Use, Care and Storage of Silica Based HPLC Columns

Silica is a well-suited stationary support for HPLC columns. It offers excellent mechanical stability and has the physicochemical properties that accommodate a variety of bonding chemistries. Silica bonded phases are compatible with a broad range of organic solvents.

pH Stability
Silica based HPLC columns are stable within a pH range of 2 to 8.Short exposure to slightly higher or lower pH conditions should not harm the column, but extended use periods outside the recommended range can damage the column irreparably and adversely affect performance.

Mechanical Stability
Bonded phase silica columns are mechanically very stable up to 6000 psi (~400 bar). Pressure shocks to the column should be avoided to prevent bed channeling which will result in peak splitting.

Mobile Phase Eluents
Silica based stationary phases are compatible with all organic solvents in the pH range of 2 to 8. Mobile phase eluents should be free of particulate matter that could foul the column resulting in a higher back pressure.Soluble impurities in the organic solvents used to prepare the mobile phase can be irreversibly adsorbed by the stationary phase. This can have an adverse effect on the selectivity and peak shape.

In gradient elution, impurities in the solvent can cause ghost peaks. Ghost peaks are peaks that always appear at the same position in the chromatogram. Their origin is not the sample, but the impurities from the solvents or solvent additives.

Guard Columns
Guard cartridge columns are recommended for samples that are known to have components that are irreversibly adsorbed by the stationary phase. Guard columns are small columns, typically 1 cm long, that are attached to the top of the column. They usually are prepared with the same packing media that is in the separation column. Guard columns are highly recommended for preparative applications.

Column Storage
Reverse Phase columns can be stored overnight in the eluent. For longer storage, RP columns should be flushed with 10 column volumes of pure water to remove buffer salts, if present, and then flushed with 10column volumes of methanol and capped. For a period longer than 60 days without use, storage in isopropanol is recommended.

Normal Phase columns can be stored overnight in the eluent. For longer storage, NP columns should be flushed with 10 column volumes of heptane and capped.

Column Regeneration
Impurities from the sample or mobile phase can adsorb to the head of the column and cause changes in selectivity, peak splitting and peak asymmetry. In many cases columns can be regenerated by the following procedures.

Reverse Phase Columns

• Flush with 20 column volumes of water
• Flush with 20 column volumes of acetonitrile
• Flush with 20 column volumes of 95/5 acetonitrile/DMF
• Flush with 20 column volumes of acetonitrile