How to Use and Maintain HPLC Columns?

Generally speaking, the reversed-phase high-performance liquid chromatography column cannot be backflushed. Only when the producer indicates that the column can be backflushed, the impurities remaining in the column header can be backflushed. Otherwise, the recoil will quickly reduce the efficiency of the HPLC columns.

Choose to use a suitable mobile phase to avoid damage to the stationary phase. Sometimes a pre-column can be connected in front of the injector. When the analytical column is bonded to silica gel, the pre-column is silica gel, so that the mobile phase can be “saturated” by the silica gel before entering the analytical column to avoid dissolution of the silica matrix in the analytical column.

Avoid directly injecting samples with complex matrices, especially biological samples, into the HPLC column. Pretreatment of the sample is required or a guard column is connected between the injector and the column. The guard column is typically a short column filled with a similar stationary phase. The guard column can and should be replaced frequently.

After the separation and analysis work is completed, it should not be stopped immediately. It is necessary to rinse the chromatographic analysis system in time, generally, 0.5h or more, to remove impurities in the HPLC columns.

Using and maintaining High-Performance Liquid Chromatography (HPLC) columns properly is essential for obtaining accurate and consistent results in analytical chemistry. Here’s a concise guide on how to use and maintain HPLC columns:

1. Column Installation:

  • Choose the right HPLC column based on sample characteristics and separation requirements.
  • Condition the column per the manufacturer’s instructions before initial use or after storage.
  • Properly connect the column to the HPLC system, ensuring no leaks.

2. Column Use:

  • Prepare samples carefully to prevent particulate matter from clogging the column.
  • Use the correct mobile phase composition and gradient program.
  • Maintain the recommended flow rate for optimal separation and column life.
  • Operate within the specified temperature range to ensure consistent results.

3. Column Maintenance:

  • Flush the column with wash solvent after each run to prevent residue buildup.
  • Consider using a guard column to protect the analytical column.
  • Monitor column backpressure for signs of clogging or deterioration.
  • Avoid mobile phases with pH values beyond the recommended range.
  • Use only high-quality and filtered solvents to prevent contamination.

(1) When loading and unloading or replacing the HPLC columns, the action should be light and the joint should be tightened properly. Strong mechanical vibration must be prevented to avoid voids in the bed.

(2) If the instrument is used for routine analysis, the sample type is limited, but the number of analyses is large, it is possible to configure a special column for each type of routine analysis, which helps to extend the life of the column.

(3) Avoid sudden changes in pressure and temperature and any mechanical shock. A sudden change in temperature or a drop in column height will affect the filling of the column; A sudden increase or decrease in the pressure of the HPLC columns can also hinder the filling of the column packing, so the flow rate of the injection valve should be adjusted slowly. The valve must not rotate too slowly.

(4) The composition of the solvent should be gradually changed, especially in reversed-phase chromatography, not directly from organic solvents to whole water, and vice versa.

(5) When using the HPLC column temperature control device, it should be noted that the temperature can be raised after the mobile phase is passed.

4. Storage:

  • Flush the column with a suitable solvent if not in use for an extended period.
  • Always use the column end cap provided by the manufacturer to prevent drying and contamination.
  • Store the column upright in a cool, dry place within the recommended temperature range.

5. Troubleshooting:

  • Address baseline drift or noise promptly, which could result from contamination, air bubbles, or solvent impurities.
  • Investigate irregular peak shapes, which may indicate issues with the column, mobile phase, or sample.
  • Monitor for loss of resolution over time, as it might signify column degradation.

6. Column Replacement:

  • Regularly assess column performance and replace it when it no longer meets separation requirements.

Remember to refer to the manufacturer’s guidelines for your specific HPLC column model, as maintenance procedures may vary. With proper care and attention, HPLC columns can provide reliable and reproducible chromatographic results for your analyses.