Management and Maintenance of High Performance Liquid Chromatography(HPLC) column

1. Factors affecting the service life of chromatographic columns

1.1 Mobile phase
When an aqueous solution is used as the mobile phase, microorganisms such as bacteria in the aqueous solution are easy to grow. When an aqueous solution of the organic acid buffer is used to protect the column, some molds may breed in the chromatographic column and block the gap between the stationary phase particles.

Due to the advent of wet packing technology, the diameter of the commonly used HPLC chromatographic column packing is generally less than 10um, and the particulate impurities in the mobile phase are easy to deposit on the column head first and then slowly block the column.

The pH value of the mobile phase also has an impact on the chromatographic column, especially for chemically bonded silica gel fillers. The optimal pH range of the aqueous solution is between 2 and 7.5. When the pH is greater than 8, silica gel will release and generate flocs to block the HPLC column, and It is difficult to recover, and the column efficiency is quickly reduced, or even completely invalid.

When an organic solvent such as methanol or acetonitrile is added to the buffer, the solubility of the salt will decrease, and the salt will precipitate and block the column. At the same time, the organic solvent and salt in the mobile phase will corrode the sieve plate of the chromatographic column head and cause the column head to sink.

The polarity of the mobile phase also has a certain effect on the column. For silica gel columns, more polar substances such as methanol, water, and glacial acetic acid will destroy the packing. On the contrary, for chemically bonded silica gel, less polar substances such as n-butanol and dichloride Methane, etc. also play the same role. The alkaline solution will damage the cation exchange resin chromatographic column, while the acid solution is easy to damage the anion exchange resin column.

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1.2 Sample and stationary phase
If a small or trace amount of sample cannot be dissolved in the mobile phase, it will block the column when passing through the stationary phase. Particulate impurities in the sample can also clog the column. Sample components such as sugars, proteins, etc. will be irreversibly adsorbed when passing through the column so that the active sites of the stationary phase are covered. The sample components will also react with the stationary phase, especially for the amino column, because the amino group is easy to form Schiff condensation with anhydrides and ketones, which reduces the active sites and shortens the retention time.

Chromatographic column bonding phase group shedding, stationary phase decomposition, and active group denaturation will affect the significant changes in column efficiency and retention time. Most of the bonded phases are connected to the silicon matrix through silicon-oxygen-silicon bonds (Si-O-Si) to form a stationary phase Si-O-Si-R with organic groups (R is CnH2n+1). The silicon-oxygen-silicon bond may be broken by hydrolysis in an aqueous solution, causing the bonding group to fall off.

2. Management of HPLC columns

2.1 Column management
Formulate and strictly implement standard operating procedures for column management. Each newly purchased chromatographic column must be registered and registered contents including production company, product name, model, specification, purchase time, start-up time, unit price, column protection solution, etc. Chromatographic columns are classified and stored, each uses a chromatographic column box, and a label is attached to the box to indicate the category, such as silica gel column, chemically bonded silica gel column, cyano or amino column, ion exchange resin column, and gel column. The column instructions are packed in a compact bag and attached to the registration book. Each time a chromatographic column is activated, a label is attached to the chromatographic column to indicate the date of activation.

Each column is maintained every two months, especially for columns that are used less frequently. Because of the long storage time, it is easy to dry the column. Make a maintenance record every time.

For columns that have really failed, make a record of decommissioning, do not discard them at will, and store them specially. If the packing of the currently used column needs to be filled, you can use the stored packing of the column. In the quality inspection department of the unit engaged in production, it can be used to detect the intermediates of the product for the column that has been used for a long time, and the efficiency is reduced.

2.2 Column maintenance
After the chromatographic column is used for a period of time, it is often encountered that the column is contaminated, and the efficiency of the column will be reduced to a certain extent. The use of appropriate methods to protect the chromatographic column can prolong the service life of the chromatographic column.

