C18 Chromatographic Column Usage Guide

Usage Guide:

  1. Before using a new column, it should be flushed with pure organic solvents (such as methanol or acetonitrile) at a low flow rate. The recommended flushing volume is 20 times the column volume to ensure that the stationary phase is fully wetted and the carbon chains are thoroughly extended, achieving peak performance of the chromatographic column.
  2. After each day’s experiments, the chromatographic column should be flushed, especially when the mobile phase contains acids or salts. It is necessary to rinse out the acids or salts from the column, and it is typically recommended to use a 10% methanol-water solution with a flushing volume of 20 times the column volume.
  3. Before long-term storage, thoroughly flush the chromatographic column for at least 3-4 hours. It is recommended to store the column in a pure organic solvent or a high proportion organic solvent solution (such as 90% methanol-water).
  4. Solvents used for daily storage (i.e., when continuing usage the next day) should preferably be the same as in step 3.
  5. Due to the strong hydrophobicity of the stationary phase in C18 columns (except for polar modified treatments), it is advisable to avoid high water phase conditions during usage. Excessive water phase ratios can cause hydrophobic collapse of the stationary phase, leading to rapid deterioration of column efficiency or even irreversible negative effects. It is recommended to keep the water phase ratio below 90% during usage.
  6. Increased column pressure and decreased column performance are often caused by contamination. Regenerating the column can restore some of its efficiency.
  7. A sudden increase in column pressure is usually caused by salt precipitation or screen blockage. If it is due to salt precipitation, flushing with a 10% methanol-water solution at a low flow rate until the pressure is restored is sufficient. If it is determined not to be salt precipitation, counter-flush the chromatographic column with pure methanol or a 10% methanol-water solution (reverse the column but do not connect to the detector). If no improvement is observed after half an hour of counter-flushing, other causes should be investigated.

Chromatographic Column Regeneration Steps:

Flush the chromatographic column in the following order, with a flushing volume of 20 times the column volume: 10% methanol-water → methanol → isopropanol (or chloroform) → methanol → 10% methanol-water. (When using isopropanol in step 3, due to its high viscosity, the flow rate should be appropriately reduced, with a recommended flow rate of 0.2 ml/min.)

Column Volume Calculation Table:

Column sizeColumn VolumeFlush volume (20 times column volume)
150×4.6 mm2.5 mL50 mL
200×4.6 mm3.3 mL66 mL
250×4.6 mm4.2 mL84 mL