Recent developments and future possibilities for polymer monoliths in separation science

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Recent developments and future possi

R. Dario Arrua,a Tim J. Causonab and Emily F. Hilder*a

Received 15th June 2012, Accepted 30th August 2012

DOI: 10.1039/c2an35804b

Within recent years there has been an increase in research focused on the design and application of

organic polymer monoliths in all areas of separation science. This is largely driven by the theoretical

and practical benefits that these materials should be able to provide, particularly in terms of improved

biocompatibility and high permeability. This review summarises recent new developments in this field

with a focus on new approaches to the design and synthesis of polymeric monolithic materials for

analytical separation science. This includes the use of alternative synthetic methodologies such as the

development of hyper-crosslinked monoliths, preparation of hybrid materials and incorporation of

nanostructures in the polymeric scaffold. New and developing approaches for the structural

characterisation of monolithic columns are also included. Finally, we critically discuss the current

chromatographic performances achieved with this column technology as well as where future

developments in this field may be directed.

1. Introduction

Monolithic polymers were introduced in the early 1990’s1 and have

become increasingly popular as a new technology for analytical

R: Dario Arrua

Dario Arrua graduated from the

National University of C�ordoba

(C�ordoba, Argentine) where he

obtained his Degree (2003) and

PhD in Chemistry (2009). He is

currently a Postdoctoral

Research Fellow in the Austra-

lian Centre for Research on

Separation Science

(ACROSS), University of Tas-

mania working under the super-

vision of Prof. Emily Hilder and

Prof. Paul Haddad. His

research interests are related

with the synthesis and chemical

surface modification of macro-

porous polymers to be used as stationary phases for the separation

of biomolecules.

aAustralian Centre for Research on Separation Science (ACROSS),School of Chemistry, University of Tasmania, Private Bag 75, Hobart,7001, Australia. E-mail: Emily.Hilder@utas.edu.au; Fax: +61 3 62262858; Tel: +61 3 6226 7670bInstitute of Analytical Chemistry, Johannes Kepler University Linz,Altenberger Str. 69, Linz, A-4040, Austria

This journal is ª The Royal Society of Chemistry 2012

chemistry and particularly for separation science. The best known

advantage they present is a rigid structure with high permeability

due to the presence of large through pores which permits the use of

high liquid flow rates at low back pressures. Another advantage of

monolithic polymers is their ease of preparation, since they can be

prepared in a single step from a homogeneous polymerization

mixture containing monomers (monomer/s with a particular

functional group and a cross-linker monomer), porogenic mixture

and a radical initiator which initiates the polymerisation reaction.

A typical scanning electron microscopy (SEM) image of a

Tim J: Causon

Tim Causon is a graduate of the

University of Tasmania where

he completed his BSc (hons) in

2008 and his PhD in 2012. He is

currently a research fellow in the

Department of Analytical

Chemistry, Johannes Kepler

University (Linz). His research

interests are in the general area

of separation science, in partic-

ular in the design and perfor-

mance optimisation of

chromatographic systems.

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monolithic polymer is shown in Fig. 1. As can be seen, the structure

generally consists of large microglobules aggregated in clusters

between which the macropores are formed. Due to their porous

properties, these polymeric materials have been extensively inves-

tigated for use as stationary phases in different chromatographic

separations,2–5 sample pre-treatment,6 as microfluidic devices,7

immobilized enzyme reactors8,9 and for solid phase chemistry.10

One of the major applications of polymer monoliths is as

chromatographic supports typically in column format. New

methods of preparation and applications have been studied

extensively since the early demonstrations of their usage in

HPLC11,12 and numerous reviews13–17 have been dedicated to

these developments. However, understanding the chromato-

graphic performance of polymer monoliths has not received so

much attention despite significant advances for silica-based

analogues.18 In this review we present the most recent trends in

the preparation and use of organic based monolithic polymers

with a focus on understanding limitations of current preparation

methods and developing a rationale for improving the support

morphology.

Fig. 1 Scanning electron micrograph of a macroporous monolithic

polymer.

Emily F: Hilder

Emily Hilder is Professor and

ARC Future Fellow in the

Australian Centre for Research

on Separation Science

(ACROSS) and School of

Chemistry at the University of

Tasmania. Her research focuses

on the design and application of

new polymeric materials, in

particular polymer monoliths, in

all areas of separation science.

She is also interested in the

development of miniaturised

analytical systems, particularly

for applications in clinical diag-

nostics and remote monitoring.

She has over 80 peer-reviewed publications and was recently rec-

ognised as the LCGC Emerging Leader in Chromatography

(2012). She is also an Editor of Journal of Separation Science.

5180 | Analyst, 2012, 137, 5179–5189

2. Preparation

Preparation of organic-based macroporous monolithic polymers

Monolithic materials can be synthesised using a number of

different approaches with the most frequently used being ther-

mally initiated2,19,20 and UV initiated polymerization

methods.2,10,21 Since these polymers adopt the format of the

mould used as the reactor for the polymerisation mixture,

monolithic materials can be prepared in different formats such as

large rod polymers using standard HPLC columns,22 capillary

columns,2–5,21 monolithic disks,23 cylinders24 and flat sheet poly-

mers.25,26 In this section we address the latest developments in the

preparation of organic polymer monoliths. If the reader is

interesting in a deeper knowledge about the different approaches

for manufacturing monolithic polymers, Svec13 recently pub-

lished a comprehensive review on this topic.

