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Innovative Food Science and Emerging Technologies xx (2007) xxx xxx www.elsevier.com/locate/ifset

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
b a Food Science Australia, Sneydes Road, Werribee, Victoria, 3030, Australia 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, biomedical, 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 closely 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
Corresponding author. Tel: +61 3 9731 3323; fax: + 613 9731 3390. E-mail address: brad.woonton@csiro.au (B.W. Woonton). 1466-8564/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2007.03.028

modes of chromatography have been developed, concurrent with advances in separation media. Martin and Synge (1941) achieved a significant breakthrough in the development of chromatography by establishing liquidliquid partition chromatography to separate various 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 chromatography 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

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2 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxxxxx Table 1 Common chromatographic techniques and their rationale for separation Chromatographic name Adsorption chromatography Ion-exchange chromatography Size-exclusion chromatography (gel filtration a) Affinity chromatography Hydrophobic (interaction) chromatography (Metal-)chelate chromatography Normal-phase chromatography Reversed-phase chromatography Mode of separation Molecular structure Surface charge Molecular size and shape Molecular structure Hydrophobicity and hydrophobic patches Complex formation with transition metals Hydrophobicity Hydrophobicity

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

antibody to its antigen. This type of chromatography was discovered in the 1930s, but the use of this technique as a routine separation 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 moieties covalently attached to an insoluble matrix. IEC has provided 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 and Lipsky (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 solubilities between the stationary and mobile phases, improving resolution during separation. Throughout the 1970s, HPLC was refined through improved column design, media, pumping systems, 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, acrylamides, polystyrene); inorganic material (porous and non-porous silica, glass, hydroxyapatite), and composite material (Hearn, 2000; Jungbauer, 2005). The first reported application of natural polymers in chromatography was by Peterson and Sober (1956) where cellulose beads were functionalized with ion-exchange groups. This development was followed by the commercialisation of dextranbased 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 structure in Sepharose (Pharmacia) is reinforced through crosslinking. 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 particle 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 polymers 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 include 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 chromatography has been associated with the introduction of bonded silica stationary phases, where the OH group is inactivated preventing interaction with proteins. Similarly, the wide application of ionexchange chromatography has been associated with the development 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 chemicals. Recent advances in continuous approaches to chromatography, 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-phase separations, 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 chromatography, the elution mobile phase usually contains high concentrations of salt which must be removed from the final product and disposed of, imposing additional equipment, processing, 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

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P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxxxxx 3 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 Poly(N-isopropylacrylamide) Environmental stimuli Temperature Induced transition Water soluble coils to water-insoluble globules and subsequent collapse of polymer or precipitation from solution or adsorption/desorption Reversible thermal precipitation Application Co-polymers used as intelligent carriers in a diverse range of applications including separations Immobilized metal affinity chromatography Reference Piskin (2004)

Co-polymers of N-vinylcaprolactam and 1-vinylimidazole

Temperature

Hydroxypropylcellulose Poly(acrylic acid) Poly(N,Ndimethylaminoethyl methacrylate) Poly(N-isopropylacrylamide) hydrogels containing ferromagnetic material Polythiophene gel

Temperature pH pH

Hydrated swollen state to dehydrated shrunken state Compact unionized state to swollen ionized state Compact unionized state to swollen ionized state Reversible collapsing of the hydrogel

Size-exclusion chromatography Colon specific drug delivery Drug delivery in the stomach

Ivanov, Kazakov, Galaev, and Mattiasson (2001) Adrados et al. (2001) Qiu and Park (2001) Qiu and Park (2001) Takahashi, Sakai, & Mizutani (1997) Irvin, Goods, & Whinnery (2001) Desponds & Freitag (2005)

Magnetic field

Gel-entrapment system in the magnetic control of immobilized enzyme reactions. Potential use as small-scale actuators and valves in microsystems application Capture of biologicals from solution mixture.

