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.

Cleaning procedure and regeneration of Reversed Phase (RP) columns

Asahipak ODP, C4P

RSpak DS

RSpak DE

Flow 50 mL of dioxane, CH3CN or methanol.

RSpak DM-614

Flow 50 mL of methanol, CH3CN or H2O.

RSpak NN-814

Inject 20 to 50 μL of 2 N nitric acid aqueous solution two or three times.

RSpak JJ-50

Reverse the direction and flow rate is 0.1 mL/min (JJ-50 2D).
(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

Silica C18M

Flow 50 mL of dioxane, THF, CH3CN or methanol.

Cleaning procedure and regeneration of HILIC columns

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

Asahipak NH2P-50

(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

Cleaning procedure and regeneration of SUGAR columns

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

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.

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.

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

OHpak LB-800

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 manual:

Troubleshooting - Abnormal peak shape

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Troubleshooting - Fluctuation in elution time

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Troubleshooting - Fluctuation in baseline

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Troubleshooting - Noise in baseline

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Troubleshooting - Rise in pressure

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