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Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 54
Development and Investigations
of Novel Sample Preparation
Techniques
Electrochemical Extraction and Evaluation
of Miniaturized Analytical Devices Coupled
to Mass Spectrometry
GUSTAV LILJEGREN
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2005
ISSN 1651-6214
ISBN 91-554-6256-1
urn:nbn:se:uu:diva-5807
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List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals:
I
Marie-Louise Nilsson, Monica Waldebäck, Gustav Liljegren,
Henrik Kylin and Karin E. Markides; Pressurized-fluid extraction
(PFE) of chlorinated paraffins from the biodegradable fraction of
source-separated household waste. Fresenius Journal of Analytical Chemistry, 2001, 370 (7), 913-918.
II
Gustav Liljegren, Jean Pettersson, Karin E. Markides and Leif
Nyholm; Electrochemical Solid-Phase Microextraction of Anions
and Cations using Polypyrrole Coatings and an Integrated ThreeElectrode Device. The Analyst, 2002, 127 (5), 591-597.
III
Gustav Liljegren and Leif Nyholm; Electrochemically Controlled
Solid-Phase Extraction and Preconcentration using Polypyrrole
Coated Microarray Electrodes in a Flow System. The Analyst,
2003, 128 (3), 232-236.
IV
Andreas Pettersson Dahlin, Magnus Wetterhall, Gustav Liljegren, Sara K. Bergström, Per Andrén, Leif Nyholm, Karin E.
Markides and Jonas Bergquist; Capillary electrophoresis coupled
to mass spectrometry
from a polymer
modified
poly(dimethylsiloxane) microchip with an integrated graphite
electrospray tip. The Analyst, 2005, 130 (2), 193 - 199.
V
Gustav Liljegren, Niklas Forsgard, Camilla Zettersten, Jean Pettersson, Malin Svedberg, Merja Herranen and Leif Nyholm; OnLine Electrochemically Controlled Solid-Phase Extraction Coupled to Electrospray and Inductively Coupled Plasma Mass Spectrometry. Manuscript.
VI
Gustav Liljegren, Andreas Dahlin, Camilla Zettersten, Jonas
Bergquist and Leif Nyholm; On-line Coupling of a Microelectrode Array equipped Poly(dimethylsiloxane) Microchip with an
Integrated Graphite Electrospray Emitter to Electrospray Ionisation Mass Spectrometry. Manuscript.
Reprints were made with kind permission from the publishers.
Author contribution
Paper I: Wrote the first draft, performed all experiments, except the Soxtec
extractions.
Paper II: Wrote the paper, performed all experiments. Jean Pettersson took
part in the ICP-MS measurements.
Paper III: Wrote the paper, performed all experiments.
Paper IV: Wrote part of the paper, performed all electrochemical experiments.
Paper V: Wrote the paper, performed almost all experiments. Niklas Forsgard took part in the initial ICP-MS measurements and Camilla Zetterberg
took part in the ESI-MS measurements. I took part in the SEM measurements, which was headed by Malin Svedberg, and the AFM measurements,
which was headed by Merja Herranen.
Paper VI: Wrote the major part of the paper, performed all experiments.
Andreas Dahlin constructed the PDMS chip and Camilla Zettersten took part
in the ESI-MS measurements.
Contents
1. Introduction.................................................................................................7
2. Sample preparation techniques ...................................................................9
2.1 Pressurized-fluid extraction..................................................................9
2.2 Solid-phase microextraction...............................................................11
2.2.1 Electrochemically controlled solid-phase microextraction.........14
2.3 Solid-phase extraction in microflow systems.....................................15
2.3.1 Electrochemically controlled solid-phase extraction in
microflow systems ...............................................................................16
3. Polypyrrole................................................................................................18
3.1 Polymerization and properties of polypyrrole....................................18
3.2 The role of polypyrrole in analytical chemistry .................................21
4. Miniaturization..........................................................................................23
4.1 Miniaturizing analytical devices ........................................................23
4.2 Use of electrodes in microflow channels............................................24
4.3 Microfabrication.................................................................................25
5. On-line coupling of electrochemical systems to mass spectrometry ........29
5.1 Electrospray ionization.......................................................................29
5.2 Sheathless electrospray ......................................................................30
5.3 On-line coupling of electrochemistry and electrospray mass
spectrometry .............................................................................................32
5.4 Inductively coupled plasma mass spectrometry .................................34
5.5 On-line coupling of electrochemistry and inductively coupled
plasma mass spectrometry........................................................................35
6. Concluding remarks and future aspects ....................................................37
7. Acknowledgements...................................................................................39
8. Summary in Swedish ................................................................................41
9. References.................................................................................................45
Abbreviations
AdSV
ASE
ASV
CV
EC
EMLC
ESI
ICP
MS
PDMS
PFE
PMMA
PPy
PPy(Br)
PPy(ClO4)
SPE
SPME
P-TAS
Adsorptive Stripping Voltammetry
Accelerated Solvent Extraction
Anodic Stripping Voltammetry
Cyclic Voltammetry
Electrochemical
Electrochemically Modulated Liquid Chromatography
Electrospray Ionization
Inductively Coupled Plasma
Mass Spectrometry
poly(dimethylsiloxane)
Pressurized-Fluid Extraction
Polymethyl methacrylate
Polypyrrole
Polypyrrole doped with bromide
Polypyrrole doped with perchlorate
Solid-Phase Extraction
Solid-Phase Microextraction
Micro Total Analysis Systems
1. Introduction
In this thesis, large scale to small scale extraction and sample preparation
techniques have been evaluated and developed. The aim has been to study
the advantages of miniaturization with the aim to develop a sample preparation technique compatible with chip-based electroanalytical analysis systems
or similar devices coupled on-line to mass spectrometric (MS) detection. An
important part of the project has been to utilize modified electrode surfaces
to increase the extraction efficiency of charged species in both static- and
microfluidic systems. Pressurized-fluid extraction (PFE), employing highdiffusion liquids, and the extraction and preconcentration of charged species
using electrochemically (EC) controlled solid-phase microextraction
(SPME) are discussed. Electrochemical sample manipulation and EC controlled solid-phase extraction (SPE), employing different polypyrrole coated
macro and microarray electrodes, have been performed in flow systems coupled on-line to different MS techniques. In addition, a microelectrode array
equipped poly(dimethylsiloxane) (PDMS) microchip with a durable integrated graphite electrospray tip has been developed and successfully coupled
on-line to electrospray ionization MS (ESI-MS).
PFE, also known as accelerated solvent extraction (ASE), is a relatively
new automated liquid extraction technique where extractions are performed
at high temperatures and pressures. The technique has been developed in an
effort to reduce the large solvent consumption, and long extraction times,
needed in traditional liquid extraction techniques. In paper I, PFE was used
to optimize the extraction conditions for chlorinated paraffins (CP) from a
very complex matrix.
In paper II, III and V, the effects of miniaturization on the extraction efficiencies were studied employing and comparing EC-SPME (II) with on-line
EC-SPE using EC detection (III) and MS detection (V). The electrochemically controlled extractions were performed using different polypyrrole coatings. Polypyrrole is a conducting polymer that is easily polymerized on conducting surfaces, and the application of an electric potential to the polymer
can be used to change its chemical and/or physical properties. The polymerization is performed electrochemically by applying a positive potential to an
electrode in a monomer solution, also containing an appropriate electrolyte.
By changing the electrolyte, it is possible to change the properties of the
polymer and thus create different conducting phases that can be used for
different analytical purposes such as separation, preconcentration and sens7
ing, in many types of analytical devices containing electrodes. The electroactivity and reversible electrochemical properties of the polymer enable both
electrochemically controlled extraction and desorption of charged species,
by simply changing the applied potential. In paper II, polypyrrole was used
as an extraction medium in SPME, employing a new compact all-in-one
electrode device. Another area of interest where sample preconcentration is
often required in order to overcome detection limit problems, or to eliminate
interferences, is in micro total analysis systems (P-TAS). Due to the flow
channel designs of these microchip devices, on-chip SPE can be difficult to
accomplish and such sample preparation steps are therefore often performed
off chip. A solution to this problem is given in paper III, where polypyrrole
coated microarray electrodes was used to perform preconcentrations in a
miniaturized flow system. The efficiency and usefulness of polypyrrole as an
extraction medium in on-line flow systems were also further evaluated in
paper V using a thin-layer cell coupled on-line to different mass spectrometric detection techniques.
In paper IV and VI, a PDMS microchip was developed and evaluated. An
on-line coupling to ESI-MS was made possible using a durable integrated
graphite electrospray tip with good electrochemical stability, as shown in
paper IV. In addition, a novel fabrication method, described in paper VI,
enabled the introduction of microelectrode arrays into the flow channel.
