Masaryk University
Faculty of Science
Department of Chemistry
Polymer-based monolithic stationary phases
in the separation of small molecules
Habilitation thesis
Brno 2017 Jiří Urban
© Jiří Urban, Masarykova univerzita, 2017
Bibliografický záznam
Autor: RNDr. Jiří Urban, Ph.D.
Přírodovědecká fakulta, Masarykova univerzita
Ústav chemie
Název práce: Separace malých molekul na monolitických stacionárních fázích
Obor: Analytická chemie
Rok: 2017
Počet stran: 45
Klíčová slova: kapilární kapalinová chromatografie, monolitické stacionární fáze, malé
molekuly, vysoké zesítění
Bibliographic Entry
Author: RNDr. Jiří Urban, Ph.D.
Faculty of Science, Masaryk University
Department of Chemistry
Title: Polymer-based monolithic stationary phases in the separation of small
molecules
Field of Study: Analytical Chemistry
Year: 2017
Number of Pages: 45
Keywords: capillary liquid chromatography, monolithic stationary phases, small
molecules, hypercrosslinking
Ivě, Kateřině a Barboře
„Challenges are what make life interesting;
overcoming them is what makes life meaningful.“
Joshua J. Marine
First and foremost, I would like to express my gratitude to my wife Iva. Without your
understanding and helpfulness this work would have never become a reality. I really appreciate
your supportive attitude, I thank you so very much!
I would also like to thank Prof. Jan Preisler and Prof. Viktor Kanický for the chance they gave
me and the confidence they showed in the realization of this work.
I was lucky to have a chance to work with many excellent scientists. Above all I would like
to name Prof. Pavel Jandera, Prof. Peter Schoenmakers, and Prof. Frantisek Svec. Each of them
taught me something, and all together, they taught me a lot.
I am grateful to my closest associates for their contribution to the papers discussed in this
work. I would like to thank Veronika Škeříková, Simona Janků, Martina Komendová, and Radovan
Metelka.
I would also like to thank the Dean of the Faculty of Science Assoc. Prof. Jaromír Leichmann
and the Czech Science Foundation for their financial support.
Jiří Urban
Brno, September 11, 2017
7
Abstract
This habilitation thesis deals with a preparation, characterization, and application of polymerbased
monolithic stationary phases with a special focus on the separation of small molecules
in capillary liquid chromatography. Work is presented as a commented collection of selected
peer-reviewed scientific papers. First, pore formation and characterization is described. Then,
protocols allowing preparation of monolithic capillary columns suitable for a separation of small
molecules are mentioned, including effect of functional monomer and post-polymerization
hypercrosslinking modification. Final part of the thesis focuses on the development of monolithic
capillary column integrating sample preparation, separation, and electrochemical detection
of polar neurotransmitters in one single unit. Conclusions and future aspects are mentioned
in the last part of the thesis.
Abstrakt
Tato habilitační práce je prezentována jako komentovaný soubor vědeckých publikací a zabývá
se přípravou a charakterizací polymerních monolitických stacionárních fází vhodných
pro separace malých molekul. První část komentáře se věnuje kontrole tvorby pórů
v monolitickém materiálu a možnostmi jejich charakterizace. Dále se práce věnuje popisu
retenčních vlastností připravených kolon, a to zejména pomocí vhodného výběru funkčního
monomeru a také využitím vysokého zesítění povrchu stacionární fáze. Poslední část komentáře
se zabývá přípravou monolitické kapilární kolony, která by integrovala přípravu vzorku nervových
přenašečů, jejich vlastní separaci a následnou elektrochemickou detekci do jednoho celku.
Možnosti budoucího vývoje monolitických stacionárních fází jsou shrnuty na konci komentáře.
8
Abbreviations
2D LC – Two-Dimensional Liquid Chromatography
HILIC – Hydrophilic Interaction Liquid Chromatography
HPLC – High Performance Liquid Chromatography
NMR – Nuclear Magnetic Resonance
RP – Reversed-Phase Retention
RSD – Relative Standard Deviation
SEC – Size-Exclusion Chromatography
9
Content
Abstract....................................................................................................................................7
Abstrakt ...................................................................................................................................7
Abbreviations...........................................................................................................................8
1 Introduction ..................................................................................................................10
1.1 Organization of thesis......................................................................................................10
1.2 Aim of the work ...............................................................................................................10
1.3 Author contribution.........................................................................................................12
1.4 Funded projects...............................................................................................................15
2 Monolithic stationary phases.........................................................................................16
2.1 Internal structure.............................................................................................................16
2.2 Preparation of polymer monoliths..................................................................................17
2.4 Pore formation ................................................................................................................19
2.5 Characterization of porous properties ............................................................................21
2.3 Repeatability of columns preparation.............................................................................24
3 Separation of small molecules .......................................................................................24
3.1 Effect of functional monomer on the retention of small molecules...............................25
3.2 Hypercrosslinking surface modification ..........................................................................29
3.2.1 Hypercrosslinked monolithic stationary phases ......................................................29
3.2.2 Surface modification of hypercrosslinked monoliths...............................................32
3.2.3 Fast separations on hypercrosslinked stationary phases.........................................33
4 Towards multifunctional monolithic capillary column ....................................................36
4.1 Sample extraction and focusing ......................................................................................36
4.2 Integrated electrochemical detector...............................................................................38
5 Conclusions & Future aspects.........................................................................................39
7 References.....................................................................................................................42
Appendix..........................................................................................................................46
10
1 Introduction
When Mikhail Semenovich Tswett described a new analytical method for the first time at the
beginning of 20th century, he used grinded calcium carbonate as a sorbent to separate plant
pigments1. The method has been named chromatography and irregularly shaped stationary
phases evolved rapidly in fine spherical particles within a well-defined diameter, porosity, specific
area, and surface chemistry. Nowadays, high performance liquid chromatography (HPLC) utilizes
cylindrical columns packed with these particles and belongs to one of the most powerful
analytical techniques allowing fast and efficient separations of complex mixtures.
For several decades, spherical particles served as stationary phases with steady optimization
of all possible physico-chemical properties but their shape. In 1990s, a new type of stationary
phases emerged that consisted of one piece of porous material that fills the whole space defined
by a confinement of column. These materials can be prepared from both inorganic and organic
precursors and are called monolithic stationary phases. Introduction of monolithic stationary
phases in column liquid chromatography is the major change in column technology since Tswett
invention2.
1.1 Organization of thesis
The thesis is presented as a commented collection of selected peer-reviewed scientific papers
published between years 2006 and 2017 that are attached in the appendix. To facilitate
orientation within the other cited works these papers are listed as the first seventeen references
and are marked bold thorough the text. Since all discussed manuscripts are attached in the
appendix, in following text I am discussing only main achievements and general conclusions.
1.2 Aim of the work
The aim of this thesis is to present the results of my research from a little bit over the last
decade concerning preparation, characterization, and application of polymer-based monolithic
1
M. S. Tsvett, Berichte der deutschen botanischen gesellschaft 24 (1906) 316 – 323.
2
G. Guiochon in J. Chromatogr. A 975 (2002) 275 – 284.
11
stationary phases with a special focus on their ability to separate small molecules in a capillary
liquid chromatography.
At first, a general introduction is presented to compare the main structural differences
in between conventional chromatographic columns packed with spherical particles and new
types of monolithic stationary phases. This part is concluded with a description of monolithic
stationary phase’s preparation with a special attention to polymer-based organic monoliths.
Pore size distribution of prepared monolithic stationary phases controls the hydrodynamic
properties of prepared capillary columns such as porosity and permeability. Therefore,
part of this chapter is also devoted to a detailed studies focused on the formation of both large
flow-through [1] and small mesopores [2] and equally important characterization of porous
properties [3] and accessibility of the mesopores for small and large molecules [4].
