Column Cleaning Procedure

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.

Category	Column name		Coulmn 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.
GPC Clean-up	CLNpak	PAE
Reversed Phase, Hydrophilic Interaction and Normal Phase (Polymer-based)	Asahipak	ODP , C8P , C4P	Flow 50 mL of dioxane, CH₃CN or methanol.
	RSpak	RP18-415 , DS
	RSpak	DE
	RSpak	DM-614	Flow 50 mL of methanol, CH₃CN or H₂O.
	RSpak	NN	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 0.5 mL/min (JJ-50 4D) or 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) Elurnt 40 min (Pollution by metar 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
	HILICpak	VG-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 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
	HILICpak	VG-50 2D 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 4D VN-50 2D	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 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
Reversed Phase, Hydrophilic Interaction and Normal Phase (Silica-based)	Silica	C18M, C18P	Flow 50 mL of dioxane, THF, CH₃CN or methanol.
	Silica	5C8 , 5C4 , 5CN	Flow 50 mL of dioxane, CH₃CN or methanol.
	Silica	5NPE
	Silica	5PYE
	SIlica	5SIL	Flow 50 mL 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.
Chiral Separation	ORpak	CRX-853	There is no appropriate cleaning method.
Ion Exchange	AXpak	WA-624	Inject 1 to 2 mL of 0.1 M NaOH aqueous solution or 30 % acetic acid aqueous solution. Connect the column in the reverse way and flow the eluent at 0.5 mL/min.
	CXpak	P-421S
	IEC	QA , DEAE SP , CM
	IEC	DEAE3N-4T SP-420N	Inject 500 μL of 0.1 M 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 0.5 mL/min.
	Asahipak	ES-502N	Flow aqueous solution of high salt concentartion or 10 mM NaOH aqueous solution for ionic adsorption and 50 mM buffer solution containing 50 % CH₃CN or methanol for hydrophobic adsorption.
	Asahipak	ES-502C	Flow aqueous solution of high salt concentartion or 0.1 N acetic acid aqueous solution for ionic adsorption and 50 mM 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 0.5 mL/min and inject 1 to 2 mL of 0.1 M NaOH or 30 % acetic acid aqueous solution several times.
Affinity	AFpak	Various	Reverse the column on the HPLC system, flow an appropriate eluent at 0.5 mL/min.
Ligand Exchange	SUGAR	SC1011, 1821 SC1011-7F SC1211	( for SC1011, 1821, SC1011-7F) Flow 50 mL of 0.1 M Ca(NO₃)₂ aqueous solution at 50 °C, 0.5 mL/min. (for 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	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	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 will be sufficient.)
	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.
	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.
	USPpak	MN-431	Flow 50 mL of 0.1 M Ca(NO₃)₂ aqueous solution at 50 °C, 0.5 mL/min.
Ion Exclusion	SUGAR	SH1011,1821	Flow 50 mL of 25 mM H₂SO₄ aqueous solution at 50 °C, 0.5 mL/min.
Ion Exclusion	RSpak	KC-811	Flow 50 mL of 25 mM H₂SO₄ aqueous solution at 50 °C, 0.5 mL/min.
Ion Chromatography	IC	I-524A	--Pollution by accumulation of metals-- 30 minutes: 0.01 M tartaric acid (pH3 to 4, adjust with 0.1 M NaOH), 1.0 mL/min --Pollution by proteins-- 150 minutes: 20 % acetic acid aq., 0.2 mL/min 150 minutes: water, 0.2mL/min --Pollution by organics-- 150 minutes: 20 % acetonitrile/water, 0.2 mL/min 150 minutes: water, 0.2 mL/min
	IC	NI-424	Flow the mixture of eluent/methanol=90/10 at 0.6 mL/min for two hours.
	IC	Y-521	Inject 100 μL of 1M HNO₃ aqueous solution several times.
	IC	YK-421	Flow 50 mL of 50 mM tartaric acid aqueous solution or 50 mM tartaric acid aqueous solution/CH₃CN=50/50 at 0.3 mL/min.
	IC	YS-50	Flow 50 mL of 4 mM Methanesulfonic acid aqueous solution /CH₃CN=70/30 at 1.0 mL/min.
	IC	SI-90 4E	--Pollution by low valency hydrophilic ions-- (flow rate 0.5 mL/min) 15 minutes: deionized water 60 minutes: 10 times concentrated eluent 15 minutes: deionized water 60 minutes: eluent --Pollution by high valency hydrophobic ions-- (flow rate 0.5 mL/min) 15 minutes : deionized water 10 minutes: 5 % acetonitrile aqueous solution 60 minutes: 100 % acetonitrile 30 minutes: deionized water 60 minutes: eluent
	IC	SI-50 4E	--Pollution by low valency hydrophilic ions-- (flow rate 0.3 mL/min) 25 minutes : deionized water 100 minutes : 10 times concentrated eluent 25 minutes : deionized water 100 minutes : eluent --Pollution by high valency hydrophobic ions-- (flow rate 0.3 mL/min) 25 minutes : deionized water 20 minutes : 5 % acetonitrile aqueous solution 100 minutes : 100 % acetonitrile 50 minutes : deionized water 100 minutes : eluent
	IC	SI-52 4E	--Pollution by low valency hydrophilic ions-- (flow rate 0.3 mL/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 0.3 mL/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 0.3 mL/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 0.3 mL/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 2B	--Pollution by low valency hydrophilic ions-- (flow rate 0.1 mL/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 0.1 mL/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-36 4D	--Pollution by low valency hydrophilic ions-- (flow rate 0.35 mL/min) 1. 25 minutes : deionized water 2. 100 minutes : 100 mM KOH or NaOH aq. solution 3. 25 minutes : deionized water 4. 100 minutes : eluent --Pollution by high valency hydrophobic ions-- (flow rate 0.35 mL/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	T-521	Flow 0.1 M NaOH aqueous solution at 0.5 mL/min for one hour and 0.1 M Nitric acid aqueous solution at 0.5 mL/min for one hour.
Aqueous SEC (GFC)	PROTEIN	KW-800	Reverse the direction and flow rate is 0.5 mL/min (KW-800 series and LW-803) or 0.15 mL/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 0.5 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
		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