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Liang Zhao - One of the best experts on this subject based on the ideXlab platform.
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preparation and performance of a novel multi mode cof 300 sio2 Chromatographic Stationary Phase
European Polymer Journal, 2019Co-Authors: Lixiao Chen, Shuqing Dong, Jie Gao, Xiaofeng Shi, Liang ZhaoAbstract:Abstract A novel multimode COF-300@SiO2 Chromatographic Stationary Phase was successfully prepared via an in situ growth strategy. Powder X-ray diffraction, solid nuclear magnetic resonance and scanning electron microscope confirmed the successful preparation of COF-300@SiO2 Chromatographic Stationary Phase. Multimode Chromatographic performance and multiple retention mechanisms were investigated. Neutral and polar compounds were efficiently separated in reverse Phase chromatography mode due to hydrophobic interaction, π-π interaction, electron-donor-acceptor interaction and size selectivity of porous structure, and the column exhibited comparable hydrophobic selectivity with C18 column. Especially, the size selectivity of porous structure promoted the separation of multicomponent compounds on the novel column. Only neutral compounds were completely or partly separated in normal Phase chromatography mode based on π-π interaction, and the column showed better separation performance than SiO2 column. Moreover, the column also presented weaker hydrophilic interaction than NH2/SiO2 column in hydrophilic Phase chromatography mode. Nucleosides, nucleobases and alkaloids were partly or completely separated on the developed column because of the presence of amino groups on the surface of COF-300. This work indicates promising application values of COFs-based Chromatographic Stationary Phases in Chromatographic separation areas.
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Preparation and performance of a novel multi-mode COF-300@SiO2 Chromatographic Stationary Phase
European Polymer Journal, 2019Co-Authors: Lixiao Chen, Shuqing Dong, Jie Gao, Xiaofeng Shi, Liang ZhaoAbstract:Abstract A novel multimode COF-300@SiO2 Chromatographic Stationary Phase was successfully prepared via an in situ growth strategy. Powder X-ray diffraction, solid nuclear magnetic resonance and scanning electron microscope confirmed the successful preparation of COF-300@SiO2 Chromatographic Stationary Phase. Multimode Chromatographic performance and multiple retention mechanisms were investigated. Neutral and polar compounds were efficiently separated in reverse Phase chromatography mode due to hydrophobic interaction, π-π interaction, electron-donor-acceptor interaction and size selectivity of porous structure, and the column exhibited comparable hydrophobic selectivity with C18 column. Especially, the size selectivity of porous structure promoted the separation of multicomponent compounds on the novel column. Only neutral compounds were completely or partly separated in normal Phase chromatography mode based on π-π interaction, and the column showed better separation performance than SiO2 column. Moreover, the column also presented weaker hydrophilic interaction than NH2/SiO2 column in hydrophilic Phase chromatography mode. Nucleosides, nucleobases and alkaloids were partly or completely separated on the developed column because of the presence of amino groups on the surface of COF-300. This work indicates promising application values of COFs-based Chromatographic Stationary Phases in Chromatographic separation areas.
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Preparation and Characterization of SiO2/SiO2 Core–Shell Microspheres as RP-HPLC Stationary Phase
Journal of Inorganic and Organometallic Polymers and Materials, 2011Co-Authors: Liu Qin, Wang Litao, Shuqing Dong, Zh.x. Zhang, Liang ZhaoAbstract:Novel SiO2/SiO2 core–shell monodisperse silica spheres for high-performance liquid chromatography packing materials are prepared by the layer-by-layer self-assembly technique. The core–shell silica spheres consist of micrometer-sized porous silica spheres as the core and a thin mesoporous silica shell formed from multilayer nanometer-sized silica particles. In addition, a reversed-Phase packing by bonding octadecyltrichlorosilane on SiO2/SiO2 particles through alkyl-modified method is prepared and characterized. The results show that the carbon content of the new reversed-Phase Stationary Phase increases by approximately 45% compared with the uncoated octadecyl-bonded SiO2 Stationary Phase. Eight kinds of tested aromatic compounds are well-separated on the packing and the peaks are symmetrical, which demonstrates that the packing acts as an excellent reversed-Phase Chromatographic Stationary Phase.
