Research Collection
Doctoral Thesis
Macro-porous chromatography resins by controlled aggregationof colloidal polymer particles
Author(s): Brand, Bastian
Publication Date: 2014
Permanent Link: https://doi.org/10.3929/ethz-a-010158880
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
DISS. ETH Nr. 21917
MACRO-POROUS CHROMATOGRAPHY RESINS BY CONTROLLED AGGREGATION
OF COLLOIDAL POLYMER PARTICLES
Abhandlung zur Erlangung des Titels
DOKTOR DER WISSENSCHAFTEN der ETH ZÜRICH
(Dr. sc. ETH Zürich)
vorgelegt von
BASTIAN BRAND
MSc ETH in Chemie- und Bioingenieurswissenschaften
geboren am 20.06.1986
Deutscher Staatsangehöriger
angenommen auf Antrag von
Prof. Dr. Massimo Morbidelli
Prof. Dr. Christophe Copéret
2014
Chapter 1
v
Summary
In this work the development of macro-porous chromatography materials using Reactive Gelation
process is described. In this process, polymeric nanoparticles are aggregated and subsequently
hardened to form highly porous materials that obey fractal mass scaling laws. Stationary phases in the
form of monoliths and particles were prepared and modified to suit ion chromatography as well as
large bio-molecule chromatography, two applications that require very different material properties.
The monoliths exhibited excellent mass transport not affected by diffusion. This is especially
important when dealing with large proteins. The particles, on the other hand, show clearly diffusion-
limited mass transport at low flow velocities, but enter the so-called perfusive mass transport mode at
high flow rates. In this mode, eluent flows convectively through the chromatographic particles,
making them very similar to monoliths. These complex kinetics have been explored using a special
chromatographic model that can account for flow through particles. A very strong perfusive behaviour
was found and attributed to good interconnectivity of the pores, originating from the fractal structure
of these materials. Encouraged by the good results obtained from ion chromatography, a way to
increase productivity was developed. For this purpose, the aggregation mechanism was totally re-
worked to be shear-induced instead of salt-induced and the process made continuous. This process was
studied in detail and the produced material properties explored. Both non-chromatographic and
chromatographic methods showed high similarity to the earlier prepared material. This last step makes
the production of these interesting materials industrially feasible.
Chapter 1
vi
Zusammenfassung
Diese Arbeit beschreibt die Herstellung von makroporösen Chromatographiematerialien mit Hilfe von
Reactive Gelation. In diesem Prozess werden polymerische Nanopartikel aggregiert und
anschliessened thermisch gehärtet, wodurch sie hochporöse Materialien formen, die fraktaler
Massenskalierung gehorchen. Es wurden Stationärphasen in der Form von Monolithen und Partikeln
erstellt und so modifiziert, dass sie für Ionenchromatographie oder der Chromatographie grosser
Biomoleküle tauglich sind – zwei Anwendungen die, wie sich herausgestellt hat, sehr unterschiedliche
Materialeigenschaften erfordern. Der hervorragende Massentransport in Monolithen wurde nicht durch
Diffusion beeinflusst, was speziell wichtig in der Chromatographie grosser Eiweisse ist. Der
Massentransport in Partikeln war bei niedrigen Flussraten klar diffusionskontrolliert, hat aber bei
höheren Flussraten schnell zu perfusivem Massentransport gewechselt. In Perfusion fliesst Eluent
konvektiv durch die Chromatographiepartikel und macht sie dadurch sehr ähnlich zu den oben
beschriebenen Monolithen. Diese komplexen kinetischen Effekte wurden mit einem speziellen
chromatographischen Modell, das Fluss durch Partikel berücksichtigt, untersucht. Es wurde sehr
starkes perfusives Verhalten gefunden und durch die gute Verbundenheit der Poren erklärt, welche
wiederum von der fraktalen Struktur der Partikel her rührt. Aufgrund der guten Resultate in den
ionenchromatographischen Tests wurde eine Methode entwickelt um dieses Material mit höherer
Produktivität herzustellen. Dazu wurde der Aggregationsmechanismus komplett überholt und auf
kontinuierliche Produktion und Aggregation durch Scherkräfte anstatt Erhöhung des Salzgehaltes
umgestellt. Dieser Prozess und das resultierende Material wurden im Detail mit chromatographischen
und nicht-chromatographischen Methoden untersucht, wobei sehr ähnliche Materialeigenschaften
festgestellt wurden. Dieser letzte Abschnitt hat die industrielle Herstellung dieser interessanten
Materialien möglich gemacht.
vii
Acknowledgements
I would like to thank Massimo Morbidelli and my supervisor Giuseppe Storti for the opportunity and
pleasure of working on such an interesting, interdisciplinary project and the helpful and encouraging
discussions throughout my stay. The entire Morbidelli group has been a great source of inspiration at
work and enjoyment outside of it, I could not have asked for a better work environment. Thanks also
to Christophe Copéret for co-refereeing my thesis. I would also like to thank my family for their
continuing, invaluable support under all circumstances. Special thanks go out to my good friends in
the politically incorrect Friday lunch group who never cease to amaze me.
Chapter 1
viii
Contents
Chapter 1 Introduction ......................................................................................................................... 1
Chapter 2 Strong Cation Exchange Monoliths for HPLC by Reactive Gelation ................................. 5
2.1 Abstract ................................................................................................................................... 5
2.2 Introduction ............................................................................................................................. 6
2.3 Experimental ........................................................................................................................... 7
2.3.1 Materials .......................................................................................................................... 7
2.3.2 Equipment ....................................................................................................................... 7
2.3.3 Latex Preparation............................................................................................................. 8
2.3.4 Monolith Preparation ....................................................................................................... 9
2.3.5 Monolith Housing and Functionalisation ...................................................................... 10
2.3.6 Characterisation ............................................................................................................. 10
2.4 Results and Discussion .......................................................................................................... 11
2.5 Conclusion ............................................................................................................................. 15
Chapter 3 Modelling the chromatographic behaviour of Reactive Gelation monoliths and micro-
clusters………. ...................................................................................................................................... 17
3.1 Introduction ........................................................................................................................... 17
3.2 Model description .................................................................................................................. 18
3.2.1 Bed equation .................................................................................................................. 18
3.2.2 Particle equation ............................................................................................................ 18
3.2.3 Micro-particle equations ................................................................................................ 20
3.3 Model analysis ....................................................................................................................... 22
3.4 Experimental verification of assumptions ............................................................................. 24
ix
Chapter 4 Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase
for ion chromatography ......................................................................................................................... 27
4.1 Abstract ................................................................................................................................. 27
4.2 Introduction ........................................................................................................................... 28
4.3 Experimental ......................................................................................................................... 31
4.3.1 Materials ........................................................................................................................ 31
4.3.2 Equipment ..................................................................................................................... 31
4.3.3 Primary Particle Preparation .......................................................................................... 32
4.3.4 Aggregate Preparation ................................................................................................... 32
4.3.5 Characterisation ............................................................................................................. 34
4.4 Results and Discussion .......................................................................................................... 35
4.5 Conclusion & Outlook ........................................................................................................... 44
Chapter 5 Shear-Induced Reactive Gelation ...................................................................................... 47
5.1 Abstract ................................................................................................................................. 47
5.2 Introduction ........................................................................................................................... 48
5.3 Experimental ......................................................................................................................... 50
5.3.1 Materials ........................................................................................................................ 50
5.3.2 Primary Particle Preparation .......................................................................................... 50
5.3.3 Aggregate Preparation ................................................................................................... 52
5.3.4 Characterisation ............................................................................................................. 53
5.4 Results and Discussion .......................................................................................................... 55
5.4.1 Method to obtain phase diagram ................................................................................... 56
5.4.2 Effect of pressure and residence time ............................................................................ 58
5.4.3 Effect of salt .................................................................................................................. 60
Chapter 1
x
5.4.4 Effect of primary particle size ....................................................................................... 61
5.4.5 Effect of post-polymerisation ........................................................................................ 62
5.4.6 Chromatographic characterisation ................................................................................. 64
5.5 Conclusion ............................................................................................................................. 66
Chapter 6 Conclusions and Outlook .................................................................................................. 67
Chapter 7 List of Figures ................................................................................................................... 71
Chapter 8 Bibliography ...................................................................................................................... 73
Chapter 9 Curriculum Vitae ............................................................................................................... 75
1
Chapter 1
Introduction
Proteins are widely used as therapeutic agents in the treatment of various disorders and diseases due to
their high specificity and low immunogenicity. Over 200 commercial products compose a $125bn
market that is expected to grow at 11% p.a. until 2017 [1]. Genetically engineered monoclonal
antibodies, the flagship of this group of drugs, use the same mechanism as our own immune system to
target any harmful entity in our body. Treating cancer by programming antibodies to attack the
afflicted cells has a very high success rate even for very complicated cancers in the metastatic stage
[2]. Consequently, the demand for more and better antibody drugs is high.
These proteins are prepared in cell cultures yielding the active ingredient in concentrations of less than
one percent. Consequently the cost of separation and purification amounts to as much as eighty
percent of the production cost [3]. Being able to run these chromatographic separation steps at higher
flow rates would save significant capital and operating expenses by downsizing the equipment and by
using less of the disposable chromatographic media, respectively. High through-put applications will
become even more important once industry's attempts at continuous upstream manufacturing succeed,
a break-through expected for a number of products in the coming ten years. This period will also see
strong growth in bio-similars (protein generics), making the production costs much more relevant for
the sale price. Similar improvements can be found during the development of the downstream process
where the screening of operating conditions can be accelerated by running experiments in shorter time.
Currently the polymeric chromatography media market can be subdivided into particle, monolith and
membrane type. Although the latter two exhibit largely flow rate-independent separation performance,
particles are still the predominant stationary phase. This can be attributed to their versatility; particles
possess most degrees of freedom in their production and thus allow for better optimisation to the
separation task. Monoliths and membranes obtain their good mass transport properties from a narrow
pore size distribution that results in uniform flow through the monolith. Particles, on the other hand,
usually exhibit two pore size distributions: one between the particles and one inside them. For gel
Chapter 1
2
particles, these pores are rather small whereas rigid polymer particles show pores that can be only a
few tens of nanometres or as large as a few hundred nanometres. These are classically prepared in
suspension polymerisation in presence of a so-called porogen, which is a solvent for the monomer and
non-solvent for the polymer. This way, during the polymerisation the polymer and porogen phase
separate and form pores. Quality and quantity of porogen can tune the pore morphology [4].
Chromatographic applications of large biomolecules like antibodies require rather large pores to allow
unrestricted access to the functional groups on their surface. By geometric constraints this imposes a
low specific surface area onto the material. A number of different functionalisations have been
developed to compensate for this loss in surface area by adding smaller pores. For example, grafting
short brushes of functional groups onto the surface creates small ‘pores’ between these brushes that
significantly increase the functional density. Alternatively, hypercrosslinking reactions can be
performed that produce a highly porous polymer network that can exhibit specific surface areas of up
to 600 g/m² [5].
This thesis describes the path to a new kind of stationary phase in a chronological manner. The
production methods for this material are, in one form or another, Reactive Gelation as first presented
by Marti et al. [6]. In this process, nanoparticles are aggregated to form highly porous clusters that can
range from only tens of micrometres to slabs of material ten centimetres large. Both materials exhibit
very similar internal morphology with macropores from hundreds of nanometres to micrometers in
diameter.
When placed in a housing, the slabs can serve as chromatographic stationary phases [7-8]. These
materials are commonly referred to as monoliths and have found increasing interest in the
chromatographic community in the last ten to twenty years because of their special mass transport
mechanism. Given a narrow enough pore size distribution and an interconnected pore network, it is
clear that a pressurised liquid will flow through all pores at similar velocity with mixing nodes at pore
junctions. This has two effects on the mass transport: a plug flow establishes across the monolith due
to frequent mixing between pores, leading to a very sharp hydrodynamic pulse response at the outlet.
The absence of diffusive pores, an untypical case in chromatography, then leads to no mass transport-
Introduction
3
related peak broadening. The result are very sharp, flow rate-independent peaks. This attractive
column efficiency lead to an industry collaboration that gave a direction to this thesis, focusing it on
industrially relevant and feasible methods.
Such a monolith was prepared and functionalised as a strong cation exchanger in Chapter 2. Its ability
to separate proteins was verified, however it could not separate small cations like sodium, potassium
etc. as was desired by the industry partner. This was attributed largely to too short columns and since
there were mechanical problems when lathing long, thin monolith rods, a considerable amount of time
was spent trying to prepare monoliths directly inside a column. However, this work yielded no
successful prototypes because of shrinkage during the post-polymerisation step, resulting in
channeling between the column housing and monolith.
During this time, another method was developed in our group in which Reactive Gelation was carried
out under shear, thus forming particulate material instead of monoliths. However, this material lacks
one of the advantages described earlier, the monodisperse diameter of channels available for flow: it
can pass between the particles or through them. This can lead to a worse hydrodynamic behaviour and
widening of the pulse response; on the other hand it is going to reduce pressure drop along the column
with wider channels available for use. It was less clear if the mass transport in and out of the particles
would worsen the peak shape, too. The large pore diameters of this material, similar to the monolith,
can result in so-called perfusive mass transport, which means that there is convective flow through the
particles that speeds up mass transport considerably. Before adapting the method to prepare particulate
material, this effect was investigated using a numerical model in Chapter 3.
Having obtained positive results from simulations, particles were produced accordingly in Chapter 4.
By preparing longer columns, first successful results were found. However, the total column capacity
was too low for small ion separations due to low surface area. Not being able to increase the surface
by the required factor, a different functionalisation was used to increase the amount of functional
groups per surface area. Charged gel nanoparticles were attached to the surface and the functionality
thus extended into the third dimension into the pores. This resulted in high enough functional density
to successfully separate ions with this new material.
Chapter 1
4
In Chapter 5, the original Reactive Gelation under shear procedure was completely reworked to be
able to quickly produce large amounts of this material. The aggregation mechanism was changed to be
shear-initiated and the aggregation process made continuous. Large amounts of precursor latex were
prepared in an easy to scale up emulsion polymerisation so that the whole process can be scaled up to
several cubic metres per day without major effort, opening up application fields beyond specialty
materials.
5
Chapter 2
Strong Cation Exchange Monoliths for HPLC by Reactive
Gelation
(this chapter was partially published in Journal of Separation Science, 2011, 34, p.2159)
2.1 Abstract
Polymeric monolithic stationary phases for HPLC can be produced by Reactive Gelation. Unlike the
conventional method of using porogens, such novel process consists of a number of separate steps,
thus enabling a better control of the quality of the final material. A suspension of polymer
nanoparticles in water is produced and subsequently swollen with hydrophobic monomers. The
particles are then destabilised (usually by salt addition) to make them aggregate into a large
percolating structure, the so-called monolith. Finally, the added monomer can then be polymerised to
harden the structure. In this chapter, a polystyrene latex is used as base material and functionalised by
introduction of epoxide groups on the surface and subsequent reaction to sulphonic acid groups,
yielding a SO3- density of 0.7 mmol/g dry material. Morphological investigations show 54% porosity
made of 300 nm large pores. Van Deemter measurements of a large protein show no practical
influence of diffusion limitations on the plate number. Finally, a separation of a test protein mixture is
shown, demonstrating the potential of using ion-exchange chromatography on Reactive Gelation
monoliths.
Chapter 2
6
2.2 Introduction
The separation of therapeutic proteins from their host cell proteins has kept gaining importance with
the progress made in biotechnology; in the production of monoclonal antibodies, the purification is
still the most important production cost factor at 50-80% and includes several time- and cost-intensive
chromatographic steps for the capture and separation of the antibody from the host cell proteins [3].
