Column Lifespan - Part 1

HPLC Troubleshooting Course

The column is a crucial component of (U)HPLC, as it is where chromatographic separation occurs. However, columns are consumable items with a limited lifespan. This is especially relevant for high-cost columns, where maximising longevity is essential. Proper handling and maintenance significantly impact both the lifespan of the column and the number of successful injections.

The first section of "Column Lifespan" focuses on general best practices for handling HPLC columns, including strategies to prevent blockages from particles and crystallisation, proactive maintenance measures, and corrective actions when issues arise.

1. Adhering to Column Specifications

Each analytical separation column has specific operating conditions that must be followed to ensure optimal performance. The key parameters include:

 

  • Pressure range
  • pH value
  • Temperature
  • Solvents

Operating outside these parameters can lead to shifts in selectivity, loss of retention time, or even permanent column damage.

Additionally, proper handling is critical to preserving column integrity. Avoid sudden pressure fluctuations, as these can cause cracks in the packing material or create dead volumes. To prevent this:

  • Always allow the HPLC system to fully depressurise before disconnecting the column.
  • When starting the system or heating the oven, equilibrate the column at a lower flow rate than the method flow rate. Higher solvent viscosity at the beginning can lead to elevated back pressure and potential pressure shocks.
  • Some HPLC systems, such as Shimadzu HPLC, feature a built-in function called FlowPilot, which gradually increases the flow rate while the system reaches the target oven temperature. This controlled ramp-up minimises stress on the column, helping to extend its lifespan. For more details, watch the video at the bottom of the page.

 

Physical damage can also shorten a column’s lifespan. Avoid dropping the column, as impact can compromise its internal structure. Always store your columns according to the manufacturer's recommendations in the appropriate solvent. Store the column tightly closed with the supplied end caps and preferably in the supplied packaging.

 

2. Column Blockage Due to Particles

 

Small dirt particles from the system, eluent, or sample can lead to column blockage, reducing performance and lifespan. The following preventive measures can help avoid such issues.

Removing Particles from Samples and Eluents

Since column longevity depends on sample quality, proper sample preparation is essential. Particles must be removed before injection. Syringe filters offer a simple and cost-effective solution, but if filtering is unsuitable for your samples, consider alternative techniques such as centrifugation or solid-phase extraction (SPE).

Similarly, the eluent should be free of particles to prevent blockages in both the column and system. Organic impurities in water can also cause unexpected ghost peaks in chromatograms. This issue was previously covered in Part 7 of this troubleshooting course.

Using Precolumns and Ghost Traps

Precolumns (guard columns) are placed before the main analytical column to trap impurities and extend column lifespan. These are typically filled with the same packing material as the main column and are no more than one-tenth its size. Precolumns are easily replaceable and far more cost-effective than replacing an analytical column. If using a precolumn, always account for its minor impact on chromatographic separation during method development.

Additionally, ghost traps can be installed between the pump and injector to remove mobile phase impurities, further protecting the system.

Preventing Crystallisation

When switching eluents, always consider their miscibility to avoid precipitation. Buffers should never be replaced directly with organic solvents, as this can lead to precipitation of buffer salts. Instead, always flush the system with water between buffer and solvent changes.

Moreover, avoid leaving an eluent mixture containing an aqueous salt solution in the system after analysis, as prolonged exposure can lead to crystallisation and potential system blockages.

Countermeasures for Column Blockages:

  • Check for Blockages in Other System Components: Before assuming the column is blocked, inspect other parts of the system, such as the tubing, frits, and injector, for obstructions.
  • Rinse with a suitable solvent: Flushing the column with an appropriate solvent can help dissolve and remove contaminants. Always refer to the column's specifications to determine compatible solvents
  • If the manufacturer allows, connect the column in the reverse direction and flush it at a low flow rate to remove insoluble substances. Ensure the eluate containing impurities is directed to waste and does not pass through the detector to prevent contamination. Always consult the column’s product information for specific guidelines.

In the next part, we will discuss further factors that can contribute to the longevity of the column.

Your Shimadzu LC Team

Related Resources