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Surface-functionalized electrospun carbon nanofiber mats as an innovative type of protein adsorption/purification medium with high capacity and high throughput
15 October 2011, 21:59:11
Publication year: 2011
Source: Journal of Chromatography A, Available online 15 October 2011
Steven Schneiderman, Lifeng Zhang, Hao Fong, Todd J. Menkhaus
Due to recent advances in the production of biotherapeutics, high capacity, high throughput adsorption media for efficient and economic separation of these medically important products are in great demand. One option that has been evaluated extensively is membrane/mat adsorption. While these media allow for rapid adsorption (due to the decreased internal diffusion) and high throughput processing (due to the open porous structure), they often suffer from low capacity and poor enrichment factors. Herein, we report the fabrication, characterization, and protein adsorption evaluation of an innovative type of membrane/mat adsorption media based on electrospun carbon nanofibers. By surface-functionalization of these nanofibers with a weak acid cation-exchange ligand, the capacity was doubled for binding a model protein (i.e., lysozyme) compared to commercial products; and the capacity value was over 200 mg lysozyme per gram of adsorption media. Meanwhile, the thin nanofibers (having diameters of ∼300 nm) along with open pores among nanofibers in the mats (having sizes of ∼10-15 μm) allowed for higher operating flow rates and lower pressure drops. Furthermore, the incorporation of higher ligand density and the addition of a non-ionic surfactant (i.e., Triton X-305) into the adsorption buffer eliminated the non-specific binding of a competing protein (bovine serum albumin). In combination, this study suggested that electrospun carbon nanofiber adsorption media would provide a promising alternative to packed resin beds for bioseparations.
Source: Journal of Chromatography A, Available online 15 October 2011
Steven Schneiderman, Lifeng Zhang, Hao Fong, Todd J. Menkhaus
Due to recent advances in the production of biotherapeutics, high capacity, high throughput adsorption media for efficient and economic separation of these medically important products are in great demand. One option that has been evaluated extensively is membrane/mat adsorption. While these media allow for rapid adsorption (due to the decreased internal diffusion) and high throughput processing (due to the open porous structure), they often suffer from low capacity and poor enrichment factors. Herein, we report the fabrication, characterization, and protein adsorption evaluation of an innovative type of membrane/mat adsorption media based on electrospun carbon nanofibers. By surface-functionalization of these nanofibers with a weak acid cation-exchange ligand, the capacity was doubled for binding a model protein (i.e., lysozyme) compared to commercial products; and the capacity value was over 200 mg lysozyme per gram of adsorption media. Meanwhile, the thin nanofibers (having diameters of ∼300 nm) along with open pores among nanofibers in the mats (having sizes of ∼10-15 μm) allowed for higher operating flow rates and lower pressure drops. Furthermore, the incorporation of higher ligand density and the addition of a non-ionic surfactant (i.e., Triton X-305) into the adsorption buffer eliminated the non-specific binding of a competing protein (bovine serum albumin). In combination, this study suggested that electrospun carbon nanofiber adsorption media would provide a promising alternative to packed resin beds for bioseparations.
Highlights
► Carbon nanofibers were fabricated and functionalized for protein purifications ► Nanofiber mats with increased ligand density do compromise mechanical integrity ► Higher ligand density and surfactant additive discourage non-specific adsorption ► Nanofiber mats have ∼10-times higher capacity than microfiber counterpart ► Nanofiber mats have over 10-times higher permeability than packed resin bedCorrelations between conformational isomerism and chromatographic diastereoselectivity of bis amino amide s-triazine derivatives
14 October 2011, 21:59:54
Publication year: 2011
Source: Journal of Chromatography A, Available online 14 October 2011
Eric Loeser, Stanislaw Babiak
NMR spectroscopy was used to probe the conformational behavior of diastereomeric s-triazine derivatives containing two chiral amino amide substituents, in order to shed light onto the mechanism of chromatographic diastereoselectivity. Utilizing the amino hydrogen signals in the proton NMR spectrum, the population of the conformations caused by rotation about the bond between the amino nitrogen and aromatic carbon atoms could be observed. The population distribution between the three possible conformations was similar but not identical between the two diastereomers, with similar trends being observed for both bis alanine amide and bis valine amide derivatives. Based on a simple model in which it is assumed that adsorption to the hydrophobic RP-LC stationary phase occurs only for the conformations having both amino amide R-groups on the same side of the triazine ring plane, the different conformation populations between the two diastereomers obtained by NMR was consistent with the observed RP-LC elution order (L-L diastereomer followed by L-D). The predicted diastereoselectivity values from NMR data were compared to RP-LC diastereoselectivity values obtained using both C18 and polymeric columns, with both acetonitrile/water and DMSO/water mobile phases. Values obtained with the polymeric column were in better agreement with calculated values than those obtained with the C18 column, suggesting that the simple adsorption model used to calculate the diastereoselectivity is more relevant towards a simple hydrophobic polymeric surface rather than a more complex C18 stationary phase. This study indicates that proton NMR is a useful tool for studying the diastereoselective mechanism of these derivatives, due to the relatively slow C-N bond rotation caused by the significant spcharacter of the amino nitrogen atoms.