The typical stationary phase for these separations, porous particles of diameters in the micrometre-
range, can only provide a quite poor compromise of through-put and peak sharpness due to diffusive
resistances inside the particles [9-10]. Monolithic columns promise to be better suited for the
separation of proteins because of their superior mass transport kinetics.
Polymeric monolithic columns are classically produced by heterogeneous bulk polymerisation
employing a porogen, which is a solvent for the monomer, but not for the polymer [11]. At the start of
the reaction, the system is homogeneous. During the course of the reaction polymer chains precipitate
to form highly cross-linked particles that aggregate and grow together as the polymerisation
progresses, building a continuous, highly porous structure. The so-formed monoliths possess a broad
pore size distribution, ranging from micron-sized channels to small mesopores inside the polymer
matrix itself [12]. Marti et al. presented an alternative route to the synthesis of polymeric porous
materials, the “Reactive Gelation” process [6]. In Reactive Gelation, the stages that occur at once in
the porogen method are separated in time in order to control them independently. First, a polymer
particle suspension (“latex”) is prepared. The polymer particles of the size of tens to hundreds of
nanometres are swollen with monomer in the second step. Then, their electrostatic stabilisation is
weakened by salt addition, leading to aggregation and finally to gelation. The resulting monolith is
held together only by weak van der Waals-forces; however, the added monomer can then be
polymerised to form strong, covalent bonds between the particles, hardening the structure. In this
chapter, the preparation and characterisation of a strong cation exchange monolith using Reactive
Gelation will be shown.
Strong Cation Exchange Monoliths for HPLC by Reactive Gelation
7
2.3 Experimental
2.3.1 Materials
The following chemicals have been employed in the work: 2-2’-azo(2-methylpropionitrile) (AIBN,
Fluka, purum), divinylbenzene (DVB, Aldrich, 80% technical), ethylene glycol dimethacrylate
(EGDMA, Merck, for synthesis), glycidyl methacrylate (GMA, Merck, for synthesis), hydrochloric
acid (HCl, Sigma-Aldrich, ≥37% purum p.a.), hydrogen chloride Titrisol (Merck, 0.1000 mol/L +/-
0.2%), methyl methacrylate (MMA, Aldrich, 99%), potassium persulphate (KPS, Fluka, puriss p.a.),
sodium chloride (VWR, 99.9%), sodium dodecyl sulphate (SDS, Fluka, ≥98%), sodium hydroxide
Titrisol (Merck, 0.1000 mol/L +/- 0.2%), sodium phosphate dibasic anhydrous (Sigma-Aldrich,
≥99.0% puriss p.a.), sodium phosphate monobasic anhydrous (Fluka, ≥99.0% purum p.a.), sodium
sulphite anhydrous (Fluka, puriss p.a.), styrene (Fisher Scientific, general purpose grade),
tetrabutylammonium hydrogensulphate (TBAHS, Merck, for synthesis). All chemicals have been used
as supplied without further purification. Ultra-pure grade water for chromatography has been prepared
by Millipore Synergy (Millipore, Billerica, MA, USA). Deionised water for synthesis has been
stripped of oxygen by degassing under vacuum and subsequent saturation with nitrogen gas.
2.3.2 Equipment
Chromatographic characterisation was carried out on an Agilent Series 1200 (Agilent Technologies
Santa Clara, CA, USA) equipped with a quaternary pump and degasser, an autosampler with
integrated cooling, a refractive index detector and a diod array detector. Van Deemter experiments
were conducted on an Äkta Basic P-903 (GE Healthcare, Little Chalfont, UK) equipped with UV-VIS
900 and conductivity/pH 900 units. SEM pictures were taken using a Gemini 1530 FEG (Carl Zeiss
AG, Oberkochen, Germany). Dynamic light scattering measurements were done on a Zetasizer nano
ZS 3600 (Malvern Instruments, Malvern, Worcestershire, UK). Thermogravimetric analysis was done
on a HG53 Halogen Moisture Analyzer (Mettler Toledo, Greifensee, Switzerland). A Hitachi L-7100
pump (Hitachi, Tokyo, Japan) was used for the semi-batch latex preparation.
Chapter 2
8
2.3.3 Latex Preparation
The monolith precursor latex was produced in two steps. In the first step a 10% cross-linked core is
prepared which is used as a seed in the second step, a seeded emulsion polymerisation. There, a 1%
cross-linked shell is prepared around the cores. Both steps are carried out in a semi-batch mode with
slow monomer feed in order to achieve a radially more homogeneous cross-linkage than would occur
in batch due to the different reactivities of divinylbenzene and styrene.
The core particles are produced by semi-batch emulsion polymerisation under nitrogen atmosphere. A
three-neck round-bottom flask is initially charged with water and surfactant (SDS) according to the
recipe reported in Table 2-1 (initial charge 1, IC1). The temperature is set to 70°C using an oil bath. In
a second flask, an emulsion of styrene, divinylbenzene, water and surfactant (SDS) is prepared
according to Table 2-1 (continuous feed 1, CF1) and kept emulsified using a stirrer. When the reactor
temperature reaches 70°C, aqueous initiator (KPS) solution is injected through a septum into the
reactor using a syringe and hypodermic needle according to Table 2-1 (Initiator solution 1, IS1) and
the emulsion is fed at 0.15 mL/min. The reaction progress is monitored with thermogravimetric dry
content analysis and dynamic light scattering. The reaction is stopped when reaching a particle size of
50 nm. The shell around the core is prepared with the same procedure, adding the seed latex in the
initial charge.
Strong Cation Exchange Monoliths for HPLC by Reactive Gelation
9
Table 2-1 Recipe for the production of the precursor core/shell latex. All numbers are target values, the actual values
varied slightly.
Core Particles Shell
IC1 CF1 IS1 IC2 CF2 IS1
H2O / g 65.0 25.0 10.0 190 65.9 10.0
Styrene / g 22.5 65.2
DVB / g 2.5 0.7
SDS / g 0.4 0.4 0.9 1.1
KPS / g 0.2 0.7
Seed latex / g 29
Diameter / nm 50 100
2.3.4 Monolith Preparation
The next step towards making a monolith is swelling the latex with a mixture of monomer, cross-
linker and initiator. For swelling, a mixture of glycidyl methacrylate (60 wt%), ethylene glycol
dimethacrylate (10 wt%), methyl methacrylate (29 wt%) and AIBN (1 wt%) was used. The desired
amount of latex is mixed with 20 wt% of swelling solution (respective to the polymer weight in the
latex) for at least 8 hours.
Before the latex can be gelled, the sodium chloride concentration in water leading to gelation in about
twenty minutes has to be determined. For this purpose, small amounts of swollen latex are mixed 1:1
with salt solutions of different concentrations until the desired aggregation speed is obtained. The gels
are then produced by slowly adding 3 mL of the salt solution to 3 mL of swollen latex in a pill flask
under vigorous stirring using a vortexer. The flask is then closed and left still for six hours.
The gel produced above is post-polymerised and hardened by heating it to 50°C in an oven for twenty-
four hours, leading to shrinkage of the gel. Afterwards, the monolith is washed in a water bath,
renewing the water several times over a period of one day.
Chapter 2
10
2.3.5 Monolith Housing and Functionalisation
In order to use the monoliths for HPLC, they are accommodated in a PEEK CIM Disk housing (BIA
Separations, Ljubljana, Slovenia). For this purpose, the monolith is fitted in a PEEK ring by lathing
the monolith to a 3° angle cone and the ring to its counter-piece. The monolith is press-fit into the ring
and cut planar on the fronts. The length of the used monolith is 8 mm, its larger diameter 10 mm. The
PEEK ring was prepared to an outer diameter of 16 mm to fit the housing.
The functionalisation is subsequently carried out by placing the housing inside a thermostat at 65°C
and circulating 25 mL of a solution 1 mol/L in sodium sulphite and 0.2 mol/L in tetra-n-
butylammonium hydrogensulphate for four hours at a flow rate of 0.5 mL/min [13]. The monolith is
then cleaned by flushing it with 30 mL of water at a flow rate of 0.5 mL/min at room temperature.
2.3.6 Characterisation
Mercury intrusion porosimetry was used to assess the pore size distribution, assuming cylindrical
pores [14]. Nitrogen adsorption was used supplementary to assess the pores below 70 nm size. The
Barret-Joyner-Halenda (BJH) equation was used to calculate the pore sizes from nitrogen adsorption,
also assuming cylindrical pores [15].
The chromatographic properties of monoliths are determined using van-Deemter measurements,
titration of the surface charge density and the separation of a protein mixture containing α-
chymotrypsinogen, cytochrome C and lysozyme. Unless stated otherwise, the eluent used was 25
mmol/L Na2HPO4-NaH2PO4 buffer at pH 6 with up to 1 mol/L NaCl. Chromatography was carried out
at 25°C. Samples were prepared in the adsorbing buffer that was used during their analysis.
The dependency of the height equivalent of a theoretical plate (HETP) on the flow rate was measured
by injecting pulses of immunoglobulin G (Erbitux, Merck, Darmstadt, Germany) under non-adsorbing
conditions (0.25 mol/L NaCl). HETP were calculated from the following formula [16]:
2
t
R
HETP cLt
( 2-1 )
where σt is the standard deviation of the peak, tR its retention time and Lc the column length.
Strong Cation Exchange Monoliths for HPLC by Reactive Gelation
11
The functional density of sulphonic acid groups is determined by titration using a method presented by
Stone and Carta [17] which was modified for flow-through in order to be non-invasive and provide
quick mass transfer kinetics [18]. For this purpose, the monolith is connected to a HPLC pump; the
flow rate is 1 mL/min throughout the experiment. The column is first acidified with 60 mL 0.5 mol/L
aqueous hydrogen chloride solution (HCl) and then cleaned with 60 mL water. Then, 25 mL 0.5 mol/L
NaCl 0.1 mol/L NaOH (Titrisol) are circulated through the system for two hours to convert the
sulphonic acid groups into the Na-form. 25 mL water are then used to flush the entire contents out of
the system into the reservoir used for circulation. The total amount of NaOH in the system is back-
titrated using 0.1 M HCl (Titrisol) and bromothymol blue as indicator. The functional density can then
be calculated using the following equation:
3
NaOH NaOH HCl HCl
SOMonolith
V c V cc
m
( 2-2 )
where VNaOH is the amount of NaOH solution circulated (0.025 L), cNaOH is the concentration of NaOH
in this solution (0.1 mol/L), VHCl the volume of HCl solution used for titration, cHCl the concentration
of HCl used for titration (0.1 mol/L) and mMonolith the dry mass of the examined monolith.
2.4 Results and Discussion
The aim of this chapter was to demonstrate that Reactive Gelation monoliths can be used for ion
exchange chromatography. Working towards the goal of separating monoclonal antibodies, the pore
structure was chosen to be accessible to large molecules and a strong cation exchange
functionalisation was introduced.
A first impression of the material is readily obtained from the SEM picture in Figure 2-1. As expected,
the particles fused with their soft shells but can still be identified. The pores are a few hundred
nanometres in diameter and appear well inter-connected.
Chapter 2
12
Figure 2-1 SEM picture of a Reactive Gelation monolith.
To quantify the observations made from the picture, the monolith morphology was examined using
mercury intrusion porosimetry. The porosity as a function of the pore diameter has been calculated
assuming cylindrical pores (see Figure 2-2), yielding a single porosity with a diameter of around 300
nm. This can be attributed to the core-shell type particles used; while rigid spheres form gaps between
them in a dense packing, the latex particles interpenetrate enough to fill this interparticle space. Below
70 nm, nitrogen adsorption was used in addition to mercury intrusion. Since nitrogen adsorption can
only measure pores smaller than the main porosity, their trend was used to continue the Hg-
porosimetry curve using its absolute value. The surface area was also calculated from nitrogen
adsorption using BET theory, yielding 15 m2/g.
Strong Cation Exchange Monoliths for HPLC by Reactive Gelation
13
Figure 2-2 Pore size distribution of a Reactive Gelation monolith. The secondary y-axis shows the percentage
contribution of each data point to the total pore volume. Squares correspond to mercury intrusion measurements,
diamonds to nitrogen adsorption measurements.
The dependence of the HETP upon the linear velocity u is given by the van Deemter equation:
HETPB
A C uu
( 2-3 )
The typical curve HETP vs. u is decreasing from infinity at u=0 to a minimum at a certain u, from
where it starts linearly increasing with a final slope of C.
Reactive Gelation monoliths show no influence of diffusive resistance, as can be seen from the typical
van Deemter plot exhibited by these materials shown in Figure 2-3. Therefore C is practically zero and
the curve asymptotically approaches A, given by Eddy diffusion. The consequence is that, contrary to
standard packings, increasing the flow rate leads to better or equal separation performance while it
increases through-put. At high flow rates, 15’300 plates/m can be achieved on an Äkta Basic.
The van Deemter plot also shows the feasibility of the conical press fitting technique introduced in the
Experimental section. There is a slight acceleration of the stationary phase along the bed length that
varies according to monolith length, diameter and angle; for the dimensions used in this work it is
about 10%. However, by operating in the nearly flat region of the van Deemter curve the acceleration
10 100 1000 100000.0
0.1
0.2
0.3
0.4
0.5
0.6
Cu
mu
lati
ve P
oro
sity
/
Pore Size / (nm)
0
5
10
15
20
Rela
tiv
e P
ore
Vo
lum
e /
(%
)
Chapter 2
14
stays unnoticed in the separation. While this simple housing technique works quickly and reliably on
the lab scale, long columns are rather difficult to produce because the cone angle has to be decreased,
increasing the impact of manufacturing deviations significantly.
Figure 2-3 Van Deemter plot for IgG on Reactive Gelation monoliths. Eluent: 25 mmol/L Na2HPO4-NaH2PO4 buffer
at pH 6 with 0.25 mol/L NaCl. The experiment was conducted on an Äkta Basic.
Having a suitable base material, a surface functionality is introduced. During the swelling, an epoxide
group is introduced, providing a highly versatile precursor group for reaction towards a number of
functional groups, including strong cation exchangers (SO3-) and strong anion exchangers (QA) [19-
20]. The monomer used to introduce the epoxide group is glycidyl methacrylate (GMA). This
chemical is about ten times as expensive as styrene and therefore a significant material cost factor in
the preparation of a stationary phase. However, in Reactive Gelation only the shell (the thickness of
which can be varied) is swollen with GMA, wasting little expensive monomer inside the matrix where
it cannot be used for functionalisation.
The functionalisation is characterised by determining the capacity of Na+ on the dry stationary phase
using titration. A functional density of 0.7 mmol Na+/g dry material was measured. CIM SO3 Disk
(BIA Separations, Ljubljana, Slovenia) feature 2.2 mmol/g dry support (technical data sheet, retrieved
June 2011). We attribute our lower value to the fact that we only introduced epoxide groups via
0 200 400 600 8000.0
0.1
0.2
0.3
0.4
HE
TP
/ (
cm
)
Linear Velocity / (cm/h)
Strong Cation Exchange Monoliths for HPLC by Reactive Gelation
15
swelling with glycidyl methacrylate. Additional epoxide groups possibly could be introduced by
preparing a thin latex shell with GMA.
Figure 2-4 shows the separation of an analytical amount of a protein mixture to demonstrate the
concept of using Reactive Gelation monoliths in ion-exchange mode. To achieve desorbing conditions
for lysozyme, 1 mol/L NaCl was necessary, reflecting the high functional density measured by
titration.
Figure 2-4 Separation of a protein mixture. A 25 mM phosphate buffer at pH 6 was used with a gradient in NaCl
concentration. Flow rate was 1 mL/min.
2.5 Conclusion
When Marti et al. [6] first proposed the Reactive Gelation process, the aim was to produce monoliths
with a higher degree of control than the conventional porogen method. They showed they could
prepare fundamentally differently structured monoliths by properly changing selected process
parameters. Bechtle et al. [7] expanded the work by changing the base matrix to PMMA instead of
polystyrene, disclosing the use of the material for hydrophobic interaction chromatography.
