Column Cleaning Procedures

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.

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 50mL of dioxane, CH3CN or methanol.
	RSpak	RP18, DS
	RSpak	DE, GOLF CARB
	RSpak	DM-614	Flow 50mL of methanol, CH3CN or H2O.
	RSpak	NN	Inject 20 to 50 µL of 2N nitric acid aqueous solution two or three times.
	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)	ODSpak	F	Flow 50mL of dioxane, THF, CH3CN or methanol.
	Silica	C18M, C18P	Flow 50mL of dioxane, THF, CH3CN or methanol.
	Silicapak	E-411	Flow 50mL each of n-hexane, dichloroehtane, methanol, dichloroethane and n-hexane.
	SIlica	5SIL
	Silica	5C8, 5C4, 5CN	Flow 50mL of dioxane, CH3CN or methanol.
	Silica	5NH	There is no appropriate washing method.
	Silica	5NPE
	Silica	5PYE
Chiral Separation	ORpak	CDA, CDB, CDC	There is no appropreate cleaning method.
Chiral Separation	ORpak	CDBS, CRX	There is no appropreate 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 500 µL 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	31 kinds	Reverse the column on the HPLC system, flow an appropriate eluent at 0.5mL/min.
Ligand Exchange	SUGAR	SC1011, 1821 SC1211	( for SC1011, 1821) 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	Flow 10mM NaOH aqueous solution at 0.5mL/min. After flowing the solvent, replace the in-column solvent completely with H2O. (Sometimes, injection of 40µL of 0.1M 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.
Ion Exclusion	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	–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	–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	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.
	IC	R-621
Aqueous SEC (GFC)	OHpak	SB-800 HQ	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.
		SB400
	PROTEIN	KW-800
		KW400
Maltimode	Asahipak	GS-HQ
Aqueous / Organic SEC	Asahipak	GF-HQ
Organic SEC (GPC)	GPC	KF-800 KF-400HQ KF-600 K-800 KD-800 LF HFIP-800 HFIP-600 HT-800 UT-800 AT-806S