Novel Chromatographic

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Novel chromatographic separation — The potential of smart polymers

Pankaj Maharjan a,b, Brad W. Woonton a,⁎, Louise E. Bennett a , Geoffrey W. Smithers a ,Kirthi DeSilva a , Milton T.W. Hearn b

a Food Science Australia, Sneydes Road, Werribee, Victoria, 3030, Australia b ARC Special Research Centre for Green Chemistry, Monash University, Clayton, Victoria, 3800 Australia

Received 11 November 2006; accepted 13 March 2007

Abstract

‘Smart ’ or stimuli-responsive polymers represent new classes of materials that are currently under development. These novel polymeric materials

undergo conformational rearrangement in response to small changes in their environment, such as temperature, pH, UV irradiation, ionic strength or

electric field. These environmental changes alter the structure of stimuli-responsive polymers and increase or decrease their overall hydrophobicity,

resulting in reversible collapse, dehydration or hydrophobic layer formation. With further research into their synthesis, behaviour and application,

these novel materials have great potential to become the ‘next generation’ of separation media for cost-effective and environmentally-friendly

extraction and purification of high value biomolecules from agri-food and other raw materials.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: Smart polymer; Temperature-responsive polymer; Poly(n-isopropylacrylamide); Bioseparation; Chromatography; Lower critical solution temperature;

Affinity separation; Bioconjugates

Industrial relevance: The growing demand for functional food ingredients is requiring the development of selective, cost-effective isolation

techniques. Chromatography is one technique employed to produce novel food ingredients. Chromatography procedures often require the use of

large quantities of solvents, which must be removed from food products, increasing processing input costs (solvent and energy), and creating an

environmental disposal issue. Smart polymers are novel materials that change phase with temperature or other types of operational conditions, and

have the potential to offer a cost and environmentally attractive means of producing functional food ingredients. This paper presents a review of

smart polymers as novel separation media, and their potential application in the food industry.

1. Introduction and background

Chromatography in its various forms is a critical separation

tool available to scientists working in the biotechnology, bio-medical, and food research fields. Today, chromatography has

been developed and refined to such a degree that it represents a

highly selective and efficient technique that can separate close-

ly related molecules from a highly complex mixture. The main

chromatographic methods used today, along with their modes of

separation, are summarised in Table 1.

Since the development of adsorption chromatography by

Tswett (1906) more than a hundred years ago, many different

modes of chromatography have been developed, concurrent with

advances in separation media. Martin and Synge (1941) achieved

a significant breakthrough in the development of chromatogra-

phy by establishing liquid–

liquid partition chromatography toseparatevarious amino acids. This innovative development,using

a solid support to create a liquid stationary phase, resulted in the

award of the Nobel Prize in 1952 and the birth of ‘normal phase’

chromatography. Boldingh (1948) reported the use of a non-polar

stationary phase in a process that has been termed ‘reversed

phase’ chromatography, and is now used extensively in biological

and chemical analysis.

Affinity chromatography is a type of adsorption chromatog-

raphy where the target molecule is reversibly adsorbed by a ligand

immobilized onto an insoluble support. The ligand is selected

based on its affinity for a biomolecule, such as the affinity of an

Available online at www.sciencedirect.com

Innovative Food Science and Emerging Technologies xx (2007) xxx–xxx

INNFOO-00466; No of Pages 11

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⁎ Corresponding author. Tel: +61 3 9731 3323; fax: + 613 9731 3390.

E-mail address: [email protected] (B.W. Woonton).

1466-8564/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.ifset.2007.03.028

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Please cite this article as: Maharjan, P., et al., Novel chromatographic separation — The potential of smart polymers, Innovative Food Science and Emerging

Technologies (2007), doi:10.1016/j.ifset.2007.03.028

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antibody to its antigen. This type of chromatography was dis-

covered in the 1930s, but the use of this technique as a routineseparation method was only established in the early 1970s.

Modern forms of ion-exchange chromatography (IEC) were

developed during the Manhattan project in the 1940s, where

technology was required to separate and concentrate radioactive

elements to be used in the atomic bomb. IEC is based on the

interaction of charged molecules with oppositely charged moi-

eties covalently attached to an insoluble matrix. IEC has pro-

vided solutions to complex separation problems in the mining,

chemical, food and pharmaceutical industries.

