Download Nanoparticles in Capillary Electrophoresis hyphenated with

Nanoparticles in Capillary Electrophoresis
hyphenated with Mass Spectrometry
-What are the benefits?
David Malmström
Department of Physical and Analytical chemistry,
Analytical Chemistry
Uppsala University
Licentiate Thesis 2011
Abstract
Malmström, David 2011. Nanoparticles in Capillary Electrophoresis hyphenated with Mass
Spectrometry – What are the benefits? Department of Physical and Analytical chemistry.
38 pp. Uppsala.
Capillary electrophoresis is a technique that separate compounds based on charge and size.
During the last two decades capillary electrophoresis (CE) and mass spectrometry (MS) has
gained in interest, become more robust to use and able to separate neutral analytes. The separation of neutral analytes was first achieved with packed columns, but several disadvantages
can be obtained with a stationary phase and the method of packing capillaries. Therefore,
pseudostationary phases became a good alternative. The risk of clogging, memory effects and
lower efficiency could be minimized with pseudophases. However, since mass spectrometry
has become the most important analytical detector, and play a key role in the search for biomarkers in clinical applications, it is important that CE can successfully be combined with
MS. To obtain this hyphenation several types of interfaces for the vital ion source exist. In
paper I an atmospheric pressure photoionization interface was investigated in order to accomplish an improved detection sensitivity. The knowledge attained with this type of interface could then be transferred to the one used in paper II, the electrospray ionization interface (ESI), where the use of a MS friendly nanoparticle based pseudostationary phase was
investigated. Both studies showed that it is still possible to improve the separation technique
and modify the ion source in order to improve the detection sensitivity for capillary electrophoresis hyphenated with mass spectrometry.
David Malmström, Department of Physical and Analytical chemistry, Box 599
SE-751 24 Uppsala, Sweden
© David Malmström
”Things should be made as simple as
possible, but not any simpler"
Albert Einstein
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
II
Axen, J., Malmström, D., Axelsson, B-O., Petersson, P.,
Sjöberg, P. J. R. (2010) Efforts to improve detection sensitivity
for capillary electrophoresis coupled to atmospheric pressure
photoionization mass spectrometry. Rapid Communication in
Mass Spectrometry, 24(9) 1260-1264
Malmström, D., Axen, J., Bergquist, J., Viberg, P., Spegel, P.
(2011) Continuous full filling capillary electrochromatography
– electrosprayingchromatographic nanoparticles. Electrophoresis, 1(32) 1-7
The author’s contribution to the papers:
Paper I: Took part in planning the study, performed the experiments
and took part in writing the paper.
Paper II: Planed the study, performed the experiments and wrote the
paper.
Reprints were made with permission from the respective publishers.
Papers not included in this thesis
i
ii
iii
†
Shevchenko, G., Sjödin, M. O. D.†, Malmström, D.†, Wetterhall, M., Bergquist, J. (2010) Cloud-Point Extraction and Delipidation of Porcine Brain Proteins in Combination with Bottom-Up Mass Spectrometry Approaches for Proteome Analysis.
Journal of Proteome Research, 9(8) 3903-3911
Eriksson, A., Bergquist, J., Edwards, K., Hagfeldt, A.,
Malmström, D., Agmo Hernaìndez, V., (2010) Optimized Protocol for On-Target Phosphopeptide Enrichment Prior to Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Using Mesoporous Titanium Dioxide. Analytical Chemistry,
82(11) 4577-4583
Eriksson, A., Bergquist, J., Edwards, K., Hagfeldt, A.,
Malmström, D., Agmo Hernaìndez, V., (2011) A novel mesoporous TiO2-based experimental layout for on-target enrichment
and separation of multi-and monophosphorylated peptides prior
to analysis with matrix assisted laser desorption-ionization mass
spectrometry. Analytical Chemistry, 83 (3) 761-766
These authors contributed equally to this manuscript
Contents
1.
Introduction ........................................................................................... 9
2.
Capillary Electrophoresis..................................................................... 12
2.1.
General concept of Capillary Electrophoresis ............................ 13
2.2.
Electroosmotic Flow................................................................... 14
2.3.
Capillary modifications .............................................................. 15
3.
Electrokinetic chromatography ............................................................ 16
3.1.
Why Pseudostationary Phases? .................................................. 16
3.2.
Micellar Electrokinetic Chromatography ................................... 17
4.
Mass Spectrometry .............................................................................. 19
4.1.
The use of Mass Spectrometry ................................................... 20
4.2.
Ion sources .................................................................................. 20
5. Modifications when hyphenating Capillary Electrophoresis to Mass
Spectrometry ............................................................................................ 22
5.1.
Tapered capillaries ..................................................................... 23
5.2.
Requirements of the interface ..................................................... 23
6. Adaptation of Electrokinetic Chromatography and Pseudostationary
Phases to Mass Spectrometry ................................................................... 26
6.1.
Traditional Micellar Electrokinetic Chromatography
adapted to Mass Spectrometric detection ................................... 27
6.2.
New Pseudostationary Phases with improved Mass
Spectrometric compatibility ....................................................... 29
6.3.
Continuous Full Filling .............................................................. 30
7.
Concluding remarks ............................................................................. 33
8.
Acknowledgments ............................................................................... 34
9.
References ........................................................................................... 35
Abbreviations
APPI
APCI
BGE
CFF
CE
CEC
CMC
CZE
EKC
EOF
ESI
HPLC
i.d.
ITP
MALDI
MEKC
MS
MS/MS
o.d.
