COLUMN OPERATION AND STORAGE

Good column care requires little additional time and will extend the lifetime of your SIELC HPLC columns. The following are general recommendations.

Mobile Phases for Column Operation and Storage

General Guidelines

• • TRY TO AVOID USING ALCOHOLS WITH Primesep (A, 100, 200, C, B2, AB and P), Promix (SP and AP), and Obelisc R columns. Alcohols can esterify carboxylic acids attached to the surface of silica, significantly affecting reproducibility and retention time of some analytes.
  • Do not use or store SIELC HPLC columns in pH outside of recommended ranges. SIELC columns require buffered or acidified mobile phases to be present in the column at all times (Table 1).
  • Do not store SIELC HPLC columns in mobile phase containing modifiers that degrade easily, such as triethylamine (TEA) tetrahydrofuran (THF) and trifluoroacetic acid (TFA). TEA and TFA are more likely to become contaminated when stored at ambient temperatures, and should be refrigerated during long storage. Contamination may change the chromatography or change the column. Use HPLC-grade solvents and additives for your chromatography.
  • Do not store SIELC HPLC columns in a mobile phase containing perchloric acid (HClO₄). As a strong oxidizer, HClO₄ is highly reactive and can cause chemical alterations to the column over time. Prolonged storage in such conditions may lead to column degradation or performance deterioration.
  • To achieve reproducible results, please use a column thermostat. Operating temperature for all SIELC columns is 0-50oC.
  • To achieve reproducible results, always equilibrate SIELC column prior to the injection. In some cases replacement of the mobile phase inside the column does not make the column sufficiently equilibrated. SIELC columns’ stationary phase retain components of the mobile phase as counter ions. To equilibrate the column in this case requires a replacement of all the ions adsorbed in the column stationary phase. Typically, 50 mM buffer would equilibrate a column in 1 hour. If only the concentration of the buffer changed, then 2-4 volumes of the column are sufficient to reach an equilibration state.
  • It is important to operate columns below the specified maximum pressure limits (5000 psi). If you exceed maximum pressure the column can be destroyed. Leaking column is an indication of excessive pressure usage.
  • Limit the use of tetrahydrofuran to 10% with SIELC columns.
  • An arrow on the column label is used to specify direction of flow through the column. Typically you should operate the column in the direction marked. If column need to be washed in opposite direction use reduced flow rate in order to not create pressure which can disturb packing.
  • Use only your fingers to tighten PEEK fittings to the column connection.

Column Cleaning

To clean the column, please refer to the table below.
The column can also be run in reverse at a reduced flow rate 2-3 times.
If your sample matrix contains many compounds, especially multi-charged compounds like proteins, we recommend the use of a guard column and replace it often as soon as you notice degradation in performance, i.e. peak shape, shift in retention, etc.
If you run a high-throughput operation, we recommend the use of FlipLC, which eliminates the use of constantly changing guard columns.

Table 1. Operational pH ranges for SIELC Columns

COLUMN OPERATION AND STORAGE

Column Cleaning by Reverse Flushing

If high backpressure, peak distortion, or loss of efficiency is suspected to be caused by particulate matter or strongly retained contaminants accumulated at the inlet frit, the column may be cleaned by reverse flushing.

Disconnect the column from the HPLC system and reconnect it in the opposite flow direction. Flush the column using the recommended cleaning mobile phase for the specific stationary phase at approximately 25–50% of the normal operating flow rate. Continue flushing for 20–60 column volumes, or until the backpressure stabilizes and the contaminating material has been removed.

After reverse flushing, reconnect the column in the normal flow direction and equilibrate it with the intended mobile phase for at least 10–20 column volumes before resuming analysis.

Recommendations:

• Always use the cleaning solvent recommended for the specific stationary phase.
• Reduce the flow rate during reverse flushing to minimize mechanical stress on the column bed and inlet frit.
• Ensure that all cleaning solvents are fully miscible with the previous mobile phase to avoid precipitation inside the column.
• If high backpressure persists after cleaning, discontinue use of the column and contact technical support for further assistance.

Other Recommendations:

Perform the column performance test using conditions described in Certificate of Analysis and compare with the result in the report. Due to the variations in particular LC systems you may observe slightly different results.