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How to maintain, regenerate and store HPLC columns correctly

Most HPLC columns feature a long service life and robustness. In addition to the fundamental quality of the columns, a corresponding maintenance of the stationary phase is of course also required. For an ideal performance of the HPLC columns, we have put together tips and tricks for a long column life with good separation performance.

Correct application of columns

pH value

The permissible pH value of the mobile phases is dependent on the respective filling material. In general, silica-based columns are stable in a range of about pH 2-8. However, we also offer columns which are stable up to pH 1 or far into the basic milieu. For the exact information about your column, please refer to the descriptions and technical details on the respective product pages.

Mobile Phases: The purity is essential

We recommend using mobile phases of high purity (HPLC grade). Mobile phases of lower purity can interfere with your analytical data caused by impurities. Possible consequences include the occurrence of ghost peaks or, in the worst case, a change in the selectivity of the column. When using self-made buffer solutions, we recommend that you filter them before usage. For columns with a particle size > 5 μm, the pore size of the filter should be 0.45 μm, for columns with a smaller particle size the pore size should be 0.2 μm. In the Analytics shop you will find a large selection of corresponding filter membranes.

Sample matrix

The lifetime of an HPLC column strongly depends on the nature of the samples which are to be tested. Drinking water samples are e.g. less problematic than complex matrix samples, like e.g. food products. Pre-analysis plays an important role in the purification of the sample. For effective sample preparation, we offer a wide range of accessories e.g. for filtration or solid phase extraction.

Application of inline filters and / or pre-columns

For a long column life, we recommend the use of inline filters or ideally guard columns. Inline filters physically retain particles that cannot be eluted due to their size. Accumulation at the column head is thus prevented. Guard columns offer the advantage, that they also retain molecules which would form irreversible bonds with the column material. Both filters and guard columns should be replaced regularly. A good indication of the time when a replacement is required, is the gradual increase in the back pressure.

Regeneration by purging

If you lose performance, you may be able to regenerate your HPLC column with a purging program. This is in particular the case, when the decrease of separation performance is caused by reversible impurities onto the stationary phase. For the wash program, analogous to the chromatographic separation: Unpolar substances are eluted by unpolar solvents and vice versa. The simplest way to purge a column is by the application of gradient with polar and unpolar runnability. Depending on the nature of the contamination as well as the column material, you can also dissolve the impurities by injecting dilute acids, bases or strongly unpolar or polar solvents. If regular purging does not help, you can also rinse the column opposite the direction of flow. However, if you are using a U(H)PLC column with a small particle size, make sure whether the above mentioned tips and tricks can be applied to the used column beforehand. Some columns have a coarser frit at the column inlet than at the column outlet. Rinsing in the opposite direction can then lead to an exit of the column material.

Storage of HPLC columns

For short-term storage, the mobile phase can remain within in the column. Alternatively, a passage of the mobile phase at very low flow is also possible. For storage over several days, we recommend using a buffer-containing eluent to rinse the column with water to prevent microbial growth.

Storing silica-based HPLC columns for an extended period of time can be done without buffer by the application of an organic solvent (for example, acetonitrile) with a maximum of 50% water content. If the previous mobile phase contained a buffer, it is recommended to rinse the column first with water in order to prevent a possible precipitation of the buffer salts in the organic solvent.

The easiest way to store your column is however by using the same solvent in which it was delivered to you. This applies in particular to stationary phases based on polymers. Depending on their material properties, these are not compatible with organic solvents, in contrast to the silica-based.

Regardless of the column material, you also have to ensure that the column is tightly closed and cannot dry out. Under these conditions, you can store your columns for a long time without compromising quality.

In general, good storage conditions for columns are:

  • Temperature: room temperature (15-30 °C)
  • Humidity: room humidity, normal laboratory conditions

Other notable points are:

  • Avoid an overflow of the column
  • Mobile phase degassing
  • Selecting the correct column and mobile phase for the analyte

Application after storage

If you continue to use the HPLC column after a (longer) storage period, you should repeat the above steps.