Best Practices for Using a HPLC System – 2

Basic Housekeeping

This is the second part of our HPLC Best Practices series. This edition will dwell on basic housekeeping such as how best to handle solvents, samples and on tasks that should be regularly carried out to get optimal performance from the HPLC.

Handling Solvents

1. Use only HPLC grade solvents and water.
2. Filter all solvents, especially organic based solvents, mixtures and aqueous buffers through 0.2um filter; otherwise, residues and/or contaminants can block filters and HPLC capillary tubing.
3. Use borosilicate glass bottles only for solvents; use of other types of bottles can leach ions which may react with the solvents.
4. Always rinse the solvent bottles with the solvent before filling it.
5. Always label solvent bottles appropriately with contents, fill and expiry dates.
6. Use amber bottles for aqueous solvents to prevent algal growth; you may also wrap the bottle with aluminum foil and avoid sunlight reaching the bottle directly.
7. Exchange water-based solvents daily to prevent algal growth and possible precipitation of insoluble salts. Both can block filters, capillaries and valves.
8. Always prepare solvents and mobile phases that can be consumed within 1-3 days.
9. Use solvent inlet filters in the solvent bottles

Handling Samples

1. Always ensure that the sample is completely soluble in the mobile phase and in the sample diluent to prevent possible precipitation.
2. Whenever possible, the mobile phase should be used as the sample diluent; where this is not possible, ensure the sample diluent is matched as closely as possible to the mobile phase or the initial mobile phase where the mobile phase composition changes with time
3. Samples should be filtered to remove any insoluble fraction. Syringe filters and syringeless filters are typically used, but centrifugation can also sometimes be used to remove insoluble fractions.

Regular Periodical Tasks

1. Replace water-based solvents daily
2. Replace organic based solvents every other day
3. Purge each channel of the HPLC to be used at a flow rate of about 5ml/min for about 5 minutes or until all the flow channels are free of bubbles and air gaps. This should be done every time before HPLC operation
4. Equilibrate the HPLC system with the mobile phase and the flow rate of the method to be run until the detector signal is stable. This should be done every time before sample injection.
5. Check the solvent inlet filters weekly for blockages or dirt; replace it if there is no solvent flow through the filter

In the next edition, we will be discussing best practices for starting up and shutting down the HPLC.

Reference: Agilent Technologies:  Best Practices – Technical Note

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Posted by Muyiwa Adebola

Muyiwa@aasnig.com