Effective Separation for New Therapeutic Modalities Utilizing Temperature-responsive Chromatography.

In recent years, drug discovery and therapeutics trends have shifted from a focus on small-molecule compounds to biopharmaceuticals, genes, cell therapy, and regenerative medicine. Therefore, new approaches and technologies must be developed to respond to these changes in medical care. To achieve this, we applied a temperature-responsive separation system to purify a variety of proteins and cells. We developed a temperature-responsive chromatography technique based on a poly(N-isopropylacrylamide) (PNIPAAm)-grafted stationary phase. This method may be applied to various types of protein and cell separation applications by optimizing the properties of the modified polymers used in this system. Therefore, the developed temperature-responsive HPLC columns and temperature-responsive solid-phase extraction (TR-SPE) columns can be an effective separation tool for new therapeutic modalities such as monoclonal antibodies, nucleic acid drugs, and cells.

Effect of Polymer Phase Transition Behavior on Temperature-Responsive Polymer-Modified Liposomes for siRNA Transfection.

Small interfering RNAs (siRNAs) have been attracting significant attention owing to their gene silencing properties, which can be utilized to treat intractable diseases. In this study, two temperature-responsive liposomal siRNA carriers were prepared by modifying liposomes with different polymers-poly(N-isopropylacrylamide-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAAm-co-DMAPAAm)) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) P(NIPAAm-co-DMAAm). The phase transition of P(NIPAAm-co-DMAPAAm) was sharper than that of P(NIPAAm-co-DMAAm), which is attributed to the lower co-monomer content. The temperature dependent fixed aqueous layer thickness (FALT) of the prepared liposomes indicated that modifying liposomes with P(NIPAAm-co-DMAPAAm) led to a significant change in the thickness of the fixed aqueous monolayer between 37 °C and 42 °C; while P(NIPAAm-co-DMAAm) modification led to FALT changes over a broader temperature range. The temperature-responsive liposomes exhibited cellular uptake at 42 °C, but were not taken up by cells at 37 °C. This is likely because the thermoresponsive hydrophilic/hydrophobic changes at the liposome surface induced temperature-responsive cellular uptake. Additionally, siRNA transfection of cells for the prevention of luciferase and vascular endothelial growth factor (VEGF) expression was modulated by external temperature changes. P(NIPAAm-co-DMAPAAm) modified liposomes in particular exhibited effective siRNA transfection properties with low cytotoxicity compared with P(NIPAAm-co-DMAAm) modified analogues. These results indicated that the prepared temperature-responsive liposomes could be used as effective siRNA carriers whose transfection properties can be modulated by temperature.

Liposomes with temperature-responsive reversible surface properties.

Liposomes composed of egg phosphatidylcholine and cholesterol were modified with the temperature-responsive polymer poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)), and exhibited reversible surface properties with temperature. Completely reversible liposome aggregation due to P(NIPAAm-co-DMAAm) hydration/dehydration was demonstrated over four successive cycles of heating and cooling. The P(NIPAAm-co-DMAAm) polymer was hydrated during cooling, which dispersed the liposomes. The rigidity of the liposomal membrane was one of the factors in the reversible aggregation, as was the modification density of the polymer on the liposomes. A low density on relatively rigid liposomes could maintain the polymer property of reversible hydrated layers below critical solution temperature (LCST) boundary. Above the LCST, temperature-responsive polymers could also transport negatively charged liposomes into cells. The reversible behavior of the temperature-responsive polymer-modified liposomes has not been reported previously and could enable new applications for switching deposit forms as alternative drug carriers.

Enhanced cellular uptake and gene silencing activity of siRNA using temperature-responsive polymer-modified liposome.