These electrodes, which were shaped as coils covering the walls of the cylindrical channels, enabled electrochemical sample modifications prior to
detection. Furthermore, the coupling of the EC controlled system to the ESIMS was simplified and improved using a wireless battery driven potentiostat.
8
2. Sample preparation techniques
2.1 Pressurized-fluid extraction
In the last few years, new solvent extraction techniques have been developed
in order to reduce the volume of solvents required for the extraction, improve the precision with respect to analyte recoveries, and to reduce extraction times and sample preparation costs. One of these techniques is pressurized-fluid extraction (PFE), also known as accelerated solvent extraction
(ASE¥, Dionex). This technique enables rapid static extractions of solid
samples using less solvents than conventional Soxhlet, Soxtec or liquidliquid extraction techniques, based on the use of elevated temperatures (40200qC) and pressures (100-140 atm) to maintain the solvent in the liquid
state.1, 2 Today, the PFE technique have been used to successfully extract a
large variety of different compounds from various solid and semisolid samples.1, 3-5
The performance of PFE mainly depends on the disruption of surface
equilibria, and solubility and mass transfer effects, caused by the elevated
temperatures and pressures.1 There are however also other advantages and
parameters to consider. With the easily automated PFE instrumentation, it is
possible to perform 24 sequential extractions with different solvents or solvent mixtures, as well as gradients, employing different extraction times.
More selective extractions can also be achieved by adding various adsorbents, filters or gels to the sample cells.6 In addition, compared to other available conventional extraction techniques, PFE enables extractions in a controlled atmosphere, which for example could be used to prevent oxidization
of certain analytes.
The dissolution rate, and the capacity of solvents to dissolve analytes,
generally increases when the temperature is increased. Furthermore, the viscosity and the surface tension of solvents and the matrix decrease at higher
temperatures. This allows better penetration of the matrix particles and facilitates the contact between the analytes and the solvent, which enhances
the extraction process, as described by Richter and co-workers.1 Thermal
energy also decrease the energy required for desorption processes by disrupting hydrogen bonding, dipole attractions, and cohesive (solute-solute) and
adhesive (solute-matrix) interactions.1 Another advantage is that the solubility of water in organic solvents generally increases with increasing tempera-
9
ture. This makes it possible for organic non-polar solvents to penetrate otherwise water-sealed pores containing analytes.1
Due to the possibility of using higher extraction temperatures and pressures, the range of solvents applicable in PFE is much wider than in for example Soxhlet extractions. During the extraction, the pressure has to be high
enough to keep the solvent in the liquid state at temperatures above the boiling point. It is, however, not clear whether a change in the pressure has any
significant effect on the recoveries, in contrast to the pronounced effect of
altering the temperature.1, 6, 7
A schematic description of a PFE instrument is showed in Figure 1. Extraction cells of different volumes are available, and they usually contain
stainless steel frits and a cellulose filter, placed at the bottom of the cell. The
sample is enclosed in the extraction cell, which is automatically filled with
an extraction solvent by opening a pump valve, and pressurized by the
pump. The cell is then heated with electronically controlled heaters and
statically extracted during a selected time. To maintain the desired pressure
during the heating step, a static valve opens periodically. After the extraction, compressed gas is used to purge the sample extract from the cell into a
collection vial. It is possible to perform one or several flushes with a selected
volume of solvent after the first extraction. In this way, a sample can be extracted a number of times with fresh solvent.
Extraction
cell
Purge valve
Pressure
relief
valve
Oven
Pump
Static valve
Solvent
Nitrogen
Vent
Waste
vial
Collection vial
Figure 1. A schematic illustration of a PFE system.
The disadvantages with PFE instruments are the initial cost of the instrument, and the fact that the extracts often need additional sample pretreatment
prior to analysis. However, the reduced solvent consumption and faster extraction times reduce the costs in the long run, and as shown in paper I, PFE
10
requires less sample clean-up than the Soxtec technique. Another disadvantage is the efficient extraction itself, as some solvents can cause partial solubilization of certain matrixes at high temperatures and pressures. These substances can then plug the system upon exiting from the extraction cell, a
phenomenon experienced at our department with plastic matrixes.
2.2 Solid-phase microextraction
Solid-phase microextraction (SPME) is a solvent free alternative sample
preparation step used to isolate, purify and concentrate analytes from a sample matrix in one single step. The method, which was developed by Pawliszyn and co-workers8 in the late 1980-ties, is a rapid, inexpensive, and easily automated technique mainly used for the extraction of organic compounds from gaseous and liquid samples. It involves an extraction and desorption of specific analytes directly from aqueous samples, or from the
headspace of such samples in closed vials, utilizing a small segment of fused
silica fiber. The fiber is coated with an appropriate material, mounted on a
syringe-like device as shown in Figure 2.
Figure 2. A schematic description of a SPME device.
11
The mass transport of the analytes from the sample to the coating is the
rate-determining step in the extraction, and in aqueous samples agitation
methods such as sonication or magnetic stirring are commonly used to
shorten the extraction times.9 Using the headspace approach can substantially reduce the extraction time since the diffusion of analytes is many times
faster in the vapor phase than in the aqueous phase.10 Another advantage
with the headspace technique is that samples with virtually any matrix, such
as soils, can be analyzed since the fiber is not in direct contact with the sample.10 After equilibration, or a well-defined adsorption time, the fiber containing the absorbed, or adsorbed analytes, is removed and usually thermally
desorbed in the hot injector of a gas chromatographic system.9 Thus far, the
technique has enabled the extraction of a vide variety of various organic
species from different sample matrixes.11-17
Although the coatings used in SPME have high affinities for organic
compounds, enabling preconcentration and good sensitivities, the analyte
partition coefficients between the coating and the sample matrix are generally not large enough to completely extract all available analytes.9 This problem is overcome by calibration, since it is an equilibrium sampling method
that can be described by the Langmuir adsorption isotherm.18 The simplest
isotherm is based on three assumptions: (1) only a monolayer of adsorbate
can be formed at the surface; (2) all sites are equivalent and the surface is
uniform; (3) there are no interactions between adsorbed molecules at neighboring sites.19
The dynamic equilibrium between a molecule (M), an empty site (S), and
the adsorbed complex (MS) is
M S œ MS
(1)
with rate constants ka for adsorption and kd for desorption.18 At equilibrium
the net rate of adsorption is zero, and thus the rate of change of surface coverage due to adsorption and desorption are equal and can be described by the
following simplified equation:
k a >M @N (1 T )
k d NT
( 2)
where N is the total number of sites i.e. the maximum surface concentration
of the analyte, T is the extent of surface coverage, and [M] is the concentration of M in the sample at the fiber surface.19 Solving T ([MS]/N) yields the
dependence of the amount of analyte adsorbed by the fiber coating on the
analyte concentration in the sample:
12
>MS @
NK A >M @
1 K A >M @
(3)
where KA is the adsorption equilibrium constant and [MS] denotes the surface concentration of the adsorbed complex.18 Although it is difficult to apply equation (3) in practice, it can be further developed to give the relationship between the initial analyte concentration in the sample and the extracted
amount, as described by Górecki et al.18
If an absorbing coating is used, the amount of analyte absorbed by the fiber coating at equilibrium is related to the analyte concentration in the sample according to equation (4)
m
KC0MVf Vs
KVf Vs
( 4)
where m is the mass of analyte absorbed by the coating, K is the partition
coefficient of the analyte between the coating and the sample solution, Vf and
Vs are the coating and sample volumes respectively, and C0M is the initial
analyte concentration.9 When extractions are performed in large sample volumes (Vs >> KVf), the extracted amount does not depend on the sample volume:9
m
KC 0MVf
(5)
Since SPME mainly is an equilibrium extraction technique, depending on
the partition coefficients, the sensitivity is determined by several different
factors affected by both the coating and the sample matrix.20 By changing
the coating material, increasing the volume of the coating, or manipulating
the matrix, the extraction efficiencies can be increased.18 It is however difficult to extract polar and charged compounds from aqueous samples using the
uncharged conventional coatings that are commercially available, as these
coatings have to have a stronger affinity for the analytes than water. This
problem is usually solved through derivatisation of either the target analytes,
to increase their volatility, or of the coating, or by the addition of complexing agents.21-23 Although derivatisation can improve the sensitivity, it is not a
process without drawbacks. It is a time consuming step that sometimes involves toxic reagents and solvents, and it is not uncommon that the derivatisation reagent reacts with matrix components to produce unstable products.