Separation of small molecules on monolithic capillary columns is a hallmark of a presented
thesis. At first, various protocols utilized for the preparation of polymer-based monolithic
stationary phases for an isocratic separation of small molecules are reviewed [5].
Then, composition of the polymerization mixture used for the preparation of stationary phases
is discussed. Change of the functional monomer is the easiest way how to control surface
chemistry of prepared stationary phases. I have studied an effect of functional monomer polarity
on the retention properties of monolithic columns in both reversed-phase [6] and hydrophilic
interaction [7] capillary liquid chromatography. I have also described a retention of small
molecules on the columns prepared with an addition of thermally responsive monomer in the
polymerization mixture at various composition of the mobile phase and working temperatures
[8].
During my post-doctoral research stay at The University of California in Berkeley I have
introduced hypercrosslinking modification to the polymer-based monoliths and used them
for the first time for the separation of small molecules [9]. In the following years, I have focused
on the optimization of the hypercrosslinking modification reaction to further improve separation
properties of hypercrosslinked stationary phases [10,11].
12
Since hypercrosslinked polymers are usually prepared from reactive 4-vinylbenzyl chloride
monomer, their surface polarity can be further alternate by a post-polymerization modification.
I have used thermally-initiated grafting [12] and nucleophilic substitution [13] to prepare
hypercrosslinked monolithic capillary columns applicable in the separation of small polar
compounds in hydrophilic interaction liquid chromatography (HILIC).
To further demonstrate applicability of hypercrosslinked stationary phases in fast separations
of small molecules I have hyphenated them for the first time with a remote nuclear magnetic
resonance (NMR) detection [14]. Last, but not least, hypercrosslinking modification was also used
to prepare monolithic capillary columns with a longitudinal gradient of porosity that were
successfully used in a two-dimensional liquid chromatography of polymers [15].
During the last couple of years, I moved my research attention towards the preparation
of monolithic capillary column applicable in the analysis of neurotransmitters, in particularly
dopamine metabolites. Dopamine belongs to one of the most important neurotransmitters
and undesired changes in its metabolism result in serious illnesses such as depression,
schizophrenia, Parkinson disease, and tumors.
I have developed an online solid-phase extraction with liquid chromatography method based
solely on polymer monoliths and used it to both determination of dopamine in urine
and continuous monitoring of dopamine in a flowing system [16]. Very recently, I have introduced
concept of an integrated monolithic capillary column that combines chromatographic separation
and electrochemical detection in one capillary device [17]. When combined with (also integrated)
sample preparation, such column can be used for unsupervised determination
of neurotransmitters in various biological samples.
1.3 Author contribution
So far, I have published 33 papers in impacted peer-reviewed journals. Seventeen of them
are selected to be part of this thesis, here I am the first author of twelve of them – including one
equal contribution of the first authors [1 – 7, 9 – 11, 14, 15], and the corresponding author of nine
of them [4, 5, 8, 11 – 13, 15 – 17].
13
The following tables summarizes my contribution to the selected works with special attention
to amount of performed experiments, supervision of students, definition of research direction,
and my contribution to manuscript preparation.
1. J. Urban, D. Moravcová, P. Jandera, A model of flow-through pore formation in methacrylate
ester-based monolithic columns, J. Sep. Sci. 29 (2006) 1064-1073.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
80 20 20 40 40
2. J. Urban, P. Jandera, P. Schoenmakers, Preparation of monolithic columns with target
mesopore-size distribution for potential use in size-exclusion chromatography,
J. Chromatogr. A 1150 (2007) 279-289.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
100 - 40 40 60
3. J. Urban, S. Eeltink, P. Jandera, P. Schoenmakers, Characterization of polymer-based
monolithic capillary columns by inverse size-exclusion chromatography and mercuryintrusion
porosimetry, J. Chromatogr. A 1182 (2008) 161-168.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
100 - 25 40 55
4. J. Urban, Pore volume accessibility of particulate and monolithic stationary phases,
J. Chromatogr. A 1396 (2015) 54-61.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
100 - 100 100 100
5. J. Urban, Current trends in the development of porous polymer monoliths for the separation
of small molecules, J. Sep. Sci. 39 (2016) 51-68.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
100 - 100 100 100
6. J. Urban, P. Jandera, P. Langmaier, Effects of functional monomers on retention behavior
of small and large molecules in monolithic capillary columns at isocratic and gradient
conditions, J. Sep. Sci. 34 (2011) 2054-2062.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
10 80 10 40 35
14
7. J. Urban, V. Škeříková, P. Jandera, R. Kubíčková, M. Pospíšilová, Preparation
and characterization of polymethacrylate monolithic capillary columns with dual hydrophilic
interaction reversed-phase retention mechanism for polar compounds, J. Sep. Sci. 32 (2009)
2530-2543.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
30 75 20 25 37.5
8. M. Chocholoušková, M. Komendová, J. Urban, Retention of small molecules
on polymethacrylate monolithic capillary columns, J. Chromatogr. A 1488 (2017) 85-92.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
10 90 100 100 75
9. J. Urban, F. Svec, J.M.J. Fréchet, Efficient separation of small molecules using a large surface
area hypercrosslinked monolithic polymer capillary column, Anal. Chem. 82 (2010) 1621-
1623.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
100 - 25 40 55
10. J. Urban, F. Svec, J.M.J. Fréchet, Hypercrosslinking: New approach to porous polymer
monolithic capillary columns with large surface area for the highly efficient separation
of small molecules, J. Chromatogr. A 1217 (2010) 8212-8221.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
100 - 25 40 55
11. J. Urban, V. Škeříková, Effect of hypercrosslinking conditions on pore size distribution
and efficiency of monolithic stationary phases, J. Sep. Sci. 37 (2014) 3082-3089.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
90 10 100 100 75
12. V. Škeříková, J. Urban, Highly stable surface modification of hypercrosslinked monolithic
capillary columns and their application in hydrophilic interaction chromatography, J. Sep. Sci.
36 (2013) 2806-2812.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
10 90 100 100 75
15
13. S. Janků, V. Škeříková, J. Urban, Nucleophilic substitution in preparation and surface
modification of hypercrosslinked stationary phases, J. Chromatogr. A 1388 (2015) 151 - 157.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
10 100 100 100 77.5
14. T.Z. Teisseyre, J. Urban, N.W. Halpern-Manners, S.D. Chambers, V.S. Bajaj, F. Svec, A. Pines,
Remotely detected NMR for the characterization of flow and fast chromatographic
separations using organic polymer monoliths, Anal. Chem. 83 (2011) 6004-6010.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
25 - 20 30 25
15. J. Urban, T. Hájek, F. Svec, Monolithic stationary phases with a longitudinal gradient
of porosity, J. Sep. Sci. 40 (2017) 1703-1709.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
90 10 90 90 70
16. S. Janků, M. Komendová, J. Urban, Development of an online solid-phase extraction
with liquid chromatography method based on polymer monoliths for the determination
of dopamine, J. Sep. Sci. 39 (2016) 4107-4115.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
20 80 100 100 75
17. M. Komendová, R. Metelka, J. Urban, Monolithic capillary column with an integrated
electrochemical detector, J. Chromatogr. A 1509 (2017) 171 – 175.
Experiments, % Supervision, % Research direction, % Manuscript, % Total, %
5 85 90 100 70
1.4 Funded projects
Since 2012 I have worked as Principal Investigator of following projects, with the final
evaluation of the first two projects being fulfilled.
