HPLC Column Care and Maintenance
Your analytical column is a consumable in your chromatography system, and it must ultimately be replaced. To maximize your column’s lifetime, it is important to take care of and maintain your column. Evidence that your HPLC problems could potentially point to your HPLC column include:
- High System Pressure
- Undesirable Peak Shape (splitting/tailing)
- Changes in Retention/Selectivity
Please note, the above listed problems may also be associated with instrument and method issues, but, in our experience, the column is typically the first to be blamed.
Large System Pressure Change, Excess Back Pressure
When your HPLC system sees a significant increase in pressure, potential problems could be a plugged inlet frit, column contamination, or blockage in the system. It is important to first check the pressure with and without the column in place, as many pressure problems are due to blockages elsewhere in the system. If the excessive pressure is isolated to the column, rinse the column (remove detector from flow path) and/or clean the column. It is best to follow your column manufacturer’s guidelines for column cleaning, but, in general, you flush with stronger solvents than your mobile phase (making sure detector is NOT in flow path).
For reversed phase columns, here is a general guideline:
Use at least ten times column volume of each solvent for analytical columns in order of increasing strength
- Mobile phase without buffer salts (water/organic)
- 100% organic (MeOH or ACN)
- Is pressure back in normal range? If not, discard column or consider more drastic conditions:
- 75% Acetonitrile:25% Isopropanol, then
- 100% Isopropanol
- 100% Methylene Methylene Chloride* Chloride*
- 100% Hexane*
*When using either Hexane or Methylene Chloride, the column must be flushed with Isopropanol before returning to your reversed phase mobile phase Isopropanol before returning to your reversed-phase mobile phase.
In order to prevent column back pressure problems, it is recommended to filter non-HPLC grade solvents, filter buffered solutions, and install an in-line filter between injector and column (trap system debris and particulates before they get to the analytical column). Chrom Tech offers these chromatography supplies, and we are happy to assist with recommending the appropriate HPLC products for your lab.
Undesirable Peak Shape and Changes in Retention/Selectivity
Split peaks can be caused by column contamination, a partially plugged inlet frit, column void in packing bed, and incompatible injection solvent effects. To minimize plugging, when analyzing samples with complex matrices, filtering solvents and good sample preparation are important for HPLC Column Care. Also, it is always recommended that the injection solvent is similar in strength (and composition) to the initial mobile phase conditions.
Changes in retention on the same column, over time, can be attributed directly to the column or other system issues: aging column, column contamination, insufficient column equilibrium, change in pump performance (mobile phase percentage, flow rate), change in column temperature, or other instrument issues.
In summary, for HPLC Column Care and Maintenance, it is important to follow your column manufacturer’s guidelines. In general, to maximize column lifetime, it is recommended to follow good sample preparation techniques, use HPLC grade solvents, filter buffered solvents, and use inline filters to trap particulates before they reach your column. If your column shows signs (high back pressure/poor chromatography) of aging, back flushing is an option. But, even with the best care and maintenance, at some point, every chromatographer will need to make the decision on when to retire their column, and when that time comes, please consider Chrom Tech as your preferred chromatography supplier.