Temperature-responsive polymer

A temperature-responsive polymer is a polymer which undergoes a physical change when external thermal stimuli are presented. The ability to undergo such changes under easily controlled conditions makes this class of polymers fall into the category of " [smart material] s] ". These physical changes can be exploited for many analytical techniques, especially for separation chemistry. After numerous investigations of poly(N-isopropylacrylamide) (poly-NIPAAm), there was a sparked interest in the applications of this and many other stimuli-responsive polymers. There has been extensive research in the applications of intelligent polymers for use as stationary phases, extraction compounds, surface modifiers, drug delivery, and gene delivery.

The focus of this article will review the introduction, development, and future of thermo-responsive polymers (TRP) in its application to chromatographic techniques and quantitative analysis. Most of this review will cover its use as a stationary phase for size-exclusion, hydrophobic interaction, ionic, and affinity chromatographic techniques due to the sheer plethora of research based on intelligent polymers [Pankaj Maharjan, Brad W. Woonton, Louise E. Bennett, Geoffrey W. Smithers, Kirthi DeSilva, Milton T.W. Hearn, Innovative Food Science and Emerging Technologies (2008), 9, 232-242.] .

Historical background of temperature responsive polymers

The effects of external stimuli on particular polymers have been investigated as early as the 1960s by Heskins and Guillet [Michael Heskins, James E. Guillet, J. Macromol. Sci. Chem. (1968), 2(8), 1441-1455.] . They established the lower critical solution temperature (LCST) for poly-N-Isopropylacrylamide (poly-NIPAAm) to be 32 ^oC. Contrary to the behavior of most compounds in aqueous solutions, thermally responsive polymers become less soluble (more hydrophobic) in water at elevated temperatures. This also does not apply to any polymer, for instance the procedure for dissolving polyethylene oxide (PEO) or polyethylene glycol (PEG) in water requires elevated temperatures and a significant amount of time under stirring conditions from personal experience. The point in which poly-NIPAAm undergoes a phase transition from soluble to insoluble has been determined to occur at 32 Celsius. This is based on standard conditions such as neutral pH and without spiking with ionic compounds. The lowering of pH and increasing ionic strength will lower the LCST and the phase transition will occur sooner. Under LCST, the polymer chains’ hydrophilic surfaces interact with water and are elongated. At this critical point, the polymer chains shrivel into an insoluble glob as the hydrophobic surfaces interact and the chains become dehydrated. The conformational changes primarily result from dehydration at the isopropyl side groups [Hideko Kanazawa, Kazuo Yamamoto, Yoshikazu Matsushima, Nobuharu Takai, Akihiko Kikuchi, Yasuhisa Sakurai, Teruo Okano, Anal. Chem. (1996), 68, 100-105.] . There were several works examining the properties of poly-NIPAAm that followed, but it wasn’t until the 1990s that the beneficial properties of this polymer and others were applied to chromatography in a surge of research, much of which took place in Japanese laboratories.

Major Breakthroughs & Novel Techniques in Separations Chemistry

Hydrophobic Interaction Chromatography

GPC

The research that appeared to spark an onslaught of modified applications was a Gel Permeation Chromatographic technique of fixing PIPA strands to glass beads and separating a mixture of dextrans, which was developed by Gewehr et al. [Markus Gewehr, Katsunori Nakamura, Norio Ise, Makromol. Chem. (1992), 193, 249-256.] . They found that between the temperatures of 25-32 ^oC, the elution time of dextrans at different molecular weights exhibited a dependence on the temperature. Dextrans of the highest molecular weight eluted first since the PIPA chains exhibit hydrophilicity at temperatures below the LCST. As the temperature of the elution increased, when the chains behave in a more hydrophobic manner, the elution times increased for each of the analytes for the given range. The trend generally applies over the entire temperature range, but there is a flattening of the curve before 25 Celsius and after 32 Celsius (the approximate LCST for this experiment). It is important to note that above the LCST, the PIPA acts as a typical nonpolar stationary phase that would be used in reverse-phased chromatography. There are also instances of the elution times increasing below 15 Celsius, which most likely can be attributed to the lower temperatures’ effects on mass transfer playing a more significant role on retention than the stationary phase behavior. This study showed that the resolution could essentially be tuned by adjusting the operating temperature. The scope of this study was limited to isothermal conditions and attaching polymer chains to glass beads. The results, however, were satisfying enough to inspire other investigations and modifications to create a more versatile stationary phase for the advancement of chromatography.

