The following procedures should rejuvenate a column whose performance has deteriorated due to sample contamination. Disconnect and reverse the column. Connect it to the pump, but not the detector.
Follow the appropriate flushing procedure in this article below, using a flow rate that results in column back pressure of 1500-4500 psi, but never higher than the maximum recommended pressure in the manufacturer’s instruction manual. If you have a SUPELCOSIL column, analyze it with the test mix and the conditions listed on the data sheet. Efficiency, symmetry, and capacity should be within 10-15% of the test sheet values. If not, repack the column inlet or replace the column.
Note: Volumes listed below are for 25 cm x 4.6 mm I.D. columns, which have a column volume of 4.15 mL. When restoring a 4.6 mm I.D. column shorter or longer than 25 cm, multiply all volumes mentioned below by the ratio of the column length to 25 (e.g., for a 15 cm column: 15/25, or 0.6 times the volumes). When restoring a column of internal diameter other than 4.6 mm, multiply all volumes mentioned below by the ratio of the square of the column I.D. to (4.6)2 (e.g., for a 3.2 mm I.D. column: (3.2)2/(4.6)2 = 10.24/21.16 = 0.48 times the values mentioned below).
Silica Column
Flush with the following:
- 50 mL hexane
- 50 mL methylene chloride
- 50 mL 2-propanol
- 50 mL methanol
- 25 mL methylene chloride
- 25 mL mobile phase
Evaluate column performance according to conditions specified by the manufacturer.
Silica-Based Reversed Phase Column
(alkyl, phenyl, or diphenyl column, SUPELCOSIL LC-PAH column)
A. Water Soluble Samples
Flush with the following:
- Flush with warm (60°C) distilled water
- 50 mL methanol
- 50 mL acetonitrile
- 25 mL methanol
- 25 mL mobile phase
Evaluate column performance.
B. Samples Not Soluble in Water
Flush with the following:
- 50 mL 2-propanol
- 50 mL methylene chloride
- 50 mL hexane
- 25 mL isopropanol
- 25 mL mobile phase
Evaluate column performance.
Polar-Bonded Phase Column
(amino, cyano, or diol column or Pirkle-type chiral columns).
For a column used in the reversed-phase mode (e.g., organic solvent/aqueous buffer mobile phase), follow the same cleanup procedure as for silica-based reversed-phase columns. For a column used with non-aqueous mobile phases, use the following scheme:
Flush with the following:
- 50 mL chloroform
- 50 mL methanol
- 50 mL acetonitrile
- 25 mL methylene chloride
- 25 mL methanol
- 25 mL mobile phase
Evaluate column performance.
SUPELCOSIL LC-PCN Column
A. To Remove Protein
Flush with 10 column volumes of acetonitrile: water, 50:50, containing 0.1% trifluoroacetic acid.
B. To Remove TCA
Flush with 10-column volumes of distilled water (adjust pH to 2.5 with H3PO4), then with 10-column volumes each of:
- Water (to remove salts)
- Methanol (to remove water)
- Methanol/Methylene Chloride, 50:50 (a general clean-up solution)
- Methanol
If column performance still is not acceptable, prepare the mobile phase buffer at 10X the concentration used for the analysis and recycle through the column overnight.* Re-equilibrate the column with the mobile phase at the normal buffer concentration and reevaluate.
* Use caution: with some buffer types and/or concentrations a 10-fold increase in concentration can cause precipitation.
Silica-Based Ion Exchange Columns
(strong or weak anion or cation exchange)
Most analyses involving ion exchange systems use ionic mobile phases. Compounds that may affect column performance are usually insoluble or only slightly soluble in water. The following procedure should be sufficient to remove these compounds.
Flush with the following:
- 50 mL hot (40-60°) distilled water
- 50 mL methanol
- 50 mL acetonitrile
- 25 mL methylene chloride
- 25 mL methanol
- 25 mL mobile phase
Evaluate column performance.
Silica-Based Columns for RPLC of Proteins and Peptides
Follow the protocol for silica-based reversed-phase columns. Alternatively, make one or more 100 μL injections of trifluoroethanol (determine the number of injections by evaluating column performance after each injection).
Evaluate column performance.
Nonbonded Silica Columns Exposed to Polar Solvent
Samples and mobile phases containing very strongly polar solvents, such as water or alcohol, can deactivate uncoated silica HPLC columns. This can drastically affect column performance, particularly solute retention and selectivity. Even prolonged column flushing with a nonpolar solvent only partially restores column performance, while wasting chemicals.
A silica regeneration solution quickly and inexpensively restores silica column performance by removing trapped polar material. Pump the solution through the affected column for 10 minutes at a rate of 4 mL/minute, then flush with the mobile phase for 10 minutes at a rate of 2 mL/minute. Evaluate column performance by using the test mixture for evaluating silica columns. Performance should be virtually the same as before the polar solvent was introduced.
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HPLC
