Capillary electrochromatography with gradient elution

A capillary electrochromatograph incorporating a gradient-forming system generally employed in HPLC is described, and the use of-gradient elution in reversed phase 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 time in a controlled manner. Thus, the composition of the mobile phase flowing through the 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 mu L mixer, From there the liquid passes at anew rate of 0.1-0.2 mL/min through the 17 mu L cavity housing the column inlet and an electrode. The now of the mobile phase was electrosmotic at an effective overall electric field strength of 500-1500 V/cm through a 50 mu m x 20/12 cm capillary column packed with 3.5 mu m octadecylated silica particles. Gradient profiles generated in this manner were highly reproducible. The same-day and day-today reproducibilities of the electrosmotic now were found to be better than 3%. The use of the capillary electrochromatographic system was demonstrated with isocratic and gradient elution for the separation of complex mixtures of biologically interesting substances. The influence of the column temperature on the electrosmotic now velocity and retention of PTH-amino acids was also investigated.