HPLC Column Care, Troubleshooting, Restoration, and Storage
HPLC Column
Care & Storage
Care & Storage
An HPLC column has a finite lifetime. It is recommended that you routinely monitor a column's retention characteristic and performance using standards. If you plan to store the column for a prolonged period of time, use a mobile phase that inhibits microbial growth (a mobile phase containing sodium azide or high concentrations of methanol, acetonitrile, etc.). For optimum performance and column life, follow these recommendations.
- Routinely monitor the column's performance.
- Switch only between miscible mobile phases.
- Avoid precipitating salts in the column.
- Use filtered and degassed mobile phases.
- Do not allow the column to dry out. Keep it tightly capped when not in use.
- For prolonged storage, use a mobile phase that inhibits bacterial and mold growth.1
- Use the column in the direction indicated on the label. An unusually high operating pressure may indicate a plugged inlet frit and may be cleared by reversing flow through the column for 10-20 mL.
- Use Hamilton guard columns to remove particulate matter or impurities that may permanently bind to the analytical column.
HPLC Column Storage
1HPLC columns can be stored in many solvents, but it is most important to store the column in a solvent mixture that will inhibit microbial growth.
Short-Term HPLC Column Storage (up to 2 weeks):
The column can be stored in the working mobile phase.
Long-Term HPLC Column Storage (2 weeks and longer):
For prolonged storage, use a storage solvent that will inhibit bacterial and mold growth such as 40% methanol and 60% water. If the mobile phase was buffer, flush all salts out before flushing with methanol/water storage solvent mixture. Pump the storage solvent mixture through the column for 5 column volumes.
Troubleshooting
Unusually high back pressure, variable retention times, poor peak shape, or diminished column performance may be the result of column fouling or contamination. To rejuvenate column performance, follow the reverse flow column flush procedure below:
1. Disconnect the tubing from the column and reattach the inlet tubing to the outlet of the column. Direct the open end of the column over a waste container; do not connect the column to the detector. This prevents accidental flushing of any loose particulates into the detector.
2. In the reverse flow direction, flush the column with about 5-10 column volumes of a solvent or buffer (the mobile phase is currently being used is a good starting point). Flushing in the reverse flow direction will expel any potential particulate/contaminant matter from the inlet frit or head of the column.
Optional: For additional column performance restoration, with the column remaining in the reverse flow direction and the outlet draining into a waste container, flush with the suggested solution for your column as defined in the Column Restoration Procedures below. NOTE: Before performing a restoration procedure, always make sure that the mobile phases are miscible and that precipitation will not occur. If necessary, wash the column with a suitable intermediate solvent before changing over to the new mobile phase. A minimum of five column volumes of intermediate solvent should be used. See below for more information.
3. After flushing, reconnect the tubing that goes from the outlet of the column to the detector. You can either leave the column in the reverse flow direction, or turn it around so that the system flow is following the flow direction arrow that is printed on the column (the column can be operated in either direction).
4. Flush the column with freshly prepared mobile phase for at least 150 column volumes or until the column is fully equilibrated (level detector baseline is observed).
Restoring Column Performance
The procedures listed below may restore the performance of a column that has become fouled, but the procedures will not improve the performance of a column that is damaged or beyond its useful life. Nor can all stationary phases be restored. The unique supports in PRP-X500 and PRP-X600 columns make it impossible to restore performance with a washing procedure. For technical assistance regarding these columns, please Contact HPLC Support. NOTE: Before performing a restoration procedure, always make sure that the mobile phases are miscible and that precipitation will not occur. If necessary, wash the column with a suitable intermediate solvent before changing over to the new mobile phase. A minimum of five column volumes of intermediate solvent should be used.
Column Restoration Procedures
Follow the appropriate restoration procedure listed below using a flow rate of 1.0 or 2.0 mL/min. unless otherwise specified. Evaluate column performance using the test mix and conditions listed on the Performance Test Report shipped with each column.
| Packing Material | Restoration Procedure | |
|---|---|---|
| PRP-C18 | Flush with 40%:40%:20% (ACN:IPA:H20) for 5-10 column volumes. Equilibrate with starting mobile phase for 5-10 column volumes. | |
| PRP-1 | Flush with 40%:40%:20% (ACN:IPA:H20) for 5-10 column volumes. Equilibrate with starting mobile phase for 5-10 column volumes. | |
| PRP-3 | Flush with40%:40%:20% (ACN:IPA:H20) for 5-10 column volumes. Equilibrate with starting mobile phase for 5-10 column volumes. | |
| PRP-h5 | Flush with 40%:40%:20% (ACN:IPA:H20) for 5-10 column volumes. Equilibrate with starting mobile phase for 5-10 column volumes. | |
| HxSil C8 | Flush with 40%:40%:20% (ACN:IPA:H20) for 5-10 column volumes. Equilibrate with starting mobile phase for 5-10 column volumes. | |
| HxSil C18 | Flush with 40%:40%:20% (ACN:IPA:H20) for 5-10 column volumes. Equilibrate with starting mobile phase for 5-10 column volumes. | |
| Pump approximately 50 mL of methanol with 1% 6 N nitric acid. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Inject several times with 100 µL of 1 N nitric acid. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Inject several times with 100 µL of 1 N nitric acid. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Inject Several times with 100 µL of 0.1 M potassium EDTA. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Flush with 50 mL 0.1 N sodium hydroxide. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Flush with 150 mL 0.1 N sodium hydroxide. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Flush with 1% calcium chloride at 0.1 mL/min overnight. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Flush with 1% hydrochloric acid at 0.2 mL/min at 80 °C overnight. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Flush with 1% calcium chloride at 0.1 mL/min at 80 °C overnight. Equilibrate with starting mobile phase for ~ 200 column volumes. | ||
| Flush with 1% lead nitrate at 0.1 mL/min at 80 °C overnight. Equilibrate with starting mobile phase for ~ 200 column volumes. |