Electric field

Swelling and deswelling

Cotelomer of N-isopropylacrylamide and N-acryloxysuccinimide with bioligand and (3-aminopropyloxy) azobenzene attached to it Dodecyl isocyanatemodified poly (ethylene glycol) grafted poly(2Hydroxyethyl methacrylate) Poly(N-isopropylacrylamide) with trisodium salt of copper chlorophyllin

UV radiation

Affinity precipitation

Ultrasound

Disrupt the orderly chains on the surface of the drug-containing polymer

Controlled drug delivery

Kwok, Mourad, Crum, & Ratner (2001)

Light

Reversible collapse of gel

Potential use as a photo-responsive artificial muscle or switch

Suzuki and Tanaka (1990)

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 in their 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 collapse of a hydrogel (Fig. 1). The driving force behind these reversible 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 efficiency of hydrogen bonding, resulting in the collapse of the hydrogel 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 temperature of the solution increases. By contrast, the solubility of temperature-responsive polymers decreases as the temperature

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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).

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 temperature-composition diagram for the polymer solution (Elias, 1984). The LCST is a distinctive property of temperature-responsive polymers. Poly(N-isopropylacrylamide) (poly-NIPAAm; Fig. 2) is the most representative and extensively studied temperatureresponsive polymer with an aqueous solution LCST of 32 C. The phase transition of poly-NIPAAm in solution is quite reversible, reproducible and sensitive to small changes in temperature. The phase transition phenomenon is accompanied by a contraction 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 hydrogen 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 the water molecule become unstable and the polymer chains contract 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 from Wiley-VCH Verlag GmbH & Co.).

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

of the monomer solution with -rays or electron beams which act as initiators (Gil and Hudson, 2004). 4. Other stimuli responsive polymers

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 polyNIPAAm with anionic acrylic acid (Kobayashi, Kikuchi, Sakai, & Okano, 2003) or cationic N,N-dimethylaminopropylacrylamide (DMAPPAm) (Ayano et al., 2006) also produces co-polymers with temperature tuneable hydrophobicity and charge density. 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 manipulated by integration of hydrophobic or hydrophilic moieties into the molecular structure. For example, the co-polymerization of NIPAAm monomers with hydrophilic monomers such as acrylamide, leads to an increase in the polymer hydrophilicity and an increase in the LCST of the co-polymer. By contrast, copolymerization of the NIPAAm monomers with more hydrophobic monomers, such as n-butyl acrylamide, increases the polymer hydrophobicity and decreases the LCST of the co polymer (Hoffman et al., 2000) (Fig. 5). Temperature-responsive materials are synthesized via two different polymerization methods. They can be prepared by radical 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

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. 6. pH dependent ionization of polyelectrolytes. (a) Poly(acrylic acid) (polyacid), and (b) Poly(N,N-diethylaminoethyl methacrylate) (polybase) (from Qiu 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 polymers composed of polyelectrolytes that demonstrate shape change (swelling, deswelling or bending) when exposed to an electric field. For example, partially hydrolyzed polyacrylamide gel 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 resulting 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 delivery systems (Yuk, Cho, & Lee, 1992). Light-sensitive hydrogels can be broadly subdivided into UV-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 molecule ionizes and leads to swelling due to an increase in the osmotic pressure within the gel. Visible light-sensitive hydrogels 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 biotechnology fields. They have been used in bioseparation, bioconjugation (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, Hadjichristidis, & 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 immunoassays (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, Sunamoto, Matsushima, Kikuchi, & Okano, 2000), ionic (Kobayashi et al., 2003), and affinity based chromatography separations (Hoffman and Stayton, 2004) using a range of different supporting materials. 5.1. Hydrophobic interaction chromatography A common approach to the use of temperature-responsive polymers is on solid supports such as silica. Poly-NIPAAmmodified silica beads showing temperature dependant hydrophobic-hydrophilic properties have been prepared and employed as novel HPLC packing materials for chromatographic separations. The stationary phase exhibits very rapid and reversible hydrophilichydrophobic changes in response to temperature, allowing temperature gradients analogous to solvent gradients in reversed-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 aminopropyl 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-dimethylaminopropylacrylamide) (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 methacrylate) 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 -endorphin fragment 127. It was found that temperature gradients could rapidly alter the stationary phase surface characteristics, resulting 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 WileyVCH Verlag GmbH & Co.). 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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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).

