Best Practices for Using a HPLC System – 3

Starting Up and Shutting Down a HPLC

This is the third part of our HPLC Best Practices series, and this edition will focus on how to start up the HPLC system, get the different HPLC modules ready for analysis and how to properly shut down at the end of the day.

Starting Up

1. First ensure that the HPLC system is in a place where the temperature is stable and within the environmental conditions recommended by the manufacturer. Also ensure that the HPLC system is not in direct sunlight.
2. Switch on all the modules of the HPLC. Modern HPLC systems are usually modular with each module having its own power supply.

Getting the Pump Ready

1. After powering up the pump, purge each pump channel with mobile phase at a flow rate of about 5ml/min for about 5 minutes or when the lines are free of bubbles and air gaps. Make sure the purge valve is open when doing this. In some models, this is automatic once you give the purge command.
2. Don’t forget to return the pump to the method’s normal flow rate before closing the purge valve
3. Always use fresh mobile phase

Getting the Autosampler Ready

1. Ensure that the sample vials have enough sample solutions for all the injections required
2. If your Sampler has a Sample Cooler or Sample Thermostat, ensure the thermostat is turned on and gets to the target temperature and is stable.
3. Always use fresh mobile phase/solvents for needle wash
4. Do not use organic solvents mixed with buffers for needle wash to avoid salts precipitation. Use Methanol, ACN, Water or their mixtures.

Getting the Detector(s) Ready

1. Ensure the detector lamp is on and allow the detector to warm up for at least 1 hour
2. The flow cell should also be thoroughly flushed while the HPLC system is equilibrating with the mobile phase and the method conditions
3. Ensure that the entire HPLC system is equilibrated with your mobile phase composition and all other instrumental conditions until the pressure signal from the pump is stable and the baseline signal from the detector is also stable. This may take up to 30 minutes or more.
4. It is only after 3 above that a sample run can be initiated

Shutting Down

1. After the analysis, first flush the column with the mobile phase
2. Then flush the entire system with Methanol/water or IPA/water mixture at 50:50.
3. If water-based buffers are used as mobile phase, it is advisable to first flush with water before moving to the methanol/water mixture
4. Flush and store the system in the 50:50 Methanol/water or IPA/Water mixture
5. Remove all samples from the autosampler tray; store/dispose as appropriate
6. Power off all the HPLC Modules.

In the next edition, we will be looking at best practices and recommendations for handling the different HPLC modules.

Reference: Agilent Technologies:  Best Practices – Technical Note

Posted by Muyiwa Adebola

Muyiwa@aasnig.com