Recently, a novel approach using photo-initiated polymerisa-

tion was demonstrated through the use of visible light. Walsh

et al.27,28 reported the photoinitiated polymerisation of mono-

lithic polymers using visible region light emitting diodes (LEDs).

This innovative preparation opens new windows with regard to

the type of mould that can be used and also demonstrates that

photo-initiated polymerisation of monomers with strong absor-

bance in the UV region, e.g. styrene-based monomers, can be

used to form rigid, porous polymers. Firstly, they reported the

polymerization of poly(glycidyl methacrylate-co-ethylene glycol

dimethacrylate) [poly(GMA-co-EDMA)] within a polyimide

coated capillary using a ternary component initiator and a 660

nm light emitting diode as the light source.27 Since the monolithic

column presented good hydrodynamic properties, the authors

showed the potential application of this polymer as an electro-

osmotic pump. The same research group has also reported the

first visible light initiated polymerization of poly(styrene-co-

divinylbenzene) [poly(Sty-co-DVB)] using a 470 nm light emit-

ting diode array.28 These capillary columns were used for a

reversed-phase (RP) gradient separation of a mixture of proteins

presenting good selectivity and a %RSD (n ¼ 3) in the retention

factor of 2.6% for the column to column variability and less than

1.73% for the run to run assays. Flook et al.29 have similarly

demonstrated that by careful consideration of wavelength,

initiator and other system components, reversed-phase columns

for the efficient separation of proteins and peptides can be

prepared using divinylbenzene as the sole monomer. In this case

UV initiation of 2-methyl-40-(methylthio)-2-morpholinopropio-

phenone at a wavelength of 350 nm was used.

The porous properties of monolithic columns, particularly the

large through pores and consequent low specific surface area,

mean that they are frequently used for the separation or purifi-

cation of large biomolecules. With the objective of expanding the

application of monolithic polymers for the separation of small

molecules, Urban et al.30,31 reported the synthesis of large surface

area poly(Sty-co-chloromethylstyrene-co-DVB) [poly(Sty-co-

CMS-co-DVB)] capillary columns. Using a monolith previously

prepared by conventional thermally initiated polymerisation

they performed a crosslinking reaction using FeCl3 as catalyst to

obtain a hyper-crosslinked material of the Davankov type. Using

this approach, the authors obtained polymers with a specific

surface area of 663 m2 g�1; one order of magnitude larger than

Fig. 2 SEM image of the 125 mm thick porous poly(butyl methacrylate-

co-ethylene dimethacrylate) monolithic layer. Reproduced from ref. 25

with permission.

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measured for the precursor support. Some separations of low

molecular weight compounds such as uracil and alkylbenzenes

compounds were demonstrated. Recently, Li et al.32 prepared

highly crosslinked monoliths for the efficient separations of alkyl

benzenes and alkyl parabenes. The polymers were obtained by

UV initiated polymerization reactions using a single monomer

preparation. The monomers used were bisphenol A dimetha-

crylate (BADMA), bisphenol A ethoxylatediacrylate (BAEDA,

EO/phenol ¼ 2 or 4) or pentaerythritoldiacrylatemonostearate

(PDAM). Using a high monomer concentration and the right

porogenic mixture the authors obtained rigid monolithic poly-

mers with high surface areas. The monolithic capillary columns

reported showed good performance in the separations of small

molecules (60 208 plates per m for pentylbenzene on poly-

BADMAmonolithic columns). The use of just a single monomer

in the polymerization mixture allowed the authors to synthesise

these monolithic columns with very high reproducibility. The

RSD (n ¼ 3) values for the run-to-run and column-to-column

reproducibility based on retention times were less than 1.2% in

both cases. In another example, Smirnov et al.33 prepared

different poly(DVB-co-ethylvinylbenzene-co-2-hydroxyethyl

methacrylate) [poly(DVB-co-EVB-co-HEMA)] monolithic

columns for the separation of aromatic compounds. The poly-

merisation reactions were carried out within vinylised glass

columns (150 mm � 3 mm I.D.) using nitrogen pressure to avoid

the polymer shrinking during its preparation. All the polymers

obtained showed high specific surface area, which is usually

desired for the separation of low molecular weight compounds,

but the pore size distribution profiles shown were heterogeneous

comparing with other monolithic polymers. The authors studied

the effect of the amount of HEMA in the polymerisation mixture

over the permeability and chromatographic performance of the

monoliths prepared. They found that higher HEMA concen-

trations resulted in an improvement in the column efficiency

(HETP of 53–83 mm for retained substituted benzenes) but a

reduction in the hydrodynamic permeability. Svobodov�a et al.34

have recently published the preparation of poly(Sty-co-DVB-co-

methacrylic acid) [poly(Sty-co-DVB-co-MAA)] capillary

columns varying the amount of MAA in the polymerisation

mixture and evaluated them in the purification of aromatic

compounds. The authors claimed that the presence of MAA in

the monolithic structure is essential for the permeability of the

support and the isocratic separations of small organic molecules.