Short interfering RNA (siRNA) delivery systems using nanoparticle carriers have been limited by inefficient intracellular delivery. One drawback is the poor cellular uptake of siRNA/particle complexes through the plasma membrane and release of the nucleic acids into the cytosol. In this study, to develop the temperature-responsive liposome as a novel carrier for siRNA delivery, we prepared lipoplexes and assessed cellular uptake of siRNA and gene silencing activity of target genes, compared with those of a commercial transfection reagent, Lipofectamine RNAiMAX, and non-modified or PEGylated liposomes. The temperature-responsive polymer, N-isopropylacrylamide-co-N,N'-dimethylaminopropylacrylamide [P(NIPAAm-co-DMAPAAm)]-modified liposome induced faster intracellular delivery because P(NIPAAm-co-DMAPAAm) exhibits a lower critical solution temperature (LCST) changing its nature from hydrophilic to hydrophobic above the LCST. The temperature-responsive liposomes showed significantly higher gene silencing activity than other carriers with less cytotoxicity. Furthermore, we showed that the temperature-responsive lipoplexes were internalized mainly via microtubule-dependent transport and also by the clathrin-mediated endocytosis pathway. This is the first report that temperature-responsive polymer-modified liposomes thermally enhanced silencing activity of siRNA. The dehydrated polymer on the liposomes, and its aggregation caused around the LCST, can probably be attributed to effective cellular uptake of the lipoplexes for gene silencing activity by interaction with the cell membrane.

Tunable Surface Properties of Temperature-Responsive Polymer-Modified Liposomes Induce Faster Cellular Uptake.

Drug delivery by nanoparticle carriers has been limited by inefficient intracellular drug delivery. Temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)-modified liposomes can release their content following heating. In this study, we synthesized the temperature-responsive polymer poly(N-isopropylacrylamide)-co-N,N'-dimethylaminopropylacrylamide (P(NIPAAm-co-DMAPAAm)) and investigated the properties of liposomes modified with P(NIPAAm-co-DMAPAAm) for intracellular drug carriers. The copolymer displayed a thermosensitive transition at a lower critical solution temperature (LCST) that is higher than body temperature. Above the LCST, the temperature-responsive liposomes started to aggregate and release. The liposomes showed a fixed aqueous layer thickness (FALT) at the surface below the LCST, and the FALT decreased with increasing temperature. Above 37 °C, cytosolic release from the temperature-responsive liposomes was higher than that from the PEGylated liposomes, indicating intracellular uptake. Here, we showed that the tunable surface properties of the temperature-responsive polymer-modified liposomes possibly enabled their dehydration by heating, which likely induced a faster cellular uptake and release. Therefore, the liposomes could be highly applicable for improving intracellular drug-delivery carriers.

Temperature-responsive molecular recognition chromatography using phenylalanine and tryptophan derived polymer modified silica beads.

Temperature-responsive polymers incorporating molecular-recognition sites were developed as stationary phases for high-performance liquid chromatography (HPLC). The grafted stationary phases consisted of functional copolymers composed of N-isopropylacrylamide (NIPAAm) and N-acryloyl aromatic amino acid methyl esters, i.e., phenylalanine and tryptophan methyl esters (Phe-OMe and Trp-OMe). Three novel temperature-responsive polymers, P(NIPAAm-co-Phe-OMe5), P(NIPAAm-co-Phe-OMe10), and P(NIPAAm-co-Trp-OMe5), were synthesized. These copolymers exhibited a reversible hydrophilic/hydrophobic phase transition at their lower critical solution temperatures (LCSTs). The polymers were grafted onto aminopropyl silica using an activated ester-amine coupling method, and were packed into a stainless steel column, which was connected to an HPLC system. Temperature-responsive chromatography was conducted using water as the sole mobile phase. More hydrophobic analytes were retained longer, and the retention times of aromatic steroids and aromatic amino acids were dramatically increased. This indicated that π-π interactions occurred between the phenyl or indole moieties of phenylalanine or tryptophan, respectively, and the aromatic compounds. Furthermore, the retention times of compounds with hydrogen bond acceptors were higher with P(NIPAAm-co-Trp-OMe5), which contained indole as a hydrogen bond donor, than with P(NIPAAm-co-Phe-OMe5). This indicated that hydrogen bonding occurred between the stationary phase and the analytes. These results indicate that hydrophobic, π-π, and hydrogen bonding interactions all affected the separation mode of the temperature-responsive chromatography, and led to selective separation with molecular recognition. Both temperature-response and molecular recognition characteristics are present in the proposed separation system that utilizes a temperature-responsive polymer bearing aromatic amino acid derivatives.

Design of Environmentally Responsive Fluorescent Polymer Probes for Cellular Imaging.

