How to Clean High Performance Liquid Chromatography Column?

Brief introduction of HPLC column

For the high performance liquid chromatography column, its main components are column, sealing ring, filter, joints, screws, and so on. There are many materials for the column tube, such as polymer, glass, stainless steel, and so on. However, it is recommended that the column tube be made of stainless steel, because stainless steel is resistant to high pressure, corrosion-resistant, and has high hardness.

Compared with materials such as glass, it is not easy to break. But, it’s required to use thick-walled glass or quartz tubes, if the pressure is not higher than 70Kg/cm2. The inner wall of the chromatographic column is required to have a high degree of smoothness, and the inner wall of the stainless steel column will be polished many times. This is to improve the column efficiency and reduce the wall effect. The 0.1μm high-finish chromatographic column tube can quickly analyze while maintaining ultra-high column efficiency and long service life. This can not only reduce the re-validation of method and condition exploration caused by frequent replacement of chromatographic columns but also greatly reduce use cost; with excellent selectivity and separation ability.

In addition to polishing, there are also fluorine plastics coated on the inner wall of the stainless steel column to improve the smoothness of the inner wall. The effect is the same as that of polishing. The column heads at both ends of the chromatographic column are equipped with sieve plates, which are usually made of stainless steel and peek. The column sieve plate that matches the chromatographic column is selected according to the needs. The pore size depends on the particle size of the filler, and the purpose is to prevent the filler from leaking. Generally, the column length, filter size, and flow rate decide the column’s inner diameter.

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High performance liquid chromatography column cleaning

Generally speaking, there are two situations:
1. If the sample analysis only uses general methanol, the mobile phase of acetonitrile, and water (including some acid added in the mobile phase), we can directly clean with 65% acetonitrile/water or pure methanol after finishing the sample 10 times the column volume can be stored (if the time is limited, the flow rate can be increased to 2ml/min within the range that the instrument can bear depending on the situation);
2. If the sample analysis uses a mobile phase containing buffer salts or acids or ion-pairing reagents, the cleaning of the sample after finishing the sample must follow the following 2 steps to clean the column (Regardless of whether your column can be resistant to pure water, a polar column or a general-purpose column).
a. The most important step-use 5% acetonitrile (methanol)/water to wash more than 20 times the column volume of solvent, 1~2ml/min (freely adjust the flow rate according to the time: if the cleaning is not so long due to limited time, the flow rate can be increased appropriately, such as 1.5 or 2ml/min; the LC/MS column should be performed at a flow rate of 0.2~0.5ml/min).
b. Use 65~90% acetonitrile (methanol)/water or pure methanol/acetonitrile, and wash 5~10 times the column volume, 1~2ml/min.

In-Depth Cleaning

Backflushing: Backflushing involves reversing the flow of the mobile phase through the column. This can help dislodge and remove any particles that may be trapped in the column. Follow the instrument and column manufacturer’s guidelines for backflushing.

Solvent Flush: Use a stronger solvent or a different solvent to flush the column. For example, if your routine mobile phase is water/acetonitrile, you might try flushing with methanol or isopropanol. Be cautious and refer to the column specifications to ensure compatibility with the chosen solvent.

pH Adjustment: In some cases, adjusting the pH of the mobile phase can aid in removing certain contaminants. For example, running a buffer at a different pH than usual can help clean the column.

Organic Solvent Soak: Soaking the column in an organic solvent (e.g., methanol or acetonitrile) can help dissolve and remove organic residues. Keep in mind the solvent compatibility with both the column material and the stationary phase.

Ultrasonic Bath: For more thorough cleaning, especially when dealing with stubborn residues, an ultrasonic bath can be used. Be cautious and ensure that the column is suitable for ultrasonic cleaning.

Column Oven Heating: Some columns can be heated in the column oven to higher temperatures to aid in cleaning. Consult the column documentation for recommended temperature limits.