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preparation and characterization of sio2 sio2 core shell microspheres as rp hplc Stationary Phase
Journal of Inorganic and Organometallic Polymers and Materials, 2011Co-Authors: Qin Liu, Shuqing Dong, Zh.x. Zhang, Litao Wang, Liang ZhaoAbstract:Novel SiO2/SiO2 core–shell monodisperse silica spheres for high-performance liquid chromatography packing materials are prepared by the layer-by-layer self-assembly technique. The core–shell silica spheres consist of micrometer-sized porous silica spheres as the core and a thin mesoporous silica shell formed from multilayer nanometer-sized silica particles. In addition, a reversed-Phase packing by bonding octadecyltrichlorosilane on SiO2/SiO2 particles through alkyl-modified method is prepared and characterized. The results show that the carbon content of the new reversed-Phase Stationary Phase increases by approximately 45% compared with the uncoated octadecyl-bonded SiO2 Stationary Phase. Eight kinds of tested aromatic compounds are well-separated on the packing and the peaks are symmetrical, which demonstrates that the packing acts as an excellent reversed-Phase Chromatographic Stationary Phase.
Hirotaka Ihara - One of the best experts on this subject based on the ideXlab platform.
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preparation and characterization of a novel hydrophilic interaction ion exchange mixed mode Chromatographic Stationary Phase with pyridinium based zwitterionic polymer grafted porous silica
IEEE Journal of Solid-state Circuits, 2018Co-Authors: Makoto Takafuji, Mohammad Shahruzzaman, Kazufusa Sasahara, Hirotaka IharaAbstract:Novel zwitterionic polymer-grafted porous silica microspheres were prepared by using sodium 2-bromoethanesulfonate to quaternize the pyridyl side chains of the polymer, which is anchored on the surface of porous silica. The separation behavior for toluene and benzoic acid proved that the zwitterionic polymer-grafted porous silica worked as a Stationary Phase in hydrophilic interaction/ion exchange mixed-mode chromatography. Compared with a conventional sulfobetaine-based zwitterion-grafted porous silica column, the prepared poly(vinylpyridinium ethanesulfonate)-immobilized porous silica afforded the highly selective separation of benzoic acid derivatives over a wide pH range, and the separation behavior was quite sensitive to the protonation state of the carboxylic acids. The high selectivity was attributed to the multiple interactions between the guest molecule and the zwitterionic groups on the polymer chains. The new zwitterionic Stationary Phase demonstrated excellent selectivity for hydrophilic biorelated molecules such as nucleobases and nucleosides.
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Preparation and characterization of a novel hydrophilic interaction/ion exchange mixed-mode Chromatographic Stationary Phase with pyridinium-based zwitterionic polymer-grafted porous silica.
IEEE Journal of Solid-state Circuits, 2018Co-Authors: Makoto Takafuji, Mohammad Shahruzzaman, Kazufusa Sasahara, Hirotaka IharaAbstract:Novel zwitterionic polymer-grafted porous silica microspheres were prepared by using sodium 2-bromoethanesulfonate to quaternize the pyridyl side chains of the polymer, which is anchored on the surface of porous silica. The separation behavior for toluene and benzoic acid proved that the zwitterionic polymer-grafted porous silica worked as a Stationary Phase in hydrophilic interaction/ion exchange mixed-mode chromatography. Compared with a conventional sulfobetaine-based zwitterion-grafted porous silica column, the prepared poly(vinylpyridinium ethanesulfonate)-immobilized porous silica afforded the highly selective separation of benzoic acid derivatives over a wide pH range, and the separation behavior was quite sensitive to the protonation state of the carboxylic acids. The high selectivity was attributed to the multiple interactions between the guest molecule and the zwitterionic groups on the polymer chains. The new zwitterionic Stationary Phase demonstrated excellent selectivity for hydrophilic biorelated molecules such as nucleobases and nucleosides.