We report the development of environmentally responsive fluorescent polymers. The reversible temperature-induced phase transition of copolymers composed of N-isopropylacrylamide and a fluorescent monomer based on the fluorescein (FL), coumarin (CO), rhodamine (RH), or dansyl (DA) skeleton was used as a molecular switch to control the fluorescence intensity. The poly(N-isopropylacrylamide) (PNIPAAm) chain showed an expanded coil conformation below the lower critical solution temperature (LCST) due to hydration, but it changed to a globular form above the LCST due to dehydration. Through the combination of a polarity-sensitive fluorophore with PNIPAAm, the synthetic fluorescent polymer displayed a response to external temperature, with the fluorescence strength dramatically changing close to the LCST. Additionally, the P(NIPAAm-co-FL) and P(NIPAAm-co-CO) polymers, containing fluorescein and coumarin groups, respectively, exhibited pH responsiveness. The environmental responsiveness of the reported polymers is derived directly from the PNIPAAm and fluorophore structures, thus allowing for the cellular uptake of the fluorescence copolymer by RAW264.7 cells to be temperature-controlled. Cellular uptake was suppressed below the LCST but enhanced above the LCST. Furthermore, the cellular uptake of both P(NIPAAm-co-CO) and P(NIPAAm-co-RH) conjugated with a fusogenic lipid, namely, l-α-phosphatidylethanolamine, dioleoyl (DOPE), was enhanced. Such lipid-conjugated fluorescence probes are expected to be useful as physiological indicators for intracellular imaging.

Temperature-responsive smart packing materials utilizing multi-functional polymers.

Polymers that respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties, are often called "smart" polymers. Poly(N-isopropylacrylamide), PNIPAAm, is one of the most representative smart polymer that exhibits a thermally reversible soluble-insoluble change in the vicinity of its lower critical solution temperature (LCST) at 32°C in aqueous solution. Temperature-responsive chromatography for the separation of biomolecules utilizing the poly(N-isopropylacrylamide) (PNIPAAm)-modified stationary phase is performed with an aqueous mobile phase without using an organic solvent. The surface properties and function of the stationary phase are controlled by external temperature changes without changing the mobile-phase composition. The separation of the biomolecules, such as nucleotides, was achieved by a dual temperature- and pH-responsive chromatography system. The electrostatic and hydrophobic interactions could be modulated simultaneously with the temperature in an aqueous mobile phase. Additionally, we also prepared functional copolymers composed of N-isopropylacrylamide (NIPAAm) and amino acid derivative or naphthyl alanine derivative, which have temperature-responsiveness and molecular recognition. These separation systems would have potential applications in the separation of biomolecules.

Temperature-Responsive Fluorescence Polymer Probes with Accurate Thermally Controlled Cellular Uptakes.

Poly(N-isopropylacrylamide) (PNIPAAm)-based temperature-responsive fluorescence polymer probes were developed using radical polymerization, with 3-mercaptopropionic acid as the chain-transfer agent, followed by activation of terminal carboxyl groups with N-hydroxysuccinimide and reaction with 5-aminofluorescein (FL). The lower critical solution temperatures (LCSTs) of the resulting fluorescent polymer probes differed depending on the copolymer composition, and had a sharp phase-transition (hydrophilic/hydrophobic) boundary at the LCST. The cellular uptakes of the fluorescent polymer probes were effectively suppressed below the LCST, and increased greatly above the LCST. In particular, the cellular uptake of a copolymer with N,N-dimethylaminopropylacrylamide, P(NIPAAm-co-DMAPAAm2%)-FL (LCST: 37.4 °C), can be controlled within only 1 °C near body temperature, which is suitable for biological applications. These results indicated that the cellular uptakes of thermoresponsive polymers could be accurately controlled by the temperature, and such polymers have potential applications in discriminating between normal and pathological cells, and in intracellular drug delivery systems with local hyperthermia.

Poly (N-isopropylacrylamide)-PLA and PLA blend nanoparticles for temperature-controllable drug release and intracellular uptake.