The development of silanised silica adsorbent material of small

pore diameter made the use of long narrow bore closed columns

possible and led to breakthroughs in high-performance liquid

chromatography (HPLC), a technique developed by Horvath andLipsky (1966). Uptake of HPLC was further assisted with the use

of gradient elution techniques developed by Arne Tiselius in the

1950s. Gradient elution created conditions for differential solu-

bilities between the stationary and mobile phases, improving

resolution during separation. Throughout the 1970s, HPLC was

refined through improved column design, media, pumping sys-

tems, and methods of detection to provide precise, repeatable, and

rapid separations.

Chromatographic media can be classified according to the

matrix material, and include natural polymers (agarose, dextran,

cellulose); synthetic polymers (modified methacrylates, acryla-

mides, polystyrene); inorganic material (porous and non-poroussilica, glass, hydroxyapatite), and composite material (Hearn,

2000; Jungbauer, 2005).

The first reported application of natural polymers in chro-

matography was by Peterson and Sober (1956) where cellulose

beads were functionalized with ion-exchange groups. This de-

velopment was followed by the commercialisation of dextran-

based media by Pharmacia (now GE Health Care). Natural

polymers such as dextran, agarose and cellulose are extremely

hydrophilic resulting in low protein adsorption and provide an

added advantage of low non-specific binding. The agarose struc-

ture in Sepharose (Pharmacia) is reinforced through cross-

linking. In Superdex (Pharmacia) cross-linked agarose is further

modified through covalent attachment to dextran. Today, there

are a range of commercial products based on modified natural

polymers for chromatographic applications.

Further advances in chromatography became possible with

the development of synthetic polymeric resins with narrow par-

ticle size ranges. Synthetic polymers have wide applications as

chromatographic media due to their resistance to chemicals,

stability at extremes of pH, and their ability to be coated or functionalized. The basic steps in the synthesis of these poly-

mers include the co-polymerization of selected monomers to

form the cross-linked matrix and the attachment of functional

groups to this matrix. Synthetic polymer supports are generally

hydrophobic and therefore need to be coated with hydrophilic

material to ensure low protein adsorption.

Silica-based adsorbents are the most widely used material in

the inorganic group although they have limited stability at high

pH. Silica-based media are functionalized by bonding different

functional groups through the formation of multilayers with

internal cross-linking. The common groups bonded to silica in-

clude phenyl, n-butyl, n-octadecyl, amino and cyano groups.Breakthroughs in different chromatographic techniques have

been facilitated by the development of chromatographic separating

media. Two of the important criteria for developing new media are

to provide high specificity and recovery for any given separation.

For example, the rapid advancement of reversed-phase chroma-

tography has been associatedwith theintroduction of bondedsilica

stationary phases, where the –OH group is inactivated preventing

interaction with proteins. Similarly, the wide application of ion-

exchange chromatography has been associated with the develop-

ment of synthetic media that is stable over a wide pH.

Development of chromatography as an industrial separation

tool was slow because the technique was inherently expensive.

Such expenses were primarily associated with low productivity,and the requirement for large volumes of solvents and chem-

icals. Recent advances in continuous approaches to chromatog-

raphy, notably simulated moving bed (SMB) chromatography,

have helped address the issues of expense and facilitated more

widespread use of the technique in manufacturing industries (eg,

food), where cost of processing must be kept to a minimum (De

Silva, Stockmann, & Smithers, 2003).

Within the pharmaceutical and food industry, biomolecules

are often separated using ion-exchange chromatography, normal

phase chromatography and reverse-phase chromatography, or

strategic combinations of these techniques. Organic solvents

(eg, acetonitrile, methanol), used in normal and reversed-phaseseparations, have a number of disadvantages that limit their use

commercially including cost, toxicity and flammability. The use

of solvents can induce protein denaturation and thus limit their

application as biological therapeutics. In ion-exchange chro-

matography, the elution mobile phase usually contains high con-

centrations of salt which must be removed from the final

product and disposed of, imposing additional equipment, pro-

cessing, and environmental costs.

Smart polymers are novel materials that operate under very

mild aqueous conditions, and have the potential to provide a

novel and cost-effective means to isolate valuable biomolecules

and pharmaceuticals from agri-food and other raw materials.

This paper presents a review of the current state of play in the

Table 1

Common chromatographic techniques and their rationale for separation

Chromatographic name Mode of separation

Adsorption chromatography Molecular structure

Ion-exchange chromatography Surface charge

Size-exclusion chromatography

(gel filtrationa

)

Molecular size and shape

Affinity chromatography Molecular structure

Hydrophobic

(interaction) chromatography

Hydrophobicity and hydrophobic

patches

(Metal-)chelate chromatography Complex formation with transition

metals

Normal-phase chromatography Hydrophobicity

Reversed-phase chromatography Hydrophobicity

Modified from Hearn (2000), and Jungbauer (2005).a Name originally used for size-exclusion chromatography.