This work is headed towards the preparation of an ion exchange material that can be used for the
purification of biopharmaceuticals. The process conditions were chosen such that a narrow porosity of
high-diameter pores was achieved, providing access to the surface even for very large biomolecules.
0 5 10 150
20
40
60
80
100
120
140
160
Ab
sorb
an
ce (
28
0 n
m)
/ (m
AU
)
Time / (min)
0.0
0.5
1.0
cN
aC
l / (
mo
l/L
)
-Chymo-
trypsinogen
Cytochrome C
Lysozyme
Chapter 2
16
Because basically only one type of pores exists, all pores are convective, showing no influence of
diffusion on the separation process and thus enabling high-throughput operation without loss in plate
count. A strong cation exchange functionality was readily introduced via epoxide groups on the
surface. Additionally, a number of other surface groups are possible in principle. The sulphonated
monolith prepared in this chapter was used to separate a model mixture of three proteins on a column
length of 8 mm, demonstrating the feasibility of Reactive Gelation monoliths for bio-separations.
17
Chapter 3
Modelling the chromatographic behaviour of Reactive
Gelation monoliths and micro-clusters
3.1 Introduction
In Chapter 2, the feasibility of Reactive Gelation monoliths for cation exchange chromatography on
proteins was demonstrated. Another project goal, the analytical separation of small ions, confronts a
stationary phase with a different separation task. In contrast to proteins, the molecules adsorb only
weakly onto ion exchangers, however they usually come in a cleaner matrix and diffuse roughly two
orders of magnitude faster.
Initially, a strong cation exchange monolith from the previous chapter was used to assess the
material’s capability in this area. Aqueous potassium chloride solution was eluted using 3 mM HNO3,
however the monolith failed to exhibit any separation of water and potassium. Combining specific
surface area measurements and ion exchange group titration, it was found that our functionalisation
was similarly efficient in introducing charged groups onto a surface. Limited by short column length
and comparably low specific surface area, the monolith only reached a hundred times lower functional
group count per column than typical commercial columns. Longer monoliths, of more than ten
centimetres, proved difficult to house reliably because of deviations in the turning process. Preparation
of monoliths directly inside their housing was unfeasible, too, because of the material shrinking during
post-polymerisation. In the PhD thesis of A. Lamprou (ETH Zurich) [21], a process is described in
which Reactive Gelation is carried out under shear, producing monolith fragments termed ‘micro-
clusters’, that share most of the monolith’s original properties like fractal mass scaling and very large
pores. Because this material can be handled easier than monoliths during functionalisation and column
packing, its feasibility for analytical ion chromatography is assessed in this part of the work. For this
Chapter 3
18
purpose, a model published by Carta and Rodrigues [22] was applied to monoliths, micro-clusters and
regular packing and the results compared.
3.2 Model description
Chromatographic models usually separate thermodynamic and kinetic effects, and distinguish the
latter between bed and particle mass transport kinetics. Whereas the mass transport in the bed is
realised convectively, molecules diffuse in and out of the particles. However, if the particle pore
diameter is large enough, a small fraction of the convective flow can pass through the particles, too.
This phenomenon is termed ‘perfusion’ and leads to peculiar mass transport kinetics that shall be
explored using Carta and Rodrigues’ model.
3.2.1 Bed equation
The model equation for the bed is not affected by perfusion and thus the ‘normal’ chromatography
equation is used:
2
2(1 )
( 0, ) 0
( , 0) ( )
( , ) finite
ax
c c c qD v
Z Z t t
c t Z
c t Z Q t
c t Z
( 3-1 )
where is the bed porosity, axD the axial dispersion coefficient, c the liquid phase concentration
outside the particles, Z the axial dimension of the column, t the time and q the average
concentration inside a particle (the reason for the double average is explained below). As can be seen
from the equation, this model accounts for axial mixing and accumulation in the stationary phase.
3.2.2 Particle equation
The particles in this model are described phenomenologically like a spherical packed bed (see Figure
3-1), consisting of ‘micro-particles’ and void space between them. These micro-particles can be
porous but also solid and are described later.
Modelling the chromatographic behaviour of Reactive Gelation monoliths and micro-clusters
19
Figure 3-1 Geometric description of a resin particle. It is composed of several micro-particles with interconnected
void space between them. η is the dimensionless axial coordinate and R the radial coordinate.
The particle model has to account for convection in direction of the column axis Z as well as radial
diffusion. Particles thus lose their spherical symmetry and need to be described using two internal
coordinates, R and or z . Consequently the equation describing the particles is quite similar to the
equation describing the bed, only without the axial dispersion but with a more complicated mass
transport term due to the two coordinates:
2 2
2 2
1 ' 1 ' ' '' (1 ) ' ' (1 ')
'( 0, , ) 0
'( , 0, ) finite
'( , , ) ( , )P
c c c c qD R u
R R R R z t t
c t R z
c t R z
c t R R z c t z
, ( 3-2 )
where 'D is the diffusion coefficient inside the particles, R the radial coordinate, z the axial
coordinate, the axial coordinate normalised by the particle radius, 'c the concentration in the liquid
phase, 'u the linear velocity inside the particle, ' the porosity between the ‘micro-particles’ and q
the average concentration inside the micro-particles.
Chapter 3
20
3.2.3 Micro-particle equations
The final and smallest entity left to be described are the microparticles composing the particles.
Composing particles out of smaller particles is not far from reality both for particles originating from
the porogen method or Reactive Gelation. In the porogen method, the precipitating polymer nucleates
into particles that later fuse together; in Reactive Gelation the particles are actually prepared from
spherical latex nanoparticles. Whereas the porogen method yields particles with a certain porosity
[12], Reactive Gelation uses latex from emulsion polymerisation which is non-porous and thus no
transport in and out of the micro-particles takes place (see Figure 2 in [8]). The transport in porous
micro-particles is described using a sol-model extended for porous media:
2
2
2c
q q qD
r r r t
, ( 3-3 )
where cD is the effective diffusion coefficient, q the micro-particle concentration and r the micro-
particle radial coordinate. The porosity is lumped into the effective diffusion coefficient according to
"
" (1 ") 'c
DD
K
, ( 3-4 )
where "D is the diffusion coefficient in the micro-particle pores, " the micro-particle porosity and
'K the distribution coefficient describing the thermodynamic equilibrium between the solid and
liquid phase inside the pores according to
' ' "q K c , ( 3-5 )
where 'q is the concentration inside the solid fraction of the micro-particles and "c the liquid phase
concentration inside the micro-particle pores. For the purpose of using a sol model, these quantities are
lumped into a micro-particle concentration q according to
" " (1 ") 'q c q . ( 3-6 )
These equations have been solved by Carta and Rodrigues using Laplace transformation, assuming
Dirac pulse injections. Using moments, the average residence time and HETP can be analytically
expressed:
Modelling the chromatographic behaviour of Reactive Gelation monoliths and micro-clusters
21
1
11 '
Lb
v
with
1 '1
'b K
( 3-7 )
and
2 2
2 1
22 2
1
1 ' ' (1 ) / 1( )
2 30 1 ' (1 ) /p
p
L B b bh A f v
R b Tv b
, ( 3-8 )
in which v is the interstitial velocity, 2 Pv R v D the reduced velocity, L the column length, '
the tortuosity of the macro-pores, cT D D the ratio of diffusional times and ( )Pf the
augmentation factor. This augmentation factor is the key concept of the model as it directly influences
the C term of the van Deemter equation, i.e. the term describing the mass transport in and out of the
resin. The shape of this function has been derived for spherical particles by Carta et al. [23] and is
plotted in Figure 3-2.
Figure 3-2 Augmentation factor as a function of the Peclet number ' 2 'P P
u R D
The augmentation factor depends only on the intra-particle Peclet number ' 2 'P Pu R D , where 'u
is the intra-particle velocity and PR the particle radius. The intra-particle velocity is evaluated from
the permeability both of the bed and the particles themselves by modelling them as two parallel
resistances for the eluent flow according to ' P
B
Bu Fu u
B , where PB is the particle permeability
10-1
100
101
102
0.2
0.4
0.6
0.8
1.0
f(
P)
/ (
)
P / ()
Chapter 3
22
and BB the bed permeability. Both permeabilities can be described using the Karman-Cozeny
equation because they are approximated as regular packings of spherical particles with known particle
diameter and packing porosity:
32
B P2
1
37.5 (1 )B R
and
32
P micro2
1 '
37.5 (1 ')B R
, ( 3-9 )
in which microR is the micro-particle diameter that is often approximated as micro pore3R R from
geometric consideration of regularly packed beds, if more accurate measurements (e.g. by TEM of
particle slices) are unavailable and fitting is undesired.
The contribution of perfusion to the mass transport can thus be discussed with the help of the intra-
particle Peclet number. For low P the augmentation factor is approximately unity and equation
( 2-8 ) is reduced to the regular van Deemter equation. Increasing P beyond unity, the augmentation
factor soon decreases linearly with the Peclet number. Due to the proportionality of 'u to the flow
velocity, Peclet linearly increases with the column flow velocity. This means that the mass transport in
and out of the particles is quickened at the same rate at which the column flow velocity quickens; the
mass transport rate stays constant with respect to flow rate. If no other mass transport (e.g. diffusion in
the micro-particles or film diffusion) becomes limiting, the van Deemter plot reaches a plateau.
3.3 Model analysis
This analysis is conveniently carried out using the characteristic times of each mass transport step (the
mass transport chain is sketched in Figure 3-3).
Figure 3-3 Diagram of the mass transport chain occuring during chromatography. u characterises the mass transport
along the column, the parallel steps of perfusion and diffusion through the particle are described by u’ and D’
respectively and finally D” characterises the last diffusion step into the micro-particles.
Modelling the chromatographic behaviour of Reactive Gelation monoliths and micro-clusters
23
Initially, the solute is transported along the column at a linear velocity u , a process with characteristic
time c L u . From there, the solute enters the particles either diffusively 2
D' P 'R D or
perfusively P P 'R u . Finally, mass transport in the micro-particles is realised diffusively with
characteristic time 2
D" c "R D . Materials prepared from Reactive Gelation, be it monoliths or
particles, do not contain pores in the micro-particles and the last step can be excluded.
In Figure 3-4 the different combinations of characteristic times are associated with a group of
columns, ignoring van Deemter’s B term (the longitudinal diffusion) because this effect only shows at
very slow flow rates that are practically not interesting. The simplest of these groups is a monolith
with a narrow pore size distribution. Only one pore size is available, so all liquid flow has to pass
through it, making the mass transport in every pore perfusive independent of flow rate. The HETP is
thus constant at a value corresponding to the axial dispersion (van Deemter’s A) of the column. The
other extreme is termed ‘regular’ packed bed, meaning that the mass transport into the resin particles
is realised by diffusion. The permeability of the resin is low compared to the bed (most often due to
the small pore diameter and/or bad pore interconnectivity), so the fraction of flow going through the
particles is very low, yielding a large P . In these conditions, c is usually much lower than D' .
Lastly, there are perfusive resins. In these resins the particle permeability is so high that under
practical conditions, the intra-particle mass transport can become convective, i.e. P is initially larger
than D' , but is decreasing with flow rate and eventually becomes the quicker of the two parallel steps.
At the same time, c is linearly decreasing with flow velocity, but so is P , not changing the
relationships between characteristic times any more. It should be noted that if there were a last,
diffusive mass transport step like micro-particle diffusion or film diffusion on the walls of the through-
pores, this would affect the final slope of the graph, attributing it a value larger than zero and smaller
than the regular bed.
Chapter 3
24
Figure 3-4 Van Deemter plot of three resin classes, describing the relationships between characteristic times of the
mass transport chain.
3.4 Experimental verification of assumptions
From Chapter 2 it is known that Reactive Gelation materials exhibit a negligible fraction of pores
smaller than 50 nm, excluding the possibility of a last, diffusive step into the material for the mass
transport chain described above. However, there is still the possibility of limiting film diffusion on the
pore walls as well as a slow ion exchange step. Both of which were assessed by injecting KCl onto a
non-porous strong cation exchange resin, a Dionex Propac SCX with 10 μm particle size, under
adsorbing conditions using 3 mM HNO3 as eluent and the resulting HETP plotted in Figure 3-5. The
investigated range of flow velocities was chosen very conservatively; typically, values above 50 cm/h
are not encountered inside even very large pores. At these flow rates film mass transport and/or ion
exchange are very quick and their contribution to the final HETP of the prepared resin is going to be
negligible, as we expect its HETP between 0.01 and 0.1 cm. According to these findings, the
assumption of ignoring the last transport step in Reactive Gelation materials can be justified.
Modelling the chromatographic behaviour of Reactive Gelation monoliths and micro-clusters
25
Figure 3-5 Van Deemter plot of K+ for a non-porous 10 μm strong cation exchange resin under adsorbing conditions.
After having prepared a first test batch of Reactive Gelation particles, the model was fit to the
recorded HETP data from dextran injections up to 12 kg/mol into a 25 mM phosphate buffer at pH 7
and the results are shown in Figure 3-6. Good agreement with the experimental data could be found
using the following set of parameters:
P
Pore
0.4 (fixed)
' 0.6
1.5
49 μm
1 μm
0 cm (fixed)
R
R
A
Due to the irregular shape and broad particle size distribution, the particle radius cannot be fixed to a
hard value like the particle’s hydrodynamic radius. In chromatographic application, both the
permeability of the packing as well as the effective diffusion distances in and out of the particles play
a crucial role, so the value was fitted and should be viewed as the equivalent diameter of a sphere in a
packing for chromatography. Similarly, the pore radius is polydisperse and greatly influences the
particle permeability and thus the extent of perfusion. Large particle diameters result in a very steep
initial slope of the van Deemter curve, resulting in a huge error when fitting A – it was consequently
fixed as 0. It is becoming clear that the values obtained from fitting a rather ideal model to such
0 100 200 300 400 5000.000
0.001
0.002
0.003
HE
TP
/ (
cm
)
Linear velocity / (cm/h)
slope: 3.2 x 10-6
Chapter 3
26
irregular particles are hardly usable. However, we learned that even for rather small molecules
perfusion can have a strong beneficial effect on the column efficiency if the material is correctly
designed, as seen in Figure 3-6. In conclusion, the model proved invaluable in understanding the
nature of perfusion and guided us throughout the following chapters by showing behavioral trends and
the similarity of Reactive Gelation monoliths and particles.
Figure 3-6 Van Deemter plot of non-adsorbing dextran tracers on Reactive Gelation particles with the described
model fitted to it.
0 1 20.00
0.02
0.04
0.06
0.08
0.10
1 kg/mol
5 kg/mol
12 kg/mol
HE
TP
/ (
cm
)
Flow rate / (mL/min)
27
Chapter 4
Macro-porous latex-coated polymer particles from
Reactive Gelation as stationary phase for ion
chromatography
(this chapter was partially published in international patent application PCT/EP2013/003532 and is to
be submitted in this form to Journal of Chromatography A)
4.1 Abstract
This chapter describes the manufacturing of a macro-porous stationary phase for ion chromatography
using Reactive Gelation, a recently proposed process to prepare porous materials as aggregates (or
clusters) of colloidal polymer particles. When applied to chromatography, large cluster diameters lead
to very low backpressure. Moreover, the large pores induce the so-called ‘perfusive’ mass transport at
high flow-rates, making the column efficiency flow rate-independent at high linear velocities, thus
eliminating the drawback typical of large particle diameters. On the other hand, a small value of
specific surface area was obtained (25 m²/g), which would bring to unacceptably low ion-exchange
capacities. This major drawback has been effectively contrasted by electrostatic decoration of the
inside of the pores by anion-exchange nanoparticles. In this way, an ion exchange capacity of 39
μmol/mL has been achieved, a satisfactory value for ion chromatography. Finally, we demonstrate the
feasibility of the concept by separating seven standard anions on such column.
