Upload
natarajantex
View
245
Download
0
Embed Size (px)
Citation preview
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 1/11
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
www.elsevier.com/locate/ifset
⁎ 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
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 2/11
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
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 3/11
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)
3 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 4/11
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).
4 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 5/11
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).
5 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 6/11
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.).
6 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 7/11
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).
7 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 8/11
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).
8 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 9/11
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.
References
Adrados,B. P., Galaev, I. Y., Nilsson, K., & Mattiasson,B. (2001). Sizeexclusion
behaviour of hydroxypropylcellulose beads with temperature-dependent
porosity. Journal of Chromatography A, 930(1–2), 73−78.
Agarwal, R., & Gupta, M. N. (1996). Sequential precipitation with revers-
ibly soluble insoluble polymers as a bioseparation strategy: Purification
of β-glucosidase from Trichoderma longibrachiatum. Protein Expres-
sion and Purification, 7 , 294−298.
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 Chro-
matography B, 76 , 1247−1254.
Anastasiadis, S. H., Retsos, H., Pispas, S., Hadjichristidis, N., & Neophytides,S.
(2003). Smart polymer surfaces. Macromolecules, 36 , 1994−1999.
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. Biomacromole-
cules, 4(2), 395−403.
Ayano, E., & Kanazawa, H. (2006). Aqueuos chromatography system using
temperature-responsive polymer-modified stationary phases. Journal of Sep-
aration Science, 29, 738−749.
Ayano, E., Nambu, K., Sakamoto, C., Kanazawa, H., Kikuchi, A., & Okano, T.
(2006). Aqueuos chromatography system using pH- and temperature-res-
ponsive stationary phase with ion-exchange groups. Journal of Chromatog-
raphy A, 1119, 58−65.
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), 281−285.
Baldi, A., Gu, Y., Lofness, P. S., Seigel, R. A., & Ziaie, B. (2003). A hydrogel-
actuated environmentally sensitive microvalve for active flow control. Journal of Michroelectromechanical Systems, 12(5), 613−621.
9 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 10/11
Boldingh, J. (1948). Application of partition chromatography to mixtures insolu-
ble in water. Experientia, 4, 270−271.
Chen, G., & Hoffman, A. S. (1993). Preparation and properties of thermo-
reversible, phase-separating enzyme-oligo( N -isopropylacrylamide) conju-
gates. Bioconjugate Chemistry, 4, 509−514.
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), 148−
152.Desponds, A., & Freitag, R. (2005). Light-responsive bioconjugates as novel
tools for specific capture of biologicals by photoaffinity precipitation. Bio-
technology and Bioengineering , 91(5), 583−591.
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 , 335−340.
Galaev, I. Y., Warrol, C., & Mattiasson, B. (1994). Temperature-induced dis-
placement of proteins from dye-affinity columns using an immobilized poly-
meric displacer. Journal of Chromatography A, 684, 37−43.
Gewehr, M., Nakamura, K., Ise, N., & Kitano, H. (1992). Gel permeation chro-
matography using porous glass beads modified with temperature-responsive
polymers. Macromolecular Chemistry, 193, 249−256.
Gil, E. S., & Hudson, S. M. (2004). Stimuli-responsive polymers and their bio-
conjugates. Progress in Polymer Science, 29, 1173−1222.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. 72−235). 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), 1478−1486.
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, 577−586.
Hoffman, A. S., & Stayton, P. S. (2004). Bioconjugates of smart polymers and
proteins: synthesis and applications. Macromolecular Symposia, 207 , 139−151.
Horvath, C. G., & Lipsky, S. R. (1966). Use of liquid ion exchange chro-
matography 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 Bioengineer-
ing , 60(5), 568−579.
Hosoya, K., Sawada, E., Kimata, K., Araki, T., Tanaka, N., & Fréchet, J. M. J.
(1994). In situ surface-selective modification of uniform size macroporous
polymer particles with temperature-responsive poly- N -isopropylacrylamide.
Macromolecules, 27 , 3973−3976.
