Suggestions for trouble free operation of your HPLC system

HPLC system

Regular maintenance is strongly recommended for a smooth and trouble-free operation of all analytical instruments. In addition, proper handling and operation will also contribute to long service life and quality of data obtained from your instruments. The recommendation is even more significant for HPLC as it is one instrument which works like a workhorse round the clock in most of the laboratories.

The article is an attempt to put across some suggestions on the operation of HPLC which will increase its working life and also place high reliability on results of the analysis.

HPLC system
HPLC system

Initial System inspection                          

It is always worthwhile to spend a few minutes before starting analysis on assessing the system suitability for the analysis task in hand. In other words is the system configuration comprising of the pump, injector/autosampler, column, and detector is appropriate for carrying out the analysis in hand. Inspect the tubings and fittings for leak free connections and if necessary replace any worn out connectors.

Filter mobile phase solvents and samples

Always make use of HPLC grade solvents for preparing mobile phases. It is advisable to filter them through 0.22µ size filter prior to mixing. Similarly, it is recommended to filter the samples before introduction into the system.

 Ensure Freedom from air bubbles

It is important to free the delivery lines of any residual air bubbles which can contribute to noisy response and flow restrictions due to the formation of voids in the flow path. Air bubbles can be removed by opening the purge valve and sucking the mobile phase with the help of syringe from the inlet tubing.


The loop of a manual injector should be flushed with isopropanol to remove any residue from previous samples analyzed. This flushing should be followed by a consistent flow of mobile phase for some time before the introduction of the sample. Similarly, when using the autosampler introduce wash cycles in between sample injections.


The column should be housed inside the thermostatted temperature control oven and should be equilibrated before making the sample and standard injections. In case buffers are part of mobile phase then after completion of analysis flush with water to prevent clogging due to salt formation on drying. When a column is required to be stored flush with original shipping solvent and affix the end caps.

Ensure degassing of the mobile phase to prevent the liberation of air under the high pressures encountered during operation.

Make use of Guard columns

Guard column cartridges fixed before the main column are helpful in extending the useful life of the col impurities.

Start-up and shut down of instrument

On start do not start the system with the final flow rate but increase the flow program in increments of 0.2ml/min steps till the final flow rate is attained.

This will result in minimal disturbance of the column packing. Next program the detector wavelength and injection volume if autosampler facility is to be used. Allow the system to equilibrate till a stable noise-free baseline is obtained. On completion of analysis switch off all the system modules except for the pump. Set the flow rate to 0.2ml/min with the mobile phase followed by the appropriate wash solvent mix until final shutdown. Remove the column and store as recommended if the same column is not required to be used for a considerable time period.

The recommended start-up and shutdown procedure will take a little more time but when adopted as a regular routine it will enhance the useful lifespan of the facility and maintain the reliability and accuracy of results

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