Chapter 4
28
4.2 Introduction
Macro-porous materials with large pores have gained considerable importance in the field of
chromatography, especially when used for separation and purification of biomolecules [24]. Different
forms of this type of packing materials are available, from monoliths to more conventional macro-
porous particles. Reactive Gelation has been recently proposed as a sequential procedure to produce
macro-porous media in the shape of monoliths [6, 8, 25]. In Reactive Gelation, the same process steps
that occur at once in the classical porogen method (e.g. [26]) are carried out in series to control them
independently. First, a colloidal suspension of polymer particles (the so-called “latex”) is prepared.
These primary polymer particles of the size of tens to hundreds of nanometres are swollen with
monomer and initiator in the second step. Then, their electrostatic stabilisation is weakened by salt
addition, leading to aggregation and finally to gelation of the whole system. A “weak” porous material
is obtained this way, since the latex particles are kept together mainly by van der Waals forces.
Therefore, the material is hardened in the last step by heating the system and post-polymerising the
earlier introduced monomer to polymer chains covalently linking the primary particles. This results in
a monolith that is mechanically stable enough to be used in high performance liquid chromatography.
These monoliths are characterised by high porosities of up to 75% (above which their mechanical
stability drops significantly) and narrow pore size distributions, with average pore sizes ranging from
few hundred nanometres up to micrometres. Notably, the final monolith exhibits negligible amount of
mesopores (< 50 nm), as shown in Figure 2 in [4].
The primary aim of this work was to synthesize the packing material for a low backpressure anion
exchange column with largely flow-rate-independent separation efficiency to be used for the analytical
separation of seven standard anions in water. Preliminary tests for small cations on our earlier
published monoliths [8] showed no retention because of the combination of two major issues, (1) short
column and (2) low capacity.
About the first issue, although we could prepare long monoliths, mechanical problems of housing
them reliably without channelling were encountered. Therefore, the original Reactive Gelation process
was modified in order to produce particles instead of monoliths by carrying out the last two steps
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
29
under shear [21] (the procedure is sketched in Figure 4-1). Namely, aggregation and post-
polymerisation are carried out in stirred tanks, leading to break-up of the gel and to the formation of
the final particles as aggregates of primary particles. In general, the final morphology of the clusters is
similar to that of our typical monoliths – the key difference, as will be shown later, is the fractal
dimension characterising their mass scaling. Since high porosity and large pore size are retained, flow-
rate-independent column efficiency at high linear velocities is anyhow expected [22]. Long columns
can thus easily be prepared by packing said particles as typically done for traditional chromatographic
materials, thus solving the issue of column length.
Figure 4-1 Process scheme of Reactive Gelation under shear
About the second issue, the reason for the low ion capacity of the monoliths is their large pore size:
while necessary for biomolecule separations to avoid exclusion, small molecule separations can be
done with much smaller pores and thus much higher surface areas, i.e. capacities. Using a packing of
particles instead of a monolith, we gain an additional degree of freedom for the column design: the
particle size. Therefore, there are two independent porosities instead of only one as for monoliths; the
intra-particle and inter-particle porosity. To retain the monolith’s flow-rate-independent column
efficiency even for small molecules in such a column, exceptionally large pores are required, as
described by Carta and Rodrigues [22]. The high particle permeability establishes a so-called perfusive
mode in which a significant, constant fraction of the liquid stream convectively flows through the
intra-particle pores. If the resulting convective transport inside the particles is quicker than the
diffusive mass transport, the intra-particle mass transport rate linearly scales with eluent velocity, thus
keeping the separation efficiency constant. Apart from the analyte diffusivity which we cannot change,
the occurrence of perfusion at a given flow rate depends on the pore size and the particle size itself,
Chapter 4
30
which determines the size of inter-particle space. If these two characteristic lengths become similar,
bed and particle permeability become also similar. The flow will then pass through and between the
particles to a comparable extent, thus mimicking a macro-porous monolith (which can abstractly be
regarded as a column where both intra- and inter-particle porosity and pore size are identical). We do
not really need to achieve such limiting condition: as stated before, to establish flow rate-independent
separation conditions it is sufficient for the convective intra-particle mass transport to be quicker than
the diffusive one at the operative flow rates; this can already be the case for few percent of the flow
permeating the intra-particle pores [22]. The larger the ratio of intra- to inter-particle channel size, the
earlier is the onset of perfusion.
Because it is essential for us to retain the flow-rate-independent column efficiency from the monolith
in the particle packing, large pores are required, posing us a challenge of low surface area. Typically
such problem is contrasted by introducing additional, smaller, usually diffusive pores, that are
connected to the large pores, e.g. via hyper-crosslinking [4]. Another method is covering the pore
walls with polyelectrolyte brushes, as is done for the very popular Fractogel resins by Merck. Both
ways would require careful material design so that the diffusion rate in and out of these secondary,
small pores or through the short brushes is faster than the mass transport by convection through the
particles: in fact, the column efficiency remains flow-rate-independent only in this case. In this work
another method is considered: a large amount of functional groups is attached to a low specific surface
area by “nanoparticle decoration”: namely, highly positively charged nanoparticles are
electrostatically bound to the inside of macropores, thus providing the required amount of functional
groups with very limited pore volume occupancy.
In this chapter we describe the evolution from a monolithic column capable of large molecule
separations to a perfusive particulate column capable of ion separations in accordance with the above
arguments. As explained, the monolith has two characteristics that make it unsuitable for ion
chromatography: its short column length and its low surface area. We solve the first by making the
support material in particulate form and the second by expanding the functionalisation into the pore
volume using decorating latex technique. While the monolith is intrinsically perfusive, here we also
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
31
have to answer the question how to transfer this essential property to the particles. To this end, we
thoroughly investigate the morphology and mass transport properties of these particles and separate a
mixture of seven standard anions present in drinking water (as defined for example in [27] and found
in column catalogues of Metrohm [28] and Dionex [29]).
4.3 Experimental
4.3.1 Materials
The following chemicals have been employed: 2-2’-azo(2-methylpropionitrile) (AIBN, Fluka, purum),
dimethylethanolamine (Fluka, ≥98.0% purum), divinylbenzene (DVB, Aldrich, 80% technical),
ethylene glycol dimethacrylate (EGDMA, Merck, for synthesis), glycidyl methacrylate (GMA, Merck,
for synthesis), nitric acid (Merck, 65%), potassium nitrate (Merck, for analysis), potassium
persulphate (KPS, Fluka, puriss p.a.), magnesium chloride chloride (VWR, 99.9%), sodium chloride
(VWR Prolabo, 99.8%), sodium dodecyl sulphate (SDS, Fluka, ≥98%), sodium hydroxide Titrisol
(Merck, 0.1000 mol/L +/- 0.2%), styrene (Fisher Scientific, general purpose grade), sulphuric acid
(Fluka, 95-97% puriss.). All chemicals have been used as supplied without further purification. Ultra-
pure grade water for chromatography has been prepared by Millipore Synergy (Millipore, Billerica,
MA, USA). Deionised water for synthesis has been stripped of oxygen by degassing under vacuum
and subsequent saturation with nitrogen.
4.3.2 Equipment
Chromatographic characterisation was carried out on a Metrohm 850 unit (Metrohm AG, Herisau,
Switzerland). SEM pictures were taken using a Gemini 1530 FEG (Carl Zeiss AG, Oberkochen,
Germany). Dynamic light scattering measurements were done on a Zetasizer nano ZS 3600 and static
light scattering was carried out on a Mastersizer 2000 (Malvern Instruments, Malvern, Worcestershire,
UK),. Thermogravimetric analysis was done on a HG53 Halogen Moisture Analyzer (Mettler Toledo,
Greifensee, Switzerland). Styrene latex synthesis was done in a Mettler Toledo Labmax with 4L
heating jacket glass reactor (Mettler Toledo, Greifensee, Switzerland). Ultrasonication was done
using a Digital Sonifier S-450D (Branson, Urdorf, Switzerland).
Chapter 4
32
4.3.3 Primary Particle Preparation
The primary particle latex was produced in two steps. In the first step a 20% cross-linked core is
prepared which is used as a seed in the second step, a seeded emulsion polymerisation. There, a 1%
cross-linked shell is grown around the core. Both steps are carried out in semi-batch mode with slow
monomer feed in order to achieve a radially more homogeneous cross-linkage than would occur in
batch due to the different reactivity of divinylbenzene and styrene [30].
The core particles are produced by semi-batch emulsion polymerisation under nitrogen atmosphere. A
4 L Mettler-Toledo LabMax is initially charged with water and surfactant (SDS) according to the
recipe reported in Table 1 (initial charge 1, IC1). The temperature is set to 70°C using the oil heating
jacket. In a second flask, an emulsion of styrene, divinylbenzene, water and surfactant (SDS) is
prepared according to Table 1 (continuous feed 1, CF1) and kept emulsified using a magnetic stirrer.
When the reactor temperature reaches 70°C, aqueous initiator (KPS) solution is injected through a
septum into the reactor using a syringe and hypodermic needle according to Table 1 (Initiator solution
1, IS1) and the monomer emulsion is fed at 1.5 mL/min. The reaction progress is monitored by
thermogravimetric dry content analysis and dynamic light scattering. The reaction is stopped when
reaching a particle size of about 100 nm. The shell around the core is prepared with the same
procedure, adding the seed latex in the initial charge.
4.3.4 Aggregate Preparation
The next step towards making micro-clusters is the latex swelling by monomer, cross-linker and
initiator. For swelling, a mixture of styrene (79 wt%), divinyl benzene (20 wt%), and AIBN (1 wt%) is
used. The desired amount of latex is mixed with 20 wt% of swelling solution (respective to the
polymer weight in the latex) for at least 4 hours. The critical coagulation concentration (ccc) of the
latex is roughly measured by mixing diluted latex and magnesium chloride solutions of different
concentrations and visually observing aggregation – the concentration at which aggregation occurs
within a few seconds is determined as ccc.
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
33
Table 4-1 Recipe for the production of the precursor core/shell latex. All numbers are target values, the actual values
varied by less than 1%.
Core Particles Shell
IC1 CF1 IS1 IC2 CF2 IS2
H2O / g 1200 539 100 166 100.0
Styrene / g 573 133
DVB / g 143 1.40
SDS / g 2.60 12.0
KPS / g 1.30 2.00
Seed latex / g 1600
Diameter / nm 105 (0.016 PDI) 120 (0.018 PDI)
The aggregation and post-polymerisation take place in a custom-made reactor with the following
characteristics: 280 mL volume, heating jacket, two cylindrical baffles, a four-blade Rushton-type
impeller and an overflow exit. First, the reactor is filled completely with latex at 1.5 wt% solid fraction
and then the stirring speed is set to 800 rpm. The aggregation is induced by slowly charging 10 mL of
MgCl2 solution at such a concentration as to reach a final MgCl2 concentration in the reactor 1.2 times
the ccc reported above. After 2 minutes, another 10 mL of MgCl2 solution (at concentration ten times
the concentration used before) are charged to be on the safe side with respect to destabilisation. After 4
hours of aggregation and breakage the reactor is heated to 70°C with the heating jacket and kept
stirring for another 4 hours, at which point the reaction is complete. The micro-clusters are now
discharged, cleaned twice with water and twice with ethanol using a cellulose filter and then cleaned
using analytical sieves. Large aggregates produced by fouling on the baffles and stirrer are removed
with a 200 μm mesh, then fines are cleaned off by washing the resin on a 20 μm mesh with plenty of
water. The particles are stored in slurry form.
Sulphonation of the polystyrene particles is then performed by slowly adding 200 mL of particle
suspension to 250 mL of gently stirred 98% sulphuric acid. This process is very exothermal and
should be done carefully; the usually preferred way of adding the acid to the aqueous solution
Chapter 4
34
produces large amounts of aggregates, however. The system is kept under stirring for 24 hours at
80°C. The particles are then thoroughly washed with water. This concludes the preparation of the
support material.
Anion exchange capacity is provided to the resin by electrostatically linking positively charged latex
particles to the pore surface. These nanoparticles are produced via emulsion polymerisation and are
subsequently aminated. The emulsion polymerisation is carried out in a 250 mL 3-neck round-bottom
flask equipped with magnetic stirrer and reflux condenser. It is charged with a solution of 0.3 g SDS in
90 mL of water that has been stripped with nitrogen for 20 minutes. Then, glycidyl methacrylate (5 g)
and ethylene glycol dimethacrylate (1 g) are added. The system is heated to 70°C and purged with
nitrogen for 10 minutes. Then, 0.2 g KPS in 10 mL water are added through a septum to start the
reaction. After 4 hours the reaction is stopped by cooling down the flask in an ice bath. After filtering
the latex through a cellulose filter, it is mixed with 100 mL of dimethylethylamine and stirred for 18
hours at 50°C. This reaction can produce small aggregates that can be easily broken down to the
original primary particles by ultrasonication (30 min, 0.5 s on / 0.5 s off cycle, 50% strength).
Combination of the anion exchange latex and the sulphonated support is achieved by diluting the
slurry such that the support is present at 5 weight-percent. 50 mL of this suspension are then gently
stirred in a beaker. 14 mL amine latex are added dropwise (1 drop per second) and then the system is
kept under stirring for another 30 min. The resulting slurry is then washed 5 times with 90 mL of
water or longer until the wash is clear of latex.
4.3.5 Characterisation
The primary particle latex (before and after shell formation) and the aminated GMA-latex were
characterised by dynamic light scattering and thermogravimetric dry content analysis.
Mercury intrusion porosimetry was used to assess the support’s pore size distribution, assuming
cylindrical pores [14]. The Brunauer-Emmett-Teller (BET) equation was used to estimate the total
surface area from nitrogen adsorption. Static light scattering (SLS) was used to characterise the
particle size of the aggregates as well as the mass scaling laws they obey.
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
35
The dependency of the height equivalent of a theoretical plate (HETP) on the flow rate was measured
by injecting 20 μL pulses of water at different flow rates into a 1.2 mM Na2CO3, 3.8 mM NaHCO3
eluent and measuring conductivity. HETP were calculated from the peak first and second moments.
The functional density of sulphonic acid groups is determined by chromatographic breakthrough
experiments carried out at 1 mL/min. The column is acidified with 10 mmol/L HNO3 for 25 minutes
and then flushed with milli-Q water for 25 minutes. Having replaced adsorbed cations by H+ and
flushed out non-adsorbed H+, the capacity is determined by desorbing the H
+ with K
+ in the form of 1
mmol/L KNO3 and integrating the conductivity from the chromatogram.
After having decorated the support with anion exchange latex, the anion exchange capacity was
determined in similar experiments, again at 1 mL/min. First, all quaternary ammonium groups are
associated with a chloride ion by flushing 50 mmol/L NaCl for 30 minutes, and then the pore void
space is flushed with milli-Q water to remove all non-adsorbed anions. The chloride ions are then
replaced by OH- using 1 mmol/L NaOH and the chromatogram is integrated to evaluate the column
capacity.
4.4 Results and Discussion
This section will focus on the characterisation of the produced material step by step using a number of
non-chromatographic techniques as well as van Deemter analysis of the column and finally a
separation of a standard seven-anion-mix to demonstrate the feasibility of the presented method.
The first step in Reactive Gelation is the production of the primary particles, the so-called latex.
Earlier works [6] showed that the aggregate morphology and strength are influenced by the primary
particle morphology. In this work, we chose to use core/shell particles based on copolymer
Styrene/DVB because their aggregates were found to be mechanically stable in chromatography and
negative charges could be readily introduced by sulphonation with sulphuric acid.