Irie, M., & Kunwatchakun, D. (1986). Photoresponsive polymers. 8: Reversible
photostimulated dilation of polyacrylamide gels having triphenylmethane
leuco derivatives. Macromolecules, 19, 2476−2480.
Irvin, D. J., Goods, S. H., & Whinnery, L. L. (2001). Direct measurement of ex-
tension and force in conductive polymer gel actuators. Chemistry of Materials,
13, 1143−1145.
Ivanov, A. E., Kazakov, S. V., Galaev, I. Y., & Mattiasson, B. (2001). Ther-
mosensitive copolymerof N -vinylcaprolactam and 1-vinylimidazole: Molecular characterization and separation by immobilized metal affinity chromatography.
Polymer , 42(8), 3373−3381.
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 Chro-
matography A, 1065, 3−12.
Kanazawa, H., Kashiwase, Y., Yamamoto, K., Matsushima, Y., Takai, N.,
Kikuchi, A., et al. (1997). Analysis of peptides and proteins by temperature-
responsive chromatographic system using N -isopropylacrylamide polymer-
modified columns. Journal of Pharmaceutical and Biomedical Analysis,
015, 1545−1550.
Kanazawa, H., Sunamoto, T., Matsushima, Y., Kikuchi, A., & Okano, T. (2000).
Temperature-responsivechromatographic separationof aminoacid phenylthio-
hydantoins using aqueous media as the mobile phase. Analytical Chemistry,
72, 5961−5966.
Kanazawa, H., Yamamoto, K., Matsushima, Y., Takai, N., Kikuchi, A., Sakurai, Y.,
et al. (1996). Temperature-responsive chromatography using poly( N -isopro-
pylacrylamide)-modified silica. Analytical Chemistry, 68(1), 100−105.
Kim, H. T., & Park, T. G. (1999). Synthesis and characterization of thermally
reversible bioconjugates composed of α-chymotrypsin and poly( N -isopro-
pylacrylamide-co-acrylamido-2-deoxy- D-glucose). Enzyme and Microbial
Technology, 25, 31−37.
Kobayashi, J., Kikuchi, A., Sakai, K., & Okano, T. (2001). Aqueous chroma-tography utilizing pH-/temperature-responsive polymer stationary phases
to separate ionic bioactive compounds. Analytical Chemistry, 73,
2027−2033.
Kobayashi, J., Kikuchi, A., Sakaia, K., & Okano, T. (2002). Aqueous chro-
matography utilizing hydrophobicity-modified anionic temperature-respon-
sive hydrogel for stationary phases. Journal of Chromatography A, 958,
109−119.
Kobayashi, J., Kikuchi, A., Sakai, K., & Okano, T. (2003). Cross-linked ther-
moresponsive anionic polymer-grafted surfaces to separate bioactive basic
peptides. Analytical Chemistry, 75(13), 3244−3249.
Kumar, A., Galaev, I. Y., & Mattiasson, B. (1998). Affinity precipitation of
α-amylase inhibitor from wheat meal by metal chelate affinity binding
using Cu(II)-loaded copolymers of 1-vinylimidazole with N -isopropyla-
crylamide. Biotechnology and Bioengineering , 59, 695−704.
Kurata, K., Shimoyama, T., & Dobashi, A. (2003). Enantiomeric separationusing temperature-responsive chiral polymers composed of L-valine diamide
derivatives in aqueous liquid chromatography. Journal of Chromatography
A, 1012, 47−56.
Kwok, C. S., Mourad, P. D., Crum, L. A., & Ratner, B. D. (2001). Self-assembled
molecularstructures as ultrasonically-responsivebarrier membranes for pulsatile
drug delivery. Journal of Biomedical Materials Research, 57 , 151−164.
Lakhiari, H.,Okano, T., Nurdin, N.,Luthi, C.,Descouts, P., Muller, D.,et al. (1998).
Temperature-responsive size-exclusion chromatography using poly ( N -isopro-
pylacrylamide) grafted silica. Biochemica et Biophysica Acta, 1379, 303−313.
