Thermoresponsive polymers in chromatography

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Thermoresponsive polymers can be used as stationary phase in liquid chromatography. [1] Here, the polarity of the stationary phase can be varied by temperature changes, altering the power of separation without changing the column or solvent composition. 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, thermoresponsive polymers allow the use of temperature gradients under purely aqueous isocratic conditions. [2] 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. [3] These developments have already introduced major improvements to the fields of hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, and affinity chromatography separations as well as pseudo-solid phase extractions ("pseudo" because of phase transitions).

Contents

Hydrophobic interaction chromatography

Gel permeation chromatography

The research that appeared to spark an onslaught of modified applications was a gel permeation chromatography technique of fixing poly(isopropyl acrylate) (PIPA) strands to glass beads and separating a mixture of dextrans, which was developed by Gewehr et al. [4] They found that between the temperatures of 25–32 °C, 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 °C and after 32 °C (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 °C, 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

Okano’s group expanded on their success by using different modifiers to enhance hydrophobicity through the attachment of butyl methacrylate (BMA), a hydrophobic comonomer. [5] 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 PNIPAAm 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 using a purely aqueous phase in HPLC. [6] Another group separated catechins using PNIPAAm. [7]

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. [8] Kanazawa’s group investigated the reversible changes of PNIPAAm 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 PNIPAAm 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. They were able to shift the LCST from 32 °C to 20 °C 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 °C, LCST=20 °C in 1M NaCl). Above the LCST (25 °C, LCST=20 °C in 1M NaCl), all of the peaks are well resolved, and there is an increasing trend of retention time versus temperature up to 50 °C.

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 PNIPAAm onto macroporous polymeric materials. [9] 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 PNIPAAm 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 used 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 PNIPAAm 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. [10] 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 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 PNIPAAm, resulting in PNIPAAm-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 °C, but decreasing the temperature to 10 °C or raising it to 50 °C 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.

Ayano et al. modified PNIPAAm with cationic N,N-dimethylaminopropylacrylamide (DMAPAAm) and hydrophobic BMA and grafted it onto silica beads to form IDB. [11] They used 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 used 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 separation 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. Chen and Hoffman used N-Hydroxysuccinimide (NHS) ester functional end group on NIPAAm to conjugate selectively with β-D-glucosidase. [12] 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 separate and purify α-glucosidase, a thermolabile compound. [13] 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 °C. 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 °C (soluble) to 8 °C (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 α-glucosidase from yeast extract under low temperature conditions. In conclusion, the pAPM was found to recover 68% of α-glucosidase 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 PNIPAAm to result in a poly(NIPAAm)-DD, and used this stationary phase to separate polyclonal antibodies from subcutaneous rabbit serum. [14] 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 °C to 33 °C. To test the success of the affinity binding, the antibodies were eluted with glycine buffer (adjusted to pH 2.6 with HCl).

Promising results were obtained in 2003 in a study that 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. [15] 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 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 PNIPAAm is modified two times, first with NHS, then with polyethylene glycol-biotin (PEG-b) resulting in PEG-b/pNIPAAm beads. The inner surface of the microfluidic channels is composed of polyethylene 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.

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">Size-exclusion chromatography</span> Chromatographic method in which dissolved molecules are separated by their size & molecular weight

Size-exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc, which have been dissolved into liquid solutions.

Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates high molecular weight or colloidal analytes on the basis of size or diameter, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964. The proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.

Micellar electrokinetic chromatography (MEKC) is a chromatography technique used in analytical chemistry. It is a modification of capillary electrophoresis (CE), extending its functionality to neutral analytes, where the samples are separated by differential partitioning between micelles and a surrounding aqueous buffer solution.

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Ion chromatography</span> Separates ions and polar molecules

Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.

<span class="mw-page-title-main">Solid-phase extraction</span> Process to separate compounds by properties

Solid-phase extraction (SPE) is a solid-liquid extractive technique, by which compounds that are dissolved or suspended in a liquid mixture are separated, isolated or purified, from other compounds in this mixture, according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue.

Chiral column chromatography is a variant of column chromatography that is employed for the separation of chiral compounds, i.e. enantiomers, in mixtures such as racemates or related compounds. The chiral stationary phase (CSP) is made of a support, usually silica based, on which a chiral reagent or a macromolecule with numerous chiral centers is bonded or immobilized.