Enhancing Hydrophobic Interaction

Kanazawa’s group expanded on their success by using different modifiers to enhance hydrophobicity through the attachment of butyl methacrylate (BMA), a hydrophobic comonomer [Hideko Kanazawa, Yuki Kashiwase, Kazuo Yamamoto, Yoshikazu Matsushima, Akihiko Kikuchi, Yasuhisa Sakurai, Teruo Okano, Anal. Chem. (1997), 69, 823-830.] . For simplification the resultant polymer has been labeled as IBc (Isopropylacrylamide butyl methacrylate copolymer). The polymers were synthesized using radical telomerization with varying BMA content. Where pure poly-NIPAAm was unable to resolve hydrophobic steroids at any temperature, IBc-grafted silica stationary phases were able to resolve steroid peaks with increasingly retarded retention times in correlation to both increased BMA content and increased temperature. They went on to develop a method to separate phenylthiohydantoin(PTH)-amino acids using their IBc stationary phase with a stronger emphasis of implementing environmentally friendly conditions by using a purely aqueous phase in HPLC [Hideko Kanazawa, Tastuo Sunamoto, Yoshikazu Matsushima, Akihiko Kikuchi, Teruo Okano, Anal. Chem. (2000), 72, 5961-5966.] . In a Japanese publication, the same group under Sakamoto separated catechins using pNIPAAm reinforcing the success of these techniques [Chikako Sakamoto, Yuji Okada, Hideko Kanazawa, Akihiko Kikuchi, Teruo Okano, Bunseki Kagaku (2003), 52(10), 903-906.] .

Modifying the LCST for Improved Experimental Parameters

Since the separation of biological molecules such as proteins would be better served by isocratic elution with an aqueous solvent, resolution of HPLC analysis should be tweaked in the area of stationary phases to elute such analytes that may be sensitive to organic solvents. Kanazawa et al. recognized the possibility of changing the LCST parameter through the addition of different moieties [Hideko Kanazawa, Kazuo Yamamoto, Yoshikazu Matsushima, Nobuharu Takai, Akihiko Kikuchi, Yasuhisa Sakurai, Teruo Okano, Anal. Chem. (1996), 68, 100-105.] . Kanazawa’s group investigated the reversible changes of poly-NIPAAm once modifying it with a carboxyl end. It was suggested that the modification leads to faster changes in conformation due to the restrictions introduced by the carboxyl group. They attached the carboxyl-terminated poly-NIPAAm chains to (aminopropyl)silica and used it as packing material for HPLC analysis of steroids. The separation took place under isocratic conditions using pure water as the mobile phase, and controlled the temperature using a water bath. One important note is that they were able to shift the LCST from 32 Celsius to 20 Celsius by making the solution 1M in NaCl concentration. Of the 5 steroids and benzene, only testosterone could be resolved from the other peaks below the LCST (5 ^oC, LCST=20 ^oC in 1M NaCl). Above the LCST (25 ^oC, LCST=20 ^oC in 1M NaCl), all of the peaks are well resolved, and there is an increasing trend of retention time versus temperature up to 50 Celsius.