While most papers have reported the use of monolithic

columns with internal diameters (I.D.) of 75 to 200 mm, re-

optimisation of the polymerisation conditions should be per-

formed to obtain monolithic polymers with smaller I.D. to retain

the same chromatographic performance as the columns prepared

in larger dimensions. Nischang et al.35 synthesised poly(butyl

methacrylate-co-EDMA) [poly(BuMA-co-EDMA)] monolithic

columns using capillaries with I.D. from 75 to 5 mm and found

that the hydrodynamic properties of the monoliths obtained

were strongly influenced by the ‘‘confinement effect’’36 of the

capillary wall. This effect is based in the reduction of the volume-

to-surface ratio as the I.D. of the capillary is decreased. Opti-

mising the conditions of the polymerisation reaction, these

monolithic columns retained the porous properties found in

larger capillaries. The chromatographic performance obtained

using these columns showed that using capillaries with low I.D.

resulted in reduced peak width and increased sensitivity for mass-

spectrometric detection of intact protein standards using

gradient elution.

On the other hand, homogeneous monolithic columns in large

format ($1 mm) should also continue to be investigated to

increase the technology uptake of these novel stationary phases.

However, there is a well known problem related with the radial

heterogeneity of monolithic columns in large formats.37 There-

fore, the preparation of monolithic columns in standard

dimensions is a challenge and new preparation methods are

needed in this sense. For example, Nesterenko et al.38 prepared

poly(BuMA-co-EDMA) monolithic columns inside 0.8 mm I.D.

micro-bore titanium columns. An advantage of using titanium

columns is that the titanium can be readily oxidized and subse-

quently modified with silane compounds to bond the polymer

network to the titanium walls, which is not possible with stainless

steel columns. At the same time the excellent thermal conduc-

tivity of titanium resulted in the formation of a temperature

gradient within the column during polymerisation, resulting in

significant radial heterogeneity in the polymer bed. This was

exacerbated by the surface pretreatment as the kinetics of the

surface polymerisation is significantly different from that

occurring in bulk within the column, significantly reducing the

overall separation efficiency that could be achieved with this

column.

Although monolithic polymers have been broadly used as

stationary phases in LC within columns of different sizes, in

recent years there has been a number of other formats

prepared.25,26 Woodward et al.25 performed separations of

peptides and oligonucleotides using flat poly(BuMA-co-EDMA)

monoliths. The polymer was prepared using photo-initiation

within two glass plates separated with a Teflon film which defined

the thickness of the monolithic layer. The structure of the

monolithic layer is shown in Fig. 2. This hydrophobic flat

polymer was used for the rapid separation of a mixture of

peptides and oligonucleotides by electrophoresis. The same

poly(BuMA-co-EDMA) planar polymers were co-photografted

with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS)

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and HEMA to obtain a surface with both ionisable and hydro-

philic groups. These modified monoliths were also used for the

separation of peptides by pressurised planar electro-

chromatography. Using a similar approach, the same research

group photografted AMPS and HEMA on poly(BuMA-co-

EDMA) planar monoliths through a mask obtaining as a result a

600 mm modified hydrophilic channel along one side of the

layer.39 Therefore, the modified polymer presented two areas

having great differences in their hydrophobicity. These planar

monoliths were used in the two dimensional thin layer chroma-

tography of peptides. The 2D separation of fluorescently labeled

peptides is shown in Fig. 3. The first dimension separation was

carried out along the hydrophilic channel in ion exchange mode

while the second dimension was performed in the reversed phase

mode through the more hydrophobic unmodified part of the

planar polymer. Maksimova et al.40 prepared different methac-

rylate-based flat monoliths having different hydrophobicity and

porous properties. These monolithic layers were successfully

used in the planar chromatography separation of low molecular

weights compounds (dyes) as well as synthetic polymers [poly-

(vinylpyrrolidone) and polystyrene]. In all cases the analysis

times were less than 9 min which gives an indication of the high

permeability of the polymers studied. Planar polymer monoliths

have also recently been introduced as an alternative to paper for

dried blood spot sampling.41 The application of polymer

monoliths to sample preparation in general is certainly not new,42

but new approaches such as this serve to further demonstrate the

advantages that these types of materials offer in this area.

While the great majority of monolithic polymers used in

separation science are prepared from a homogeneous polymeri-

sation mixture using heat or radiation as initiation source, there

are other types of organic-based monolithic materials which

Fig. 3 Two-dimensional TLC separation of a mixture of labeled

peptides on 50 mm thick monolithic polymer layer with dual chemistry

using UV detection. Reproduced from ref. 39 with permission.

5182 | Analyst, 2012, 137, 5179–5189

present a different porous architecture. Amongst these materials

both poly(high internal phase emulsions) polymers (polyHIPEs)

and cryogels appear to be promising.

polyHIPEs are prepared by polymerisation of an emulsion and

are characterised by high porosities (more than 70%) and large

spherical cavities interconnected one each other by holes called

‘‘windows’’.43 A possible disadvantage of polyHIPEs is that they

present a low specific surface area (as a consequence of the low

monomer concentration used in the polymerization mixture) and

this limits the use of these polymers in separation science.

