How to ensure trouble-free HPLC System Operation

How to ensure trouble-free HPLC System Operation
HPLC Preventive Maintenance

HPLC is the major support system in laboratories dealing with separation and identification of complex organic compounds. In industrial sector some laboratories are equipped with several HPLC systems in Quality control and R&D sections which run like workhorses 24 X7 hours for 365 days in a year. Downtime of these systems proves to be very expensive in terms of production and loss of man-hours so It becomes extremely important to keep HPLC systems up and running.

The HPLC system comprises of different modules and things can go wrong unexpectedly. It becomes essential to follow preventive maintenance schedules religiously even when the system is giving excellent performance. Many comprehensive HPLC texts are available which deal with pages and pages on the subject of preventive maintenance but the objective of this short article is to provide some valuable tips that can ensure trouble-free system operation.

Mobile phase

Mobile phase is the lifeline of your HPLC. Any disruptions in the flow can result in unstable baseline and erratic fluctuations in retention time of peaks.

  • Use high purity grade solvents and HPLC grade water for preparing mobile phase mixtures
  • Mobile phase should be prepared fresh every time.
  • It is advisable to filter even HPLC grade mobile phase solvents prior to use under vacuum using 0.45 µ filter
  • Clean or replace online filters regularly as a practice
  • Degas the mobile phase on preparation even if your system has inbuilt degassing facility.
  • When mobile phase requires to be changed flush the system with the solvent having intermediate polarity and then only run on the new mobile phase
  • Flush the system with at least 200 ml of water after using buffered mobile phases. Buffers if allowed to dry out lead to formation of solid crystalline deposits which can damage pistons and plunger seals.


  • Try dissolving sample in mobile phase. If this is not possible then try dissolving in a solvent of close polarity
  • Filter sample prior to injection
  • Rinse sample vials with sample solution before loading onto the autosampler.

Column Care

Column is a critical component and needs proper attention and care :

  • Avoid damage to packing by exposure to thermal or mechanical shocks
  • Avoid sudden change of operating conditions such as flow rate and pressure
  • Use guard columns ahead of the main column
  • Before storage purge out buffers or mobile phase. For long storage purge and store with the manufacturer recommended solvent and close the end caps
  • Use columns within the pH range 2 – 8.
  • It is a good idea to keep a stock of frequently used columns

Pump care

The efficiency of your system will depend on the capability of the pump to always give consistency of flow rates.

  • Maintain stock of recommended essential spares such as o-rings, plungers, check valves, etc
  • Operate well within the specified pressure limits to prevent damage to the component parts
  • Do not allow buffers to dry inside the pump
  • Clean seals and o-rings periodically as per supplier recommendations
  • Clean online filters at regular intervals
  • Prime pump with new mobile phase to remove previous mobile phase components

Injector care

  • In manual injection rinse sample loop with sample to prevent memory effects
  • Flush with iso- propanol in between injections and also when using the auto sampler

Detector care

Detector response decides the final output of your results. Care and maintenance of detector has same importance as any other HPLC component :

  • With continuous use the lamp can contribute to baseline noise and drift. Keep in stock a spare lamp and replace as per supplier recommendations
  • User HPLC grade solvents for mobile phase preparation. Always degas solvents to prevent bubble formation inside the detector cells
  • Clean cell off-line when required with 10% nitric acid or chromic acid. Flush with several volumes of water and finally with the mobile phase
  • Do not exceed the prescribed pressure limits to prevent damage to the cell walls

The tips provided can ensure long trouble-free operation of the HPLC system. We shall appreciate receiving comments based on your experience on this important topic.

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