Size Exclusion Chromatography

Prior to these studies, HPLC analyses were tuned by modifying the mobile and stationary phases only. Gradient elution for HPLC merely meant changing the ratio of solvents to improve column efficiency, and this requires the use of sophisticated solvent pumping mechanisms along with extra steps and precautions in the chromatographic analysis. Enlightened by the prospect of using temperature gradient elutions for HPLC analyses, Hosoya et al. sought to make surface modification of HPLC stationary phases more accessible. Their study utilizes graft-type copolymerization of poly-NIPAAm onto macroporous polymeric materials [Ken Hosoya, Etsuko Sawada, Kazuhiro Kimata, Takeo Araki, Nabuo Tanaka, Jean M.J. Fréchet, J. Macromol. (1994), 27, 3973-3976.] . The in situ preparation compared the use of cyclohexanol and toluene as porogens in the preparation of the modified polystyrene seeds. Reverse-phased size exclusion chromatography (SEC) revealed pore size and pore size distribution of the particles and its dependence on temperature. Cyclohexanol acted as a successful porogen showing a dependent relationship of pore size to temperature. The use of toluene as a porogen gave results that were similar to unmodified macroporous particles. This indicates that poly-NIPAAm can be successfully grafted onto the surface and within the pores of macroporous materials. The application of this preparatory technique gives rise to tunable pore sizes. Temperature gradient elutions can be utilized to improve column efficiency through the changing of pore size in SEC. The mechanism of the change in pore size is simple, the pores are smaller under LCST due to the elongated chains of poly-NIPAAm within the pores, as temperature increases to and above LCST, the chains retract into a globular formation increasing the pore size.

Ion-Exchange Chromatography

Modification had also been extended past hydrophobic and hydrophilic attachments, charged compounds have also been introduced to TRPs. Kobayashi et al. had previously performed successful modifications to separate bioactive ionic compounds, and continued on that success to improve separation efficiency of bioactive compounds [Jun Kobayashi, Akihiko Kikuchi, Kiyotaka Sakai, Teruo Okano, Anal. Chem. (2003), 75, 3244-3249.] . Common methods of separating angiotensin peptides had involved reverse-phased high performance liquid chromatography (RP-HPLC) and cation-exchange chromatography. RP-HPLC requires the use of organic solvents, which is not favored and current trends are moving away from that. Hydrophobic interaction chromatography requires high concentration salt elutions and requires eluent cleaning to remove the salt. To address the shortcomings of the previous methods, Kobayashi’s group grafted acrylic acid (anionic acrylate under neutral conditions) and tert-butylacrylamide (hydrophobic) monomers onto poly-NIPAAm, resulting in poly-NIPAAm-co-AAc-co-tBAAm (IAtB) onto silica beads as a stationary phase medium. The reason for incorporating both ionic and hydrophobic compounds is multifaceted. The ionic compound improves interactivity with the ionic species, but raises the LCST significantly. The hydrophobic addition counteracts against the raise in LCST and lowers it to a more standard value, but also interacts with the hydrophobic surfaces of biological compounds. This resulted in successful and resolved elution of angiotensin peptides. Additionally, they were able to tune the retention factor for the analytes through isocratic temperature gradient elution. Ideal elutions occurred at 35 ^oC, but decreasing the temperature to 10 ^oC or raising it to 50 ^oC caused faster elutions either way. This is a strong indication that electrostatic and hydrophobic interactions can be similarly affected by changes in temperature. The major advantages from applying these success of this study include stationary phase versatility and maintaining bioactivity of the analytes.

In a separate study, Ayano et al. modified poly-NIPAAm with cationic N,N-dimethylaminopropylacrylamide (DMAPAAm) and hydrophobic BMA and to form IDB grafted it onto silica beads [Eri Ayano, Kyoko Nambu, Chikako Sakamoto, Hideko Kanazawa, Akihiko Kikuchi, Teruo Okano, J. Chromatogr. A (2006), 1119, 58-65.] . The research parallels that of Kobayashi’s group, but also utilized pH changes to adjust the LCST. The effect of pH on the LCST is as follows, from a plateau value between pH 4.5 and pH 6.0, the LCST decreased up to pH 9 and below pH 4.5. This can be interpreted as requiring slightly basic or moderately acidic conditions, as the 4.5-6.0 pH region holds a maximum value of the LCST, an unfavorable condition. They utilized these properties to separate several non-steroidal anti-inflammatory drugs (NSAIDs). The analysis of acidic drugs (salicylic acid: BA; SA; MS; and As) was performed below pH 4.5. MS is hydrophobic only its retention time was affected by an increase in temperature on the column without a terminally modified anion-exchanger (IB column). However, with an anion-exchanger present, dissociated acidic drugs were retained longer at temperatures below LCST, and shorter at temperatures above LCST. When the IBD column compared to recently established PNIPAAm columns, electrostatic forces show remarkably higher retention ability of charged compounds than its hydrophilic predecessor. A single stationary phase can accomplish pharmaceutical separations based on hydrophobic interactions, hydrophilic interactions, and electrostatic interactions merely by adjusting the temperature (while adjusting pH to tweak the LCST).