PF
PSP
RM
SDS
Atmospheric Pressure Photoionization
Atmospheric Chemical Iionization
Background electrolyte
Continuous Full Filling
Capillary Electrophoresis
Capillary Electrochromatography
Critical Micelle Concentration
Capillary Zone Electrophoresis
Electrokinetic Chromatography
Electroosmotic Flow
Electrospray Ionization
High Performance Liquid Chromatography
Inner dimension
Isotachophoresis
Matrix Assisted Laser Desorption/Ionization
Micellar Electrokinetic Chromatography
Mass Spectrometry
Tandem Mass Spectrometry
Outer dimension
Partial Filling
Pseudostationary Phase
Reverse Migrating
Sodium Dodecyl Sulfate
1. Introduction
Electrophoresis, which is the separation of charged molecules based on different migration in an applied electric field, has since it was introduced by
Tiselius [1] in the 1930’s and later on adapted into capillary formats by
Hjertén and others [2-4] developed into a robust separation technique. John
Craig Venter et al. [5] showed in 2001 the great power of capillary electrophoresis (CE) when the human genome was sequenced well ahead of schedule. However, there are some limitations with CE, such as the inability to
separate nonionic solutes and separate analytes with the same size-to-charge
ratio. There have been ways to solve these limitations such as packed capillaries, capillary electrochromatography (CEC), and the use of pseudostationary phases (PSP). The basic principal of CEC is that a fused silica capillary
is packed with a stationary phase. The packing materials are typically conventional high pressure liquid chromatography (HPLC) materials such as
bonded silica and ion exchangers or monoliths. The most obvious advantage
of CEC is that it combines the features of CE, with its flat-flow velocity
profile generated by the electroosmotic flow (EOF) that reduces peak broadening, with the selectivity in HPLC. However, ever since the introduction
of CEC it faced some major challenges. For instance, the method of packing
the capillaries was problematic, since bubble formations and frit problems
arises and can lead to column-to-column variation. This will affect the EOF
since it is controlled mainly by the surface of the packing material. Due to
the difficulties in the packing of the capillaries and thereby varying EOF
CEC can be seen as irreproducible in a larger perspective. There have been
many different way of improve the packing of solid particles into the capillaries: capillaries with pressurized ends [6], sol-gel methods with in-situ polymerization [7] and gel based CEC [8]. But the challenges with producing
good frits still exist. [9] Despite the challenges, the development in CEC is
still active and developing since its breakthrough in the second half of the
1990’s as can be seen in Figure 1.
9
Figure 1. Number of published articles with a topic containing (A) capillary electrophoresis or (B) capillary electrochromatography (source ISI Web of knowledge).
Another way to circumvent the limitations of packed columns was to use a
pseudophase. This interesting technique was introduced by Terabe and others in 1984 where a solution of ionic micelles was introduced in the background electrolyte [10-14]. This separation principle first mentioned by Nakagawa [15] and is based on that micelles can migrate in an aqueous solution
by electrophoresis.
The use of mass spectrometry (MS) has during the past 25 years reached an
outstanding position among analytical methods due to its sensitivity and low
detection limits. One of the areas where mass spectrometry has become the
method of choice is in the enormous field of complex protein samples – proteomics [16]. MS has also been recognized by the Nobel Committee by giving the Nobel Prize in chemistry in 2002 for the ESI and laser desorption
ionization. Olivares et al. [17] and Smith et al. [18] were the first to combine
the powerful technique of mass spectrometry with capillary electrophoresis.
The interface was based on the electrospray ionization (ESI) technique initially developed by Dole et. al. [19, 20] and Fenn et al. [21, 22]. Due to their
great work CE-MS is now routinely used in areas such as metabolome analysis [23].
During the last decade, nanoparticles have been a hot topic, including as PSP
in CE (see Figure 10). The use of nanoparticles as PSP´s, in combination
with continuous full filling (CFF) and an orthogonal ESI-interface, has
shown to be a good way to separate neutral analytes without affecting the
electrospray process or contaminate the ion source [24, 25]. An orthogonal
method of coupling MEKC and MS utilize an alternative ionization source –
atmospheric pressure chemical ionization (APCI) and atmospheric pressure
photoionization (APPI) [26]. In these methods the ionization efficiency has
been shown to be unaffected by the surfactant SDS using both APCI and
APPI [26-28].
10
This licentiate thesis is based on fundamental studies showing how the separation as well as the coupling of CE with MS can be improved. Paper I
shows how the detection sensitivity in APPI-MS was optimized by varying
different ion source parameters. The results indicated that a specially designed APPI interface for low flow rates would be favorable. In paper II, a
pseudostationary phase involving nanoparticles and the continuous full filling technique was investigated. Both separation and ion source parameters
were investigated and optimized leading to improvements in column-tocolumn variability and detection limits.
11
2. Capillary Electrophoresis
Capillary electrophoresis (CE) is an umbrella term for many different methods. The general benefit of CE is the highly efficient separations for all the
electrophoretic driven separations in CE. However, the inability to separate
neutral analytes with traditional capillary zone electrophoresis (CZE) is a
major limitation. But capillary electrochromatography (CEC), which is a
packed capillary with the same stationary phase as in HPLC, could solve this
challenge. Although CEC and HPLC are very similar, the main difference is
the flat flow velocity profile generated by the EOF in CEC, in comparison to
the pressure driven parabolic flow velocity profile in HPLC. This difference
in front profile lead to the highest separation efficiencies of all time. But
unfortunately, the reproducibility was a major challenge in CEC. The problem turned out to depend on the heterogeneous surface charges resulting in
local variations in EOF. Despite several approaches to minimize the problems by improving the packing methods, improved frit fabrications and insitu polymerization the reproducibility still remain a challenge [9].
In the predecessor of capillary electrophoresis, gel electrophoresis, the gel
was a good way of avoiding joule heating, but unfortunately lead to convection and band broadening. When trying to exclude the gel for a liquid based
system it turned out to be impossible to separate neutral analytes and analytes with the same size-to-charge ratio. Gel filled capillaries has also been
investigated, similar to traditional gel electrophoresis. However, producing
and using gel filled capillaries was often quite difficult due to bubbles
present inside the gel. The challenge was elegantly solved by using noncross-linked gels because the gel could be injected into the capillary and
after separation discarded [29, 30]. Apart from the non-cross linked acrylamide gels, other types of non-trapped stationary phase have been used in CE
and CEC (and will be discussed in chapter 3).
12
2.1.