2 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx

ARTICLE IN PRESS







area of ‘smart polymers’ and highlights the potential of these

materials in ‘next generation’ bioseparations.

2. Types of smart polymers

Polymeric materials that undergo fast, reversible changes intheir structure and function in response to external physical,

chemical or electrical stimuli are termed ‘smart ’ or ‘intelligent ’

polymers. As shown in Table 2, various types of polymeric

materials fall into this category and various stimuli, such as

temperature, pH and light, have been investigated. ‘Smart

polymers’ may be cross-linked to form hydrogels, immobilized

or grafted on solid surfaces, or dissolved in aqueous solutions.

Upon stimulation of smart polymers (eg, raising temperature

above a certain critical value), the polymer chains change from

water soluble to water-insoluble, resulting in conversion of the

polymeric material from a hydrophilic to hydrophobic state

(Piskin, 2004; Hoffman and Stayton, 2004). Depending on the

polymeric system, the response may be precipitation, gelation,

adsorption, collapse of the polymer attached to a surface or col-

lapse of a hydrogel (Fig. 1). The driving force behind these re-

versible transitions varies with the stimulus. For instance, a pH

shift causes the neutralization of charged groups, whereas an

increase in the temperature or ionic strength reduces the ef-

ficiency of hydrogen bonding, resulting in the collapse of thehydrogel and an interpenetrating polymer network. Because of

their potential and application in the isolation of valuable

components from various agri-food streams (including waste),

and the large body of research literature, this paper will focus on

temperature and pH-responsive ‘smart polymers’.

3. Temperature-responsive polymers

Temperature is the most widely studied stimulus in ‘smart

polymer ’ systems. In chemical terms and as a general guide, the

solubility of solids in solution usually increases as the tem-

perature of the solution increases. By contrast, the solubility of

temperature-responsive polymers decreases as the temperature

Table 2

Various polymeric materials that behave as ‘smart polymers’ when subjected to environmental stimuli, and their induced transitions and applications, both established

and potential

Polymeric material Environmental stimuli Induced transition Application Reference

Poly( N -isopropylacrylamide) Temperature Water soluble coils to water-insoluble

globules and subsequent collapse of

polymer or precipitation fromsolution or adsorption/desorption

Co-polymers used as intelligent

carriers in a diverse range of

applications including separations

Piskin (2004)

Co-polymers of

N -vinylcaprolactam

and 1-vinylimidazole

Temperature Reversible thermal precipitation Immobilized metal affinity

chromatography

Ivanov,

Kazakov,

Galaev, and

Mattiasson

(2001)

Hydroxypropylcellulose Temperature Hydrated swollen state to dehydrated

shrunken state

Size-exclusion chromatography Adrados et al.

(2001)

Poly(acrylic acid) pH Compact unionized state to swollen

ionized state

Colon specific drug delivery Qiu and Park

(2001)

Poly( N , N ′-

dimethylaminoethyl

methacrylate)

pH Compact unionized state to swollen

ionized state

Drug delivery in the stomach Qiu and Park

(2001)

Poly( N -isopropylacrylamide)

hydrogels containingferromagnetic

material

Magnetic field Reversible collapsing of the hydrogel Gel-entrapment system in the

magnetic control of immobilizedenzyme reactions.

Takahashi, Sakai, &

Mizutani(1997)

Polythiophene gel Electric field Swelling and deswelling Potential use as small-scale

actuators and valves in microsystems

application

Irvin, Goods, &

Whinnery (2001)

Cotelomer of

N -isopropylacrylamide and

N -acryloxysuccinimide

with bioligand and

(3-aminopropyloxy)

azobenzene attached to it

UV radiation Affinity precipitation Capture of biologicals from solution

mixture.

Desponds & Freitag

(2005)

Dodecyl isocyanate-

modified poly

(ethylene glycol)

grafted poly(2-Hydroxyethyl

methacrylate)

Ultrasound Disrupt the orderly chains on the

surface of the drug-containing

polymer

Controlled drug delivery Kwok, Mourad,

Crum, & Ratner

(2001)

Poly( N -isopropylacrylamide)

with trisodium salt of

copper chlorophyllin

Light Reversible collapse of gel Potential use as a photo-responsive

artificial muscle or switch

Suzuki and Tanaka

(1990)


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increases. The lower critical solution temperature (LCST) is the

phase transition temperature of the thermo-sensitive polymer

and is the lowest phase separation temperature on the tem-

perature-composition diagram for the polymer solution (Elias,1984). The LCST is a distinctive property of temperature-res-

ponsive polymers.

