Abstract
A home-made gradient microbore high-performance liquid chromatographic (HPLC) system was designed using two Varian 8500 stepper motor-driven syringe pumps. The pump delivery of the mobile phase solvents in the 10 μl/min flow-rate range is quite pulsed in nature. Static and dynamic mixing devices were evaluated in terms of their ability to homogenize the slugs of solvent delivered by the pumps. Poor mixing was obtained when a simple tee was used to combine the solvents; this incomplete mixing resulted in increased baseline noise, poorer retention time reproducibility, and a significant decrease in column plate count. The use of static mixing tubes did not yield a completely homogenized mobile phase. A stirred mixing chamber with an internal volume of 80 μl gave the best results, and chromatographic performance was not degraded through use of this mixer. Stirred mixing chambers do distort the linearity of a gradient through exponential dilution; a computer simulation of a stirred mixing chamber was developed in order to study the dependency of exponential dilution effects upon mixing-chamber volume, ramp-rate, and flow-rate. A simple formula was obtained that allows one to use these variables to calculate the extent of gradient distortion. Finally, comparison of gradient microbore and conventional HPLC for polymer fractionations showed that the two methods performed equally well.
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