General concept of Capillary Electrophoresis
The separation by electrophoresis is based on different movement of charged
compounds (ions) by attraction or repulsion in an electric field [2, 3]. In CE,
the electrophoretic separation is taking place in a thin glass capillary made of
fused silica. The inner diameter of the capillary is typically 25-75 μm, which
can be compared to a human hair shaft, typically at 100 μm in diameter. The
outer surface of the capillary is coated with a polymeric substance, polyimide, which gives a tremendous flexibility to the otherwise very fragile capillary. In comparison to traditional gel electrophoresis, CE is filled with a
buffer. The choice of buffer has a major influence since it will connect the
applied high electric field. In gels this applied current is in higher degree
limited by Joule heating, the heating of a conducting medium as current
flows through it. This will have an effect on the speed of the separation due
to the limitation in using a higher potential. Electric fields between 100 and
500 V/cm is possible to use with only minimal heath generation. With higher
electric fields come shorter analysis time, higher peak efficiencies and better
resolution. The inner diameter (i.d.) of the capillary also has a large effect on
the limitations. An increase in i.d. lead to a lowering of the surface-tovolume ratio. A capillary with 50 μm in i.d. would have around 78 times
higher surface-to-volume ratio compared to a standard slab gel and 1.5 times
higher than the capillary with 75 μm in i.d. The high surface-to-volume ratio
will allow for very efficient dissipation of Joule heat generated from large
applied electric fields. As a result, electrophoretic separations in capillary
electrophoresis can easily be performed at up to 30 000 volts.
The simplest form but also the most commonly utilized type of CE is capillary zone electrophoresis (CZE). The capillary is filled with an appropriate
separation buffer with a desired pH, sample is injected in the inlet (normally
the anode end) while the other end of the capillary is grounded either by
placing it in another buffer vial or adding an extra liquid flow (sheath liquid)
(see Figure 2). As a high voltage is applied into the two buffer reservoirs,
ionic species in the sample plug will start to migrate with an electrophoretic
mobility determined by their charge and mass. However, if the applied electric field were the only driving force acting on the ions, just net positivelycharged compounds (cations) would be separated along the capillary while
neutral compounds would remain static and net negatively-charged compounds (anions) would be driven back into the buffer vial in the inlet end,
making CZE quite limited in use. Fortunately, a force called electroosmotic
flow (EOF) creates a movement of all components in the capillary towards
the cathode (under normal conditions).
13
Figure 2. The principal of CE where the negatively charged silica surface in combination with the applied electric current creates the EOF indicated with a blue arrow.
Analytes will be migrating with an electrophoretic mobility determined by their
charge and mass which then will lead to a separation of the analytes. On the capillary wall the static Stern Layer can be seen, as well as the moving Outer Helmholtz
Plane in the middle of the capillary.
A marker is useful to add as an internal standard in order to determine relative migration times for the compounds. A neutral compound, e.g. dimethyl
sulfoxide (DMSO) is used as only charged species can be separated in CZE
while neutral analytes will migrate with the EOF. Herein lay also one major
problem with CZE, which lead to the development of electrokinetic chromatography, EKC, including micelles and nanoparticles.
2.2.
Electroosmotic Flow
The silanol groups (SiO-) on the inside wall of the capillary (see Figure 2)
will attract cationic species from the buffer. This attracted ionic layer has a
positive charge that will decrease exponentially as the distance from the
capillary wall increases. The double layer that is formed closest to the surface is named Stern Layer or Inner Helmholtz [31] and can be seen as a static layer. A mobile and more diffused layer is formed distal to the Stern
Layer, called Outer Helmholtz Plane (OHP). When a field is applied, cations
will start to move in the OHP towards the cathode but carrying water molecules at the same time. The cohesive nature of the hydrogen bonding of the
14
waters of hydration to the water molecules of the bulk solution will result in
the entire buffer solution being pulled towards the cathode. This bulk flow is
called electroosmotic flow (EOF) and can be seen as a pumping mechanism
that will carry all analytes. A flat flow front profile will be created by the
EOF in contrast to a parabolic flow created by a pressurized driven flow.
Due to the flat flow profile all analytes will be affected in the same way
leading to sharp peaks in contrast to the parabolic flow typically present in
HPLC. The BGE will have a large effect on the EOF due to the pH dependency of the silanol groups at the capillary inner wall. Apart from pH, the
ionic strength will have a large effect on the EOF. High ionic strength, or
electrolyte concentration, will compress the Stern Layer, decrease the zeta
potential (the potential between two layers of the opposite charge) and thereby reduce the mobility of EOF. By optimizing the EOF it is then possible to
have a short analysis time while still having well separated peaks.
2.3.
Capillary modifications
Basic proteins and peptides have a high tendency to interact with the capillary wall creating a problem in protein and peptide analysis due to low efficiencies, low recovery, poor reproducibility, and decreased sensitivity [32,
33]. Many ways of modifying the capillary in order to improve the separation and sensitivity have therefore been tried, such as extreme pH values,
high salt concentrations and different additives into the BGE [34-38]. However, when using ESI/MS the preferable strategy has been to permanently
coat the inner capillary wall. The permanently coatings are attached to the
capillary wall prior to analysis and are therefore excluded from the buffer
electrolyte making it suitable with ESI/MS. However, despite the regeneration of the coatings by flushing the capillary in between injections with a
diluted solution in order to overcome stability issues and repeatability, the
time and cost required to prepare the coating could be a challenge to overcome. Coatings could be used in order to reduce, neutralize or reverse the
EOF as well. Gilges et al. [39] showed for instance how polyvinyl alcohol
and other polymers could be used as a dynamic coating and effectively suppress the EOF and thereby focus the different zones, as in isoelectric focusing (IEF). Except modifications on the inside of the capillary wall, coatings
on the tip and outside wall in order to secure the electrical connection between the capillary end ESI source using metalized coatings and emitters has
been developed [40-46]. This type of modification has shown an important
role in sheathless ESI, which will be discussed in chapter 5.2.