=Affinity Chromatography=

Selective enzyme and antibody separations can be achieved with the use of specific end groups that conjugate with the specific compounds. This results in a formation of a polymer-enzyme conjugate which can be reversibly precipitated and dissolved by changing the temperature. In Chen and Hoffman’s study, 1993, N-hydroxysuccinimide (NHS) ester functional end group on NIPAAm is used to conjugate selectively with β-D-glucosidase [Guohua Chen, Allan S. Hoffman, Bioconjugate Chem. (1993), 4, 509-514.] . They found that the conjugated enzyme could be repeatedly precipitated and dissolved in solution and still maintain sufficient enzymatic activity.

In a study that was published in 1998, Hoshino et al. prepared a TRP with a maltose ligand, evaluated it with concanavalin A (Con A), and attempted to purify/separate α-glucosidase (AG), a thermolabile compound [Kazuhiro Hoshino, Masayuki Taniguchi, Taichi Kitao, Shoichi Morohashi, Toshisuke Sasakura, Biotech. & Bioeng. (1998), 60(5), 568-579.] . Since the goal is to selectively isolate a thermolabile enzyme, a TRP with a small LCST value is desired. To fit this condition, the selected TRP was poly(N-acryloylpiperidine)-cysteamine (pAP), which has an LCST of 4 ^oC. The terminally bound maltose moiety maintains affinity for both analytes, thus the modified TRP, pAPM, met critical conditions of external temperature requirements and affinity for both target analytes. The solubility properties changed from 4 ^oC (soluble) to 8 ^oC (insoluble). Several reagents were tested for the recovery of Con A by desorption which had higher binding affinities to Con A than maltose. These reagents were α-D-glucopyranoside, D-mannose, methyl α-D-mannopyranoside, and glucose. α-D-mannopyranoside was the most effective for desorbing Con A from pAPM at virtually 100% after 1 hour. As a control, pAPM was used to bind Con A from a crude extract, which found the pickup of several impurities but still managed to recover 80% of Con A. This exemplifies the need for selective moieties, maltose not residing among them. Finally, the application of pAPM was tested by attempting to separate AG from yeast extract under low temperature conditions. In conclusion, the pAPM was found to recover 68% of AG activity tested against, maltose being the selected desorption reagent.

Another interesting development for AC was involved with antibody separation using another TRP-ligand combination. Anastase-Ravion et al. attached a dextran derivative to the classic poly-NIPAAm to result in a poly(NIPAAm)-DD, and used this stationary phase to separate polyclonal antibodies from subcutaneous rabbit serum [S. Anastase-Ravion, Z. Ding, A. Pellé, A.S. Hoffman, D. Letourneur, J. Chromatogr. B (2001), 761, 247-254.] . From the study, the dextran derivative of choice was carboxymethyl dextran benzylamide sulfonate/sulfate, and when bound to the TRP was labeled poly(NIPAAm)-CMDBS. The LCST for the poly(NIPAAm)-CMDBS was raised from 32 ^oC to 33 ^oC. To test the success of the affinity binding, the antibodies were eluted with glycine buffer (adjusted to pH 2.6 with HCl).

