Download NOVEL ON-LINE MID INFRARED DETECTION STRATEGIES IN CAPILLARY ELECTROPHORETIC SYSTEMS Malin Kölhed

NOVEL ON-LINE
MID INFRARED DETECTION STRATEGIES
IN CAPILLARY ELECTROPHORETIC SYSTEMS
Malin Kölhed
Doctoral Thesis
Department of Analytical Chemistry
Stockholm University
2005
i
Akademisk avhandling framlägges för avläggande av filosofie
doktorsexamen vid Stockholms universitet. Försvaras offentligen
torsdagen den 22 september 2005, kl. 13:30 i Magnélisalen, Kemiska
övningslaboratoriet, Svante Arrhenius väg 12.
Avhandlingen kommer att försvaras på engelska.
ISBN 91-7155-102-6 (pp i-xiv, pp 1-60)
© Malin Kölhed, 2005
Intellecta DocuSys AB, Nacka, Sverige 2005
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Till Robban
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iv
KORT SAMMANFATTNING
I det infraröda området av det elektromagnetiska spektrumet finner man
strukturell information om ett stort antal föreningar, alltifrån små joner
till stora biologiska molekyler. Faktum är att alla molekyler som har ett
dipolmoment som förändras då atomerna vibrerar är infrarödaktiva. Det
infraröda (IR) spektrumet kan vidare indelas i far-, mid- och näraområden. Fokus i denna avhandling ligger på mid-IR området. I detta
område finns grundvibrationerna för de flesta organiska föreningar vilket
möjliggör positiv identifiering av dessa analyter. IR-tekniken är direkt
tillämpbar då man undersöker ett fåtal enkla molekyler i okomplicerade
provsystem. Problem kan emellertid uppstå då antalet olika analyter ökar
och/eller då komplexiteten i provmatrisen ökar. Här kan en ihopkoppling
med en separationsmetod före detektion med IR utgöra en elegant lösning
på ett komplicerat analytiskt problem. Artikel I, bilagd i denna avhandling,
presenterar (för första gången) on-line ihopkopplingen av Fourier transform infraröd spektroskopi, FTIR, och kapillär frizonelektrofores, CZE.
CZE är en mycket effektiv separationsteknik som separerar joner med
avseende på deras laddning/massa-förhållande. CZE utförs vanligen i
vattenbaserade buffertar och i kapillärer gjorda av silika. Då dessa
kapillärer absorberar i stort sett allt IR-ljus var utvecklingen av en IRtransparent flödescell nödvändig. I vidare studier (Artikel II) har
tillämpbarheten av CZE utvidgats till att inkludera även neutrala analyter.
Genom att tillsätta miceller till bufferten, och tillämpa tekniken micellär
elektrokinetisk kromatografi, MEKC, kunde denna teknik ihopkopplas
on-line med en FTIR-detektor för första gången. Artikel III beskriver en
tillämpning av on-line CZE-FTIR tekniken då icke-UV-absorberande
analyter i en komplex provmatris först separerades och därefter
sekventiellt identifierades och kvantifierades i en och samma körning.
Att mäta på vattenlösningar i mid-IR området är synnerligen
problematiskt då vattnet självt delvis eller helt absorberar IR-ljuset i detta
område. En mid-IR detektor kan därmed inte urskilja de enskilda
analyternas absorbansbidrag. Det är av denna anledning som quantum
kaskadlasrar är intressanta. Dessa lasrar representerar en ny klass av
mid-IR halvledarlasrar vilka kan ge hög ljusstyrka tack vare en genial
v
design. Laseraktionen sker inom ett konduktivt band och lasrarna kan
konstrueras så att de emitterar ljus i hela mid-IR området genom
användning av ett och samma halvledarmaterial. Dessa lasrar har använts
tillsammans med ett flödessystem för att undersöka deras potential att
öka den optiska strålgången vid mätning på vattenlösningar (Artikel IV),
och erfarenheterna från detta arbete utnyttjades vid ihopkopplingen av
dessa lasrar med ett CZE system (Artikel V).
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ABSTRACT
Infrared absorption spectra can provide analytically useful information
on a large variety of compounds, ranging from small ions to large biological molecules. In fact, all analytes that possess a dipole moment that
changes during vibration are infrared-active. The infrared (IR) spectrum
can be subdivided into far-, mid- and near- regions. The focus of attention in this thesis is the mid-IR region, in which the fundamental vibrations of most organic compounds are located, thus providing scope for
positive structural identification. However, while such near-ubiquitous
signals can be very useful for monitoring simple molecules in simple systems, they can be increasingly disadvantageous as the number of analytes
and/or the complexity of the sample matrix increases. Thus, hyphenation to a separation system prior to detection is desirable. Paper I appended to this thesis presents (for the first time) the on-line hyphenation
between Fourier transform infrared spectroscopy, FTIR, and capillary zone
electrophoresis, CZE. CZE is a highly efficient separation technique that
separates ionic analytes with respect to their charge-to-size ratio. It is
most commonly performed in aqueous buffers in fused silica capillaries.
Since these capillaries absorb virtually all infrared light an IR-transparent flow cell had to be developed. In further studies (Paper II) the applicability of CZE is expanded to include neutral analytes by the addition of
micelles to the buffer, and micellar electrokinetic chromatography, MEKC,
was successfully hyphenated to FTIR for the first time. Paper III describes
an application of the on-line CZE-FTIR technique in which non-UVabsorbing analytes in a complex matrix were separated, identified and
quantified in one run.
Measuring aqueous solutions in the mid-IR region is not straightforward since water absorbs intensely in this region, sometimes completely,
leaving no transmitted, detectable light. For this reason, quantum cascade lasers are interesting. These lasers represent a new type of mid-IR
semiconducting lasers with high output power due to their ingenious
design. The laser action lies within one conduction band (intersubband)
and can be tailored to emit light in the entire mid-IR region using the
same semiconducting material. To investigate their potential to increase
vii
the optical path length in aqueous solutions, these lasers were used
with an aqueous flow system (Paper IV), and the experience gained
in these experiments enabled hyphenation of such lasers to a CZE
system (Paper V).
viii
PAPERS INCLUDED IN THIS THESIS
This thesis is based on the following papers, which are referred to in the
text by the corresponding Roman numerals:
I
On-line Fourier transform infrared detection in capillary
electrophoresis
Malin Kölhed, Peter Hinsmann, Peter Svasek, Johannes Frank,
Bo Karlberg and Bernhard Lendl
Analytical Chemistry (2002), 74(15), 3843-3848
II
Micellar electrokinetic chromatography with on-line Fourier
transform infrared detection
Malin Kölhed, Peter Hinsmann, Bernhard Lendl and Bo Karlberg
Electrophoresis (2003), 24, 687-692
III
Capillary electrophoretic separation of sugars in fruit juices using
on-line mid infrared Fourier transform detection
Malin Kölhed and Bo Karlberg
Analyst (2005), 130(5), 772-778
IV
Assessment of quantum cascade lasers as mid infrared light
sources for measurements of aqueous solutions
Malin Kölhed, Michael Haberkorn, Viktor Pustogov,
Boris Mizaikoff, Johannes Frank, Bo Karlberg and Bernhard Lendl
Vibrational Spectroscopy (2002), 29(1-2), 283-289
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V
On-line hyphenation of quantum cascade laser and capillary
electrophoresis
Malin Kölhed, Stefan Schaden, Bo Karlberg and Bernhard Lendl
Journal of Chromatography A (2005), 1083(1-2), 199-204
The author was responsible for all (Paper III), for major parts (Papers I, II
and IV) and for some (Paper V) of the experimental work described in
these papers, and either all (Paper III) or substantial parts (the others) of
their writing.
Permission to reprint the articles was kindly granted by the publishers.
PAPER NOT INCLUDED IN THIS THESIS
On-line infrared detection in aqueous micro-volume systems
Malin Kölhed, Bernhard Lendl and Bo Karlberg
Analyst (2002), 128(1), 2-6 (Highlight paper)
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ABBREVIATIONS
ATR
BGE
CE
CEC
CGE
CIEF
CIRCLE
CITP
CMC
CZE
DAD
DTGS
EOF
FFT
FIA
FP
FTIR
HPLC
IR
MBE
MCT
MEKC
MS
NMR
QC
QCL
RP
SDS
S/N
UV
Attenuated total reflection
Background electrolyte
Capillary electrophoresis
Capillary electrochromatography
Capillary gel electrophoresis
Capillary isoelectric focusing
Cylindrical internal reflectance
Capillary isotachophoresis
Critical micelle concentration
Capillary zone electrophoresis
Diode array detection
Deuterated tri-glycine sulfate
Endoosmotic flow
Fast Fourier transform
Flow injection analysis
Fabry Pérot
Fourier transform infrared spectroscopy
High performance liquid chromatography
Infrared
Molecular beam epitaxy
Mercury cadmium telluride
Micellar electrokinetic chromatography
Mass spectrometry
Nuclear magnetic resonance
Quantum cascade
Quantum cascade laser
Reversed phase
Sodium dodecyl sulphate
Signal-to-noise
Ultraviolet
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xii
TABLE OF CONTENTS
About this thesis
1
1
3
Capillary electrophoresis
1.1
1.2
1.3
1.4
1.5
1.6
1.7
2
Infrared spectroscopy
2.1
2.2
2.3
3
What is capillary electrophoresis? . . . . . . . . . . . . . . . . . . . . . . 3
Instrumentation in CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Separation mechanisms and endoosmotic flow . . . . . . . . . . . 5
Modes of operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.4.1 Micellar electrokinetic chromatography . . . . . . . . . . . . . . . . 8
1.4.2 Other modes of CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.5.1 Hydrodynamic injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.5.2 Electrokinetic injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Electrodispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.6.1 Zone-broadening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.6.2 Sample stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
On-line detection techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 13
15
The electromagnetic spectrum . . . . . . . . . . . . . . . . . . . . . . . . 15
Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Quantitative measurements . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Fourier Transform infrared spectroscopy
3.1
3.2
3.3
3.4
3.5
3.6
3.7
21
Development of FTIR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
The Michelson interferometer . . . . . . . . . . . . . . . . . . . . . . . . . 21
Advantages of FTIR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Limitations of the Michelson interferometer . . . . . . . . . . . . 26
Apodization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.6.1 The DTGS detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.6.2 The MCT detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Mid-IR light sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
xiii
4
Quantum cascade lasers
4.1
4.2
4.3
5
31
Basic semiconductor theory . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.1.1 Semiconducting lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Fundamentals of quantum cascade lasers . . . . . . . . . . . . . . . 32
Applications of QC lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
CE-FTIR hyphenation
5.1
5.2
35
Why hyphenate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
On-line and off-line hyphenation strategies . . . . . . . . . . . . . 37
5.2.1 Solvent elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5.2.2 Transmission flow cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5.2.3 Attenuated total reflection flow cells . . . . . . . . . . . . . . . . . 42
6
On-line QC laser hyphenations
43
7
Comments on the optical set-ups
45
7.1
7.2
The CE-FTIR set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
QC laser set-ups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
8
The past and a future perspective
49
9
Acknowledgements - Tack!
