Capillary Electrochromatography with Gradient Elution

摘要

A capillary electrochromatograph incorporating a gradient-forming system generally employed in HPLC is described, and the use of gradient elution in reversed plume electrochromatography is demonstrated by the separation of PTH-amino acids and steroid hormones. The gradient former employs two reciprocating displacement pumps to control the composition of the eluent in the reservoir at the column inlet with lime in a controlled manner. Thus, the composition of the mobile phase flowing through file column and driven by electrosmotic forces can be changed with time in a controlled fashion as customary in HPLC with gradient elution. The design of the system allows also for isocratic elution by pumping the eluent of constant composition through the cavity at the column inlet and thus continuously supplying fresh buffer. The eluent gradient is generated by the two pumps and a 10 /μL mixer. From there the liquid passes at a flow rate of 0.1-0.2 mL/rain through the 17 #L cavity housing the column inlet mad an electrode. The flow of the mobile phase was electrosmotic at an effective overall electric field strength of 500-1500 V/em through a 50 #m x 20/12 cm capillary column packed with 3.5#m octadecylated silica particles. Gradient profiles generated in this manner were highly reproducible. The same-day and day-to-day reproducibilities of the electrosmotic flow were found to be better than 3%. The use of the capillary electro-chromatographic system was demonstrated with isocratic and gradient elution for the separation of complex mix-tares of biologically interesting substances. The influence of the column temperature on the electrosmotic flow velocity and retention of PTH-amino acids was also investigated.