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2.3 Sample and mobile phase handling
The dissolved sample is filtered with a filter membrane before injection to remove insoluble matter. If necessary, sample pretreatment is required. Impure substances in the mobile phase will affect the efficiency, so try to use the chromatographic grade solvent as much as possible, at least the analytical grade, and filter the mobile phase with a microporous membrane before using it.
Install a filter before the infusion tube of the mobile phase. The filter is cleaned regularly with methanol ultrasonic, if the effect is not good, it can be soaked and cleaned with 10% dilute nitric acid. For severely clogged filters, they can be cleaned after burning the flame.

2.3.1 Use of guard column (pre-column)
For more expensive chromatographic 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 preparative column, the use of a guard column is particularly important because of its large injection volume.

2.3.2 Routine treatment
After each use of silica gel, alumina, and polar bonded phase chromatographic columns, flush them with a solvent such as dichloromethane or an n-hexane at a low flow rate for a long time; for the bonded phase silica gel chromatographic columns, ion-exchange chromatography columns, and gel chromatographic columns, first rinse with distilled water, then with methanol, and then with a solution of methanol and distilled water mixed in a certain ratio and rinse overnight.

For the chromatographic column whose column efficiency has decreased after being used for a period of time, the following method can be used for regeneration.

The silica gel, alumina, and polar bonded phase chromatographic columns are washed in the following order: base pentane or hexane ← → trichloroethane ← → ethyl acetate ← → acetone ← → ethanol ← → water. As for the bonded phase silica gel, the column is washed with distilled water and then methanol. During the washing process, we can add a small amount of dimethylformamide.

2.3.3 Ion exchange resin column
Most of the resin can be regenerated by the NaCl solution with high concentration (1-2molL). A few substances that are tightly bound to the resin such as grease can be washed with a low-concentration alkaline solution (such as 0.1molL NaOH solution), and acidic organic matter is adsorbed on the stationary phase. Rinse with low pH buffer, alkaline organics with high pH buffer, and then with distilled water←→methanol←→dichloromethane←→methanol←→distilled water.

2.3.4 Gel chromatography column
Based on the separated substances’ molecular weight, the gel column separates the substances. In general, wash it with non-ionic detergent (0.2%-1% NP-40 or Lubrol) or dilute sodium hydroxide to get rid of most of the binding substances. It is usually washed with dilute sodium hydroxide or non-ionic detergents (0.2%-1% NP-40 or Lubrol) to remove most of the bound substances.

If some contaminants still cannot be removed, rinse with 24% or 30% acetonitrile overnight to remove hydrophobins, 30%-50% acetic acid can remove hydrophilic proteins and treatment with proteolytic enzymes can decompose the remaining traces of pepsin in the gel, and then rinse with distilled water ←→methanol ←→distilled water.

2.3.5 Disposal of contaminated column
Due to the contamination of strong retained substances, the insoluble matter in the mobile phase or sample is deposited on the head of the column. The following methods can be used to wash: Remove lipids can be washed with C4H8O, acetonitrile, or methanol; Remove proteins can be eluted with acetonitrile, propanol, and 1% trichloroacetic acid; Some highly hydrophobic compounds can be eluted with acetonitrile or methanol while repeatedly injecting 100-200ml of C4H8O.

2.3.6 Head of column treatment
For chromatographic columns where the column head is seriously clogged and polluted and the solvent is ineffective, only open the column to remove the packing on the top of the column and reinstall: first remove the stainless steel sintered filter, check the column bed, common dents or contaminated colored packing, remove the irregular bed and colored fillers, make the column bed appear white and completely horizontal, then use methanol as the paste filler homogenate, drop the paste filler homogenate on the column and drain the methanol solution from the homogenate by gravity. Repeat the above steps until reaching the filling level.

2.3.7 Column storage
First, the column must be cleaned before it can be stored. If it is stored in aqueous solvents or water, the column may cause the growth of microorganisms.

Polar chromatographic columns can be flushed with appropriate solvents such as dichloromethane;

Flush the bonded phase chromatography column with methanol; The anion exchange chromatography column is flushed with 0.002% chlorhexidine (chlorhexidine) buffer, and the cation exchange chromatography column is flushed with 0.005% thimerosal buffer; The gel chromatography column is flushed with 0.02% sodium nitride or 20% ethanol in the buffer. Then seal both ends of the column to prevent the evaporation of the solvent to dry the column and cause the geometrical change of the column structure.