51
10
Bibliography
55
xiv
ABOUT THIS THESIS
This thesis consists of three parts, concerned with separation (Chapter 1),
detection (Chapters 2-4) and hyphenation (Chapters 5-7). The first part
describes the capillary electrophoresis (CE) system and references are
given, where appropriate, to informative books and reviews for readers
who are not familiar with the concepts of capillary zone electrophoresis
or micellar electrokinetic chromatography. In the second part the underlying concepts of Fourier transform infrared spectroscopy (FTIR) and
quantum cascade (QC) lasers are explained in detail and a fundamental
description of infrared radiation is provided. The final part of the thesis
is concerned with hyphenations between CE separation systems and both
an FTIR instrument and a QC laser system, respectively. Overall, focus is
on on-line hyphenation since it represents an elegant and desirable solution to coupling problems. As the work has been quite technical this part
ends with a chapter covering my own reflections on the optical set-ups
used. This thesis concludes with comments on the potential utility of online CE-FTIR and CE-QCL hyphenations.
1
2
Chapter 1 Capillary electrophoresis
1 CAPILLARY ELECTROPHORESIS
This chapter covers fundamental principles of the two modes of capillary
electrophoretic systems employed in my studies, namely capillary zone
electrophoresis and micellar electrokinetic chromatography. The electrophoretic separation mechanisms and the different instrumental components are described in detail.
1.1
What is capillary electrophoresis?
Electrophoresis is defined as the migration of analytes in an electric field
and was first introduced as a separation technique by Arne Tiselius in
the 1930s. Electrophoresis, as a separation technique, was initially limited by removal of the large amounts of heat generated during separation and problems associated with the stabilising media (such as polymer gels). These obstacles were solved by Hjertén [1] who showed that it
was possible to perform electrophoresis in a rotating, 300 µm, glass tube.
The breakthrough for capillary electrophoresis came when Jorgenson and
Lukacs further decreased the inner diameter of an open tubular glass
capillary (to 75 µm) and demonstrated the simplicity of the experimental
set-up and the high resolving power of capillary electrophoresis [2,3].
Capillary electrophoresis (CE) represents a merger of technologies derived from traditional electrophoresis and high performance liquid chromatography (HPLC). On-line detection systems are generally used in both
CE and HPLC analyses, and there are CE modes which involve a pseudostationary phase (micellar electrokinetic chromatography, see Chapter 1.4.1). Nevertheless, the fundamental difference between CE and HPLC
derives from the separation mechanism, which is always electrophoretic
in CE and chromatographic in HPLC.
Frequently cited advantages of CE compared to traditional separation
techniques include its high separation efficiency, short analysis times,
the extremely small sample volumes required (1-50 nL) and the simplicity of varying the selectivity and developing methods. However, detecting CE-separated analytes is a significant challenge because of the narrow bore of the capillary. Since its conception several good books [4-7]
3
and reviews [8,9] have been written on this subject and they are recommended to interested readers for a more comprehensive explanation of
the scope of CE.
1.2
Instrumentation in CE
The straightforwardness of CE instrumentation facilitates hyphenation
to other techniques. CE is generally performed in narrowbore tubes
(i.d. ranging between 20 and 100 µm) or on microfabricated chips [10].
The discussion below focuses on CE in fused silica capillaries.
A schematic diagram of a typical CE set-up made in-house is shown
in Figure 1.1A. It consists of a fused silica capillary, a high voltage power
supply, two electrodes and two buffer vials. Each of the buffer vials is
filled with a background electrolyte (BGE).
Fused silica capillary
A
Pt electrode
Detection point
Pt electrode
High voltage
power supply
EOF
μe-
B
μe+
Diffuse layer
+
Stern layer
+
+
+
+
+
+
+
N
- -
- +
+
+ +
+
+
+
N
+
+ +
+
+
+
+
+
+
+
+
+
+
N
N
+
+ +
+ +
+
+
+
+
+
+
+
+ +
+
+
+
+ + + +
+
+ +
+
+ +
+
++ +
+ + +
+
+
+
+
+
+
+
+
+
+
Internal surface of a fused silica capillary
Figure 1.1 (A) A schematic representation of a CE system built in-house. (B)
Diagram of the inside of a fused silica capillary and a hypothetical CE separation
of anions, neutrals and cations.
The inevitable variability of inexpensive CE set-ups made in-house imposes some limitations, for instance on the reproducibility of the injection volumes and the constancy of the capillary temperature (which is
4
Chapter 1 Capillary electrophoresis
especially critical when working with high field strengths and currents).
These problems can be avoided (or at least accounted for) by using a
dedicated, commercially available instrument, such as those supplied by
Agilent Technologies and Beckman Coulter. However, the importance
placed on safety and robustness in the design and construction of these
instruments limits the scope to try new concepts and technical solutions
using them. Thus, the CE hyphenations described in my papers have all
been accomplished using a CE system made in-house, although the
optimisation of the separations were generally performed using a commercially available instrument.
1.3
Separation mechanisms and endoosmotic flow
Electrophoretic separation mechanisms are based on differences in analyte
migration velocities in an electric field. When a charged species is exposed to an electric field it experiences an electrical force, Fe, proportional to the effective ion charge, q, and the electric field strength, E. The
translational movement of an ion is opposed by a retarding frictional
force, Ff, proportional to the ion velocity,νe, and the friction coefficient, ƒ.
Under steady state conditions these two forces balance each other
f
Equation 1.1
The friction coefficient, ƒ, of a moving spherical ion is related to its
hydrodynamic radius, r, and the viscosity, η, of the surrounding BGE.
Rearranging the equation above for the ion velocity (see Equation 1.4)
gives the following formula for the ion mobility
Equation 1.2
f
5
Hence, a small, highly charged ion will have a high mobility whereas a
large, minimally charged ion will have lower mobility. The direction of
the mobility is dependent on the charge of the species. Included in
Figure 1.1B is a typical, hypothetical CE separation.
The inner surface of a fused silica capillary is covered with silanol
groups that have pKa-values ranging from 2 to 5. When the capillary is
filled with a BGE at a pH exceeding the pKa of the silanol groups, the
inner surface becomes negatively charged, counterbalanced by positive
ions from the BGE forming a double (Stern) layer and a diffuse layer
(see Figure 1.1B). If an electric field is then applied across the capillary,
cations in the diffuse layer will move towards the cathode, dragging the
entire bulk solution with them due to the viscosity of the BGE. This phenomenon is referred to as endoosmotic flow (EOF). Thus, regardless of
the analytes’ individual migration direction, the EOF is stronger and will
sweep all of them towards the cathode, passing the point of detection on
the way.† A flat velocity profile is formed due to the fact that the driving
force of the EOF is evenly distributed along the capillary, in contrast to
the parabolic velocity profile generated by mechanical pumping. The flat
velocity profile is beneficial since it does not directly contribute to the
dispersion of the analyte. However, the flat flow profile will be disrupted
if the inner diameter of the capillaries exceeds 200-300 µm [4].
The velocity of the EOF, νEOF , is given by
Equation 1.3
and the velocity of an ion, νe, by
Equation 1.4
making the apparent migration velocity, νapp, of an ion
Equation 1.5
†
Assuming a negatively charged inner surface, moderately charged and sized ions and no inner
surface modifications.
6
Chapter 1 Capillary electrophoresis
where E is the electric field strength, i.e. simply a function of the applied
voltage divided by the total capillary length. The ion mobility, —e, is a
factor that is characteristic for the specific ion in a specific medium. The
EOF mobility, —EOF, is influenced by many factors, including the viscosity,
η, the dielectric constant, ε, of the medium and the zeta potential, ζ, at the
capillary-BGE interface.
Equation 1.6
The zeta potential is in turn governed largely by the electrostatic nature
of the capillary surface and to a minor extent by the ionic nature of the
BGE. At low pH the EOF mobility is diminished since there is no zeta
potential. The EOF mobility will also decrease as ionic strength increases
due to compression of the double layer and can be suppressed by coating
the capillary inner surface.
1.4
Modes of operation
The main advantage of CE is that the separation conditions and, thus,
the selectivity can be very easily varied. When no modification has
been applied to the capillary inner surface and no additives have been
introduced into the BGE the technique is hereafter referred to as
capillary zone electrophoresis (CZE). The addition of different
surfactants to the BGE can change the selectivity of the system. Added
surfactants adsorb to the inner surface, mostly via hydrophobic and/
or ionic interactions. A cationic surfactant can form a double layer on
the inner surface of the capillary thereby creating a positively charged
surface, resulting in a reversal of the EOF direction. Anionic surfactants
can increase the EOF and neutral surfactants decrease the EOF by
shielding the inner surface. If the concentration of the added surfactant
is higher than its critical micelle concentration (CMC) micelles will be
formed in the BGE, resulting in the formation of a media that can act
as a chromatographic pseudo-stationary phase. This mode of CE is
7
called micellar electrokinetic chromatography, MEKC, and is described
in further detail below.
