7 misunderstandings of HPLC liquid chromatography column usage, have you been hit?

7 misunderstandings in the use of liquid chromatography columns, have you been hit?

HPLC columns are the core components of liquid chromatographic analysis. Proper use and maintenance of the column are critical. Improper use will reduce the column efficiency, shorten the service life, or even cause damage. The following seven issues should be focused on during the usage of HPLC columns.

1. Pressure and Temperature

Sudden changes in temperature or dropping the column from a high point can affect the condition of the packing materials inside the column. Sudden increases or decreases in column pressure can also affect the packing inside the column. Therefore, the flow rate should be adjusted slowly to maintain the stability of the packing inside the column.

2. Composition Change of Solvent

If you need to change the composition of the solution of the test sample, you need to change the composition of the solvent gradually. In particular, for reversed-phase chromatography, does not directly change all organic solvents to water. And when using an eluent with a high sub-elution capacity to flush the column, the replacement of the mobile phase in the flow path system should be gradually transitioned with a solvent that is miscible. The volume of each mobile phase should be about 20 times the volume of the column, i.e. 50~75ml is required for routine analysis.

3. Column Backflush

Some HPLC columns cannot be backflushed. Only if the column instruction manual indicates that the column can be backflushed. Backflush can remove the impurities left on the column head.

4. Improper Usage of Mobile Phase

Choose a suitable mobile phase (especially pH) to avoid the destruction of the stationary phase. A pre-column can be attached to the front of the injector. When the analytical column is bonded silica-gel, the pre-column could be silica, which allows the mobile phase to be "saturated" with silica before entering the analytical column, avoiding the dissolution of the silica matrix in the analytical column.

5. Direct injection of samples into the column without treatment

Avoid injecting complex samples, especially biological samples, directly into the HPLC column. It is necessary to pre-treat the sample or connect a protective column between the injector and the column. The guard column is usually a short column packed with a similar stationary phase. The guard column needs to be replaced frequently.

6. Store the buffer in HPLC column for a long time

When storing the HPLC column, the column should be filled with acetonitrile or methanol, and the column joints should be tightened to prevent the solvent from evaporating and drying. It is absolutely forbidden to leave the buffer solution in the column overnight or even longer time.

7. Store HPLC columns without treatment

HPLC columns should be cleaned with a suitable solvent after use. ODS columns should be rinsed with methanol until baseline equilibrium. When a salt buffer solution is used as the mobile phase, the column should be rinsed with a salt-free mobile phase after use. Compounds containing halogenated elements (fluorine, chlorine, bromine) may corrode stainless steel tubing and should not be exposed to them for long periods of time. Columns mounted on liquid chromatographs that are not used frequently should be opened and flushed for 15 minutes every 4-5 days.

Note: The test conditions in this article are only according to common experience. They need to be adjusted based on specified conditions. We are not responsible for any adverse factors arising from this article.

GALAK ChromatographyAuthor
Tian Jing

Manager & Engineer in GALAK Chromatography. Master of Chemical Engineering.
During my college study, I found liquid chromatography to be a profound subject. I know the painful struggle a novice needs to go through to get started. I share this article to help you solve your problems quickly.

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