Column cleaning and troubleshooting
When a column is deteriorated by the foreign substances remaining in the column or adsorbed by the packing material, the substances might be washed out by the procedure described below. However, sometimes the procedure is not sufficient to regenerate the column and, in such case, it is necessary to replace the column with a new one. And, even if the column can be regenerated, the column performance may be not so good as before.
After cleaning the column, be sure to replace the in-column solvent to the normal solvent. The use of a guard column at the up-stream of the main column is recommended to prevent the main column from the deterioration by the foreign substances. When a guard column is used, first, the guard column will be deteriorated by the foreign substances and it is possible to prevent the main column from the deterioration by replacing the guard column regularly.
When cleaning a guard column, be sure to disconnect the main column before the cleaning. If a guard column and a main column are connected in series and the cleaning procedure is performed, foreign substances in the guard column may move to the main column and, as the consequence, the main column may be deteriorated.
For more information, please also read and download the column manuals:
Cleaning procedure and regeneration of Reversed Phase (RP) columns
Asahipak ODP-50 series
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the flow direction and use the flow rate at less than half of the recommended flow rate. Introduce 5 to 10 times column volume of the washing solvent.
-
Method 1: Basic solvent wash
Apply after using the column under acidic to neutral condition for an extended time period. Introduce 0.1 M aqueous sodium hydroxide solution/acetonitrile = 80/20.
-
Method 2: Acidic solvent wash
Apply after using the column under basic to neutral condition for an extended time period. Introduce 50 mM aqueous phosphoric acid solution/acetonitrile = 90/10.
-
Method 3: Organic solvent wash
Apply after using the column under highly aqueous condition for an extended time period. Introduce water/acetonitrile = 20/80.
Asahipak C4P
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the flow direction and use the flow rate at less than half of the recommended flow rate. Introduce 5 to 10 times column volume of the washing solvent.
-
Method 1: Basic solvent wash
Apply after using the column under acidic to neutral condition for an extended time period. Introduce 0.1 M aqueous sodium hydroxide solution/acetonitrile = 80/20.
-
Method 2: Acidic solvent wash
Apply after using the column under basic to neutral condition for an extended time period. Introduce 50 mM aqueous phosphoric acid solution/acetonitrile = 90/10.
-
Method 3: Organic solvent wash
Apply after using the column under highly aqueous condition for an extended time period. Introduce water/acetonitrile = 20/80.
RSpak DE series
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the flow direction and use the flow rate at less than half of the recommended flow rate. Introduce 5 to 10 times column volume of the washing solvent.
-
Method 1: Basic solvent wash
Apply after using the column under acidic to neutral condition for an extended time period. Introduce 10 mM aqueous sodium hydroxide solution/acetonitrile = 80/20.
-
Method 2: Acidic solvent wash
Apply after using the column under basic to neutral condition for an extended time period. Introduce 50 mM aqueous phosphoric acid solution/acetonitrile = 90/10.
-
Method 3: Organic solvent wash
Apply after using the column under highly aqueous condition for an extended time period. Introduce water/acetonitrile = 20/80.
RSpak DS series
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the flow direction and use the flow rate at less than half of the recommended flow rate. Introduce 5 to 10 times column volume of the washing solvent.
-
Method 1: Basic solvent wash
Apply after using the column under acidic to neutral condition for an extended time period. Introduce 10 mM aqueous sodium hydroxide solution/acetonitrile = 80/20.
-
Method 2: Acidic solvent wash
Apply after using the column under basic to neutral condition for an extended time period. Introduce 50 mM aqueous phosphoric acid solution/acetonitrile = 90/10.
-
Method 3: Organic solvent wash
Apply after using the column under highly aqueous condition for an extended time period. Introduce water/acetonitrile = 20/80.
RSpak DM-614
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the flow direction.
Method: Set the flow rate at 0.3 mL/min, and introduce 50 mL of methanol, acetonitrile or water.
RSpak NN-814
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) In case adsorption of ionic substances is suspected, inject 20-50 μL of 2 M nitric acid aqueous solution several times while running the eluent of analysis.
RSpak JJ-50 2D
Reverse the direction and flow rate is 0.1 mL/min.
-
(Pollution by low valency hydrophilic ions)
1) H2O 6 min, 2) 0.1 M NaOH aq. 75 min, 3) H2O 12 min, 4) Eluent 40 min
-
(Pollution by metal ions)
1) H2O 6 min, 2) 0.1 M HNO3 aq. 75 min, 3) H2O 6 min, 4) 0.1 M NaOH aq. 75 min, 5) H2O 12 min, 6) Eluent 40 min
-
(Pollution by high valency hydrophobic ions)
1) H2O 25 min, 2) 5 % acetonitrile aq. 20 min, 3) 100 % acetonitrile 100 min, 4) H2O 50 min
ODP2 series
See column manual for basic, acidic or organic solvent wash.