▪ Improving the performance of hypercrosslinked monolithic stationary phases and their
application in separations of polar compounds, Czech Science Foundation, postdoctoral
project P206/12/P049 (2012 – 2014).
16
▪ Development of a multifunctional monolithic capillary column with integrated sample
focusing, separation, and on-column electrochemical detection, Czech Science Foundation,
standard project 14-22426S (2014 – 2016).
▪ Tailoring selectivity of polymer-based monolithic stationary phases, Czech Science
Foundation, standard project 17-11252S (2017 – 2019).
2 Monolithic stationary phases
2.1 Internal structure
The most important advances in chromatography have been always related
to the development of novel separation media featuring higher efficiency and better selectivity.
The development of high performance liquid chromatography was triggered by the introduction
of a broad range of small-particle separation media prepared from silica gel, polymers,
and inorganic oxides. To minimize the pore diffusion and mass transfer resistance, non-porous
and superficially porous particles with a solid central core and a very thin porous outer layer of the
stationary phase were also introduced [18].
Figure 1. Comparison of an internal structure for packed (A, top) and monolithic (A, bottom) stationary phase [19],
together with representation of organic polymer-based (B) and inorganic silica-based (C) monolithic stationary phase
[20,21].
In recent years, continuous separation media have attracted considerable attention because
of the advantages they offer over packed columns [5,22,23]. This research resulted in two useful
monolithic material types, the first based on modified silica gel and the second on organic
17
polymers. The monoliths consist of a single piece of highly porous material and can be compared
to a single large “particle” that does not contain interparticular voids typical of packed beds.
Figure 1 highlights difference in between packed and monolithic stationary phases and compares
an internal structure of organic and inorganic monolithic stationary phase.
2.2 Preparation of polymer monoliths
Monolithic stationary phases based on organic polymers are generally prepared by a free
radical polymerization although several other approaches were also introduced [24].
The polymerization mixture contains radical initiator to start the reaction, functional and/or
crosslinking monomer, and pore forming solvents (e.g. short alcohols) that do not participate
in the polymerization reaction but are responsible for phase separation during the reaction
and formation of the resulting polymer [25]. Generally, a homogenous polymerization mixture
is filled in the capillary where monolithic material is formed by a free radical reaction initiated
by an elevated temperature or a UV light [18,26]. Figure 2 shows several examples of both
methacrylate and styrene-based functional and crosslinking monomers and radical initiators used
for the preparation of polymer monoliths.
The formation and growth of polymeric monolithic stationary phase was already described
in 1995 [27] and recently further studied by thermal analysis and scanning electron microscopy
[28]. During the early stages of the polymerization reaction, polymer growth and branching
is homogeneously initiated throughout reaction medium leading to a formation of compact
microgel particles that crosslink and form polymer microglobule.
At this stage, phase separation occurs, which might happen as little as 10 minutes when the
polymerization reaction is running at 70 °C. Then, pre-gel network is formed by interconnected
globules and at around 45 – 60 minutes of polymerization reaction macroscopic network
of interconnected globules is already formed throughout the capillary.
18
Figure 2. Examples of functional and crosslinking monomers, together with radical initiators, used for the preparation
of polymer-based monolithic stationary phases.
19
After a couple of hours, previously formed gel links together and continuously fills the capillary
confinement with a macroscopic network structure. At this point, polymer microglobules further
grow by a coalescence with newly formed microglobules and reduce the size of flow-through
pores. The total conversion at this stage of the polymerization reaction is generally over 60% and
reaches 95% after 24 hours [28].
Compared to that, inorganic silica monoliths are prepared by the classical sol–gel process
of sequential hydrolysis and polycondensation of organo-silicium compounds, as the same
protocol is used for the preparation of fine silica particles packed in currently available
conventional HPLC columns [23].
2.4 Pore formation
Pores inside the monolith are open, forming a highly interconnected network of channels.
There are two main types of pores in the structure of monolithic columns, i.e. (i) flow-through
pores enabling an easy flow of the mobile phase and (ii) micropores and mesopores filled
with a “stagnant” mobile phase in which the solute molecules migrate to access the adsorption
sites. According to the IUPAC classification, micropores are pores with a diameter smaller
than 2 nm, mesopores have diameters between 2 and 50 nm, and flow-through pores are larger
than 50 nm [29].
Pore size distribution of monolithic stationary phases can be controlled in experimental
conditions including polymerization reaction and time, and by the composition
of the polymerization mixture. We have thoroughly investigated the effect of polymerization
mixture composition on the formation of both flow-through pores [1] and mesopores [2]
in polymethacrylate polymer monoliths.
We systematically varied the concentrations of butyl methacrylate as functional and ethylene
dimethacrylate as crosslinking monomer in the presence of 1,4-butanediol and 1-propanol
as the porogen solvents to find significant factors affecting pore formation in monolithic capillary
columns. To distinguish the role of the four components of the polymerization mixture in the pore
formation process, multivariate analysis of polynomial models allows investigation not only of the
20
separate effects of the individual components, but also of the combined synergistic
or antagonistic effects [1,2]. After a mathematical optimization of polynomial models, the mean
error of prediction was lower than 8% for flow-through pore formation [1] while it was 32% for
model describing formation of mesopores [2]. It should be pointed out, however, that absolute
values of mesopore volume are significantly lower than those of flow-through pores.
According to the models derived based on multivariate data analysis, individual components
of the polymerization mixture provide complementary contribution of the formation of the pores.
Increasing the sum of the concentrations of the porogen solvents (1,4-butanediol and 1-propanol)
in the polymerization mixture increases the content of flow-through pores in the monolithic
separation medium. On the other hand, increased concentrations of butyl methacrylate
monomer and of 1-propanol show a combined negative effect on the flow-through pore
formation. Hence, the concentration of 1,4-butanediol in the porogen solvent mixture is the main
factor enhancing the flow-through pore formation [1]. Oppositely, the main role in the mesopore
formation plays the combination of the hydrophobic butyl methacrylate monomer and less polar
1-propanol as the porogen solvent. Increasing concentration of 1-propanol and decreasing
concentration ratios of the cross-linker to monomer and of 1,4-butanediol to 1-propanol solvents
leads to higher incorporation of the mesopores in the monolithic capillary columns [2].
The pore formation is controlled by a phase separation governed by a polarity of the
polymerization mixture. With higher polarity of the mixture the phase separation starts at early
stages of the reaction allowing diffusion of monomers over a longer distance to the first regions
of a polymeric phase, so that the polymerization can proceed preferentially at the phase
interface. Consequently, a network with relatively large flow-through pores is formed.
On the other hand, later phase separation provides denser network of crosslinked polymers
and therefore denser polymer with a lower concentration of flow-through pores arises.
The presented models can be used to predict the porosity of flow-through pores
and mesopores within a limited, well defined space of polymerization mixture composition.
Alternatively, they can be also applied in a tailored preparation of monolithic capillary columns
with desired pore size distribution. For example, monolithic stationary phase with a dominant
flow-through pores and absent mesopores can be successfully used in fast gradient separations
21
of both small and large molecules [1]. On the other hand, monolith with a substantial volume
of mesopores might provide selectivity necessary for a size-exclusion type of the separation.
Hence, we have prepared monolithic columns with large volume of mesopores, however further
optimization is necessary to prepare columns with mesopore distribution suitable for efficient
and selective practical SEC separation of polymers according to their size in specific molar mass
range [2].
2.5 Characterization of porous properties
Various methods have been used to determine the porous properties of chromatographic
stationary phases. The most common methods include nitrogen adsorption using the Brunauer,
Emmett, Teller (BET) equation and mercury-intrusion porosimetry [3]. Alternatively, inverse sizeexclusion
chromatography that utilizes a set of well-defined molecular probes with widely varying
sizes can be used to determine pore dimensions [30].
