8 Misunderstandings in the Use of HPLC Columns

There are several main components for the HPLC columns, such as the column tube, ferrule (sealing ring), pressure cap, joints, sieve plate (filter), and so on. The most frequently used is stainless steel column tube and for thin tubular columns, there are also fused silicon or glass lining used. There are joints with sieve plates at column ends. It aims to prevent the packing from leaking.

The HPLC column plays a separate role in the chromatography analysis system and is the core component of chromatographic analysis. Correct use and maintenance of the chromatographic column are particularly important. Improper use will reduce the efficiency, shorten the service life and even damage the column. HAWACH introduces 8 common problems that should be paid attention to during the use of the HPLC column. Hawach provides CN HPLC column, NH2 HPLC column, SCX HPLC column, etc. for your choice.

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1. Sudden changes of temperature and pressure should be avoided
It will affect the column packing conditions, if there is a sudden change in the temperature, column pressure, or the chromatographic column is dropped from a high place. Therefore, we should adjust the flow rate slowly to maintain the inside of the column and the stability of the filler.

2. Avoid directly changing the composition of the solvent
When it is necessary to change the composition of the test sample solution, the composition of the solvent should be gradually changed, especially in reversed-phase chromatography, it should not be directly changed from an organic solvent to all water, and vice versa. And when washing the HPLC column with the eluent with strong secondary elution capacity, the displacement of the mobile phase in the flow path system should be gradually transitioned with miscible solvents. Each mobile phase’s volume needs to be about 20 times the column volume. That is, 50~75ml is required for routine analysis.

3. Avoid column backflush
Generally speaking, a chromatographic column cannot be backflushed. Only when the column’s operating instructions indicate that the column can be backflushed can backflush remove impurities left on the head of the column. Otherwise, the effect of backflushing the HPLC column is to rapidly reduce column efficiency.

4. Avoid improper use of mobile phase and damage to the stationary phase
How to avoid damage to the stationary phase should be considered, when choosing a suitable mobile phase (especially pH). If necessary, connect a pre-column to the front of the sampler. To prevent the silica matrix in the analytical column from being dissolved, the mobile phase can be “saturated” with silica gel before entering the analytical column, if the pre-column is silica gel and the analytical column is bonded silica gel.

5. It is forbidden to enter the HPLC column without processing the sample
Injecting complex matrices to samples into the column directly should be avoided, especially for the biological samples. The samples need to be pretreated or connected to a guard column, which is generally packed with a similar stationary phase, between the injector and column. Please note: replace the guard column frequently.

6. It is forbidden to store the buffer solution in the HPLC column for a long time
When storing the chromatographic column, the column should be filled with acetonitrile or methanol, and the column connector should be tightened to prevent the solvent from evaporating and drying. Do not leave the buffer solution in the column overnight or longer.

7. It is forbidden to seal the HPLC column without treatment
After the chromatographic column is used, it must be cleaned with a suitable solvent. For example, the ODS column should be flushed with methanol to balance the baseline; when a salt buffer solution is used as the mobile phase, after use it should be flushed with mobile phase that is salt-free; It should not be in contact with them for a long time, as it may corrode stainless steel pipes if it contains halogen elements (fluorine, Chlorine, bromine) compounds; if the HPLC column installed on the liquid chromatography is not used frequently, it should be turned on and flushed for 15 minutes every 4-5 days.

8. It is forbidden to use contaminated column and treat it well to extend the service life
Operators often get such problems as contaminated columns and reduced efficiency of the column after the HPLC column is used for a period of time. We can prolong the HPLC column service life by taking suitable methods to protect it. The use of guard column (pre-column): For more expensive chromatographic columns, you can add a guard column before the column to prevent impurities in the sample from contaminating the analytical column. For preparative columns, the use of guard columns is particularly important because of the large injection volume.

Sometimes, the mobile phase or insoluble matter in the sample is deposited on the head of the column, because of the strong retention substances’ contamination. It’s forbidden to use contaminated columns. If the columns have been contaminated, the following methods can be used to wash: To remove lipids, you can wash with tetrahydrofuran, acetonitrile, or methanol; To remove proteins, you can use acetonitrile, propanol, and 1 % Trichloroacetic acid for gradient elution; some highly hydrophobic compounds can be eluted with acetonitrile or methanol, while repeated injection of 100-200ml of tetrahydrofuran.

Column head treatment: For chromatographic columns that are seriously clogged and contaminated with solvents and are not washed with solvents, only open the column and remove the packing on the top of the column to reload. First, remove the stainless steel sintered filter, check the column bed, common dented or contaminated colored packing, remove the irregular bed and colored packing, make the column bed appear white and completely horizontal. Then use methanol as the paste filler homogenate, on the column drop the paste filler homogenate and drain the methanol solution by gravity from the homogenate, and repeat until reaching the filler level.

When we use a new column, we should perform a performance test on our own liquid chromatography, that is, use the test conditions and samples on the inspection report attached to the column to determine the efficiency of the column. And, in future use, the HPLC column should be tested from time to time.