Poly( N -isopropylacrylamide) (poly-NIPAAm; Fig. 2) is

the most representative and extensively studied temperature-

responsive polymer with an aqueous solution LCST of 32 °C.

The phase transition of poly-NIPAAm in solution is quite revers-

ible, reproducible and sensitive to small changes in temperature.

The phase transition phenomenon is accompanied by a con-

traction of the polymer chains, called coil-globular transition

(Ayano and Kanazawa, 2006). Below the LCST, the amide

group of the poly-NIPAAm and a water molecule form a hy-

drogen bond causing solubilization of the poly-NIPAAm. When

the temperature is increased above the LCST, the hydrogen

bonds between the amide group of the poly-NIPAAm and thewater molecule become unstable and the polymer chains con-

tract and enter a globular state (Fig. 3).

For separation applications, enhanced thermosensitivity (ie, the

rate of polymer phase transition and subsequent polymer swelling

or de-swelling rate) is critical for faster phase transition so as to

improve resolution, and enhance selectivity and throughput. To

Fig. 2. Structural formula of N -isopropylacrylamide (NIPAAm). LCST = lower critical solution temperature.

Fig. 3. Coil to globule transition and subsequent solution turbidity change when

poly-NIPAAm is heated above the lower critical solution temperature (LCST)

(adapted from Ayano and Kanazawa (2006), reprinted with permission fromWiley-VCH Verlag GmbH & Co.).

Fig. 1. Schematic representation of the various types of induced transitions that ‘smart polymers’ undergo in response to environmental stimuli (from Hoffman (2000);

reprinted with permission from the American Association of Clinical Chemistry).


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impart desired thermosensitivity, and enhance the hydrophilicity,

N -alkylacrylamides are often co-polymerized with hydrophilic

monomers such as acrylic acid or methyl acrylic acid. Apart

from enhancing thermosensitivity, co-polymerization of poly-

NIPAAm with anionic acrylic acid (Kobayashi, Kikuchi, Sakai, &

Okano, 2003) or cationic N , N ′-dimethylaminopropylacrylamide

(DMAPPAm) (Ayano et al., 2006) also produces co-polymerswith temperature tuneable hydrophobicity and chargedensity. The

thermosensitivity of the polymer system can also be increased by

preparing comb-type polymers instead of linear structures

(Yoshida et al., 1995; Annaka et al., 2003). The grafted side

chains in the polymer network in comb-type structures create

hydrophobic regions that aid faster expulsion of water from the

network during collapse. A schematic illustration of normal and

comb-type polymeric structures is shown in Fig. 4.

The LCST of temperature-responsive polymers can be ma-

nipulated by integration of hydrophobic or hydrophilic moieties

into the molecular structure. For example, the co-polymeriza-

tion of NIPAAm monomers with hydrophilic monomers such asacrylamide, leads to an increase in the polymer hydrophilicity

and an increase in the LCST of the co-polymer. By contrast, co-

polymerization of the NIPAAm monomers with more hydro-

phobic monomers, such as n-butyl acrylamide, increases the

polymer hydrophobicity and decreases the LCST of the co poly-

mer (Hoffman et al., 2000) (Fig. 5).

Temperature-responsivematerials are synthesized via two dif-

ferent polymerization methods. They can be prepared by radi-

cal polymerization of the temperature-responsive monomers

(eg, NIPAAm, N -vinylisobutyramide, etc.) with a cross-linking

agent such as ethylene glycol dimethacrylate. Alternatively, they

can be prepared by introducing cross-links to a polymer solution

via chemical reaction of functional side groups or by irradiation

of the monomer solution withγ-rays or electron beams which act

as initiators (Gil and Hudson, 2004).

4. Other stimuli responsive polymers

Although temperature-responsive polymers are the most

widely studied, ‘smart polymers’ that respond to other external

stimuli such as pH, electric field and light (Table 2) are also of

interest for the separation of valuable molecules from agri-food

and other raw materials. The pH-responsive polymers constitute

ionizable pendant groups that are either acidic, such as

carboxylic acid, or basic, such as amine groups, that can accept or donate protons in response to variations in environmental pH

(Fig. 6). These polymers can be broadly categorized into

polyacids such as poly(acrylic acid) or polybases such as poly

(4-vinylpyridine). Polyacids ionize at a high pH (Philippova,

Hourdet, Audebert, & Khokhlov, 1997) whereas polybases

ionize at a low pH (Pinkrah et al., 2003). The electrostatic

repulsion among charges present on the polymer chains is the

primary driving force that governs precipitation/solubilization

Fig. 4. Schematic illustration of (a) normal type and (b) comb-type polymer

structures.

