Do and Donts IN HPLC.ppt
- 1. DO AND DON”T FOR UPLC/HPLC NRI Institute of Research & Technology-Pharmacy, Bhopal Sophisticated Analytical Instruments Laboratory
- 2. GENERAL • Please sign-in the logbook placed near the instrument . • Always wear apron inside the laboratory. • Do not drink or eat inside the laboratory. • Do not kept any drinking or eating material into the laboratory. • Prepare the sample in the appropriate solvent. Ensure there is no particulate by filtering the sample solution through a membrane. • Before start any analysis, purge/ prime of the system must be done. • Record the pressure before and after analysis.
- 3. Mobile Phase and Solvents • Filter all solvents – Water, Buffer, organic solvents through 0.22 micron (PTFE filters) for UPLC and 0.45 micron for HPLC. • Prepare fresh mobile phase (specially for buffers) everyday. • When using at lower wavelengths below 220nm, usage of JT Baker solvents are recommended. • The Seal wash line should be placed in 90:10 (water: methanol) and primed before every analysis (for UPLC).
- 4. Sample and Needle wash solutions • Check for sample precipitation in mobile phase or crystallization. • Filter all samples and standards to be injected in the UPLC column through a 0.22 micron filter. • For best results, weak wash solution should be equivalent to the (excluding buffers) Mobile Phase composition. • Do not use salt buffers in wash solvents.
- 5. Column • Columns are unidirectional. The flow direction is engraved on the column. These columns should not be used in the reverse direction. • If ion pairing reagents are used then it is highly recommended to dedicate the column to such analysis. • Column Washing – Use only volumes of 90% water to wash the column after buffer use for a period of 45 mins. for HPLC and 30 mins. for UPLC, then follow it up by increase volumes of organic to store the column.
- 6. Detector • It is necessary to pass solvent through the flow cell before you switch ON the detector. This is necessary as the flow cell is a light guided flow cell and require solvent flow to pass calibration.
- 7. System Flushing • It is of utmost importance to flush the buffer lines and the system before an analysis changeover ,or system shut down. • Prepare a solution of 90:10 (water: methanol / Acetonitrile). Dip the A1 or A2 line (which contains buffer) and prime the system for 5 minutes. • Do the same for weak wash and strong wash lines. • If planning a shutdown, ensure that all solvent lines and system are in 50:50 (water: methanol/acetonitrile).