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. |
| CLNpak | PAE | ||
| Reversed Phase, Hydrophilic Interaction and Normal Phase (Polymer-based) |
Asahipak | ODP, C8P, C4P | Flow 50mL of dioxiane, 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 micro-L of 2N nitric acid aqueous solution two or three times. | |
| Asahipak | NH2P-50 4E | Reverse the column on chromatograph and flow 5mL of H2O, 60mL of 0.1M HClO4 aq., 5mL of H2O, 60mL of 0.1M NaOH aqueous solution and then 5mL of H2O at the flow rate of 0.5mL/min. | |
| Reversed Phase, Hydrophilic Interaction and Normal Phase (Silica-based) |
ODSpak | F | Flow 50mL of dioxiane, THF, CH3CN or methanol. |
| Silica | C18M, C18P | ||
| 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. |
| 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 micro-L 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 50deg-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 50deg-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 deg-C, 0.5mL/min. Then inject of 50micro-L 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 40micro-L 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 micro-L 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 50deg-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 50deg-C, 0.5mL/min. | |
| Ion Exclusion | SUGAR | SH1011,1821 | Flow 50mL of 25mM H2SO4 aqueous solution at 50deg-C, 0.5mL/min. |
| RSpak | KC-811 | Flow 50mL of 25mM H2SO4 aqueous solution at 50deg-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 100micro-L 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 |
|