1.4.1 Micellar electrokinetic chromatography
Micellar electrokinetic chromatography, MEKC, was first introduced by
Terabe and co-workers in 1984 [11] and is a CE mode that can separate
neutral analytes. The mechanism involved in such separations can be
described according to chromatographic theory, i.e. it is based on the differential partitioning of analytes between a “stationary” phase (the micellar phase) and a mobile phase (the surrounding BGE). The micelles are
usually spherical and consist essentially of aggregates of monomeric surfactants that arrange themselves, at concentrations higher than the CMC,
with the hydrophobic tail facing the centre of the micelles and the hydrophilic head facing the BGE (see Figure 1.2). The hydrophobic tail may be
a straight or branched chain of carbons, whereas the hydrophilic head
may be cationic, anionic, zwitterionic or neutral.
Figure 1.2 Schematic diagram of a micelle.
Although MEKC in many ways resembles HPLC, a fundamental difference is that the “stationary” phase in MEKC, i.e. the micelles, are moving.
The micelles are often anionic, that is, migrating in the opposite direction
to the EOF, but at neutral and basic pH the migration of the EOF is stronger and consequently the net flow is towards the cathode. In MEKC there
is a separation window (see Figure 1.3) between analytes completely dissociated in the BGE (and hence elute with the EOF at t0) and analytes that
8
Chapter 1 Capillary electrophoresis
Signal
always associate with the micelles (consequently eluting with the micelles at tmc). Separation takes place between these two extremes.
b
a
tmc
t0
Elution window
Time
Figure 1.3 Schematic illustration of a hypothetical MECK electropherogram of
neutral analytes (a and b) showing the elution window, where markers for the
EOF (t0) and micelle (tmc) elution times have been added to the BGE.
The main advantage of MEKC, in comparison to CZE, is that neutral
analytes can be separated.† However, it can also separate ionic analytes
in the same run. An ionic analyte will have a migration velocity that
is usually different from that of the micelles. Furthermore, the velocity of an ionic analyte will be affected whenever it is interacting with
the micelles.
The selectivity can easily be varied in MEKC simply by changing the
surfactant. In addition, the use of organic modifiers such as methanol or
acetonitrile can weaken hydrophobic interactions between the analyte
and the micelles. The addition of chiral selectors to the BGE in either CZE
or MEKC also enables the separation of racemic solutions. Due to the
versatility of MEKC it can be employed to separate a variety of analytes
in diverse pharmaceutical, clinical, environmental and other applications.
For further details on current developments and trends in MEKC readers
are advised to read [12-16].
† Neutral analytes have no migration velocity of their own, µ = 0, making µ
e
app= µEOF (see Equation 1.5)
9
1.4.2 Other modes of CE
In addition to the previously mentioned CZE and MEKC, CE encompasses several other electrokinetic techniques, including the size-based
separation of macromolecules such as oligonucleotides and proteins by
capillary gel electrophoresis (CGE) and the isoelectric point-based separation of peptides and proteins by capillary isoelectric focusing (CIEF).
Capillary isotachtophoresis (CITP) is a moving boundary method that is
sometimes used as a sample pre-concentration step. A recent trend is
capillary electrochromatography (CEC), performed in open-tubular, LC
stationary phase-packed or monolithic columns. CEC combines the high
efficiency of normal CE with the high selectivity of HPLC. However, these
techniques are beyond the scope of this thesis and interested readers can
find more information in almost any good monograph covering CE.
1.5
Injection
Samples are injected in CE by temporarily replacing one of the BGE vials
with a sample vial. The volume injected is controlled either by electrokinetic or hydrodynamic forces, or both.
1.5.1 Hydrodynamic injection
Hydrodynamic injection is accomplished by applying a pressure difference over the capillary, either by creating a low pressure at the exit end
or by elevating the injection side and thus creating a siphoning force. In
either case the sample quantity loaded is independent of the sample matrix co-ions. The injected sample volume, V, will be a function of the applied pressure, ΔP, the duration it is applied, t, the viscosity, η, of the
BGE and the capillary dimensions (d, inner diameter and L, total capillary length) and can be calculated using the Hagen-Poiseuille equation.
Equation 1.7
10
Chapter 1 Capillary electrophoresis
When using siphoning† the applied pressure, ΔP, is calculated from
the density of the BGE, ρ, the gravitational constant, g, and the height
difference, ΔH, introduced between the inlet and outlet vial during
the injection
Equation 1.8
1.5.2 Electrokinetic injection
Electrokinetic injection is performed by applying a potential difference over the capillary. The voltage used is usually 3-5 times lower
than the separation voltage [4]. The molar amount of each analyte
injected depends on several factors, including the apparent velocity,
the injection duration, the conductivity ratio between the sample and
the BGE, the concentration of the analyte and the capillary radius.
The amounts injected of specific analytes will vary since they will all
have different mobilities.
1.6
Electrodispersion
No chapter covering the basic principles of CE would be complete without mentioning electrodispersion. Under ideal CE conditions the only
factor influencing zone broadening is longitudinal molecular diffusion.‡
However, when the conductivities between the analyte zone and the surrounding BGE zone differ, skewed peaks will appear and sample stacking can occur.
1.6.1 Zone-broadening
Let us assume that anions are being separated and that we have an EOF
towards the cathode. Ions in a sample zone that have a higher conductiv-
†
Siphoning is the most common way to introduce samples in a CE system made in-house.
In accordance with the Van Deemter equation; The plate height, H=A+B/ν+(CM+CS)ν. In CE the
terms A, multiple path diffusion, and C, resistance to mass transfer between phases, are cancelled.
Hence the term B, longitudinal diffusion, inversely proportional to the linear velocity, ν (or more
appropriate νEOF), is the factor that contributes to zone broadening under ideal CE conditions.
‡
11
ity than that of the ions in the BGE will then experience a higher field
strength when entering the BGE, hence their velocity will increase
(see Figure 1.4). When ions present at the front of the sample zone (with
respect to the EOF direction) diffuse into the BGE the increase in their
velocity will cause them to rapidly return to the sample zone (since anions have an opposite migration direction than the EOF flow). Here, their
velocity will decrease again, resulting in a sharp concentration edge.
Detection point
EOF
A
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Detection point
EOF
-
B
-
+
-
- - -
-
-
-
-
-
-
-
-
Figure 1.4 Electrodispersion of anionic analytes. The direction of the EOF is indicated by the arrow. (A) Ions with higher mobility than the surrounding BGE.
(B) Ions with lower mobility than the BGE.
Anions in the trailing side of the sample zone will, of course, also
experience a higher field strength when they diffuse into the BGE,
here the increase in velocity will cause them to migrate further away
from the sample zone, resulting in peak tailing in the electropherogram
(see Figure 1.4A and Figure 1.5).
For anions with lower conductivity than that of the BGE the opposite is true (see Figure 1.4B). Therefore, when ions in the rear side of
the sample zone diffuse into the BGE they will slow down and the
sample zone ions will quickly catch up to form a sharp side. The ions
present in the front zone will also slow down when they diffuse into
the BGE. Consequently, a fronting peak shape will occur in the electropherogram (Figure 1.5).
12
Signal
Chapter 1 Capillary electrophoresis
μe μBGE
μe= μBGE
μe μBGE
Time
Figure 1.5 Detector response of anionic analytes having lower, equal or higher
mobility than the BGE.
Neutral ions are unaffected by these conductivity differences. For cations, the forces will act in opposite directions, and hence the overall effects will be reversed.
1.6.2 Sample stacking
The simplest way to enhance sensitivity by on-capillary pre-concentration
(stacking) is to keep the concentration of the sample significantly lower
than the BGE concentration (as in the lower conductivity case above).
When the voltage is applied in such cases the field strength across the
sample zone will be high, promoting migration of the analyte ions by
enhancing their velocity. When the ions then reach the surrounding BGE
they will be slowed down, thereby forming a sharp band. Other ways to
perform sample stacking can be found in the literature [17-19].
1.7
On-line detection techniques
Detecting CE-separated analytes is challenging because of the narrow
bore of the capillaries. Although the injection volume in CE is in the
nanolitre range, it is not a trace analysis technique since it requires
relatively concentrated samples or pre-concentration methods [20]. UVvisible detection systems are the most commonly used. The main
advantages attributed to these detectors are their convenience and
flexibility. The disadvantage is that not all compounds absorb UV
radiation. The most sensitive available detectors are fluorescence monitors,
but their use generally requires sample derivatisation. Electrochemical
detectors, such as amperometric and conductivity monitors, have also
been used with CE. One problem with electrochemical detectors has been
13
that of isolating the detectors’ electrodes from the high potential required
for separation. For information covering the different types of detection
systems used in CE readers are advised to read [6,7,21,22]. Notably, none
of the CE detectors mentioned provide real structural information or other
means of positive identification. UV-diode array detection (DAD) offers
a spectrum for each separated analyte though the peaks are often broad
and, as mentioned above, limited to UV absorbing compounds. Mass
spectrometry, MS, [23] and now also Fourier transform infrared
spectroscopy, FTIR, [Paper I] can provide complementary information
for these purposes. Both of these relatively recent CE detection techniques
require an interface between the CE system and the MS or FTIR
instrument, which must be able to handle the low flow rates from the CE
capillary, the electric potential applied across the capillary and BGEs with
different additives. The hyphenation between CE and FTIR is described
and discussed in further detail in Chapter 5.
14
Chapter 2
Infrared spectroscopy
2 INFRARED SPECTROSCOPY
Spectroscopy is essentially the measurement of spectra, or distinctive
patterns, arising from interactions between matter and various types of
radiation or forces (here principally infrared, IR, electromagnetic radiation). This chapter is devoted to the nature of IR, and its use in analytical
contexts. The discussion starts with an introduction to the electromagnetic spectrum, continues with an explanation of vibrations and ends with
a quantitative perspective of IR radiation. Standard references include
the books written by Ingle and Crouch [24] and by Chalmers and
Griffiths [25]. The following two chapters focus on Fourier transform infrared spectroscopy (Chapter 3) and quantum cascade lasers (Chapter 4).
2.1
The electromagnetic spectrum
Who would have thought that something as fundamental as light could
have been so difficult to describe, historically? The properties of radiation, or photons, are unique since they behave both as particles,
carrying discrete energy quanta, and as longitudinal, electromagnetic
waves. The energy, E, associated with electromagnetic radiation follows the expression
Equation 2.1
where h is Planck’s constant and ν the frequency. Depending on its appearance or effect on human senses, electromagnetic radiation is classified as light, heat, X-rays etc. The only differences in these forms of radiation lie in their frequencies or wavelengths.