15
3. Electrokinetic chromatography
“Never, never, never give up"
Winston Churchill
Since CZE cannot separate neutral analytes due to the lack of electrophoretic
mobility, the technique Electrokinetic chromatography (EKC) was developed. In 1982 Terabe and co-workers [47] published the first successful separation with sodium dodecyl sulfate (SDS) as an additive in the buffer. The
technique was later called micellar electrokinetic chromatography (MEKC).
Apart from micelles, other types of additives have been developed and used
in EKC. How the additives are introduced into the flow has been varied in
order to use mass spectrometry as detection without ion suppression and ion
source contamination.
3.1.
Why Pseudostationary Phases?
The most frequently used separation technique for analysis in liquid phases
is HPLC. However, since it requires large sample and buffer volumes, miniaturized μLC and nanoLC systems [48, 49] have gained in popularity. To
improve the separation efficiency, the particle size in the stationary phase
has been decreased in parallel to the instrumentation miniaturization [50].
Due to the nature of a stationary phase the risk of sample adsorption and
column clogging could get higher with the decreased column- and particle
size. The problem with memory effects in the column due to “sticky samples” is another problem existing with stationary phases e.g. HPLC and
CEC. It is therefore not a coincidence why the use of “moving stationary
phases” i.e. pseudostationary phases, as in paper II, has gained in popularity
[47, 51].
16
3.2.
Micellar Electrokinetic Chromatography
Terabe was one of the pioneers in introducing selectivity in CE with the
technique electrokinetic chromatography [10-14]. However, it was later
called micellar electrokinetic chromatography (MEKC) to specify the use of
micelles. These initial reports by Terabe and co-workers resulted in an explosion of studies during the 1990’s with the use of different surfactants,
polymers, macromolecules and particles as PSP’s [14, 47, 52, 53].The technique was based on partitioning analytes between two phases, a pseudostationary phase composed of charged micelles and the surrounding electrolyte
phase migrating with different velocities. Terabe generated anionic micelles
out of SDS and showed that the micelles had an electrophoretic mobility
directed towards the positively charged anode. Despite the electrophoretic
mobility out of the capillary the micelles will migrate towards the detector
due to the high EOF. Analytes would be portioned between the micellar
phase and the aqueous phase. A neutral analyte, which will spend no time
with the micelles, will travel with a migration time of t0, while the neutral
analytes that will spend all of its time with the micelles with the migration
time tmc. Other neutral analytes will partition themselves in between the two
phases and thereby migrate at intermediate times (see Figure 3).
Figure 3. Schematic figure of the separation mechanism in MEKC using anioic
micelles. The principle is similar to the traditional liquid chromatography, i.e. the
sample is distributed between two phases migrating with different speeds. But in
contrast to traditional chromatography, the pseudostationary phase in EKC has an
electrophoretic mobility. Despite the fact that the micelles are strongly negatively
charged and therefore should migrate towards the injector end, the net mobility will
be towards the detector side due to the high EOF under neutral and alkaline conditions.
17
By adding SDS to the BGE, anionic, cationic, and neutral analytes can be
separated in the same run. The separation of neutral analytes will be based
on the differences in hydrophobicity and hydrogen bonding, and can be controlled by varying surfactant concentration, pH and the organic additives e.g.
methanol or acetonitrile. The surfactant or micelle in MEKC can be compared to the stationary phase in conventional liquid chromatography.
The selectivity in MEKC depends on the structure of the surfactant molecule. The selectivity can therefore be manipulated by varying the polar head
group [54] or mixing different surfactants. However, the surfactant concentration, buffer, pH of electrolyte solution, or the temperature are other parameters to manipulate in order to vary the selectivity. But the most convenient
way is to incorporate or modify an organic solvent to the BGE. That was
also shown by S.K. Poole and C.F. Poole [54, 55] to be the most efficient
way to manipulate the selectivity. The buffer composition and concentration,
pH, temperature, and voltage only had a small influence on selectivity [56].
Coupling MEKC with a mass spectrometric detection has proven to be a
great challenge. The main reason is due to that the most commonly used
surfactant, sodium dodecyl sulphate (SDS), cause massive ionization suppression, contamination of the ion source and increased background signals
when using ESI/MS [28, 57]. Nonetheless, there have been reports on the
direct coupling of MEKC with ESI/MS utilizing SDS as a surfactant [27,
58]. Even though they could obtain the required LOD for the application,
significant ionization suppression was found. Numerous attempts have been
made to solve these problems, such as the partial filling (PF) technique in
combination with MEKC [59] or reverse the migrating micelles to prevent
the surfactants from ionizing and entering the ion source.
18
4. Mass Spectrometry
”Intelligence is the ability to adapt to change"
Stephen Hawking
Since the 1980’s mass spectrometry has reached an unsurpassed status as
analytical tool because of its universality, sensitivity, selectivity and ability
to give the elemental composition, structural information and qualitative and
quantitative information of the analyte [60-64]. Although several different
types of MS instrumentations exist, they all include three main parts. The
first part is an ionization source where the analytes are ionized and transferred into the gas phase, a mass analyzer that will sort the analyte ions by
their mass-to-charge (m/z) ratio, and finally a detector which will monitor
the separated ions. Tandem mass spectrometry (MS/MS) involves an extra
step. An extra mass analysis will occur after the ions with a specific m/z are
fragmented. Fragmentation can occur through addition of energy or by collision with a gas. The benefit of MS/MS is the valuable information given
about the molecular structures of an unknown analyte due to the often
unique fragmentation pattern for a specific analyte. Other benefits of MS/MS
are the possibilities of higher selectivity and sensitivity. [65, 66]
19
4.1.
