High Performance Liquid Chromatography (HPLC) – Operations by Dr. Sejal P. Gandhi

Dr. Sejal P. Gandhi introduces the HPLC demonstration, discussing the instrument facility at AIIMS Pimpri-Pune and outlining the various steps for mobile phase preparation and system setup.

Prepare the mobile phase by filtering through 0.05 micron fritters and 0.22 micron membrane filters. Degas the mobile phase using an ultrasonic water bath to remove air bubbles from the solvent system.

Power on the HPLC instrument and begin the pump operation. Allow the system to equilibrate with the mobile phase, setting appropriate temperature and flow rate parameters for 5-10 minutes.

Prepare working standard solution of paracetamol at 250 micrograms per mL and sample solutions from paracetamol tablets dissolved in equal quantities of distilled water at appropriate concentrations.

Double-click the HPLC software to launch the analysis program. Set instrument parameters including data acquisition time (50 minutes), flow rate, wavelength detection (254 nm), and save the method file.

Conduct system suitability testing by injecting the standard paracetamol solution. Verify system readiness, then proceed with the single standard injection after obtaining acceptable chromatographic parameters.

Inject the paracetamol sample solution and collect chromatographic data. View the resulting chromatogram and spectrum, noting the retention time and peak area of the drug.

Use the peak areas from standard and sample injections to calculate paracetamol content using the calibration formula. Report the assay result in milligrams and compare against USP limits to complete the analysis.