We designed a temperature-responsive and biodegradable novel drug-delivery carrier. A block copolymer, poly (N-isopropylacrylamide-dl-lactide) (PNIPAAm-PLA), was synthesized by the ring-opening polymerization of dl-lactide, and used as a carrier for a drug-delivery system. In this study, temperature-responsive nanoparticles (NPs) encapsulating betamethasone disodium 21-phosphate (BP) were prepared from a blend of PLA homopolymer and block copolymers by an oil-in-water solvent-diffusion method in the presence of zinc ion (PLA/PNIPAAm-PLA (NPs)). The resulting NP size was around 140 nm. The drug release from temperature-responsive NP could be controllable by changing the temperature. Moreover, a murine macrophage-like cell line, RAW 264.7 cells, was used to measure and image the cell uptake of fluorescent PLA/PNIPAAm-PLA NPs at 30 °C and 37 °C on the boundary of LCST (34 °C). Below the LCST, cellular uptake was not observed, but contrary to cellular uptake it was clearly observed above the LCST. Moreover, we found this effect to be useful for controlling the stealthiness by changing the temperature. Present temperature-responsive NPs have successfully exhibited thermo-responsive drug release and intracellular uptake while possessing a biodegradable character.

Separation of phosphorylated peptides utilizing dual pH- and temperature-responsive chromatography.

The phosphorylation of a peptide is considered to be one of the most important post-translational modification reactions that can alter protein function in mammalian cells. To separate and purify, we developed a dual temperature- and pH-responsive chromatography based on terpolymer composed of N-isopropylacrylamide, N,N'-dimethylaminopropylacrylamide and butylmethacrylate. The property of the surface of the terpolymer-grafted stationary phase altered from hydrophilic to hydrophobic, and from changed to non-charged by changes in the temperature and the pH, respectively. In addition, it was possible to appear and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena resulted from changes in the charge and the hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface by controlling the temperature. In the developed environmental-responsive chromatographic system, the ionizable dimethylamino group of N,N'-dimethylaminopropylacrylamide in terpolymer played a key role for the separation. We applied the developed chromatographic system to the separation of phosphorylated compounds, such as phospho-tyrosine, phosphopeptide and oligonucleotides. At a low column temperature, the electrostatic interaction plays a predominant role for retain anionic phosphorylated compounds, because of the strong interaction between the cationic dimethylamino group in the stationary phase and the anionic phosphoric group in the analyte. On the contrary, the hydrophobic interaction became predominant upon increasing the temperature. The results showed that both the electrostatic and the hydrophobic interactions became controllable with a temperature change during the chromatographic process. Dual pH- and temperature-responsive chromatography would be very useful for biomacromolecules separation and purification.

Polymeric nanoparticles encapsulating betamethasone phosphate with different release profiles and stealthiness.

The purpose of this study was to engineer nanoparticles with various sustained profiles of drug release and prolonged circulation by blending poly(D,L-lactic acid)/poly(D,L-lactic/glycolic acid) (PLA/PLGA) homopolymers and poly(ethylene glycol) (PEG)-block-PLA/PLGA copolymers encapsulating betamethasone disodium 21-phosphate (BP). Nanoparticles of different sizes, drug encapsulation/release profiles, and cellular uptake levels were obtained by mixing homopolymers and block copolymers with different compositions/molecular weights at various blend ratios by an oil-in-water solvent diffusion method. The in vitro release of BP increased with nanoparticles of smaller size or of PLGA homopolymers instead of PLA homopolymers. Furthermore, the uptake of nanoparticles by macrophage-like cells decreased with nanoparticles of higher PEG content, and nanoparticles of PEG-PLGA block copolymers were taken up earlier than those of PEG-PLA block copolymers after incubation with serum. In addition, prolonged blood circulation was observed with nanoparticles of smaller size with higher PEG content, and nanoparticles of PEG-PLA block copolymers remained longer in circulation than those of PEG-PLGA block copolymers. Analysis of BP concentration in organs revealed reduced liver distribution of blended nanoparticles compared with PLA nanoparticles. This is the first study to systematically design and characterize biodegradable PLA/PLGA and PEG-PLA/PLGA-blended nanoparticles encapsulating BP with different release profiles and stealthiness.

Analysis of melatonin using a pH- and temperature-responsive aqueous chromatography system.