Preparation of the core Styrene/DVB latex was done using a straightforward semi-batch emulsion
polymerisation and lead to satisfactory latex quality with particle diameter of 105 nm and
polydispersity index (PDI) of 0.016. The shell was grown around the core particles in a seeded semi-
Chapter 4
36
batch emulsion polymerisation, resulting in particles of 120 nm and PDI of 0.018, with a final dry
content of 23%. The soft shell leads to partial coalescence, providing a much stronger contact after
post-polymerisation than the point-to-point contact typical of rigid, non-interpenetrating spheres. From
a number of unpublished experiments, the ratio of shell to core thickness of 1:10 was found to yield
mechanically stable particles. A relatively thicker shell results in more stable particles but with lower
specific surface area [21].
The latex was then aggregated and post-polymerised inside a stirred tank. During this process, little
fouling was observed on the impeller and baffles. The aggregates are opaque, white particles of
varying size suspended in clear liquid. A closer impression of the material can be obtained by
scanning electron microscopy (see Figure 4-2). The primary particles making up the clusters can still
be distinguished and one can see that the formed pores are several hundred nanometres in diameter (cf.
Figure 2 of a Reactive Gelation monolith in [8]: the morphology is very similar). The conversion to
aggregates as well as their size mass scaling laws was assessed using static light scattering. Figure
4-3a shows the structure factor as a function of the scattering vector q. This graph quantifies a key
characteristic of the material. The structure factor S q scales, in fact, with q according to the power
law:
fdS q q ( 4-1 )
over a wide range of q, showing that the aggregate weight scaling exhibits a fractal dimension
f 2.76d . Similar values have been previously reported for porous materials synthesized by this
process [21]; the variation can be explained by the difference in size and core/shell thickness ratios of
the primary particles [31]. The fractal mass scaling of the particles implies that their porosity and
average pore size is higher on the outside than on the inside. The concept of large pores splitting into
smaller ones seems advantageous for mass transport and is in fact identical to how convective fluid
transport is realised in nature, e.g. in blood vessels or trees.
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
37
a)
b)
Figure 4-2 SEM picture of the plain support . The primary particles composing the particles can still be identified
because of the limited sintering due to the core/shell morphology
a) b)
Figure 4-3 Static light scattering analysis of the plain support. a) structure factor ( )S q as a function of the
scattering vector q , b) particle size distribution calculated from this data using Malvern Mastersizer 2000 v5.40.
The porosity has been more thoroughly assessed using mercury intrusion porosimetry (see Figure 4-4).
Compared to earlier works [6, 8, 21], the pores are even larger and there are no pores below 2 μm –
the overall pore size distribution is extremely narrow, ranging only from 2-6 μm, possibly due to the
narrow primary particle size distribution, with a total intrusion volume of 0.72 cm3/g. The large pore
diameters will allow the decorating latex to enter the pores during the functionalisation step and still
provide high pore permeability, as is necessary to achieve perfusive flow mode. Nitrogen adsorption
10-5
10-4
10-3
10-2
10-5
10-4
10-3
10-2
10-1
100
S(q
) /
(
)
q / (1/nm)
df=2.76
100
101
102
103
104
0
2
4
6
8
10
V
olu
me /
(%
)
Particle diameter / (m)
Chapter 4
38
yielded a BET surface area of 25 m²/g, a typical value for macro-porous materials made by Reactive
Gelation. The rough pore surface created by the primary particles results in higher specific surface
areas than expected from mercury intrusion porosimetry, where cylindrical pores are assumed.
Figure 4-4 Mercury intrusion porosimetry of the plain support.
The next step of the process, sulphonation of the polystyrene support, introduces a large amount of
negative charges per surface area and thus provides a deep potential well for the following
electrostatic anchoring of positively charged latex particles. The sulphonation was carried out under
harsh conditions of 80 wt% sulphuric acid at 80°C. For this step, a linear dependence of introduced
SO3- groups on the time of reaction up to 24 hours was found (Figure 4-5). After this time, the material
looks slightly brownish, suspended in grey aqueous solution; after washing with large excess of water,
the particles stay light brown, as opposed to the bright white plain polystyrene resin.
10-2
10-1
100
101
0.0
0.2
0.4
0.6
0.8
Hg
In
tru
ded
Vo
lum
e /
(cm
3/g
)
Pore diameter / (m)
0
2
4
6
8
10
12
dV
/d(l
n(D
)) /
(cm
3/g
)
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
39
Figure 4-5 Amount of SO3- groups introduced as a function of reaction time by sulphonation of the support at 80°C
and H2SO4 concentration of 80 % wt.
The second part of the functionalisation involves latex nanoparticles based on glycidyl methacrylate
and functionalised with dimethylethanolamine, having had short-term colloidal stability issues with
vinylbenzyl chloride based latexes. With reference to Pohl et al. [32], a relatively high cross-linking
degree was chosen to avoid high swelling, keeping the particle size low to make diffusion into the
support’s pores easier. The latex was aminated using dimethylethanolamine (DMEA) for symmetrical
peak shapes and decent ability to separate fluoride from the water peak [32]. The copolymerisation of
glycidyl methacrylate and ethylene glycol dimethacrylate was carried out using batch emulsion
polymerisation after having estimated similar reactivity ratios of GE 0.85r and
EG 1.12r via the Q-e
scheme [33]. Before amination the latex particles have z-average diameter of 60 nm (0.040 PDI); after
amination, the z-average diameter increases to 75 nm (0.056 of PDI). The final latex is colloidally
stable for several weeks (no sedimentation and no average size increase by dynamic light scattering
over a period of 6 months). The initially not strongly hydrophobic repeating unit is made hydrophilic
by the amination, thus resulting in a better compatibility with water, as proved by the increased
swelling of the gel by a factor 2 in volume.
Decoration of the support with the aminated latex particles is achieved by slowly dripping the latex
into the support suspension. In this step, the SO3- groups on the surface electrostatically bind to NR3
+
0 5 10 15 20 250
1
2
3
4
Ch
arg
e d
en
sity
/ (
mo
l S
O3
- /mL
)
Time / (h)
Chapter 4
40
residues of the gel, immobilising them on the surface. No aggregate formation has been observed
during this step, and the solution viscosity did not change significantly. After gently stirring for thirty
minutes, the liquid was filtered off and the resin gently re-dispersed in water for five minutes, then
again filtered. After the third cycle of such washing, the filtered liquid was clear. Further 2 cycles were
carried out afterwards to ensure a thoroughly clean product.
The prepared resin was re-suspended in 20% vol. ethanol and packed into a non-commercial PEEK
column of size 4 x 250 mm. During the packing at a maximum flow rate of 3 mL/min the pressure
stayed below 0.1 MPa, the sensitivity limit of our equipment. The anion exchange capacity was
determined in a similar breakthrough measurement as before the cation exchange capacity. The
resulting value of 39 μeq/mL is around ten times larger than the previous cation exchange capacity and
about twice as large as a typical commercial material (Metrohm ASupp15, 17 μeq/mL). This material
is 5 μm in particle diameter and has pores smaller than 100 nm, making it impossible for the latex
particles to enter: therefore, the geometric external surface area of the particles is the only possible
locus of attachment for the decorating nanoparticles. Taking the amount of anion exchange groups per
unit area that can be loaded onto this commercial resin as base case, it is possible to determine if the
latex entered the pores of our perfusive resin. While the commercial material has 22 μeq/m², a 16-fold
higher value has been evaluated for the perfusive resin (353 μeq/m² of geometric surface area). This
large difference could be explained by a much larger amount of gel nanoparticles per geometric
surface area or by the penetration of the decorating particles into the support’s pores. This second
explanation is indeed much more reasonable, considering that 16 times more decorating particles is
unlikely (the same surface chemistry is involved) and that the pores of the perfusive material are
significantly larger than the latex particles.
The support presented in this work shows a rough surface and considerable polydispersity in particle
size – both factors could affect the results of the previous paragraph to some extent. The rough surface
leads to a higher external surface area and the geometric surface area of a polydisperse sample is
larger than the geometric surface area of a monodisperse sample at the same volumetric mean. To
estimate the impact of such non-idealities, a popular commercial packing material, POROS R1 – a
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
41
spherical, comparably smooth and uniform in size resin with particle size of 50 μm and pore size of
400 nm – was used to run comparative experiments. Apart from the shape, this material is chemically
identical to the support developed in this work: plain Poly(Sty/DVB). It was thus identically
sulphonated and decorated using the same nanoparticles and the same methodology. Although
somewhat smaller, the pores in this material are still large enough to host the decorating particles.
After the treatment, the anion exchange capacity of the commercial particles was evaluated by titration
as 34.6 μeq/mL. Normalised to the geometric surface area (for this material known with much larger
accuracy), a value quite similar to the one estimated for our clusters could be obtained: 480 μeq/m²
instead of 353 μeq/m². The larger value can be readily explained by the larger specific pore surface
area due to the smaller pore diameters.
The column efficiency was determined from the peak moments of a non-adsorbing species, after
subtracting equipment-caused peak broadening measured at every examined flow rate. These data
were made dimensionless and are shown in Figure 4-6. Initially quickly increasing reduced plate
heights, caused by large particle diameters, soon reach a plateau and become independent of the flow
velocity. This is because the mass transport inside the particles changes from diffusive to convective –
at high flow rates the convective transport of the solute in the particle through-pores is quicker than
that by diffusion. Since the flow velocity inside the particles linearly scales with the flow velocity in
the bed, further increasing the flow rate through the column increases the liquid-solid mass transport
rate by the same amount, resulting in flow-rate independent column efficiency at high flow rates.
Chapter 4
42
Figure 4-6 Reduced van Deemter plot obtained from water injections.
This transition from diffusive to convective mass transport can be demonstrated using the intra-
particle Peclet number λP and the concept of ‘enhanced’ mass transport introduced by Carta and
Rodrigues [22]. Fitting the ratio of flow velocities inside and between the resin particles, 'uFu
(where u’ is the velocity inside the particles and u the linear bed velocity), the measured HETP data
can be compared to Carta and Rodrigues’ function describing the enhanced mass transport P( )f as
follows:
p
P P2 2 2P P P eff3 3 3
'3 1 1 with
tanh 2
u Rf
D
( 4-2 )
where RP is the particle size and Deff the effective diffusion coefficient inside the particles. This
enhancement function extends the van Deemter equation to become:
PHETP A B u C u f (4-3)
Figure 4-7 shows the value of the enhancement function Pf over a range of Peclet numbers
encountered under typical operating conditions. While initially (0.2 mL/min) diffusion is clearly the
dominant mass transport mechanism ( P 1f ), at 1 mL/min the Peclet number values are already 5
0 20 40 60 80 1004
6
8
10
12
Red
uced
HE
TP
/ (
)
Reduced velocity / ()
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
43
and the Pf function in its linear regime, thus indicating the column is in perfusive operation
mode.
Figure 4-7 Enhanced mass transport function, Pf , as a function of Peclet number, P (cf. [22]). The curve
shows the theoretical solution for Pf and the squares the experimental values obtained by fitting the ratio
between inter- and intra-particle flow to the HETP data obtained from water injections.
Having established that the resin possesses the desired mass transport properties, the quality of the
nanoparticle functionalisation was finally tested by running a typical ion-chromatography separation.
A mixture of seven standard anions (2 ppm fluoride, 5 ppm chloride, 5 ppm nitrite, 10 ppm phosphate,
10 ppm bromide, 10 ppm nitrate and 10 ppm sulphate in water) was injected and eluted with a weak
standard eluent composed of 1.2 mM Na2CO3, 3.8 mM NaHCO3. As shown in Figure 4-8, all seven
peaks are baseline separated from each other and the negative water peak is almost resolved from the
fluoride signal, making the determination of fluoride concentrations possible.
10-1
100
101
102
0.2
0.4
0.6
0.8
1.0
f(
P)
/ (
)
P / ()
Chapter 4
44
Figure 4-8 Chromatogram of a mixture of seven standard ions at 1 mL/min with sodium carbonate eluent (1.2 mM
Na2CO3, 3.8 mM NaHCO3). The peaks are: 1 = 2 ppm fluoride, 2 = 5 ppm chloride, 3 = 5 ppm nitrite, 4 = 10 ppm
phosphate, 5 = 10 ppm bromide, 6 = 10 ppm nitrate, 7 = 10 ppm sulphate).
4.5 Conclusion & Outlook
In this chapter, the complete synthesis of a novel kind of stationary phase combining the advantages of
large diameter, macro-porous supports with those of latex ion exchangers is shown. First, the Reactive
Gelation technique is used to prepare mechanically stable porous particles made of PSDVB which are
then densely covered with negative charges by sulphonation. The synthesis procedure is divided into a
number of consecutive steps, allowing for independent adjustment of material characteristics. The
support, both on the outside and inside its large pores, is subsequently decorated with anion-exchange
latex based on glycidyl methacrylate aminated with dimethylethanolamine. Flow rate-independent
column efficiency at high linear velocities was shown using careful peak moment analysis and the
resulting data compared to an established model for perfusive columns, demonstrating the strongly
perfusive nature of this resin. Having determined the anion exchange capacity of this novel packing
material under chromatographic conditions, normalising this value by the particle geometric surface
area and comparing it to surface-only decorated resins shows that the latex nanoparticles enter the
pores and stick to their inside. This material was then used to separate a mixture of seven standard
anions to prove its feasibility.
0 10 20 30 40 50 60 70
1.5
2.0
2.5
3.0
3.5
Co
nd
ucti
vit
y /
(
S/c
m)
Time / (min)
1
2
3
4 56 7
Macro-porous latex-coated polymer particles from Reactive Gelation as stationary phase for ion chromatography
45
The main advantage of this new material compared to existing ones is its very low pressure drop while
still providing narrow enough peaks for baseline separations due to the perfusive mass transport. This
material is thus well suited to fill a niche application, the separation of uncomplicated systems with
cheap and simple, low-pressure equipment. Having focused on one type of material to show the
detailed preparation and characterization, a wide range of modifications is possible due to the modular
synthesis procedure. Future works are going to investigate different functionalisations to address
specific, common separation problems and the optimisation of eluents for such problems to improve
peak shape and reduce analysis times.
47
Chapter 5
Shear-Induced Reactive Gelation
(this chapter was partially published in international patent application PCT/EP2013/003532)
5.1 Abstract
This chapter describes a method for the continous production of porous polymer materials using the
principle of Reactive Gelation. Starting from a poly(styrene-co-divinylbenzene) latex with particle
diameter of one hundred nanometres, aggregates are prepared in a continuous aggregation reactor by
shear rates on the order of 500’000 1/s. These aggregates are then thermally hardened to strengthen
their mechanical resilience. Their mass scaling was found to obey a power low with fractal dimension
of 2.4-2.7, depending on process conditions and the effect of process parameters on conversion and
material properties investigated. BET specific surface areas ranged from 20 to 40 m2/g. The material
was packed into columns and tested chromatographically. The column efficiency was found to be
strongly perfusive, i.e. independent of linear velocity, at high reduced velocities as is the case for large
biomolecules.
Chapter 5
48
5.2 Introduction
Porous polymers are employed for a variety of different applications ranging from catalysis [34-35]
over thermal insulators [36] to scaffolding in medical tissue engineering [37]. Each application
demands different properties and different manufacturing methods depending on the amounts and
morphology of material to be produced. Most polymeric materials are prepared using pore-generating
systems [36], usually in the form of a porogen. These porogens are solvents for the monomers but not
the polymers and thus segregate from the forming polymer phase along the course of the reaction. This
means that during polymerisation, a dispersed phase of polymer forms in a process termed nucleation.