Fig. 5. Effect of co-polymerization of poly-NIPAAm with hydrophilic

acrylamide (AAm) or hydrophobic N -tert -butylacrylamide ( N -tBAAm) on the

lower critical solution temperature (LCST) (from Hoffman et al. (2000),

reprinted with permission from John Wiley & Sons, Inc.).

Fig. 6. pH dependent ionization of polyelectrolytes. (a) Poly(acrylic acid)

(polyacid), and (b) Poly( N , N ′

-diethylaminoethyl methacrylate) (polybase) (fromQiu and Park (2001), reprinted with permission of Elsevier Science).


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of the polymer, swelling or deswelling of hydrogels, or the

hydrophobic or hydrophilic characteristics of surfaces (Gil and

Hudson, 2004).

Electrofield-responsive polymers are pH-responsive poly-

mers composed of polyelectrolytes that demonstrate shape

change (swelling, deswelling or bending) when exposed to an

electric field. For example, partially hydrolyzed polyacrylamidegel in contact with both the anode and cathode electrodes

undergoes extensive shrinkage of volume even by a small

change in electric potential across the polymer. When potential

is applied, hydrated H+ ions migrate towards the cathode re-

sulting in loss of water at the anode side. At the same time,

electrostatic attraction of negatively charged acrylic acid groups

towards the anode surface creates an unaxial stress along the

polymer axis. These simultaneous events lead to shrinking of

the polymer structure (Qiu and Park, 2001). The design of

electro-sensitive hydrogels has mainly been for use as drug de-

livery systems (Yuk, Cho, & Lee, 1992).

Light-sensitive hydrogels can be broadly subdivided intoUV-sensitive and visible light sensitive. UV-sensitive hydrogels

have been synthesized by incorporating leuco dye derivative

molecules into the polymer matrix (Irie and Kunwatchakun,

1986). Upon UV irradiation, the neutral leuco derivative mol-

ecule ionizes and leads to swelling due to an increase in the

osmotic pressure within the gel. Visible light-sensitive hydro-

gels can be prepared by introducing a visible light-sensitive

chromophore (eg, trisodium salt of copper chlorophyllin) to the

poly-NIPAAm (Suzuki and Tanaka, 1990). Visible light causes

phase transition in these polymer systems due to an extremely

fast direct heating process (Suzuki and Tanaka, 1990).

5. Application of temperature-responsive polymers

‘Smart polymers’ have found use in an array of biotechnol-

ogy fields. They have been used in bioseparation, bioconjuga-

tion (Galaev and Mattiasson, 1999; Hoffman et al., 2000), drug

delivery (Qiu and Park, 2001), as immobilized biocatalysts

(Park and Hoffman, 1993), as thermo-responsive surfaces

(Tsuda et al., 2004; Anastasiadis, Retsos, Pispas, Hadjichristi-

dis, & Neophytides, 2003), in protein renaturation (Roy and

Gupta, 2003), as biomimetic actuators (Osada, Okuzaki, &

Hori, 1992; Ueoka, Gong, & Osada, 1997), as chemical valves

(Baldi, Gu, Lofness, Seigel, & Ziaie, 2003) and in immunoas-

says (Malmstadt, Hoffman, & Stayton, 2004). In the separations

field, poly-NIPAAm and related polymers have been used to

generate temperature-responsive stationary phases for size

exclusion (Hosoya et al., 1994; Adrados, Galaev, Nilsson, &

Mattiasson, 2001), hydrophobic interaction (Kanazawa, Suna-

moto, Matsushima, Kikuchi, & Okano, 2000), ionic (Kobayashi

et al., 2003), and affinity based chromatography separations

(Hoffman and Stayton, 2004) using a range of different sup- porting materials.