However different research groups have recently reported the

preparation of polyHIPEs with very high surface areas which

could have potential application as chromatographic

supports.44,45 Using the same approach30 as for conventional

organic-based monoliths, Schwab et al.45 prepared a hyper-

crosslinked polyHIPE of the Davankov type. Firstly, the authors

synthesised poly(Sty-co-CMS-co-DVB) polymers with very low

surface areas (less than 20 m2 g�1). Then, the network was

swollen with 1,2-dichloroethane and crosslinked using FeCl3 as

catalyst. Using this approach, monolithic HIPE polymers were

obtained with surface areas as high as 1210 m2 g�1. Although

these materials were used for gas adsorption, they seem to be

interesting supports for potential use in separation science.

An important difference between these supports and conven-

tional monoliths is that polyHIPEs can easily be prepared in

analytical scale HPLC columns since the monomer concentration

used in the preparation of poly(HIPE) is lowand consequently the

heat produced during the polymerisation reaction is easily dissi-

pated by the solvent phase. Yao et al.46 prepared poly(GMA-co-

EDMA) macroporous polymers within stainless steel HPLC

columns (50 � 4.6 mm I.D.) by controlling supramolecular self-

assembly polymerisation using a tri-block copolymer (Pluronic

F127) as director and stabiliser. The surface morphology of these

polymers is shown in Fig. 4. These HPLC monolithic columns

showed a very high dynamic binding capacity (42.5mgmL�1) and

good chromatographic performance for a fast gradient separation

of a protein mixture. Although the authors claim that these

monoliths ‘‘have homogeneous sub-micrometer skeletons’’, the

porous size distribution curves presented for these supports are

broader than those commonly observed for conventional organic-

based monoliths. Recently, a similar porous size distribution

profile was obtained for poly(isodecylacrylate-co-DVB) [poly-

(IDA-co-DVB)] HIPE monolithic columns used for the separa-

tion of alkylbenzenes by CEC obtaining good resolution.47

Therefore, an improvement in the preparation of these materials

to obtain more homogeneous structures will have a consequent

improvement in the separation efficiency increasing the potential

application of these alternative materials.

In cases where the remotion of the surfactant used is a

problem, polyHIPEs can also be prepared by Pickering emulsion

polymerisation.48,49 In this approach, the emulsion droplets are

stabilised by solid nanoparticles instead of using conventional

surfactants. The use of particles to stabilise the emulsion poly-

merisation mixture has some advantages such as the possibility

to functionalise the cell walls of the porous polymers with a layer

of solid nanoparticles bearing different functional groups.

Although the chromatographic application of these types of

polyHIPE materials has not yet been demonstrated, these kinds

of porous polymers offer great potential in this area.

Fig. 4 SEM images of polyHIPE’s prepared using different amount of

aqueous phase in the polymerization mixture. (A) 88% pore volume, (B)

80.0% pore volume, (B1) magnified five times of (B), (C) 75.0% pore

volume, (D) 70.0% pore volume. Reproduced from ref. 46 with

permission.

Fig. 5 (A) A picture of carriers with different cryogels. The gels were

prepared in Kaldnes carriers as protective shells. SEM images of (B)

polyacrylamide plain cryogel, (C) inorganic adsorbent in polyacrylamide

cryogel, (D) MIP adsorbent in polyacrylamide cryogel. The scale bar

shows 50 mm in all three images. The arrows in image (A) show which

carrier belongs to which SEM image. Reproduced from ref. 54 with

permission.

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A second interesting type of macroporous monoliths are the

superporous cryogels which are prepared from different water

soluble monomers at sub-zero temperatures where a macro-

porous structure is formed from a semi-frozen aqueous media

utilizing ice crystals as a porogen.50,51 To complete the poly-

merisation reaction, the polymer is placed at room temperature

and the ice crystals melts leaving a polymer network with high

porosity (up to 90%) and very large pores (0.1 to 200 mm). This

characteristic macroporosity of cryogels permits them to be used

in the purification of particulate systems such as crude broths or

crude cell homogenates. Wang et al.52 performed a direct isola-

tion of cytidine triphosphate from unclarified broth of yeast cells

using anion exchange cryogels. Firstly, they prepared a cryogel

using acrylamide (Aam) and N,N-methylene-bis-acrylamide

(BIS) as a crosslinker to obtain a highly permeable polymer with

a porosity of 85%. Then the cryogel surface was grafted with

N,N-dimethylaminoethyl methacrylate (DMAEMA) and this

support was used for the isolation of the target biomolecule.

The highly porous structures found in cryogels also permit

them to be used as high-throughput SPE supports to treat large

volumes of effluent samples at high flow rates. Recently,

composite cryogels consisting of conventional cryogels and

molecular imprinting particles (MIP) were prepared and used as

SPE supports of different pollutants.53–55 Baggiani et al.53

prepared composite cryogels of micrometer-size MIP particles

embedded in poly(Aam-co-BIS) cryogels for the solid phase

extraction of bisphenol A from river water and wine samples.