Given enough monomer, these nuclei grow and connect to each other, forming a continuous polymer
phase. At the end of the reaction, the porogen is extracted from the pores using an appropriate solvent
[12, 34, 38-41]. This method allows tuning pore size and morphology by choice of porogen and
reaction conditions. The initial system of porogen and monomer can be dispersed in a non-compatible
continuous phase to form droplets that later turn into particles, too [12, 41-42]. It is becoming clear
that in this process a number of phenomena occur at once and are still not well understood [12, 41-43].
More control over the process could be achieved by separating the process steps in time [6].
Accordingly, over the last years, a porogen-free method called Reactive Gelation has been developed
in our research group [6-8, 21]. It is a process to prepare macro-porous polymers in a very controlled,
step-wise manner, that mimics the different steps of the porogen method to some extent and only
produces brine as waste-product. Both slab-shaped and micro-particulate matter have been prepared
and functionalised in a number of ways to obtain chromatographic media – other applications have not
been explored yet. The two preparation methods are similar: a polymer latex is prepared, the polymer
swollen in additional monomer and initiator, aggregated and finally hardened by post-polymerisation
through heating. The last two steps can be carried out in stagnant conditions (thus preparing slabs), or
with agitation where aggregation and breakage by shear yield particles on the micrometre scale. These
particulate materials, are commonly produced in rather diluted conditions, i.e. dry content around
1wt%, leaving room for further process intensification. To overcome this limitation we developed a
continuous aggregation step, utilising a high-pressure pump to force the latex through a narrow
channel with sharp bends that applies high shear stress. To control the aggregation, it is convenient if
Shear-Induced Reactive Gelation
49
it only occurs in the micro-channel and not before or after. This can be ensured by staying in the
reaction limited cluster aggregation (RLCA) regime, far from the critical coagulation concentration,
during the entire process. The micro-channel provides the particles with enough energy to overcome
practically any potential barrier between them [44] and afterwards there is the possibility to carry out
the post-polymerisation step in lower shear and at higher solid fraction than in the original stirred tank
process without the system gluing together to form one block.
This versatile process can be applied to the preparation of very different materials both in chemistry
and morphology. Since one of the prime interests of our group lies in chromatography, especially for
large biomolecules, a material to serve as chromatographic support is synthesised in this chapter.
Commonly, protein chromatography is carried out on particulate supports made from polymer for its
ability to withstand caustic soda in cleaning steps. Monolithic materials still only make up a rather
small fraction of the market [10]. Particulate resins are prepared in variety of methods like suspension
polymerisation [45], the Ugelstad method [46] or the staged template suspension polymerisation by
Frechet et al. [4]. For our purpose, the material should be chemically resistant to most solvents,
withstand at least 1 M sodium hydroxide and be mechanically strong. The particle size should be
rather large to obtain low back pressures and the pores wide in diameter to grant the proteins access to
the entire surface area and possibly induce perfusion [22].
In this chapter, we describe the new, continuous preparation process and investigate the effects of
substrate properties as well as process parameters on the final product. Characterisation of the
resulting aggregates, herein termed ‘micro-clusters’, is carried out mostly using static light scattering,
but also nitrogen adsorption and scanning electron microscopy where possible. Finally, the material is
packed into an HPLC column and its chromatographic efficiency evaluated using tracer injections.
Chapter 5
50
5.3 Experimental
5.3.1 Materials
The following chemicals have been employed in the work: 2-2’-azo(2-methylpropionitrile) (AIBN,
Fluka, purum), divinylbenzene (DVB, Aldrich, 80% technical), potassium persulphate (KPS, Fluka,
puriss p.a.), magnesium chloride (VWR, 99.9%), sodium chloride (Merck, for analysis), sodium
dodecyl sulphate (SDS, Fluka, ≥98%), styrene (Sty, Fisher Scientific, general purpose grade), sodium
phosphate monobasic (Fluka, purum p.a. anhydrous, ≥99.0%), sodium phosphate dibasic (Fluka,
purum p.a. anhydrous, ≥98.0%). All chemicals have been used as supplied without further
purification. Ultra-pure grade water for chromatography has been prepared by Millipore Synergy
(Millipore, Billerica, MA, USA). Deionised water for synthesis has been stripped of oxygen by
degassing under vacuum and subsequent saturation with nitrogen gas.
5.3.2 Primary Particle Preparation
The primary particle latexes E1-E3 were produced in two steps. In the first step a 20% cross-linked
core particles are produced by semi-batch emulsion polymerisation under nitrogen atmosphere. A 4 L
Mettler-Toledo LabMax is initially charged with water and surfactant (SDS) according to the recipe
reported in Tables 5-1 to 5-3 (initial charge 1, IC1). The temperature is set to 70°C using the oil
heating jacket. In a second flask, an emulsion of styrene, divinylbenzene, water and surfactant (SDS)
is prepared according to Tables 5-1 to 5-3 (continuous feed 1, CF1) and kept emulsified using a
magnetic stirrer. When the reactor temperature reaches 70°C, aqueous initiator (KPS) solution is
injected through a septum into the reactor using a syringe and hypodermic needle according to tables
5-1 to 5-3 (Initiator solution 1, IS1) and the monomer emulsion is fed. In cases with reaction times
significantly longer than the half-life time of KPS, initiator solution was fed using a syringe pump
(IF1). The reaction progress is monitored with thermogravimetric dry content analysis (HG53 Halogen
Moisture Analyzer (Mettler Toledo, Greifensee, Switzerland)) and dynamic light scattering (Zetasizer
nano ZS 3600 (Malvern Instruments, Malvern, Worcestershire, UK)). The reaction is stopped when
reaching the desired core size.
Shear-Induced Reactive Gelation
51
Such particles are consequently used as a seed in the second step, a seeded emulsion polymerisation.
There, a 1% cross-linked shell is prepared around the core using a semi-batch mode with slow
monomer feed to achieve a radially more homogeneous cross-linkage [33].
Table 5-1 Recipe for the production of the precursor core/shell latex E1. All numbers are target values, the actual
values varied by less than 1%.
Core Particles Shell
IC1 CF1 (10h) IS1 IF1 (10h) IC2 CF2 (5h) IS2
H2O / g 1575 525 75.0 75.0 2590 100
Styrene / g 420 266
DVB / g 105 2.68
SDS / g 8.40 3.00
KPS / g 2.00 2.20 3.00
Seed latex / g 1446
Diameter / nm 60 (0.097 PDI) 80 (0.102 PDI)
Table 5-2 Recipe for the production of the precursor core/shell latex E2. All numbers are target values, the actual
values varied by less than 1%.
Core Particles Shell
IC1 CF1 (14h) IS1 IC2 CF2 (5h) IS2
H2O / g 1200 100 166 100
Styrene / g 573 133
DVB / g 143 1.40
SDS / g 2.60 12.0
KPS / g 1.30 2.00
Seed latex / g 1600
Diameter / nm 105 (0.016 PDI) 125 (0.018 PDI)
Chapter 5
52
Table 5-3 Recipe for the production of the precursor core/shell latex E3. All numbers are target values, the actual
values varied by less than 1%.
Core Particles Shell
IC1 CF1 (22h) IS1 IF1 (22h) IC2 CF2 (14h) IS2 IF2 (14h)
H2O / g 1000 862 40.0 100 1185 109 150
Styrene / g 917 795
DVB / g 229 6.00
SDS / g 2.10 19.2
KPS / g 1.00 3.20 3.00 6.00
Seed latex / g 1446
Diameter / nm 180 (0.010 PDI) 200 (0.005 PDI)
5.3.3 Aggregate Preparation
Depending on the experiment, the latex was taken either plain or swollen beforehand – if not stated
otherwise, the latex was unswollen. Where applicable, swelling was carried out by preparing a mixture
of hydrophobic monomers Sty and DVB and hydrophobic initiator AIBN and subsequently adding it
slowly into the latex. The suspension was then agitated for at least four hours.
Aggregation of the latex was carried out using a high-shear device HC-2000 from Microfluidics
(Newton, MA, USA) equipped with a L30Z micro-channel and is sketched in Figure 5-1, from hereon
called ‘micro-channel’.
Shear-Induced Reactive Gelation
53
Figure 5-1 Scheme of the micro-channel equipment used. The pump exerts a pressure of 160 bar.
Unless stated otherwise, the latex was filtered through a cellulose filter to remove aggregates that form
when the latex dries on the storage bottle wall and neck and then added to a well stirred salt solution or
water, depending on experiment. The suspension is then quickly transferred into the micro-channel’s
reservoir. Between experiments, the micro-channel was filled with water, so the first half of the
leaving product was discarded and the sample taken from the middle of the latex plug in order to
correctly resemble the product obtained from steady-state operation and not the diluted product at the
start. Swollen latex was aggregated in an identical way as described before and subsequently post-
polymerised over night at 70°C in a gently stirred batch reactor. Formed aggregates were characterised
using static light scattering Mastersizer 2000 (Malvern Instruments, Malvern, Worcestershire, UK).
5.3.4 Characterisation
Two samples from the aggregate plug’s centre were collected from the micro-channel exit and stored
in a pill flask. One sample was transferred to the rheometer (equipped with cone/plate geometry
stator/rotor, respectively) and the viscosity measured at shear rates of 15.85 1/s. The second sample
was diluted as a whole until the obscuration was in the limits of the Mastersizer 2000 used for their
analysis. This ensured representative sampling where neither large nor small particles are preferred.
The diluted sample was then slowly passed through the Mastersizer 2000 to prevent sedimentation. In
all steps of the process, utmost care was taken in order to avoid large shear rates that could break the
aggregates.
Membrane
Pump
Latex
Reservoir
Micro-channel
Chapter 5
54
Static angle light scattering (SALS) was used to characterise the particle size of large aggregates as
well as their (fractal) internal morphology. The average structure factor ( )S q of the produced
aggregates was evaluated from the scattered light intensity ( )I q according to [47-48]
(0) ( )( )
( )
I P qS q
I q
, ( 5-1 )
where ( )P q is the primary particles’ form factor and q the scattering vector amplitude defined as:
4 sin2
nq
, ( 5-2 )
Here n refers to the refractive index of the continuous phase (in all cases water), is the laser
wavelength and is the scattering angle. The number of measurements points therefore corresponds
to the number of detection angles and is 44 for the equipment used.
The Guinier approximation was used to relate the structure factor with the radius of gyration:
2 2
g ( )( ) exp
3
S qq R
S q
, for g ( )
1S q
q R and 2 2 2
g g g,p( )S qR R R , ( 5-3 )
with g,p p3 5R R used as the radius of gyration of primary particles. It is worth noting that most
aggregates studied in this work were several tens of micrometres in diameter, making the contribution
of primary particles rather insignificant.
Because the used systems obey the Rayleigh-Debye-Gans theory [47, 49] additional information about
the internal structure of formed aggregates or their fractal dimension can be obtained from light
scattering analysis. When plotting the average structure factor ( )S q as a function of q according to
f( ) dS q q , for pg
1 1q
RR ( 5-4 )
the slope of ( )S q vs q in a double logarithmic plot is equal to fd . Once the average radius of
gyration has been determined for a cluster population, multiplying q with gR and plotting ( )S q
against this product yields a graph that is normalised by aggregate size. This is very useful to obtain an
Shear-Induced Reactive Gelation
55
impression of the similarities and differences between populations, not taking into account their size,
e.g. when evaluating polydispersity and fractal dimension.
The hardened aggregates allow for mechanically more demanding tests, so the Brunauer-Emmett-
Teller (BET) equation was used to estimate the total surface area from nitrogen adsorption
measurements. The dependency of the height equivalent of a theoretical plate (HETP) on the flow rate
was measured by injecting pulses of dextrans at different flow rates into a 25 mM, pH 7 phosphate
buffer using an Agilent Series 1200 (Agilent Technologies, Santa Clara, CA, USA) equipped with a
quaternary pump and degasser, an autosampler with integrated cooling, a refractive index detector and
a diode array detector.. HETP were calculated from the refractive index peak’s first and second
moment.
For visual inspection of the formed clusters SEM pictures were taken using a Gemini 1530 FEG
(Zeiss, Oberkochen, Germany).
5.4 Results and Discussion
In this chapter, a process for the preparation of porous materials in a continuous manner is presented.
Starting from a colloidal precursor system, aggregates are formed and hardened. The production of
polymer latex is well understood [50] and will not be discussed in detail. For our purpose, a core/shell
latex is produced because, as shown in earlier publications from our group [6, 21] it allows us to tune
the final material properties. These works demonstrate that the amount of cross-linking determines if
particles can interpenetrate after aggregation or not. Fully ‘soft’, i.e. little cross-linked, particles will
sinter together when heated close to their glass transition temperature and lose their shape, whereas
fully hard latex will only touch in one point, resulting in weak bonding between particles that form the
aggregates. In the first case, tough material with low surface area is formed while the latter one results
in mechanically weak material with larger surface area. Working with particles composed of a hard
core and a soft shell, the degree of sintering can be controlled by the thickness of the shell. Therefore,
the material can be designed strong enough for a specific purpose, maximising the specific surface
area while fulfilling the stiffness constraint.
Chapter 5
56
The aggregation step is characterised by a number of process parameters and substrate latex properties
such as dry content, applied pressure (and thus shear rate) and ionic strength. The product leaving the
aggregation reactor, on the other hand, can be described by its viscosity that changes because of the
increasing effective occupied volume fraction when porous aggregates form. Given enough dry
content and high conversion to aggregates, the liquid latex is transformed into a solid extruded paste,
strong enough to be handled with pincers [51].This phenomenological description was quantified by
construction of a phase diagram in which the phase boundary separates a liquid suspension from a gel.
The extent of particle aggregation has to be characterised as a function of particle stability, dry content
and shear rate in the aggregation reactor to understand how to prepare a material of desired properties.
For further processing, the ideal consistency of the material is a slurry with as high dry content as
possible, but not forming a gel. At the same time, high conversion of primary particles to aggregates is
desired. The first part of the results section will therefore demonstrate a method how to obtain a phase
diagram for a given latex at the example of E2.
5.4.1 Method to obtain phase diagram
The purpose of the phase diagram is to predict the consistency of the product leaving the micro-
channel for a combination of applied pressure and salt content at given dry content for a given latex.
According to the particle stability theory [52] [53] adding a suitable electrolyte results in reduced
colloidal stability leading to the formation of aggregates. In contrast, as demonstrated by Zaccone et
al. [44] applying high shear rates could induce the aggregation process by supplying the particles with
enough kinetic energy to overcome the potential barrier originating from the charges on their surface.
Lastly, dry content is not going to influence the particle stability, but speed up aggregation simply due
to aggregation being dependent on two particles colliding, thus being a second order kinetics with
respect to particle concentration. In conclusion, the phase diagram shall be in the plane of salt
concentration and shear rate. Two points in this plane are essential: 1) the salt concentration at which
gelation occurs in the absence of shear (i.e. in a stagnant system) and 2) the shear rate at which
gelation occurs in the absence of salt. The first point is simply the latex critical coagulation
concentration which can be easily obtained from aggregates size evolution measured under static
Shear-Induced Reactive Gelation
57
conditions [21]. The critical coagulation concentration (ccc) was measured as 0.01±0.001 M MgCl2
for all three latexes. The second point is theoretically obtained by varying the shear rate at fixed dry
content (or vice versa) in the absence of salt. Practically one cannot know if the maximum pressure of
the equipment is going to reach this point. In our case the second approach was applied where the
maximum shear rate as determined by the operating pressure was set to the maximum and the dry
content varied to find the minimum dry content at which gelation occurs. To determine the gelation
point the material leaving the micro-channel is collected and its viscosity measured at a given shear
rate in a rheometer. It is worth noting that the applied shear rate during viscosity measurement is
orders of magnitude lower than that in the microchannel so any additional aggregation can be
neglected. The measured viscosity is then plotted against dry content and the viscosity divergence
defines the gel point. This point was found to lie between 13 and 14 percent dry content by connecting
the three low-end values with a straight line and the three high end values with a different line; the
intercept was defined as the point of divergence. Due to the low sensitivity of the used rheometer the
viscosity for low dry content was calculated from the Einstein relationship [54]. The procedure is
shown for one data point in Figure 5-2.
