Temperature-responsive polymers
Encyclopedia
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)
(PNIPAAm), 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.
) in water at elevated temperatures. This also does not apply to any polymer, for instance dissolving polyethylene oxide or polyethylene glycol
(PEG) in water requires elevated temperatures and stirring for significant time. In neutral pH
and without spiking with ionic compounds PNIPAAm undergoes a phase transition from soluble to insoluble at 32 °C. The lowering of pH and increasing ionic strength will lower the LCST and the phase transition will occur sooner. Under LCST, the hydrophilic
surfaces of polymer chains interact with water and are elongated. At and above the LCST, 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. Several groups examined the properties of PNIPAAm, but it wasn’t until the 1990s that the beneficial properties of this polymer were applied to chromatography
in a surge of research.
technique of fixing poly(isopropyl
acrylate
) (PIPA) strands to glass beads and separating a mixture of dextran
s, which was developed by Gewehr et al. 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.
(BMA), a hydrophobic comonomer. 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 steroid
s 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. Another group separated catechin
s using PNIPAAm.
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.
peptide
s 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. 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).
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
. 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. 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
. 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. 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 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.
(TEMED), imidazole
, carboxylic acid
(COOH), iminodiacetic acid
(IDA), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC). The resulting extractions found PNIPAAm-imidazole, were relatively successful for extracting all metals except for cadmium(II). The most efficient extraction was achieved by PNIPAAm-IDA by recovering greater than 95% of all tested metals. In order to analyze the extract, the polymer was dissolved in dimethylformamide
. The results were compared to traditional methods of ion-exchange chromatography. A disadvantage of the method is the use of an organic solvent, but at small volumes this is a minor issue.
Another example demonstrates the TRP assisted precipitation of acidic macromolecules for the application of extracting RNA and glycosaminoglycans from cultured cells. An elaborate procedure is used to modify PNIPAAm with poly-L-lysine
moiety, resulting in the PL-polymer (PNIPAAm-PL). In a 0.5M saline solution
, PNIPAAm 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).
More recent developments have seen the rise of molecular imprinted polymer
s. This takes advantage of natural molecular recognition between biological compounds, as well as the swelling and contracting properties of particular TRPs. One study combined molecular imprinting
techniques with the advancement in research of TRPs to design a template that can successfully extract polar dopamine
from urine
samples. Molecular imprinting mostly involves the use of N-substituted polyacrylamide
s. 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 °C. 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.
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. 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. 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" because of phase transitions). The growth of TRP applications is also merging with new technologies in the case of molecular imprinted polymer
and nanotechnology.
Polymer
A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...
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
Smart material
Smart materials or designed materials are materials that have one or more properties that can be significantly changed in a controlled fashion by external stimuli, such as stress, temperature, moisture, pH, electric or magnetic fields....
s. These physical changes can be exploited for many analytical techniques, especially for separation chemistry. After numerous investigations of poly(N-isopropylacrylamide)
Poly(N-isopropylacrylamide)
Poly is a temperature-responsive polymer that was first synthesized in the 1950s. It can be synthesized from NIPAM which is commercially available....
(PNIPAAm), 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.
History
The effects of external stimuli on particular polymers have been investigated as early as the 1960s by Heskins and Guillet. They established the lower critical solution temperature (LCST) for PNIPAAm to be 32 °C. Contrary to the behavior of most compounds in aqueous solutions, polymers that exhibit LCST behaviour become less soluble (more hydrophobicHydrophobe
In chemistry, hydrophobicity is the physical property of a molecule that is repelled from a mass of water....
) in water at elevated temperatures. This also does not apply to any polymer, for instance dissolving polyethylene oxide or polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
(PEG) in water requires elevated temperatures and stirring for significant time. In neutral pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
and without spiking with ionic compounds PNIPAAm undergoes a phase transition from soluble to insoluble at 32 °C. The lowering of pH and increasing ionic strength will lower the LCST and the phase transition will occur sooner. Under LCST, the hydrophilic
Hydrophile
A hydrophile, from the Greek "water" and φιλια "love," is a molecule or other molecular entity that is attracted to, and tends to be dissolved by water. A hydrophilic molecule or portion of a molecule is one that has a tendency to interact with or be dissolved by, water and other polar substances...
surfaces of polymer chains interact with water and are elongated. At and above the LCST, 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. Several groups examined the properties of PNIPAAm, but it wasn’t until the 1990s that the beneficial properties of this polymer were applied to chromatography
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
in a surge of research.