These side reactions complicate the extraction process, and can also effect
the sensitivity.22, 23
13
2.2.1 Electrochemically controlled solid-phase microextraction
In paper II, a novel approach of performing SPME under electrochemical
control is presented. In this study, polypyrrole was used as a conductive
coating and instead of using a normal SPME device as described in Figure 2,
a compact three-electrode device, shown in Figure 3, was employed. This
set-up has several advantages compared to the more traditional electrode setups employed in other recent studies in which electrochemically modified
electrodes were used to perform SPME.22, 24 First of all, the electrode device
is compact which enables electrochemically controlled extractions and desorptions in small sample volumes. In previous studies, employing traditional three-electrode set-ups and electrochemically modified metal fibers as
working electrodes, successful desorptions have not been performed in small
(PL) volumes.22, 25, 26 This is a major drawback since the desorption volume
affects the sensitivity. However, in a recent study Tamer et al.27 performed
electrochemically aided SPME of neutral analytes using conducting polymer
coated platinum wires. The latter authors performed the desorptions in a 250
Pl three-electrode cell.
Figure 3. Cross-sectional representation of the manufactured SPME electrode device
in the (a) extraction and (b) transfer mode. 1, stainless steel counter electrode; 2,
polyethylene insulation; 3, silver quasi reference electrode; 4, PTFE insulation; and
5, glassy carbon working electrode.
Another advantage with the compact three-electrode device is that the individual parts of the electrode device can be moved relative to each other.
14
By pulling the working electrode inside the counter electrode, the extracted
species can be transferred between solutions under controlled potential conditions, due to a small amount of capillary held solvent remaining between
the electrodes. This eliminates open circuit problems such as oxidation, and
spontaneous charging and discharging processes of the polymer, and makes
it possible to fully control the entire extraction and desorption process.
The most promising application of combining electrochemistry with
SPME is perhaps the fact that both the extraction and desorption can be performed by simply changing the applied potential. This totally eliminates the
use of different desorption solvents and the commonly used thermally desorption techniques. Combining the use of electrodes with a conducting
polymer coating such as polypyrrole further increases the possibilities and
enables the extraction of charged species. In paper II, two types of polypyrrole coatings were used in order to extract both anions and cations according
to the principle described in section 3.1. The results demonstrate the capability and potential of this new SPME approach, but also points out some of the
difficulties associated with preconcentrations using this extraction and desorption technique. The simplest way to increase the sensitivity is to reduce
the desorption volume and to perform the entire sample preparation step online in a miniaturized flow system, as described in paper III.
2.3 Solid-phase extraction in microflow systems
Solid-phase extraction is traditionally a time consuming off-line procedure
that may require large volumes of samples and solvent.28 Integrating this
sample preparation step within a miniaturized flow system, enabling extraction, desorption and detection, can eliminate sampling handling losses, contamination problems and greatly simplify the overall procedure. During recent years, on-chip SPE has received increased attention since it is often
required to improve the selectivity, sensitivity and for the removal of interferences in association with very small sample volumes.29 There are, however, several problems associated with the use of SPE in miniaturized systems, mainly due to the small dimensions of the microfabricated channels
used in these devices.
Today new techniques to perform SPE in miniaturized systems have been
developed. A common technique to perform an increase in concentration, or
to purify an analyte on-chip, is by use of sample stacking by zone electrophoresis or iso-electric focusing.30, 31 The drawback of these procedures is
that they require the presence of an electrodriven system such as a capillary
electrophoresis microchip. Another approach is to extract and preconcentrate
the sample on a microfabricated material with a high affinity for the analyte.
Yu and co-workers32 successfully performed SPE using monolithic porous
polymers prepared by photoinitiated polymerization inside microchip chan15
nels, and Kutter and co-workers33 used C-18 as a stationary phase to modify
microchip channels. The preparation of these phases is more tedious compared to for example electropolymerization, and in order to achieve desorption and release the extracted species, the solvent has to be replaced by a
stronger eluent. Different types of coated microspheres have also been used
as on-chip SPE materials.34 Oleschuk et al.34 presented an exchangeable microchip cavity structure in which octadecylsilane coated silica beads were
trapped and used for preconcentration.
2.3.1 Electrochemically controlled solid-phase extraction in
microflow systems
As described in paper II, III and V, polypyrrole coated electrodes can be
used to extract and desorb a range of analytes. By performing these extractions in miniaturized flow systems, preconcentrations are possible since the
desorption step automatically occurs within a very small volume. This approach was investigated in paper III employing an array of microelectrodes
for both the extraction/desorption and the detection of a model analyte in a
miniaturized flow system. The principle of this type of preconcentration is
the same as for regular stripping analysis in a flow system. First analytes are
accumulated on the polymer during a certain period of time, and by then
changing the applied potential the analytes are desorbed. There is however
an important difference between SPE employing a conducting polymer and
related stripping analysis techniques; the active conducting surface of the
polymer coated electrode is significantly larger. When regular electrodes are
used for preconcentrations of for example organic substances, using adsorptive stripping voltammetry (AdSV), the dynamic range is usually limited as
only the bare electrode surface is available for adsorption.35 One way of
overcoming this problem is to coat the electrode surface with a conducting
polymer such as polypyrrole. This increases the number of charged sites
available for extraction and thus the dynamic range.
The SPE technique described in paper III has several advantages. Electrodes are today readily incorporated and used in conjunction with micro
analytical devices,36 as described in paper VI, and their performance is also
enhanced upon miniaturization.37, 38 Another advantage is that a conducting
polymer such as polypyrrole can be easily electropolymerized in small systems containing electrodes, from a monomer solution. Finally, using a conducting polymer should make it possible to prepare fine structures inside
small channels depending on the thickness of the polymer and its morphology. The surface structure of the polymer depends on the electropolymerization process, as is described in section 3.1.
The results presented in paper III show that EC controlled SPE can be
used as an effective preconcentration step in miniaturized analytical flow
16
systems. Significant preconcentration was possible using extraction times of
only a few minutes and a good linearity between the extraction time and the
detection response was present both for mM and PM sample concentrations.
The preconcentration factor could be increased by a factor of 210, compared
to the results presented in paper II, due to the use of a miniaturized flow
system.
In paper III, the detection was performed using one of the individually
addressable microelectrodes available on the microarray electrode. Although
EC detection is very suitable for microanalytical devices, as further described in section 4.2, the detection is generally limited to electroactive species. The on-line coupling of EC controlled SPE, employing conducting
polymers as extraction phases, to mass spectrometric detection is therefore
of great interest. The latter combination was studied in paper V using a thinlayer cell, in which the working electrode was coated with a polypyrrole
film. Although the on-line EC/SPE-MS combination significantly increases
the analytical possibilities, there are difficulties associated with the combination of the two techniques. In section 5, the on-line coupling of EC/SPE with
both electrospray ionization and inductively coupled plasma MS detection is
described and evaluated.
17
3. Polypyrrole
3.1 Polymerization and properties of polypyrrole
Polypyrrole and its derivatives are today, mainly due to their high electrical
conductivity, facile synthesis, and relatively good stability of the oxidized
state of the polymer, one of the most studied groups of conducting polymers.
The polymer is easily prepared by either an oxidative chemical polymerization, or using electrochemical oxidation in aqueous or organic solutions by
applying a positive potential to a monomer solution containing an appropriate supporting electrolyte.39 The electrochemical oxidation of pyrrole, which
involves the formation of radical cations and subsequent polymerization, is
illustrated in Figure 4.40
A
+
.
+
Oxidation (-e)
n
N
H
A
-
A
-
Polymerization
H
H
N
N
N
N
N
H
H
H
n
+
A
-
n
Figure 4. Suggested polymerization reaction of pyrrole to yield polypyrrole.
In its oxidized state, it has been found that polypyrrole contains one positive charge per three or four pyrrole rings, while the polymer is neutral in its
reduced state.41 During the polymerization, anions in the electrolyte solution
are incorporated in the polymer film to maintain the charge balance. The
presence of these so called dopants greatly effect the properties of the polymer, and by using different counter ions during the polymerization it is possible to create polymers for many different applications.42-59 If a small anion
with a high mobility inside the polymer film is used as a counter ion during
the polymerization, it will be expelled when the polymer is reduced, and thus
the film becomes an anion exchanger. By incorporating a large immobile
anion, the film can be used as a cation exchanger in its reduced state, since
cations will be incorporated in the film to maintain electroneutrality, as
shown in Figure 5. A polypyrrole (PPy) film doped with the anion (A) is
often denoted PPy(A).
18
+
+A
N
ox.
n
H
A
N
red.
n
H
+
AA C +
N
ox.
n
H
AA + C +
N
red.
n
H
Figure 5. Ion exchange properties of polypyrrole films doped with a small mobile
anion (A-) and a large immobile anion (AA-), respectively.