The use of Mass Spectrometry
Mass spectrometry has gained an enormous impact in important fields such
as proteomics, peptidomics, and metabolomics [67, 68]. This is due to the
introduction of soft ionization techniques, electrospray ionization (ESI) and
Matrix Assisted Laser Desorption/Ionization (MALDI). These techniques
made it possible to analyze intact biological molecules such as proteins, peptides, and polymers. The use of mass spectrometry has become an important
part in the large scale study on the proteome, proteomics. Except the use of
MS to discovering potential biomarkers for severe diseases like Alzheimer’s
and Parkinson’s disease [69], MS has been used in other fields such as forensic science. The use of gas chromatography/mass spectrometry have
shown to be an important tool when detecting drug abuse in athletes, or
detect accelerants in arson investigations. Modified MS instrumentations, socalled space MS is valuable in NASA’s Origins theme, and have been traveling in space since early 1960s and have returned information about the solar
wind isotopes and other isotopic ratios. [66] Since MS can be used in so
many different fields of interest, high demands are put on the instrumentation. The MS need to be fast in order to keep up with hyphenated techniques
and be sensitive enough to detect low amounts, but at the same time give
reproducible results.
4.2.
Ion sources
In 1963, Richardson [20] developed a technique where he sprayed a diluted
polymer solution onto a carbon coated film. The solvent was allowed to
spread out in the carbon surface and thereafter be evaporated. The remaining
single solid entities on the carbon surface could then be photographed in an
electron microscope. This turned out to be a forerunner to the most commonly used ionization technique today, electrospray ionization (ESI). In the late
1980’s ESI was combined with MS due to the work by Fenn et al. ESI as
well as thermospray (TS) and aerospray (AS) all share the same basic principles. A sample solution enters the ion source through a stainless steel
needle. The needle is maintained at a few kilovolts relative to the surrounding chamber. As a result, the electric field at the needle tip will charge the
introduced liquid. Due to Coulomb forces the liquid will disperse into a fine
spray of charged droplets. The applied drying gas will evaporate the solvent,
leading to smaller droplets but with higher charge density at the surface. This
may then lead to a Coulomb explosion where the droplet tears apart. This
progression of events can be repeated until ions exist in ambient gas. The
electric field will let the droplets and ions migrate towards the inlet of the
MS.
20
Figure 4. Two commonly used interfaces in MS; to the left is the APPI ion source
with the photoionization lamp seen in the window underneath the heater. To the
right is the ESI interface with the spray needle tip visible at the entrance to the mass
spectrometer.
One of the latest among the soft ionization techniques in mass spectrometry
is the APPI ion source [70]. This ionization technique has shown less dependency on buffer composition compare to the ESI source, and works good at
low flow rates. This indicates that APPI would be suitable with CE, due to
the commonly used non-volatile buffers and low flow rates [71]. In contrast
to ESI the spray needle is introduced into a heater where the sample is vaporized (Figure 4). The ionization process will then be initiated by a discharge
lamp. One of the major benefits with APPI is the possibility to discriminate
between the analyte and solvent ions. By selecting a suitable UV source, so
that the selected photon-emission energy is higher than the ionization energies (IEs) of the target molecules, but lower than the IEs of the solvent and
surrounding air it is possible to take full advantage of the APPI source.
Therefore, the Krypton lamp (10.0 eV) is probably the most suitable UV
source since it has a photon energy lower than the major components of air
and the most commonly used solvents. For instance, hexane, isopropanol,
methanol, oxygen, acetonitrile, water, and nitrogen all have higher IE than
the energy of the emitted photons by the Krypton lamp and are therefore not
ionized. The probability to directly ionize an analyte molecule is not very
likely to occur, and therefore can a so-called dopant be introduced. This was
shown by Sprangler et al. [72] in where introducing a suitable substance
could significantly increase the number of ions. A dopant is effective if it is
photoionizable and can function as an intermediate between the photons and
the analyte, like the typical dopant acetone (9.70 eV).
21
5. Modifications when hyphenating Capillary
Electrophoresis to Mass Spectrometry
When hyphenating CE to MS one has to consider several aspects, such as the
ionization technique used. APPI and APCI, for instance, have a tendency to
be more mass flow sensitive than ESI. This indicates that injecting a large
volume of sample will give a higher response in MS. However, the low sample loading abilities in CE becomes a limitation. The injection volume in CE
is limited to approximate 3 % of the capillary, leading to that the only possibility is to increase the inner diameter or increase the capillary length in order to increase the injected sample volume. On the other hand, APPI has a
tendency to be more suitable with nonpolar compounds and BGEs with high
salt content.
In protein and peptide analysis ESI/MS and MALDI/MS are the most commonly used techniques. Since MALDI operates under high vacuum, transferring solutions from a liquid based separation to the high vacuum in the ion
source has been a challenge. The method of choice has been an off-line system where a fraction collector has been used to spot down the liquid on a
plate before analyzing with MALDI. However, due to the high efficiency of
CE, large demands are put on the fraction collector.
Coupling MEKC with a mass spectrometric detection has proven to be a
great challenge. The main reason is due to that the most commonly used
surfactant, sodium dodecyl sulphate (SDS), cause massive ionization suppression, contamination of the ion source and increased background signals
when using ESI/MS [28, 57]. Nonetheless, there have been reports on the
direct coupling of MEKC with ESI/MS utilizing SDS as a surfactant [27,
58]. Even though they could obtain the required LOD for the application,
significant ionization suppression was found. Numerous of attempts have
been made to solve these problems, such as the partial filling (PF) technique
in combination with MEKC [59] or reverse the migrating (RM) micelles to
prevent the surfactants from ionizing and entering the ion source. These
techniques therefore require specific optimization, with large restrictions in
the background electrolyte (BGE) composition. Other procedures reported
have involved complicated capillary connections constructed to dilute or
remove surfactants from the separation capillary effluent [73-75].Yet, other
22
approaches have involved volatile surfactants or high molecular weight
pseudostationary phases (PSP’s) [76-78]. Despite these technical developments, coupling of MEKC with ESI-MS has so far been shown to require
careful optimization and suffer from limitations in selecting interaction
phase.
5.1.