A new method for the qualitative and quantitative analysis of an intracerebral hormone, such as melatonin, has been proposed, utilizing newly designed copolymers that include ion-exchange groups. These copolymers responded to both the temperature and the pH, and the copolymers were modified with cross-linked hydrogel applied onto aminopropyl silica beads. The products were evaluated as HPLC packing materials for a pH- and temperature-responsive chromatography. The property of the surface of the stationary phase was altered from hydrophilic to hydrophobic, and from charged to non-charged by changes in both the temperature and the pH. In the chromatographic system, we investigated how to change the retention of melatonin by varying the temperature. A pH- and temperature-responsive chromatography is expected to be useful for the separation of pharmaceuticals and biomolecules.

Aqueous chromatography system using temperature-responsive polymer-modified stationary phases.

Extensive research has been carried out on functional polymers which are currently playing important roles in various fields such as medicine and engineering. Such functional polymers which respond to various kinds of stimuli are termed 'intelligent materials'. Poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer, was utilized as a chromatography column matrix modifier for a novel chromatographic approach in which only aqueous media are used as a mobile phase. The ability of the developed temperature-responsive chromatography system to separate solutes without using an organic solvent is advantageous from the point of view of maintaining the structure and activity of bioactive compounds. Recently, we designed and synthesized a new pH- and temperature-responsive copolymer as a representative of such environment-responsive polymers and grafted it onto aminopropyl silica beads. The products were evaluated as HPLC packing materials for separation systems based on a new concept, according to which the properties of the stationary phase surface are altered by external stimuli such as pH and temperature. This chromatography system utilizing the PNIPAAm copolymer is very useful for the separation of bioactive substances, such as proteins and peptides, because separation in the aqueous mobile phase is controlled solely by changing the temperature. This analytical system reduces organic waste because no organic solvent is used to separate the solutes and can therefore be classified as environmentally friendly. Future medical and pharmaceutical applications are expected.

Separation of nucleotides with an aqueous mobile phase using pH- and temperature-responsive polymer modified packing materials.

A new method for the qualitative analysis of adenosine nucleotides (AMP, ADP, and ATP) and synthetic oligonucleotides has been proposed, utilizing a pH- and temperature-responsive polymer of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm) as the stationary phase of HPLC. In the chromatographic system using the copolymer with ionizable groups of modified packing materials, we investigated how to separate adenosine nucleotides and oligonucleotides by temperature. The properties of the surface of the copolymer-grafted stationary phase altered from hydrophilic to hydrophobic and from charged to non-charged due to changes in the temperature and in the pH, respectively. In addition, it is possible to exhibit and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena result from changes in the charge and hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface with the controlling temperature. A pH- and temperature-responsive chromatography would be greatly useful for biopolymer and nucleotide separation and purification.

Aqueous chromatography system using pH- and temperature-responsive stationary phase with ion-exchange groups.

We report on the development of a novel analytical HPLC technique of nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, ketoprofen and naproxen, with an isocratic aqueous mobile phase. In this study, we designed a new pH- and temperature-responsive copolymer of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm). The copolymer was modified with cross-linked poly(NIPAAm-co-BMA-co-DMAPAAm) (IBD) hydrogel on to aminopropyl silica beads, and the products were evaluated as HPLC packing materials for an ion-exchange- and temperature-responsive chromatography. The property of the surface of the stationary phase was altered from hydrophilic to hydrophobic, and from charged to non-charged by changes in the temperature and pH. In addition, it is possible that ion-exchange groups can appear or be hidden on the polymer chain surface by temperature changes. The interactions of NSAIDs with this stationary phase were controlled by the temperature and the pH with a constant aqueous mobile phase. PH- and temperature-responsive chromatography is expected to be useful for the separation of pharmaceuticals and biomolecules.

Study of temperature-responsibility on the surfaces of a thermo-responsive polymer modified stationary phase.

