions. Under the applied electric field, the negatively charged analytes residing in
the buffer would tend to migrate toward the anode. Meanwhile, the EOF would tend to
drive them toward the cathode. Since EOF is generally stronger than electrophoretic
mobility, overall the analytes will move toward the cathode, pass through the on-column
UV detector window and get detected.
156
a. Filling the capillary with the sample and analytes will be extracted on the capillary surface.
b. Applying a high electric field V= +9 kV to generate EOF
Anode Cathode
c. Buffer solution is introduced into the capillary by EOF and a dynamic pH junction is formed to desorb extracted analytes out of the capillary.
Anode Cathode
EOF
Cathode
EOF
anioncationneutral analyte
Anode
EOF window
window
dynamic pH junction
d. Desorbed analytes are focused at the border of pH junction and are carried to pass through the detector window.
Figure 5.3 Illustration of on-line preconcentration using a negatively charged sol-gel
column
157
5.3.4 On-line preconcentration of zwitterionic molecules
5.3.4.1 On-line preconcentration of amino acids using sulfonated sol-gel columns
A sol-gel coated column (74% MPTMS) was used to preconcentrate some test
amino acids: argnine, lysine, asparagine and histidine using preconcentration method 1
described in Chapter Four. Table 5.4 lists the chemical structures and some physical
properties of these test amino acids.
Briefly, a sample was introduced into sol-gel column for an extended period (180
s.) which approximately corresponded to 25 capillary volumes. Then, sample matrix was
removed from the column by a flow of deionized water while most of the extracted
amino acid molecules remained attached to the capillary surface due to electrostatic
attractive forces between the positively charged solute ions and the negatively charged
capillary surface. After this, a high voltage was applied and electroosmotic flow was
generated in a basic buffer solution, which formed a dynamic pH junction (the buffer
between deioinized water) facilitating local pH change. The local pH change reversed the
electrical charge on the extracted cations and led to the desorption from the surface due to
electrical repulsive forces. The high voltage focused the desorbed analytes into narrow
zones and detected when passing through the on-column optical window. Figure 5.4
presents an electropherogram representing the preconcentration of arginie on a sol-gel
sulfonated column. As shown in Figure 5.4, remarkably high sample concentration
sensitivity was achieved with the described method using the sulfnoated sol-gel column.
It should be pointed out that the sample concentration was 5000 times higher for the used
uncoated fused silica capillary column for which conventional injection was used. Except
the injection protocol, all other operation conditions were the same. The
electropherogram of arginine obtained with the preconcentration method shows 1382
mAU peak height, 54607 arbitrary units for peak area and 4472 arbitrary units for
corrected peak area. While the electrophorogram obtained on an uncoated fused silica
capillary column shows a peak with 3014 mAu peak height, 304799 arbitrary units for
peak area and 97692 arbitrary units for corrected peak area.
158
Table 5.4 Chemical structures and some physical properties of the test amino acids in this
work
Sample name Chemical structure Molecular weight
(daltons)
pI*
Arginine
H2N CH CCH2
OHO
CH2CH2NHCNH2
NH
174.20
10.8
Asparagine
H2N CH CCH2
OHO
CNH2
O
132.12
5.4
Lysine
H2N CH CCH2
OHO
CH2CH2CH2NH2
146.19
9.5
*: Data were calculated according to the procedure described in literature 70
159
-2
-1
0
1
2
3
4
0 5 10 15 20
min.
mA
U
A
B
Figure 5.4 Illustration of zwitterionic sample preconcentration on a negatively charged
sol-gel sulfonated column using arginine as a test sample. Operation conditions for
electropherogram A: sample 50 mM arginine, uncoated column 50 cm x 50 um, buffer 10
mM Tris-base pH 8.86, injection 10 psi ⋅ sec, V = +15.00 kV, polarity + to -, wavelength
214 nm. Conditions for electropherogram B: sample 10 µM arginine, coated column 50
cm x 50 µm, buffer 10 mM Tris-base pH 8.86, injection 100 psi for 180 s, UV detection
at 214 nm. sample matrix was removed by deionized water, V = + 15.00 kV, polarity + to
-.