The doping and undoping process of polypyrrole is presently not completely understood, and reports indicate that both anions and cations, and
accompanying water, can enter the polymer upon the oxidation and reduction.60-63 A simple way of studying the redox properties of many conducting
polymers involves the use of cyclic voltammetry (CV), as described in paper
V. When using this reversal technique, the applied potential is varied linearly
with time, while the current is recorded as a function of the potential. An
example of how CV can be employed to study the doping and undoping of a
polypyrrole film in two different pH 4 solutions is given in Figure 6.
1,2
i/mA
0,8
(b)
0,4
0
(a)
-0,4
-0,8
-1
-0,5
0
0,5
E/V
Figure 6. Cyclic voltammograms of polypyrrole in two different pH 4 ~50 mM
buffer solutions containing; (a) potassium phthalate/biphthalate and (b) sodium
acetic/acetic acid, in addition to 0.5 M NaNO3, scan rate 100 mV/s (n = 3).
The difference between the (a) and (b) voltammograms can be ascribed to
the different buffer solutions (~50 mM), despite the fact that both voltammograms were recorded in the presence of 0.5 M NaNO3. In (a) the buffer
solution contained potassium phthalate/biphthalate, which was found to significantly suppress the doping-dedoping of nitrate, probably due to an ad19
sorption of the large organic phthalate ion to the polymer surface. On the
other hand, the use of a sodium acetic/acetic acid buffer (b) resulted in a
rather typical polypyrrole voltammogram, with broad oxidation and reduction peaks due to the incorporation and expulsion of the nitrate anion.
The use of different counter ions during polymerization also significantly
alters the conductivity, flexibility, elasticity, permeability, morphology and
other physical and mechanical properties of the film.39, 58, 64-67 This in combination with the natural ability of the polymers to interact with many different
compounds, make it possible to introduce a certain degree of selectivity with
respect to various types of analytes in the film.68-70 An example of the effect
of dopants on the polypyrrole morphology is shown in Figure 7, in which
scanning electron micrographs of polypyrrole films doped with perchlorate
and bromide ions are compared at different degrees of magnification.
(a)
(b)
2k
34k
148k
Figure 7. Scanning electron micrographs of (a) PPy(ClO4) and (b) PPy(Br) coatings
at three different degrees of magnification.
20
3.2 The role of polypyrrole in analytical chemistry
Since polypyrrole is electrically conducting, the redox switching of the
polymer can be electrochemically controlled or obtained employing reducing
and oxidizing solutions. This makes it highly interesting for the development
of controlled release devices and separation processes in novel analytical
systems. In electrochemically modulated liquid chromatography, polypyrrole
coated stationary phases under potentiostatic control have been used to perform separations of different types of analytes.71 These polypyrrole columns
have the ability to alter their retention properties and thus their molecular
interaction capabilities during operation by merely changing the oxidationstate of the polymer. This can be performed using either direct electrochemical control or a concentration gradient of a redox reagent.72 Porter and coworkers71 have successfully constructed electrochemically modulated liquid
chromatography (EMLC) columns in which polypyrrole coated graphitic
particles have been used as conductive supports i.e. the working electrode/stationary phase.
Polypyrrole has also been shown to be an excellent coating for in-tube
SPME, an on-line extraction technique in which analytes are extracted and
concentrated on an extraction phase coated on the inner surface of an open
tubular capillary positioned between a sample loop and an injection device.26,
70
Pawliszyn and co-workers70 have shown that polypyrrole coated capillaries show higher extraction efficiency for polar and aromatic compounds
compared with the commercial extraction phases currently used for in-tube
SPME. More recently, electrodes coated with polypyrrole have been used in
SPME; a technique that traditionally utilizes thin film coated fused silica
fibers as extraction phases.25, 54 The main advantage of using a conducting
polymer when performing these extractions is that charged species can be
extracted without having to perform derivatisations or employing complexing agents. In addition, desorption can be achieved by simply changing the
applied potential as described in paper II.
Polypyrrole and related polymers can also be used as freestanding functional membranes or as coatings on porous supports.65, 73 The different applications and functions of these membranes, which can be used for both liquid
and gas separations, have been reviewed by Lewis56 and Wang et al.39 In
principle, the transport of charged species as well as the transport rate and
the selectivity of these membranes can be controlled by applying different
potentials, and by using different dopants during the polymerization. As
described above, the properties of a polypyrrole film depends on the mobility of the incorporated dopants. Ehrenbeck and Jütter74 utilized this possibility and prepared a polypyrrole membrane by copolymerization of modified
pyrrole monomers of n-sulfopropyl-pyrrole, containing both mobile and
immobile anions. This resulted in a membrane that could be electrochemically redox switched between both anion and cation selectivity.
21
By monitoring the current accompanying the incorporation and expulsion
of electroinactive species in a polypyrrole film, it is also possible to perform
amperometric detection using the polymer as the sensor.52, 75 In the simplest
case, the polymer is doped with a mobile anion and functions as an ion exchange sensor. Since the doping, undoping and redoping process of the
polymer is only to a certain degree selective with respect to the size of the
incorporated anion used during the polymerization, the polymer films are
usually used as a sensor subsequent to a separation in flow systems.50, 52
There are however many different parameters that have to be considered if
these sensors are to function properly, since small changes in the current are
being monitored. Variations in the conductivity, e.g. due to an eluent, can for
example result in a change of the effective applied potential and thus the
electrochemical switching potential affecting the doping level of the polymer.51 It is also possible to incorporate different biochemical recognition
species such as enzymes76, 77, antibodies78, 79 or metal complexing groups45
capable of extremely specific molecular and ionic recognition. Another advantage of using a conducting polymer such as polypyrrole as an electrochemically modified electrode is that it is easily electrodeposited on electrodes in micro fluidic channels from a monomer solution. This, and the
wide range of applications, makes polypyrrole an interesting tool for micro
total analysis systems (P-TAS).80
22
4. Miniaturization
4.1 Miniaturizing analytical devices
The interest in miniaturized analytical techniques is growing rapidly and
such systems have in recent years become very attractive because of the
number of advantages they posses over traditional analytical techniques.
Some advantages such as reduced sample and reagent consumption, which
often are of primary importance in environmental or biomedical sciences,
and faster separations and reduced analysis times, are evident.81 There are
however additional benefits with chip based devises such as reducing the
cost per reaction and an improvement in the reaction kinetics on for example
biological assays.82 An increased sample throughput is also possible as
miniaturized systems facilitate parallel and automated operations. Disposable lab-on-a-chip devices should also significantly reduce cross contamination and carryover effects in analytical chemistry. Today, the miniaturization
of electronic and optical devices, sensors and actuators for mechanical,
chemical and biological applications, has led to the development of P-TAS,
as described in a recent review by Manz and co-workers.83, 84 These systems,
although generally expensive to produce initially, can be mass-produced at
low cost for many different applications such as capillary electrophoresis
separations,85 ion chromatography,86 enzymatic analysis,87 hydrodynamic
chromatography,88 electrochromatography,89 liquid chromatography90 and
capillary electrochromatography91.
In P-TAS, different analytical standard operations such as sample preparation, injection, manipulation, separation and detection, as well as applications such as DNA separation,92 DNA sequencing93 and clinical diagnostics,94 can be performed using a single device.84 It is however worth mentioning that most analytical chip devices are produced for a specific application. Some of the steps mentioned above are more difficult to perform
mainly due to the large surface-to-volume ratios originating from the open
channel architecture employed in the microfluidic systems. Since conducting
polymers have a high surface-to-volume ratio, are inexpensive and simple to
produce, they are an interesting alternative for these micro devices. Conducting polymers can also be modified in a variety of ways, and are easily prepared and incorporated in microstructures.53
The small dimensions of the channel structures used in P-TAS devices,
often varying between 50 nm and 100 Pm in depth and height, and the small
23
sample and reagent volumes, down to pL95, pose new challenges regarding
both detection and sample preparation. There are several ways of performing
detection on-chip, and some of the most common techniques are chemiluminescence,96 electrochemiluminiscence,97 electrochemical detection,98 fluorescence,99 and other optical measurements such as UV absorption100. Most of
these methods are only suitable for a narrow range of analytes, or relatively
high concentrations, and generally require complicated and expensive
equipment. Another factor that limits the detection is the small amount of
sample present due to the very small sample volumes. Electrochemical detection by the use of microelectrodes is, however, comparably simple and
also exhibit improved performance upon miniaturization, but is generally
limited to electroactive species. The combination of mass spectrometry with
analytical chip devices is therefore of interest, as it may provide highthroughput sample identification, quantification and low detection limits.101
4.2 Use of electrodes in microflow channels
Reducing the size of a working electrode from millimeter to micrometer
dimensions has several important advantages that makes so-called microelectrodes very suitable for miniaturized analytical devices.37, 51 As the electrode area is decreased the measured current (i), typically pA-nA for microelectrodes, decreases. This reduces the ohmic potential drop (iR), where R
denotes the cell resistance. The ohmic potential drop acts to weaken the
working potential and can cause significant errors in amperometric detection
if allowed to become too large.38 Due to the small currents associated with
microelectrodes, it is also possible to use a combined reference and counter
electrode in a two-electrode system.102 This further simplifies the experimental set-up in a miniaturized analytical system. An enhanced mass transport is
likewise obtained since the spherical diffusion rapidly becomes dominant
over planar diffusion when using for example disk microelectrodes.103 The
faradic-to-charging current ratio and the response time of the working electrode are also improved, as the charging current and double layer capacitance decreases with decreasing electrode area.38 A consequence of this is
the possibility to perform very rapid scanning of the applied potential. All
these factors contribute to improve the quality of experimental data; steadystate signals are established rapidly and the signal-to-noise ratio can be improved.37 In addition, due to the advantages mentioned above, microelectrodes also enable measurements in low conductivity media, i.e. solutions
with high resistances such as organic solvents. This makes microelectrodes
versatile tools for amperometric detection in a wide range of sample matrixes. Microelectrodes are thus advantageous in many areas of electroanalytical chemistry and, due to their size, a perfect choice for miniature cell
designs.
