How to Properly Maintain and Regenerate the HPLC Column?

High performance liquid chromatography column is the core of chromatographic separation. A suitable, stable, and efficient chromatographic column is indispensable for the establishment of a universal and reproducible method. The stainless steel column tube is frequently used. But when the pressure is not higher than 70bar, thick-walled glass or quartz tube can also be used, and the inner wall of the tube requires a high degree of smoothness. Polish the stainless steel column inner wall to reduce the wall effect and improve column efficiency. There are also those using fused silicon or glass lining for thin tubular columns. Today, HAWACH will discuss how to maintain and regenerate the HPLC column.

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How to properly maintain and store HPLC columns?

Correct maintenance and storage of HPLC columns:
All chromatographic columns have a certain service life. It is wise to routinely monitor the retention time and performance of each column using appropriate standards. When the storage time of the chromatographic column exceeds two weeks, it is necessary to use a mobile phase that inhibits the growth of microorganisms. It is recommended to use a solvent mobile phase containing sodium nitrate or a high concentration of methanol or acetonitrile. When using, the following precautions should be taken to achieve a longer product life:

1. Routinely monitor the performance of the chromatographic column.
2. It can only switch between mutually miscible mobile phases.
3. Avoid salt precipitation in the chromatographic column.
4. Use only filtered and degassed mobile phase.
5. It is forbidden to empty the chromatographic column.
6. When it is out of service, install a protective cap at the end.
7. When storing for a long time, use a mobile phase that can inhibit the growth of bacteria and molds.
8. Abnormally high working pressure means that the inlet filter is blocked, and it may be necessary to use 5-10 times the volume of the flow to flush instead.
9. It is strongly recommended to use a guard column to remove particles or impurities that may be attached to the polymer matrix in the analytical chromatographic column for a long time.

How to regenerate the HPLC column?

Because the chromatographic column is consumable, as the time of use or the number of injections increases, the chromatographic peak height will decrease, the peak width will increase or shoulder peaks will appear. Generally speaking, the column efficiency will decrease. At this time, the chromatographic column needs to be regenerated:

(1) Regeneration of the reverse phase column: Use methanol: water = 90: 10 (V/V), pure methanol, isopropanol, dichloromethane, and other solvents as mobile phases, and wash sequentially, and the amount of each mobile phase flowing through the chromatographic column is 20 to 30 times column volume. Then flush the column in the reverse order.

(2) Normal phase column regeneration: Wash the chromatographic column sequentially with n-hexane, isopropanol, dichloromethane, and methanol as mobile phases. Pay attention to the order of balancing solvents at each step and do not reverse it. The amount of each mobile phase flowing through the chromatographic column is 20-30 times the column volume (due to the high viscosity of isopropanol, please pay attention to adjust the flushing flow rate during flushing). After flushing with methanol, flush the chromatographic column in reverse order. Remember to dehydrate the above solvents strictly. In addition, please read the column instructions carefully before cleaning to understand which solvents are not available.

(3) Regeneration of ion exchange column: Long-term use in high pH and high ionic strength buffer solutions will result in reduced ion exchange capacity of the chromatographic column. Washing with dilute acid buffer solution can regenerate the cation column; conversely, washing with dilute alkali buffer solution can regenerate the anion column. The above chromatographic column regeneration method is not an absolute standard method. Users can choose a suitable solvent that can dissolve the contaminants in the column as the mobile phase according to their actual experience and purpose, and flush in the forward or reverse direction.

However, it should be pointed out that no matter what method is used to regenerate the chromatographic column, it is impossible to completely restore the column efficiency or other parameters to the new column level. In addition, for current UPLC columns, as the volume of the chromatographic column is getting smaller and smaller, the influence of extra-column effects is more significant. A detector with a smaller cell volume (even on-column detection) is required, and column connectors and connection parts with a smaller dead volume are required. The supporting equipment should have the following performance: the infusion pump can accurately output a low flow rate of 1-100μL/min and can provide higher pressure, and the injection valve can accurately and repeatedly inject small volumes of samples. It would require a high-sensitivity detector due to the small injection volume.