Gel permeation chromatography
The research that appeared to spark an onslaught of modified applications was a gel permeation chromatographyGel Permeation Chromatography
Gel permeation chromatography is a type of size exclusion chromatography , that separates analytes on the basis of size. 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...
technique of fixing poly(isopropyl
Isopropyl
In organic chemistry, isopropyl is a propyl with a group attached to the secondary carbon. If viewed as a functional group an isopropyl is an organic compound with a propyl group attached at its secondary carbon.The bond is therefore on the middle carbon....
acrylate
Acrylate
The acrylate ion is the ion of acrylic acid.Acrylates are the salts and esters of acrylic acid. They are also known as propenoates ....
) (PIPA) strands to glass beads and separating a mixture of dextran
Dextran
Dextran is a complex, branched glucan composed of chains of varying lengths...
s, which was developed by Gewehr et al. They found that between the temperatures of 25–32 °C, the elution
Elution
Elution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent ....
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
Operating temperature
An operating temperature is the temperature at which an electrical or mechanical device operates. The device will operate effectively within a specified temperature range which varies based on the device function and application context, and ranges from the minimum operating temperature to the...
. 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 methacrylateMethacrylate
Methacrylates are the salts or esters of methacrylic acid.Methacrylates contain methyl-vinyl groups, that is, two carbon atoms double bonded to each other, directly attached to the carbonyl carbon, and wherein the vinyl group is substituted with a non-terminal methyl group.Methacrylates are common...
(BMA), a hydrophobic comonomer. 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 steroid
Steroid
A steroid is a type of organic compound that contains a characteristic arrangement of four cycloalkane rings that are joined to each other. Examples of steroids include the dietary fat cholesterol, the sex hormones estradiol and testosterone, and the anti-inflammatory drug dexamethasone.The core...
s 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. Another group separated catechin
Catechin
Catechin is a natural phenol antioxidant plant secondary metabolite. The term catechins is also commonly used to refer to the related family of flavonoids and the subgroup flavan-3-ols ....
s using PNIPAAm.
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. 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. The in-situ preparation compared the use of cyclohexanolCyclohexanol
Cyclohexanol is the organic compound with the formula 5CHOH. The molecule is related to cyclohexane ring by replacement of one hydrogen atom by a hydroxyl group. This compound exists as a deliquescent colorless solid, which, when very pure, melts near room temperature...
and toluene
Toluene
Toluene, formerly known as toluol, is a clear, water-insoluble liquid with the typical smell of paint thinners. It is a mono-substituted benzene derivative, i.e., one in which a single hydrogen atom from the benzene molecule has been replaced by a univalent group, in this case CH3.It is an aromatic...
as porogens in the preparation of the modified polystyrene
Polystyrene
Polystyrene ) also known as Thermocole, abbreviated following ISO Standard PS, is an aromatic polymer made from the monomer styrene, a liquid hydrocarbon that is manufactured from petroleum by the chemical industry...
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. Common methods of separating angiotensinAngiotensin
Angiotensin, a peptide hormone, causes blood vessels to constrict, and drives blood pressure up. It is part of the renin-angiotensin system, which is a major target for drugs that lower blood pressure. Angiotensin also stimulates the release of aldosterone, another hormone, from the adrenal cortex...
peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
s had involved reverse-phased high-performance liquid chromatography
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
(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
Acrylic acid
Acrylic acid is an organic compound with the formula CH2=CHCO2H. It is the simplest unsaturated carboxylic acid, consisting of a vinyl group connected directly to a carboxylic acid terminus. This colorless liquid has a characteristic acrid or tart smell. It is miscible with water, alcohols,...
(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. 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
Salicylic acid
Salicylic acid is a monohydroxybenzoic acid, a type of phenolic acid and a beta hydroxy acid. This colorless crystalline organic acid is widely used in organic synthesis and functions as a plant hormone. It is derived from the metabolism of salicin...
: 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
Ion exchange
Ion exchange is an exchange of ions between two electrolytes or between an electrolyte solution and a complex. In most cases the term is used to denote the processes of purification, separation, and decontamination of aqueous and other ion-containing solutions with solid polymeric or mineralic 'ion...
