How to Solve the Problem of HPLC Column Aging?

In the process of gas analysis, the HPLC column is the “heart” of the gas chromatograph and plays a vital role in qualitative and quantitative analysis. Today we are going to share some questions about HPLC column aging. Column aging, in short, is the process of baking the column at high temperatures and using the carrier gas to bring the unstable stationary phase, possible impurities, and pollutants out of the column to restore the column’s efficiency. Hawach provided CN HPLC columns, NH2 HPLC columns, and SCX HPLC columns for your choice.

High-Performance Liquid Chromatography (HPLC) column aging is a common issue that can lead to a decrease in chromatographic performance over time. Several factors contribute to column aging, including sample matrix components, mobile phase composition, and the types of analytes being analyzed. Here are some strategies to address and minimize the effects of HPLC column aging:

1. Optimize Mobile Phase Composition:

  • Solvent Selection: Choose appropriate solvents and buffers based on the characteristics of the analytes. Incompatible solvents or buffers can contribute to column degradation.
  • pH Control: Maintain consistent pH levels, as extreme pH conditions can lead to column degradation. Use buffers within the recommended pH range for the column.

2. Use Guard Columns:

  • Employ HPLC guard columns to protect the analytical column from contamination and matrix components. Guard columns can be replaced or cleaned more frequently than the analytical column.

3. Sample Filtration:

  • Filter samples before injection to prevent particulate matter and contaminants from entering the column. Particulate matter can contribute to increased backpressure and column deterioration.

4. Prevent Overloading:

  • Avoid overloading the column with excessive sample amounts. Overloading can lead to irreversible damage and decreased separation efficiency.

5. Regular Maintenance:

  • Implement a routine maintenance schedule, including flushing the column with compatible solvents and following manufacturer recommendations for column care.

6. Use of Additives:

  • Incorporate appropriate additives, such as ion-pairing reagents or chelating agents, to enhance column stability and prevent metal ion contamination.

7. Optimize Flow Rate:

  • Avoid using flow rates outside the recommended range for the column. Excessive flow rates can lead to increased backpressure and column stress.

8. Temperature Control:

  • Maintain consistent temperature control during HPLC runs. Temperature fluctuations can affect column efficiency and reproducibility.

9. Column Conditioning:

  • Condition the column regularly with appropriate solvents before and after each use to ensure a stable and reproducible baseline.

What are the conditions for the aging treatment of the chromatographic column?

1. Newly purchased column: For the new column, before the initial use, the aging treatment can remove the stationary solution with unstable bonding in the column or the residual solvent in the packed column, so as to ensure the stability of the new column’s performance.
2. For the chromatographic column that needs to be restarted after being idle for a long time, the aging of the chromatographic column can remove the pollutants, oxygen, and moisture caused by the long-term placement of the chromatographic column. and restore the properties of the column.
3. Long-time use or dealing with complicated analysis
Through the aging treatment, the active sites on the inner wall of the column are covered again by using the thermal expansion and contraction characteristics of the stationary phase.

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How to operate and what details should be paid attention to during the aging process?

1. Selection of aging temperature

In general, the aging temperature can be controlled between the higher analytical temperature of the method and the upper limit of the column thermostat. The aging temperature can also be set to be 20-30°C lower than the upper limit of the column constant temperature, or 20-30°C higher than the higher analysis temperature of this method.

Each HPLC column has a temperature limit that it can withstand. Before aging, read the oven or manual carefully. During the aging process, once the column temperature limit is exceeded, the stationary phase will be lost and the service life will be shortened. Therefore, under the premise of ensuring the removal of impurities and contaminants, the aging temperature can be reduced as much as possible.

2. Aging time

The specific aging process is generally set to programmed heating (slowly heating up to the set aging temperature), such as initial temperature 35°C, 2-5 °C/min, heating to the aging temperature, and maintaining 0.5-2h. We can decide the aging time based on the impurity removal effect.

3. Prevent oxygen from entering the column

Because of the presence of oxygen at high temperatures, the fixative is easily oxidized, and the stable silane bond will be broken, resulting in serious column loss and decreased column efficiency.
Therefore, in order to avoid oxygen entering the chromatographic column, the following points should be paid attention to:

a.Connect the gas inlet of the chromatographic column to the inlet of the instrument. Place the end of the column in alcohol, take the carrier gas and observe whether there are continuous bubbles. If not, check the column for mid-section breaks, installation, and system leaks.
b.Set the carrier gas flow rate to the normal working value, set the inlet, column oven, and detector (if connected to the detector) to 35℃, or turn off the instrument temperature control system. The carrier gas is available for 15-30 minutes at room temperature to replace the oxygen that may be present in the instrument system and column.
c.Make sure that the other joints of the instrument are installed correctly and pass the leak test.

4. Avoid pollution

In general, to avoid contamination of the detector, it is not recommended to connect the detector during the burn-in process. Normal operation is to plug the lower end of the detector with a solid compression ring and shut down the temperature. In addition, the use of hydrogen to age columns is prohibited, i.e. to avoid hydrogen leakage and accumulation in the column oven, which could lead to explosions.

5. Maintenance should be carried out before aging

For columns that have been used for a long time or have analyzed complex matrices and multiple samples, conditioning is to remove some residual contaminants. These contaminants tend to be higher boiling compounds that accumulate at the top of the column, such as proteins, pigments, gels, etc. in the sample. In order to prevent the contaminants from continuing to transfer to the column when the contaminants in the HPLC column cannot be completely discharged from the column, a part of the column should be properly cut off before the column is aged.

In addition, the injector liner, quartz wool, and injector separator should be well maintained to avoid secondary pollution to the chromatographic column during the aging process. In conclusion, it makes sense to age the column after removing the source of contamination.