Tapered capillaries
Tapered and narrowed restrictor capillaries have shown to be an aid in suppressing the bubble formation and an interesting alternative to frits in CEC
[6]. Tapering the capillary tip in CE has shown to be especially good when
working with low flow rates and with sheathless interfaces. The tapered capillary permit a more stable electrospray and make it possible to operate at
lower ESI voltages, and give better signal-to-noise ratios [79]. The tapering
of the tip can be made in different ways, like pulling during heat, etching
with hydrofluoric acid or by mechanical grinding. However, to reproduce a
tapered capillary tip by mechanical grinding could be a challenge.
5.2.
Requirements of the interface
The interfaces for MS are originally designed to be used with higher liquid
flows, commonly achieved with HPLC. Since CE uses significantly lower
volumes and losses of analytes to the walls due to adsorption will be more
crucial, it is therefore reasonable to believe that the ion sources need to be
modified. This has been indicated in several studies and shown as well in
paper I [71, 80, 81].
Figure 5. Modifications of the interface can be necessary when hyphenating CE with
MS. Paper I showed an improved signal intensity when moving the sprayer closer
to the ionization region.
23
One of the major requirements of the interface is that the current from the
CE is maintained or the separation will be interrupted. There are three main
ways of securing the electrical circuit; liquid junction, sheath liquid interface, and sheathless interface (see Figure 6). In a liquid junction interface a
stainless steel union serves as a point for the high voltage contact. A nebulizer gas and an extra liquid could be introduced in order to create a good
spray. Even though this is an easy and excellent way of define the electric
field some negative aspects exist. Due to the electrode reactions occurring
inside the stainless steel union gas bubble formation could interfere with the
separation and spray formation. The distance between the two capillaries and
the sheath liquid/nebulizing gas tubing will create a small volume, leading to
band broadening in the separation. Despite the mentioned challenges and the
fact that the electric field is not defined at the end of the capillary, leading to
a laminar flow beyond that point, liquid junction is commonly used.
Figure 6. Three variations of ESI needles for CE-MS: liquid junction (left), sheath
flow (top), and sheathless interface (right).
In both paper I and II, a sheath flow interface was used. In this type of interface the sheath liquid and nebulizer gas is introduced at the tip of the capillary by concentrically stainless steel tubes. This will thereby minimize the
risk of gas bubble formations, avoiding any post-column region with laminar
flow, and give a more robust interface and reproducible results. Studies have
also shown an increase in theoretical plate numbers with the coaxial sheath
liquid interface compared to the liquid junction interface. One other advantage with the coaxial interface over the liquid junction interface is that the
sheath liquid is delivered independently of the BGE. This will provide more
24
flexibility in the type of BGE used. [82, 83] However, since sheath liquid is
introduced, a dilution of the sample is unavoidable.
The sheathless ESI interface on the other hand does not dilute the sample
since no extra liquid is introduced. Sheathless ESI uses a thin layer of a conductive material on the outside of the emitter end in order to secure the electric circuit. Even though several challenges arise with this method, such as
instability of the conductive coating and the total reliance on the eluting solution to create the spray, sheathless ESI has shown a great potential.
The choice of metal coating onto the fused silica has a large influence. A
silver layer have shown to be unstable under red-ox conditions at the ESI tip,
and sputtered gold have a finite lifetime before the electrical contact is lost
[41, 46]. However, other coatings such as graphite-polyimide mixtures have
shown a good performance and stability for a long time under oxidative
stress [44] especially in combination with a tapered capillary. One major
difference between sheathless ESI and sheath flow and liquid junction interfaces is the added organic modifier to the separation buffer in order to facilitate the ESI process. However, an organic modifier can be added to the BGE
for other reasons than facilitate a good spray. In paper II the organic modifier was added in order to vary the interaction between neutral analytes and
the nanoparticles. The type and amount of organic modifier have shown to
largely impact the electrospray stability with both the sheath liquid and
sheathless ESI interface. A careful optimization to avoid a decrease in EOF
by too high concentrations or disrupting the electrospray process is therefore
needed [25, 84].
Figure 7. Paper II showed that the amount of organic modifier in the BGE and the
sheath liquid had a large affect on the ionization efficiency.
25
6. Adaptation of Electrokinetic
Chromatography and Pseudostationary
Phases to Mass Spectrometry
On-column UV-detection is by far the most prevalent detection technique
applied in CE and it is also the detection technique with which most of the
commercially available CE instruments are equipped. MEKC was invented
on such equipment and for this reason most of the PSPs in use today are UVtransparent. However, whereas coupling of CE with MS detection was fairly
easy, a number of problems were identified. A major problem was the analyte ionization suppression, high background signals and contamination of
the ion-source with non-volatile PSP’s [28, 57]. Three different solutions to
solve these problems will be discussed in this chapter. The first solution
involved adaptation of the traditional MEKC protocols to MEKC/MS. This
involved adjustments of buffer composition, surfactant concentration, partial
filling (PF) of the PSP, and reverse migrating (RM) PSPs. The second solution was to design new types of PSPs with improved MS compatibility. This
involved high molecular weight as well as semi-volatile PSPs. The third
solution involved instrumental modifications including inventions in the
electrospray interface as well as application of other ionization sources such
as APPI and atmospheric pressure chemical ionization (APCI). Inventions in
MEKC/MS within these three categories will be reviewed in the following
sections.
26
6.1.
Traditional Micellar Electrokinetic
Chromatography adapted to Mass Spectrometric
detection
The important advantages of MEKC over HPLC are the higher efficiencies achievable as well as selectivity originating from both electrophoretic and chromatographic mechanisms. Furthermore, as the PSP is replenished after every separation, memory effects, stationary phase ageing
and column clogging are minimized. However, the compatibility of MEKC
with MS detection has, as previously mentioned, been poor. Still, studies
were conducted in which the buffer composition and surfactant concentration was altered to allow for MS detection [52, 58]. Although the LODs reported were high, some analytes could still be detected. Ionization suppression, source contamination and high background signals were observed in all
studies and the obvious solution to these problems was to exclude the surfactants from entering the ion source. Lamoree and co-workers [85] suggested
that coupling of MEKC with MS detection could be realized by transferring
the analyte zones to a capillary free of SDS by voltage switching. However,
this technique requires a home built capillary coupling system. Furthermore,
the efficiencies were about two-fold lower than for MEKC/UV, which was
attributed to band broadening due to the application of a lower separation
voltage and extra band-broadening in the coupling device. Nevertheless, the
authors described a fascinating system for on-line heartcutting of sample
zones in MEKC.