5.1. Hydrophobic interaction chromatography

A common approach to the use of temperature-responsive

polymers is on solid supports such as silica. Poly-NIPAAm-

modified silica beads showing temperature dependant hydro-

phobic-hydrophilic properties have been prepared and employed

as novel HPLC packing materials for chromatographic separa-

tions. The stationary phase exhibits very rapid and reversible

hydrophilic–hydrophobic changes in response to temperature,

allowing temperature gradients analogous to solvent gradients inreversed-phase HPLC. Poly-NIPAAm-modified silica beads are

prepared either by radical co-polymerization at the surfaces of

initiator immobilized silica beads or by modification of ami-

nopropyl silica with NIPAAm co-polymer by activated ester

amine coupling. Co-polymers that have been grafted onto silica

beads include:

• poly(NIPAAm-co-butyl methacrylate) (Kanazawa et al., 1997;

Kanazawa et al., 2000; Sakamoto et al., 2004);

• poly(NIPAAm-co-acrylic acid) (Kobayashi, Kikuchi, Sakai,

& Okano, 2001);

• poly(NIAAm-co-butyl methacrylate-co- N , N -dimethylami-

nopropylacrylamide) (Sakamoto et al., 2004; Ayano et al.,2006); and

• poly(NIPAAm-co-acrylic acid-co- N -tert -butylacrylamide)

(Kobayashi, Kikuchi, Sakaia, & Okano, 2002; Kobayashi

et al., 2003).

The temperature-responsive poly(NIPAAm-co-butyl meth-

acrylate) terminally-modified silica beads (Fig. 7) prepared by

Kanazawa et al. (1997) have been used to separate a mixture of

three peptides-insulin chain A, insulin chain B and β-endorphin

fragment 1–27. It was found that temperature gradients could

rapidly alter the stationary phase surface characteristics, re-

sulting in temperature-modulated peptide elution from the

Fig. 7. Aminopropyl-silica modified with Poly(NIPAAm-co-butyl methacrylate) (adapted from Ayano and Kanazawa (2006), reprinted with permission from Wiley-VCH Verlag GmbH & Co.).


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column. It is possible that the optimization of silica-grafted

‘smart polymers’ and temperature gradients for particular

separation tasks could remove or reduce the requirement for

gradient changes in the mobile phase composition during HPLCor other gradient-dependent separation.

Poly-NIPAAm-modified silica has been used as a HPLC

stationary phase for the separation of five different steroids with

water as the only mobile phase (Kanazawa et al., 1996). Al-

though the steroids were not resolved at a temperature lower

than the LCST (32 °C; when the poly-NIPAAm was hydrated

and hydrophilic), an excellent resolution was achieved above

the LCST (Fig. 8), possibly due to hydrophobic interaction

between the hydrophobic steroids and the poly-NIPAAm sta-

tionary phase at the higher temperatures.

Enantiomer separation of racemic N -(3,5-dinitrobenzoyl

(DNB))amino acid isopropyl esters was achieved by temperature-responsive liquid chromatography using chiral stationary phases

(silica gel modified with acryloyl-L-valine N -methylamide and its

N , N -dimethylamide analogue) (Kurata, Shimoyama, & Dobashi,

2003). During chromatography, enantioselectivity and retentivity

for solute enantiomers were controlled by column temperature,

which changed the aggregation and extension states of the chiral

polymers depending upon their interior hydrophobic nature.

Retention of the amino acid derivatives and enantioselectivity

was prolonged with an increase in column temperature.

Temperature-responsive chromatography with a stationary

phase made from poly-NIPAAm-modified silica has been de-

veloped and employed as a simple and rapid method to separate

and analyze herbicides (five sulfonylurea and three urea her-

bicides) in water (Ayano et al, 2005). At low temperature (10 °C),

the peaks of the various analytes overlapped. After raising the

column temperature the retention times increased and the her-

bicides could be resolved from one another.

5.2. Ion-exchange chromatography

A pH and temperature-responsive co-polymer of poly

(NIPAAm-co-AAc-co-tBAAm) grafted onto silica beads has

been evaluated as a anionic temperature-responsive chromatog-

raphy medium (Kobayashi et al., 2002; Kobayashi et al., 2003).

The polymer grafted stationary phase showed simultaneous

thermally modulated changes in charge density and hydropho-

bicity due to incorporation of AAc as the anionic exchange

group and hydrophobic tBBAm into the NIPAAm sequence.

Effective separation of basic bioactive peptides under exclu-sively aqueous conditions was attained using anionic tempera-

ture/pH-responsive polymer-modified surfaces (Fig. 9).

Similarly, silica beads grafted with poly(NIPAAm-co-BMA-

co-DMAPAAm) has been evaluated as a cationic temperature-

responsive chromatography medium (Ayano et al., 2006). The

medium was effective for the separation of bioactive compounds

and pharmaceuticals using isocratic aqueous mobile phases.