(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 enzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
and antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
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
N-Hydroxysuccinimide
N-Hydroxysuccinimide is a compound with a molecular weight of 115.09 and a melting point of 95 °C.As it is slightly acidic, it is an irritant to skin, eyes and mucous membranes....
(NHS) ester functional end group on NIPAAm to conjugate selectively with β-D-glucosidase
Glycoside hydrolase
Glycoside hydrolases catalyze the hydrolysis of the glycosidic linkage to release smaller sugars...
. 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
Maltose
Maltose , or malt sugar, is a disaccharide formed from two units of glucose joined with an αbond, formed from a condensation reaction. The isomer "isomaltose" has two glucose molecules linked through an α bond. Maltose is the second member of an important biochemical series of glucose chains....
ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
, evaluated it with concanavalin A
Concanavalin A
Concanavalin A is a lectin originally extracted from the jack-bean, Canavalia ensiformis. It binds specifically to certain structures found in various sugars, glycoproteins, and glycolipids, mainly internal and nonreducing terminal α-D-mannosyl and α-D-glucosyl groups...
(Con A), and attempted to separate and purify α-glucosidase
Glycoside hydrolase
Glycoside hydrolases catalyze the hydrolysis of the glycosidic linkage to release smaller sugars...
, a thermolabile
Thermolabile
Thermolabile refers to a substance which is subject to destruction/decomposition or change in response to heat. This term is often used to describe biochemical substances....
compound. 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
Desorption
Desorption is a phenomenon whereby a substance is released from or through a surface. The process is the opposite of sorption . This occurs in a system being in the state of sorption equilibrium between bulk phase and an adsorbing surface...
which had higher binding affinities to Con A than maltose. These reagents were α-D-glucopyranoside
Glucoside
A glucoside is a glycoside that is derived from glucose. Glucosides are common in plants, but rare in animals. Glucose is produced when a glucoside is hydrolysed by purely chemical means, or decomposed by fermentation or enzymes....
, D-mannose
Mannose
Mannose is a sugar monomer of the aldohexose series of carbohydrates. Mannose is a C-2 epimer of glucose. It is not part of human metabolism, but is a component of microbial cell walls, and is therefore a target of the immune system and also of antibiotics....
, 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
Dextran
Dextran is a complex, branched glucan composed of chains of varying lengths...
derivative to the classic PNIPAAm to result in a poly(NIPAAm)-DD, and used this stationary phase to separate polyclonal antibodies from subcutaneous
Subcutaneous tissue
The hypodermis, also called the hypoderm, subcutaneous tissue, or superficial fascia is the lowermost layer of the integumentary system in vertebrates. Types of cells that are found in the hypodermis are fibroblasts, adipose cells, and macrophages...
rabbit serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
. From the study, the dextran derivative of choice was carboxymethyl dextran benzylamide sulfonate
Sulfonate
A sulfonate is a salt or ester of a sulfonic acid. It contains the functional group R-SO2O-.- Sulfonate salts:Anions with the general formula RSO2O− are called sulfonates. They are the conjugate bases of sulfonic acids with formula RSO2OH. As sulfonic acids tend to be strong acids, the...
/sulfate
Sulfate
In inorganic chemistry, a sulfate is a salt of sulfuric acid.-Chemical properties:...
, 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
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
buffer (adjusted to pH 2.6 with HCl
Hydrochloric acid
Hydrochloric acid is a solution of hydrogen chloride in water, that is a highly corrosive, strong mineral acid with many industrial uses. It is found naturally in gastric acid....
).
Promising results were obtained in 2003 in a study that merged the newer developments in affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
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. 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 PNIPAAm is modified two times, first with NHS, then with polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
-biotin (PEG-b) resulting in PEG-b/pNIPAAm beads. The inner surface of the microfluidic channels is composed of polyethylene terephthalate
Polyethylene terephthalate
Polyethylene terephthalate , commonly abbreviated PET, PETE, or the obsolete PETP or PET-P, is a thermoplastic polymer resin of the polyester family and is used in synthetic fibers; beverage, food and other liquid containers; thermoforming applications; and engineering resins often in combination...