Using these composite cryogels the authors reported the

extraction of the target compound in highly diluted samples (ng

L�1) with recoveries above 75%. In a similar approach, Hajiza-

deh et al.54 recently prepared two types of composite cryogels for

the removal of bromate from drinking water. Firstly, they

reported the synthesis of molecular imprinted chitosan-based

beads using bromate as template. Secondly, ion-exchange

particles (Fe2O3$Al2O3$xH2O) were prepared using the sol–gel

method to investigate adsorption of bromate by ionic interac-

tion. Finally, both kinds of adsorbents were incorporated into

the acrylamide-based monomer mixture and the composite cry-

ogel was produced at sub-zero temperatures inside plastic

carriers used as protective shells. The use of plastic should permit

them to be used in large scale applications. The morphology of

these composite cryogels can be seen on Fig. 5. Using these

materials, the content of bromate in treated samples could be

determined below the required level of 10 mg L�1 although this

good performance did not appear to be maintained after subse-

quent extraction cycles.

Since most cryogels are prepared with a low crosslinker

concentration, they do not present good mechanical properties

and therefore their use as a stationary phase in HPLC is limited.

In order to produce cryogels with improve mechanical proper-

ties, Chen et al.56 prepared poly(MAA-co-polyethylene glycol

diacrylate) [poly(MAA-co-PEGDA)] cryogels with a high

crosslinker concentration inside a stainless steel column. The

monolithic cryogel was then used in the separation of a protein

mixture and water soluble nanoparticles. Although this short

communication seemed to be an interesting contribution toward

the preparation of monolithic cryogels to be used in the sepa-

ration of biomolecules in adsorption mode, no new articles have

yet been published using this kind of cryogel.

We have recently introduced a new approach to the synthesis

of cryogels demonstrating that high performance separations are

possible with these types of materials. Composite cryopolymers

were synthesised based on poly(PEGDA) cryostructures con-

taining embedded polystyrene or poly(EDMA) nanoparticles,

prepared at sub-zero temperatures using ice crystals as porogen.

Using this approach we achieved both high permeabilities and

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separation performance similar to that achieved using conven-

tional rigid polymer monoliths, as shown in Fig. 6 for proteins in

the hydrophobic interaction chromatography mode. With the

addition of charged nanoparticles, separations can also be ach-

ieved in the ion-exchange mode.

Novel polymer monolithic and hybrid structures

Due to the mostly mediocre efficiency obtained for conventional

organic monolithic polymers utilised in isocratic liquid chro-

matography, alternative syntheses of monolithic materials have

been investigated by a number of authors. Bechtle et al.57

prepared porous methacrylate-based monoliths by the reactive

gelation process, which involves a series of independent steps to

reach a monolithic polymer network based on monodisperse

nanoparticles. As one of the main advantages, the authors

claimed that this approach is a good alternative to prepare large

scale monoliths without the well known problems of heat

removal present in the synthesis of conventional monolithic

polymers. In this approach the last step of the preparation uses a

large percentage of water which has a high heat capacity.

Although the materials prepared by this process showed good

hydrodynamic properties, high efficiencies and good adsorption

capacities, numerous steps are needed in the preparation of these

monolithic columns making the process time intensive. At the

same time, another drawback could be that polymerisation is not

performed within the final mould (as can be done with conven-

tional monolithic polymers) since these polymers need to be later

placed in a suitable housing to be used as a chromatographic

column. This creates additional challenges in terms of elimi-

nating unwanted wall effects, which will have a detrimental

impact on separation efficiency.

Watanabe et al.22 reported the preparation of a novel mono-

lithic column called ‘‘spongy monoliths’’ since these materials

presented a high permeability (compared with the permeability

of 30 mm particles). The procedure to obtain this monolithic

column is apparently easy since it consists of melting EVA resin

(a copolymer of ethylene and vinyl acetate) at 130 �C in the

presence of pentaerythritol as a pore template. The resultant

flexibility of these materials permits them to be packed in an

Fig. 6 SEM image of monolithic cryopolymer based on poly(PEGDA) conta

mixture under HIC mode using the same monolithic column. Conditions: 1

myoglobin (1), ribonuclease A (2), lysozyme (3) and a-chymotrypsinogen A (

buffer, pH 6.9. Eluent B: 0.1 M phosphate buffer, pH 6.9. 1 min isocratic elutio

to 100% B, and then isocratic elution with 100% B for 5 min.

5184 | Analyst, 2012, 137, 5179–5189

untreated HPLC column housing. Although these polymers

showed good results as pre-concentrators for bisphenol A, they

have some disadvantages such as their solubility in non-polar

solvents which limits the application of this support as a

stationary phase and the low surface area due to the lack of meso

and micropores.