24
If an array of individually addressable electrodes is used for detection, both the sensitivity and the selectivity can be enhanced. The sensitivity
is increased by employing every second band as a collector or generator
electrode, respectively,104 as shown in Figure 8.
Ox
Red
Ox
Red
Ox
Red
Ox
Red
Ox
Figure 8. The principle of redox cycling using array electrodes in a flow system.
The individual bands may also be used to detect different analytes by applying different potentials to different bands, thus increasing the selectivity.37
Since the technique is improved by the degree of redox cycling, i.e. the average number of times a molecule is reduced and reoxidized, it is well adapted
for the low flow rates used in common microsystem.105
4.3 Microfabrication
Many of the fabrication techniques used to create microstructures, such as
lithography and molding, demands access to clean room facilities, involves
expensive techniques, and are time consuming.106 Another difficulty with
applying the fabrication processes and materials often originally developed
for the microelectronics industry to analytical devises, is that the surfaces of
the employed materials become more important upon miniaturization. The
advantage of microfabrication is that as soon as a fabrication procedure is
complete, it allows rapid replication and thus relatively cheap massfabrication.
Since the initial production steps of new microanalytical fluidic devices
often require evaluations of many different prototypes, it is important to
develop simple and fast fabrication methods. To meet these demands, a variety of plastic materials that allows for low cost fabrication in the absence of
clean room facilities have been used to produce prototypes.107 One such
polymer is poly(dimethylsiloxane) (PDMS), a biocompatible commercially
available physically and chemically stable material used in paper IV and VI.
Other important properties of PDMS are low water absorption, high optical
transparency, high stretch ability and low curing temperature.108 The drawbacks with this material include swelling, hydrophobicity, diffusivity and a
relatively poor mechanical strength.108, 109 However, the synthetic polymer
has a backbone of repeating units of silicon and oxygen that can be modified
25
by the introduction of various types of functional groups. This is a simple
way of changing the mechanical and viscous properties of the polymer,
which increases the versatility of the material.109
The most common way to produce microchannels in plastic materials like
PDMS is to employ a procedure requiring several steps such as casting onto
a master wafer, molding and bonding.108 The molding procedure creates
three of the channel walls and a bonding step is then used to seal the channel
to a flat surface. The sealing can be done reversible by van der Waals contact
or irreversible by the use of an air-plasma, which produces a channel that
can withstand significantly higher pressures.110 Compared to other materials,
such as glass or thermoplastics, the PDMS sealing process is simpler, and as
PDMS is an elastomer it can also seal to non-planar surfaces.110
In paper IV and VI, a new approach for the fabrication of microchannels
in PDMS is presented. A schematic description of the PMMA mould used to
create the chip device is shown in Figure 9.
Figure 9. Schematic description of the PMMA mould used to produce the PDMS
device. a-e represents the different modules defining the final structure of the microchip. The inset shows the coil array with isolating wires (in black) separating the
electrodes from each other. A) stainless steel wire, B) fused silica capillaries, C)
PEEK-tubing and D) isolating wires.
By employing this fabrication method both a flow channel111 and a threedimensional emitter could be fabricated in a one step procedure. Furthermore, the stainless steel metal wire used to create the channel could be used
to introduce electrodes into the channel wall, as described in paper VI. This
26
novel approach enabled the introduction of arrays of gold coil electrodes by
simply twining a gold wire around the steel wire. The distance between the
individual coil electrodes significantly affected the EC performance, as
shown in Figure 10. In order to reduce the interelectrode distance, and consequently decrease the iR drop, isolating wires was twined between all coils.
This prevented shortcuts, and supported and stabilized each coil when the
channel wire was removed after the curing. A small interelectrode distance
of 100 Pm inside the channel was necessary to obtain a cyclic voltammogram resembling that of a conventional three-electrode set-up.
0,4
(a)
(b)
0,4
0,2
0,2
i/PA
i/PA
0
0
-0,2
-0,2
-0,4
-0,6
-0,4
-2
-1,6 -1,2 -0,8 -0,4
0
0,4
0,8
-1
1,2
-0,5
0
E/V
2
0,5
1
E/V
2
(c)
(d)
1
1
i/PA
i/PA
0
0
-1
-2
-1
-3
-2
-4
-1
-0,5
0
0,5
E/V
1
1,5
-1
-0,5
0
0,5
1
1,5
E/V
Figure 10. Voltammograms obtained with the micro gold coil electrodes (a) o (c)
vs. a gold coil pseudo reference electrodes in 25 mM H2SO4. The distance between
working and reference electrode was 4 mm (a), 1 mm (b), and 100 Pm (c). (d) shows
a voltammogram obtained with a gold wire vs. a gold wire pseudo reference electrode using a conventional three-electrode set-up in 25 mM H2SO4.
A way to create the three-dimensional emitters needed to combine microsystems with MS is by fitting a spray capillary to the outlet channel end.112
This method is robust, but often increases the dead volume prior to the emitter tip, which is avoided with the procedure described in paper IV. The introduction of electrodes into a microflow channel is often performed by sealing the channel replica to a flat surface containing microband electrodes.
Today a vide variety of materials and fabrication techniques have been used
27
to integrate electrodes in lab-on-a-chip devices.113 The advantage with the
technique described in paper VI is that it is inexpensive, relatively simple
and demands no advanced equipment. This means that the procedure is well
suited for the fabrication of chip prototypes.
28
5. On-line coupling of electrochemical systems
to mass spectrometry
5.1 Electrospray ionization
The electrospray ionization (ESI) technique was used for the first time in the
late 1960s,114 and is based upon the transfer of ions from a liquid phase into
the gas phase. ESI usually operates at ambient temperature and atmospheric
pressure, and is a soft ionization technique by which complex and fragile
molecules can be ionized without extensive fragmentation. It is most suitable
for medium to polar compounds, and both small and large molecules can be
analyzed. Multiple charging of large analytes reduces their mass-to-charge
(m/z) ratio, which simplifies the detection of macromolecules. The electrospray is formed by applying a high electric potential of several kV, between a
hollow emitter and a counter electrode plate. The principle of positive electrospray, with a positive potential applied to the emitter, is shown in Figure
11.
Figure 11. Schematic description of the electrospray ionization process in the positive mode. (Figure provided by Andreas Dahlin)
29
As a solvent passes through the emitter, the high electric field forces the
liquid to form a so-called Taylor cone,115 due to the electrostatic attraction
between the counter electrode and ions of opposite charge. If the potential
difference is sufficiently high the apex of the cone disperses into charged
droplets which then travel towards the counter electrode. During this process
the solvent evaporates from the droplets and gaseous ions are produced.116
5.2 Sheathless electrospray
There are several types of interfaces used in ESI-MS, and the most common
approaches when combining ESI with low flow rate applications using capillaries are the sheath-flow interface,117 the liquid junction interface118 and the
sheathless interface119. The sheathless interface, shown in Figure 12, is well
suited for polymeric microfluidic devices, and a common way of integrating
the ES tip is to attach spray capillaries at the exit of the microchannel.120-122
It is however also possible to fabricate the emitter at the outlet of the microchannel from the bulk material of the chip.123 This significantly reduces the
dead volume and increases the sensitivity, as there is no additional dilution
of the eluting sample. The conductive surface of the emitter can also be used
as one of the electrodes in CE separations. This enables electrophoretic separation to be performed all the way to the electrospray, as described in paper
IV. When the emitter is fabricated from the non-conductive polymeric bulk
material of a chip, or when a fused silica spray capillary is used, a durable
conductive coating has to be applied. The stability of this conductive coating, and the fact that it is impossible to control the performance of the spray
using a sheath or make up liquid, are the main limitations of sheathless ESI
from miniaturized devices.