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a new route for synthesis of n methylimidazolium grafted silica Stationary Phase and reevaluation in hydrophilic interaction liquid chromatography
Talanta, 2017Co-Authors: Jia Chen, Zhan Li, Makoto Takafuji, Hirotaka IharaAbstract:Abstract In this work, a “solvent-free” and green method for controllable and quantitative preparation of Chromatographic Stationary Phase has been proposed. About the method, the largely excess of functional monomer and strict control of the proportion between silane and silica are the two most important points. Due to the existence of excessive functional monomer, silane can be utilized at the maximum extent, thereby achieving quantitative and reproducible bonding. Meanwhile, the excessive monomer can also be well recycled. By this new method, we controllably synthesized three Sil-MIm columns with various bonding amounts to investigate how the bonding amounts influence separation performances. The experiment results stated in some cases the columns with appropriate bonding amounts rather than maximum, were the best to fulfil some specific tasks. Meanwhile, it also demonstrated how important it was to control the bonding amounts of Stationary Phase.
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synthesis and characterization of poly ionic liquid grafted silica hybrid materials through surface radical chain transfer polymerization and aqueous anion exchange
Materials Letters, 2010Co-Authors: Tsuyoshi Sawada, Shengxiang Jiang, Hirotaka IharaAbstract:Poly(ionic liquid)-grafted silica materials were firstly synthesized by polymerization of 1-vinyl-3-butylimidazolium bromide as a new ionic liquid monomer on mercaptopropylated silica by surface radical chain-transfer polymerization. The bromide counterion was exchanged with three other inorganic anions including tetrafluoroborate, hexafluorophosphate, and trifuoromethanesulfonate through simple aqueous anion-exchange reaction. The obtained poly(ionic liquid)-grafted silica materials were characterized by elemental analysis, infrared spectra, thermogravimetric analysis, and X-ray fluorescence. The wettabilities of the materials with different counterions were verified by static water contact angle measurement. This kind of new materials may have some potential in applied fields such as used as a catalyst, an extractant, a Chromatographic Stationary Phase, etc.
Ruin Moaddel - One of the best experts on this subject based on the ideXlab platform.
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stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter 1 determined using cellular membrane affinity chromatography
Analytical Biochemistry, 2010Co-Authors: Ruin Moaddel, Rika Yamaguchi, Sharvil Patel, F Bighi, Sarangan Ravichandran, Irving W. WainerAbstract:Abstract Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488 M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid Chromatographic Stationary Phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1 G465R ) and CMAC(hOCT1 R488M ). Columns were created using both Stationary Phases, and frontal displacement chromatography experiments were conducted using [ 3 H] MMP + (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The Chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.
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development of an immobilized gpr17 receptor Stationary Phase for binding determination using frontal affinity chromatography coupled to mass spectrometry
Analytical Biochemistry, 2009Co-Authors: Caterina Temporini, Ruin Moaddel, Stefania Ceruti, Enrica Calleri, Silvia Ferrario, Maria P Abbracchio, Gabriella MassoliniAbstract:Abstract A liquid Chromatographic Stationary Phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)–IAM and GPR17(−)–IAM Stationary Phases. Frontal chromatography experiments on both GPR17(+)–IAM and GPR17(−)–IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)–IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (Kd) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y12/P2Y13 antagonist), MRS2179 (a P2Y1 receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated Kd values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)–IAM in ranking affinity studies for the selection of new potential candidates.
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development and characterization of immobilized human organic anion transporter based liquid Chromatographic Stationary Phase hoat1 and hoat2
Journal of Chromatography B, 2007Co-Authors: T. Kimura, J. Perry, N. Anzai, J.b. Pritchard, Ruin MoaddelAbstract:This paper reports the development of liquid Chromatographic columns containing immobilized organic anion transporters (hOAT1, hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid Chromatographic Stationary Phase. The resulting Stationary Phases were characterized by frontal affinity chromatography, using the marker ligand [ 3 H]-adefovir for the hOAT1 and [ 14 C]- p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (Kd) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r2= 0.688 (p < 0.05) and r2=0.9967 (p<0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.
