Activation Method & Precautions For HPLC Column 

This article includes a brief introduction to the activation methods for HPLC Column.

  • Precautions for the usage of HPLC columns
  • Mobile phase
  • Sample
  • Storage of HPLC columns

Precautions for the usage of HPLC columns

The storage solution of HPLColumn is the mobile phase shown in the evaluation report(if no special instruction). Before usage, please pay attention to the mutual solubility of the storage solution of the HPLC column and the mobile phase of the analysis sample. Solvent miscibility rules- chart for mobile phase.

In reversed-phase chromatography, use about 10% low-concentration of organic phase eluent as a transition, if you need to use a high concentration of salt or buffer as eluent. Otherwise, the salt in the buffer will easily precipitate out in the high concentration of organic phase and block the column.

Solvent miscibility rules- chart for mobile phase

Mobile phase

The organic solvents used in the mobile phase should be chromatographic purity. The water used for the mobile phase prefers ultrapure water or double-distilled water in all-glassware.

The mobile phase could be filtered through a 0.45 μm membrane, especially for mobile phases containing salt. In addition, devices such as containers for the mobile phase and in-line filters in the chromatography system should be cleaned or replaced regularly.

If you have both LC-MS and LC, we recommend the mobile phase in LC-MS should not pass through the membrane, as it is prone to cross-contamination. You could use a special membrane for the instrument.

Using the same membrane device for LC-MS and LC system will cause triethylamine to enter the mass spectrum, and the response will suddenly drop.

The pH range of conventional silica-based packing material is usually 2.0-8.0. It is essential to use HPLC columns under the boundary conditions of the pH range. Wash the column immediately after each use with a solvent suitable for storage and soluble with the mobile phase used, and completely replace the mobile phase used.

The range of pH and column temperature is different for different packed columns.


We recommend that samples are pretreated with filters or sample pretreatment columns (SPE) to keep them as clean as possible. Use a guard column, if the samples are inconvenient to handle. When analyzing samples by normal phase chromatography, all solvents and samples should be strictly dehydrated.

If the handle costs are to high to make every sample filter with membrane or SPE, you could use high-speed centrifugation. Pay attention, do not remove the precipitated material below when removing the supernatant. The pre-column and in-line filters are preferable before the HPLC column.

Storage of HPLC columns

(1) Normal-phase HPLC columns

Normal-phase HPLC column is recommended to be stored in n-hexane.

HILIC HPLC column is recommended to be stored in pure acetonitrile or 90% acetonitrile/10% 0.1M ammonium acetate, pH=5.


  • Make sure that all buffer salts have been flushed from the column before flushing with organic solvents. Buffer salts are insoluble in organic solvents, they can clog lines and columns.
  • Primary amine-based columns (i.e., amino columns) should not contact ketones.
  • In HILIC mode, if the mobile phase contains buffer salts, first rinse with 5 times the column volume of 20% acetonitrile/80% deionized water, and then rinse with 10 times the column volume of pure acetonitrile and store.

(2) Reversed-phase HPLC columns

Short-term storage: Storage in the mobile phase.

Long-term storage: Storage in 80% or more aqueous methanol or acetonitrile. (Note: C4, C4-T, phenyl, and PFP are stored in pure acetonitrile or methanol)


  • Make sure all buffer salts have been flushed from the column before flushing with organic solvents. Buffer salts are insoluble in organic solvents and can clog lines and columns.
  • When the mobile phase pH < 2.5 or pH > 7.0, use the long-term storage method.

(3) Ion-exchange HPLC columns

It can be stored in water (with preservative sodium azide or thimerosal) and plugged with the plug included with the purchase of a new column. The storage temperature should preferably be room temperature.

(4) Maintenance of the reversed-phase column after each day's experiment

Using buffer or mobile phase containing salt, the column should be rinsed with 10% methanol/water for 30 minutes after the experiment is completed to wash off the salt in the column, and then rinsed with methanol for 30 minutes.

Note: The column should not be rinsed with pure water, and 10% methanol should be added to the water to prevent collapsing the packing.

DO NOT use a mobile phase with a pure water mobile phase unless it is the special hydrophilic column.

Take care of your column, clean it more often. If the column loses its temper, all projects will ruin. ^-^

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