CAPILLARY ELECTROCHROMATOGRAPHY WITH GRADIENT ELUTION

A capillary electrochromatograph incorporating a gradient-forming syst em generally employed in HPLC is described, and the use of-gradient el ution in reversed phase electrochromatography is demonstrated by the s eparation of PTH-amino acids and steroid hormones, The gradient former employs two reciprocating displacement pumps to control the compositi on of the eluent in the reservoir at the column inlet with time in a c ontrolled manner. Thus, the composition of the mobile phase flowing th rough the column and driven by electrosmotic forces can be changed wit h time in a controlled fashion as customary in HPLC with gradient elut ion, The design of the system allows also for isocratic elution by pum ping the eluent of constant composition through the cavity at the colu mn inlet and thus continuously supplying fresh buffer. The eluent grad ient 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 cavi ty housing the column inlet and an electrode. The now of the mobile ph ase was electrosmotic at an effective overall electric held strength o f 500-1500 V/cm through a 50 mu m x 20/12 cm capillary column packed w ith 3.5 mu m octadecylated silica particles, Gradient profiles generat ed in this manner were highly reproducible. The same-day and day-today reproducibilities of the electrosmotic now were found to be better th an 3%. The use of the capillary electrochromatographic system was demo nstrated with isocratic and gradient elution for the separation of com plex mixtures of biologically interesting substances. The influence of the column temperature on the electrosmotic now velocity and retentio n of PTH-amino acids was also investigated.