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 also required. Here are some tips and tricks to ensure a long column life with an ideal performance of the HPLC columns.

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 exact information on your column, please refer to the descriptions and technical details in the respective product information.

Mobile Phases: Purity is essential

We recommend using mobile phases of high purity (HPLC grade). Mobile phases of lower purity can interfere with your analytical data due to 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 use. 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. Find a large selection of corresponding membrane filters in the Analytics shop.

Sample matrix

The lifespan of an HPLC column highly depends on the nature of the samples to be tested. Drinking water samples are e.g. less problematic than complex matrix samples, like 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 pre-columns

For a long column life, the use of inline filters or ideally guard columns is recommended. 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 when a replacement is required, is the gradual increase in the back pressure.

Regeneration by purging

If performance results worsen, regenerate your HPLC column by purging. This is highly recommended when ineffective separation results are caused by reversible impurities onto the stationary phase. During purging, similar to chromatographic separation, non-polar substances are eluted by non-polar solvents and vice versa. The simplest way to purge a column is by the application of gradient with polar and non-polar 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 non-polar or polar solvents. If regular purging does not help, you may rinse the column in the opposite direction of the flow. However, if you are using a U(H)PLC column with a small particle size, make sure beforehand that the above-mentioned tips and tricks can be applied to the used column. Some columns have a coarser frit at the column inlet than at the column outlet. Rinsing in the opposite direction may then lead to an expulsion of the column material.

Storage of HPLC columns

For short-term storage, the mobile phase can remain in the column. Alternatively, the mobile phase may pass through at a very low flow rate. For storage over several days, a buffer-containing eluent is recommended to rinse the column with water to prevent microbial growth.

Storing silica-based HPLC columns for an extended period of time may be done without buffer by applying an organic solvent (e.g. acetonitrile) with a maximum of 50 % water content. If the previous mobile phase contains 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 by using the same solvent in which it was delivered to you. This applies particularly to stationary phases based on polymers. Depending on their material properties, these are not compatible with organic solvents, in contrast to the silica-based ones.

Regardless of the column material, ensure that the column is tightly closed and will not dry out. Under these conditions, columns may be stored 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 points to note:

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

Application after storage

If you continue to use the HPLC column after a (long) storage period, the above steps should be repeated.