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Column Cleaning Procedure

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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 suffcient to regenarate the column and, in such case, it is necessary to replace the column with a new one. And, even if the column can be regenarated, 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.

CategoryColumn nameCoulmn cleaning procedure
GPC Clean-up CLNpak EV Reverse the column on the HPLC system, flow the eluent at 80% 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 PAE
Reversed Phase,
Hydrophilic Interaction
and Normal Phase
(Polymer-based)
Asahipak ODP , C8P , C4P Flow 50mL of dioxane, CH3CN or methanol.
RSpak RP18-415 , DS
RSpak DE
RSpak DM-614 Flow 50mL of methanol, CH3CN or H2O.
RSpak NN Inject 20 to 50μL of 2N nitric acid aqueous solution two or three times.
RSpak JJ-50 Reverse the direction and flow rate is 0.5mL/min (JJ-50 4D) or 0.1mL/min (JJ-50 2D).
(Pollution by low valency hydrophilic ions)
1) H2O 6min, 2) 0.1M NaOH aq. 75min, 3) H2O 12min, 4) Elurnt 40min
(Pollution by metar ions)
1) H2O 6min, 2) 0.1M HNO3 aq. 75min, 3) H2O 6min, 4) 0.1M NaOH aq. 75min, 5) H2O 12min, 6) Eluent 40min
(Pollution by high valency hydrophobic ions)
1) H2O 25min, 2) 5% acetonitrile aq. 20min, 3) 100% acetonitrile 100min, 4) H2O 50min
HILICpak VG-50 4E (Standard cleaning)
Reverse the direction and flow rate is 0.5mL/min.
1) H2O 10min, 2) 0.1M NaOH aq. 120min, 3) H2O 20min, 4) Eluent 60min
(Cleaning metal ions)
Reverse the direction and flow rate is 0.5mL/min.
1) H2O 10min, 2) 0.1M HNO3 aq. 60min, 3) 1) H2O 10min, 4) 0.1M NaOH aq. 120min, 5) H2O 20min, 6) Eluent 60min
HILICpak VG-50 2D
VT-50 2D
(Standard cleaning)
Reverse the direction and flow rate is 0.1mL/min.
1) H2O 6min, 2) 0.1M NaOH aq. 75min, 3) H2O 12min, 4) Eluent 40min
(Cleaning metal ions)
Reverse the direction and flow rate is 0.1mL/min.
1) H2O 6min, 2) 0.1M HNO3 aq. 75min 3) 1) H2O 6min, 4) 0.1M NaOH aq. 75min, 5) H2O 12min, 6) Eluent 40min
HILICpak VC-50 2D Reverse the direction and flow rate is 0.1mL/min.
1) H2O 10min, 2) 25mM HNO3 aq./CH3CH=70/30 60min, 3) H2O 10min, 4) Eluent 60min
HILICpak VN-50 4D
VN-50 2D
Reverse the direction and flow rate is 0.3mL/min (VN-50 4D) or 0.1mL/min (VN-50 2D).
1) H2O 6min, 2) 0.1M HNO3 aq. 75min, 3) H2O 6min, 4) 0.1M NaOH aq. 75min, 5) H2O 12min, 6) Eluent 40min
Asahipak NH2P-50 4E (Standard cleaning)
Reverse the direction and flow rate is 0.5mL/min.
1) H2O 10min, 2) 0.1M NaOH aq. 120min, 3) H2O 20min, 4) Eluent 60min
(Cleaning metal ions)
Reverse the direction and flow rate is 0.5mL/min.
1) H2O 10min, 2) 0.1M HClO4 aq. 60min, 3) H2O 10min, 4) 0.1M NaOH aq. 120min, 5) H2O 20min, 6) Eluent 60min
Reversed Phase,
Hydrophilic Interaction
and Normal Phase
(Silica-based)
Silica C18M, C18P Flow 50mL of dioxane, THF, CH3CN or methanol.
Silica 5C8 , 5C4 ,
5CN
Flow 50mL of dioxane, CH3CN or methanol.
Silica 5NPE
Silica 5PYE
SIlica 5SIL Flow 50mL each of n-hexane, dichloroehtane, methanol, dichloroethane and n-hexane.
Silica 5NH There is no appropriate washing method.
Chiral Separation ORpak CDBS-453 There is no appropriate cleaning method.
ORpak CRX-853 There is no appropriate cleaning method.
Ion Exchange AXpak WA-624 Inject 1 to 2 mL of 0.1M NaOH aqueous solution or 30% acetic acid aqueous solution. Connect the column in the reverse way and flow the eluent at 0.5mL/min.
CXpak P-421S
IEC QA , DEAE
SP , CM
IEC DEAE3N-4T
SP-420N
Inject 500μL of 0.1M NaOH aqueous solution, 50% formic acid aqueous solution, 20% CH3CN aqueous solution or 20% dimethyl sulfoxide aqueous solution several times. Flow the eluent at 0.3 to 0.5mL/min.
Asahipak ES-502N Flow aqueous solution of high salt concentartion or 10mM NaOH aqueous solution for ionic adsorption and 50mM buffer solution containing 50% CH3CN or methanol for hydrophobic adsorption.
Asahipak ES-502C Flow aqueous solution of high salt concentartion or 0.1N acetic acid aqueous solution for ionic adsorption and 50mM buffer solution containing 50% CH3CN aqueous solution or methanol for hydrophobic adsorption.
Hydrophobic HIC PH-814 Reverse the column on the HPLC system, flow the eluent at 0.5mL/min and inject 1 to 2 mL of 0.1M NaOH or 30% acetic acid aqueous solution several times.
Affinity AFpak Various Reverse the column on the HPLC system, flow an appropriate eluent at 0.5mL/min.
Ligand Exchange SUGAR SC1011, 1821 SC1011-7F
SC1211
( for SC1011, 1821, SC1011-7F)
Flow 50mL of 0.1M Ca(NO3)2 aqueous solution at 50°C, 0.5mL/min.
(for SC1211)
After replacing in-column solvent completely with H2O, flow 30mL of 0.1M Ca(NO3)2 aqueous solution at 50°C, 0.5mL/min. Then, replace the in-column solvent completely with H2O and gradually increase the organic solvent concentration to the concentration to be used.
SUGAR SP0810

Flow of H2O at 70 - 95°C, 0.5mL/min. Then inject of 50μL of 0.2M Pb(NO3)2 aqueous 3 or 4 times.