<span class="mw-page-title-main">Fast protein liquid chromatography</span>

Fast protein liquid chromatography (FPLC) is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid (the mobile phase) and a porous solid (the stationary phase). In FPLC the mobile phase is an aqueous buffer solution. The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

Reversed-phase Liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using High Performance Liquid Chromatography-HPLC in recent years are done using the Reversed Phase mode. In the Reversed Phase mode, the sample components are retained in the system, the more hydrophobic they are. 

Mixed-mode chromatography (MMC), or multimodal chromatography, refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and analytes in order to achieve their separation. What is distinct from conventional single-mode chromatography is that the secondary interactions in MMC cannot be too weak, and thus they also contribute to the retention of the solutes.

<span class="mw-page-title-main">Hydrophilic interaction chromatography</span> Type of chromatography

Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state whereby bulk liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The idea of liquid and gas properties convergence was first envisioned by Giddings.

<span class="mw-page-title-main">Elution</span> Extraction of a material by washing with a solvent

In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.

Displacement chromatography is a chromatography technique in which a sample is placed onto the head of the column and is then displaced by a solute that is more strongly sorbed than the components of the original mixture. The result is that the components are resolved into consecutive “rectangular” zones of highly concentrated pure substances rather than solvent-separated “peaks”. It is primarily a preparative technique; higher product concentration, higher purity, and increased throughput may be obtained compared to other modes of chromatography.

Poly(N-isopropylacrylamide) (variously abbreviated PNIPA, PNIPAM, PNIPAAm, NIPA, PNIPAA or PNIPAm) is a temperature-responsive polymer that was first synthesized in the 1950s. It can be synthesized from N-isopropylacrylamide which is commercially available. It is synthesized via free-radical polymerization and is readily functionalized making it useful in a variety of applications.

<span class="mw-page-title-main">Temperature-responsive polymer</span> Polymer showing drastic changes in physical properties with temperature

Temperature-responsive polymers or thermoresponsive polymers are polymers that exhibit drastic and discontinuous changes in their physical properties with temperature. The term is commonly used when the property concerned is solubility in a given solvent, but it may also be used when other properties are affected. Thermoresponsive polymers belong to the class of stimuli-responsive materials, in contrast to temperature-sensitive materials, which change their properties continuously with environmental conditions. In a stricter sense, thermoresponsive polymers display a miscibility gap in their temperature-composition diagram. Depending on whether the miscibility gap is found at high or low temperatures, either an upper critical solution temperature (UCST) or a lower critical solution temperature (LCST) exists.

A monolithic HPLC column, or monolithic column, is a column used in high-performance liquid chromatography (HPLC). The internal structure of the monolithic column is created in such a way that many channels form inside the column. The material inside the column which separates the channels can be porous and functionalized. In contrast, most HPLC configurations use particulate packed columns; in these configurations, tiny beads of an inert substance, typically a modified silica, are used inside the column. Monolithic columns can be broken down into two categories, silica-based and polymer-based monoliths. Silica-based monoliths are known for their efficiency in separating smaller molecules while, polymer-based are known for separating large protein molecules.

Chiral analysis refers to the quantification of component enantiomers of racemic drug substances or pharmaceutical compounds. Other synonyms commonly used include enantiomer analysis, enantiomeric analysis, and enantioselective analysis. Chiral analysis includes all analytical procedures focused on the characterization of the properties of chiral drugs. Chiral analysis is usually performed with chiral separation methods where the enantiomers are separated on an analytical scale and simultaneously assayed for each enantiomer.