A study with very promising results came out of the Department of Engineering at the University of Washington, 2003. This study by Malmstadt et al. merged the newer developments in affinity chromatography with microfluidic devices. Upon the development of microfluidic technology, coupling it with affinity chromatography meant modifying channel surfaces, packing coated beads, or packing with coated porous material, neither of which allow for replenishing the columns [Noah Malmstadt, Paul Yager, Allan S. Hoffman, Patrick S. Stayton, Anal. Chem. (2003), 75, 2943-2949.] . This produces limitations that prevent the packing material from being changed or the column being regenerated. The approach they took to address those challenges meant incorporating TRP particles as a reversibly immobilized stationary phase. What separates this development from other AC methods is the that the beads on which the modified TRP are attached can reversibly adhere to the inner surfaces of the microfluidic channels. The formulation of the smart bead matrix is a little complex, but in general poly-NIPAAm is modified two times, first with NHS, then with poly(ethylene glycol)-biotin (PEG-b) resulting in PEG-b/pNIPAAm beads. The inner surface of the microfluidic channels is composed of poly(ethylene terephthalate), to which the PEG-b/pNIPAAm beads reversibly bind above the LCST. When the sample solution is passed through the channels, the target analyte binds to the biotin ligand. The temperature can then be brought below the LCST to dissociate and become removed from the inner channels. This allows for a system adept to being reloaded with stationary phase under mild conditions. They successfully separated and eluted Streptavidin. Further application of these procedures allow for portable AC columns which can be packed on site and used for local or clinical analytical separations of complex biological fluids.

Developments in Extraction and Preconcentration

TRPs need not be viewed as simple chromatographic stationary phases, they can also be employed as chelating extraction compounds. With proper modification of the poly-NIPAAm or some other TRP, the moiety can complex with the target analytes, then the solution temperature can be brought above the LCST at which point the modified TRP complexed with the analyte will precipitate. That precipitate can then be collected and reintroduced to a solution of much smaller volume. At that point the temperature of the smaller volume solution should be below LCST, which will solubilize the modified TRP, and reagents that take advantage of chemical equilibria could force the analytes back into solution. An example of this type of application was demonstrated by a method explored by Saitoh et al. [T. Saitoh, F. Satoh, M. Hiraide, Talanta (2003), 61, 811-817.] . Several moieties were testing for the extraction of several heavy metal cations, copper(II), nickel(II), cobalt(II), lead(II), and cadmium(II). Some of the moieties tested were N,N,N”,N”-tetramethylethylene-diamine (TEMED), imidazole (Im), carboxylic acid (COOH), iminodiacetic acid (IDA), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The resulting extractions found poly-NIPAAm-Im, were relatively successful for extracting all of the metals except for cadmium(II). The most efficient extraction was achieved by poly-NIPAAm-IDA by recovering greater than 95% of all tested metals. In order to analyze the extract, the polymer was dissolved in N,N-dimethylformamide (DMF). The results were compared to traditional methods of ion-exchange chromatography. The only major disadvantage of the method is the use of an organic solvent, but at small volumes it is a relatively small factor.

Another example demonstrates the TRP assisted precipitation of acidic macromolecules for the application of extracting RNA and glycosaminoglycans from cultured cells [Tomonori Hayashi, Shin-ichi Yasueda, Yasuharu Nakanishi, Hiroko Ohta, Mitsuhiro Kinoshita, Yasuyoshi Miki, Takashi Masuko, Kazuaki Kakehi, Analyst (2004), 129, 421-427.] . An elaborate procedure is used to modify poly-NIPAAm with poly-L-lysine moiety, resulting in the PL-polymer (poly-NIPAAm-PL). In a 0.5M saline solution, poly-NIPAAm is introduced and then brought several degrees Celsius above the LCST, when the polymer precipitates with the captured acidic macromolecules. The precipitate can then be placed into a smaller volume at a higher saline concentration (above 1M) and below the LCST, at which point the analytes will be released. After this, a desalting procedure is required, a disadvantage, but still does not require the use of organic solvents. The results were analyzed by agarose gel electrophoresis to verify the presence of the target analytes against a standard run. This study has a potentially wide application of removing interference from biological studies by selectively removing the target analytes. Further studies would investigate other moieties for the extraction of macromolecules with different properties (as in non multi-anionic macromolecules).