We investigated a thermo-sensitive polymer, poly(N-isopropylacrylamide) (PNIPAAm), which is the basis of an HPLC stationary phase. We prepared a PNIPAAm terminally-modified surface. In this study, we investigated the effect of PNIPAAm on the surface of a stationary phase on separation based on changes of the retention time with the temperature step gradient. As the temperature changed the surface property of the stationary phase switched from hydrophilic to hydrophobic. The retention on the polymer-modified stationary phase remarkably changed upon changing the temperature. Using a column packed with PNIPAAm-modified silica, the separation of steroids was carried out by changing the temperature. With increasing temperature, an increased interaction between solutes and PNIPAAm-grafted surfaces of the stationary phases was observed. A temperature-dependent resolution of steroids was achieved using only water as a mobile phase. The PNIPAAm-modified surface of the stationary phase exhibited temperature-controlled hydrophilic-hydrophobic changes. The drastic and reversible surface hydrophilic-hydrophobic property alteration for PNIPAAm terminally-grafted surfaces should be due to rapid changes in the polymer hydration state around the polymer's transition temperature. A solvent gradient elution-like effect could be achieved with a single mobile phase by programmed temperature changes during chromatographic runs. This system should be highly useful to control the function and property of the stationary phase for HPLC only by changing the temperature with an aqueous solvent.

Temperature-responsive stationary phase utilizing a polymer of proline derivative for hydrophobic interaction chromatography using an aqueous mobile phase.

A new method of chromatography is proposed, utilizing a thermo-responsive polymer carrying an amino acid ester residue for the stationary phase of high-performance liquid chromatography (HPLC). We have been investigating the new concept of chromatography, a temperature-responsive chromatography, using temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)-modified surface for HPLC with a constant aqueous media as the mobile phase. In this study, we designed and synthesized thermo-responsive poly(acryloyl-L-proline methyl ester) and its copolymer with N-isopropylacrylamide (NIPAAm). Homopolymers of acryloyl-L-proline methyl ester and copolymer were prepared by the reaction of radical telomerization. These polymers underwent a reversible phase transition from water-soluble forms into aggregates by changing the temperature, similar to PNIPAAm. The surface properties and functions of stationary phases modified with poly(acryloyl-L-proline methyl ester) were controlled by the external temperature. In the chromatographic system, we separated steroids and amino acids with a variety of hydrophobicities using a sole aqueous mobile phase. In contrast to a PNIPAAm-modified surface, a poly(acryloyl-L-proline methyl ester)-modified surface showed a greater affinity for hydrophobic amino acids.

Analysis of herbicides in water using temperature-responsive chromatography and an aqueous mobile phase.

A simple and rapid method has been developed for herbicides in water using temperature-responsive liquid chromatography (LC) and a column packed with poly(N-isopropylacrylamide) (PNIPAAm), a polymer anchored on the stationary-phase surface of modified silica. PNIPAAm reversibly changes its hydrophilic/hydrophobic properties in water in response to temperature. The method was used to determine five sulfonylurea and three urea herbicides. Separation was achieved with a 10 mM ammonium acetate (pH 3.0) isocratic aqueous mobile phase, and by changing the column temperature. The analytes were extracted from water by off-line solid-phase extraction (SPE) with an N-vinyl-pyrrolidone polymer cartridge. The average recoveries of the eight herbicides from spiked pure water, tap water and river water were 70-130% with relative standard deviations (RSDs) of <10%. The limits of quantitation (LOQ) of the eight herbicides were between 1 and 4 microg l(-1).

Temperature- and pH-responsive aminopropyl-silica ion-exchange columns grafted with copolymers of N-isopropylacrylamide.

We have designed copolymers of N-isopropylacrylamide, environmentally-responsive polymers, which respond to temperature and other external stimuli. In this study, we designed and synthesized copolymers that introduced ion-exchange groups. These copolymers responded to the temperature and the pH, and the copolymer-grafted aminopropyl silica beads were used as HPLC packing materials. This stationary phase altered the properties from hydrophilic to hydrophobic and from charge to non-charge by temperature and pH changes. We studied the separations of organic acids and phenylthiohydantoin-amino acids using environmentally-responsive chromatography, and confirmed the effects of the ion-exchange groups. The elution behaviors of these samples were controlled by the temperature changes without organic solvents in the mobile phase. It was confirmed that the interactions between the solute and stationary phase could be freely controlled by the temperature and the pH. Environmentally-responsive chromatography is expected to be applicable to the separation of pharmaceuticals and biomolecules, such as peptides, proteins and nucleic acids.