The first technique being more general for rendering a standard MEKC protocol MS compatibility was PF of the PSP (Figure 8.). With this technique
only a section of the capillary at the injection side is filled with a PSP. Subsequently, the sample is injected and when a voltage is applied over the capillary the sample migrates through the PSP and reaches the detector ahead
of the PSP. The analysis is then stopped before the PSP reaches the detector,
thereby avoiding introduction of the PSP into the ion source. Drawback with
the PF technique includes the required introduction of additional variation
with the extra injection of PSP. Furthermore, the electrolyte becomes discontinuous resulting in EOF variations between the PSP plug and the surrounding electrolyte, causing pressure variations and ultimately introduction of
laminar flows [86, 87]. Furthermore, the net velocity of the analytes is generally higher outside the PSP plug resulting in further band broadening.
Another problem is that in the border between the PSP plug and the surrounding electrolyte the concentration of surfactants gradually drops due to
diffusion and laminar flow effects ultimately resulting in a micelle gradient.
27
Due to these problems and due to that the PSP plug generally is kept very
short to avoid elution of surfactants into the ionization source, the resolution
is always lower than for traditional MEKC [88]. Obviously, method development is therefore more complicated in PF-MEKC than in traditional
MEKC.
Figure 8. (A) Schematic illustration of PF-EKC. Step 1: A plug of PSP is injected
prior to injection of the sample. Step 2: As the voltage is applied, the analytes start
to migrate through the PSP plug thereby becoming separated. Step 3: The analytes
reaches the electrolyte phase prior to detection. The separation is stopped when all
analytes have been detected and before the PSP reaches the detector. (B) Schematic
illustration of RM-EKC. Step 1: The capillary is filled with PSP prior to injection of
sample. Step 2: As the voltage is applied, the analytes start to migrate through the
PSP and thereby becoming separated. The PSP migrates in the opposite direction
into the inlet vial, thereby efficiently avoiding elution of surfactants into the ionization source. Step 3: The separation is stopped when all analytes have been detected.
In PF, the longest applicable plug length would be realized with a very low
EOF allowing for a net reverse migration of the micelles. Thereby the theoretical plug length would be equal to the total capillary length. Highly negatively charged PSPs, with low pH electrolytes or neutral capillary coatings
have made it possible to realize such long plug length and the technique has
been named RM-EKC (described in Figure 8B) [73, 89]. Due to the very low
or absent EOF in RM-EKC, the technique is mainly applicable for positively
charged analytes, although some positively charged PSPs have been applied
in reverse polarity for separation of negatively charged analytes. However,
due to the risk of contaminating the ionization source the whole capillary is
rarely filled with PSP and therefore in practice very similar to PF. Despite
the development of techniques that enable the use of traditional PSPs in
EKC/MS, the counter ions of the PSP may still reach the detector causing
ionization suppression. Thus, PSPs designed for use with MS detection and
innovations in MS detection are required.
28
6.2.
New Pseudostationary Phases with improved
Mass Spectrometric compatibility
In the development of new PSPs with improved MS compatibility two main
approaches can be identified. On one hand, researchers have strived to reduce ionization suppression by developing high molecular weight PSPs with
a CMC close to or equal to zero [77, 78, 90]. With these PSPs, accumulation
of surfactants at the water-air-interface is reduced causing less ionization
suppression. Furthermore, electropherograms with less background signals
in the low m/z-range are generated and the high molecular weight PSPs are
in less extent ionized. Also, method development is simplified as the electrolyte composition no longer affects the integrity of the PSP. On the other
hand, reduced contamination of the ion source has been assessed by the use
of semi-volatile surfactants [58, 91]. These two approaches are thus very
similar to traditional MEKC. However, several new types of phases, not
falling into these categories, have also been developed. Magnetic nanoparticles have been applied in a variety of analytical applications, such as in
vivo examination via magnetic resonance imaging (MRI) [92] and in chip
based systems (microfluidics) [92, 93]. The main characteristics of these
particles are that they can be functionalized on the bead surface while still
having the embedded magnetic properties, allowing for manipulation of the
particle with permanents magnets or electromagnets. Such magnetic beads
have been applied in sample preparation and pre-concentration [94, 95]. In
1997 Rashkovetsky et al. [96] performed an enzymatic assay, affinity adsorption and isotachophoretic (ITP) focusing using magnetic beads in a CE
system. In that application a small amount of magnetic beads, containing
immobilized antibodies, was injected and trapped by a magnet in a neutral
hydrophilic-coated fused-silica capillary. Subsequently, the sample, containing the antigen, was injected and the antigen was trapped onto the beads. The
beads could then be washed and the antigen eluted by a change in the buffer
pH. Finally, the separation voltage was switched on and ITP was performed.
An interesting application of nanoparticles was presented by Kan and Barron
[97] and later by others [98][97][96][96][96]. In their application, poly(Nisopropylacrylamide) (PNIPAAm) modified temperature-sensitive latex
beads were trapped in the capillary by an increase in the capillary temperature. Thereby, these particles could be used to trap analytes in the capillary.
Lowering the temperature brought the beads back into suspension and the
analytes could in that way be eluted from the capillary. These particles have
also shown to be useful as coating in CE capillaries [97, 99]. The magnetic
beads and the temperature sensitive latexes are not PSPs per se, but, they do
29
fulfill one of the requirements of a PSP, being a non-immobilized interaction
phase. Even though these particles have not yet been applied in CEC/MS,
they do present an interesting means of combining CEC with a non-trapped
stationary phase with MS detection.
6.3.