Present and future direction

Newer investigations have explored the other conformational changes that TRPs experience. These polymers do not only change their solubility state, but can also change their size in solution. Castellanos et al. used lithography to pattern poly-NIPAAm hydrogel to solid surfaces. As the temperature changes, the hydrogel can swell (take in more solvent) or collapse (expel solvent) in a calculated relationship with temperature [Alexandro Castellanos, Samuel J. DuPont, August J. Heim II, Garrett Matthews, Peter G. Stroot, Wilfrido Moreno, Ryan G. Toomey, Langmuir (2007), 23, 6391-6395.] . Selectivity to target analytes depend on the size of the gaps created in the hydrogel, which can swell up to 70% in the lateral direction. This reduces the gap size in the hydrogel. The gap sizes can range from 3 μm below LCST to 12 μm above LCST. The method was titled as a “catch and release” technique. To test the method, the group attempted to selectively separate 2 sizes of polystyrene microspheres, with diameter sizes of 6 μm and 20 μm. The method was validated by several microscopic imaging techniques to accommodate the setup, and it was verified that after raising the temperature above LCST, and lowering it below LCST again, the hydrogel effective trapped the smaller 6 μm particles and not the larger 20 μm particles. Further development would require reducing gap sizes to a controllable nanoscale range.

More recent developments have seen the rise of molecularly imprinted polymers (MIP). This takes advantage of natural molecular recognition between biological compounds, as well as the swelling and contracting properties of particular TRPs. One study by Suedee et al. (2006) combines molecular imprinting techniques with the advancement in research of TRPs to design a template that can successfully extract polar dopamine from urine samples [Roongnapa Suedee, Vatcharee Seechamnanturakit, Bhutorn Canyuk, Chitchamai Ovatlarnporn, Gary P. Martin, J. Chromatogr. A (2006), 1114, 239-249.] . Molecular imprinting mostly involves the use of N-substituted polyacrylamides. In this study, they used (N,N-methylene-bis-acrylamide cross-linked) polymer. The template molecule in this case was dopamine. It was shown to effectively bind and release dopamine, epinephrine, isoproterenol, salbutamol, and serotonin by adjusting the temperature in a solid phase extraction system. The LCST of the polymer is not established, as it is a continuous conformational change and there is no critical temperature for a major phase change. The experiment tested efficiency over the range of 25-70 ^oC. Selective adsorption can be achieved by choosing an extraction temperature that is most efficient for the target analyte, since not all compounds are extracted most efficiently at the same temperature.

Conclusive Notes

When introduced to the possibility of having available several versatile separation and extraction techniques that only require easily controllable external variables to change properties that are more ideal for analyte specific analysis, and that those same techniques are environmentally friendly, then you are peering into the future of analytical chemistry. Many developments have paved the way for not only ready-to-use applications, but also easily adaptable research for limitless possibilities. Analysis has benefited largely from the properties of poly(N-isopropyl acrylamide) (PNIPAAm) due it having the fastest and most distinguished phase transition of all thermoresponsive polymers [Hideko Kanazawa, Anal. Bioanal. Chem. (2004), 378, 46-68.] .

Additionally, the thermally related benefits of gas chromatography can now be applied to classes of compounds that are restricted to liquid chromatography due to their thermolability. In place of solvent gradient elution, TRPs allow the use of temperature gradients under purely aqueous isocratic conditions [Hideko Kanazawa, J. Sep. Sci. (2007), 30, 1646-1656.] . The versatility of the system is controlled not only through changing temperature, but through the addition of modifying moieties that allow for a choice of enhanced hydrophobic interaction, or by introducing the prospect of electrostatic interaction [Eri Ayano, Hideko Kanazawa, J. Sep. Sci. (2006), 29, 738-749.] . These developments have already introduced major improvements to the fields of HIC, SEC, IEC, and AC separations as well as pseudo solid phase extractions (pseudo due to phase transitions). The growth of TRP applications is also merging with new technologies in the case of MIP and nanotechnology. There is no doubt that temperature responsive polymers will grow increasingly popular as environmental regulations become more stringent and researchers discover the simplicity of controlling fewer chromatographic conditions upon exploration of the topic.

References