160
Preconcentrations of lysine and asparagines on a sol-gel sulfonated column are
demonstrated in Figure 5.5 and 5.6, respectively. The repeatability of the sample
preconcentration method was examined by a series of experiments and shown in terms of
relative standard deviation of the migration time, peak height, peak area as well as
corrected peak area. Table 5.5 shows the repeatability of the preconcentration method in
five replicate experiments. As shown in Table 5.5, quite good repeatability was obtained
considering that these RSD values pertain to a sample preparation technique. The RSD
values of migration times are in the range of 2.2% - 5.9%. The RSD values in terms of
peak height are no more than 7.5%. The RSD values of peak area and corrected peak area
are in the range of 6.2% to 8.7%.
For the presented method, the sensitivity enhancement factors (SEF) were
calculated according to the method described by Quirino and Terabe 67, using the test
amino acid samples. The equation is as follows.
factordilution methodration preconcent without obtainedparameter peak
methodration preconcent with obtainedparameter peak SEF ×=
The SEFs calculated for the test amino acid analytes are presented in Table 5.6.
The SEF values were calculated by peak height and peak area, obtained from
preconcentration and traditional method. As can be seen in this table, remarkably high
sample enrichment factors were achieved using the described sample preconcentration
method. For example, the SEF value for analyte lysine reaches 7381 in terms of peak
height.
161
Figure 5.5 Illustration of zwitterionic sample preconcentration on a negatively charged
sol-gel sulfonated column using lysine as a test sample. Operation conditions for
electropherogram A: sample 50 mM lysine, uncoated column 50 cm x 50 µm, buffer 10
mM Tris-base pH 8.86, injection 10 psi ⋅ sec, V = +15.00 kV, polarity + to -, UV
detection wavelength 214 nm. Conditions for electropherogram B: sample 10 µM lysine,
sol-gel sulfonated column 50 cm x 50 µm, buffer 10 mM Tris-base pH 8.86, injection
100 psi for 180 s, UV detection 214 nm, sample matrix was removed by deionized water,
V = + 15.00 kV, polarity + to -.
162
-3
-1
1
3
5
7
9
0 5 10 15
min.
mA
U
A
B
Figure 5.6 Illustration of zwitterionic sample preconcentration on a negatively charged
sol-gel sulfonated column using asparagine as a test sample. Operation conditions for
electropherogram A: sample 50 mM lysine, uncoated column 50 cm x 50 µm, buffer 10
mM Tris-base pH 8.86, injection 10 psi ⋅ sec, V = +15.00 kV, polarity + to -, UV
detection wavelength 214 nm. Conditions for electropherogram B: sample 10 µM
asparagine, sol-gel sulfonated column 50 cm x 50 µm, buffer 10 mM Tris-base pH 8.86,
injection 100 psi for 180 s, UV detection 214 nm, sample matrix was removed by
deionized water, V = + 15.00 kV, polarity + to -.
