A counteracting chromatographic electrophoresis(CACE)separation device has been success-fully built and tested.Experiments are performed with protein mixtures of BSA,Hg,Mg and Cyto-cto study the separation performance...A counteracting chromatographic electrophoresis(CACE)separation device has been success-fully built and tested.Experiments are performed with protein mixtures of BSA,Hg,Mg and Cyto-cto study the separation performances,concentration profiles,start-up methodology as well as influ-ences of pH and ionic strength on the separation process.Mathematical models based on the fluiddynamics of the porous medium are proposed.Experiments indicate that the CACE process has agood commercial potential in protein engineering and fine chemicals purification.展开更多
Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affini...Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 μmol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE mass spectrometry (CE MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE MS.展开更多
This work presents an approach to build a high-performance, low-viscous and replaceable separation matrix, semi-crosslinked polyacrylamide （semi-CPA） capillary gel electrophoresis. Non- denatured basic proteins, suc...This work presents an approach to build a high-performance, low-viscous and replaceable separation matrix, semi-crosslinked polyacrylamide （semi-CPA） capillary gel electrophoresis. Non- denatured basic proteins, such as lysozyme, cytochrome C, ribonuclease A and trypsin were separa- ted. The impacts of monomer and cross-linker concentrations on protein separation were studied, and the ability of dynamic capillary inner wall coating was demonstrated. The UV absorption interfer- ence by semi-CPA gel matrix was successfully overcome by a partial filling technique, which results in sensitivity 20 times higher than other protein separation method. The excellent separation ability, reproducibility and dynamic coating ability made semi-CPA an ideal separation media in both capillar- y electrophoresis and microfluidic chip separation scheme.展开更多
8-Gluconolactone was covalently coupled to aminopropyl derivatized capillary, which created hydrophilic brushes on the inner wall of the capillary. The coated capillary was shown to generate a stable electroosmotic fl...8-Gluconolactone was covalently coupled to aminopropyl derivatized capillary, which created hydrophilic brushes on the inner wall of the capillary. The coated capillary was shown to generate a stable electroosmotic flow （EOF） in the investigated pH range of 2.0-9.0 and to suppress effectively the adsorption of proteins. And it enabled separation of some biopolymer mixtures including basic proteins, DNA and tryptic digested bovine serum albumin （BSA） within 15 rain with efficiencies up to 450,000 plates/m. The intra- and inter-day reproducibility of the coating referring to the retention times of proteins were satisfactory with mean relative standard deviations （R.S.D.） of 0.8 and 1.7%, respectively. 2009 Yin Mao Wei. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All fights reserved.展开更多
This paper demonstrated simultaneous separation of acidic and basic proteins using cationic gemini surfactants as buffer additives in capillary electrophoresis. We showed that even at a low concentration (0.1 mmol...This paper demonstrated simultaneous separation of acidic and basic proteins using cationic gemini surfactants as buffer additives in capillary electrophoresis. We showed that even at a low concentration (0.1 mmol·L-1) of alkanediyl-α,ω-bis(dimethyloctadecylammonium bromide) (18-s-18), the wall adsorption of both acidic and basic proteins could be effectively suppressed under acidic conditions. Smaller micelle size (e.g., s=5-8) is more effective for the separation of acidic proteins than larger micelle size (e.g., s<4 or >10). Varying the spacer length of gemini surfactants can influence the electrophoretic mobility and selectivity of proteins to achieve the desired separation. Under the optimized conditions, RSDs of the migration time were less than 0.8% and 2.2% for run-to-run and day-to-day assays, respectively, and protein recoveries ranged from 79% to 100.4%. Furthermore, we also investigated the use of gemini surfactant-capped gold nanoparticles (gemini@AuNPs) as buffer additives in protein separation. Introduction of AuNPs into the buffer shortened the analysis time and slightly improved the separation efficiencies. Finally, we presented the applications of this method in the analysis of bio-logical samples, including plasma, red blood cells and egg white.展开更多
Fused-silica capillaries used in capillary zone electrophoresis were statically coated with γ- glycidoxypropyltrimethoxysilane and epoxy polymer in order to suppress wall adsorption in the separation of proteins. It ...Fused-silica capillaries used in capillary zone electrophoresis were statically coated with γ- glycidoxypropyltrimethoxysilane and epoxy polymer in order to suppress wall adsorption in the separation of proteins. It has been shown that a significant decrease in adsorption was obtained and eletroosmotic flow was the diminished in the pH range 3-5. However with higher pH values, appreciable peak deformation and decreases in the resolving power were observed. Under pH 5, the epoxy polymer coating was shown to be quite stable and exhibited reproducible separations from run-to-run and day-to-day over a period of time.展开更多
Separation of basic proteins was performed using a homemade field-modulated capillary electrophoresis system. The resolution. elution and even wall adsorption can be regulated by ad-lusting the radial rather than axia...Separation of basic proteins was performed using a homemade field-modulated capillary electrophoresis system. The resolution. elution and even wall adsorption can be regulated by ad-lusting the radial rather than axial voltage applied. Selection of running buffer and pH was found to be critical.展开更多
This paper describes non-gel capillary sieving electrophoresis employing semi-crosslinked polyacrylamide as a high performance and low viscous replaceable separation matrix for separation of non-denatured protein sepa...This paper describes non-gel capillary sieving electrophoresis employing semi-crosslinked polyacrylamide as a high performance and low viscous replaceable separation matrix for separation of non-denatured protein separation. Arising from the fine sieving and dynamic coating ability of this polymer, a mixture of basic proteins lysozyme, cytochrome C, ribonuclease A, and trypsin was resolved with excellent reproducibility. Mixing different semi-crosslinked polyacrylamides together further improves the separation. The separtion mechanism was analyzed. With network structure developed to an intermediate state between crosslinked gel and linear polymer solutions, these semi-crosslinked polyacrylamide polymers demonstrate a promise as a new class of size sieving separation medium, not only in capillary electrophoresis, but also in microfluidic chip separation schemes.展开更多
基金Supported by National Natural Science Foundation of China
文摘A counteracting chromatographic electrophoresis(CACE)separation device has been success-fully built and tested.Experiments are performed with protein mixtures of BSA,Hg,Mg and Cyto-cto study the separation performances,concentration profiles,start-up methodology as well as influ-ences of pH and ionic strength on the separation process.Mathematical models based on the fluiddynamics of the porous medium are proposed.Experiments indicate that the CACE process has agood commercial potential in protein engineering and fine chemicals purification.