Figure 5-2 Viscosity of the slurry leaving the micro-channel at 160 bar with no salt addition. The diamonds are
calculated values from Einstein equation and the squares measured values, both at shear rates of 15.85 1/s.
0.10 0.12 0.14 0.1610
-3
10-2
10-1
/ (P
a.s
)
Dry content / ()
Chapter 5
58
From this point on, the phase diagram can be rendered more precise by measuring additional data
points in between the extremes of the phase boundary to determine the exact shape of the curve. In this
case, one additional point was added at 1/3 critical coagulation concentration (ccc). Here, the dry
content from gelation without salt was assumed resulting in the reduction of applied shear rate. It is
worth noting that due to the addition of salt leading to reduced stability of latex particles the critical
shear rate at this salt concentration will be be equal or lower than without salt. The final phase diagram
is shown in Figure 5-3 together with a quadratic fit going through the points. It should be noted that, in
theory, this graph should be possible to build from DLVO theory – however, in practice, this did not
match the experimental data points. Most likely this is because the shear rates in the micro-channel are
not homogeneous and one pass through it does not lead to equilibrium between aggregation and
breakage (see chapter 5.4.2).
Figure 5-3 Phase diagram for latex E2 at 13% dry content.
5.4.2 Effect of pressure and residence time
As explained in section 5.4.1, shear rate is the driving force for aggregation in this process by
providing the substrate particles with enough energy to overcome the potential barrier between them.
Consequently, it is expected that reducing the shear rate is going to reduce the fraction of particles
with enough energy to aggregate and thus lower conversion. Additionally, shear rate is also going to
10-7
10-6
10-5
10-4
10-3
10-2
10-1
100
100
101
102
103
PRIMARY PARTICLES
AND SMALL AGGREGATES
GEL
Pre
ssu
re /
(b
ar)
Ionic Strength / (mol/L)
Shear-Induced Reactive Gelation
59
impose a maximum aggregate size to the system: if a particle is large enough, it is going to be broken
by the shear forces acting on it [55]. Thus, it is expected that, given sufficient residence time in the
high-shear environment, an equilibrium size establishes from breakage and re-aggregation of the
aggregates [56]. Because the force required to break an aggregate depends on its internal strength,
stronger aggregates are going to survive this process longer, leading to a strengthening of the average
aggregate. This rearrangement to tougher structures becomes readily visible in the fractal dimension
describing the aggregates’ internal morphology; the strengthening originates from a densification of
the clusters that is visible in an increasing fractal dimension.
Figure 5-4 Effect of pressure and residence time onto fractal dimension and conversion of E3. All experiments at 1%
dry content. a) Full squares indicate 160 bar, empty triangles 120 bar and empty circles 80 bar. b) Additionally a
sample that was passed through the micro-channel twice at 160 bar is shown, indicated by full diamonds. For better
comparison, all samples were normalised by the average particle size.
Figure 5-4 a) shows the effect explained above by plotting the structure factors as a function of q for
latex E3 passed through the micro-channel at different pressures. At 160 bar, the fractal dimension is
equal to 2.4, as has been previously observed for latex aggregated in the micro-channel [55]. Reducing
the pressure to 120 bar yields a similar result, although with less conversion, as can be observed from
the higher tail at higher q-values that correspond to small aggregates and primary particles. Reducing
pressure to 80 bar, an interesting phenomenon occurs: the conversion decreases slightly, as expected,
but the fractal dimension increases significantly to 2.7. This value was reported before for a different
system [21, 57-59] in which equilibrium between aggregation and breakup was reached in a stirred
10-6
10-5
10-4
10-3
10-2
10-5
10-4
10-3
10-2
10-1
100
a)
-2.7
S(q
)
q / (1/nm)
-2.4
10-1
100
101
102
103
10-5
10-4
10-3
10-2
10-1
100
qRG / ()
b)S
(q)
-2.7
Chapter 5
60
tank. In those experiments, the particles were fully destabilised and shear was only used to break the
forming aggregates over a time frame of several hours. What is observed in our experiments is a
similar effect: it appears that because of the lower pressure, the residence time in the high shear zone
is long enough to break and aggregate the particles over and over again, yielding the densification
explained above. To verify this theory, the same latex was passed through the micro-channel twice at
160 bar to obtain a similar residence time as at 80 bar. In fact, as can be seen in Figure 5-4 b), the two
curves follow the same scaling with a fractal dimension of 2.7. Due to higher shear rate the lower tail
at high qRG values is consistent with significantly higher conversion for 160 bar. From these
experiments it can be concluded that one pass through the micro-channel does not result in an
equilibrated material, but rather one that is more open as densification has not progressed to
equilibrium. However, this knowledge cannot be exploited directly, as passing aggregates through the
pump again (i.e. recycling) quickly leads to leakage through the pump seals (they can be fully
recovered by cleaning out all aggregates from between them). Therefore, one has to reside to using
low pressures in combination with salt to achieve high fractal dimensions if desired (see section 5.4.3
for the effect of salt). Alternatively, setting up two micro-channels in series with a sufficiently strong
pump should have the same effect; this has not been explored in this work, though.
5.4.3 Effect of salt
All latex particles employed in this work are charge-stabilised with a combination of sulphate groups
from the initiator KPS and the surfactant SDS. Increasing the ionic strength in the aqueous phase
results in a compression of the electrical double layer on the particles’ surface and thus reduction of
magnitude and reach of the potential barrier. Viewing an aggregation event as a reaction with certain
activation energy and the particles’ kinetic energy distributed around an average, it becomes clear that
the fraction of particles with sufficient energy to overcome this barrier is a function of the magnitude
of the barrier, the average particle energy and the width of its distribution. It is difficult to influence
the width of the energy distribution because this is directly given by the distribution of shear rate that
in turn depends on the micro-channel geometry. Unfortunately, this information is not provided by the
manufacturer and cannot be changed therefore it was not investigated further.
Shear-Induced Reactive Gelation
61
Having looked at changing the average energy in section 5.4.2, this chapter treats the effect of
changing the barrier magnitude. Decreasing this barrier by increasing the ionic strength is useful when
low conversions are achieved – it will have almost no effect when most primary particles are already
incorporated into aggregates. Therefore, the three experiments treated in this section are carried out at
80 bar pressure. As a reference, E3 was passed through the micro-channel without salt and a dry
content of 1%. The high tail visible at high qRG values again shows a significant fraction of primary
particles and very small aggregates. This problem can be solved in one of two ways: 1) the dry content
is increased, in this case to 5%, which leads to better conversion because of more collisions between
particles or 2) by adding salt and keeping the dry content constant at 1%, thus increasing the fraction
of collisions that lead to an aggregation event while keeping constant the number of collisions. This
modification has no effect on fractal dimension and thus allows the production of the same aggregates,
yet increasing conversion.
Figure 5-5 Effect of dry content and ionic strength onto E3. Empty squares indicate 80 bar, 1% dry content without
salt, empty circles 80 bar, 5% dry content without salt and full triangles 80 bar, 1% with ionic strength at ½ ccc of the
latex.
5.4.4 Effect of primary particle size
In the introduction to this chapter, the primary effects of changing the precursor latex in Reactive
Gelation were elaborated based on earlier works. By choosing very similar latexes in morphology that
are only different in size, similar behaviour during the aggregation can be expected. This short section
10-1
100
101
102
103
10-5
10-4
10-3
10-2
10-1
100
S(q
)
qRG / ()
-2.7
Chapter 5
62
therefore only verifies that the three latexes in fact aggregate in a similar way. In fact, in Figure 5-6 it
can be seen that the fractal dimension after one pass is 2.4 in all cases. Additionally, having
normalised the graphs by the aggregate size through multiplying the x-axis with the average particle
size, the curvature around qRG=1 can be associated with the polydispersity; the sharper the bend, the
more narrow the distribution. This shows that the polydispersity for all samples is similar.
Figure 5-6 Comparison of aggregates made from all three precursor latexes. Empty triangles denote E1, full squares
E2 and full circles E3. All experiments were carried out at 5% dry content, ½ ccc and 160 bar for high conversion.
5.4.5 Effect of post-polymerisation
Figure 5-7 Comparison of post-polymerised aggregates from all three precursor latexes. Empty squares denote E1,
empty triangles E2 and full circles E3. All experiments were carried out at 2% dry content, ½ ccc and 160 bar.
10-1
100
101
102
10-5
10-4
10-3
10-2
10-1
100
S(q
)
qRG / ()
-2.4
10-1
100
101
102
10-5
10-4
10-3
10-2
10-1
100
S(q
)
qRG / ()
-2.4
Shear-Induced Reactive Gelation
63
Figure 5-8 SEM micrographs of post-polymerised aggregates from all three latexes. The varying degree of
interpenetration can be clearly identified between E1 (top), E2 (middle) and E3 (bottom).
All aggregates investigated so far were only characterised with light scattering due to their fragile
nature that originates from the weak van-der-Waals forces holding them together. Application of this
Chapter 5
64
kind of material only becomes feasible when these aggregates are hardened. This is carried out using a
method called Reactive Gelation [6]. In this method, primary particle latex is swollen with
hydrophobic monomer and hydrophobic thermal initiator before aggregation. That way, the aggregates
can be linked together with polymer chains in the ‘post-polymerisation’ step which is initiated by
heating the aggregates. In this chapter, aggregates from all three latex types were prepared from
swollen latex (no significant difference to unswollen material was observed in fractal dimension or
size). These were then transferred to a gently agitated tank and heated overnight. The resulting
material is mechanically strong and can be characterised by SEM, nitrogen adsorption and even
chromatographically. A first, visual impression can be obtained from the SEM pictures in Figure 5-8.
A clear difference in particle interpenetration can be observed from the two smaller latexes (E1 and
E2) to the rather large latex E3 which shows almost unchanged spheres. This is because the relative
thickness of the shell varies from latex to latex, as the total diameter is changed between 80 nm and
200 nm, but the absolute shell thickness is constant at 20 nm. This effect of difference in particle
interpenetration can be measured by nitrogen adsorption, using the BET equation to estimate the
aggregate surface area. As shown in Table 5-4, both of the smaller particles lose about half of their
surface area during the post-polymerisation, whereas the largest particles only lose a third.
Nevertheless, the aggregates from the smallest primary particles exhibit the highest absolute specific
surface area with 40 m2/g.
Table 5-4: Quantification of particle interpenetration by specific surface area measurements
E1 E2 E3
Primary particle diameter / (nm) 80 120 200
Primary particle specific surface area (calculated) / (m2/g) 75 50 30
Measured specific surface area of aggregates / (m2/g) 39 26 20
Loss in specific surface area / (%) 48 48 33
5.4.6 Chromatographic characterisation
For a last analysis of the aggregates prepared in this chapter, they are characterised in terms of
chromatographic efficiency. As reported in earlier works [21, 60], macro-porous particles exhibit a
peculiar behaviour of the column efficiency as the flow rate increases. Due to the high hydrodynamic
Shear-Induced Reactive Gelation
65
permeability of the material, a significant fraction (often a few percent) of the liquid passes
convectively through the particles. This has a very beneficial effect on mass transport into the
particles, especially for otherwise slowly diffusion species like large proteins. As the linear velocity in
the bed increases, so does the flow velocity inside the particles because they are related by the ratio of
permeabilities of the support and the bed, which is constant in the laminar flow regime. This flow
convectively transports the tracer molecules into the particles, in parallel to the regular diffusion.
When the convective transport becomes dominant, the mass transport rate inside the particles scales
linearly with the flow rate through the column, resulting in constant column efficiency – the height
equivalent of a theoretical plate (HETP) does not change anymore with flow rate. This effect allows
for constant separation quality even at very high flow rates. For a mathematical description of the
phenomenon, please consult the article by Carta and Rodrigues [22] that comprehensively treats this
effect.
In this chapter, the HETP was measured by packing the aggregates into a GE Healthcare Tricorn glass
column of dimensions 5 mm i.d. x 50 mm and injecting dextran tracer pulses into a 25 mM phosphate
buffer at pH 7. Their refractive index signal after the column was recorded and its moments calculated.
The HETP was then derived from the moments according to
,col ,tot ,eqi i i and ( 5-5 )
2,col col
2
1,col
HETPL
, ( 5-6 )
where i denotes the i-th moment of the recorded peak of the entire setup or just the equipment
without column. The column’s moments were then calculated from the differences. The HETP could
finally be derived from the column’s moments and the column length colL .
The results are shown in Figure 5-9. An initial decrease of HETP due to van Deemter’s B term can
usually not be observed in HETP due to the slow longitudinal diffusion, as in this case. Therefore, the
HETP initially increases as expected, but soon the slope decreases and the curve reaches a plateau. At
this point, convection dominates the mass transport and there will be no further increase in HETP as
Chapter 5
66
long as film diffusion inside the pores does not become dominant. This effect was never observed
during our experiments. This is most likely due to the still quite low flow velocities inside the pores.
Figure 5-9 Van Deemter plot of post-polymerised aggregates from latex E2. Dextrans of different molecular weights
were injected into the column under non-adsorbing conditions.
5.5 Conclusion
In this chapter we have described a way to efficiently produce large amounts of macro-porous
polymeric clusters without use of any organic solvent, only producing salt water as a waste product.
Due to space constraints we have not diverged into its applicability to other monomers, but it was
successful with any latex tried so far, including poly-HEMA (hydroxyethylmethacrylate), poly-MMA
(methyl methacrylate), poly-VBC (vinyl benzyl chloride) and poly-VAc (vinyl acetate). Therefore, we
are convinced that this is a truly universal process, as also all theory would suggest. A method has
been shown to evaluate the behaviour of any charge-stabilised particle suspension in the micro-
channel, obtaining a phase diagram that can be used for experiment design and finding suitable
aggregation conditions for the studied latex. Methods to affect the particle morphology in terms of
fractal dimension and specific surface area have been studied and explained. The chromatographic
behaviour of these materials was investigated as a possible application field and it was found that they
exhibit highly favourable, ‘perfusive’ mass transport properties.
0 1x104
2x104
3x104
0
10
20
30
1 kg/mol
5 kg/mol
150 kg/mol
410 kg/molRed
uced
pla
te h
eig
ht
h /
(-)
Reduced velocity v / (-)
67
Chapter 6
Conclusions and Outlook
The goal of this thesis was the development of chromatographic materials suitable for different
chromatographic applications. One of these applications was given through the collaboration with an
industry partner looking for a new stationary phase that can be used in the analytical separation of ions
contained in drinking water. There the aim was to design a column that can be used as the core of a
cheap ion chromatography unit that can be used for simple separations in uncomplicated matrices.
Therefore, the pressure drop should be very low to be able to save equipment costs and the column
itself very affordable, too. Additionally, the prepared material could be used for purification of bio-
molecules and should be shortly examined in that respect.
Starting in the first chapter with the same base material as already used by Marti et al. [6], polystyrene,
core-shell precursor particles were designed to reach a mechanically and chemically very stable
monolith that was then functionalised to bear strong cation exchange groups. Due to the high degree of
functionalisation, proteins adsorbed very strongly by ionic interaction and a mixture of proteins was
successfully separated on this monolith.