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 HPLC or 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). Although 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 stationary phase at the higher temperatures. Enantiomer separation of racemic N-(3,5-dinitrobenzoyl (DNB))amino acid isopropyl esters was achieved by temperatureresponsive 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 developed 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 herbicides 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 chromatography medium (Kobayashi et al., 2002; Kobayashi et al., 2003). The polymer grafted stationary phase showed simultaneous thermally modulated changes in charge density and hydrophobicity due to incorporation of AAc as the anionic exchange group and hydrophobic tBBAm into the NIPAAm sequence. Effective separation of basic bioactive peptides under exclusively aqueous conditions was attained using anionic temperature/pH-responsive polymer-modified surfaces (Fig. 9). Similarly, silica beads grafted with poly(NIPAAm-co-BMAco-DMAPAAm) has been evaluated as a cationic temperatureresponsive 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 media. Poly-NIPAAm grafted onto silica beads coated with dextran

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

and diethylaminoethyl groups have been synthesized and studied 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, possibly 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 molecular masses b 20 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 prepared and employed as a column packing material for gel permeation size-exclusion chromatography (Gewehr, Nakamura, Ise, & Kitano, 1992). Poly-NIPAAm was end-functionalized through telomerization polymerization of NIPAAm with mercaptopropionic 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-NIPAAmmodified 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 sizeexclusion chromatography. With beads modified with cyclohexanol 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, thereby 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-solubilization behaviour of the bioconjugates in the aqueous solution.

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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 -chymotrypsin (Kim and Park, 1999), -glucosidase (Chen and Hoffman, 1993; Agarwal and Gupta, 1996), thermolabile -glucosidase (Hoshino, Taniguchi, Kitao, Morohashi, & Sasakura, 1998), -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 dyeaffinity agarose modified by physically adsorbing Poly(Nvinylcaprolactam) [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 to 23 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 sensitive polymers have been developed into smart 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 chromatographic affinity separation matrix, capable of 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 microfluidic chromatography 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 array of fields including bioseparation has been reviewed. Although there are many different types of external stimuli, temperatureresponsive polymers made from poly(NIPAAm) and its co-polymers have been the most widely studied for the effective separation of biomolecules using hydrophobic interaction chromatography, ion-exchange chromatography, size-exclusion chromatography and affinity based separations. These smart polymers offer promise 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 growing, 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 employing 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 assistance from the State Government of Victoria, Australia (Department of Industry, Innovation and Regional Development), Monash University (Centre for Green Chemistry), Melbourne, Australia, and Food Science Australia. References
Adrados, B. P., Galaev, I. Y., Nilsson, K., & Mattiasson, B. (2001). Size exclusion behaviour of hydroxypropylcellulose beads with temperature-dependent porosity. Journal of Chromatography A, 930(12), 7378. Agarwal, R., & Gupta, M. N. (1996). Sequential precipitation with reversibly soluble insoluble polymers as a bioseparation strategy: Purification of -glucosidase from Trichoderma longibrachiatum. Protein Expression and Purification, 7, 294298. Anastase-Ravion, S., Ding, Z., Pelle, A., Hoffman, A. S., & Letourneur, D. (2001). New antibody purification procedure using a thermally responsive poly(N-isopropylacrylamide)-dextran derivative conjugate. Journal of Chromatography B, 76, 12471254. Anastasiadis, S. H., Retsos, H., Pispas, S., Hadjichristidis, N., & Neophytides, S. (2003). Smart polymer surfaces. Macromolecules, 36, 19941999. Annaka, M., Matsuura, T., Kasai, M., Nakahira, T., Hara, Y., & Okano, T. (2003). Preparation of comb-type N-isopropylacrylamide hydrogel beads and their application for size-selective separation media. Biomacromolecules, 4(2), 395403. Ayano, E., & Kanazawa, H. (2006). Aqueuos chromatography system using temperature-responsive polymer-modified stationary phases. Journal of Separation Science, 29, 738749. Ayano, E., Nambu, K., Sakamoto, C., Kanazawa, H., Kikuchi, A., & Okano, T. (2006). Aqueuos chromatography system using pH- and temperature-responsive stationary phase with ion-exchange groups. Journal of Chromatography A, 1119, 5865. Ayano, E., Okada, Y., Sakamoto, C., Kanazawa, H., Okano, T., Ando, M., et al. (2005). Analysis of herbicides in water using temperature-responsive chromatography and an aqueous mobile phase. Journal of Chromatography A, 1069(2), 281285. Baldi, A., Gu, Y., Lofness, P. S., Seigel, R. A., & Ziaie, B. (2003). A hydrogelactuated environmentally sensitive microvalve for active flow control. Journal of Michroelectromechanical Systems, 12(5), 613621.

