Important role of preventive maintenance in smooth functioning of HPLC systems

Important role of preventive maintenance in smooth functioning of HPLC systems
Important role of preventive maintenance in smooth functioning of HPLC systems
HPLC manual injector

Preventive maintenance plays a key role in trouble-free operation of laboratory instruments. It is even more critical for instruments that are required to operate round the clock. HPLC is one such instrument which is expected to run continuously in commercial testing laboratories and industries.

In pharma and food manufacturing industries quality of products is required to be monitored at regular time intervals and there is need for the system to meet the requirements of online quality control. In case it runs into problems there will be delays in taking decisions on product quality. The same is true in laboratories involved in clinical tests or R&D activities.

Trouble free HPLC operation can be ensured thorough regular maintenance schedules. It may not be possible to follow such schedules on daily basis but if carried out weekly or at regular time intervals they can contribute to significant increase in analysis of samples and loss of valuable time.

The article covers some of the important areas that can be looked into even if the system is running without problems.

Mobile Phase

Mobile phase is the life line of your HPLC

  • Use high purity grade solvents, water and reagents for mobile phase preparation
  • Filter mobile phase under vacuum using  0.22/0.45μ size filter to remove solid suspensions, if any
  • Use online filters in mobile phase containers to make doubly sure that no solid suspended particles reach the column
  • Mobile phase should always be degassed to remove dissolved air by sonication, helium sparging or online degassing, if available
  • At time of changing mobile phases take out inlet filter from reservoir and put in fresh mobile phase .Open purge valve and purge of the earlier mobile phase completely out of flow line. Close the valve to allow mobile phase to pass to injector and flush it. Finally connect to column to wash it with the new fresh mobile phase
  • Use washing solution such as iso-propyl alcohol that is miscible with both previous and next mobile phase. Finally replace intermediate washing solution with fresh mobile phase
  • Pass HPLC grade water to wash out buffers. Use of buffers can result in crystalline deposits. The salts can damage plunger or seal and shorten the useful lifetime of the column.

Sample Preparation

  • Check solubility of samples in mobile phase before moving to solvents of similar polarity
  • Optimize concentration for injection so as to prevent overloading of the column
  • Filter sample prior to injection using  0.22/0.45μ size filter


HPLC columns are expensive and if you take precautions in usage and storage you should get long service from them

  • Always avoid overloading the column. For analytical columns the sample load should not be more than 500 μg.
  • Avoid sudden changes in operating conditions such as pressure, flow rate, etc
  • Always choose guard columns for protection of columns and on changing columns change to recommended guard column
  • At end of day remember to carry out washing of columns
  • In case column is taken out of system do remember to fix the end caps
  • Before storage purge off solid salt deposits and buffers and for long time storage always store column in manufacturer recommended solvent and close the end caps
  • Use columns within the pH range 2-8 as columns begin to deteriorate faster outside this range


  • Operate the system within the recommended flow rates and pressure limits to prevent damage to the seals and O-rings of the pump
  • Never leave corrosive solvents or buffers in systems when pump is not running
  • Change seals and O-rings periodically as recommended by the supplier. Sonicate inlet filters regularly to prevent any suspended particles from blocking the column
  • Prime pump with mobile phase to remove remnants of earlier analysis components
  • After using buffer solutions as mobile phases run sufficient volumes of water to remove any salt deposits


  • Flush the injector with mobile phase to remove memory effects and eliminate associated impurity peaks
  • Flush with isopropanol in between analysis
  • Keep the injector in load position only at time of sample introduction when using a manual injector
  • Rinse automated injection systems, needles with water/ isopropanol wash cycles to remove previous sample contamination


  • The detector cell can be cleaned of residues by running water
  • Do not exceed the recommended pressure limits to avoid damage to detector cell walls

Column Oven

Column oven maintains constant temperature which prevents drift of retention times of the peaks. The set temperature should be maintained within limits recommended by supplier. Keep the oven clean and periodically check the connections to ensure that there is no blockage of air flow to the oven.

A well maintained HPLC system will give trouble-free operation for continuous operation thereby avoiding breakdowns and help maintain your high sample analysis throughputs.

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