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Development and Characterization of Immobilized Human Organic Anion Transporter Based Liquid Chromatographic Stationary Phase: hOAT1 and hOAT2
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2007Co-Authors: T. Kimura, J. Perry, N. Anzai, J.b. Pritchard, Ruin MoaddelAbstract:This paper reports the development of liquid Chromatographic columns containing immobilized organic anion transporters (hOAT1, hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid Chromatographic Stationary Phase. The resulting Stationary Phases were characterized by frontal affinity chromatography, using the marker ligand [ 3 H]-adefovir for the hOAT1 and [ 14 C]- p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (Kd) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r2= 0.688 (p < 0.05) and r2=0.9967 (p
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Direct Chromatographic determination of dissociation rate constants of ligand-receptor complexes: assessment of the interaction of noncompetitive inhibitors with an immobilized nicotinic acetylcholine receptor-based liquid chromatography Stationary p
Analytical chemistry, 2005Co-Authors: Ruin Moaddel, Rika Yamaguchi, Krzysztof Jozwiak, Irving W. WainerAbstract:A liquid Chromatographic Stationary Phase containing immobilized membranes from a cell line expressing the alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) has been used to assess dissociation rate constants (kd) of 12 noncompetitive inhibitor-nAChR complexes. The pharmacological effects of the noncompetitive inhibitors, expressed as percent recovery of activity at 7 min and 4 h postexposure to the inhibitor, were also determined. The results demonstrate that the kd values correlated with the pharmacological effect and that this approach can be used to identify molecular structures associated with differences in kd values. The method can be adapted for use with membrane-bound receptors, ion channels, and transporters and represents a direct and facile technique for the assessment of dissociation rate constants (kd) of ligand-receptor complexes.
Shuqing Dong - One of the best experts on this subject based on the ideXlab platform.
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preparation and performance of a novel multi mode cof 300 sio2 Chromatographic Stationary Phase
European Polymer Journal, 2019Co-Authors: Lixiao Chen, Shuqing Dong, Jie Gao, Xiaofeng Shi, Liang ZhaoAbstract:Abstract A novel multimode COF-300@SiO2 Chromatographic Stationary Phase was successfully prepared via an in situ growth strategy. Powder X-ray diffraction, solid nuclear magnetic resonance and scanning electron microscope confirmed the successful preparation of COF-300@SiO2 Chromatographic Stationary Phase. Multimode Chromatographic performance and multiple retention mechanisms were investigated. Neutral and polar compounds were efficiently separated in reverse Phase chromatography mode due to hydrophobic interaction, π-π interaction, electron-donor-acceptor interaction and size selectivity of porous structure, and the column exhibited comparable hydrophobic selectivity with C18 column. Especially, the size selectivity of porous structure promoted the separation of multicomponent compounds on the novel column. Only neutral compounds were completely or partly separated in normal Phase chromatography mode based on π-π interaction, and the column showed better separation performance than SiO2 column. Moreover, the column also presented weaker hydrophilic interaction than NH2/SiO2 column in hydrophilic Phase chromatography mode. Nucleosides, nucleobases and alkaloids were partly or completely separated on the developed column because of the presence of amino groups on the surface of COF-300. This work indicates promising application values of COFs-based Chromatographic Stationary Phases in Chromatographic separation areas.