163
Table 5.5 Repeatability of sample preconcentration on a sulfonated sol-gel column using amino acids as test solutes
Arginine Lysine Asparagine
tR Peak
Height
Peak
Area
Corrected
Area
tR Peak
Height
Peak
Area
Corrected
Area
tR Peak
Height
Peak
Area
Corrected
Area
(min) (mAU) (arbitrary
unit)
(arbitrary
unit)
(min) (mAU) (arbitrary
unit)
(arbitrary
unit)
(min) (mAU) (arbitrary
unit)
(arbitrary
unit)
Run #1 12.71 1141 62093 4885 10.03 1848 71277 7106 9.63 2102 68133 7075
Run #2 12.33 1273 52539 4261 9.96 1935 69586 6987 9.72 2197 70607 7264
Run #3 12.07 1285 51592 4274 9.61 1965 62053 6457 8.94 2222 76197 8523
Run #4 11.85 1368 50200 4236 9.56 1995 66662 6973 8.85 2325 63798 7209
Run #5 12.21 1382 54607 4472 9.92 2138 68470 6902 10.13 1977 70850 6994
RSD(%) 2.6 7.5 8.7 6.2 2.2 7.0 4.7 3.6 5.9 5.9 6.5 8.3
Operation conditions: 50 cm x 50 µm i.d. sulfonated sol-gel column, 10 mM Tris buffer pH 8.8, 10 µM arginine, lysine, and
asparagines samples. Samples were injected at 100 psi for 180 s. Sample matrix was removed by DI water at 100 psi for 30 sec. V = +
15.00 kV, polarity + to -, UV detection wavelength 214 nm
164
Table 5.6 Sensitivity enhancement factors obtained on a sulfonated sol-gel columns using
amino acids as test solutes
SEF
Sample By height By area By corrected area
Arginine 5310 659 145
Asparagine 2010 1343 422
Lysine 7381 1518 364
Operation conditions for preconcentration experiment: 50 cm x 50 µm i.d. sulfonated sol-
gel column, 10 mM buffer pH 8.8, 10 µM arginine, lysine, and asparagine. Samples were
injected at 100 psi for 180 s. Sample matrix was removed by DI water at 100 psi for 30
sec. V = + 15.00 kV, polarity + to -, wavelength 214 nm. Conditions for traditional
injection method: 50 cm x 50 µm i.d. uncoated fused silica capillary column, 10 mM
buffer pH 8.8, 50 mM arginine, lysine, and asparagine. Samples were injected at 10 psi ⋅
sec. V = + 15.00 kV, polarity + to -, wavelength 214 nm.
165
In the foregoing experiments, deionized water was used to remove the sample
matrix after extraction was accomplished. In this process, a portion of the analytes
extracted on the sol-gel surface of the column could have been rinsed out with sample
matrix. To prevent this loss, a second preconcentration method was designed without the
removal of matrix. The method was illustrated in section 5.3.3 of this chapter.
Figure 5.7 illustrates two electropherograms of asparagine obtained on (a) a sol-
gel sulfonated column using the preconcentration method and (b) an uncoated column
with traditional injection. The asparagine sample enriched on the sol-gel coated column
had a concentration 1000 times smaller than that used on the uncoated column. The peak
height obtained on the sol-gel column was 8.9 mAU, and the corrected peak area was
2.01 x 106 arbitrary units. The electropherogram shown in Figure 6b was obtained on an
uncoated column with traditional injection. The peak height and corrected peak area were
2.0 mAu and 2.74 x 105 arbitrary units, respectively. The sensitively was greatly
increased by using the presented preconcentration method. SEF based on peak height was
4450, and SEF based on corrected peak area was 7335. Compared with the SEF of
asparagine by preconcentration method with the removal of sample matrix (2010 by peak
area and 422 by corrected peak area), it clearly shows that the preconcentration method
without the removal of sample matrix is more effective in sensitivity enhancement.
166
-5
0
5
10
0 5 10 15 20min.
mA
U.
a
b
Figure 5.7 Preconcentration of asparagine on negatively charged sol-gel coated column.
Running buffer 50 mM Tris-Base (pH = 8.8), V = +9 kV, UV detector wavelength 214
nm. (a) injection for 30 sec at 100 psi, sample concentration 10 µM (pH = 7.1); 60%
MPTMS sol-gel coated 30 cm x 50 µm ID colum; (b) injection at 10 psi⋅sec, 30 cm x 50
µm ID uncoated column, sample concentration 10 mM.
167
5.3.4.2 On-line preconcentration of proteins using sulfonated sol-gel columns
The 74% MPTMS sol-gel coated column was used to preconcentrate protein test
samples. Figure 5.8 and 5.9 illustrate the comparison of two electropherograms obtained
on a sol-gel coated column by preconcentration method without the removal of sample
matrix and an uncoated fused silica capillary column with traditional sample injection.
