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...

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometryPeptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH 4 HF 2 . The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 507C, even 20 fmol cytochrome c could be well digested and detected.

A mode of capillary electrochromatography (CEC), based on the dynamical adsorption of surfactants on the uncharged monolithic stationary phases has been developed. The monolithic stationary phase, obtained by the in situ polymerization of butyl methacrylate with ethylene dimethacrylate, was dynamically modified with an ionic surfactant such as the long-chain quaternary ammonium salt of cetyltrimethylammonium bromide (CTAB) and long-chain sodium sulfate of sodium dodecyl sulfate (SDS). The ionic surfactant was adsorbed on the surface of polymeric monolith by hydrophobic interaction, and the ionic groups used to generate the electroosmotic flow (EOF). The electroosmotic mobility through these capillary columns increased with increasing the content of ionic surfactants in the mobile phase. In this way, the synthesis of the monolithic stationary phase with binary monomers can be controlled more easily than that with ternary monomers, one of which should be an ionic monomer to generate EOF. Furthermore, it is more convenient to change the direction and magnitude of EOF by changing the concentration of cationic or anionic surfactants in this system. An efficiency of monolithic capillary columns with more than 140000 plates per meter for neutral compounds has been obtained, and the relative standard deviations observed for to and retention factors of neutral solutes were about 0.22% and less than 0.56% for ten consecutive runs, respectively. Effects of mobile phase composition on the EOF of the column and the retention values of the neutral solutes were investigated. Simultaneous separation of basic, neutral and acidic compounds has been achieved.