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

ARTICLE IN PRESS
10 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxxxxx Kanazawa, H., Yamamoto, K., Matsushima, Y., Takai, N., Kikuchi, A., Sakurai, Y., et al. (1996). Temperature-responsive chromatography using poly(N-isopropylacrylamide)-modified silica. Analytical Chemistry, 68(1), 100105. Kim, H. T., & Park, T. G. (1999). Synthesis and characterization of thermally reversible bioconjugates composed of -chymotrypsin and poly(N-isopropylacrylamide-co-acrylamido-2-deoxy-D-glucose). Enzyme and Microbial Technology, 25, 3137. Kobayashi, J., Kikuchi, A., Sakai, K., & Okano, T. (2001). Aqueous chromatography utilizing pH-/temperature-responsive polymer stationary phases to separate ionic bioactive compounds. Analytical Chemistry, 73, 20272033. Kobayashi, J., Kikuchi, A., Sakaia, K., & Okano, T. (2002). Aqueous chromatography utilizing hydrophobicity-modified anionic temperature-responsive hydrogel for stationary phases. Journal of Chromatography A, 958, 109119. Kobayashi, J., Kikuchi, A., Sakai, K., & Okano, T. (2003). Cross-linked thermoresponsive anionic polymer-grafted surfaces to separate bioactive basic peptides. Analytical Chemistry, 75(13), 32443249. Kumar, A., Galaev, I. Y., & Mattiasson, B. (1998). Affinity precipitation of -amylase inhibitor from wheat meal by metal chelate affinity binding using Cu(II)-loaded copolymers of 1-vinylimidazole with N-isopropylacrylamide. Biotechnology and Bioengineering, 59, 695704. Kurata, K., Shimoyama, T., & Dobashi, A. (2003). Enantiomeric separation using temperature-responsive chiral polymers composed of L-valine diamide derivatives in aqueous liquid chromatography. Journal of Chromatography A, 1012, 4756. Kwok, C. S., Mourad, P. D., Crum, L. A., & Ratner, B. D. (2001). Self-assembled molecular structures as ultrasonically-responsive barrier membranes for pulsatile drug delivery. Journal of Biomedical Materials Research, 57, 151164. Lakhiari, H., Okano, T., Nurdin, N., Luthi, C., Descouts, P., Muller, D., et al. (1998). Temperature-responsive size-exclusion chromatography using poly (N-isopropylacrylamide) grafted silica. Biochemica et Biophysica Acta, 1379, 303313. Malmstadt, N., Hoffman, A. S., & Stayton, P. S. (2004). Smart mobile affinity matrix for microfluidic immunoassays. Lab Chip, 4, 412415. Malmstadt, N., Yager, P., Hoffman, A. S., & Stayton, P. S. (2003). A Smart microfluidic affinity chromatography matrix composed of poly(N-isopropylacrylamide)-coated beads. Analytical Chemistry, 75, 29432949. Martin, A. J., & Synge, R. L. (1941). A new form of chromatogram employing two liquid phases: A theory of chromatography. 2. Application to the microdetermination of the higher monoamino-acids in proteins. The Biochemical Journal, 35(12), 13581368. Osada, Y., Okuzaki, H., & Hori, H. (1992). A polymer gel with electrically driven motility. Nature, 355, 242244. Park, T. G., & Hoffman, A. S. (1993). Thermal cycling effects on the bioreactor performances of immobilized beta-galactosidase in temperature-sensitive hydrogels beads. Enzyme and Microbial Technology, 15, 476482. Peterson, E. A., & Sober, H. A. (1956). Chromatography of proteins. I. Cellulose ion-exchange adsorbants. Journal of the American Chemical Society, 78, 751. Philippova, O. E., Hourdet, D., Audebert, R., & Khokhlov, A. R. (1997). pHresponsive gels of hydrophobically modified poly(acrylic acid). Macromolecules, 30, 82788285. Pinkrah, V. T., Snowden, M. J., Mitchell, J. C., Seidel, J., Chowdhry, B. Z., & Fern, G. R. (2003). Physicochemical properties of poly(N-isopropylacrylamide-co-4-vinylpyridine) cationic polyelectrolyte colloidal microgels. Langmuir, 19, 585590. Piskin, E. (2004). Molecularly designed water soluble, intelligent, nanosize polymeric carriers. International Journal of Pharmaceutics, 277, 105118. Qiu, Y., & Park, K. (2001). Environment-sensitive hydrogels for drug delivery. Advanced Drug Delivery Reviews, 53, 321339. Roy, I., & Gupta, M. N. (2003). pH-responsive polymer-assisted refolding of urea- and organic solvent-denature -chymotrypsin. Protein Engineering, 16(12), 11531157. Sakamoto, C., Okada, Y., Kanazawa, H., Ayano, E., Nishimura, T., Andob, M., et al. (2004). Temperature- and pH-responsive aminopropyl-silica ionexchange columns grafted with copolymers of N-isopropylacrylamide. Journal of Chromatography A, 1030, 247253. Suzuki, A., & Tanaka, T. (1990). Phase