Figure 5.8 shows the results obtained from a protein test sample conalbumin, while
Figure 5.9 demonstrates the results from myoglobin. Both figures indicate significant
sensitivity enhancement using the preconcentration method on sulfonated sol-gel
columns. For instance, the sol-gel column preconcentrated a 3.6 ppm conalbumin sample
and gave a peak height of more than 3.5 mAU in Figure 5.8 (B). While with an uncoated
column and conventional mode of sample injection, a 1797 ppm conalbumin sample gave
an absorbance signal with peak height of around 1.5 mAu (shown in Figure 5.8 A). In
Figure 5.9, the peak height obtained from a 2195 ppm (125 µM) myoglobin sample was
only 4.1 mAU (Figure 5.9 A). This value increased to more than 39 mAU (Figure 5.9 B)
on sulfonated sol-gel column with a 21.95 ppm (1.25 µM) myoblobin sample
preconcentrated by the described method. Based on the results shown in Figure 5.8 and
Figure 5.9, sensitivity enhancement factors (SEFs) were calculated. For conalbumin, the
SEFs were 1143, 1012 and 2436 in terms of peak height, peak area and corrected peak
area, respectively. For myoglobin, SEFs were 973, 3769, 3104 in terms of peak height,
peak area and corrected peak area, respectively.
168
-1
0
1
2
3
4
5
6
0 5 10 15 20
min.
mA
U
A
B
Figure 5.8 Preconcentration of conalbumin on a negatively charged sol-gel coated
column. Running buffer 50 mM Tris-base (pH = 8.8), UV detector: 214 nm. A. uncoated
fused silica capillary column 30 cm x 50 µm i.d., sample 1797 ppm conalbumin solution
(pH = 7.0), sample injection 10 psi⋅sec. V = +9 kV. B. Sulfonated sol-gel column 30 cm x
50 µm i.d., sample 3.6 ppm conalbumin solution (pH = 7.0), sample injection 100psi for
30 sec. V = +9 kV.
169
-10
-5
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20
min.
mA
U
A
B
Figure 5.9 Preconcentration of myoglobin on a negatively charged sol-gel coated column.
Running buffer 50 mM Tris-base (pH = 8.8), UV detection at 214 nm. A. uncoated fused
silica capillary column 30 cm x 50 µm i.d., sample 2195 ppm (125 µM) myoglobin
solution (pH = 7.1), sample injection 10 psi⋅sec. V = +9 kV. B. Sulfonated sol-gel column
30 cm x 50 µm i.d., sample 21.95 ppm (1.25 µM) myoglobin solution (pH = 7.1), sample
injection 100psi for 60 sec. V = +9 kV.
170
In order to investigate the influence of MPTMS content in the sol-gel coating
solution preconcentration effect, three sulfonated sol-gel columns with different MPTMS
composition were used to preconcentrate a protein sample (myoglobin) under the
identical operation conditions. Figure 5.10 presents the electropherograms of myoglobin
obtained after preconcentration on three different sulfonated sol-gel columns prepared by
the described method using sol solutions containing (a) 40%, (b) 60%, and (c) 74% of
MPTMS. For comparison, under identical conditions a myoglobin sample of 100-fold
higher concentration was injected into an uncoated fused silica capillary using a
conventional electromigration injection (Figure 5.10 d). In the obtained electropherogram
(shown in Figure 5.10 d), the peak height and corrected peak area were 4.1 mAU and
3.32 x 104 arbitrary units, respectively. With the increase of the MPTMS content in the
sol solution, the active extraction sites on the sol-gel coating also increased. Therefore,
the preconcentration effect was enhanced. It should be pointed out that the sample
concentration applied to the sol-gel coated columns was 100 times less than that of
applied on the uncoated column. The peak height obtained on column “a” (prepared by
using 40% MPTMS in the sol solution) was 4.0 mAU. This value increased to 39.9 mAU
on column “c” (prepared by using 74% MPTMS in the sol solution). For these two
columns, the corrected peak area (in arbitrary unit) for myoglobin increased from 7.68 x
104 (Figure 5.10a) to 1.03 x 106 (Figure 5.10c).