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Preparation and performance of a novel multi-mode COF-300@SiO2 Chromatographic Stationary Phase
European Polymer Journal, 2019Co-Authors: Lixiao Chen, Shuqing Dong, Jie Gao, Xiaofeng Shi, Liang ZhaoAbstract:Abstract A novel multimode COF-300@SiO2 Chromatographic Stationary Phase was successfully prepared via an in situ growth strategy. Powder X-ray diffraction, solid nuclear magnetic resonance and scanning electron microscope confirmed the successful preparation of COF-300@SiO2 Chromatographic Stationary Phase. Multimode Chromatographic performance and multiple retention mechanisms were investigated. Neutral and polar compounds were efficiently separated in reverse Phase chromatography mode due to hydrophobic interaction, π-π interaction, electron-donor-acceptor interaction and size selectivity of porous structure, and the column exhibited comparable hydrophobic selectivity with C18 column. Especially, the size selectivity of porous structure promoted the separation of multicomponent compounds on the novel column. Only neutral compounds were completely or partly separated in normal Phase chromatography mode based on π-π interaction, and the column showed better separation performance than SiO2 column. Moreover, the column also presented weaker hydrophilic interaction than NH2/SiO2 column in hydrophilic Phase chromatography mode. Nucleosides, nucleobases and alkaloids were partly or completely separated on the developed column because of the presence of amino groups on the surface of COF-300. This work indicates promising application values of COFs-based Chromatographic Stationary Phases in Chromatographic separation areas.
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Preparation and Characterization of SiO2/SiO2 Core–Shell Microspheres as RP-HPLC Stationary Phase
Journal of Inorganic and Organometallic Polymers and Materials, 2011Co-Authors: Liu Qin, Wang Litao, Shuqing Dong, Zh.x. Zhang, Liang ZhaoAbstract:Novel SiO2/SiO2 core–shell monodisperse silica spheres for high-performance liquid chromatography packing materials are prepared by the layer-by-layer self-assembly technique. The core–shell silica spheres consist of micrometer-sized porous silica spheres as the core and a thin mesoporous silica shell formed from multilayer nanometer-sized silica particles. In addition, a reversed-Phase packing by bonding octadecyltrichlorosilane on SiO2/SiO2 particles through alkyl-modified method is prepared and characterized. The results show that the carbon content of the new reversed-Phase Stationary Phase increases by approximately 45% compared with the uncoated octadecyl-bonded SiO2 Stationary Phase. Eight kinds of tested aromatic compounds are well-separated on the packing and the peaks are symmetrical, which demonstrates that the packing acts as an excellent reversed-Phase Chromatographic Stationary Phase.
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preparation and characterization of sio2 sio2 core shell microspheres as rp hplc Stationary Phase
Journal of Inorganic and Organometallic Polymers and Materials, 2011Co-Authors: Qin Liu, Shuqing Dong, Zh.x. Zhang, Litao Wang, Liang ZhaoAbstract:Novel SiO2/SiO2 core–shell monodisperse silica spheres for high-performance liquid chromatography packing materials are prepared by the layer-by-layer self-assembly technique. The core–shell silica spheres consist of micrometer-sized porous silica spheres as the core and a thin mesoporous silica shell formed from multilayer nanometer-sized silica particles. In addition, a reversed-Phase packing by bonding octadecyltrichlorosilane on SiO2/SiO2 particles through alkyl-modified method is prepared and characterized. The results show that the carbon content of the new reversed-Phase Stationary Phase increases by approximately 45% compared with the uncoated octadecyl-bonded SiO2 Stationary Phase. Eight kinds of tested aromatic compounds are well-separated on the packing and the peaks are symmetrical, which demonstrates that the packing acts as an excellent reversed-Phase Chromatographic Stationary Phase.
Irving W. Wainer - One of the best experts on this subject based on the ideXlab platform.
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stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter 1 determined using cellular membrane affinity chromatography
Analytical Biochemistry, 2010Co-Authors: Ruin Moaddel, Rika Yamaguchi, Sharvil Patel, F Bighi, Sarangan Ravichandran, Irving W. WainerAbstract:Abstract Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488 M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid Chromatographic Stationary Phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1 G465R ) and CMAC(hOCT1 R488M ). Columns were created using both Stationary Phases, and frontal displacement chromatography experiments were conducted using [ 3 H] MMP + (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The Chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.