A major question in characterizing the porous properties of monolithic beds is, however,
the extent to which the porous properties of the “dry” monoliths are indicative
of the chromatographic performance under “wet” (swollen) conditions. Therefore, we have
prepared polymethacrylate monolithic stationary phases and determined their porous properties
by inverse size-exclusion chromatography and mercury-intrusion porosimetry method [3].
Both techniques are complementary; while mercury-porosimetry measures the entire range
of pore sizes and provides more physical information on the monoliths, inverse size-exclusion
chromatography is suitable for determining the size of mesopores in the swollen monoliths.
Additionally, inverse size-exclusion chromatography has some advantages in comparison with
mercury intrusion porosimetry such as limited accuracy of the mercury porosimetry at elevated
pressures (for characterizing small pores) and the high toxicity of mercury can be avoided by using
inverse size-exclusion chromatography.
As expected, the porosities, permeabilities, flow-through pore diameters, pore volumes,
surface areas, and polymer densities were found to depend on the composition
of the polymerization mixture. The concentration of the porogen solvents in the mixture affected
22
the total and flow-through porosities most significantly. With higher concentrations
of the porogen solvents in the mixture, very porous monolithic materials with high permeabilities
and large flow-through pore diameter were obtained.
The total porosity of polymer monolith measured with inverse size-exclusion chromatography
decreased in increasing concentration of the monomers in the polymerization mixture. However,
the decrease in the total porosity determined using mercury intrusion porosimetry was much less
significant. The differences between the results obtained using the two techniques may be
possibly attributed to swelling of the monolithic beds when tetrahydrofuran is used as the mobile
phase in inverse-size exclusion chromatography and confirms once again the importance
of determination of pore characteristics directly from the size-exclusion data in the wet (swollen)
state, like the conditions of practical use of the columns for chromatographic separations [3].
In size-exclusion chromatography, compounds are eluted with respect to their size that
controls accessible pore volume. While small molecules penetrate to all pores and elute last, large
molecules are excluded from the pores smaller than their hydrodynamic diameter and elute first.
I have used size-exclusion chromatography to determine the pore volume fractions of various
stationary phases that are accessible to small and large molecules [4], i.e. accessible porosity,
and correlated it with the size of the pores from which individual compounds are excluded
as schematically shows Figure 3.
Such evaluation allows simple and straightforward comparison of various types of stationary
phases with different structural, porous, and hydrodynamic properties. To prove this concept,
I have first determined pore volume accessibility of commercially available columns packed with
fully and superficially porous particles, as well as with silica-based monolithic stationary phase.
Pore volume accessibility correlated well with the internal structure of both particulate
and monolithic stationary phases highlighting morphological differences of individual stationary
phases.
23
Then, I have used this approach
to study pore formation in polymerbased
monoliths. In polymethacrylate
monolithic stationary phases,
the mesopores were formed at the
very beginning of the polymerization
reaction followed by the
development of micro-pores during
later stages of the polymerization
reaction. The same applies also for
hypercrosslinking modification,
where mainly mesopores were
formed during the first 60 min
of hypercrosslinking and micropores
were formed when the
hypercrosslinking modification was
carried out for more than 60 min.
When hypercrosslinking modification
followed early termination
of the polymerization reaction, the pore volume accessibility decreased with longer time
of the polymerization reaction for styrene-based polymerization mixture [4].
I believe, that this simple protocol can be applied not only to studies concerned with pore
volume of stationary phases for liquid chromatography but also for non-destructive
characterization of any porous material such as heterogeneous catalysts and adsorbents.
Additionally, in case of polymer-based monoliths, this method provides fast, simple,
and straightforward information about pore formation and efficiency during the fabrication
process.
Figure 3. Schematic representation of pore volume accessibility
determination. Size of the molecule is expressed
as a hydrodynamic diameter of the molecule (nm), while accessible
porosity is determined as a fraction of internal pores that
are accessible for individual compounds (%) [4].
24
2.3 Repeatability of columns preparation
Repeatability and mechanical stability of polymer monoliths have always been considered less
robust when compared to both silica-based monoliths and particulate stationary phases.
However, several studies already demonstrated that the repeatability of the preparation
and mechanical stability of polymer-based monolithic stationary phases is (at least) comparable
to other types of the stationary phases conveniently used in liquid chromatography [31-33].
We have showed, that hypercrosslinked polymer monoliths with grafted polar functionality
do not change its chromatographic properties even after 10 000 injections of test compounds
[12]. Additionally, we also demonstrated that a combination of an early termination
of polymerization reaction and post-polymerization surface modification provides repeatable and
robust column properties with plate height RSD lower than 6.5% [13].
Most articles about polymer monoliths published nowadays provide a part devoted to columns
preparation repeatability showing that polymer-based monoliths are very robust and stable
stationary phases.
3 Separation of small molecules
Since their introduction, the main application area of porous polymer monoliths has been the
fast gradient separations of synthetic and natural polymers. On the other hand, preparation
of polymer monoliths providing column efficiency comparable with particulate and monolithic
silica-based stationary phases has been proven to be difficult.
During the last decade, several experimental approaches were performed that aimed
to improve this property of polymer monoliths which I have summarized in recent review [5].
These protocols include fine tuning of the polymerization mixture composition, preparation
of monolithic stationary phases at limited conversion of the polymerization reaction, application
of novel, highly ordered, nanomaterials, and/or hypercrosslinking surface modification
controlling the crosslink density of the prepared monoliths. By using some of these approaches,
25
monolithic stationary phases with column efficiency reaching 200 000 plates/m
for low-molecular-weight compounds have been prepared [5].
This part of the thesis focuses on two approaches leading to the preparation of polymer
monoliths suitable for an isocratic separation of small molecules: tuning a composition
of the polymerization mixture and hypercrosslinking modification of polymer surface.
3.1 Effect of functional monomer on the retention of small molecules
The composition of the polymerization mixture is the easiest way to change and control the
properties of polymer-based monoliths. While the concentration and polarity of pore forming
solvents affects phase separation hence porous and hydrodynamic properties of the prepared
monoliths, type of the functional and crosslinking monomer controls the surface chemistry
of the stationary phase that is responsible for chromatographic selectivity.
The disadvantage is the time-consuming optimization of experimental conditions. Even small
changes in the concentration of individual parts of the mixture provide the monolithic material
with different properties. Changes in the concentration, or even a replacement of monomer
or pore forming solvent must be followed by a new optimization of preparation and experimental
conditions to provide stationary phase with the desired properties.
By a proper selection of functional monomers monolithic capillary columns applicable
in reversed-phase [6] and hydrophilic interaction [7] liquid chromatography can be prepared.
It is also possible to utilize a thermally-responsive monomer to prepare monolithic stationary
phases suitable for the analysis of small molecules [8].
To compare polarity and efficiency of polymethacrylate monolithic capillary columns, we have
prepared stationary phases with butyl, cyclohexyl, 2-ethylhexyl, lauryl, and stearyl methacrylate
functional monomers and tested them in reversed-phase chromatography of low-molecular
alkylbenzenes and in gradient elution of proteins [6].
The non-polar chromatographic selectivity can be tuned by choosing the alkylmethacrylate
monomer used. The lauryl methacrylate column showed the lowest methylene and phenyl
26
selectivity for small molecules, whereas the butyl methacrylate column showed high phenyl
selectivity and the column with stearyl methacrylate had a high methylene selectivity.
On the other hand, there were no direct correlations between the size of the functional monomer
and the methylene and phenyl selectivity. This may be possibly caused by the differences
in the skeleton conformation (folding) affecting the shape and accessibility of pores
of the monolithic media for alkylbenzenes.