Figure 2.1 shows the electromagnetic spectrum, in which infrared
radiation, the region of interest in this thesis, is surrounded by the
visible light region to the higher energy side and microwaves to the
lower energy side.
15
or
RADIOWAVES MICROWAVES
INFRARED
Wavelength
300 mm
1000 μm
Frequency (Hz)
109
1011
Wavenumber (cm-1) or
Energy (eV)
UV-VISIBLE
GAMMA
100 Å
0.05 Å
1014
1016
1019
100 eV
105 eV
12800 cm-1, 1 eV
10 cm-1
X-RAY
770 nm
Energy
Figure 2.1 Illustration of the electromagnetic spectrum. The infrared region is
associated with molecular bendings and vibrations. The UV-visible and X-ray
regions change the electron distribution (valence- resp. K and L shell electrons),
the microwaves the molecular rotation and the radiowave region the electron and
nuclear spin. The gamma region changes the conformation of the nuclei.
Electromagnetic radiation is characterized by either its frequency, ν, or
wavelength, λ. The frequency is defined as the number of oscillations per
second, and is expressed in s-1, often referred to as Hertz, Hz. The wavelength is typically expressed in metres and is defined as the distance between two successive crests. The wavelength and frequency are related
to each other by the speed of the radiation’s propagation, i.e. the speed of
light, c, according to
Equation 2.2
The infrared frequencies are causally related to the different vibrational
motions in molecules. However, due to its large values (ca. 1013) this parameter is seldom used in the mid-IR region. Instead the wavenumber,
υ~, is employed, expressed as the number of waves per unit length, cm-1.
The wavenumber is related to the wavelength and frequency by,
Equation 2.3
16
Chapter 2
Infrared spectroscopy
Just beyond the red part of the visible light spectrum we find the infrared
radiation. Although we can’t see it we can sense it as heat. Photons in the
infrared region can excite vibrations in molecules, and a vibration is said
to be infrared-active if IR energy absorption causes a vibrational excitation that induces a change in the dipole moment. Hence, nearly all
polyatomic molecules absorb in the IR region, exceptions being diatomic
homonuclear molecules such as O2 and N2 since they do not possess a
dipole moment.† For molecules that are free to move in three spatial directions consisting of n atoms, the possible number of normal vibrational
modes is 3n-6 (3n-5 for a linear molecule).‡ In general, a normal vibration
is defined as a molecular motion in which all atoms oscillate at the same
frequency and pass through their equilibrium positions simultaneously.
Absorption bands may be regarded as arising from stretching or deformation vibrations. These can, in some cases, be considered as symmetric or anti-symmetric motions. Stretching vibrations involve a change
in the bond-length and often require high energy since the bonding force
directly opposes the change. Deformation vibrations involve a change in
the bond angle of the group and can be classified as scissoring, wagging,
rocking or twisting. Ring compounds can also undergo a symmetric
stretching vibration, called breathing. The possible normal vibrations of
a tri-atomic, non-linear molecule are shown in Figure 2.2.
Symmetric stretching
Asymmetric stretching
Bending (scissoring)
Figure 2.2 The possible normal vibrations of an AX2 tri-atomic non-linear molecule.
†
The Raman effect is worth mentioning here; inelastic scattering that requires a molecule that distorts under an electric field, temporarily separating the positive and negative charge, and thus producing an induced electric dipole moment, — = α E, α = polarisability.
‡ Three motions are due to movement of the entire molecule without changes in shape (translational
motions in the X, Y and Z directions of the Cartesian coordinates) and three motions are rotations of
the entire molecule around the X, Y and Z axes.
17
The infrared region is often further subdivided into far-, mid- and near-IR.
In the mid-IR region (2.5 to 50 —m, 4000 to 200 cm-1) the fundamental
vibrations of organic compounds occur. Bands in the near-IR region
(0.78 to 2.5 —m, 12800 to 4000 cm-1) are due to overtones or combinations
of two or more fundamental vibrations. The near-IR peaks are usually
broad, and overlapping spectra are often observed. In the far-IR region
(50-1000 —m, 200-10 cm-1) information about the fundamental vibrations
of many organo-metallic and inorganic compounds can be obtained, due
to the heavy atoms and weak bonds in these compounds. In addition,
lattice vibrations of crystals are localised here. The part of the spectrum of interest in this thesis (and thus the region focused on) is the
mid-IR region.
2.2
Identification
The mid IR-spectra of simple molecules are correspondingly simple, but
the spectra rapidly increase in complexity as the size and complexity of
the analytes increase, due to the many possible vibrations and resulting
overtones, combination bands and difference bands. However, the spectra of certain functional groups seem to be almost unaffected by the rest
of the molecule, these are called group frequencies. They are important
for identifying compounds, and most are found in the region from
3600 to 1250 cm-1, which is also known as the group frequency region.
Although group frequencies occur within a narrow and well-defined frequency range, interference from neighbouring electronegative groups or
atoms (in particular H) may shift the characteristic band (i.e. vibrational
coupling). Similar shifts may also result from the spatial geometry of the
molecule. These shifts are helpful for identification. In the fingerprint
region, 1250–700 cm-1, vibrational frequencies affect the entire molecule
and (like a fingerprint of a person) the IR spectrum is unique and characteristic for a substance and can thus be used to identify it. Both the positions and intensity of absorption bands are extremely specific to that substance. The book by Socrates [26] is recommended for structural identification of the characteristic group frequencies. For identification in the
fingerprint region, use of a pure reference spectrum taken under identical conditions to the sample is recommended.
18
Chapter 2
2.3
Infrared spectroscopy
Quantitative measurements
Quantitative measurements are based on Beer’s approximation, which
states that the absorbance, A, of a given substance at a specific wavelength is proportional to the molar concentration, c, of the substance, the
optical path length, b, of the sample compartment and the molar absorption coefficient, ε, according to
Equation 2.4
The output from an IR-spectrometer is a sample single beam spectrum, I.
This spectrum is divided by a single beam background spectrum, I0, to
achieve a transmission spectrum, T.† However, since it is the absorbance
that is linearly related to the concentration, the transmission needs to be
converted to absorption, using the expression
Equation 2.5
In some situations Beer’s approximation is no longer linear, for instance
when the sample concentration is too high, chemical interactions within
the sample cause the concentration to vary or the system exceeds the
linear range of the instruments used.
†
i.e. the light that was not absorbed by the molecules is detected.
19
20
Chapter 3
Fourier transform infrared spectroscopy
3 FOURIER TRANSFORM INFRARED SPECTROSCOPY
The purpose of this chapter is to outline the theory underlying Fourier
transform infrared spectroscopy, FTIR, and illustrate the advantages that
this technique offers compared to dispersive IR spectroscopy. Further, a
brief description of the different instrumental parts of an interferometer
and spectrometer are presented. For a more thorough presentation of the
theory and concepts of FTIR, readers are referred to the books by Griffiths
and De Haseth [27] and Chalmers and Griffiths [25]. However, this chapter begins with a brief history of the development of the FTIR technique.
3.1
Development of FTIR
If it hadn’t been for the progress in interferential infrared techniques that
eventually led to FTIR instruments largely replacing traditional dispersive IR instruments, infrared spectroscopy would probably have negligible use in the array of techniques available for analytical chemists, and
would surely have been surpassed by NMR and MS. The development
of FTIR started in 1880 with the invention of the Michelson interferometer by Albert Abraham Michelson. Michelson used his interferometer
for various applications and contributed to our knowledge about light.†
However, the FTIR technique was not readily accepted, since it is not an
intuitive technique and it wasn’t until the development of the fast Fourier transform (FFT) algorithm by Cooley and Tukey, together with the
advances in calculating capacities in minicomputers made in the 1950s,
that modern FTIR systems began to be developed. The first commercial
instrument was produced in the early 1960s and in the 1980s chemists
adopted the technique. Today, FTIR is a mature technique that is frequently used for fast, robust identification purposes, and for quantification. Applications in the latter area are still growing [29].
3.2
The Michelson interferometer
A schematic diagram of a Michelson interferometer and its essential parts
is depicted in Figure 3.1. It consists of a broadband light source, a detec†
One of the most famous negative results was obtained by Michelson and his interferometer when
the question whether or not light needed a medium (i.e. the existence of “aether”) in which to travel
was answered [28].
21
tor, a beam splitter and two mirrors, one of which is movable and the
other fixed. In addition, a HeNe laser is needed.
g
Fixed mirror
Light source
BMS
Movable mirror
Detector
Figure 3.1 A schematic diagram of a Michelson interferometer. The radiation coming from the light source is divided into two parts by the beam splitter (BMS).
After recombination again at the beam splitter the light exits to the detector.
When the incoming radiation hits the beam splitter it is divided into two
parts. Approximately 50% of the light is transmitted through the beam
splitter, and continues to the movable mirror. The other half of the light
is reflected at the beam splitter and travels to the fixed mirror. After reflection at the two mirrors the two beams are recombined at the beam
splitter and half of the light is transmitted back to the source, while the
other half continues to the sample cell and the detector. A HeNe laser
light also passes through the interferometer and is detected by a photodiode detector. The laser has multiple purposes, it is used for precise
control of the mirror displacement (constant mirror velocity), to trigger
the sampling of interferograms and to ensure that interferograms from
consecutive scans are added coherently [25]. The fact that the HeNe laser
is used as an internal wavelength calibration standard gives the Fourier
transform spectrometer its inherent wavelength stability.†
To simplify, let us consider a situation in which the broadband light
source has been changed to a monochromatic one and assume a perfect
beam splitter. The difference in distance that the light has to travel is
†Also
known as the Connes advantage.
