Chromatographic columns are the heart of any chromatographic system, whether it be gas chromatography (GC), high-performance liquid chromatography (HPLC), or other types of chromatography. These columns are responsible for the separation of complex mixtures into their individual components, making them indispensable tools in analytical chemistry, pharmaceuticals, environmental analysis, and many other fields.
Given their critical role, proper use and maintenance of chromatographic columns are essential to ensure optimal performance, longevity, and reliable analytical results. In this comprehensive blog post, we will explore eight key points on the use and maintenance of chromatographic columns, providing practical tips and best practices to help you get the most out of your columns and maintain the integrity of your analytical workflows.
1. Avoid drastic changes in pressure and temperature and any mechanical vibration. Sudden changes in temperature or dropping the chromatographic column from a high place will affect the filling condition in the column; sudden increases or decreases in column pressure will also affect the filling material in the column. Therefore, the flow rate should be adjusted slowly, and the valve should not be rotated too slowly when the valve is injected.
2. The composition of the solvent should be changed gradually, especially in reversed-phase chromatography, and should not be changed directly from organic solvent to all water, or vice versa.
3. Generally speaking, the chromatographic column cannot be backflushed. Only when the manufacturer indicates that the column can be backflushed, can the impurities left at the column head be backflushed to remove them. Otherwise, backflushing will quickly reduce the column efficiency.
4. Choose to use a suitable mobile phase (especially pH) to avoid the stationary phase from being destroyed. Sometimes a pre-column can be connected in front of the injector. When the analytical column is bonded silica gel, the pre-column is silica gel, which can make the mobile phase “saturated” with silica gel before entering the analytical column to avoid the silica gel matrix in the analytical column from being dissolved.
5. Rinse the chromatographic column frequently with a strong solvent to remove impurities retained in the column. When cleaning, the replacement of the mobile phase in the flow system should be gradually transitioned with miscible solvents, and the volume of each mobile phase should be about 20 times the volume of the column.
The following are some cleaning solvents and sequences for chromatographic columns for reference:
Silica gel columns are washed with n-hexane (or heptane), dichloromethane and methanol in sequence, and then washed in reverse order. All solvents must be strictly dehydrated. Methanol can wash away residual strong polar impurities, and hexane reactivates the silica gel surface. Reverse phase columns are washed with water, methanol, acetonitrile, monochloromethane (or chloroform) in sequence, and then washed in reverse order. If the mobile phase used for the next analysis does not contain buffer, the final water washing step can be omitted. A mixed solution of tetrahydrofuran and acetonitrile or methanol can remove lipids. Sometimes dimethyl sulfoxide is also injected several times. In addition, gradient elution with acetonitrile, acetone and trifluoroacetic acid (0.1%) can remove protein contamination. The cation exchange column can be rinsed with dilute acid buffer, and the anion exchange column can be rinsed with dilute alkali buffer to remove salts with strong exchange performance, and then rinsed with water, methanol, dichloromethane (to remove organic matter adsorbed on the surface of the stationary phase), methanol, and water in sequence.
6. When storing the chromatographic column, the column should be filled with acetonitrile or methanol, and the column joint should be tightened to prevent the solvent from volatilizing and drying. It is absolutely forbidden to leave the buffer solution in the column overnight or longer.
7. During the use of the chromatographic column, if the pressure increases, one possibility is that the sintered filter is blocked, and the filter should be replaced or removed for cleaning; another possibility is that macromolecules enter the column and contaminate the column head; if the column efficiency is reduced or the chromatographic peak is deformed, the column head may collapse and the dead volume will increase. In the latter two situations, carefully unscrew the column joint, use a clean small steel to remove the column head filler to a height of 1 to 2 mm (note to remove the contaminated filler) and then level the filler in the column. Then fill the chromatographic column with a stationary phase moistened with an appropriate solvent (the same as in the column), flatten it, and tighten the column joint. After such treatment, the column efficiency is improved, but it is difficult to restore to the level of a new column. The column usually fails at the end of the column. Installing a short column (5-30mm) with the same stationary phase as the analytical column in front of the analytical column can protect and extend the life of the column. The use of a protective column will lose a certain amount of column efficiency, which is worth it. Usually, the life of a chromatographic column can reach more than 2 years when used correctly. Fillers based on silica gel can only be used in the pH range of 2-9. After the column has been used for a period of time, some substances with strong adsorption may remain at the top of the column, especially some colored substances are easier to see adsorbed on the fillers at the top of the column.
8. After a period of use, the top filler of a new chromatographic column may collapse, causing the column efficiency to decrease. At this time, fillers can also be added to restore the column efficiency. After each work, it is best to rinse with an eluent with strong elution ability. For example, the ODS column should be rinsed with methanol until the baseline is balanced. When a salt buffer solution is used as the mobile phase, it should be rinsed with a salt-free mobile phase after use. Compounds containing halogen elements (fluorine, chlorine, and bromine) may corrode stainless steel pipes and should not be in contact with them for a long time. If the column installed on the HPLC instrument is not used frequently, it should be turned on and flushed for 15 minutes every 4 to 5 days.
Want to know more about Chromatographic Columns, please check this article: How to Extend the Lifetime of Your HPLC Columns