Even though the efficiencies of most polymethacrylate columns prepared with various
functional monomers for alkylbenzenes were low, the monolithic columns prepared with lauryl
methacrylate monomer showed markedly better efficiency than the other columns, which might
be possibly attributed to different degree of lauryl methacrylate stationary phase swelling
and hence its accessibility for low molecular alkylbenzenes.
Generally, the organic polymer monolithic columns show much better performance
(efficiency) for biopolymers than for low-molecular samples. The linear solvent strength gradient
model showed good fit to the retention data of proteins in gradient elution. We have found that
the retention and the concentration of acetonitrile at the time of elution of proteins increased
for functional monomers with longer alkyl chains, especially for large proteins. The model allowed
simple prediction of the effects of the gradient program on the elution volumes and bandwidth
in gradient liquid chromatography.
All the tested columns could be successfully used for fast gradient separations of proteins.
However, the lauryl methacrylate column showed the lowest hydrophobicity and best efficiency
for alkylbenzenes and proteins [6].
Previously discussed monolithic capillary columns were applied in the separations
of moderately polar compounds following reversed-phase liquid chromatography retention
mechanism. However, very polar compounds do not provide any retention in reversed-phase
chromatography and therefore it is very difficult to separate them properly.
One of the possibilities of how to overcome this drawback is to utilize polar stationary phase
together with highly organic mobile phase and separate polar compounds in HILIC [34].
27
Thus, we aimed to prepare monolithic capillary column based on zwitterionic
[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide monomer suitable
for the separation of polar phenolic compounds [7]. The columns prepared with methanol as the
only pore forming solvent [35] did not provide satisfactory permeability. Therefore, we have
tested various polar porogen solvents including methanol, ethanol, tetrahydrofuran, 1-propanol,
1,4-butanediol, and their mixtures with water to prepare monolithic HILIC columns. We found
that addition of water to the porogen solvent mixture enhances the solubility of the zwitterionic
sulfobetaine monomer and therefore permits the use of organic solvents less polar than
methanol to adjust the porosity [7].
Polymethacrylate monolithic capillary columns with zwitterionic sulfobetaine groups
incorporated into the polymer structure were prepared using binary or ternary porogen solvent
mixtures containing water. Porosity, permeability, selectivity, and retention characteristics
of prepared columns depended on the composition of the polymerization mixture. The main
factors affecting the selectivity of monolithic stationary phase were the concentration
of zwitterion monomer and the composition of the porogen solvents in the polymerization
mixture.
The retention of phenol and phenolic acids on the monolithic columns showed a dual RP-HILIC
separation mechanism, depending on the concentration of acetonitrile in the mobile phase
(reversed-phase in water-rich mobile phases and HILIC at high concentrations of acetonitrile
in aqueous–organic mobile phases) that could be successfully described by the model assuming
the additivity of the HILIC and RP contributions to the retention.
The monolithic sulfobetaine columns show a separation selectivity for phenolic acids
comparable to that of the commercial column with sulfobetaine stationary phase chemically
bonded to silica gel particles in the HILIC mode, but provided significantly higher retention and
separation selectivity than the silica-based column in the reversed-phase mode in highly aqueous
mobile phases.
Monolithic capillary columns with a dual retention mechanism offer the possibility of selecting
between the two retention modes with different separation selectivity on a single column,
28
just by changing the composition of the mobile phase, which is advantageous for separations
of samples containing compounds with significant differences in polarities.
N-isopropylacrylamide belongs to the family of monomers for the preparation of thermally
responsive materials that change their solvation and associated polarity based on external
temperature: when heated in an aqueous solution over 32°C (lower critical solution temperature,
LCST) it reversibly loses over 90% of volume and changes from a swollen hydrated (hydrophilic)
monomer to a shrunken dehydrated (hydrophobic) one [36]. Unfortunately, this behavior
is observed mainly in pure water and presence of high concentration of organic solvent eliminates
the effect.
We have explored an effect of composition of the polymerization mixture, concentration
of acetonitrile in the mobile phase, and working temperature on the retention of small molecules
on polymethacrylate monolithic capillary columns prepared with N-isopropylacrylamide
in the polymerization mixture [8]. Optimization of a second-order polynomial retention model
revealed that the most significant parameter controlling the retention of small molecules
is the concentration of acetonitrile in the mobile phase, and this effect is more dominant for less
polar compounds.
Working temperature and combined parameter of acetonitrile concentration and elevated
temperature contribute only a little to the retention. The possible explanation is that quite low
concentrations of added N-isopropylacrylamide were unable to form sufficiently long polymer
sequences inside the monolithic phase. This drawback can be easily overcome by an application
of a grafting reaction and attach poly(N-isopropylacrylamide) chains at the surface of generic
monolith.
This chapter confirms that by a proper optimization of polymerization mixture composition,
particularly by a selection of type and chemistry of functional monomer, monolithic capillary
columns applicable in reversed-phase and hydrophilic interaction liquid chromatography of small
molecules can be successfully prepared [6-8].
It should be pointed out again however, that when functional monomer in the polymerization
mixture is replaced by another one, new optimization must be performed to prepare monolithic
29
stationary phase with desired efficiency and selectivity. To avoid this, generic monolithic capillary
column allowing efficient separation of small molecules should be prepared and its chemistry
(eventually) controlled by a post-polymerization surface modification either via direct chemical
modification or by surface initiated grafting of selected functional groups. Preparation of such
generic columns allows for example post-polymerization hypercrosslinking modification
that is thoroughly discussed in next chapter of this thesis.
3.2 Hypercrosslinking surface modification
3.2.1 Hypercrosslinked monolithic stationary phases
Several decades ago, Davankov prepared large surface area materials from preformed polymer
precursors in a technique termed “hypercrosslinking” [37]. The original implementation used
linear polystyrene, which was crosslinked via Friedel-Crafts alkylation to afford materials
containing mostly small pores. This approach was later extended to crosslinked porous
poly(styrene-co-divinylbenzene) particles and led to products containing both the original pores
and an extensive network of additional micro- and mesopores generated during hypercrosslinking
[38].
Hypercrosslinking modification allows tailored preparation of functional materials suitable
for various applications, including HPLC, biomedical applications in hemodialysis, solid-phase
extraction, and large-scale liquid adsorption chromatography [39-41].
In 2010, we demonstrated for the first time the preparation of hypercrosslinked porous
polymer monoliths exhibiting a large surface area and their use in capillary columns
for the separation of small molecules as well as for rapid size-exclusion chromatography [9].
Since then, we have further optimized the experimental conditions to improve column efficiency
[10,11], tuned surface polarity of hypercrosslinked monoliths [12,13], coupled hypercrosslinked
monolithic columns with an online NMR detection [14], and utilized hypercrosslinking
modification to prepare monolithic capillary columns with a longitudinal gradient of small
pores [15].
30
Figure 4. Individual steps in the preparation of hypercrosslinked monolithic stationary phases. In the first step,
generic monolith is formed by a free-radical polymerization. Then, stationary phase is swollen in thermodynamically
good solvents and loose polymer chains are fixed by Friedel-Crafts alkylation [9,10].
Hypercrosslinking modification utilizes the fact, that bifunctional divinyl monomer polymerizes
faster and hence the surface of the monolith at the end of the polymerization reaction is formed
mainly by slightly cross-linked chains attached to the surface of highly crosslinked microglobular
scaffolds. After the swelling of a monolith in a thermodynamically good solvent, the loose
polymeric chains at the surface of monolithic stationary phases are fixed by a nucleophilic
substitution reaction catalyzed by a Lewis acid. A thin layer of small pores is then formed
at the surface of the monolithic material, thus providing a higher column efficiency and selectivity
in the separation of small molecules. Figure 4 provides description of individual steps in the
preparation of hypercrosslinked monolithic stationary phases.