However, these monoliths were unsuitable for the application targeted by our industry partner: the
analytical separation of small ions. This is because these ions are mono- or divalent and thus do not
adsorb as strongly onto the surface as proteins do far from their isoelectric point, requiring a higher
amount of functional groups to separate them. Therefore, the production of longer columns was
attempted. While larger monoliths could be prepared without issues, their mechanical treatment
afterwards (lathing, fitting) proved to be prone to error and was thus considered unfeasible with the
tools available. Instead, longer columns could be prepared by packing porous particles into a regular
column. These particles originated from a modified version of the Reactive Gelation process [21]
employing shear to break up forming aggregates, thus establishing an equilibrium size. Before
Chapter 6
68
modifying this process to fit the desired chemical specifications, the feasibility of this material was
verified with a model that can account for the perfusive mass transport exhibited by these particles.
Having obtained positive results from the model, the particles were prepared according to the model
and sulphonated. While there was some separation of ions, the functional density was still on the
same, low value as before and the columns could not be made longer due to equipment dimensions.
The functional density per surface area was as high as for good commercial resins, pinpointing the
problem on the specific surface area of the produced materials. Increasing the specific surface area is
possible with Reactive Gelation, as shown in Chapter 5, but not by a factor 20 as would be required to
reach acceptable functional densities with the same functional density per surface area. In this work,
the type of functionalisation was therefore changed to covering the surface with highly charged gel
nanoparticles, thus switching from a 2D-functionalisation on the surface to a 3D-functionalisation that
reached into the pores. Because the ions could easily diffuse in and out of the gel due to its thickness
of only 100 nm, the perfusive mass transport was retained while the amount of charged groups per
surface area could be increased by the required factor. This resulted in a very low pressure, flow rate-
independent analytical column for the separation of the seven standard anions that can serve as the
core of a very cheap ion chromatography system employing low-pressure pumps and parts.
The possibility of industrial application of the designed material necessitated a higher productivity
than was thus far achieved. However, the scale-up of Reactive Gelation under shear is difficult due to
the low particle volume fraction of 1% and the high, narrowly distributed shear rates required to obtain
low particle polydispersity. The scale-up of this process was thus carried out by completely re-
working the aggregation step to be shear-induced instead of salt-induced. This entailed that after the
now continuous aggregation step the particles do not aggregate further and can thus be hardened under
gentle conditions and at higher dry contents. This process was found to yield more defined material,
both in particle polydispersity and pore size distribution, at very high productivities and thus widened
the field of application to non-specialty materials like thermal insulators.
As has been shortly mentioned before, a number of different chemistries have already been
successfully used for particle preparation with the continuous aggregation method, including pre-
Conclusions and Outlook
69
functional monomers like 4-vinylbenzyl chloride or vinyl acetate. It remains to apply these materials
to real separation tasks now that large amounts can be prepared easily. For this purpose, a palette of
ligands and functionalisations should be attached to these pre-functional particles and used for their
respective separation problems. In this work, a cation exchange material was prepared by sulphonating
polystyrene, and an anion exchange material by electrostatic attachment of highly positively charged
gel nanoparticles. The latter cannot be used for protein chromatography; the high ionic strength in
eluting buffers and cleaning agents would quickly desorb these nanoparticles. Therefore, a good first
step in the direction of preparing an anion exchanger for protein chromatography is the substitution of
chloride in a vinylbenzylchloride-based material by some form of quarternary amine. This would lead
to a mechanically strong material that does not possess any base-sensitive linkers, thus being well-
suited for protein chromatography.
Furthermore, this process can be used to prepare materials that can be tested for entirely different
applications, like spongy polymer gels, e.g. from poly(HEMA) or poly(HEMA-co-MMA). Such soft
materials have been prepared but not thoroughly characterised in this work; they prepared
homogeneous looking, soft monoliths whose stiffness increased with MMA content. A possible
application is in the field of tissue scaffolding, where large pores could improve transport of nutrients
into the scaffold.
71
Chapter 7
List of Figures
Figure 2-1 SEM picture of a Reactive Gelation monolith. .................................................................... 12
Figure 2-2 Pore size distribution of a Reactive Gelation monolith. ...................................................... 13
Figure 2-3 Van Deemter plot for IgG on Reactive Gelation monoliths. ............................................... 14
Figure 2-4 Separation of a protein mixture ........................................................................................... 15
Figure 3-1 Geometric description of a resin particle. ............................................................................ 19
Figure 3-2 Augmentation factor as a function of the Peclet number ..................................................... 21
Figure 3-3 Diagram of the mass transport chain occuring during chromatography .............................. 22
Figure 3-4 Van Deemter plot of three resin classes .............................................................................. 24
Figure 3-5 Van Deemter plot of K+ for a non-porous 10 μm strong cation exchange resin. ................. 25
Figure 3-6 Van Deemter plot of non-adsorbing dextran tracers on Reactive Gelation particles........... 26
Figure 4-1 Process scheme of Reactive Gelation under shear ............................................................... 29
Figure 4-2 SEM picture of the plain support ......................................................................................... 37
Figure 4-3 Static light scattering analysis of the plain support. ............................................................ 37
Figure 4-4 Mercury intrusion porosimetry of the plain support. ........................................................... 38
Figure 4-5 Amount of SO3- groups introduced as a function of reaction time. ..................................... 39
Figure 4-6 Reduced van Deemter plot obtained from water injections. ................................................ 42
Figure 4-7 Enhanced mass transport function, Pf , as a function of Peclet number, P ................ 43
Figure 4-8 Chromatogram of a mixture of seven standard ions ............................................................ 44
Figure 5-1 Scheme of the micro-channel equipment used. The pump exerts a pressure of 160 bar. .... 53
Figure 5-2 Viscosity of the slurry leaving the micro-channel at 160 bar with no salt addition ............ 57
Figure 5-3 Phase diagram for latex E2 at 13% dry content. .................................................................. 58
Figure 5-4 Effect of pressure and residence time onto fractal dimension and conversion of E3 .......... 59
Figure 5-5 Effect of dry content and ionic strength onto E3. ................................................................ 61
Figure 5-6 Comparison of aggregates made from all three precursor latexes. ...................................... 62
Chapter 7
72
Figure 5-7 Comparison of post-polymerised aggregates from all three precursor latexes .................... 62
Figure 5-8 SEM micrographs of post-polymerised aggregates from all three latexes .......................... 63
Figure 5-9 Van Deemter plot of post-polymerised aggregates from latex E2. ...................................... 66
73
Chapter 8
Bibliography
1. http://www.marketsandmarkets.com/Market-Reports/protein-antibody-engineering-market-
898.html [cited 2014].
2. Baselga, J., Cortes, J., Kim, S.B., Im, S.A., Hegg, R., Im, Y.H., Roman, L., Pedrini, J.L.,
Pienkowski, T., Knott, A., Clark, E., Benyunes, M.C., Ross, G., Swain, S.M. and Grp, C.S.,
New England Journal of Medicine, 366 (2012) 109-119.
3. Roque, A.C.A., Lowe, C.R. and Taipa, M.A., Biotechnology Progress, 20 (2004) 639-654.
4. Wang, Q.C., Hosoya, K., Svec, F. and Frechet, J.M.J., Analytical Chemistry, 64 (1992) 1232-
1238.
5. Urban, J., Svec, F. and Frechet, J.M.J., Analytical Chemistry, 82 (2010) 1621-1623.
6. Marti, N., Quattrini, F., Butte, A. and Morbidelli, M., Macromolecular Materials and
Engineering, 290 (2005) 221-229.
7. Bechtle, M., Butte, A., Storti, G. and Morbidelli, M., Journal of Chromatography A, 1217
(2010) 4675-4681.
8. Brand, B., Krattli, M., Storti, G. and Morbidelli, M., Journal of Separation Science, 34 (2011)
2159-2163.
9. Guiochon, G., Felinger, A., Shirazi, D.G. and Katti, A.M., Fundamentals of Preparative and
Nonlinear Chromatography, Academic Press, San Diego, CA 2006
10. Guiochon, G., Journal of Chromatography A, 1168 (2007) 101-168.
11. Svec, F. and Frechet, J.M.J., Analytical Chemistry, 64 (1992) 820-822.
12. Okay, O., Progress in Polymer Science, 25 (2000) 711-779.
13. Paul, S. and Ranby, B., Macromolecules, 9 (1976) 337-340.
14. Washburn, E.W., Physical Review, 17 (1921) 273.
15. Barrett, E.P., Joyner, L.G. and Halenda, P.P., Journal of the American Chemical society, 73
(1951) 373-380.
16. Schulte, M. and Epping, A., Fundamentals and General Terminology, Wiley-VCH Verlag
GmbH & Co. KGaA, 2005
17. Stone, M.C. and Carta, G., Journal of Chromatography A, 1146 (2007) 202-215.
18. DePhillips, P.L., I.; Färenmark, J.; Lenhoff, A.M.;, Analytical Chemistry, 76 (2004) 5816-
5822.
19. Hutchinson, J.P., Hilder, E.F., Shellie, R.A., Smith, J.A. and Haddad, P.R., Analyst, 131
(2006) 215-221.
20. Tsuneda, S., Saito, K., Sugo, T. and Makuuchi, K., Radiation Physics and Chemistry, 46
(1995) 239-245.
21. Lamprou, A., Synthesis and application of novel polymeric materials: short surfactants via
ATRP and functional macroporous particles via reactive gelation under shear. Diss. ETH No.
20571., in Department of Chemical and Bio-Engineering, 2012, Swiss Federal Institute of
Technology Zurich.
22. Carta, G. and Rodrigues, A.E., Chemical Engineering Science, 48 (1993) 3927-3935.
23. Carta, G., Gregory, M. E., Kirwan, D. J. and Massaldi, H. A., Sep. Technol, 2 (1992.
24. Jungbauer, A., Journal of Chromatography A, 1065 (2005) 3-12.
25. Bechtle, M., Butté, A., Storti, G. and Morbidelli, M., Journal of Chromatography A, 1217
(2010) 4675-4681.
26. Svec, F. and Frechet, J.M.J., Biotechnology and Bioengineering, 48 (1995) 476-480.
27. Schminke, G. and Seubert, A., Journal of Chromatography A, 890 (2000) 295-301.
28. http://metrohm.ch/Produkte2/IC/Columns.html [cited 2013].
29. http://www.dionex.com/en-us/webdocs/84509-Bro-Dionex-IC-Solutions-18Dec2009-
LPN2405.pdf [cited 2013].
30. Storey, B.T., Journal of Polymer Science Part A: General Papers, 3 (1965) 265-282.
Chapter 8
74
31. Wu, H., Lattuada, M. and Morbidelli, M., Advances in Colloid and Interface Science, 195
(2013) 41-49.
32. Pohl, C.A., Stillian, J.R. and Jackson, P.E., Journal of Chromatography A, 789 (1997) 29-41.
33. Polymer Handbook, Wiley, 1999
34. Sherrington, D.C., Chemical Communications, 21 (1998) 2275-2286.
35. Ford, W.T., Lee, J. and Tomoi, M., Macromolecules, 15 (1982) 1246-1251.
36. Hentze, H.-P. and Antonietti, M., Porous Polymers and Resins, Wiley-VCH Verlag GmbH,
2008
37. Liu, X. and Ma, P., Annals of Biomedical Engineering, 32 (2004) 477-486.
38. Winter, J.S., J.; Malinský, J.; Dušek, K.; Heitz, W., Fortschritte der Hochpolymeren-
Forschung, 5 (1967) 113-213.
39. Guyot, A. and Bartholin, M., Progress in Polymer Science, 8 (1982) 277-331.
40. Cheng, C.M., Vanderhoff, J.W. and El-Aasser, M.S., Journal of Polymer Science Part a-
Polymer Chemistry, 30 (1992) 245-256.
41. Svec, F. and Frechet, J.M.J., Chemistry of Materials, 7 (1995) 707-715.
42. Svec, F. and Frechet, J.M.J., Macromolecules, 28 (1995) 7580-7582.
43. Svec, F., Journal of Chromatography A, 1217 (2010) 902-624.
44. Zaccone, A., Wu, H., Gentili, D. and Morbidelli, M., Physical Review E, 80 (2009.
45. Millar, J.R., Marr, W.E., Kressman, T.R. and Smith, D.G., Journal of the Chemical Society,
(1964) 2740.
46. Ugelstad, J., Kaggerud, K.H., Hansen, F.K. and Berge, A., Makromolekulare Chemie-
Macromolecular Chemistry and Physics, 180 (1979) 737-744.
47. Kerker, M., The scattering of light, Academic Press, New York 1969
48. Bohren, D.R. and Huffman, C.F., Absorption and Scattering of Light by Small Particles,
Wiley-Interscience, New York 1983
49. Jones, A.R., Progress in Energy and Combustion Science, 25 (1999) 1-53.
50. Chern, C.S., Progress in Polymer Science, 31 (2006) 443-486.
51. Wu, H., Lodi, S. and Morbidelli, M., Journal of Colloid and Interface Science, 256 (2002)
304-313.
52. Russel, W.B., Saville, D.A. and Schowalter, W.R., Colloidal Dispersions, Cambridge
University Press, 1989
53. Israelachvili, J.N., Intermolecular and Surface Forces. 3rd ed., Academic Press London,
2011
54. Einstein, A., Annalen der Physik, 324 (1906) 289-306.
55. Xie, D., Wu, H., Zaccone, A., Braun, L., Chen, H. and Morbidelli, M., Soft Matter, 6 (2010)
2692-2698.
56. Soos, M., Moussa, A., Ehrl, L., Sefcik, J., Wu, H. and Morbidelli, M., Journal of Colloid and
Interface Science, 319 (2008) 577-589.
57. Ehrl, L., Soos, M. and Morbidelli, M., Langmuir, 24 (2008) 3070-3081.
58. Harshe, Y.M., Lattuada, M. and Soos, M., Langmuir, 27 (2011) 5739-5752.
59. Soos, M., Ehrl, L., Bäbler, M.U. and Morbidelli, M., Langmuir, 26 (2010) 10-18.
60. Coquebert de Neuville, B., Modeling and experimental characterization of protein mass
transfer and adsorption in preparative chromatography, in Department of Chemical and Bio-
Engineering, 2013, Swiss Federal Institute of Technology Zurich.
Curriculum Vitae
75
Chapter 9
Curriculum Vitae
Bastian Brand
04/2010-04/2014 PhD studies
Institute for Chemical and Bioengineering,
Prof. Massimo Morbidelli, ETH Zurich, Switzerland
10/2009-02/2010 Master thesis
Institute for Chemical and Bioengineering,
Prof. Massimo Morbidelli, ETH Zurich, Switzerland
09/2008-02/2010 MSc Chemical and Bio-Engineering
ETH Zurich, Switzerland
10/2005-08/2009 BSc Chemical Engineering
ETH Zurich, Switzerland
09/2003-06/2005 Secondary education
Xàbia International College
Jávea, Spain
20/06/1986 Born in Ulm, Germany
Chapter 9
76
Publications
2010 Magnetic Gelation: a new method for the preparation of polymeric anisotropic
porous materials. Furlan M., Brand B., Lattuada M. Soft Matter, 2010, 6,
5636-5644
2011 Strong cation exchange monoliths for HPLC by Reactive Gelation. Brand B.,
Krättli M., Storti G., Morbidelli M. Journal of Separation Science, 2011, 34,
2159-2163
2013 Method for the preparation of macroporous particles and macroporous
particles obtained using such a method – International patent application
Scientific Presentations
2010 Two poster presentations at MSS, Portorož, Slovenia
2010 Invited oral presentation at General Assembly of Association of Swiss Process
and Chemical Engineers (SGVC), Visp, Switzerland
2010 Poster Presentation at annual assembly of Material Research Center, Zürich,
Switzerland
2011 Oral presentation at IPSCSS, Wernigerode, Germany
2011 Oral presentation at PREP, Boston, MA, USA
2012 Oral presentation at IPSCSS, Lauterbad, Germany
2012 Oral presentation at EUPOC, Gargnano, Italy
2012 Oral presentation at the University of Bologna, Italy
2013 Oral presentation at AICHE Full Meeting, San Francisco, CA, USA