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Direct Chromatographic determination of dissociation rate constants of ligand-receptor complexes: assessment of the interaction of noncompetitive inhibitors with an immobilized nicotinic acetylcholine receptor-based liquid chromatography Stationary p
Analytical chemistry, 2005Co-Authors: Ruin Moaddel, Rika Yamaguchi, Krzysztof Jozwiak, Irving W. WainerAbstract:A liquid Chromatographic Stationary Phase containing immobilized membranes from a cell line expressing the alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) has been used to assess dissociation rate constants (kd) of 12 noncompetitive inhibitor-nAChR complexes. The pharmacological effects of the noncompetitive inhibitors, expressed as percent recovery of activity at 7 min and 4 h postexposure to the inhibitor, were also determined. The results demonstrate that the kd values correlated with the pharmacological effect and that this approach can be used to identify molecular structures associated with differences in kd values. The method can be adapted for use with membrane-bound receptors, ion channels, and transporters and represents a direct and facile technique for the assessment of dissociation rate constants (kd) of ligand-receptor complexes.
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Development and characterization of an immobilized human organic cation transporter based liquid Chromatographic Stationary Phase.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2005Co-Authors: Ruin Moaddel, Rika Yamaguchi, Sharvil Patel, C.-p. Hsu, V. Subrahmanyam, Irving W. WainerAbstract:Abstract Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid Chromatographic Stationary Phase to form the hOCT1(+)-IAM Stationary Phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(−)-IAM Stationary Phase. Columns were created using both Stationary Phases, and frontal displacement chromatography experiments were conducted using [3H]-methyl phenyl pyridinium ([3H]-MPP+) as the marker ligand and MPP+, verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The Kd values calculated from the Chromatographic studies correlated with previously reported Ki values (r2 = 0.9987; p
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G-Protein-Coupled Receptor Chromatographic Stationary Phases. 2. Ligand-Induced Conformational Mobility in an Immobilized β2-Adrenergic Receptor
Analytical chemistry, 2004Co-Authors: Farideh Beigi, Khalid Chakir, Rui-ping Xiao, Irving W. WainerAbstract:Membranes from a HEK-293 cell line expressing the β2-adrenergic receptor (β2-AR) have been immobilized on an artificial membrane liquid Chromatographic Stationary Phase. The resulting Phase was packed into a glass column (1.8 × 0.5 (i.d.) cm) and used in on-line Chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociation constants (Kd) of CGP 12177A (552.6 nM) and (S)-propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemic mixtures of the antagonists nadolol and propranolol demonstrated that the immobilized β2-AR retained its ability to specifically bind these compounds. Similar experiments with (R)- and (S)-propranolol demonstrated that the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 marker to a great extent that the (R)-enantiomer. The addition of the agonist butoxamine to the mobile Phase increased the retention of the CGP-12177A as did the addition of the agon...
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Syntheses of immobilized G protein-coupled receptor Chromatographic Stationary Phases: characterization of immobilized mu and kappa opioid receptors.
Analytical chemistry, 2003Co-Authors: Farideh Beigi, Irving W. WainerAbstract:Opioid receptors are members of the superfamily of G protein-coupled receptors (GPCRs). Liquid Chromatographic Stationary Phases containing either the human mu or kappa opioid receptor subtypes have been developed to study the binding between the opioid receptors and their ligands. The receptors were obtained from Chinese hamster ovary cells stably transfected with human mu or kappa receptor subtypes. The receptors were isolated through partial solubilization with sodium cholate detergent, and the partially purified receptor complex was immobilized in the phospholipid analogue monolayer of an immobilized artificial membrane liquid Chromatographic Stationary Phase. The resulting Phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in the on-line Chromatographic determination of drug/ligand binding affinities to the immobilized opioid receptors. Preliminary on-line binding studies employing frontal Chromatographic techniques were conducted with the known mu antagonist (naloxone) and a kappa agonist (U69593). The calculated dissociation constants (Kd) were 110 nM for naloxone and 84 nM for U69593. The results indicate that the immobilized receptors retained their ability to specifically bind ligands and were active for 1200 column volumes applied over two weeks. Zonal Chromatographic experiments were also conducted, and competitive displacements of the marker ligands were observed. The results suggest that the immobilized opioid receptor Stationary Phases can be used to qualitatively assess the binding affinities of compounds to the immobilized receptors and represent the first example of the use of immobilized GPCRs in a Chromatographic system.