Preservation and Regeneration of the HPLC Column

Liquid chromatography is an important branch of chromatography. With liquid as the mobile phase, a high-pressure infusion system is used to pump a single solvent with different polarities or a mixed solvent of different proportions, buffer solution, and other mobile phases into the high-performance liquid chromatography column equipped with a stationary phase. After the components in the column are separated, they enter the detector for detection, thereby realizing the analysis of the sample.

High-performance liquid chromatography has high separation efficiency, fast speed, a wide range of mobile phase options, high sensitivity, reusable chromatographic columns, and easy collection of effluent components, which can be used for separation and quantification. It is used in food safety testing and The range of applications for analytics will continue to grow.

The liquid chromatographic column is a key product and consumable for the separation of samples on the liquid chromatograph. When the number of uses increases, the column efficiency will decrease, the peak height will decrease, the peak width will increase, or the peak shoulder condition will occur. General users may replace the chromatographic column immediately. In fact, some problems can prolong the service life of the HPLC column through regular cleaning and regeneration.

1. The preservation of the HPLC column

(1) Maintenance of the reversed-phase column after the daily experiment:
The buffer or saline mobile phase was used and rinsed with 10% methanol/water for 30 minutes after the experiment was completed. The salt in the column was washed off and rinsed with methanol for 30 minutes.

(2) Long-term preservation of the HPLC column:
If the column is to be stored for a long time, it must be stored in a suitable solvent. The reverse phase column can be stored in pure methanol or acetonitrile. The normal phase column can be stored in pure n-hexane after dehydration. The ion exchange column can be stored in water (containing preservative sodium azide or thimerosal). Plug the plug that came with the purchase of the new column. The stored temperature is preferably room temperature.

2. Regeneration of the HPLC column

Because the HPLC column is consumable, as the time of use or the number of injections increases, the peak height decreases, the peak width increases, or the shoulder peak appears. Generally, the efficiency may be reduced.

(1). Washing and regeneration method of the reversed-phase HPLC column:

Use methanol: water = 90: 10, pure methanol, dichloromethane, and other solvents to fill the mobile phase, and rinse in sequence. Each mobile phase flows through the column with no less than 20 times the volume of the chromatographic column. Then rinse in reverse order.

(2). Cleaning and regeneration method of normal phase HPLC column:

Use n-hexane, isopropanol, dichloromethane, methanol, and other solvents as mobile phases, wash in sequence, and each mobile phase flows through the column volume of not less than 20 times the column volume (isopropanol has a high viscosity, so the flow rate can be reduced), to avoid excessive pressure). Be careful not to reverse the order of using solvents. After washing with methanol, wash to n-hexane in reverse order. All mobile phases must be strictly dehydrated.

(3). Regeneration of ion exchange HPLC column:

Using and injecting samples in a buffer solution for a long time will lead to a decrease in the ion exchange capacity of the chromatographic column. Washing with a dilute acid buffer solution can regenerate the cation column, conversely, washing with a dilute alkali buffer solution can regenerate the anion column.

Users can also choose solvents that can dissolve the pollutants in the column to wash the mobile phase in the forward and reverse directions. However, the column efficiency of the regenerated chromatographic column cannot be restored to the level of the new column.

If the column is reversed, it can be adjusted back, but it may cause the carrier in the column to collapse. Try not to reverse the column as a last resort.

(4). Regeneration of the reversed-phase HPLC column:

Sequentially using 20-30 times the column volume of methanol: water = 10:90 (V/V), acetonitrile, and isopropanol as the mobile phase to wash the column, and then Rinse the column in the reverse order.

(5). Regeneration of normal phase HPLC column:

The column is washed with n-hexane, isopropanol, dichloromethane, and methanol as the mobile phase in the order of 20-30 times column, and then the column is washed in the reverse order. It is to be noted that the above solvents must be strictly dehydrated.