transition in polymer gels induced by visible light. Nature, 346, 345347. Boldingh, J. (1948). Application of partition chromatography to mixtures insoluble in water. Experientia, 4, 270271. Chen, G., & Hoffman, A. S. (1993). Preparation and properties of thermoreversible, phase-separating enzyme-oligo(N-isopropylacrylamide) conjugates. Bioconjugate Chemistry, 4, 509514. De Silva, K., Stockmann, R., & Smithers, G. W. (2003). Isolation procedures for functional dairy components Novel approaches to meeting the challenges. Australian Journal of Dairy Technology, 58(2), 148152. Desponds, A., & Freitag, R. (2005). Light-responsive bioconjugates as novel tools for specific capture of biologicals by photoaffinity precipitation. Biotechnology and Bioengineering, 91(5), 583591. Elias, H. (1984). Macromolecules: Synthesis, materials and technology. 2nd end. Vol 2. New York: Plenum Press. Galaev, I. Y., & Mattiasson, B. (1999). Smart polymers and what they could do in biotechnology and medicine. Tibtech, 17, 335340. Galaev, I. Y., Warrol, C., & Mattiasson, B. (1994). Temperature-induced displacement of proteins from dye-affinity columns using an immobilized polymeric displacer. Journal of Chromatography A, 684, 3743. Gewehr, M., Nakamura, K., Ise, N., & Kitano, H. (1992). Gel permeation chromatography using porous glass beads modified with temperature-responsive polymers. Macromolecular Chemistry, 193, 249256. Gil, E. S., & Hudson, S. M. (2004). Stimuli-responsive polymers and their bioconjugates. Progress in Polymer Science, 29, 11731222. Hearn, M. T. W. (2000). Physicochemical factors in polypeptide and protein purification and analysis by high performance chromatographic techniques: Current status and challenges for the future. In S. Ahuja (Ed.), Handbook of Bioseparation (pp. 72235). San Diego: Academic Press. Hoffman, A. S. (2000). Bioconjugates of intelligent polymers and recognition proteins for use in diagnostics and affinity separations. Clinical Chemistry, 46(9), 14781486. Hoffman, A. S., Stayton, P., Bulmus, V., Chen, G., Chen, J., Cheung, C., et al. (2000). Really smart bioconjugates of smart polymers and receptor proteins. Journal of Biomedical Materials Research, 52, 577586. Hoffman, A. S., & Stayton, P. S. (2004). Bioconjugates of smart polymers and proteins: synthesis and applications. Macromolecular Symposia, 207, 139151. Horvath, C. G., & Lipsky, S. R. (1966). Use of liquid ion exchange chromatography for the separation of organic compounds. Nature, 211, 5050. Hoshino, K., Taniguchi, M., Kitao, T., Morohashi, S., & Sasakura, T. (1998). Preparation of a new thermo-responsive adsorbent with maltose as a ligand and its application to affinity precipitation. Biotechnology and Bioengineering, 60(5), 568579. Hosoya, K., Sawada, E., Kimata, K., Araki, T., Tanaka, N., & Frchet, J. M. J. (1994). In situ surface-selective modification of uniform size macroporous polymer particles with temperature-responsive poly-N-isopropylacrylamide. Macromolecules, 27, 39733976. Irie, M., & Kunwatchakun, D. (1986). Photoresponsive polymers. 8: Reversible photostimulated dilation of polyacrylamide gels having triphenylmethane leuco derivatives. Macromolecules, 19, 24762480. Irvin, D. J., Goods, S. H., & Whinnery, L. L. (2001). Direct measurement of extension and force in conductive polymer gel actuators. Chemistry of Materials, 13, 11431145. Ivanov, A. E., Kazakov, S. V., Galaev, I. Y., & Mattiasson, B. (2001). Thermosensitive copolymer of N-vinylcaprolactam and 1-vinylimidazole: Molecular characterization and separation by immobilized metal affinity chromatography. Polymer, 42(8), 33733381. Just-food.com (2006). Global market review of functional foods Forecasts to 2012. Report #44028, August 2006. Jungbauer, A. (2005). Chromatographic media for bioseparation. Journal of Chromatography A, 1065, 312. Kanazawa, H., Kashiwase, Y., Yamamoto, K., Matsushima, Y., Takai, N., Kikuchi, A., et al. (1997). Analysis of peptides and proteins by temperatureresponsive chromatographic system using N-isopropylacrylamide polymermodified columns. Journal of Pharmaceutical and Biomedical Analysis, 015, 15451550. Kanazawa, H., Sunamoto, T., Matsushima, Y., Kikuchi, A., & Okano, T. (2000). Temperature-responsive chromatographic separation of amino acid phenylthiohydantoins using aqueous media as the mobile phase. Analytical Chemistry, 72, 59615966.