References

  1. ↑ Irene Tan, Farnoosh Roohi, Maria-Magdalena Titirici, Thermoresponsive polymers in liquid chromatography, Analytical Methods, 2012, Volume 4, pages 34-43.
  2. ↑ Hideko Kanazawa (2007). "Thermally responsive chromatographic materials using functional polymers". J. Sep. Sci. 30 (11): 1646–1656. doi:10.1002/jssc.200700093. PMID   17623446.
  3. ↑ Eri Ayano; Hideko Kanazawa (2006). "Aqueous chromatography system using temperature-responsive polymer-modified stationary phases". J. Sep. Sci. 29 (6): 738–749. doi:10.1002/jssc.200500485. PMID   16830486.
  4. ↑ Gewehr, Markus; Nakamura, Katsunori; Ise, Norio; Kitano, Hiromi (1992). "Gel permeation chromatography using porous glass beads modified with temperature-responsive polymers". Die Makromolekulare Chemie. 193 (1): 249–256. doi:10.1002/macp.1992.021930123. ISSN   0025-116X.
  5. ↑ Hideko Kanazawa; Yuki Kashiwase; Kazuo Yamamoto; Yoshikazu Matsushima; Akihiko Kikuchi; Yasuhisa Sakurai; Teruo Okano (1997). "Temperature-responsive liquid chromatography. 2. Effects of hydrophobic groups in N-isopropylacrylamide copolymer-modified silica". Anal. Chem. 69 (5): 823–830. doi:10.1021/ac961024k. PMID   9068270.
  6. ↑ Hideko Kanazawa; Tastuo Sunamoto; Yoshikazu Matsushima; Akihiko Kikuchi; Teruo Okano (2000). "Temperature-responsive chromatographic separation of amino acid phenylthiohydantions using aqueous media as the mobile phase". Anal. Chem. 72 (24): 5961–5966. doi:10.1021/ac0004658. PMID   11140763.
  7. ↑ Chikako Sakamoto; Yuji Okada; Hideko Kanazawa; Akihiko Kikuchi; Teruo Okano (2003). "Separation of catechins by temperature-responsive chromatography". Bunseki Kagaku. 52 (10): 903–906. doi: 10.2116/bunsekikagaku.52.903 .
  8. ↑ Hideko Kanazawa; Kazuo Yamamoto; Yoshikazu Matsushima; Nobuharu Takai; Akihiko Kikuchi; Yasuhisa Sakurai; Teruo Okano (1996). "Temperature-Responsive Chromatography Using Poly(N-isopropylacrylamide)-Modified Silica". Anal. Chem. 68 (1): 100–105. doi:10.1021/ac950359j. PMID   21619225.
  9. ↑ Ken Hosoya; Etsuko Sawada; Kazuhiro Kimata; Takeo Araki; Nabuo Tanaka; Jean M.J. Fréchet (1994). "In situ Surface-Selective Modification of Uniform Size Macroporous Polymer Particles with Temperature-Responsive Poly-N-isopropylacrylamide". J. Macromol. 27 (14): 3973–3976. Bibcode:1994MaMol..27.3973H. doi:10.1021/ma00092a042.
  10. ↑ Jun Kobayashi; Akihiko Kikuchi; Kiyotaka Sakai; Teruo Okano (2003). "Cross-linked thermoresponsive anionic polymer-grafted surfaces to separate bioactive basic peptides". Anal. Chem. 75 (13): 3244–3249. doi:10.1021/ac026364m. PMID   12964775.
  11. ↑ Eri Ayano; Kyoko Nambu; Chikako Sakamoto; Hideko Kanazawa; Akihiko Kikuchi; Teruo Okano (2006). "Aqueous chromatography system using pH- and temperature-responsive stationary phase with ion-exchange groups". J. Chromatogr. A. 1119 (1–2): 58–65. doi:10.1016/j.chroma.2006.01.068. PMID   16460743.
  12. ↑ Guohua Chen; Allan S. Hoffman (1993). "Preparation and properties of thermoreversible, phase-separating enzyme-oligo(N-isopropylacrylamide) conjugates". Bioconjugate Chem. 4 (6): 509–514. doi:10.1021/bc00024a013. PMID   8305520.
  13. ↑ Kazuhiro Hoshino; Masayuki Taniguchi; Taichi Kitao; Shoichi Morohashi; Toshisuke Sasakura (1998). "Preparation of a new thermo-responsive adsorbent with maltose as a ligand and its application to affinity precipitation". Biotechnology and Bioengineering. 60 (5): 568–579. doi:10.1002/(SICI)1097-0290(19981205)60:5<568::AID-BIT7>3.0.CO;2-V. PMID   10099465. S2CID   29656259.
  14. ↑ S. Anastase-Ravion; Z. Ding; A. Pellé; A.S. Hoffman; D. Letourneur (2001). "New antibody purification procedure using a thermally responsive poly(N-isopropylacrylamide)–dextran derivative conjugate". J. Chromatogr. B. 761 (2): 247–254. doi:10.1016/S0378-4347(01)00336-X. PMID   11587355.
  15. ↑ Noah Malmstadt; Paul Yager; Allan S. Hoffman; Patrick S. Stayton (2003). "A smart microfluidic affinity chromatography matrix composed of poly(N-isopropylacrylamide)-coated beads". Anal. Chem. 75 (13): 2943–2949. doi:10.1021/ac034274r. PMID   12964737.
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