Capillary Electrochromatography for Separation of Peptides Driven with Electrophoretic Mobility on Monolithic Column

Abstract

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.Capillary electrochromatography (CEC) is a hybrid method that combines features of both high-performance capillary electrophoresis (HPCE) and high performance liquid chromatography (HPLC). 1-3 To date, in most applications of CEC, neutral and hydrophobic compounds have been separated under conditions similar to those employed in reversed-phase HPLC. However, for CEC to become a widely used analytical technique, it is also necessary to offer a means to separate charged biomolecules with selectivity different from that obtained in HPLC or HPCE. Nonetheless, this aspect of CEC has not received much attention, probably because it requires novel separation systems especially tailored to take advantage of the peculiar features of CEC.Furthermore, an understanding of the separation mechanism greater than presently available is needed to exploit the full potential of CEC.The most widely used columns in CEC are packed with an alkyl-silica stationary phase, and frits are prepared to prevent the loss of the stationary phase from the capillary. These stationary phases have an abundance of silanol groups, which are usually negatively charged in contact with neutral or alkaline mobile phase. They are responsible for the generation of an electroosmotic flow in a high electric field. However, a drawback with this kind of CEC column is that frits can result in the formation of bubbles, leading to loss of electroosmotic flow. [4][5][6] Although no frits are used in open tubular CEC (OT-CEC) columns, the relatively low phase ratio in OT-CEC restricts its further developments and applications. Recently, the monolithic columns 7-25 in CEC have attracted increasing attention because of their...

The separation of biomolecules using capillary electrochromatography

“…[21] and Wu et al [27] have achieved separations of peptides on the uncharged monolithic columns with poly (styrene-codivinylbenzene) and poly (lauryl methacrylate-co-ethylene dimethacrylate) rods. It was observed that peptides were separated on the basis of their difference in electrophoretic mobility and hydrophobic interaction with the stationary phase, and all of the basic peptides containing histidine and lysine showed good peak symmetry [27].…”

“…It was observed that peptides were separated on the basis of their difference in electrophoretic mobility and hydrophobic interaction with the stationary phase, and all of the basic peptides containing histidine and lysine showed good peak symmetry [27].…”

“…Gusev et al [21] and Wu et al [27] have achieved separations of peptides on the uncharged monolithic columns with poly (styrene-codivinylbenzene) and poly (lauryl methacrylate-co-ethylene dimethacrylate) rods. It was observed that peptides were separated on the basis of their difference in electrophoretic mobility and hydrophobic interaction with the stationary phase, and all of the basic peptides containing histidine and lysine showed good peak symmetry [27].In another example, a porous organic±inorganic hybrid monolith modi®ed with silane containing large steric groups was prepared by photopolymerization of sol-gel [28], and the cationic peptides were separated on the column because the electrostatic interaction between the cationic peptides and the anionic monolith was largely eliminated. …”

On the nature of the forces controlling selectivity in the high performance capillary electrochromatographic separation of peptides

“…(9)], can be defined by analogy to the capacity factor, kЈ, in HPLC, as cec ϭ ͑t cec Ϫ t eo ͒ t eo (9) where t cec is the apparent migration time of the peptide and t eo is the migration time of the EOF marker. Alternative definitions have been proposed, [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] e.g., the apparent migration time of the peptide can also be related to the applied voltage E (in kV/m) and capillary length, or in the case of pressure-assisted electrochromatographic separations, 28 according to…”

Charged porous membrane structures for separation of biomolecules

“…The second group of CEC columns are continuous beds, also called monoliths [27][28][29][30][31][32][33][34][35][36][37][38][39][40]. Compare with packed capillaries, they are much easier to prepare.…”