Figure 12. Schematic description of the sheathless ESI interface. (Figure provided
by Andreas Dahlin)
30
During ESI measurements there is a constant charge transfer between the
emitter and the counter electrode, and when the analyte solution reaches the
sheathless emitter only the tip of the thin conductive layer provides the electrical contact. In principle, the interface functions as a two-electrode controlled current EC cell where the steady state current is carried by the
charged droplets formed at the working electrode i.e. the ES emitter.124 In
positive ESI, an oxidation takes place at the emitter and a reduction consequently occurs at the counter electrode plate to maintain the charge balance.
This means that the electrochemical stability of the emitter becomes very
important as it determines the lifetime of an integrated sheathless interface.
There are several methods available for the application of a conductive
layer on spray capillaries and integrated emitters. The most common coatings are metal layers, with a relative short lifetime of less than 100 hours,
applied by techniques such as sputtering, vapor deposition and electroplating.125 However, the so-called Fairy Dust techniques, by which gold particles
are immobilized onto fused silica emitters using different types of glue can
produce metal coatings with an excellent stability.126-128 Nilsson and coworkers, employing a graphite-polyimide mixture that produced durable socalled Black Dust coatings, further developed this technique.129 A similar
coating procedure using a silicon-based resin as the glue, also containing
graphite powder, was developed by Svedberg et al.130 The latter coating
technique is compatible with the thermally unstable surface modifications
commonly needed in PDMS devices, as silicon polymerizes at temperatures
lower than 70qC. In paper IV, a thin layer of non-polymerized PDMS was
used to glue graphite powder to a integrated three-dimensional PDMS emitter tip. The long-term stability of this coating was evaluated using off-line
ES, during which the spray current was found to be stable for over 180 h,
and chronoamperometry. Chronoamperometry allows for faster stability
evaluations, as described by Nilsson et al.131 and Wetterhall et al.,132 and is
hence a convenient way to study how the current density changes with time
for different coatings. Although the current density for the graphite coating
was found to decrease after 40 min, probably due to the formation of a passivating oxide layer, the emitter could be regenerated by reducing the coating as is shown in Figure 13. The extent of the regeneration most likely depends on the reduction time as the passivation layer was allowed to form
during 40 min.
31
300
200
i ( A)
100
0
2.5 V
2.5 V
-100
-200
-2.5 V
-300
0
-2.5 V
10
20
30
40
Time (min)
Figure 13. Chronoamperometric plot depicting the regeneration of the oxidized
graphite coating using potential steps between 2.5 and –2.5 V vs. Ag/AgCl in 0.5 %
nitric acid.
5.3 On-line coupling of electrochemistry and
electrospray mass spectrometry
Bruckenstein and Gadde, employing a porous electrode to study reactions
involving volatile analytes, performed the first successful coupling of EC
with MS.133 This and similar methods were later used and further developed
by several groups,134-136 but the technique was initially limited to the detection of volatile species. The first EC-MS report regarding the detection of
non-volatile analytes was published by Hambitzer and Heitbaum in the middle of the 1980s.137 Since then different types of two-138, 139 and threeelectrode140, 141 flow cell systems have been coupled on-line to MS using
various types of ionization techniques, including ESI. As ESI is a soft ionization technique often producing multiple charge ions of large analytes, it is
becoming one of the most important ionization techniques in mass spectrometry. The EC-MS combination further increases the analytical applications.
When coupling an EC system to ESI there are several issues that have to
be considered.142 The ES usually operates at kV potentials, which can severely affect the EC instrumentation and the electrochemical measurements.
As reported by Van Berkel et al.142, different approaches can be used to successfully combine the two techniques. The entire EC system has to be either
floating at the potential induced by the ES potential or be decoupled from the
latter. In the first case, the induced potential depends on the applied ES volt32
age and the resistance between the EC cell and the electrospray high voltage
electrode. A decoupling can therefore be obtained by increasing the distance
between the EC cell and the ES contact, which however unfortunately increases the delay time and the band broadening. Another alternative is to
introduce a ground point between the EC cell and the ESI high voltage electrode.142, 143 The main drawback with this method is that electrochemical
reactions will occur at the ground point, which of course can cause severe
interferences.144 In the floating mode, the EC system can be based either on a
battery driven potentiostat or an isolation transformer, as in paper V.140, 142,
144
The floating mode allows for short connections between the EC cell and
the ES emitter, which significantly reduces delay times and improves the
detection of unstable species. The drawback with this mode of operation is
that the absence of a ground point increases the risk of electrical shocks, and
as a result the handling of the EC instrumentation becomes more difficult. A
promising solution to this problem was examined in paper VI employing a
wireless battery-powered potentiostat. In this study, the electrode equipped
PDMS chip device shown in Figure 14 was employed to further reduce the
transfer times. As is seen in this figure, the leads used to connect the gold
wires to the potentiostat had to be shielded from the ESI high voltage using a
protective PDMS cage to prevent electrical discharges.
Figure 14. Schematic picture of the electrode equipped PDMS-ESI emitter device.
A) denotes the protective PDMS cage, B) the leads connected to electrode array, C)
the PDMS microchip with the integrated graphite coated electrospray emitter, D) the
metal plate used to apply the high voltage to the graphite tip, E) ESI high voltage
contact and F) graphite ESI emitter.
Another factor that needs to be considered is the EC cell design. An appropriate EC cell design can minimize influences from the counter electrode
reactions, as described by Bökman and et al. in a recent study.144 The choice
of electrolyte can also be problematic, as most electrolytes commonly used
in EC measurements are more or less incompatible with ESI. Non-volatile
33
electrolytes easily form gas-phase cluster ions that often suppress the analyte
signal. These salts can also cause additional problems such as plugging of
the emitter tip and contaminating the curtain plate. Some lithium salts have
however been found to reduce suppression effects and is therefore an interesting electrolyte alternative.142, 145
5.4 Inductively coupled plasma mass spectrometry
Inductively coupled plasma mass spectroscopy (ICP-MS), which was developed in the 1980s,146, 147 is a speciation technique employing a plasma source
for the atomization, excitation and ionization of elemental species.148 As ESI,
the ICP technique has high requirements regarding sample introduction, as
the liquid has to be introduced in the form of fine droplets in order to fully
desolvate and atomize. Sample delivery is commonly performed using a
peristaltic pump, but due to the high sensitivity and selectivity of the technique it is also commonly used in conjunction with different chromatographic separation techniques.149 Aerosol generation of the sample is often
accomplished utilizing a pneumatic nebulizer connected to a spray chamber.
There are many types of nebulizers and spray chambers commercially available, adapted for different flow rates, all having different effects on the analyte transport efficiency and sensitivity.150 When the sample solution exits
from the nebulizer it is mixed with a nebulizer gas (usually argon), and is
converted into finely divided droplets. Approximately 1 to 3 % of the aerosol
passes through the spray chamber into the torch body where it is mixed with
an argon auxiliary gas.151 The nebulizer efficiency can however be significantly higher depending on parameters such as flow rate and solvent type.
The droplets that are too large for the plasma are removed via a drain at the
bottom of the spray chamber. A schematic description of the nebulizer, spray
chamber and plasma is shown in Figure 15.
A typical plasma torch consists of three concentric quartz tubes, where the
inner tube contains the sample aerosol. The two surrounding tubes are used
for auxiliary gas introduction and coolant gas. By applying a radio-frequency
current to the induction tubes, wrapped around the quartz tubes, an oscillating magnetic field is created. This magnetic field and the argon gas flow
sustain the plasma, which is initiated by the ionization of argon using a
spark. The high temperature of the plasma (6000-10000 K) effectively removes any remaining solvent and causes sample atomization, ionization and
excitation, which is used in ICP-atomic emission spectroscopy. In addition,
due to the very high temperature molecular interferences are significantly
reduced, but not completely eliminated.148
34
Radio-frequency
induction coil
Coolant gas
Auxiliary gas
Plasma
Sample aerosol
Spray
chamber
Aerosol
Magnetic field
Sample
liquid
Nebulizer gas
Drain
Figure 15. Schematic description of a nebulizer, spray chamber and a typical inductively coupled plasma.
