Effects of the length and modification of the separation channel on microchip electrophoresis–mass spectrometry for analysis of bioactive compounds

Analyses of amino acids and peptides were performed using a quartz microchip and an interface for microchip electrophoresis–electrospray ionization mass spectrometry (MCE–ESI–MS). In MCE–ESI–MS, negative pressure caused by ESI increased band broadening and deteriorated separation. We tried to suppress the negative pressure and improve separation using a microchip with a long separation channel. Separations of peptide standards were compared using two microchips with long separation channel (58.9 mm) and short one (22.9 mm). Theoretical plate numbers and resolution were improved significantly using the former. The theoretical plate numbers of [Val4]angiotensin was 8600 using the former and 1700 using the latter. When background electrolytes of low pH were used in an uncoated quartz microchip, electrokinetic injection was difficult because of weak electroosmotic flow. The use of successive multiple ionic polymer layers coating of the microchip channel stabilized electrokinetic injection and permitted analysis of amino acids and peptides even under low pH conditions. Separation of amino acids was successfully performed using formic acid solution (pH 2.5) as background electrolyte.