Silica C18M series
(1) Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.
(2) Follow below cleaning steps for adsorbing components. For an efficient cleaning, reverse the flow direction and use the flow rate at less than half of the recommended flow rate. Introduce 5 to 10 times column volume of the washing solvent.
-
Apply after using the column under highly aqueous conditions. Introduce water/acetonitrile = 20/80.
-
Apply to samples containing protein. Introduce water/acetonitrile = 50/50 - 30/70 (containing 0.1 % TFA)
Cleaning procedure and regeneration of
HILIC columns
Asahipak NH2P-50 4E
(Standard cleaning)
Reverse the direction and flow rate is 0.5 mL/min.
1) H2O 10 min, 2) 0.1 M NaOH aq. 120 min, 3) H2O 20 min, 4) Eluent 60 min
(Cleaning metal ions)
Reverse the direction and flow rate is 0.5 mL/min.
1) H2O 10min, 2) 0.1 M HClO4 aq. 60 min, 3) H2O 10 min, 4) 0.1 M NaOH aq. 120 min, 5) H2O 20 min, 6) Eluent 60 min
HILICpak VG-50
(Standard cleaning) VG-50 4E
Reverse the direction and flow rate is 0.5 mL/min.
1) H2O 10 min, 2) 0.1 M NaOH aq. 120 min, 3) H2O 20 min, 4) Eluent 60 min
(Cleaning metal ions)
Reverse the direction and flow rate is 0.5 mL/min.
1) H2O 10 min, 2) 0.1 M HNO3 aq. 60 min, 3) 1) H2O 10 min, 4) 0.1 M NaOH aq. 120 min, 5) H2O 20 min, 6) Eluent 60 min
(Standard cleaning) VG-50 2D
Reverse the direction and flow rate is 0.1 mL/min.
1) H2O 6 min, 2) 0.1 M NaOH aq. 75 min, 3) H2O 12 min, 4) Eluent 40 min
(Cleaning metal ions)
Reverse the direction and flow rate is 0.1 mL/min.
1) H2O 6 min, 2) 0.1 M HNO3 aq. 75 min 3) 1) H2O 6 min, 4) 0.1 M NaOH aq. 75 min, 5) H2O 12 min, 6) Eluent 40 min
HILICpak VT-50 2D
(Standard cleaning)
Reverse the direction and flow rate is 0.1 mL/min.
1) H2O 6 min, 2) 0.1 M NaOH aq. 75 min, 3) H2O 12 min, 4) Eluent 40 min
(Cleaning metal ions)
Reverse the direction and flow rate is 0.1 mL/min.
1) H2O 6 min, 2) 0.1 M HNO3 aq. 75 min 3) 1) H2O 6 min, 4) 0.1 M NaOH aq. 75 min, 5) H2O 12 min, 6) Eluent 40 min
HILICpak VC-50 2D
Reverse the direction and flow rate is 0.1 mL/min.
1) H2O 10 min, 2) 25 mM HNO3 aq./CH3CH=70/30 60 min, 3) H2O 10 min, 4) Eluent 60 min
HILICpak VN-50
Reverse the direction and flow rate is 0.3 mL/min (VN-50 4D) or 0.1 mL/min (VN-50 2D).
1) H2O 6 min, 2) 0.1 M HNO3 aq. 75 min, 3) H2O 6 min, 4) 0.1 M NaOH aq. 75 min, 5) H2O 12 min, 6) Eluent 40 min
Cleaning procedure and regeneration of
SUGAR columns (Ligand Exchange)
SUGAR SP0810
Flow of H2O at 70 - 95°C, 0.5 mL/min. Then inject of 50 μL of 0.2 M Pb(NO3)2 aqueous 3 or 4 times.
SUGAR SC1011
SUGAR EP SC1011-7F
USPpak MN-431
Flow 50 mL of 0.1 M Ca(NO3)2 aqueous solution at 50°C, 0.5 mL/min.
SUGAR SC1211
After replacing in-column solvent completely with H2O, flow 30 mL of 0.1 M Ca(NO3)2 aqueous solution at 50°C, 0.5 mL/min. Then, replace the in-column solvent completely with H2O and gradually increase the organic solvent concentration to the concentration to be used.
