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HPLC Column 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.
  • 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.

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 MaterialRestoration 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-1Flush 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 with 40:40:20 (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.

PRP-X100

Pump approximately 50 mL of methanol with 1% 6 N nitric acid. Equilibrate with starting mobile phase for ~ 200 column volumes.

PRP-X200

Inject several times with 100 µL of 1 N nitric acid. Equilibrate with starting mobile phase for ~ 200 column volumes.

PRP-X300

Inject several times with 100 µL of 1 N nitric acid. Equilibrate with starting mobile phase for ~ 200 column volumes.

PRP-X400

Inject Several times with 100 µL of 0.1 M potassium EDTA. Equilibrate with starting mobile phase for ~ 200 column volumes.

RCX-10

Flush with 50 mL 0.1 N sodium hydroxide. Equilibrate with starting mobile phase for ~ 200 column volumes.

RCX-30

Flush with 150 mL 0.1 N sodium hydroxide. Equilibrate with starting mobile phase for ~ 200 column volumes.

HC-40

Flush with 1% calcium chloride at 0.1 mL/min overnight. Equilibrate with starting mobile phase for ~ 200 column volumes.

HC-75 H+

Flush with 0.1 N sulfuric acid at 0.1 mL/min overnight. Equilibrate with starting mobile phase for ~ 200 column volumes.

HC-75 Ca+2

Flush with 1% calcium chloride at 0.1 mL/min overnight. Equilibrate with starting mobile phase for ~ 200 column volumes.

HC-75 Pb+2

Flush with 1% lead nitrate at 0.1 mL/min overnight. Equilibrate with starting mobile phase for ~ 200 column volumes.