SUGAR KS-800 series Flow 0.2M NaOH aqueous solution at 0.5mL/min. After flowing the solvent, replace the in-column solvent completely with H2O. (Sometimes, injection of 40μL of 1M NaOH aqueous solution is will be sufficient.)
RSpak DC-613 Flow 50mL of Methanol, Ethanol, CH3CN, or 5mM NaOH aqueous solution. Or, inject 20 to 50 μL of 0.5M NaOH aqueous solution.
SUGAR SZ5532 After replacing in-column solvent completely with H2O, flow 30mL of 0.1M Zn(NO3)2 aqueous solution at 50°C, 0.5mL/min. Then, replace the in-column solvent completely with H2O and gradually increase the organic solvent concentration to the concentration to be used.
USPpak MN-431 Flow 50mL of 0.1M Ca(NO3)2 aqueous solution at 50°C, 0.5mL/min.
Ion Exclusion SUGAR SH1011,1821 Flow 50mL of 25mM H2SO4 aqueous solution at 50°C, 0.5mL/min.
RSpak KC-811 Flow 50mL of 25mM H2SO4 aqueous solution at 50°C, 0.5mL/min.
Ion Chromatography IC I-524A --Pollution by accumulation of metals--
30minutes: 0.01M tartaric acid (pH3 to 4, adjust with 0.1M NaOH), 1.0mL/min
--Pollution by proteins--
150 minutes: 20% acetic acid aq., 0.2mL/min
150 minutes: water, 0.2mL/min
--Pollution by organics--
150 minutes: 20% acetonitrile/water, 0.2mL/min
150 minutes: water, 0.2mL/min
IC NI-424 Flow the mixture of eluent/methanol=90/10 at 0.6mL/min for two hours.
IC Y-521 Inject 100μL of 1M HNO3 aqueous solution several times.
IC YK-421 Flow 50mL of 50mM tartaric acid aqueous solution or 50mM tartaric acid aqueous solution/CH3CN=50/50 at 0.3mL/min.
IC YS-50 Flow 50mL of 4mM Methanesulfonic acid aqueous solution /CH3CN=70/30 at 1.0mL/min.
IC SI-90 4E --Pollution by low valency hydrophilic ions--
1. 15 minutes: deionized water
2. 60 minutes: 10 times concentrated eluent
3. 15 minutes: deionized water
4. 60 minutes: eluent
--Pollution by high valency hydrophobic ions--
1. 15 minutes : deionized water
2. 10 minutes: 5% acetonitrile aqueous solution
3. 60 minutes: 100% acetonitrile
4. 30 minutes: deionized water
5. 60 minutes: eluent
IC SI-50 4E --Pollution by low valency hydrophilic ions--
(flow rate 0.3mL/min)
1. 25 minutes : deionized water
2. 100 minutes : 10 times concentrated eluent
3. 25 minutes : deionized water
4. 100 minutes : eluent
--Pollution by high valency hydrophobic ions--
(flow rate 0.3mL/min)
1. 25 minutes : deionized water
2. 20 minutes : 5% acetonitrile aqueous solution
3. 100 minutes : 100% acetonitrile
4. 50 minutes : deionized water
5. 100 minutes : eluent
IC SI-52 4E --Pollution by low valency hydrophilic ions--
(flow rate 0.3mL/min)
1. 25 minutes : deionized water
2. 100 minutes : 10 times concentrated eluent
3. 25 minutes : deionized water
4. 100 minutes : eluent
--Pollution by high valency hydrophobic ions--
(flow rate 0.3mL/min)
1. 25 minutes : deionized water
2. 20 minutes : 5% acetonitrile aqueous solution
3. 100 minutes : 100% acetonitrile
4. 50 minutes : deionized water
5. 100 minutes : eluent
IC SI-35 4D --Pollution by low valency hydrophilic ions--
(flow rate 0.3mL/min)
1. 15 minutes : deionized water
2. 60 minutes : 10 times concentrated eluent
3. 15 minutes : deionized water
4. 60 minutes : eluent
--Pollution by high valency hydrophobic ions--
(flow rate 0.3mL/min)
1. 15 minutes : deionized water
2. 15 minutes : 5% acetonitrile aqueous solution
3. 60 minutes : 100% acetonitrile
4. 30 minutes : deionized water
5. 60 minutes : eluent
IC T-521 Flow 0.1M NaOH aqueous solution at 0.5mL/min for one hour and 0.1M Nitric acid aqueous solution at 0.5mL/min for one hour.
Aqueous SEC (GFC) PROTEIN KW-800 Reverse the direction and flow rate is 0.5mL/min (KW-800 series and LW-803) or 0.15mL/min (KW400 series). 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 0.5M 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
  KW400
OHpak SB-800 HQ
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.
Multimode Asahipak GS-HQ
Aqueous / Organic SEC Asahipak GF-HQ
Organic SEC (GPC) GPC KF-800
KF-400HQ
KF-600
HK-404L
K-800
KD-800
LF
HFIP-800
HFIP-600
HT-800
UT-800
AT-806S