SUGAR KS-800 series
Flow 0.2 M NaOH aqueous solution at 0.5 mL/min. After flowing the solvent, replace the in-column solvent completely with H2O. (Sometimes, injection of 40 μL of 1 M NaOH aqueous solution is sufficient.)
SUGAR SZ5532
After replacing in-column solvent completely with H2O, flow 30 mL of 0.1 M Zn(NO3)2 aqueous solution at 50°C, 0.5 mL/min. Then, replace the in-column solvent completely with H2O and gradually increase the organic solvent concentration to the concentration to be used.
RSpak DC-613
Flow 50 mL of Methanol, Ethanol, CH3CN, or 50 mM NaOH aqueous solution. Or, inject 20 to 50 μL of 0.5 M NaOH aqueous solution.
Cleaning procedure and regeneration of
Ion Exclusion columns (organic acids)
SUGAR SH1011
SUGAR SH1821
Flow 50 mL of 25 mM H2SO4 aqueous solution at 50°C, 0.5 mL/min.
RSpak KC-811
Flow 50 mL of 25 mM H2SO4 aqueous solution at 50°C, 0.5 mL/min.
Cleaning procedure and regeneration of aqueous SEC columns
PROTEIN KW-800
series
Reverse the direction and flow rate is 0.5 mL/min. The volume of the cleaning solvent required is 5 to 10 times the column volume.
1) In case where protein has been adsorbed :
Use a solvent containing 0.5 M salt or phosphate buffer (about pH3.0).
2) In case where a hydrophobic substance has been adsorbed :
Use a solvent containing 10 to 20 % acetonitrile or methanol.
PROTEIN LW-803
Reverse the direction and flow rate is 0.5 mL/min. The volume of the cleaning solvent required is 5 to 10 times the column volume.
1) In case where protein has been adsorbed :
Use a solvent containing 0.5 M salt or phosphate buffer (about pH3.0).
2) In case where a hydrophobic substance has been adsorbed :
Use a solvent containing 10 to 20 % acetonitrile or methanol.
PROTEIN KW-400 series
Reverse the direction and flow rate is 0.15 mL/min. The volume of the cleaning solvent required is 5 to 10 times the column volume.
1) In case where protein has been adsorbed :
Use a solvent containing 0.5 M salt or phosphate buffer (about pH3.0).
2) In case where a hydrophobic substance has been adsorbed :
Use a solvent containing 10 to 20 % acetonitrile or methanol.
PROTEIN LW-403 4D
Reverse the direction and flow rate is 0.15 mL/min. The volume of the cleaning solvent required is 5 to 10 times the column volume.
1) In case where protein has been adsorbed :Use a solvent containing 0.5 M salt or phosphate buffer (about pH3.0).
2) In case where a hydrophobic substance has been adsorbed :Use a solvent containing 10 to 20 % acetonitrile or methanol.
OHpak SB-800 HQ series
OHpak LB-800 series
Reverse the column on the HPLC system, flow the eluent at 50 % of ordinary flow rate. This is effective for the foreign substances remaining in the column, however not effective for the substances adsorbed to the packing material.
Cleaning procedure and regeneration of
organic SEC columns
GPC KF-800 series
Reverse the column on the HPLC system, flow the eluent at 50 % of ordinary flow rate. This is effective for the foreign substances remaining in the column, however not effective for the substances adsorbed to the packing material.
GPC KF-400HQ
Reverse the column on the HPLC system, flow the eluent at 50 % of ordinary flow rate. This is effective for the foreign substances remaining in the column, however not effective for the substances adsorbed to the packing material.
GPC KD-800 series
Reverse the column on the HPLC system, flow the eluent at 50 % of ordinary flow rate. This is effective for the foreign substances remaining in the column, however not effective for the substances adsorbed to the packing material.
GPC HK-400 series
Reverse the column on the HPLC system, flow the eluent at 50 % of ordinary flow rate. This is effective for the foreign substances remaining in the column, however not effective for the substances adsorbed to the packing material.
GPC LF series
Reverse the column on the HPLC system, flow the eluent at 50 % of ordinary flow rate. This is effective for the foreign substances remaining in the column, however not effective for the substances adsorbed to the packing material.
CLNpak EV series
Reverse the column on the HPLC system, flow the eluent at 50 % of ordinary flow rate. This is effective for the foreign substances remaining in the column, however not effective for the substances adsorbed to the packing material.
For more information, please also read and download the column manuals:
Troubleshooting - Abnormal peak shape
Troubleshooting - Fluctuation in elution time
Troubleshooting - Fluctuation in baseline
Troubleshooting - Noise in baseline
Troubleshooting - Rise in pressure