22
Chapter 3
Fourier transform infrared spectroscopy
referred to as the optical retardation. Now, assuming that there is no displacement of the movable mirror, the two beams will be in phase as they
recombine at the beam splitter and constructive interference will occur. If
the movable mirror has been shifted ¼λ the optical retardation will be
½λ at the beam splitter and total destructive interference thus occurs.‡
If the mirror is continuously moved at a constant rate, the intensity at the
detector will alternate, as illustrated in Figure 3.2A. Maxima will occur
when the optical retardation is an integer number of the wavelength and
minima whenever the optical retardation is a half-integer number of the
wavelength. If we alter the wavelength of the monochromatic light, to
say 3λ, then the response at the detector will be different, see Figure 3.2B.
C
λ+3λ
B
3λ
A
λ
Figure 3.2 Modulation of (A) λ, (B) 3λ and (C) their superposition, λ+3λ.
‡
The optical retardation is two times the mirror displacement since the light travels there and back.
23
Using these two different wavelengths simultaneously in the FTIR,
the detector response, Figure 3.2C, will be the superposition of these
responses.
The constructive and destructive interference that occurs in the interferometer affects the light intensity of a given wavelength as if a shutter
were opening and closing in the light beam, alternatively blocking the
beam and letting light through. Therefore, a beam of light that passes
through an interferometer is said to be modulated. The number of times
per second the light switches from light to dark is referred to as the Fourier frequency and can be calculated by use of the moving mirror velocity
and the wavenumber.
Let us return to the broadband light source. Each wavelength could
be considered as a cosine oscillation (or modulation) and the resulting
output from the interferometer will consist of the superposition of all
these wavelengths, i.e. an interferogram (intensity vs. mirror position) is
created (see Figure 3.3A). The resulting interferogram is then transformed
by use of the FFT algorithm into the original frequency spectrum (intensity vs. wavelength) of the radiation. For the case with only two wavelengths (λ and 3λ) the resulting spectrum will show only two peaks, one
at λ and the other at 3λ. A frequency spectrum from a broadband light
source is shown in Figure 3.3B.
3.3
Advantages of FTIR
FTIR instruments have several advantages over traditional dispersive IR
instruments. Dispersive instruments use a monochromator and a grating
to produce a spectrum in which each spectral point is measured sequentially. An FTIR, on the other hand, measures all spectral points simultaneously. Suppose that the measurement time and resolution are the same
for the two instruments, then the FTIR instrument will give increases in
signal-to-noise, S/N, ratios that are proportional to the square root of M,
where M is the number of resolution elements. The improvement in S/N
is due to the fact that the FTIR uses all of the available time for all spectral
points. This is referred to as the multiplex (or Fellgett) advantage. In addition, the required measurement time is much shorter in an FTIR com-
24
Chapter 3
Fourier transform infrared spectroscopy
A
B
Figure 3.3 (A) Interferogram, (B) transmission spectrum.
25
pared to a dispersive instrument, hence more spectra can be co-added in
the time a dispersive instrument measures just one spectrum. Another
advantage is the throughput (or Jacquinot) advantage arising from the
fact that the resolution in FTIR results from mirror-displacement, thereby
eliminating the need for input and exit slits as used in dispersive instruments and thus allowing a circular beam shape.
The combination of these advantages results in FTIR instruments with
high improvements in S/N in comparison to dispersive instruments in
the mid-IR region.
3.4
Limitations of the Michelson interferometer
Moving the mirror without tilting it is a problem in FTIR, especially when
using fast scan speeds and/or high resolution. However, interferometers
have developed greatly since the invention of the Michelson interferometer, and nowadays several types are available. The double pendulum,
or wishbone, interferometer developed by Kayser-Threde GmHb
(Munich, Germany) [30] and currently used by Bomem (Québec, Canada),
optically compensates for the tilt, by exchanging the two plane mirrors
used in a normal Michelson interferometer for two cube corner mirrors
Detector
BMS
Cube coner mirror
Light source
Pivot point
Cube corner mirror
Rotating arm
Figure 3.4 Simple representation of a double pendulum interferometer with two
cube corner mirrors rotating around a pivot point.
26
Chapter 3
Fourier transform infrared spectroscopy
attached to the same arm, rotating around a pivot point (see Figure 3.4).
The interferometer optically compensates for shear of the mirror assembly in all three axes and rotation about the axis parallel to the line connecting the two mirrors. These motions will change neither the optical
retardation nor the alignment of the interferometer. Rotation in an axis
parallel to the line connecting the pivot point and the beam splitter will,
however, affect the alignment of the interferometer.
The interferometer used in the studies described in Papers I-III was of
the double pendulum type, but an extra folding mirror was included on
each arm of the interferometer (see Figure 3.5) [31]. This has the advantage of making the arm holding the cube corner mirrors smaller and
lighter, thus enabling higher scan speeds. Further, these folding mirrors
facilitate the alignment of the interferometer and shift the centre of mass
of the mirror assembly to coincide with the pivot point.
Detector
BMS
Light source
Plane mirror
Plane mirror
Cube corner
mirror
Cube corner mirror
Rotating arm
Figure 3.5 The interferometer used in studies described in Papers I-III: a double
pendulum type, but with two extra plane mirror inserted into the optical path.
3.5
Apodization
The spectral resolution depends on the maximum retardation of the movable mirror in the interferometer. In order to record all wavelengths from
a broadband light source the mirror displacement must be infinite. Since
27
this is impossible, the interferogram must be truncated. Truncation can
lead to undesirable sidelobes around large absorption peaks in the spectrum. Apodization (“foot” removal) is a mathematical compromise that
compensates for incomplete data. Apodization is used to remove these
undesirable sidelobes and can be considered as a smoothing of the spectrum by gradually adjusting the endpoints in the interferogram to zero.
As in all smoothing techniques, the resolution is affected, hence there is a
trade-off between smoothing and resolution. Consequently, several different apodization algorithms are now available.
3.6
Detectors
Detection is the final step performed by the FTIR instrument, and before
the infrared intensity can be Fourier transformed into a spectrum the signal must be converted to an electric signal. Infrared detectors can be divided into two types: thermal and quantum [32].
3.6.1 The DTGS detector
The most frequently used thermal detector is the DTGS (deuterated triglycine sulfate) detector. When photons hit the DTGS element they change
its temperature, which alters its surface polarisation and the resulting
capacitance change can be measured as a voltage. The DTGS detector is
robust and simple, it has a wide dynamic range and can be operated at
room temperature. However, it is relatively insensitive and has a slow
response time. Thus, it is unsuitable for on-line hyphenations to a fast
separation system.
3.6.2 The MCT detector
When detecting analytes in a moving stream (as in CE) the scan speed of
the interferometer should be high and the detector’s response time short.
The MCT (mercury cadmium telluride) detector has an element made of
a semiconducting n-type material (see Chapter 4.1) which exhibits a wavelength cut-off proportional to the alloy composition. Photons in the midinfrared region with energy greater than the semiconductor cut-off can
excite electrons from the valence band into the conducting band thereby
28
Chapter 3
Fourier transform infrared spectroscopy
increasing the conductivity of the material. Since the energy required for
excitation is low the detector surroundings can influence the performance.
For this reason, the MCT detector must be operated at liquid nitrogen
temperature.
Specific detectivity, D*
The composition of the MCT mixture can be varied, and hence the
band gap (or cut-off). The specific detectivity is a measure of the sensitivity of a given detector and Figure 3.6 illustrates how the specific detectivity
(D*) varies as a function of wavelength both for a DTGS and two MCT
detectors having different alloy composition. As can be seen only the
MCT detectors are dependent on the wavelength.
“narrow band” MCT
“broad band” MCT
DTGS
Wavelength
Figure 3.6 Specific detectivity as a function of wavelength for MCT (solid lines)
and DTGS (dashed line) detectors.
MCT detectors intended for hyphenation with a separation system should
have a small detector element since the cross-section area of the transmitted radiation that is focused onto the detector element is usually smaller
than 1 mm2 [27]. If the detector element is unfilled, the unexposed areas
generate noise which degrades the interferometric signal.
3.7
Mid-IR light sources
The radiation source in an FTIR instrument is often a rod of silicon carbide,
called a globar. It is a blackbody radiation source powered by electricity,
which heats the rod and causes it to emit radiation at temperatures
exceeding 1000 °C. An ideal blackbody absorbs and emits light at the
29
same frequency. Increasing the temperature of the globar will result in higher
spectral intensity. However, a shift will also occur towards higher energy
emission (to the blue) in accordance with Planck’s radiation law [24]. The
intensity of the radiation from this light source is one of the limiting factors
when measuring analytes separated in an aqueous solvent due to the
strong absorption of water (the subject of Chapter 5.1). This is why the
recent introduction of quantum cascade lasers as powerful mid-infrared
light sources is interesting. The next chapter will present the basic theory
of these lasers and their applications.
30
Chapter 4 Quantum cascade lasers
4 QUANTUM CASCADE LASERS
There has been tremendous progress in the development of high performance mid-infrared light sources since the first presentation of a working quantum cascade (QC) laser by Faist et al in 1994 [33]. This chapter
provides an introduction to basic semiconducting theory, as well as describing the fundamental principles of a QC laser model and its advantages over normal semiconducting lasers.
4.1
Basic semiconductor theory
A semiconductor is a solid crystal in which each atom bonds to neighbouring atoms, and interactions between them cause the energy level to
split into two quantized bands. The valence band is occupied by the
valence (outermost) electrons and the conducting band by electrons that
are free to move in the crystal. The distance between these two bands is
called the band gap. In a conductor (i.e. a metal, Figure 4.1A), the valence
band is fully populated and the conducting band is always partially occupied, allowing electrons to move easily in this type of material. In contrast, in insulators (such as glass or CaF2, Figure 4.1B) the valence band
is fully populated, but the conducting band is empty and the band
Conducting
band
- - - - - - - - - Band gap
Valence
band
Band gap
Band gap
- - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - -
A
B
C
Figure 4.1 Electron distribution in (A) a conductor, (B) an insulator and (C) a
semiconductor.
31
gap between these two states is large, hence the flow of current is limited. A semiconductor (Figure 4.1C) resembles an insulator, but the band
gap is smaller, allowing electrons to jump to the conducting band if energy is added, leaving a hole in the valence band in the process.
A semiconducting crystal can be modified by doping it with impurities and thus creating a crystal with excess electrons (n-type) or excess holes (p-type). When n-type and p-type semiconductors are
brought into contact a pn-junction is formed by the recombination of
electrons and holes.
