Maintenance of HPLC Column

Maintenance of HPLC Column

After the column is used for a period of time, the column is often contaminated, and the column efficiency will be reduced to some extent. The column can be protected by an appropriate method to extend the service life of the column.

Treatment of sample and mobile phase: The dissolved sample is filtered through a filter before injection to remove insolubles. Pretreatment of the sample is required if necessary. If there are impurities in the mobile phase that will affect the efficiency of the column, the solvent should be chromatographed as much as possible, at least the analytical grade, and the mobile phase should be filtered with a microporous membrane before use. Install a filter in front of the infusion tube of the mobile phase. The filter is periodically cleaned with methanol, and if the effect is not good, it can be washed with 10% dilute nitric acid. For heavily clogged filters, burn on the flame before cleaning.

Use of guard column (pre-column): For more expensive columns, a guard column can be added in front of the column to prevent impurities in the sample from contaminating the analytical column. For the preparation of the column, it is especially important to use a guard column because of its large injection volume.

Routine treatment: silica gel, alumina, polar bonded phase chromatography column after each use, rinse with a low flow rate of a long time dichloromethane or n-hexane solvent; bonded phase silica gel column, ion exchange chromatography column and gel The column was first rinsed with distilled water, rinsed with methanol, and then rinsed overnight with a solution of methanol and distilled water in a certain ratio.

Columns with reduced column efficiency after a period of use can be regenerated as follows. The regeneration treatment includes activation (right, left) and purification (left and right). The silica gel, alumina, and polar bonded phase columns were washed in the following order: trimethylpentane or hexane, trichloroethane, ethyl acetate, acetone, ethanol, water. Bonded phase silica gel column: distilled water, methanol (a small amount of dimethylformamide can be added during the washing). Ion exchange resin column: high concentration of NaCl solution (1-2molL of NaCl solution) can regenerate most of the resin, and a small amount of substances closely combined with the resin can be washed with a low concentration alkali solution (such as 0.1molL NaOH solution). The organics are adsorbed in the stationary phase and rinsed with a low pH buffer. The alkaline organics are rinsed with high pH buffer and then rinsed with distilled water, methanol, dichloromethane, methanol, distilled water. Gel column: Since the gel column is separated according to the molecular weight of the substance to be separated, it is usually rinsed with dilute sodium hydroxide or non-ionic detergent (0.2%-1% NP-40 or Lubrol). Remove most of the bound material. If some contaminants still cannot be removed, the hydrophobin can be removed by rinsing with 24% or 30% acetonitrile overnight. The hydrophilic protein can be removed with 30%-50% acetic acid and decomposed by treatment with proteolytic enzyme. The trace amount of pepsin remaining in the gel is then rinsed with distilled water, methanol, and distilled water.

Treatment of contaminated columns: Insoluble matter in the mobile phase or sample is deposited on the column head due to the retention of strong material contamination. It can be washed by the following methods: removing the lipids can be washed with tetrahydrofuran, acetonitrile or methanol; removing the protein can be acetonitrile, propanol and 1 Gradient elution of % trichloroacetic acid; some highly hydrophobic compounds can be eluted with acetonitrile or methanol while repeatedly injecting 100-200 ml of tetrahydrofuran.

Stigma treatment: For the case where the stigma is heavily contaminated and the solvent is used to flush the invalid column, only the column is opened to remove the packing at the top of the column. Reinstall the stainless steel sintered filter, check the column bed, common pits or contaminated color. Filler, remove irregular bed layer and colored filler, make the bed white and completely horizontal, then use methanol as paste-like filler homogenate, and paste the paste filler homogenate on the column to remove methanol from the homogenate by gravity. The liquid is repeated until the filler level.

2018-10-15T01:24:44+00:00 October 15th, 2018|