Best Practices: Taking Care of Chromatography Columns

A column is the heart of any chromatographic separation. Taking good care of that column will help ensure that your investment has the longest possible lifetime and will be ready for your samples. It isn’t hard to ensure your LC and SFC columns are used, stored, and cleaned correctly, but mistakes can be costly.

Your Reversed Phase, Normal Phase, Chiral, and SFC columns need some care at various phases of the lifecycle, including installation, storage, and cleaning.

Here are some simple tips to help you keep your columns in tiptop shape to maximize the value and life of these critical items.

Mobile Phase Considerations for All Column Types:

• Use only the highest grade/purity chemicals and reagents
• Degas and filter all mobile phases prior to use
• Trace impurities present in mobile phases or additives can dramatically degrade columns
• When changing to a different mobile phase, make sure the solvents and buffers are miscible
• Using solvents that are immiscible with the solvent in the column can permanently damage the column. Salt and buffer precipitation from the mobile phase can permanently damage the column
• Always check sample solubility and, if possible, use the initial mobile phase composition as the sample diluent (solvent)

Reversed Phase Columns

• Installation:
  • Flush HPLC pumps and lines thoroughly with filtered and degassed mobile phase (without any buffers). Make sure there are no air bubbles in the system
  • Connect the column to the injector corresponding to the direction of the flow label (located on the column). Leave the outlet of the column unattached
  • Set the pump to flow at 0.1 mL/min (or lowest setting) and increase to your typical operational flow rate for at least 5 minutes
  • Stop the flow when there is a free flow of solvent from the column outlet. Wipe the end of the column with a lint-free technical wipe and attach it to the detector
  • Equilibrate the column by passing approximately 10-30 column volumes of mobile phase at your operational flow rate through the column
  • Avoid any sudden pressure changes

• Storage:
  • Note! Storage conditions affect column lifetimes
  • Never store columns filled with mobile phase containing buffers
  • Flush with 5 column volumes of a mobile phase without buffers to remove any buffers or salts
  • Store the column in 65% acetonitrile/35% water
• Cleaning (if high backpressure is observed):
  • Rinse with 10 column volumes each of:
    • 95% water/5% acetonitrile (for buffer removal)
    • THF
    • 95% water/5% acetonitrile
    • Mobile phase
    • If using a PKI Epic LC column, you may reverse flush the column if high backpressure is still observed

Normal Phase Columns

• Installation:
  • Flush HPLC pumps and lines thoroughly with filtered and degassed mobile phase (without any buffers). Make sure there are no air bubbles in the system
  • Connect the column to the injector corresponding to the direction of the flow label (located on the column). Leave the outlet of the column unattached
  • Set the pump to flow at 0.1 mL/min (or lowest setting) and increase to your typical operational flow rate for at least 5 minutes
  • Stop the flow when there is a free flow of solvent from the column outlet. Wipe the end of the column with a lint-free technical wipe and attach it to the detector
  • Equilibrate the column by passing approximately 10-30 column volumes of mobile phase at your operational flow rate through the column
  • Avoid any sudden pressure changes
• Storage:
  • Note! Storage conditions affect column lifetimes
  • Never store columns filled with mobile phase containing buffers
  • Flush with 5 column volumes of a mobile phase without buffers to remove any buffers or salts
  • Store the column in 100% hexane
• Cleaning:
  • We recommend you contact our LC column technical assistance at +1-800-356-6140 if you are interested in cleaning your normal phase column

Supercritical Fluid (SFC) Columns

• Installation:
  • Flush HPLC pumps and lines thoroughly with filtered and degassed mobile phase (without any buffers). Make sure there are no air bubbles in the system
  • Connect the column to the injector corresponding to the direction of the flow label (located on the column). Leave the outlet of the column unattached
  • Set the pump to flow at 0.1 mL/min (or lowest setting) and increase to your typical operational flow rate for at least 5 minutes
  • Stop the flow when there is a free flow of solvent from the column outlet. Wipe the end of the column with a lint-free technical wipe and attach it to the detector
  • Equilibrate the column by passing approximately 10-30 column volumes of mobile phase at your operational flow rate through the column
  • Avoid any sudden pressure changes
• Storage:
  • Note! Storage conditions affect column lifetimes
  • Never store columns filled with mobile phase containing buffers
  • Flush with 5 column volumes of a mobile phase without buffers to remove any buffers or salts
  • Store the column in 100% ethanol
• Cleaning:
  • We recommend you contact our LC column technical assistance at +1-800-356-6140 if you are interested in cleaning your supercritical fluid column

Chiral Columns

NOTE! Residual solvents that remain in the HPLC or SFC systems (e.g. acetone, chloroform, ethyl acetate, DMF, DMSO, dichloromethane, THF, and water) will destroy a chiral column. Therefore, ensure the system is flushed with IPA or ethanol before connecting a chiral column.

• Installation and use parameters:
  • NOTE! If the chiral column is to be used for supercritical fluid chromatography (SFC), rinse thoroughly with 100% ethanol before use
  • Flow direction: the flow direction for chiral columns will typically be indicated by an arrow on the column label
  • Flow rate and pressure: to maximize column lifetime, flow rates should be adjusted so that the backpressure does not exceed 6,300 psi (≈ 450 bar)
  • Temperature range: 0 – 40 ◦C
  • Mobile phase: typical mobile phases include hexane/IPA and hexane/ethanol. All samples and mobile phases should be filtered through a membrane filter
  • Modifiers: If needed, DEA, TEA, TFA, and acetic acid can be used to improve peak shape for highly basic or acidic compounds. Note! Modifiers will cause a memory effect on the column
• Storage:
  • Columns should be flushed and stored in 90% hexane/10% IPA with the end plugs secured to each end of the column

Following these guidelines will help ensure your LC and SFC columns are ready for their first separation and for many more after that.  Samples can sometimes be hard enough on a column, no one needs additional problems. These steps will help set you up for reliable results.

Browse our full line of LC and SFC columns here!