4.1.1 Semiconducting lasers
The key requirement for laser action is to create a population inversion such that coherent light is generated when emission is stimulated. Since a population inversion is contrary to the normal behaviour
of electrons one must use some sort of pumping mechanism to accomplish this. A number of pumping techniques have been developed, including electrical, thermal and chemical approaches and the
use of intense flashes of light [34,35].
In a semiconducting laser the population inversion occurs between
the conducting electrons and the valence band holes of a degenerately
doped pn-junction (interband recombination) and the feedback is generated by an optical cavity. The frequency of the emitted light is directly
proportional to the band gap energy. By changing the composition of the
semiconductor material, the band gap energy (and hence the laser emission frequency) is altered [35,36].
4.2
Fundamentals of quantum cascade lasers
Quantum cascade (QC) lasers are a new type of semiconducting lasers in
which the optical transitions take place within the conducting band
(intersubband). Consequently, carriers of just one type, typically electrons,
are responsible for the laser action (unipolar lasers). As early as 1971 it
was predicted that light could be amplified by intersubband transitions [37]. The reason why it took a further 23 years until Faist et al [33]
were able to realise this prediction was mainly due to the ultra-fast car-
32
Chapter 4 Quantum cascade lasers
rier relaxations that occur within a band of a semiconductor (of the order
of a picosecond) thereby limiting the success of attempts to achieve population inversions. The population inversion problem was solved by careful band-structure engineering at monomolecular resolution, thereby
forming a heterostructure of alternating “well” and “barrier” layers. Such
artificial semiconducting crystals are grown with monomolecular layer
resolution by molecular beam epitaxy (MBE) techniques [38]. As each
electron remains in the conduction band after emitting a photon it can be
recycled, emitting another photon as it travels down the quantum well
structure. This cascading property is another fundamental difference from
normal lasers and the reason why QC lasers can deliver such high output
powers. Another fundamental difference from conventional lasers is that
they can be programmed to emit light in the entire mid-IR region by
merely adjusting the thickness of the materials in the active region. The
composition of this material remains the same (e.g. AlInAs/GaInAs/InP
or GaAs/AlGaAs) [39,40]. A simple schematic view of the general structure of a QC laser is presented in Figure 4.2. It is composed of an active
and an injector-relaxation region. These two regions make up one stage
and a QC laser typically consists of 30 (but sometimes up to 100) stages.
3
2
1
Active region
Relaxation and injector region
Figure 4.2 General structure of the QC laser. Each stage involves an active region
followed by a relaxation-injector region. The population inversion takes place between levels 3 and 2 in the active region.
33
The active region is where the population inversion and gain takes place.
The active region can have various designs, but the basic one resembles
the three-level laser type, in which the laser action occurs between levels
3 and 2. The injector-relaxation region performs several tasks, the most
important of which is to build a minigap and a miniband. The minigap is
needed to prevent electrons from escaping from level 3 into the continuum.
The miniband helps to extract electrons from the lower laser subband
(level 1) in the active region and to efficiently inject them via a tunnel
barrier to an active downstream region thereby creating population inversion.
For a more thorough explanation of QC laser theory readers are
recommended to read the articles by Cho et al [39,41-43], Hofstetter and
Faist [44] and Sirtori and Nagle [45]. In addition, the book edited by Liu
and Capasso [46] gives a comprehensive review of various topics related
to intersubband transitions in quantum wells.
4.3
Applications of QC lasers
Since the first report describing QC lasers was published their performance has been rapidly improved, prompted mainly by the need for
mid-IR lasers sources in trace gas spectroscopy [44]. Their operational
range now spans the mid-IR and into the far-IR region, and QC lasers
based on a variety of semiconducting materials are available [40,47], some
can be operated continuously at room temperature [48,49] and distributed feedback techniques have led to the development of single-mode
tuneable QC lasers [50]. The heterostructure nature of the QC lasers allows construction of two- [51] and even multi-wavelength laser operation [43,52]. Within a decade of their invention QC lasers have now become commercially available [53].
34
Chapter 5
CE-FTIR hyphenations
In this part of the thesis the hyphenations between CE and FTIR
(Chapter 5 and Papers I-III) and the QC laser hyphenations (Chapter 6 and Papers IV-V) are discussed, together with references to
similar, relevant hyphenations. The section ends with my personal reflections on optical set-ups (Chapter 7) and future perspectives (Chapter 8).
5 CE-FTIR HYPHENATION
Why is the idea to hyphenate CE and FTIR so appealing? And why do
this on-line? Have hyphenations between CE and FTIR been performed
in some other way, and why is water such a challenging medium to work
with in the mid-IR? These, and further, questions will be addressed here.
5.1
Why hyphenate?
From chapter 2.2 we learned that mid-IR radiation provides group frequency and fingerprint identification for almost all organic compounds.
However, while such near-universal signals can be very useful for monitoring simple molecules in simple systems, they can be increasingly disadvantageous with larger numbers of analytes and/or more complex
sample matrices. This is because the spectrum will be crowded with peaks
or the solvent matrix will absorb all of the light. One way to handle the
problems associated with multiple analytes in a sample is by adding a
separation system prior to detection. Several attempts have been made
to hyphenate FTIR with a separation system, and a thorough discussion of all these attempts is beyond the scope of this thesis. Instead,
attention will be focused on reversed phase (RP) HPLC-FTIR [54-57]
and the hyphenation of CE to FTIR [58], since aqueous solvents are
usually used in both of these separation techniques. However, an explanation of why water is such a challenging matrix for mid-IR analyses needs to be provided.
35
Intensity [arbitrary units]
7
6
5
4
3
2
1
0
3700
3200
2700
2200
1700
1200
700
Wavenumbers [cm-1]
Figure 5.1 Water transmission spectrum obtained using a 25 —m CaF2 flow cell.
A water transmission spectrum in the mid-IR obtained using a 25 —m
transmission flow cell is illustrated in Figure 5.1. As can be seen, there
are several regions where the transmission is zero and hence no light is
transmitted through the flow cell. These regions are primarily located
around 3700-2900 cm-1 (symmetric and anti-symmetric O-H stretching
vibration), around 1640 cm-1 (bending O-H vibration) and around 950 cm-1
(libration modes) [26].
On the other hand, there are regions, referred to as the water windows, where the transmission is high and can therefore be employed
for identification purposes. These windows are located in the regions
around 1600-950 cm -1 and 2900-1700 cm-1, i.e. in both the fingerprint
and group frequency region (Chapter 2.2). To increase the S/N ratio
and the optical throughput in the spectral region of interest, optical
filters can be used that cut-off frequency regions that do not contain
relevant information.
36
Chapter 5
5.2
CE-FTIR hyphenations
On-line and off-line hyphenation strategies
Two basic strategies for hyphenating FTIR and a separation technique
can be distinguished: one based on solvent elimination and the other
on flow cells. Both of these approaches have specific advantages and
limitations.
5.2.1 Solvent elimination
The obvious reason for eliminating the solvent (here water) is that it absorbs intensely in the IR region, as illustrated in Figure 5.1. However, as
water is a non-volatile solvent, ensuring its complete and reproducible
evaporation is not straightforward, and quite sophisticated elimination
strategies are required. The elimination interface must completely evaporate the eluent coming from the column while maintaining the chromatographic resolution. Important factors in this context include the flow rate
(which must be neither too high nor too low), solvent composition, the
nature of the sample (which must be less volatile than the solvent) and
the IR substrate (which must reusable and easily cleansed). Another vital
requirement for maximizing the sensitivity is to adapt the size of the
evaporated spots to the available FTIR probe area [54]. Furthermore, the
quality of the resulting IR spectra will be affected by the morphology of
the evaporated spots. Some analytes will be deposited as crystals, others
as amorphous layers, smooth films, or irregular clusters [55]. Particle
beam interfacing [59,60], ultrasonic nebulisation [61], concentric flow
nebulisation [62,63] and, recently, flow-through picolitre dispensation [64]
are a few examples of the elimination strategies that have been coupled
to RP-HPLC. However, the abovementioned elimination methods cannot easily handle non-volatile buffers (or have not yet been used with
buffers) due to the strong co-deposition of buffer salts. In order to circumvent this problem, Somsen and co-workers inserted a liquid-liquid
extraction module between the LC column and the IR substrate [65].
When using a solvent elimination approach for hyphenating FTIR and
CE instead of HPLC all of the above mentioned problems remain. However, in addition, the flow rates are dramatically decreased (from ml/min
or µL/min in µHPLC columns to less than 100 nl/min in CE) and the
37
electrical contact necessary for separation must be maintained throughout the course of the analysis. Despite these problems, De Haseth et al have
presented a CZE-FTIR hyphenation approach in which a volatile and rather
IR-transparent BGE was sprayed via a glass [66] or metal [67] nebulizer onto
a CaF2 substrate, which was subsequently placed in an FTIR microscope.
This method could not handle non-volatile BGE as the ones commonly used
in CE (like borate and phosphate buffers). Further, the deposits of analytes
showed residuals also from the non-volatile BGE utilized [67].
5.2.2 Transmission flow cells
The aforementioned examples of FTIR hyphenations are all based on
solvent elimination, a technique that inherently implies off-line hyphenation. If a transmission flow cell approach is utilised instead, an
on-line arrangement is possible, together with real-time detection and
the option to add further detectors after the FTIR device. Disadvantages mentioned regarding the flow cell arrangement include difficulties
in subtracting the eluent’s absorption from the IR signals and in applying
elution gradients. A further problem cited is the need to use short optical
path lengths, ranging from a few to maximum 50 —m, depending on the
solvents employed and spectral regions of interest. Such short optical
path lengths inevitably reduce the sensitivity of the analysis in accordance with Beer’s approximation (Equation 2.4). However, when hyphenating FTIR and CE, the short optical path length required can be advantageous since it is fully compatible with the narrow capillaries normally
employed, and it helps to prevent excessive dispersion of the separated
analyte zones [Paper I].