5.3. Size-selective separation

There have been attempts to employ temperature-sensitive

polymers in developing improved size selective separation me-

dia. Poly-NIPAAm grafted onto silica beads coated with dextran

Fig. 8. Chromatograms showing the separation of a mixture of five steroids and benzene on a poly-NIPAAm-modified silica column with water as the only mobile

phase at (a) 5, (b) 25, (c) 35, and (d) 50 °C. Peaks: 1, benzene; 2, hydrocortisone; 3, prednisolone; 4, dexamethasone; 5, hydrocortisone acetate; and 6, testosterone

(from Kanazawa et al. (1996), reproduced with permission from American Chemical Society).


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and diethylaminoethyl groups have been synthesized and stud-

ied as a stationary phase for high-performance size-exclusion

chromatography (Lakhiari et al., 1998). It was observed that at

low temperature there was a higher resolution of proteins, pos-

sibly due to the hydrophilic properties of poly-NIPAAm at low

temperatures, improving the porosity of the support. At higher

temperatures, the hydrophobic properties of poly-NIPAAm

produced interactions with proteins and a slight retardation in

some of the component elution times. Adrados et al. (2001) prepared hydroxypropylcellulose (HPC) beads that exhibited

temperature-dependent porosity, and evaluated these beads as a

chromatographic material. At room temperature the beads were

swollen with large pores that could resolve proteins with mo-

lecular masses b20 kDa. At elevated temperatures the shrunken

HPC beads with smaller pores excluded proteins as small as

14 kDa.

Poly-NIPAAm grafted onto porous glass beads has been pre-

pared and employed as a column packing material for gel per-

meation size-exclusion chromatography (Gewehr, Nakamura,

Ise, & Kitano, 1992). Poly-NIPAAm was end-functionalized

through telomerization polymerization of NIPAAm with mer-captopropionic acid used as chain transfer agent. Porous glass

beads were firstly aminated with 3-aminopropyltriethoxysilane

followed by conjugation of poly-NIPAAm with active ester

chain ends through amide bond formation. The poly-NIPAAm-

modified glass beads were packed into columns and elution of

dextrans with various molecular weights was examined with

changing column temperature. The elution time of the dextrans

was substantially altered between 25 and 32 °C due to a change

in the effective pore size via the transition of the poly-NIPAAm

chains from coils to globules on the surface of the pores of the

glass beads (Gewehr et al., 1992).

Porous polystyrene beads grafted with poly-NIPAAm have

been synthesized and used as a stationary phase in HPLC with

temperature tuneable pore size (Hosoya et al., 1994). The

polymerization of NIPAAm was carried out using cyclohexanol

or toluene as the porogen agents in water. When cyclohexanol

was used as the porogen, the entire surface of the porous beads

was covered with poly-NIPAAm and the beads were relatively

homogeneous. When using toluene as the porogen, only the

external bead surface was grafted with poly-NIPAAm and the

beads had rough surface morphology. The surface structure had

an influence on the elution behaviour of dextrans during size-exclusion chromatography. With beads modified with cyclo-

hexanol as the porogen agent, an increase in temperature

prolonged the elution time of higher molecular weight dextrans.

This behaviour may have been due to high temperature collapse

of poly-NIPAAm chains causing the pore size to expand, there-

by permitting the dextrans to penetrate the pores. In contrast,

beads modified with poly-NIPAAm using toluene as the

porogen agent, faster elution at higher temperatures may have

been the result of a reduction in the pore size through shrinkage

of the surface grafted poly-NIPAAm chains. This research

highlights that the separation mode can be influenced through

the synthetic pathway used to manufacture the ‘smart polymer ’.Research on cyclohexanol or toluene leaching from these resins

after manufacturing has not been reported.

5.4. Affinity separations

Smart polymers can be conjugated to biomolecules such as

proteins and peptides, sugars, oligonucleotides, simple lipids and

phospholipids, and an array of recognition ligands (Hoffman,

2000). A number of studies have explored the application of

randomly conjugated ‘smart polymers’ to proteins for affinity

separations. A stepwise thermal cycling operation below and

above the LCST induces reversible precipitation-solubiliza-

tion behaviour of the bioconjugates in the aqueous solution.

Fig. 9. Chromatograms showing the separation of peptides on a poly(NIPAAm-co-acrylic acid-co- N -tert -butylacrylamide) grafted silica column at 10, 30 and 50 °C.

