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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

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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



4.5 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



5.4.1 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.


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.


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.


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)


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:


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.


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.


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


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.


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%.


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.


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.


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.


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.


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

)


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


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 / ()


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


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


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.


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%.


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





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


Chapter 5

52




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


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’.


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).


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


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).


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


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)


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.


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


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


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

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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

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