FTIR has been successfully hyphenated to RP-HPLC utilising standard, commercially available, transmission flow cells. Vonach et al [68]
used a transmission flow cell, available from Perkin-Elmer, consisting of
two IR-transparent CaF2 windows and a polymer spacer, yielding a flow
cell with an optical path length of 25 —m and an inner cell volume of
10 —L. With this system sugars in non-alcoholic beverages could be quantified and the presence of minor constituents such as taurin and ethanol
could be confirmed. The HPLC system comprised an ion-exchange column and distilled water served as the mobile phase. The same group
38
Chapter 5
CE-FTIR hyphenations
also determined carbohydrates, alcohols and organic acids in wine [69].
By applying a thin polymer film as a coating on the CaF2 flow cell, an
acidic mobile phase could be employed. De la Guardia and co-workers
used a micro-flow through cell with ZnSe windows and a lead spacer of
~50 —m for determining cholesterol in animal grease [70]. The use of such
a long optical path length was enabled by selecting a relatively IR-transparent mobile phase consisting of chloroform and acetonitrile (45:55). Alternatively, deuterated solvents can allow longer optical paths by avoiding
spectral overlap between the eluent and the analytes. Such solvents are expensive, but their cost may be justified when using microbore columns [71].
For capillary electrophoresis, the inner volume of these commercially
available flow cells is often too large. Furthermore, the connection between the flow cell and the fused silica capillary must be isolated and
tight. In Paper I we described the first successful on-line hyphenation of
capillary zone electrophoresis with FTIR detection utilising a custom-built,
IR-transparent flow cell and a non-volatile, aqueous (phosphate) BGE. In
Paper II it was proved that the presence of non-volatile eluents, such as
micelles (in this case of sodium dodecyl sulphate, SDS) did not cause any
major problems. The fused silica capillaries employed start to absorb IR
radiation at around 3300 cm-1 and at 2500 cm-1 there is no transmission at
all, which is why an IR-transparent flow cell was needed and had to be
developed. The flow cell used in the studies reported in both of these
IR-light
Calcium fluoride disk
UV curing epoxy adhesive
SU8 photoresist layer
Titanium layer
Calcium fluoride disk
Flow
Figure 5.2 Schematic illustration of a dismantled mid-IR flow cell.
39
A
B
Figure 5.3 (A) SEM micrograph showing the capillary with epoxy o-ring as used
in studies described in Papers I and II. (B) Microscopic view of the commercial oring and the recently developed cell holder described in Papers III and V.
papers, and Papers III and V, was constructed of two CaF2 plates forming
a wafer (see Figure 5.2). On one of these discs a thin layer (200 nm) of
titanium was deposit by evaporation, providing an optical aperture.† The
titanium was isolated from the BGE to prevent electrical discharges. The
separation channel was created by applying two streaks of SU-8 epoxybased photo-resist to each disk, producing a 150 —m wide, 15 —m thick
† The flow cell used in paper V omitted this titanium layer, instead Al-foil was glued on top of the cell.
40
Chapter 5
CE-FTIR hyphenations
(60 —m in paper V) and 2 mm long flow channel. The area outside the
flow channel was filled with UV-curing epoxy adhesive. The two disks
were bonded together by cross-linking the SU-8 during a hard-baking
process (200 ºC, 1h), giving the flow cell the required chemical resistance
and mechanical strength. Using this technique, 40 flow cells could be constructed in a single batch.
The choice of CaF2 as flow cell material was based not only on its transparency in the mid-IR region, but also its mechanical strength and its property to withstand the aqueous BGEs commonly used in CE separations. Further, CaF2 acts as an insulator so it will not be influenced by the high voltages applied during CE separations. To achieve a sealed connection between
the flow cell and the capillary, an epoxy adhesive was added to the sharply
cut capillary end while air was passed through the capillary, Figure 5.3A.
The flow cell and the capillary were mounted in a specially constructed cell
holder. The epoxy adhesive was exchanged in paper III and V to a commercially available o-ring and a new cell holder was constructed, with a movable sledge, that allowed applying external pressure, Figure 5.3B.
Paper III presents the first application of the on-line CZE-FTIR technique; the separation of non-UV absorbing compounds, sugars, in natural fruit juices, using a non-volatile sodium carbonate BGE (see Figure 5.4).
Figure 5.4 CZE-FTIR separation of natural sugars in commercially available fruit
juices.
41
5.2.3 Attenuated total reflection flow cells
Another approach is to use a flow cell based on attenuated total reflection (ATR). Here the optical path length is dependent on the number of
internal reflections, allowing the path lengths to be very short. In ATRbased flow cells only a small fraction of the available cell volume is probed
by the IR light. If the optical path length is increased too far the cells
become excessively large. One example of an ATR-cell is the CIRCLE
(cylindrical internal reflectance) cell [72-74], consisting of a cylindrical
ZnSe crystal with cone-shaped ends mounted in a stainless steel cell. The
solution to be analysed bathes the exposed area of the ZnSe crystal. The
IR beam is focused on the entrance cone and reflected several times (typically 10) along the crystal. The resulting light is then collected and guided
to the detector. Louden and co-workers used a micro CIRCLE flow cell
with a 25 —L cell volume for the multiple on-line hyphenations of reversed phase HPLC to UV, MS, FTIR and NMR using deuterated superheated D 2 O as the mobile phase for the on-line analysis and
characterisation of ecdysteroids in plant extracts [75] and model pharmaceuticals [76]. Edelmann et al hyphenated a diamond ATR cell with nine
internal reflections with a HPLC system for the determination of carbohydrates, alcohols and organic acids in red wine and employed post-run
chemometric treatment to resolve overlapping peaks [77]. Another alternative has been presented by Patterson et al [78]. They placed a hemispherical germanium internal reflection element, which only permits one
internal reflection, at the terminus from a capillary HPLC column and
focused an FTIR microscope on this spot achieving injection mass detection limits in the low microgram range. One problem with this set-up
was that the sample could pass the detector point unnoticed, but this
could be solved by further reducing the column diameter. Recently the
capillary HPLC column was replaced with a CE fused silica capillary
(i.d. 25, 50 or 75 —m) and separation of different test analytes, with sodium chloride as BGE, was achieved utilizing a separation program varying between pressure- and voltage-driven flow [79]. The main beneficial
feature of this system is that the IR path length is independent of the
capillary dimensions.
42
Chapter 6
On-line QC laser hyphenations
6 ON-LINE QC LASER HYPHENATIONS
QC lasers have primarily been adapted for use as chemical gas sensors
for detecting and monitoring toxic and dangerous compounds, including various pollutants and exhaust emissions [80-82]. The use of QC lasers in optical free-space communications in mid-infrared frequencies has
also been considered [83]. Their potential utility as powerful light sources
in the condensed phase has only recently been demonstrated. Lendl
et al [84] utilized a Fabry-Pérot (FP) QC laser in a flow analysis system
(FIA) and showed that this arrangement could yield S/N ratios that were
50-fold higher than those provided by a state-of-the-art FTIR instrument.
Phosphate in diet Coke was determined using an optical path length that
could be increased to 100 —m. Edelmann et al [85] used a HPLC system
and a distributed feedback QC laser emitting light at 1067 cm-1 for the
functional group detection of carbohydrates in wine.
In the study reported in Paper IV two FP-QC lasers were used in conjunction with a micro flow system and aqueous samples containing a
nucleoside, xanthosine, or a nucleic base, adenine (see Figure 6.1).
Adenine
Xanthosine
Carbohydrate group
Figure 6.1 Structural formulas of adenine and xanthosine.
One laser emitted light at 1080 cm-1, in the water window and the band
where the carbohydrate group (e.g. C-O stretch) of the nucleoside absorbs, whereas the other laser emitted light at 1650 cm-1, where water has
43
strong IR absorption (e.g. O-H bending, Chapter 5.1) and the
heteroaromatic ring structure of both analytes under investigation is located. With this configuration functional group specific detection could
be manifested. Further, the results showed that by having a strong midIR light source the optical path length at 1650 cm-1 could be extended
from the normal 10-15 —m to approximately 60 —m, and the optical path
length in the water window could be increased to 150 —m; the limit of the
available fibre optic flow cell.
In Paper V the hyphenation between an FP-QC laser and a CZE
separation system is presented for the first time. The IR-transparent
flow cell was similar to the one described in Papers I-III, but without
the titanium layer and with the optical path length increased to 60 —m
instead of 15 —m. Functional group specific detection (carbohydrate,
laser emission = 1080 cm-1) was achieved for the analytes under investigation. The CZE system included a common, non-volatile background
electrolyte (10 mM borate at pH 9.3). Special focus was placed on
minimising the system background noise, resulting mainly from general
electronic problems. Although the results are only preliminary and the
true potential of QC laser as a powerful mid-infrared light source has not
yet been realised, I strongly believe that when the lasers’ performance
has been stabilised and fine-tuned (which is only a matter of time) the
utility and scope of CE-QC laser hyphenation will be greatly enhanced.
44
Chapter 7
Comments on the optical set-ups
7 COMMENTS ON THE OPTICAL SET-UPS
During the course of my PhD studies I have been involved in constructing
several optical set-ups, in both Stockholm, Sweden, and Vienna, Austria.
The purpose of this chapter is to describe these set-ups a little more
thoroughly.
7.1
The CE-FTIR set-up
There are several aspects to consider when constructing a functioning
CE-FTIR instrument. The speed of the CE separation imposes demands
on the scanning velocity of the interferometer. This velocity is, in turn,
governed by the desired number of co-additions of scans per spectrum
and the spectral resolution. The residence time in the flow cell for an
analyte is typically 1-5 s under optimised CE separation conditions, hereby
limiting the time available for data acquisition. Under such conditions
there must be a trade-off between accumulating many scans to improve
the S/N ratio and the spectral resolution between subsequent spectra.
The cube corner interferometer, depicted in Figure 3.5, offers high scanning frequencies. In addition to a fast interferometer one must have a fast
and highly sensitive detector, like the MCT detector described in Chapter 3.6. In order to focus the light coming from the interferometer onto
the developed IR-transparent flow cell a beam condenser was constructed.