Another promising alternative toward the preparation of new

stationary phases is the development of hybrid monolithic

materials (monoliths + particles) which combine the well known

high mass transport and stability of monolithic polymers and the

retention mechanism of different functionalised particles

commonly used in chromatographic separations. Functionali-

sation of the pore surface with nanoparticles was first demon-

strated several years ago58 based on electrostatic attachment of

charged nanoparticles to an oppositely charged polymer mono-

lith. Separations of a range of species have been demonstrated

including carbohydrates,58 inorganic ions59 and oligonucleo-

tides.60 More recently Krenkova et al.61 incorporated commercial

50 nm hydroxyapatite nanoparticles into a poly(HEMA-co-

EDMA) monolithic capillary column and this support was used

in the separation of a model protein mixture, detection of

aggregates of monoclonal antibodies and enrichment of phos-

phopeptides from complex protein digests. The binding capacity

reached using these hybrid capillary columns were in the same

order with that of commercial hydroxyapatite packed columns

and the separation time was around five times lower than the

packed columns. This is an example of the favourable properties

presented by such hybrid materials. As noted already in this

review, the incorporation of a range of polymer nanoparticles

into cryopolymers can also be used to improve both the sepa-

ration performance and add new selectivity to these materials.

In another approach Jandera et al.62 prepared hybrid silica

particle-monolithic columns so that ‘‘the polymerization mixture

fills the space in between the particles in the column and glues the

entrapped sorbent particles together and to the fused silica

capillary walls’’. The rationale in this case was to synthesis

materials resistant to shrinkage and where voids between the

monolithic bed and the capillary wall should not form. The

authors carried out the polymerisation of poly(lauryl methac-

rylate-co-EDMA) [poly(LMA-co-EDMA)] within a capillary

ining 10% poly(DVB) nanoparticles and separation of a standard protein

mL of protein mixture was injected containing 0.2 mg mL�1 each of

4). Flow rate: 4 mL min�1. Eluent A: 3 M (NH4)2SO4 in 0.1 M phosphate

n with 100% eluent A, followed by a 15 min linear A–B gradient from 0%

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pre-packed with 3–5 mm C18 or aminopropyl silica bonded

particles. These hybrid monolithic columns were used in the RP

mode for the separations of aromatic compounds and a mixture

of proteins and also for the separation of a mixture of phenolic

acids using the hydrophilic interaction (HILIC) mode. For these

columns, silica particles with different functionalities were

embedded in a polymerisation mixture composed of the zwit-

terionic methacrylate monomer (MEDSA) cross-linked with

ethylene dimethacrylate. Although, the authors found that in

general the efficiency of these novel columns were between the

efficiency of particle-packed and standard monolithic columns

the hybrid columns presented in this work allowed good sepa-

ration times and resolution for the mixtures studied.

Using a different strategy, Wu et al.63 synthesised hybrid

monolithic columns using a polyhedral oligomeric silsesquioxane

(POSS–MA) reagent as the inorganic–organic hybrid crosslinker

and a synthesized aliphatic long chain methacrylate quaternary

ammonium salt as the monomer. The polymerization reaction is

shown in Fig. 7. These novel materials were used as stationary

phase in both mHPLC and CEC and showed that the combina-

tion of the inorganic–organic crosslinker and organic monomers

gave this material the advantages of organic based monoliths and

silica based monoliths, which are high efficiencies, good

mechanical properties and a large pH stability range (pH 1–11).

Since POSS reagents are commercially available with different

chemistries, new hybrid columns could be synthesised looking

for high efficiency polymer monolith stationary phases. More

recently Nischang et al.64 have also demonstrated the facile,

single-step preparation of high surface area porous materials

based on POSS, although application of these materials as

chromatographic supports was not included.

3. Structural characterisation

As has been highlighted by a number of authors37,65,66 one of the

limiting factors in preparing reproducible monoliths with good

chromatographic performance is the degree of bed heterogeneity.

A number of methods for examining monolithic structures have

been presented in the literature with scanning (or transmission)

electron microscopy (SEM/TEM) and indirect methods such as

mercury intrusion porosimetry (MIP),67 inverse size exclusion

chromatography (ISEC)68 and adsorption/desorption of

nitrogen continuing to be the most commonly employed.

However, the aforementioned approaches are not entirely

adequate for comprehensive morphological characterisation due

Fig. 7 Schematic preparation of MDOA–POSS hybrid monolithic

column using polyhedral oligomeric silsesquioxane (POSS–MA) as cross-

linker and MDOAB as monomer. Reproduced from ref. 63 with

permission.

to the limited information that can be obtained from a single

analysis (as is the case with SEM/TEM) and the underlying

assumptions of indirect methods (i.e. an open-pore cylindrical

structure). Moreover, to carry out characterisation studies by

MIP and BET, a relatively large amount of sample is required.

Therefore, in the case of the synthesis of capillary monolithic

columns it is usually common to prepare larger bulk polymers

(using the same polymerisation mixture) to perform porosity

studies. However as long speculated by many working in the

field, Bystr€om et al.69 have recently demonstrated that bulk

polymerised monoliths present different porous characteristics

from those monoliths prepared within capillary columns.