文摘Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 μmol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE mass spectrometry (CE MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE MS.
基金Supported by the Key Project in the National Science ＆ Tech- nology Pillar Program During the Eleventh Five-Year Plan Pe- riod （2009BAK59B02）
文摘This work presents an approach to build a high-performance, low-viscous and replaceable separation matrix, semi-crosslinked polyacrylamide （semi-CPA） capillary gel electrophoresis. Non- denatured basic proteins, such as lysozyme, cytochrome C, ribonuclease A and trypsin were separa- ted. The impacts of monomer and cross-linker concentrations on protein separation were studied, and the ability of dynamic capillary inner wall coating was demonstrated. The UV absorption interfer- ence by semi-CPA gel matrix was successfully overcome by a partial filling technique, which results in sensitivity 20 times higher than other protein separation method. The excellent separation ability, reproducibility and dynamic coating ability made semi-CPA an ideal separation media in both capillar- y electrophoresis and microfluidic chip separation scheme.
基金National Natural Science Foundation of China(Nos.20875075 and 20875074)
文摘8-Gluconolactone was covalently coupled to aminopropyl derivatized capillary, which created hydrophilic brushes on the inner wall of the capillary. The coated capillary was shown to generate a stable electroosmotic flow （EOF） in the investigated pH range of 2.0-9.0 and to suppress effectively the adsorption of proteins. And it enabled separation of some biopolymer mixtures including basic proteins, DNA and tryptic digested bovine serum albumin （BSA） within 15 rain with efficiencies up to 450,000 plates/m. The intra- and inter-day reproducibility of the coating referring to the retention times of proteins were satisfactory with mean relative standard deviations （R.S.D.） of 0.8 and 1.7%, respectively. 2009 Yin Mao Wei. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All fights reserved.
基金Supported by the National Natural Science Foundation of China (No. 20575019)the National Basic Research Program of China (973 Program, No. 2006CB504701)
文摘This paper demonstrated simultaneous separation of acidic and basic proteins using cationic gemini surfactants as buffer additives in capillary electrophoresis. We showed that even at a low concentration (0.1 mmol·L-1) of alkanediyl-α,ω-bis(dimethyloctadecylammonium bromide) (18-s-18), the wall adsorption of both acidic and basic proteins could be effectively suppressed under acidic conditions. Smaller micelle size (e.g., s=5-8) is more effective for the separation of acidic proteins than larger micelle size (e.g., s<4 or >10). Varying the spacer length of gemini surfactants can influence the electrophoretic mobility and selectivity of proteins to achieve the desired separation. Under the optimized conditions, RSDs of the migration time were less than 0.8% and 2.2% for run-to-run and day-to-day assays, respectively, and protein recoveries ranged from 79% to 100.4%. Furthermore, we also investigated the use of gemini surfactant-capped gold nanoparticles (gemini@AuNPs) as buffer additives in protein separation. Introduction of AuNPs into the buffer shortened the analysis time and slightly improved the separation efficiencies. Finally, we presented the applications of this method in the analysis of bio-logical samples, including plasma, red blood cells and egg white.
文摘Fused-silica capillaries used in capillary zone electrophoresis were statically coated with γ- glycidoxypropyltrimethoxysilane and epoxy polymer in order to suppress wall adsorption in the separation of proteins. It has been shown that a significant decrease in adsorption was obtained and eletroosmotic flow was the diminished in the pH range 3-5. However with higher pH values, appreciable peak deformation and decreases in the resolving power were observed. Under pH 5, the epoxy polymer coating was shown to be quite stable and exhibited reproducible separations from run-to-run and day-to-day over a period of time.
文摘Separation of basic proteins was performed using a homemade field-modulated capillary electrophoresis system. The resolution. elution and even wall adsorption can be regulated by ad-lusting the radial rather than axial voltage applied. Selection of running buffer and pH was found to be critical.
基金Supported by the National Natural Science Foundation of China (Grant No. 20705005)
文摘This paper describes non-gel capillary sieving electrophoresis employing semi-crosslinked polyacrylamide as a high performance and low viscous replaceable separation matrix for separation of non-denatured protein separation. Arising from the fine sieving and dynamic coating ability of this polymer, a mixture of basic proteins lysozyme, cytochrome C, ribonuclease A, and trypsin was resolved with excellent reproducibility. Mixing different semi-crosslinked polyacrylamides together further improves the separation. The separtion mechanism was analyzed. With network structure developed to an intermediate state between crosslinked gel and linear polymer solutions, these semi-crosslinked polyacrylamide polymers demonstrate a promise as a new class of size sieving separation medium, not only in capillary electrophoresis, but also in microfluidic chip separation schemes.