Apart from the previously mentioned focusing, this beam condenser offers optical alignment of the flow cell by use of an XYZ-stage with
microscaling and proper focusing of the transmitted light onto the detector element. The beam condenser was placed inside a plexiglas hood with
entrance and exit holes for the capillary. This hood also provided a means
for maintaining a controlled atmosphere. Since the CE separation is carried out throughout the entire capillary these lengths should be kept as
short as possible, making the distance between the inlet vial and the
XYZ-stage critical. Figure 7.1 shows photographs of the on-line CE-FTIR
optical set-ups, which fulfilled these requirements, used in the studies
described in Papers I and II (A) and Paper III (B).
45
A
B
Figure 7.1 Photographs of the CE-FTIR set-ups used in studies reported in (A)
Papers I and II and (B) Paper III.
In a further study of the influence of the scanning velocity, expressed as
the Fourier frequency of the HeNe laser used to determine the optical
path difference, we concluded that increasing the scanning velocity from
80 kHz to 200 kHz only reduced the time required for the acquisition of
50 co-additions, with a spectral resolution of 16 cm-1, from 3.5 s to 2.2 s
[Paper I]. This indicates that the time-consuming step between consecu-
46
Chapter 7
Comments on the optical set-ups
tive scans was the reversal of the moving mirror. Developments towards
ultra-rapid scanning interferometers are therefore interesting in this context. Griffiths and Manning [86] proposed a tilt-compensated interferometer in which the optical path difference can be generated by a rotating mirror. The mirror rotates at a maximum rate of 500 kHz, producing
two interferograms per revolution and, thus, 1000 scans per second.
7.2
QC laser set-ups
The process of constructing a working QC laser set-up involves not only
the production of an optical set-up but also intense work on the signalprocessing procedures. In the two papers concerning QC laser two
different set-ups were utilized. In both cases the laser was housed in a
water-cooled unit with a Peltier element and special driving units
controlled the laser’s performance. In the first case [Paper IV] ZnSe lenses
were used to focus the laser-light onto the fibre optic flow cell and,
subsequently, the transmitted light onto the detector element (see Figure 7.2). The signal from the MCT detector was acquired using a lock-in
amplifier and digitalised using a digital multimeter.
Figure 7.2 Photograph of the FIA-QCL set-up, showing the QC laser housing, the
fibre optic flow cell and the MCT detector.
47
In the second case [Paper V] a home-made software package (Sagittarius
v. 3.0) [87] had been developed enabling, in combination with a computer interface, control of the pulse duration and amplitude of the QC
laser as well as the A/D conversion of the detector signal. In addition,
gold-coated mirrors replaced the ZnSe lenses and a boxcar averager processed the signal from the detector. A photograph of the CZE-QC laser
hyphenation, illustrating the optical set up, the cell holder, laser housing
and the CE system is presented in Figure 7.3. The signal processing units,
the driving units for the laser and the separation system are not included
in this picture.
Figure 7.3 The CZE-QCL optical set-up.
48
Chapter 8 The past and a future perspective
8 THE PAST AND A FUTURE PERSPECTIVE
The work underlying this thesis started as an abstract concept, envisaged
by Professor Bo Karlberg, to hyphenate QC lasers with a CE separation
system. However, since QC lasers at that time had only very recently
become commercially available many problems had to be overcome to
construct a system in which they could work effectively, without creating
too much electrical noise. In the process, the idea of hyphenating FTIR
and CE was conceived. Now, five years later, I can say that I have had the
privilege to work in an exciting field in which few have worked before.
We were, to our knowledge, the first to on-line hyphenate CZE-FTIR
[Paper I], MEKC-FTIR [Paper II] and CZE-QCL [Paper V]. For CE-FTIR
hyphenations I see a bright future, with many potential improvements,
including more elegant flow cells, better connections to the capillary, more
sophisticated signal processing systems and a greater range of
applications, especially where a near-universal detection system with
positive identification capacities is required. CE-QC laser hyphenations
are in their infancy, and many further developments are still needed.
However, I have faith that the results will be enhanced tremendously if
the system can be improved and optimized. Possibly, we may soon be
able to buy QC laser arrays and couple them on-line to a highly efficient
separation system. It is therefore my hope that someone will continue
the work I have outlined.
Malin Kölhed
2005-07-26
49
50
Chapter 9
Acknowledgements
9 ACKNOWLEDGEMENTS - TACK!
Under min tid som doktorand på Stockholms universitet har jag haft turen
och privilegiet att träffa en rad trevliga och duktiga personer som jag nu
vill ta tillfället i akt att tacka ordentligt.
Först och främst så vill jag tacka min handledare och mentor, Bo Karlberg.
Tack vare din vishet och personlighet har jag lärt mig hur livet ska passeras
på trevligast möjliga sätt. Tack för alla tillfällen vi har haft att fira med en
lunch på Skafferiet!
During the course of my PhD studies I also had the privilege to spend
some time at the Vienna Universiy of Technology, Austria, under the
excellent guidance of Bernhard Lendl. Thank you Bernhard for welcoming me to your group, for your great spectroscopy knowledge and for pushing me to do my best!
I also want to acknowledge Peter Hinsmann for all the time we spent
together by the CE-FTIR instrument, for the coffee breaks and for never
giving up- “I feel good and I’m worth it...today”. Mike, thanks for
organising everything when I first arrived to Vienna, unforgettable, and
thanks for encouraging me when the lasers just never worked. And the
rest of the Vienna group, Sepp the big organizer, for caring and for all the
cooking sessions, Andrea, for all our nice conversations, Eva, for taking me
out dancing (sometimes on the tables…), Almudena, my sweet Spanish sister
without whom I don’t know what I would have done. I am blessed to have you
all as friends and can’t think of any of you without a smile in my heart.
Johannes, the guy with the magical hands, thanks for taking time and
building me all the things I needed. What would I have done without
you? Thanks also to Peter S for constructing the CaF2 cells, Markus for
fixing the MCTs, Viktor, for introducing me to the QC laser world and to
Stefan S, for helping me finalizing the CE-QCL paper.
51
Jag vill tacka mina gamla rumskompisar, Stina CB, för att du kan allt
som har med CE att göra och alltid hade tid att lyssna och Magnus Å, för
att du alltid var glad och villig att hjälpa till. Jenny, som jag nu lämnar
över stafettpinnen till, tack för din energi och för alla trevliga samtal. Till
dem som nu tillkommit, Helena H, vi blev vänner för livet en sen natt på
torget i Madrid, och Leila, för att du har varit med om så mycket spännande
och gärna hjälper till. Tack alla ni för att ni gjorde/ gör vårt rum så mysigt!
Vidare så vill jag tacka nuvarande doktorander, Helena I, för ett härligt
humör och för all omtanke. Stina M, för alla tokiga upptåg, skämt och
allvarliga samtal. Johanna, för alla inrednings- och modesamtal, skönt
med en som talar samma språk. Thorvald, för skränande radiopop och
björnkramar. Nana, for you help and interest in my process och Caroline,
för ett härligt humör.
Och till doktorer som dröjer sig kvar; Petter, för att det är så lätt att
göra dig förskräckt och Ralf, för hjälp med kemometri och spektroskopi.
Till andra doktorander som passerat i korridoren; Jonas B, Sindra,
Yvonne, Ove, Anders Ch, Magnus E, Ludde, Gunnar, Magnus A, Kent,
Helena B, Bodil, Cristina och Kakan, tack för att ni gjorde att man ville
stanna kvar!
Till doktorander som jag inte hann lära känna, Davide, Christoffer,
Ragnar, Shahram och Leonard önskar jag en händelserik och rolig
doktorandtid.
Ett speciellt tack vill jag ge till, Jonas R, för att du alltid är lugn då
datorn strular, för all hjälp med layouten av denna avhandling samt för
alla trevliga pratstunder and to John Blackwell, for linguistic correction
of this manuscript and all my papers.
Hillevi, vill jag ge ett fång rosor för all hjälp med att hitta även de mest
omöjliga referenserna.
Jag vill också tacka övrig personal; Anita, för all trevlig undervisningstid vi hade tillsammans, Ann-Marie, för att du fixar alla konstiga fakturor
och reseräkningar och Lena, för att du ser till att vi ser hyfsade ut då vi
52
Chapter 9
Acknowledgements
springer runt i våra labbrockar! Och alla andra, Anders, Conny, Leopold,
Ulla, Björn, Sven, Roger, Håkan, Carlo (special thanks for all the Italian
specialities), Ulrika, Rune (för att du kan fixa allt), Eva och Ramon som
gör/ har gjort Analytisk Kemi till en så bra arbetsplats.
Till hela min släkt och alla mina kompisar som inte bryr sig ett dugg om
Kemi- jag kan inte namnge er alla men ni är alla ovärdeliga! Ett speciellt
tack vill jag dock ge till Mormor och Morfar samt till Pernilla, Matilda,
Jenny och Helena, mina underbara tjejkompisar som jag kan dela allt med,
och till mina vapendragare i gymnasiet, Anna och Caroline, för konkurrens
om betygen!
Ett stort tack för allt stöd och uppmuntran, för böner, kramar och tårar
vill jag ge till mina kära föräldrar, Beyron och Birgitta, och till mina bröder,
Magnus, för att du lovade att hjälpa mig om jag valde natur på gymnasiet
och Mats, för att du finns. Tack också till min svägerska Marie, för att
du är den storasyster jag aldrig hade och för att du också jobbar med
kemi. Mina söta brorsbarn, Maximilian och Filippa, vill jag tacka för
alla bus och härliga skratt.
Jag vill även tacka min andra familj, Inna och Pelle, och mina svägerskor
Ida, Lina och Sofia. Tack för att ni välkomnat mig in i er familj, för alla
härliga grillningar och för att ni alltid ställer upp. Jag älskar er alla.
Och slutligen så vill jag tacka Robban, vad livet vore utan dig vågar
jag inte ens föreställa mig. Tack för att du är min allra bästa vän, min
make och den jag vill spendera resten av mitt liv med. Jag älskar dig.
The studies underlying this work were financed by Foss Analytical,
Denmark, the European union Marie Curié training site on advanced
and applied vibrational spectroscopy (contract HPMT-CT-2000-00059)
and AstraZeneca, Södertälje, Sweden.
53
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