Separation of peptides by pressurized capillary electrochromatography

A pressurized electrochromatography (pCEC) instrument with gradient capability was used in this work for separation of peptides. Three separation modes, namely, pCEC, high-performance liquid chromatography and capillary electrophoresis can be carried out with the instrument. In pCEC mode, the mobile phase is driven by both electroosmotic flow and pressurized flow, facilitating fine-tuning in selectivity of neutral and charged species. A continuous gradient elution can be carried out conveniently on this instrument, which demonstrates that it is more powerful than isocratic pCEC for separation of complicated samples. The effects of applied voltage, supplementary pressure and ion-pairing agents on separation of peptides in gradient pCEC were investigated. The effects of flow-rate of the pump and the volume of the mixer on resolution were also evaluated. (C) 2002 Elsevier Science B.V. All rights reserved.

Two-dimensional capillary electrophoresis involving capillary isoelectric focusing and capillary zone electrophoresis

Capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) was on-line hyphenated by a dialysis interface to achieve a 2D capillary electrophoresis (CE) system. The system was used with just one high-voltage power supply and three electrodes (one cathode shared by the two dimensions). The focused zone in the first dimension (i.e. the cIEF) was driven to the dialysis interface by electroosmotic flow (EOF), besides chemical mobilization from the first anode to the shared cathode. And then in the second dimension (i.e. the CZE), the separated zone was further separated and driven by an inverted EOF, which originated from the charged layer of a cationic surfactant adsorbed onto the inner wall of the capillary. Finally, a solution of ribonuclease was rapidly separated to assess the feasibility of the two-dimensional CE implement. (C) 2003 Elsevier B.V. All rights reserved.

Pressurized capillary electrochromatography separation of peptides with strong cation exchange and hydrophilic interaction

A pressurized capillary electrochromatography (pCEC) instrument with solvent gradient capability has been used for the separation of a peptide mixture. Retention mechanism and selectivity of the peptides were studied by pCEC using a strong cation exchange (SCX) column. The effects of applied voltage, supplementary pressure, organic modifier concentration, ionic strength,, and pH value on pCEC separation were investigated. It was found that the retention mechanism of the peptides in this system is based on a mixed mode of hydrophilic interaction, strong cation exchange, and electrophoresis. Compared with the separation results obtained by reverse phase pCEC and capillary electrophoresis (CE), this mixed-mode pCEC is more powerful for the separation of hydrophilic peptides with similar charge-to-mass ratio.

Determination of arsenic species by capillary zone electrophoresis with large-volume field-amplified stacking injection

Determination of arsenic species by large-volume field amplified stacking injection-capillary zone electrophoresis (LV-FASI-CZE) is reported in this paper. Whole column injection was employed. The optimum buffer pH for the separation of weak acids was discussed. It was found that the optimum buffer to analyze the stacked arsenate (As(V)), monomethylarsonate (MMA), and dimethylarsinate (DMA) was 25 mm phosphate at pH 6.5. However, the optimum buffer to analyze the concentrated arsenite (As(III)) was 20 mm phosphate - 10 mm borate at pH 9.28. The limits of detection of the method developed were 0.026 mg/L for As(III), 0.023 mg/L for As(V), 0.043 mg/L for MMA, and 0.018 mg/L for DMA. An enrichment factor of 34-100 for several arsenic species was obtained. In the end, this method was applied to determine the arsenic concentration in the environmental reference materials to show the usefulness of the method developed.

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C-12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150 000 plates/meter) and good reproducibility (migration time with RSD < 0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.

Migration of neutral solutes by double stepwise gradient elution in capillary electrochromatography

Characteristics of electroosmotic flow (EOF) and the migration of neutral solutes under double stepwise gradient elution in capillary electrochromatography were studied systematically. EOF velocity proved to be the function of operation time changing with the introduction of the second mobile phase. Accordingly, the retention of components also changed. The migration of neutral solutes was studied under the following three situations; A, components eluted when the column was filled only with the first kind of mobile phase; B, solutes eluted still in the first kind of mobile phase while at that time two kinds of mobile phase coexisted in the column and C, samples eluted in the second kind of mobile phase. Equations to describe the retention times of components under these three kinds of conditions were deduced and applied to predict the retention times of 12 aromatic compounds. Relative errors between experimental and calculated values were below 5.0%, which proved the reliability of the equations. In addition, parameters that might affect the retention time of solutes, such as the transferring time of mobile phase vials, the capacity factors of components and EOF velocities two steps were studied systematically (C) 2001 Elsevier Science B.V. All rights reserved.