Repetitive injection method: A tool for investigation of injection zone formation and its compression in microfluidic liquid chromatography

Sample introduction in microfluidic liquid chromatography often generates wide zones rather than peaks, especially when a large sample volume (relative to column volume) is injected. Formation of wide injection zones can be further amplified when the sample is dissolved in a strong eluent. In some cases sample breakthrough may occur, especially when the injection is performed into short trapping columns. To investigate the band formation and subsequent zone focusing under gradient elution in situations such as these, we developed the Repetitive Injection Method (RIM), based on the temporally resolved introduction of two discrete peaks to a column, mimicking both the leading and trailing edges of a larger, singly injected sample zone. Using titanium microfluidic 0.32 mm I.D. columns, the results of RIM experiments were practically identical to injection of a correspondingly larger single zone volume. It was also experimentally shown that zone width (spacing between two injected peaks) decreases during gradient elution. We utilized RIM experiments to investigate wide sample zones created by strong sample solvent, and subsequent gradient zone focusing for a series of compounds. This experimental work was compared with computationally simulated chromatograms. The success of sample focusing during injection and gradient elution depends not only on an analyte's absolute retention, but also on how rapidly the analyte's retention changes during the mobile phase gradient.