, 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 PNIPAAm 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. 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 tetramethylethylenediamineTetramethylethylenediamine
Tetramethylethylenediamine is a chemical compound with the formula 2NCH2CH2N2. This species is derived from ethylenediamine by replacement of the four N-H groups with four N-methyl groups. Its odor is remarkably similar to that of fish.-As a reagent in organic and inorganic synthesis:TMEDA is...
(TEMED), imidazole
Imidazole
Imidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...
, carboxylic acid
Carboxylic acid
Carboxylic acids are organic acids characterized by the presence of at least one carboxyl group. The general formula of a carboxylic acid is R-COOH, where R is some monovalent functional group...
(COOH), iminodiacetic acid
Iminodiacetic acid
Iminodiacetic acid, HN2, often abbreviated to IDA, is an dicarboxylic acid amine. The iminodiacetate anion can act as a tridentate ligand to form a metal complex with two, fused, five membered chelate rings...
(IDA), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EDC is a water soluble carbodiimide usually obtained as the hydrochloride. It is typically employed in the 4.0-6.0 pH range. It is generally used as a carboxyl activating agent for the coupling of primary amines to yield amide bonds...
(EDC). The resulting extractions found PNIPAAm-imidazole, were relatively successful for extracting all metals except for cadmium(II). The most efficient extraction was achieved by PNIPAAm-IDA by recovering greater than 95% of all tested metals. In order to analyze the extract, the polymer was dissolved in dimethylformamide
Dimethylformamide
Dimethylformamide is an organic compound with the formula 2NCH. Commonly abbreviated as DMF , this colourless liquid is miscible with water and the majority of organic liquids. DMF is a common solvent for chemical reactions...
. The results were compared to traditional methods of ion-exchange chromatography. A disadvantage of the method is the use of an organic solvent, but at small volumes this is a minor issue.
Another example demonstrates the TRP assisted precipitation of acidic macromolecules for the application of extracting RNA and glycosaminoglycans from cultured cells. An elaborate procedure is used to modify PNIPAAm with poly-L-lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
moiety, resulting in the PL-polymer (PNIPAAm-PL). In a 0.5M saline solution
Saline (medicine)
In medicine, saline is a general term referring to a sterile solution of sodium chloride in water but is only sterile when it is to be placed intravenously, otherwise, a saline solution is a salt water solution...
, PNIPAAm 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).
Research directions
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 PNIPAAm 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. 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 named a “catch and release” technique and tested on separation of 6 μm and 20 μm polystyrene spheres. After raising the temperature above LCST, and lowering it below LCST again, the hydrogel effective trapped the smaller 6 μm particles but 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 molecular imprinted polymer
Molecular imprinted polymer
A Molecularly Imprinted Polymer , or plastic antibody is a polymer that is formed in the presence of a molecule that is extracted afterwards, thus leaving complementary cavities behind. These polymers show a certain chemical affinity for the original molecule and can be used to fabricate sensors,...
s. This takes advantage of natural molecular recognition between biological compounds, as well as the swelling and contracting properties of particular TRPs. One study combined molecular imprinting
Molecular imprinting
In chemistry, molecular imprinting is a technique to create template-shaped cavities in polymer matrices with memory of the template molecules to be used in molecular recognition . This technique is based on the system used by enzymes for substrate recognition, which is called the "lock and key"...
techniques with the advancement in research of TRPs to design a template that can successfully extract polar dopamine
Dopamine
Dopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...
from urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
samples. Molecular imprinting mostly involves the use of N-substituted polyacrylamide
Polyacrylamide
Polyacrylamide is a polymer formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N-methylenebisacrylamide. Polyacrylamide is not toxic...
s. 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 °C. 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.
Importance of thermoresponsive polymers in medicine
Thermoresponsive polymers can be used for various biomedical applications including drug delivery, tissue engineering and biofunctional molecular techniques for smarter behavior. Many developments have paved the way for ready-to-use applications using the fast and pronounced phase transition of PNIPAAm.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. 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. 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" because of phase transitions). The growth of TRP applications is also merging with new technologies in the case of molecular imprinted polymer
Molecular imprinted polymer
A Molecularly Imprinted Polymer , or plastic antibody is a polymer that is formed in the presence of a molecule that is extracted afterwards, thus leaving complementary cavities behind. These polymers show a certain chemical affinity for the original molecule and can be used to fabricate sensors,...
and nanotechnology.