Source: Journal of Chromatography A, Available online 14 October 2011
Eric Loeser, Stanislaw Babiak
NMR spectroscopy was used to probe the conformational behavior of diastereomeric s-triazine derivatives containing two chiral amino amide substituents, in order to shed light onto the mechanism of chromatographic diastereoselectivity. Utilizing the amino hydrogen signals in the proton NMR spectrum, the population of the conformations caused by rotation about the bond between the amino nitrogen and aromatic carbon atoms could be observed. The population distribution between the three possible conformations was similar but not identical between the two diastereomers, with similar trends being observed for both bis alanine amide and bis valine amide derivatives. Based on a simple model in which it is assumed that adsorption to the hydrophobic RP-LC stationary phase occurs only for the conformations having both amino amide R-groups on the same side of the triazine ring plane, the different conformation populations between the two diastereomers obtained by NMR was consistent with the observed RP-LC elution order (L-L diastereomer followed by L-D). The predicted diastereoselectivity values from NMR data were compared to RP-LC diastereoselectivity values obtained using both C18 and polymeric columns, with both acetonitrile/water and DMSO/water mobile phases. Values obtained with the polymeric column were in better agreement with calculated values than those obtained with the C18 column, suggesting that the simple adsorption model used to calculate the diastereoselectivity is more relevant towards a simple hydrophobic polymeric surface rather than a more complex C18 stationary phase. This study indicates that proton NMR is a useful tool for studying the diastereoselective mechanism of these derivatives, due to the relatively slow C-N bond rotation caused by the significant spcharacter of the amino nitrogen atoms.
Highlights
► Diastereomeric bis amino amide s-triazine derivatives exhibit conformational isomerization observable by H-NMR spectroscopy. ► The relative populations of conformational isomers are not equal. ► NMR data suggests the relative populations of conformers plays an important role in the diastereoselectivity.Fluorosurfactant-prepared triangular gold nanoparticles as postcolumn chemiluminescence reagents for high-performance liquid chromatography assay of low molecular weight aminothiols in biological fluids
14 October 2011, 21:59:54
Publication year: 2011
Source: Journal of Chromatography A, Available online 14 October 2011
Qianqian Li, Fei Shang, Chao Lu, Zhixia Zheng, Jin-Ming Lin
Our recent study demonstrates the synthesized triangular gold nanoparticles (AuNPs) by trisodium citrate reduction of HAuCl4in presence of nonionic fluorosurfactant (FSN) could display stronger catalytic activity towards luminol-chemiluminescence (CL) than spherical AuNPs. Ultratrace aminothiols may cause a great decrease in CL intensity of the triangular AuNPs-luminol CL system. In this article, we utilize the as-prepared triangular AuNPs as novel postcolumn CL reagents to explore a simple high-performance liquid chromatography (HPLC)-CL method for the determination of low molecular weight aminothiols (i.e.,cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine). The as-prepared triangular AuNPs were easier to synthesize, stable at a wider pH range and high ionic strength, and highly selective and sensitive toward reduced aminothiols. The detection limits at a signal-to-noise ratio of 3 for cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine were 0.016, 0.08, 0.1, 0.04 and 0.1 pmol, respectively. Recoveries from spiked urine and plasma samples were 95.7-104.3%. The applicability of the proposed method has been validated by determining these low molecular weight aminothiols in human urine and plasma samples with satisfactory results, and thus it will have great potential application in clinical diagnosis.
Source: Journal of Chromatography A, Available online 14 October 2011
Qianqian Li, Fei Shang, Chao Lu, Zhixia Zheng, Jin-Ming Lin
Our recent study demonstrates the synthesized triangular gold nanoparticles (AuNPs) by trisodium citrate reduction of HAuCl4in presence of nonionic fluorosurfactant (FSN) could display stronger catalytic activity towards luminol-chemiluminescence (CL) than spherical AuNPs. Ultratrace aminothiols may cause a great decrease in CL intensity of the triangular AuNPs-luminol CL system. In this article, we utilize the as-prepared triangular AuNPs as novel postcolumn CL reagents to explore a simple high-performance liquid chromatography (HPLC)-CL method for the determination of low molecular weight aminothiols (i.e.,cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine). The as-prepared triangular AuNPs were easier to synthesize, stable at a wider pH range and high ionic strength, and highly selective and sensitive toward reduced aminothiols. The detection limits at a signal-to-noise ratio of 3 for cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine were 0.016, 0.08, 0.1, 0.04 and 0.1 pmol, respectively. Recoveries from spiked urine and plasma samples were 95.7-104.3%. The applicability of the proposed method has been validated by determining these low molecular weight aminothiols in human urine and plasma samples with satisfactory results, and thus it will have great potential application in clinical diagnosis.
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