Mobile phase used is phosphate-citrate buffer; (a) pH 4.0 (b) pH 7.0 and ionic strength 0.1 and (c) pH 7.0 and ionic strength 0.5. Peaks: 1,3,4-dihydroxy- L-

phenylalanine; 2, adrenaline; 3, dopamine, and 4, tyramine (from Kobayashi et al. (2002), reprinted with permission from Elsevier Science).


ARTICLE IN PRESS







Bioconjugate formation with ‘smart polymers’ can therefore be

used as simple one-step bioseparation processes based on a cyclical

operation of heating and cooling. Specific examples include the

separation of α-chymotrypsin (Kim and Park, 1999), β-glucosi-

dase (Chen and Hoffman, 1993; Agarwal and Gupta, 1996),

thermolabile α-glucosidase (Hoshino, Taniguchi, Kitao, Moroha-

shi, & Sasakura, 1998), α-amylase inhibitor (Kumar, Galaev, &Mattiasson, 1998), and lysozyme (Vaidya, Lele, Kulkarni, &

Mashelkar, 2001). Further, a novel chromatographic media using a

PNIPAAm-dextran derived conjugate has been developed for the

rapid, sensitive and inexpensive purification of antibodies

(Anastase-Ravion, Ding, Pelle, Hoffman, & Letourneur, 2001).

Temperature triggered enzyme (lactate dehydrogenase, LDH)

separation from porcine muscle has been achieved using a dye-

affinity agarose modified by physically adsorbing Poly( N -

vinylcaprolactam) [PVCL] (Galaev, Warrol, & Mattiasson,1994).

LDH from porcine muscle was bound to the PVCL shielded

column at 40 °C. At this temperature LDH could not be eluted

from the column with 0.1 M KCl. A decrease in temperature to23 °C resulted in LDH elution with 0.1 M KCI. This appears to be

the first reported successful enzyme purification in which a

temperature shift was used as the only eluting factor, without

changing the buffer composition.

Temperature sensitivepolymershavebeendevelopedintosmart

beads that can be reversibly immobilized on microfluidic channel

walls to capture and release target molecules (Malmstadt, Yager,

Hoffman, & Stayton, 2003). In one example, latex beads (100 nm)

were modified with poly-NIPAAm and biotin. When a suspension

of the modified beads flowed through the microfluidic channel

constructed of poly(ethylene terephthalate) at a temperature above

the LCST, the modified beads adhered to the channel walls and

functioned as a chromatographicaffinity separation matrix, capableof binding streptavidin. Once streptavidin was bound, cooling to

below the poly-NIPAAm LCST, the beads and the captured

streptavidin were dissolved and eluted from the channel walls. In

this example, the ability to easily remove the matrix allows for

straightforward renewal of a microfluidicchromatography column,

improving the reusability and flexibility of the device. Further,

reversible matrix formation simplifies the elution process and also

eliminates the need for harsh chemical eluents.

6. Conclusions

The potential applications of ‘smart polymers’ in a wide arrayof fields including bioseparation has been reviewed. Although

there are many different types of external stimuli, temperature-

responsive polymers made from poly(NIPAAm) and its co-poly-

mers have been themost widely studied for theeffective separation

of biomolecules using hydrophobic interaction chromatography,

ion-exchange chromatography, size-exclusion chromatography

and affinity based separations. These ‘smart polymers’ offer pro-

mise in the cost-effective isolation of valuable components (eg,

functional (bioactive) ingredients) from agri-food raw materials

and other complex feeds, in an environmentally-friendly manner.

Demand for economical functional ingredients is large and grow-

ing, reflected in the burgeoning functional foods market (current

market value ∼$73.5 billion, with a projected market size N$100

billion by 2012 (Just-food.com, 2006)). However, before employ-

ing these novel separation media in food industry applications,

further research into a number of important issues is required.

Some of the critical aspects that require attention include:

• Separation efficiency and capacity of the media,

• Cost-effective use of the media in food component isolation,• Resistance of the media to common cleaning agents used in

the food industry,

• Extent to which the media can be reused,

• Ease and economy of scaling the media manufacturing

process,

• Stability of the media during long term use, and

• Toxicity of any potential leakage products from the media.

These aspects of ‘smart polymer ’ use in food applications are

currently being investigated by our research team, and answers

to these questions will dictate whether the potential of ‘smart

polymers’

in food bioseparation is real and how quickly that potential can be captured by the industry.

Acknowledgments

The authors would like to acknowledge the financial assis-

tance from the State Government of Victoria, Australia (De-

partment of Industry, Innovation and Regional Development),

Monash University (Centre for Green Chemistry), Melbourne,

Australia, and Food Science Australia.

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