The main advantages with ICP-MS for speciation analysis are the low detection limits, high selectivity and a dynamic range that can cover up to eight
orders of magnitude.149 Some of the difficulties with the ICP-MS technique
are isobaric interferences, caused by equal mass isotopes of different elements, and polyatomic (or molecular) interferences due to recombination of
ions, and plasma instabilities due to changes in the solvent composition
and/or due to organic solvents.152 For example, in paper V, 81Br was overlapped by the polyatomic species 40Ar 40Ar 1H.153 The latter problem has to
be considered when coupling ICP-MS to chromatographic techniques, and it
can be reduced by for example employing different types of interfaces and
voltages, and by optimizing the gas flow rates.150 Furthermore, introducing
buffers and high concentrations of different salts can result in partial or total
clogging of the sampler cone, and signal suppression due to space charge
effects.151, 154
5.5 On-line coupling of electrochemistry and
inductively coupled plasma mass spectrometry
The on-line coupling of an EC cell to ICP-MS is uncomplicated compared to
the EC/ESI-MS combination, and the coupling can readily be achieved using
a small piece of tubing or a capillary. Commonly, the EC/ICP-MS combination is used for sample pretreatment such as preconcentrations and matrix
elimination employing anodic155-157 or adsorptive158 stripping voltammetry.
35
In anodic stripping voltammetry (ASV) electroactive species are selectively
accumulated by applying a suitable deposition potential. After the accumulation it is easy to change the solvent to a solution containing a minimal
amount of interfering species, and the analyte is then stripped from the working electrode back into the flow system for detection. Both flow-through EC
cells employing porous electrodes159, 160 and thin-layer EC cells157, 161 have
successfully been coupled to ICP-MS for the determination of a wide range
of compounds. The advantage of the flow-trough cells is the high deposition
efficiency, while the drawback is the dilution of the accumulated sample
upon stripping due to the large cell volume.156 Thin-layer cells have proven
to be more advantageous with respect to signal enhancement, but the very
small cell volume and the low flow rates increase the demands on the nebulizer and spray chamber to reduce dispersion effects.156, 157
In paper V, a thin-layer EC cell coupled on-line with ICP-MS was used to
perform preconcentrations utilizing polypyrrole coated electrodes. The advantage with this approach is that the amount of charged sites available for
extraction can be altered by increasing or decreasing the polymer thickness.
In addition, it enables the extraction of species unsuitable for adsorptive and
anodic stripping voltammetry. A drawback with the use of a thick polymer
coating and long extraction times is however the prolonged desorption process, which increases the dilution and dispersion of the released analytes. This
effect could be reduced by turning the flow off while performing the desorptions. Figure 16 shows the dependence of the analyte signal response on the
extraction time, employing the stopped flow desorption technique.
(e)
8,E+05
(d)
Inte nsity
6,E+05
(c)
4,E+05
2,E+05
(b)
(a)
0,E+00
0
5
10
15
T ime /min
Figure 16. A comparison of bromide ion peaks obtained using the stopped flow
desorption technique, employing the following extraction times: (a) 5 min, (b) 10
min, (c) 20 min, (d) 30 min and (e) 40 min.
36
6. Concluding remarks and future aspects
Different sample preparation steps prior to detection are often time consuming and essential parts of analytical chemistry. Today many of the techniques
used to prepare a sample for analysis consumes large quantities of solvents
and materials, and they are often difficult to automate and to perform online. In paper I, the relatively new automated PFE technique was evaluated
using samples with a very complex organic matrix. The results of these experiments show that PFE enable selective and effective extractions, using
less solvent and a significantly reduced sample preparation time. Much research and development is also performed to enable fast and automated sample preparation systems suitable for miniaturized analytical devises. In such
devices, separations and preconcentrations are often performed using chemically modified surfaces and different eluents. The uptake and release of
charged and neutral species can be electrochemically controlled using conducting polymers as a stationary phase. This was studied in paper II, employing polypyrrole as a stationary phase in an all-in-one SPME electrode
device, and in paper III, where microarray electrodes were used for both
extraction and detection in a flow system. The results show that polypyrrole
coated electrodes can be used for extractions of charged species, and that
increased preconcentration factors can be obtained by miniaturization i.e. by
reducing the desorption volume.
A common problem for many different detection techniques is interferences that, for example, distort and/or overlap with the analyte signal, or
otherwise negatively affect the general performance of the detection technique. In addition, as the sample volumes are reduced and the interest in
detecting lower and lower analyte concentrations increases, on-line sample
preparation and manipulation becomes more challenging. Furthermore, to
fully benefit from the small dimensions in lab-on-a-chip devices it is vital to
minimize the dilution between separation/preparation steps and detection.
One way to reduce some of these problems is to introduce filters or to perform some kind of analyte extraction and preconcentration prior to detection.
Electrochemical detection is a very versatile technique for the detection of
electroactive species in micro flow channels, but on-line couplings to different MS techniques are often also desirable to increase the range of analytical
applications. The use of polypyrrole as a general preconcentration device in
on-line MS systems was therefore studied, as described in paper V. A difficulty with the integration of the latter two techniques was that the electro37
lytes commonly used to ensure optimal EC control of the electrodes are not
always compatible with MS detection. In addition, charged species used to
increase the conductivity of the sample solutions might compete with the
analyte for the charged sites in the polymer, and thus reduce the extraction
efficiency. The results did however show that polypyrrole can be used as an
EC controlled preconcentration step prior to detection, but further studies are
needed to completely evaluate the usefulness of the technique in combination with ESI-MS.
In paper IV, a novel approach was employed to fabricate a hybrid capillary-PDMS microchip with a durable integrated three-dimensional ESI emitter. This analytical device enabled on-line coupling of capillary electrophoresis to ESI-MS detection. No measurable band broadening effects, compared to a fused silica capillary sheathless interface, was observed due to the
hybrid PDMS device. The fabrication method was further developed in paper VI, in which gold coil electrodes were incorporated to the micro flow
channel. These electrodes could be used to perform on-line EC manipulations of a model analyte, using a battery-driven wireless potentiostat that
greatly simplified the EC-MS coupling. The results of these studies show the
successful combination of EC and ESI-MS employing a miniaturized analytical device, which further developed and explored could become an important contribution to the lab-on-a-chip field.
38
7. Acknowledgements
I want to express my gratitude to all friends and colleagues at the department
for their support, and for helping my finishing this thesis. I especially want
to thank:
My supervisor Leif Nyholm, for encouraging me, and for always being there
when I needed support. No one can critically review manuscripts like you,
and without your electrochemical knowledge this work would have been a
lot more difficult. I cannot imagine anyone having a better supervisor!
Jonas Bergquist for your support at the end when I really needed it!
Karin Markides for accepting me as a graduate student in analytical chemistry.
Jean Pettersson for fruitful discussions and helping me with everything
related to ICP-MS.
Co-authors Marie-Louise Nilsson and Monica Waldebäck for their support
in the beginning and for introducing me to PFE.
My other collaborators: Niklas Forsgard, Andreas Dahlin and Camilla
Zettersten, for your help in this work. It was fun working with you, and I
wish you all the best of luck with your own research!
Björn “capo di tutti capi” Arvidsson, for being a good friend and roommate. One day you might be the King of the Lab!
Oliver Klett, my first room-mate, for your assistance and interesting discussion when I started working at the department.
All past and present colleagues at the department. Special thanks to Magnus, Stefan, Ardeshir, Joakim, Sara B, Sara U, Emilia, Nina, and my
teachers Marit, Rolf, Roland, Eva and Per for your support and fruitful
discussions about chemistry and research.
39
Yngve Öst, Erik Forsman, Lasse Svensson, Bosse Fredriksson and Barbro Nelson, for helping me with technical and practical issues.
Captain and crew of the greatest clan in NZ history.
All my friends!
My mother, father and sister for your never-ending support and encouragement.
40
8. Summary in Swedish
Utveckling och undersökning av nya provupparbetningstekniker
Elektrokemisk extraktion och utvärdering av miniatyriserade analytiska plattformar kopplade till masspektrometri
Syftet med denna avhandling har varit att undersöka olika provupparbetningsmetoder lämpade för chip-baserade elektroanalytiska analyssystem
eller motsvarande system baserade på masspektrometrisk detektion. En viktig del av projektet har bestått i att undersöka hur man med olika extraktionsmetoder kan uppnå en uppkoncentrering av analyter. Tonvikten har
lagts på att använda modifierade elektrodytor i flödessystem för att elektrokemiskt kunna styra både extraktions- och desorptionsprocesserna. Detta
arbete har även inkluderat utveckling och utvärdering av ett poly(dimetylsiloxan) (PDMS) mikrochip med en integrerad elektrosprayelektrod och en flödeskanal innehållande elektroder.
I artikel I användes en relativt ny statisk extraktionsmetod för att extrahera klorerade paraffiner från ett fast prov med en komplex organisk matrix.