171
Figure 5.10 Influence of sol solution MPTMS composition on the preconcentration of
myoglobin on a negatively charged sol-gel column. Running buffer: 50 mM tris-base (pH
= 8.8), UV detection at 214 nm. a: 40% MPTMS 30 cm x 50 µm ID column. b: 60%
MPTMS 30 cm x 50 µm ID column. c: 74% MPTMS 30 cm x 50 µm ID column.. a, b
and c used sol-gel columns and the described preconcentration method. Sample: 1.25µM
myoglobin d: uncoated 30 cm x 50 µm ID column, conventional injection (10 psi ⋅ s).
sample: 125 µM myoglobin.
172
Based on the results shown in Figure 5.10, sensitivity enhancement factors (SEFs)
were calculated relative to an uncoated column operated using traditional injection.
Calculation based on peak heights provided SEF values of 97, 549, and 973 for column
prepared by using sol solution containing 40%, 60% and 74% MPTMS, respectively.
Analogous calculation made on the basis of corrected peak areas gave SEF values of 232,
1739, and 3104, respectively. The preconcentration effect of the negatively charged sol-
gel coating on the test protein samples is summarized in Table 5.7.
It is noticed that larger SEFs were obtained for the amino acid sample asparagine
compared with those for the protein analyte. This may be explained by the differences in
molecular sizes of these analytes. Being smaller in size, more amino acid molecules can
be extracted on the surface of sol-gel coating than the much larger protein molecules.
The run-to-run repeatability of the extraction process for myoglobin was
investigated in terms of migration time, peak area, corrected peak area (peak area divided
by migration time), and peak height RSDs. The RSD values in terms of migration time,
peak heights, peak areas, and corrected peak areas were 5.1%, 6.0%, 3.4% and 7.8%,
respectively. Figure 5.11 illustrates extraction repeatability using electropherograms for
myoglobin obtained in replicate extraction experiments. The repeatability of
preconcentration effect for protein test sample conalbumin is listed in Table 5.8. The
presented RSD values (4.1% ~ 8.3%) indicate that the repeatability of the
preconcentration method is quite good, considering that these RSD values pertain to a
sample preparation technique and that the used solute is a biological macromolecule.
173
Table 5.7 Sensitivity enhancement factors (SEFs) obtained on sol-gel coated columns
using proteins as test solutes
SEF
sample column by peak
height
By peak area by corrected
peak area
myoglobina 40%MPTMS sol-gel
column
97 215 232
myoglobina 60%MPTMS sol-gel
column
549 1539 1739
myoglobina 74%MPTMS sol-gel
column
973 3769 3104
Conalbuminb 74%MPTMS sol-gel
column
1143 1012 2436
a Operation conditions: Preconcentration of myoglobin on negatively charged sol-gel
columns 30 cm x 50 µm i.d. Running buffer 50 mM tris-base ( pH = 8.80 ), V = + 9.0 kV,
injection for 30 s at 100 psi, UV detection at 214 nm, sample concentration 21.95 ppm
(1.25 µM) ( pH = 7.1 ). bOperation conditions: Preconcentration of conalbumin on a negatively charged sol-gel
column 30 cm x 50 µm i.d. Running buffer 50 mM tris-base ( pH = 8.80 ), V = + 9.0 kV,
injection for 30 s at 100 psi, UV detection at 214 nm, sample concentration 3.6 ppm ( pH
= 7.0 ).
174
Figure 5.11 Repeatability of myoglobin preconcentration on a negatively charged sol-gel
sulfonated column. Operation conditions: 40% MPTMS sol-gel sulfonated 30 cm x 50
µm i.d. column, buffer 50 mM tris-base (pH = 8.8), applied voltage + 9 kV, injection for
30 s at 100 psi, UV detection at 214 nm, sample myoglobin, concentration 1.25 µM (pH
= 7.1).