Despite its recent introduction to the field of polymer monoliths [9], hypercrosslinking
modification has already attracted considerable attention [42]. The hypercrosslinked stationary
phases have been used for sample clean up [43], to separate compounds in capillary LC [44]
and capillary electrochromatography [45], modified with gold nanoparticles [46], used
as stationary phases in TLC of peptides and proteins [47] or as a media for a gas storage [48].
We have illustrated the significant effects of the polymerization conditions and the postpolymerization
hypercrosslinking process on the chromatographic performance of the resulting
monolithic poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) stationary phase [10].
31
The comprehensive optimization based on mathematical design of the composition
of the polymerization mixture used to prepare the precursor monoliths, and temperature,
at which the hypercrosslinking is carried out, led to an equation which facilitated the calculation
of conditions that should afford columns with predetermined efficiencies. Specifically,
this approach enabled the reproducible preparation of a column for (i) isocratic reversed phase
separation of alkylbenzenes characterized by its high plate count, (ii) gradient elution of peptides,
and (iii) very efficient size-exclusion chromatography of polystyrene standards in organic solvent.
The use of the high temperature and of a ternary mobile phase permits a significant acceleration
of the separations.
The proper combination of the swelling solvent and Friedel-Crafts catalyst can be used
for the preparation of hypercrosslinked monolithic stationary phases with controlled
hydrodynamic and porous properties for a separation of small molecules [11]. Three dihalogenic
solvents differing in the length of alkyl chain (1,2-dichloroethane, 1,4-dichlorobutane,
and 1,6-dichlorohexane) with three Friedel–Crafts alkylation catalysts varying in reactivity
(AlCl3 > FeCl3 > SnCl4) have been used to prepare hypercrosslinked poly(styrene-co-vinylbenzyl
chloride-co-divinylbenzene) columns.
The column permeability is significantly affected by the combination of swelling solvent and
reaction catalyst applied. With increasing reactivity of the alkylation catalysts and increasing
length of alkyl chain in the swelling solvent, the column permeability decreases. The column
efficiency is higher for columns modified with highly reactive catalyst in the presence
of the solvent with the longest alkyl chain. The column efficiency however decreases with
the higher catalyst reactivity for 1,4-dichlorobutane. The column efficiency also increases
for columns with longer time of hypercrosslinking modification, lower concentration of reaction
catalyst, and at elevated reaction temperature.
The pore volume of micropores smaller than 2 nm can be successfully linked to the efficiency
of hypercrosslinked stationary phases. The higher their pore volume in the monolithic material,
the better was the column efficiency. This pore volume segment is probably associated
to so-called gel porosity, introduced earlier [49] as a general property responsible for an efficiency
of organic polymer-based monoliths in the separation of small molecules [50].
32
3.2.2 Surface modification of hypercrosslinked monoliths
Due to the low polarity of scaffold styrenic monolith, hypercrosslinked monolithic stationary
phases have been mainly used in reversed-phase liquid chromatography retention mechanism
[9-11,44,45]. Since not all chloromethyl groups of generic hypercrosslinked monolith are involved
in Friedel-Crafts alkylation, residual reactive groups can be utilized for further surface
modification.
A two-step surface modification of poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene)
monolithic stationary phases, including hypercrosslinking and grafting of [2-(methacryloyloxy)
ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide, provided hypercrosslinked monolithic
capillary columns for the isocratic separation of small polar compounds in HILIC [12].
According to response surface methodology, the main factor in the surface modification
of hypercrosslinked monoliths was the synergistic effect of grafting time. Modified columns
provided a dual retention mechanism, including RP and HILIC, which can be controlled
by the composition of the mobile phase. Hypercrosslinked stationary phases with zwitterion
functionality have been used for isocratic separation of polar phenolic acids in HILIC, and applied
in one- and two-dimensional liquid chromatography.
We also demonstrated an application of nucleophilic substitution reaction in the preparation
of hypercrosslinked monolithic stationary phases [13]. We have used linear diaminoalkanes
differing in the number of methylene units in between terminal amino groups to crosslink swollen
polymer material. The column efficiency of the prepared columns increased with the length
of diaminoalkane. We have optimized the modification reaction conditions, such as time,
temperature, and concentration of the crosslinking agent. To improve the permeability
of prepared columns, we have hypercrosslinked monolithic material after
only 2 h of polymerization reaction.
Furthermore, we have modified the polymer hypercrosslinked by nucleophilic substitution
reaction with 2-aminoethanesulfonicacid (taurine) to prepare capillary columns suitable
for analysis of low-molecular polar compounds. We have optimized reaction conditions and used
prepared capillary columns in the isocratic separation of polar phenolic compounds [13].
33
Although presented protocol is more complex than a single-step polymerization reaction,
our results clearly demonstrate the versatility of hypercrosslinked monolithic stationary phases
for separation of small molecules with superior stability and robustness.
3.2.3 Fast separations on hypercrosslinked stationary phases
Hypercrosslinked modification allows preparation of stationary phases providing fast
and efficient separation. Hence, they find applications in an online hyphenation of LC
with a remote NMR detection [14] or industrial applications where fast isocratic separation
of polymers is needed, such as the second dimension in 2D LC [15].
Although UV-visible spectroscopy is the most common method of monitoring chromatographic
separations in real-time, it requires well resolved analyte peaks and the use of chemical standards
to correlate species as they elute from the column. Mass spectrometry can also be used for realtime
detection, providing an additional method to confirm peak assignments. However,
the chemical information provided through mass spectrometry is limited to the mass-to-charge
ratio of the individual compounds, unless secondary processes such as fragmentation are utilized.
Neither detection method can perform multidimensional imaging of flow or separations directly
on the column. One powerful alternative to these detection methods is nuclear magnetic
resonance.
As the sensitivity of NMR detection is directly proportional to the filling factor of the coil
(the fraction of the coil volume occupied by the sample), a microsolenoid with diameter
comparable to the inaccessible microporous features of the column will afford the highest
sensitivity. As an alternative, remotely detected NMR provides an improved filling factor
and a tremendous sensitivity enhancement when detecting small volumes of flowing liquid.
Remote detection separates and optimizes the components of a magnetic resonance experiment
(polarization, encoding, and detection). This is possible because the steps are correlated in both
space and time by the flowing fluid, whose spin degrees of freedom act like a magnetic recording
tape in which information can be stored, at one time and place, and read out later (Figure 5A).
34
Figure 5. (A) Illustration of the remote detection experiment, as applied to an organic polymer monolithic column,
(B) Axial images illustrating intensity and velocity for acetonitrile flowing through the open capillary (top) and
monolith (bottom), (C) Two-dimensional plot illustrating the separation of small molecules using a hypercrosslinked
monolithic chromatography column. The horizontal axis corresponds to the NMR chemical shift, while the vertical
axis represents the transit time of compounds [14].
We have demonstrated an application of remotely detected magnetic resonance imaging
for the characterization of flow and the detection of fast, small molecule separations within
hypercrosslinked polymer monoliths [14]. Magnetic resonance imaging enabled high resolution
intensity and velocity-encoded images of mobile phase flow through the monolith. The images
confirm that the presence of a polymer monolith within the capillary disrupts the parabolic
laminar flow profile that is characteristic of mobile phase flow within an open tube. As a result,
the mobile phase and analytes are equally distributed in the radial direction throughout
the monolith (Figure 5B). We have also shown in-line monitoring of chromatographic separations
35
of small molecules at high flow rates (Figure 5C). These experiments demonstrate the unique
power of the magnetic resonance, both direct and remote, in studying chromatographic
processes. Hypercrosslinked monoliths shown to be robust media for the rapid separation of
small molecules with a transit time relative standard deviation of less than 2.1%, even after more
than 300 column volumes were pumped through at high pressure and flow.
