HPLC and UHPLC Column Care

HPLC and UHPLC column care and maintenance

An HPLC column is a fragile laboratory consumable. Therefore, special care must be taken to avoid early and costly replacement of the columns and to obtain consistently reliable data. On this page we provide general precautions to take when working with HPLC or UHPLC columns and we provide detailed descriptions for cleaning, regeneration, rehydration and information on how to check performance parameters of columns. The latter is required in order to decide whether or not special action has to be taken.

Column Regeneration Column Washing

Column Rehydration

Performance parameters

How to handle the column outside the system

  • Prevent the column from mechanical shocks
  • Store the column in the column packing provided by the manufacturer
  • Storage solvent is described in the operating and conditions sheet for each column type
  • Make sure plugs are tightened in order to prevent dehydration of the column

How to install an HPLC or UHPLC column

1.     Preparation of the mobile phase

Information on the mobile phases compatible with the column is found in the operating and conditions sheet. In order to prevent air bubbles from entering the column, the mobile phase needs to be degassed before use. Additionally, small particles need to be removed by vacuum filtration through a 0.2 µm or 0.45 µm nylon membrane.

2.     Prepare column installation

Before installing the column, make sure the pump flow does not exceed the maximal flow rate of the column. The information is given in the operating and conditions sheet. Furthermore, look out for the arrow on the column, which indicates the flow direction.

3.     Connect the HPLC column to the injector and detector

After manufacturing and quality control, the column has been flushed with storage solvent and closed with caps to prevent evaporation of the solvent. As mentioned above, it is important to prevent air from entering the column. Remove the cap from the column inlet side. The solvent should be visible at the inlet fitting. Before connecting the column to the injector, slightly untighten the end plug without completely removing it. Then the column can be connected to the injector and the pump started at a low flow rate. Make sure to insert the capillary tubing fully into the column end fitting before setting the ferrule by tightening the nut (Figure 1).

Connecting the column to the instrument:  Capillary tubing fully inserted into the column end fitting before setting the ferrule by tightening the nut.

Remove the end cap and wait for liquid flowing out the exit. Then, the column can likewise be connected to the detector inlet. 

Considerations for HPLC column use

Running conditions

In the operating and conditions sheet available for each column type, the conditions for maximum temperature, maximal flow rate and pressure as well as for chemical compatibility are outlined. Deviating conditions can lead to changes in column performance.

Sample preparation for HPLC

If possible, always dissolve your sample in mobile phase or the starting mobile phase when operating under gradient conditions. Alternatively, try to match the pH, salt concentration and organic solvent of the sample with those of the mobile phase and run a test to ensure that no precipitate, suspension or flocculate is formed. Finally, before making an injection, filter the sample through a 0.20-0.45 micron porosity membrane. 

Testing HPLC column performance

Analyzing standards in regular intervals helps to determine changes in column performance. Thus, measures such as column regeneration can be taken to counteract the degradation of the column. It is possible to use chemical standards or a sample component for regular testing. As the column will always be tested in a specific system (HPLC instrument), a testing procedure is also referred to as system suitability test (SST). First of all, you must establish baseline data when the column is new and performing well. After establishing that the column performs properly, using standard test probes, calculate the asymmetry factor, theoretical plates and resolution of one or more of your sample components or chemical standard. Also note the retention time. This becomes your „baseline test mix“ with which you can later compare.

If you find this information helpful, find it with additional details on how to take of HPLC column in the TSKgel HPLC Instruction manual.