Further shortfalls of this approach are also apparent from a

number of recent publications from Nischang and Br€ugge-

mann,70 providing detailed characterisation of organic polymer

monoliths in the solvated state (i.e. chromatographic operating

conditions). In this work the presence of inherent gel porosity is

clearly demonstrated, correctly questioning the validity of

measurements such as surface area being made in only the dry

state. That is, it is clear from this work that the typically low

surface areas reported for polymer monoliths are not necessary

an accurate reflection of the accessible surface area under chro-

matographic conditions. For the reasons mentioned above, new

non-invasive methods for characterising the morphology of

monoliths (particularly chromatographic columns) have become

a recent focus for some research groups.

One alternative approach demonstrated by Petter et al.71 uti-

lised near-infrared spectroscopy for determination of pore size,

pore volume, total porosity and surface area in a single analysis.

While this might have some advantageous over MIP and BET

(which are destructive methods), this approach still does not

provide detailed information on fine morphological details

including potential wall defects and the degree of radial hetero-

geneity, which are particularly important for studying chro-

matographic supports.

Perhaps the best known non-invasive characterisation tech-

nique, scanning coupled contactless conductivity (sC4D)

methods were first demonstrated by Gillespie et al.72 in 2006 and

have been most commonly applied for ion-exchange monolithic

columns to investigate porosity73 and axial variation of column

functionality incorporated via photografting74,75 or surface

coating.72 This non-invasive approach uses a cell containing a

bore of two ring electrodes via which the signal is capacitively

coupled through walls of insulating tubing.76 The major advan-

tages of this approach are that the method is non-invasive (the

cell is contactless) and can be easily used to interrogate the length

of a capillary column in a single analysis. However, this approach

has not been successfully employed to characterise radial heter-

eogeneity in capillary columns, which requires a high level of

resolution to observe small deviations in structural

morphology.37

The structure of monolithic columns has also been studied

using confocal laser scanning microscopy.77–85 This technique

yields similar visual information to that obtained from SEM/

TEM, but allows rapid acquisition of geometric information for

both axial and radial features (Fig. 8). A recent advance in this

technique has been the incorporation of statistical analysis

measures including chord length distributions to allow quantifi-

able, shape-independent information to be obtained.84,86 This

Analyst, 2012, 137, 5179–5189 | 5185

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approach requires a surface modification procedure to covalently

attach a fluorescent dye to the surface of the monolith. The

method is particularly advantageous for understanding perfor-

mance characteristics of monolithic columns as individual

contributions to eddy dispersion (e.g. wall defects)85 can be

rapidly identified. The introduction of other new techniques such

as magnetic resonance imaging87 and small angle neutron scat-

tering88 also offers opportunities to gain a more complete

understanding of the monolith morphology and consequent

separation performance.

4. Performance characterisation

As chromatographic supports, polymer monoliths offer several

practical advantages including the wide range of available

organic monomers, structural integrity, low flow resistance and

stability under extreme pH and temperature conditions.4,6,89

While this list of advantages is attractive, it must be tempered

with examination of the quality of separation that can be

obtained using this column format. Some examples of

biopolymer separations have demonstrated the excellent

performance of this column format in gradient mode.2,90–92

However, compared to silica monoliths, which yield excellent

isocratic separations for small molecules and rival particulate

columns for chromatographic performance,18 separation

performance for polymeric monoliths is generally poor, partic-

ularly for separations of small molecules30,70,93 despite the oft-

cited advantage of improved mass transfer characteristics.94

Theoretical direction for improving the performance of

monolithic supports is provided from a combination of detailed

experiment,95,96 computational fluid dynamics65,97 and an

understanding of kinetic performance characterisation.98 The

overwhelming conclusion of these studies is that currently

available monoliths generally suffer from a broad pore-size

distribution and poor bed homogeneity. Simply put, monoliths

with a reduced domain size and improved bed homogeneity will

benefit most from the enhanced permeability of this column

format by virtue of reduced eddy dispersion.97 This background

has provided impetus for research developing modified sol–gel

approaches for manufacturing silica monoliths with smaller

domain sizes and improved kinetic performance for small

molecule separations.18 Conversely, little attention has been

Fig. 8 Raw CLSM images of the 100 mm i.d. fluorescently labelled silica

monolithic capillary column: (A) longitudinal (i.e., along the column

axis) central section (x–y slice, 100 mm distance from wall to wall); (B)

cross-sectional (x–z) slice. Reproduced from ref. 84 with permission.

5186 | Analyst, 2012, 137, 5179–5189

paid to polymeric monoliths with only some research on

shortened polymerisation times,70 variation of polymerisation

conditions99,100 and secondary hypercrosslinking30 currently

published. While these approaches provide some performance

improvement (although none have characterised kinetic

performance) these columns still lag far behind silica monoliths

and particulate columns as shown by comprehensive studies

mostly yielding minimum plate heights (Hmin) of >20 mm for

retained compounds when practical chromatographic condi-

tions are employed. The importance of the degree of retention

and the physicochemical conditions employed has been recently

highlighted in the work of Nischang,101 which demonstrated the

wide range of efficiency values that can be obtained for alkyl

benzenes with a typical poly(styrene-co-divinylbenzene) column

simply by adjustment of the isocratic mobile phase composition

and thus retention factor. This result is supported by other

studies that have also reported a large variance in efficiency for

numerous probe solutes under practical chromatographic

conditions. For example, Li et al.32 reported efficiencies in the

range of 14 900–21 100 plates for a homologous series of

alkylbenzenes on a 16 cm � 75 mm I.D. polyBADMA mono-

lithic column. Thus the influence of the physicochemical

conditions should be considered in characterising new polymer

monolith columns and ultimately better understood to improve

the chromatographic performance.