Furthermore, in separating the polarization, encoding, and detection steps of an NMR
experiment, remote detection enables development of truly portable LC-NMR instrumentation
when advantageously coupled with nonmetallic monolithic capillary columns.
Generally, we use isotropic columns in LC and control their separation performance by tuning
external conditions. However, because of the simple and straightforward preparation, monolithic
stationary phases offer elegant way how to prepare columns with longitudinal gradients
of column properties.
We demonstrated the preparation
of monolithic stationary phases with
longitudinal gradient of porosity. For this, we
used hypercrosslinking modification and
controlled the extent of formation of small
pores by the change in reaction time [15].
Segments of five columns hypercrosslinked for
30–360 min were coupled via zero-volume
unions to prepare columns with segmented
porosity gradients. The quality of isocratic
separation for both low-molecular weight
alkylbenzenes and high molar mass
polystyrene standards improved significantly
with the steepness of porosity gradient.
We have also prepared two individual columns with continuous gradient of porosity differing
in the steepness. Column with a shallower porosity gradient enabled separation of polystyrene
Figure 6. Online 2D LC of polystyrene standards using
completely hypercrosslinked monolithic column
combined with monolithic column with a gradient
of porosity [15].
36
standards comparable to that of hypercrosslinked column with no porosity gradient. However,
compared to completely hypercrosslinked column, due to higher pore volume of permeable
macropores, column with porosity gradient exhibited 1.5 times better column permeability and
allowed fast separation of high molar mass polystyrenes.
Finally, we coupled completely hypercrosslinked column with porosity gradient column
in 2D LC and demonstrated suitability of these columns for application in 2D separation
of polymers combining on-column precipitation–redissolution and SEC separation modes
as demonstrates Figure 6.
4 Towards multifunctional monolithic capillary column
Neurotransmission is a chemical communication which is controlled by small molecules called
neurotransmitters that travel across the synaptic gap to transmit the desired information to other
neurons. Catecholamine dopamine belongs to one of the most important neurotransmitters since
it plays a major role in reward-motivated behavior. Undesired changes in dopamine metabolism
result in serious illnesses such as depression, schizophrenia, Parkinson’s disease, and tumors
[51,52]. Therefore, it is necessary to develop fast, simple, and reliable analytical methods
for its precise determination in various biological samples.
To reach this goal, numerous protocols are developed daily that include several subsequent
steps and apply various experimental methods. One of the current trends in analytical chemistry
is to integrate individual steps of sample analysis into one online and comprehensive workflow,
which eliminates tedious and expensive sample manipulation. Yet, another trend is to miniaturize
these methods and systems, and to develop online miniaturized capillary and microfluidic
analytical systems.
4.1 Sample extraction and focusing
We have used polymer monoliths to develop an online solid-phase extraction with liquid
chromatography method for determination of dopamine in a urine sample [16]. A polymerization
37
mixture containing 4-vinylphenylboronic acid monomer has been used to prepare a trapping
column. Boronic acid functionality covalently bonds to cis-diol functionality at neutral pH, while
acidic pH allows reversible de-attachment of selectively trapped compounds. Additionally,
a monolithic stationary phase with zwitterion functionality has been used to prepare capillary
column for the separation of dopamine.
One-variable-at-a-time approach has been used to optimize experimental conditions including
molarity, pH, and flow rate of the loading buffer together with a time switching valve to provide
the highest dopamine recovery. The best results were achieved for dopamine loaded
at 0.5 L/min in 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer with
pH 7. By using these conditions, the concentration of dopamine in urine sample was determined
to be 1.19 and 1.28 mg/L with a calibration curve method and a standard addition method,
respectively.
In the next step, an experimental design has been used to optimize an online coupling of two
extraction loops with a separation monolithic capillary column to allow comprehensive
monitoring of dopamine in a flowing system. Sample loading flow rate and a flow rate of buffer
back-flush were determined as the most significant variables. Finally, the degradation of
unstabilized dopamine in a buffer was continuously studied by an optimized experimental setup
that allowed the determination of both rate and half-time of dopamine degradation.
Although presented for dopamine only, other cis-diol containing compounds can be also
determined. Optimized results will be used in the development of monolithic capillary column
integrating both sample focusing and separation of cis-diol neurotransmitters in a single capillary
unit. For that, we plan to attach 4-vinylphenylboronic acid monomer at the surface of a generic
monolithic stationary phase by a spatially controlled UV-initiated grafting. Our yet unpublished
results show that modification of one third of the column length with 4-vinylphenylboronic acid
monomer allows fabrication of dual-function monolithic capillary column providing both sample
focusing (extraction) of the desired compounds and their subsequent separation in one single
analysis.
38
4.2 Integrated electrochemical detector
Very recently, we have introduced a concept of an integration of an electrochemical detector
inside the monolithic capillary column [17]. Both carbon fiber and silver microwire were
used as a working and a pseudoreference electrode, respectively, and inserted into the ending
of capillary to prepare a monolithic capillary column with an integrated electrochemical detector.
Carbon fiber as a working electrode and silver microwire as a pseudoreference electrode
with a diameter of 7 m and 25 m, respectively, were attached to contact silver-plated wires
using conductive silver paint and allowed to dry at room temperature. Afterwards,
the microelectrodes were cut to desired length with a razor blade and fixed in parallel on ceramic
slides using cyanoacrylate adhesive (Figure 7A). The capillary monolithic column was carefully
slid to the required length onto the working and reference electrode with the aid
of micromamipulator under microscopic observation using an optical stereomicroscope
(Figure 7B). The position of the capillary with embedded microelectrodes on a ceramic support
was fixed afterwards with cyanoacrylate adhesive (Figure 7C).
Prepared capillary devices offered stable and robust results with relative standard deviations
of retention, resolution, and detection signal lower than 1.5, 5.5, and 5.0%, respectively.
To further increase the sensitivity of developed electrochemical microdetector, a multiple pulse
amperometry detection mode has been used. An optimized integrated device provided a reliable
chromatographic separation of mixture of neurotransmitters (Figure 7D) with a calibration curve
for dopamine linear from 0.5 to 20.0 mg/L and an instrumental limit of detection as low as 24 pg
of injected dopamine.
Finally, a developed capillary column was applied to successfully determine the dopamine
in a human urine. By using both a calibration curve and a standard addition method,
the dopamine level was determined to be 0.74 ± 0.03 mg/L and 0.71 ± 0.02 mg/L, respectively.
Triplicates of dopamine analysis provided relative standard deviations lower than 2.7%
for intraday analyses, while interday relative standard deviations were lower than 3.6% for five
consecutive days.
39
Figure 7. Preparation of integrated electrochemical detector: (A) working (carbon fiber, up) and pseudoreference
(silver microwire, down) electrodes glued to the silver-plated wires, (B) insertion of microelectrodes into the end of
fused-silica capillary with monolithic stationary phase, and (C) microelectrodes inside the end of the capillary. (D)
Separation of mixture of neurotransmitters on monolithic capillary column with an integrated electrochemical
detector in 5% acetonitrile with 0.1% TFA. Analytes: (1) epinephrine, (2) dopamine, (3) serotonin,
(4) homovanillic acid, (5) 5-hydroxyindol-3-acetic acid, (6) 3,4-dihydroxyphenyl acetic acid [17].