Malmstadt, N., Hoffman, A. S., & Stayton, P. S. (2004). Smart mobile affinity
matrix for microfluidic immunoassays. Lab Chip, 4, 412−415.
Malmstadt, N., Yager, P., Hoffman, A. S., & Stayton, P. S. (2003). A Smart
microfluidic affinity chromatography matrix composed of poly( N -isopro-
pylacrylamide)-coated beads. Analytical Chemistry, 75, 2943−2949.
Martin, A. J., & Synge, R. L. (1941). A new form of chromatogram employingtwo liquid phases: A theory of chromatography. 2. Application to the micro-
determination of the higher monoamino-acids in proteins. The Biochemical
Journal , 35(12), 1358−1368.
Osada,Y., Okuzaki, H.,& Hori, H. (1992). A polymer gel with electricallydriven
motility. Nature, 355, 242−244.
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, 476−482.
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). pH-
responsive gels of hydrophobically modified poly(acrylic acid). Macromo-
lecules, 30, 8278−8285.
Pinkrah, V. T., Snowden, M. J., Mitchell, J. C., Seidel, J., Chowdhry, B. Z., &
Fern, G. R. (2003). Physicochemical properties of poly( N -isopropylacryla-mide-co-4-vinylpyridine) cationic polyelectrolyte colloidal microgels.
Langmuir , 19, 585−590.
Piskin, E. (2004). Molecularly designed water soluble, intelligent, nanosizepoly-
meric carriers. International Journal of Pharmaceutics, 277 , 105−118.
Qiu, Y., & Park, K. (2001). Environment-sensitive hydrogels for drug delivery.
Advanced Drug Delivery Reviews, 53, 321−339.
Roy, I., & Gupta, M. N. (2003). pH-responsive polymer-assisted refolding of
urea- and organic solvent-denature α-chymotrypsin. Protein Engineering ,
16 (12), 1153−1157.
Sakamoto, C., Okada, Y., Kanazawa, H., Ayano, E., Nishimura, T., Andob, M.,
et al. (2004). Temperature- and pH-responsive aminopropyl-silica ion-
exchange columns grafted with copolymers of N -isopropylacrylamide.
Journal of Chromatography A, 1030, 247−253.
Suzuki, A., & Tanaka, T. (1990). Phase transition in polymer gels induced by
visible light. Nature, 346 , 345−347.
10 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
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
8/3/2019 Novel Chromatographic
http://slidepdf.com/reader/full/novel-chromatographic 11/11
Takahashi, F., Sakai, Y., & Mizutani, Y. (1997). Immobilized enzyme reaction
controlled by magnetic heating: γ-Fe2O3-loaded thermosensitive polymer
gels consisting of N -isopropylacrylamide and acrylamide. Journal of
Fermentation and Bioengineering , 83, 152−156.
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, 70−
78.Tswett, M. (1906). Adsorptionsanalyse und chromatographische Methode. Anwen-
dung an die Chemie des Chlorophylls. Berichte der Deutschen botanischen
Gesellschaft , 24, 385.
Ueoka, Y., Gong, J., & Osada, Y. (1997). Chemomechanical polymer gel withfish-
like motion. Journalof intelligent materialsystems and structures, 8, 465−471.
Vaidya, A. A., Lele, B. S., Kulkarni, M. G., & Mashelkar, R. A. (2001).
Thermoprecipitation of lysozyme from egg white using copolymers of N -
isopropilacrylamide and acidic monomers. Journal of Biotechnology, 87 ,
95−107.
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, 240−242.
Yuk, S. H., Cho, S. H., & Lee, H. B. (1992). Electric current-sensitive drugdelivery systems using sodium alginate/polyacrylic acid composites. Phar-
maceutical Research, 9(7), 955−995.
11 P. Maharjan et al. / Innovative Food Science and Emerging Technologies xx (2007) xxx – xxx
ARTICLE IN PRESS
Pl it thi ti l M h j P t l N l h t hi ti Th t ti l f t l I ti F d S i d E i