Continuous Full Filling
Today, ESI is the dominating ionization technique used in liquid phase separations coupled with MS detection. As ESI originally was developed for
HPLC, the performance of many of the earlier separations published on
MEKC/MS were hampered due to the use of ESI interfaces not suited for the
capillary format. As the ESI interfaces were miniaturized and adapted to the
capillary format significant improvements in MEKC/MS were achieved.
Further improvement was obtained with the orthogonal ESI-interfaces.
These interfaces, spraying at an angle to the mass spectrometer inlet, had a
greatly improved tolerance to non-volatile electrolyte constituents [100].
However, further developments are required to realize the full potential of
CE/MS [71, 80, 101]. Viberg [24] elegantly showed that nanoparticles could
be used as a PSP in a continuous full filling (CFF) technique using an orthogonal ESI interface (Figure 9) in the same manner as in paper II. In CFF the
PSP is suspended in the electrolyte similar to traditional MEKC. Thus, the
electrolyte is continuous, avoiding the problems resulting from discontinuous electrolytes in e.g. PF-EKC. Due to an orthogonal ESI interface the
nanoparticles was exclude from entering the MS. The particles could not,
due to their larger momentum, deviate from the off-axis spray direction. If
these highly concentrated nanoparticles in electrolyte suspensions would be
sprayed on-axis several microgram per minute of non-volatile nanoparticles
would risk entering the mass spectrometer. Several studies, mainly involving
improvements in the nanoparticle based PSP, followed this proof of principle
report [25, 102-108]. The later studies, including paper II, showed that CFF
with nanoparticles could be performed without any negative effects on the
analyte signal, without any contamination of the mass spectrometer and with
efficiencies as high as one million plates per meter [25, 106, 107].
30
Figure 9. A schematic picture showing the principal of CFF-CEC/ESI-MS,
where the added nanoparticles to the BGE are transported through the open
capillary. This will allow for a separation of neutral analytes as well due to
the analytes different partitioning to the nanoparticles. The electroosmotic
flow is sufficiently high to generate a net transport towards the cathode despite any anodic migration of the nanoparticles.
31
The number of publications involving nanoparticles in EKC and related
techniques has increased exponentially during the past two decades
(Figure 10). Besides their ESI-MS compatibility, these phases also have
beneficial mass transfer and a zero CMC. Despite developments of new
PSP’s and ESI-interfaces, the full potential of EKC/MS may not yet have
been realized. Recent developments in ionization techniques indicate that
other ionization sources may be better suited for coupling of EKC with MS
detection. Recently, two conceptually different ionization techniques were
applied in MEKC/MS. Atmospheric pressure chemical ionization (APCI)
and atmospheric pressure photo ionization (APPI) applies electrical discharges and photons, respectively, to ionize the analytes in the gas phase.
Interestingly, these two techniques were found to be virtually insensitive to
the presence of surfactants in the BGE. Analogous to the improvements in
ESI with orthogonal spraying, orthogonal APCI was also found superior to
on-axis APCI [80]. However, although proven to possess an extensive potential in EKC/MS, ESI-MS is generally still more sensitive [80, 109-111].
Figure 10. The number of published articles with a topic concerning nanoparticles
and CE and the sum of PSP-CE related techniques (source ISI Web of knowledge).
32
7. Concluding remarks
”If you can’t explain it simply,
you don’t understand it well enough"
Albert Einstein
Since CZE is unable to separate neutral analytes, the use of pseudostationary
phases is an excellent way of combining the high efficiency achieved in CE
with the selectivity in HPLC. As paper II indicates, the new ESI/MS friendly pseudostationary phases could be a good way of solving limitations with
complex matrixes using CE-ESI/MS without tedious modification steps or
loosing sensitivity due to ion suppression. Despite the different ionization
techniques in APPI and ESI the same spray needle is used. Therefore, several of the drawn conclusions could be used in paper II as well. Paper I indicated that optimizing the protruding capillary end from the stainless steel
sprayer and that the nebulizing gas flow had a large influence on the detection sensitivity. It could also be concluded that the available APPI interface
is not fully optimized for CE-MS. However, small modifications and miniaturization of the interface, as well as changing the capillary inner diameter,
are some ways of optimize the conditions for CE-APPI/MS.
Based on paper I and II, and all the number of publications during the last
decade, there are definitely a high interest in CE and CE-related techniques.
But more research is needed to fully use their potentials. However, the use of
MS friendly pseudostationary phases will lead to higher interest in the technique and hopefully become even more appreciated and used in important
field such as clinical applications. The use of nanoparticles in sheathless ESI
would be an excellent way of combining ESI friendly pseudostationary
phases with the good properties of the sheathless interface, since paper II
showed the importance of organic modifier in the BGE when working with
nanoparticles in CE. When working with samples with high salt content,
nanoparticles in CE-APPI/MS could however be the method of choice.
33
8. Acknowledgments
”As we express our gratitude, we must never forget that the
highest appreciation is not to utter words, but to live by them"
John F. Kennedy
There are many I would like to show my appreciation, especially my family
and friends for all their love and support!
I would like to show my biggest gratitude to my supervisors, Jonas Bergquist and Charlotta Turner for giving me this opportunity to pursue as a
Ph.D. student at department; guiding me into the world of science.
Marcus Sjödin, you have given me a great time with many laughs both at
and outside work - one can’t ask for a better friend and colleague!
This work couldn’t have been possible without Peter Spégel, Jakob Axén,
Peter Viberg and all my other excellent collaborators! Thank you for all
the scientific discussions and practical help you have given me. Peter Spégel,
I would in particular show my appreciations to you and I hope we will have
many more projects together in the future. I would also like to give many
thanks to Nanosep AB (PerÅke Oldentoft) for giving me this opportunity
to work with your nanoparticles and thereby getting into the field of pseudostationary phases in CE.
Magnus Wetterhall, thank you for the all the help you have given me. I’m
looking forward to continue our projects together, and I’m very happy to
start having you as a supervisor.
Last but not least, a gratitude to all at the department, in particular Jörg Hanrieder and Roland Pettersson.
34
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