175
Table 5.8 Illustration of the preconcentration repeatability on a sol-gel sulfonated column
using conalbumin as a test sample
tR (min)
Peak Height
(mAU)
Peak Area
(arbitrary units)
Corrected Area
(arbitrary units)
Run #1 1.99 3593 361531 189323
Run #2 2.01 3889 440466 206512
Run #3 2.01 3772 415090 219137
Run #4 1.99 3548 376752 181674
Run #5 2.01 3554 377366 187744
RSD(%) 0.55 4.1 8.3 7.9
176
Further experiments were carried out to investigate the sorption/desorption of the
ionic analytes on sol-gel sulfonated surface. Sample of asparagine was injected
hydrodynamically for 180 seconds at 100 psi. This means that more than 25 column
volumes of sample were passed through the column. This was followed by removing the
sample matrix with over 4 column volumes of either deionized water (pH = 7.0) or 10
mM neutral buffer (pH = 7.0) or 10 mM running buffer (pH = 8.8) or dilute NaOH
solution in water (pH = 8.8). Finally, an electric field was applied using basic running
buffer (pH = 8.8) to desorb, focus, and carry the preconcentrated analyte to the detector.
Figure 5.12 shows the results. As shown in Figure 5.12, a peak for asparagine was
obtained in the electropherograms only when the sample matrix was removed by
deionized water (Figure 5.12d). On the other hand, no sample peaks could be detected
when a basic running buffer (pH = 8.8) was used to remove the sample matrix (Figure
5.12a). An incomplete desorption of the extracted asparagine was observed when a
neutral buffer (pH = 7.0) or an aqueous NaOH solution (pH = 8.8) was used (Figure
5.12b and 5.12c).
177
Figure 5.12 Effect of matrix removing media on preconcentration effectiveness using a
sol-gel coated column. Operation conditions: column 50 cm x 50 µm i.d. sol-gel
sulfonated column prepared using 74% MPTMS in the coating solution; running buffer
10 mM tris-base (pH = 8.8); V = +15kV; sample 10 µM asparagine; UV detection at 214
nm; sample injection 180 s at 100 psi; sample matrix was removed for 30 s at 100 psi by
(A) 10 mM basic buffer (pH 8.8), (B) 10 mM neutral buffer (pH = 7.0), (C) a dilute
NaOH solution (pH = 8.8), and (D) deionized water (pH = 7.0).
178
5.4 Conclusions A method was developed for the preparation of capillary columns with a sol-gel
coating intrinsically carrying negatively charged sulfonic acid groups. Microextreaction
has been successfully combined with dynamic pH junction to perform on-line
preconcentration of zwitterionic biomolecules using proteins and amino acids as test
samples. The sorption/desorption of the extracted analytes may be attributed to
electrostatic interaction between the zwitterionic analytes and the negatively charged
surface of the capillary as well as the effects arising from the dynamic pH junction and
ion-exchange of the buffer. The presented on-line preconcentration method provided a
significant enhancement in detection capability of a standard CE system equipped with a
UV detector. On-line preconcentration and analysis results obtained on sulfonated sol-gel
columns were compared with those obtained on an uncoated fused silica capillary of
identical dimensions using conventional sample injections. Using UV detection, the
sample preconcentration technique demonstrated here provided a sensitivity enhancement
factor of more than 3,000 for myoglobin and more than 7,000 for asparagine. On-line
preconcentration of zwitterionic molecules by using a negatively charged sol-gel coated
column may prove to be a promising approach in trace analysis by CE. In addition, the
sulfonated sol-gel column was successfully used to separate a protein mixture. The
results indicate the preconcentration method developed here and coating procedure
possess quite high run-to-run and column-to-column reproducibilities.
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ABOUT THE AUTHOR Wen Li was born in Dalian, Liaoning Province, China. She obtained her
Bachelor’s degree in Chemical Engineering from Beijing Institute of Technology. In
the Fall of 2000, she enrolled at the University of South Florida and joined to Dr.
Abdul Malik’s research group in the Spring of 2001. The work presented here was
conducted under the guidance of Dr. Abdul Malik. During her study at USF, she
published several works relating to her research.
