If You Love Flower Gardening, You Know Different Flowers Have Different Needs; Similarly, 6 Things to Consider When Using Liquid Chromatography Columns, to Keep Them in Optimal Condition and Avoid Damaging Them.
01 Filter the Mobile Phase and Samples
Filtering is necessary for the mobile phase and samples used in liquid chromatography. The mobile phase is usually a mixture of water or buffer solution and organic solvents. In principle, all mobile phases passing through the chromatography column should be filtered, but in practice, it depends on the specific circumstances. When using chromatography-grade organic solvents and quartz sub-boiling water or other ultrapure water for the water component, filtering may not be necessary if the quality is ensured and not contaminated.
When the mobile phase contains buffer salts, filtering is necessary. As mentioned earlier, buffer solutions should be filtered again before use if they have been stored for a while, as the presence of buffer salts can lead to bacterial growth. It is recommended to prepare buffer solutions as needed to avoid this issue.
Samples to be injected into the system should be filtered through a 0.45μm (or preferably 0.22μm) membrane filter before being added. It is important to distinguish between water-based and oil-based samples, and not to mix them to prevent membrane dissolution and contamination.
The containers used to hold the mobile phase and the online filters in the chromatography system should be regularly cleaned and replaced to prevent overloading and ensure effective filtering.
02 Purifying the Sample
When a sample contains chemical components that can damage the chromatography column, such as proteins, sugars, other organic molecules that cause permanent adsorption, strongly adsorptive substances, and ion groups that may damage the column packing material, it is necessary to separate and purify the sample as much as possible before injection to remove excess impurities.
03 Avoiding Excessive Column Pressure
Although high-performance liquid chromatography columns can withstand pressures up to 6000 psi (pounds per square inch), excessive and rapid pressure changes can shorten the lifespan of the chromatography column. To avoid this, the following methods are recommended:
- When the column pressure is too high, use a lower flow rate to keep the pressure below half of the maximum allowable pressure.
- When using a high flow rate, apply a flow rate gradient to gradually increase the flow rate.
- When using a manual injection valve, switch the valve as quickly as possible to avoid pressure shocks that can cause the column to fail. When using multiple columns in series, avoid switching columns under high pressure.
04 Pay Attention to Temperature and pH
Chromatography columns based on silica gel matrix generally have a maximum recommended temperature of 60℃, with temperatures below 40℃ being preferable. It is especially important to reduce the temperature when the pH of the mobile phase approaches the upper limit.
High temperatures can accelerate the hydrolysis of the bonding phase and the dissolution of silica gel, leading to changes in the properties of the filler, column bed collapse, decreased column efficiency, and altered peak shapes.
Please pay attention to the pH range specified in the chromatography column manual. Most silica-based chromatography columns on the market have a pH range of 2-10, but extreme pH conditions should be avoided in practice. (For pH≤2, it is recommended to use the Shimadzu Ultimate® LP series column; for pH≥8, it is recommended to use the Shimadzu Xtimate® series column). If your existing chromatography column is used under alkaline conditions (pH≥8), the following methods can be used to avoid column damage:
- Install a pre-column (saturated column) between the pump and injection valve to saturate the mobile phase.
- Lower the operating temperature.
- Immediately clean the column thoroughly with a “mild solvent” that is miscible with the mobile phase used after the analysis is complete.
05 Correct and Timely Column Wash
Before starting the experiment, it is important to know what solvent is currently in the column. For new chromatography columns, the default solvent is usually the mobile phase used in the evaluation report unless otherwise specified. The current solvent in the column must be miscible with the mobile phase used for the sample analysis. If they are not miscible, a third solvent that is miscible with both should be used for transition.
It is recommended to wash the column before and after each use to ensure proper column performance and prevent contamination. For example, the column can be washed with a strong solvent to remove residual components, followed by a weaker solvent to remove impurities that may have accumulated during use. The washing procedure should be carried out carefully to avoid damaging the column or causing bed collapse. The specific washing procedure may vary depending on the type of column and the nature of the sample being analyzed. It is important to follow the manufacturer’s instructions and use appropriate protocols to ensure optimal column performance.
06 Adding a guard column
The guard column is used to filter out chemical “junk” from the mobile phase and sample, as well as effectively removing insoluble particles from both. Although modern chromatographs are equipped with online filters of various pore sizes at the mobile phase inlet, sample injection valve, and both ends of the chromatographic column, these filters can only remove insoluble particles and cannot remove chemical contamination. Therefore, adding a guard column is necessary, especially when analyzing complex mixtures such as traditional Chinese medicine, Chinese patent medicine, and biological samples, which is an essential measure to protect the analytical column.
The packing material of the guard column should be consistent with that of the analytical column, and the particle size can be larger than that of the analytical column. The guard column is a consumable, and after a certain number of sample analyses (50-100 times), if there is a significant increase in column pressure, deterioration in peak shape, or baseline drift, the guard column should be replaced.