The presented instrumental limit of detection calculated from a regression line is slightly
higher when compared to other HPLC systems with electrochemical detection where limits
of detection are determined from the noise of the baseline [53]. However, in this proof-ofconcept
study we aimed mainly in the utilization of straightforward preparation of monolithic
stationary phases and simple miniaturization of an electrochemical detection.
Advantageous combination of these techniques allows further development of tailored
analytical systems. For example, the coupling of an integrated electrochemical detector
to a dual-function monolithic capillary column mentioned in Chapter 4.1 provides multifunctional
three-in-one monolithic capillary column offering sample focusing, separation, and separation
in one single analysis.
5 Conclusions & Future aspects
Since their introduction in the 1990s, monolithic stationary phases caught considerable
attention as an alternative to widespread particulate stationary phases. The internal porous
40
structure of an interconnected network of channels allows convective flow of the mobile phase
through the column and provides fast isocratic and gradient separations of both small and large
molecules. While inorganic-based silica monoliths excel in the isocratic separations of small
molecules, the power of polymer-based monoliths is in the fast gradient separations of large
molecules. Up to now, various preparation protocols have been introduced to increase
the efficiency of polymer monoliths in the separation of small molecules, as also demonstrated
in this thesis.
Nowadays, the efficiency of polymer-based monolithic stationary phases cannot compete
with commercially available particulate stationary phases, especially those with superficially
porous particles. One of the possibilities of how to improve efficiency of monolithic stationary
phases is a utilization of highly ordered materials, such as polyhedral oligomeric silsesquioxanes
or metal-organic frameworks and prepare homogeneous composite stationary phases [5].
A very promising technique for future fabrication of monolithic stationary phases is additive
manufacturing, also known as 3D printing, especially when combined with post-processing
surface modification with a polymer layer providing desired selectivity.
On the other hand, applicability of current polymer monoliths is in their very simple
preparation and straightforward surface modification. Without exaggerating, almost
any functional moiety can by attached at the surface of monolith. Additionally, spatial control
of UV-initiated grafting allows segmented modification leading to multifunctional devices.
Possible control of monolithic stationary phase selectivity via tailored preparation might provide
– after thorough optimization of experimental conditions – solution for almost any separation
problem.
Another advantage of polymer monoliths is that they can be very easily prepared in any size
and shape, including capillary, chip, and planar format. This makes them ideal materials
for development of lab-on-chip systems integrating all necessary steps of sample analysis
(sampling, sample transport, filtration, dilution, chemical reactions, separation, and detection)
in one integrated total analytical system. Such miniaturized and portable systems offer analysis
results at the point of interest and eliminate time-consuming, long-distance, and expensive
sample transport and preparation.
41
I believe that my involvement in polymer-based monolithic stationary phases including
a description of pore formation and its characterization, preparation of polymer monoliths
for analysis of polar compounds, introduction of hypercrosslinked monolithic stationary phases,
and the development of an integrated monolithic capillary column significantly contributed to the
current state of the art of polymer-based monolithic stationary phases research.
42
7 References
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in methacrylate ester-based monolithic columns, J. Sep. Sci. 29 (2006) 1064-1073.
[2] J. Urban, P. Jandera, P. Schoenmakers, Preparation of monolithic columns with target
mesopore-size distribution for potential use in size-exclusion chromatography,
J. Chromatogr. A 1150 (2007) 279-289.
[3] J. Urban, S. Eeltink, P. Jandera, P.J. Schoenmakers, Characterization of polymer-based
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porosimetry, J. Chromatogr. A 1182 (2008) 161-168.
[4] J. Urban, Pore volume accessibility of particulate and monolithic stationary phases,
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[7] J. Urban, V. Škeříková, P. Jandera, R. Kubíčková, M. Pospíšilová, Preparation and
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[8] M. Chocholouskova, M. Komendova, J. Urban, Retention of small molecules
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Appendix
- 1 J.
Urban, D. Moravcová, P. Jandera, A model of flow-through
pore formation in methacrylate ester-based monolithic
columns, J. Sep. Sci. 29 (2006) 1064 – 1073.
- 2 J.
Urban, P. Jandera, P. Schoenmakers, Preparation
of monolithic columns with target mesopore-size distribution
for potential use in size-exclusion chromatography,
J. Chromatogr. A 1150 (2007) 279 – 289.
- 3 J.
Urban, S. Eeltink, P. Jandera, P. Schoenmakers,
Characterization of polymer-based monolithic capillary columns
by inverse size-exclusion chromatography and mercuryintrusion
porosimetry, J. Chromatogr. A 1182 (2008) 161 – 168.
- 4 J.
Urban, Pore volume accessibility of particulate and monolithic
stationary phases, J. Chromatogr. A 1396 (2015) 54 – 61.
- 5 J.
Urban, Current trends in the development of porous polymer
monoliths for the separation of small molecules,
J. Sep. Sci. 39 (2016) 51 – 68.
- 6 J.
Urban, P. Jandera, P. Langmaier, Effects of functional
monomers on retention behavior of small and large molecules
in monolithic capillary columns at isocratic and gradient
conditions, J. Sep. Sci. 34 (2011) 2054 – 2062.
- 7 J.
Urban, V. Škeříková, P. Jandera, R. Kubíčková, M. Pospíšilová,
Preparation and characterization of polymethacrylate
monolithic capillary columns with dual hydrophilic interaction
reversed-phase retention mechanism for polar compounds,
J. Sep. Sci. 32 (2009) 2530 – 2543.
- 8 M.
Chocholoušková, M. Komendová, J. Urban,
Retention of small molecules on polymethacrylate monolithic
capillary columns, J. Chromatogr. A 1488 (2017) 85 – 92.
- 9 J.
Urban, F. Svec, J.M.J. Fréchet, Efficient separation
of small molecules using a large surface area hypercrosslinked
monolithic polymer capillary column, Anal. Chem. 82 (2010)
1621 – 1623.
- 10 J.
Urban, F. Svec, J.M.J. Fréchet, Hypercrosslinking:
New approach to porous polymer monolithic capillary columns
with large surface area for the highly efficient separation
of small molecules, J. Chromatogr. A 1217 (2010) 8212 – 8221.
- 11 J.
Urban, V. Škeříková, Effect of hypercrosslinking conditions
on pore size distribution and efficiency of monolithic stationary
phases, J. Sep. Sci. 37 (2014) 3082 – 3089.
- 12 V.
Škeříková, J. Urban, Highly stable surface modification
of hypercrosslinked monolithic capillary columns and their
application in hydrophilic interaction chromatography,
J. Sep. Sci. 36 (2013) 2806 – 2812.
- 13 S.
Janků, V. Škeříková, J. Urban, Nucleophilic substitution
in preparation and surface modification of hypercrosslinked
stationary phases, J. Chromatogr. A 1388 (2015) 151 - 157.
- 14 T.Z.
Teisseyre, J. Urban, N.W. Halpern-Manners, S.D. Chambers,
V.S. Bajaj, F. Svec, A. Pines, Remotely detected NMR
for the characterization of flow and fast chromatographic
separations using organic polymer monoliths,
Anal. Chem. 83 (2011) 6004 – 6010.
- 15 J.
Urban, T. Hájek, F. Svec, Monolithic stationary phases
with a longitudinal gradient of porosity,
J. Sep. Sci. 40 (2017) 1703 – 1709.
- 16 S.
Janků, M. Komendová, J. Urban, Development of an online
solid-phase extraction with liquid chromatography method
based on polymer monoliths for the determination
of dopamine, J. Sep. Sci. 39 (2016) 4107 – 4115.
- 17 M.
Komendová, R. Metelka, J. Urban, Monolithic capillary
column with an integrated electrochemical detector,
J. Chromatogr. A 1509 (2017) 171 – 175.