Chromatographic columns are precious consumables in chromatographic analysis. In the face of column contamination, we always want to restore their performance by cleaning and regeneration. Welch’s technicians recommend that teachers follow the instruction manual for flushing, but there are always teachers who want more ways to bring the column back to life.

Today, Welch will bring you an upgraded version of the liquid chromatography column regeneration method. Please pay attention. Although this article is not a rumor, it is also an unofficial method. Please consider it before using it.

Cleaning and Regeneration of Silica-Based Reversed-Phase Chromatographic Columns

  1. Routine sample contamination column cleaning and regeneration

There are various cleaning and regeneration methods for silica-based reversed-phase columns contaminated by routine samples. Welch generally recommends that customers wash with methanol-acetonitrile-isopropanol-acetonitrile for 20 times the column volume in the abnormal flushing of ordinary conventional reversed-phase columns.

There are also some companies on the market that recommend the use of stronger solvents for elution. For example, use dichloromethane:methanol (96:4, V/V), add 1% ammonium hydroxide to rinse, and the added ammonium hydroxide has a good cleaning effect on the pollutants accumulated at the column head. For another example, while washing the chromatographic column with water, inject 4 equal portions of 200 μL dichloromethyl sulfone, and then wash with methanol, chloroform and methanol in sequence. Rinse the chromatographic column with N,N-dimethylformamide and acetone in sequence. These methods are time-consuming, but the special solvents used, such as ammonium hydroxide, dichloromethyl sulfone, N, N-dimethylformamide, etc., all have certain damage to the chromatographic stationary phase.

  1. Cleaning of metal ion contaminated chromatographic column

When the chromatographic column is contaminated by metal ions, general cleaning methods cannot be effective, and special cleaning methods are required. Phosphoric acid and 0.1mol/L citric acid have a good cleaning effect on the metal ions adsorbed on the chromatographic column, so they are often used to clean the chromatographic column contaminated by metal ions [1] ethylenediaminetetraacetic acid (EDTA) can Many metal ions form complexes, and 0.05mol/L EDTA aqueous solution can also be used to remove metal ion pollutants in the chromatographic column.

Although these reagents can remove metal pollutants, they also have some defects, such as the poor wettability of aqueous solution to the stationary phase, the cleaning effect is not ideal, phosphoric acid has a certain destructive effect on the bonded phase on the stationary phase, EDTA brings sodium ions, which affect the performance of the stationary phase.

  1. Cleaning of protein-contaminated chromatographic column

Proteins have large molecular weights, have both hydrophilic and hydrophobic properties, and are sensitive to many physical and chemical factors. In general, pure organic solvents such as acetonitrile or methanol cannot dissolve peptides and proteins well and cannot effectively clean the chromatographic column, so some special cleaning methods are required.

The common method now is to wash 20 times the column volume with the mobile phase without buffer salt – 0.1% TFA (trifluoroacetic acid) aqueous solution – acetonitrile: isopropanol (1:2, V/V, containing 0.1% TFA), Or wash the column with 0.1% TFA (trifluoroacetic acid): acetonitrile (1:1, V/V) at a flow rate of 0.2 mL/min overnight.

When the above methods are ineffective, some harsh reagents, such as guanidine hydrochloride, dimethylformamide and surfactants, can be used. For example, when washing with 50% isopropanol aqueous solution, mix 6 mol/L guanidine hydrochloride aqueous solution and isopropanol at a ratio of 1:1, inject 250 μL of the sample, or wash with 30% dimethylformamide aqueous solution for no more than 30 minutes. Then rinse with water and methanol repeatedly. When washing the chromatographic column with conventional mobile phase, inject 500 μL of 1% sodium dodecyl sulfate (SDS), and then wash with a gradient of 5% to 95% acetonitrile water (containing 0.1% TFA), and the effect of removing protein contaminants is also good . [2] If the type of protein contamination has been determined, the corresponding proteolytic enzyme (such as 1% trypsin or papain) can be injected into the chromatographic column to hydrolyze the protein into peptides, and then eluted with salt solution.

Protein contamination of the chromatographic column, whether it is cleaned with high-concentration phosphate solution or urea or guanidine, will cause greater damage to the chromatographic column and damage the chromatographic instrument at the same time. Among them, the injection of 1% SDS method to clean the chromatographic column not only saves time, but also obtains better results, which is a better choice among the existing cleaning methods.

Cleaning and Regeneration of Ion Exchange Columns

Ion-exchange chromatographic columns are used for a long time under extreme pH range and high salt concentration conditions, or after analyzing biological samples such as proteins, salt ions and proteins will be adsorbed on the surface of the stationary phase, resulting in a decrease in the separation ability of the chromatographic column and an increase in column pressure. Poor peak shape.

For contaminated ion-exchange columns, Welch’s Ultisil® XB-SCX and Ultisil® XB-SAX recommend flushing 20 column volumes with a buffer-free mobile phase – 10% methanol.

If the conventional method cannot improve the performance of the chromatographic column, according to the nature of the chromatographic column and the specific pollution situation, choose a strong acidic or alkaline solution, a high salt solution, a buffer solution containing an organic solvent, a solvent such as urea, or a surfactant ( Non-ionic surfactant), buffered saline solution, certain protein denaturants (such as guanidine hydrochloride and urea, etc.), and one or a combination of proteases for cleaning and regeneration.

Cleaning and Regeneration of Size Exclusion Chromatography Columns

Size exclusion chromatography is a mode of chromatography that separates substances based on molecular size.

For Welch Xtimate® SEC columns, it is recommended to wash 20 times the column volume with 10% acetonitrile or 5% acetonitrile-90% acetonitrile for daily flushing. However, some samples may adsorb to the inlet frit or packing materials after repeated use. When accumulated to a certain extent, there will be an abnormal phenomenon of pressure increase and peak broadening, which requires special flushing. The basic principles of cleaning solution selection for this rinse: 1) low pH salt solution helps to remove basic proteins, 2) organic solvents help to remove hydrophobic proteins, 3) co-solvents help to remove Strongly adsorbed substances (such as through hydrogen bonding, etc.).

Note: Use co-solvents (such as 6-8M urea or 0.2-0.3% sodium lauryl sulfate) only if neutral salt solutions or organic solvents have no obvious improvement.

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