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

ARTICLE IN PRESS
P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxxxxx Takahashi, F., Sakai, Y., & Mizutani, Y. (1997). Immobilized enzyme reaction controlled by magnetic heating: -Fe2O3-loaded thermosensitive polymer gels consisting of N-isopropylacrylamide and acrylamide. Journal of Fermentation and Bioengineering, 83, 152156. Tsuda, Y., Kikuchi, A., Yamato, M., Sakurai, Y., Umeju, M., & Okano, T. (2004). Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces. Journal of Biomedical Materials Research, 69A, 7078. Tswett, M. (1906). Adsorptionsanalyse und chromatographische Methode. Anwendung an die Chemie des Chlorophylls. Berichte der Deutschen botanischen Gesellschaft, 24, 385. Ueoka, Y., Gong, J., & Osada, Y. (1997). Chemomechanical polymer gel with fishlike motion. Journal of intelligent material systems and structures, 8, 465471. 11 Vaidya, A. A., Lele, B. S., Kulkarni, M. G., & Mashelkar, R. A. (2001). Thermoprecipitation of lysozyme from egg white using copolymers of Nisopropilacrylamide and acidic monomers. Journal of Biotechnology, 87, 95107. Yoshida, R., Uchida, K., Kaneko, Y., Sakai, K., Kikuchi, A., Sakurai, Y., et al. (1995). Comb-type grafted hydrogels with rapid de-swelling response to temperature changes. Nature, 374, 240242. Yuk, S. H., Cho, S. H., & Lee, H. B. (1992). Electric current-sensitive drug delivery systems using sodium alginate/polyacrylic acid composites. Pharmaceutical Research, 9(7), 955995.

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