Basic Guide on How to Use the HPLC

Learn how the Agilent 1260 Infinity 2 reverse-phase HPLC works by separating compounds based on polarity through hydrophobic interactions. Identify key parts including the injector, pump, column, detector, fraction collector, and solvents.

Set up the HPLC method by clicking Instrument > Set Up Instrument Method. Configure solvent volumes (water with 0.1% TFA and acetonitrile with 0.1% TFA), adjust column settings, set detection wavelength, and verify all solvents are filtered and pure.

Set up the fraction collector panel for automatic or manual collection. Enter sample name with initials and date in Sample Info, select the correct folder for data storage, and optionally create a new folder.

Filter the sample to remove all solid particles and debris. Select a syringe matching the loop size, rinse it with the sample solvent, and uptake the filtered sample while ensuring no air bubbles are present.

Position the injector lever in Load position, inject the sample into the injector, then move the lever to Inject position. As the HPLC runs and peaks appear on the graph, click Collect Fraction at desired peaks and ensure vials are positioned in the collector.

Review the chromatogram as it appears on screen. Optionally refine the data by clicking Integration to delete unnecessary peaks, then save the modified graph and print or save as PDF for your records.

Run the same method without sample to flush the column with solvent. Optionally perform this wash before your sample run to verify the baseline and ensure any peaks observed are from your sample only.