This ostensibly leads to two important research goals: (1)

improving the structural homogeneity of polymer monoliths

and (2) demonstrating applications where these columns

provide a clear advantage over alternative technologies. In the

case of the former, the task is challenging as it will require novel

polymerisation methods perhaps using templates102,103 or silica–

polymer hybrids104 to improve the structural homogeneity and

reduce the contribution of eddy dispersion to band broadening.

In this regard, it is also important to consider that quantifica-

tion of performance improvement for monolithic columns

requires measurement of both the efficiency (via the conven-

tional van Deemter approach) and the bed permeability to allow

an unbiased estimate of the optimum column performance.98 In

doing so, this avoids erroneous over-estimation of column

performance by use of measures such as plates per m as

practical column lengths and analysis times can be

properly considered. The impact of the nanoscale porosity of

the support also appears to be a dominant factor in modulating

chromatographic performance. This is a substantial challenge

for development of polymer monoliths for liquid

chromatography.

In terms of applications, polymer monoliths already service

the analysis of large biomolecules and offer a number of practical

advantages for typical analysis and purification methods.

However, research in this area needs to be continued to cope with

the growing demands of the biopharmaceutical industry, in

particular for the analysis of monoclonal antibodies,105 oligo-

nucleotides60 and intact cells.106 In this case, monolithic columns

offer distinct advantages in terms of their fast binding kinetics for

these large molecules and relatively high permeability, allowing

them to be operated at high separation speeds. Practical benefits

such as the ability to synthesise materials with sufficiently large

pores to allow the direct separation of intact cells, or cell debris

without column clogging are also significant.

Fig. 9 (a) SEM image showing the cross-sectional view of a porous-shell pillar-array column. The estimation of the porous-layer thickness was based on

the difference in pillar diameter before (b) and after (c) silica-layer deposition. Reproduced from ref. 102 with permission.

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5. Future trends in the preparation of high efficiencymonolithic structures

The future of polymer monoliths as chromatographic supports is

inextricably linked to improving the regularity of the bed

morphology. The practical advantages in using an organic

polymer will become much more attractive as chromatographic

performance is improved. One recent example is the use of long

(>4 m) monolithic porous layer open tubular (PLOT)

columns.107,108 This approach provides very good chromato-

graphic performance for proteomic applications as the phase

ratio in 10 mm i.d. columns is suitable for liquid phase separa-

tions coupled to MS detection with high sensitivity.

Is well known that to get higher efficiencies with the monolithic

technology, new highly ordered structures are needed and

consequently this is related with novel polymerisation methods

which we expect to be found in other fields not related to sepa-

ration science. An example is the recent publications reported by

Sandhage’s research work in the preparation of highly ordered

titania-based structures using 3D nanostructured bioorganic

templates (butterfly wings).109,110 Although these materials were

not used in separation science, approaches like these could

contribute to the development of new homogeneous monolithic

structure for analytical applications. Another approach is that

developed by Desmet et al.102,103 where they prepared highly

ordered silica-based monolithic pillar-array columns reaching

very high efficiency when they were used as stationary phase in

chip-based liquid chromatography (Fig. 9). Although in these

publications the authors prepared silica based monolithic

columns the development of pillar-array monolithic columns

based on organic polymers could be adapted, increasing the

chemistry of the chromatographic media and consequently the

range of applications.

6. Conclusions

While the range of column chemistries for chromatographic

applications has been well studied, less progress has been made in

improving the chromatographic performance of this support

format for typical applications. Fortunately, the theoretical

background required to guide future developments can be

assumed from the vast amount of research on silica monolithic

columns.18,37,95–97,103,111,112 Developing polymer monoliths with

improved chromatographic performance remains a practical

challenge for polymerisation chemistry as the range of structural

morphologies from conventional methods have been almost

exhausted and innovation is required. This is the area in which

we can see significant future developments occurring in this field,

especially as other recent developments in chromatographic

stationary phases (e.g. superficially porous particles) continue to

demonstrate that separation performances exceeding those ach-

ieved with conventional polymer monoliths can be readily

accessed.

At the same time, the range of applications of organic polymer

monoliths continues to grow and it may be the case that these

materials offer the greatest advantages in areas of separation

science beyond high performance separations. With a number of

publications already demonstrating the real advantages of

organic polymer monoliths in a range of modes of sampling and

sample preparation,41,42,113,114 this is certainly a field where we

expect significant new developments to also occur.

Acknowledgements

This work was supported by the Australian Research Council’s

Discovery funding scheme (DP0987318). E.F.H. is the recipient

of an ARC Future Fellowship (FT0990521). We gratefully

acknowledge technical support from the Central Science Labo-

ratory, University of Tasmania.

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