Metoden kallas trycksatt vätskeextraktion (PFE), och det är en automatiserad
teknik där upp till 24 prov kan extraheras i sekvens. Proven placeras i stålcylindrar vilka sedan fylls med önskat lösningsmedel och placeras i en ugn. Ett
högt gastryck används för att behålla lösningsmedlet flytande vid temperaturer över dess kokpunkt. Detta förbättrar förutsättningarna för snabb och effektiv extraktion då ökad temperatur bland annat ökar substansers upplösningshastighet, samt minskar vätskors viskositet och ytspänning, vilket gynnar extraktionsprocessen. Detta har i sin tur lett till att extraktionstiden
och/eller lösningsmedelskonsumptionen kan minskas i förhållande till andra
konventionella tekniker. Resultaten från vår studie visar tydligt hur olika
extraktionsparametrar som temperatur, tid och lösningsmedel påverkar både
selektiviteten och utbytet. I jämförelse med Soxtec-tekniken kunde extraktionstiden, samt den efterföljande provupparbetningen och lösningsmedelskonsumptionen reduceras signifikant med hjälp av PFE.
En annan statisk extraktionsteknik kallad fastfasmikroextraktion (SPME)
studerades i artikel II. Denna teknik bygger på fördelningen av analyter mellan ett polymermaterial applicerat på en fiber, och själva provlösningen.
Extraktionsprocessen går till på så sätt att en polymertäckt fiber med en hög
41
affinitet till analyten sänks ned i en provlösning under en bestämd till. Uppkoncentrering och desorption sker sedan genom att t.ex. flytta över fibern till
en liten volym av ett rent lösningsmedel med än ännu högre affinitet för
analyten. Tekniken skulle kunna vara ett attraktivt alternativ till de provupparbetningssteg som ofta används efter extraktionstekniker som PFE och Soxtec. Vanligtvis används SPME för att extrahera olika organiska substanser,
då de kommersiella polymermaterialen ofta fungerar mindre bra för polära
och laddade substanser. Genom att täcka fibern med en elektriskt ledande
polymer, som t.ex. polypyrrol, kan dock tekniken användas för att extrahera
både negativt och positivt laddade analyter.
I artikel II konstruerades ett kompakt tre-elektrod-SPME-instrument som
möjliggjorde elektrokemiska (EC) extraktioner under potentialkontroll. Treelektrodinstrumentet var uppbyggt av individuellt rörliga små rör vilket
gjorde det möjligt att dra in arbets- och referenselektroden inuti motelektroden efter extraktionen. Den pålagda potentialen kunde därför bibehållas även
när elektroderna transporterades mellan olika vätskor, p.g.a. den medföljande kapillärt bundna vätskevolymen. Då SPME-instrumentet var uppbyggt av
elektroder kunde både extraktion och desorption styras genom att ändra potentialen på den polypyrroltäckta extraktionsfibern, d.v.s. arbetselektroden.
Vid en positiv potential oxideras polypyrrol och vissa negativt laddade substanser i provlösningen kommer att vandra in i polymeren för att neutralisera
de positiva laddningarna. Genom att sedan flytta över elektroden till en ny
vätska och reducera polymeren kan de negativt laddade substanserna desorberas elektrokemiskt.
Eftersom oxiderad polypyrrol är positivt laddad, och reducerad polypyrrole är oladdad, måste polymeren modifieras för att man skall kunna extrahera
positivt laddade substanser. Detta görs genom att under den elektrokemiska
polymerisationen dopa polymeren med en tillräckligt stor negativt laddad
molekyl som byggs in i polymermaterialet. Polypyrrol kan appliceras på en
elektrod genom att man oxiderar pyrrol monomerer till katjonradikaler, som
kombineras, deprotoneras och bildar polymeren. Denna s.k. elektropolymerisering skapar positivt laddade polymerkedjor på elektrodytan. Under denna
process blir polymeren dopad med de anjoner som finns i lösningen, d.v.s.
ledelektrolytens anjoner. Är anjonen relativt liten och mobil i polymeren
kommer den att bli utstött vid reduktionen av polymeren, och polymeren kan
användas som en anjonbytare. Vissa stora anjoner kommer dock att bli steriskt hindrade från att vandra ut och in genom polymeren. Detta innebär att
polymeren inte blir neutral vid reduktionen, utan får en negativ nettoladdning vilket kompenseras genom ett upptag av katjoner. Två olika polypyrrolfilmer användes i artikel II för att elektrokemiskt extrahera både anjoner och
katjoner. Polypyrrol dopad med perklorat fungerade som en anjonbytare och
paratoluensulfonat dopad polypyrrol kunde användas som en katjonbytare.
Möjligheten att använda polypyrrol som ett uppkoncentreringssteg för
analyter i miniatyriserade flödessystem studerades i artikel III. I detta arbete
42
placerades parallella rader av plana mikroelektroder, vilka fungerade som
arbetselektroder, vid utloppet av en kapillär. Den första elektroden täcktes
med polypyrrol och användes för att extrahera och desorbera analyter, medan den efterföljande elektroden användes för elektrokemisk detektion. Resultatet av studien visade att man kunde extrahera anjoner från vätskeflödet
genom att lägga på en positiv potential på den polymertäckta elektroden.
Mängden extraherad analyt ökade för längre uppkoncentreringstider. Desorptionen av den extraherade substansen genomfördes genom att polymeren
reducerades, d.v.s. både extraktion och desorption kunde åstadkommas elektrokemiskt. Samma provupparbetningsprincip tillämpades i artikel V, där en
elektrokemisk tunnskiktscell med en polypyrroltäckt arbetselektrod kopplades mot dels elektrosprayjonisation-masspektrometri (ESI-MS) och dels
induktivt kopplat plasma (ICP) MS.
Att direktkoppla ett elektrokemiskt system till ESI-MS innebär att man
måste ta hänsyn till en hel del olika parametrar. Hela det elektrokemiska
systemet måste skyddas från den höga (kV) potential som används för elektrosprayjonisering, och eventuell ledelektrolyt måste väljas noggrant då
många salter har en negativ inverkan på joniseringsprocessen, och kan störa
både känsligheten och spraystabiliteten. I artikel V, användes en isolationstransformator för att hålla all utrustning flytande på den p.g.a. elektrosprayspänningen inducerade potentialen. Extraktionsexperimenten visade sig dock
vara svåra att genomföra då höga uppkoncentreringar ledde till att elektrosprayen kollapsade. Detta kan ha berott på att lösningens sammansättningen
förändrades för mycket vid desorptionen. Kopplingen till ICP-MS var betydligt enklare då nebuliseringsprocessen inte påverkar EC-systemet, och eftersom ICP-tekniken och förstoftarna var mindre känsliga för icke-flyktiga
mobilfaskomponenter. Dessa experiment visade att polypyrrol kan användas
som ett generellt uppkoncentreringssteg för en analyt som inte lämpar sig för
vanlig stripping- eller adsorptions-voltammetri. En modifierad cell användes
även för att studera extraktion från lösningar innehållande olika analytkoncentrationer, och en ökad mängd interfererande anjoner. Resultaten visade
att mängden extraherad analyt ökade med ökad koncentration. Polypyrrol
har en ganska dålig selektivitet för anjoner av liknande karaktär. Det är därför ett material som med fördel kan användas för en generell uppkoncentrering av joner i flödessystem.
Till sist utvecklades också ett PDMS mikrochip med integrerade guldelektroder. Utmaningarna med utvecklingen av detta miniatyriserade analyssystem var dels att integrera elektrosprayspetsen i chipets bulkmaterial, belägga spetsen med ett stabilt ledande skikt, samt att introducera elektroder i
flödeskanalen. Utvecklingen av spetsen och de elektrokemiska stabilitetstesterna beskrivs i artikel IV. Som ledande material användes grafit, vilket visade sig vara mycket stabilt. Kronoamperometriexperiment visade att grafitmaterialet var stabilt i 40 minuter varefter strömtätheten minskade. Under
ES-förhållanden tog det över 180 timmar innan elektrosprayströmmen avtog.
43
Strömminskningen berodde förmodligen på bildandet av ett passiviserande
oxidskikt, och genom att reducera spetsen kunde strömmen regenereras. I
artikel VI, redovisas en ny mikrofabriceringsteknik för att introducera elektroder i ett PDMS-chips kanalväggar. Elektroderna tillverkades genom att
linda guldtrådar runt den stråltråd som användes för att gjuta själva kanalen.
Detta visade sig vara ett snabbt och reproducerbart tillverkningsätt, vilket
bekräftades i elektrokemiska utvärderingar av elektrodareorna. Direktkopplingen av chipet till ESI-MS, och elektrokemisk manipulation av en modellsubstans, dopamin, möjliggjordes genom att använda en batteridriven Blåtandsbaserad potentiostat. Denna direktkopplingslösning var mycket fördelaktig då potentiostaten kunde hanteras utan risk för elektriska stötar. Systemet visade sig även vara mycket effektivt, med en bra elektrokemisk
konverteringsgrad och en mycket kort responstid.
44
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Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
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