HPLC Tutorial Part 2_Sample Analysis

Turn on the computer and boot the Shimadzu prominence HPLC system. Sequentially power on pump A, pump B, auto sampler, column chamber, UV detector, and communication bus, allowing green lights to appear after each activation.

Open the LCsolution software on the computer and double-click the instrument to establish a connection between the software and the HPLC system. Wait for the confirmation sound indicating successful connection.

Open the pump A and pump B lines by turning the valves counter-clockwise, then press the pull buttons for both lines to flush the system for several minutes. Tighten the lines clockwise when complete to prepare for sample injection.

Open the auto sampler tray and insert the sample vial into well number 1 of tray 3-1. Push the tray back into the auto sampler and gently close the door.

Remove the protective seals from the symmetry C18 5-micrometer column, insert it into the column chamber following the flow direction arrow, and tighten the connections at both the lower and upper ends to prevent leakage.

Switch on the instrument from the software, set the mobile phase composition to 50% organic and 50% aqueous solvent, and run the flow for 10-20 minutes to equilibrate and clean the column. Return to 80% water and 20% organic solvent.

Set the column temperature to 35°C and verify all method parameters including flow rate, injection volume (10 µL), analysis time (10 minutes), and detection wavelength (254 nm). Select the appropriate sample vial and tray location.

Click OK to start the single run analysis. Monitor the run progress on the software display, then access the results through the LCsolution main function to view the chromatogram and data.