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Information Column Cleaning Procedures88Column Cleaning ProceduresChange in peak shapes, elution timing, and the elevated column pressure may be resolved by cleaningthe column. This section describes general indications of column deterioration and column cleaningprocedures. The details of column cleaning procedures should be referred to each column’s operationmanual.Typical indicators of column deterioration1. Elevated column pressure2. Abnormal peak shapes (broad, leading, or tailing) and split peaks3. Change in retention time4. Unstable baselineSelection guide to the cleaning solventChoose a solvent capable of dissolving the adsorbed substances.Choose the strong eluent for the choice of column.*Use the solvent specified in the operation manualStandard cleaning proceduresFor an efficient cleaning, reverse the direction and reduce the flow to 1/3 of the regular flow rate.Reversed phase columnsIon exchange chromatographyClean the columns with solvent containing higher concentration of organic solvent such asmethanol, acetonitrile, or THF.(In case of using buffer as a mobile phase, miscibility of the buffer solution and the organicsolvents need to be checked)• Contamination caused by ionic adsorptionUse solvent with higher salt concentration or solvent with different pH from the mobilephase.• Contamination caused by hydrophobic adsorptionUse solvent containing small percentage of organic solvent.(In case of using buffer as a mobile phase, miscibility of the buffer solution and the organicsolvents need to be checked)• Contamination caused by proteinsInject 1-2 mL of 0.1 M NaOH (aq) or 30% (v/v) acetic acid several times.Sugar analysis columnsHydrophobic interaction columnsAqueous SEC(GFC) columns[Ligand exchange columns (SUGAR series)]• In case of counter-ion detachmentFlush or inject solvent containing the salt corresponding to the modified counter-ligand.[Polymer-base amino columns (NH2P series)]• To neutralize the acidic form of amino function groupFlush with solvents in the following sequence: water, 0.1M perchloric acid, water, 0.1MNaOH (aq), water, and mobile phase.• Contamination caused by proteinsInject 1-2 mL of 0.1 M NaOH (aq) or 30% (v/v) acetic acid several times.• Contamination caused by ionic adsorptionUse solvent with higher salt concentration or solvent with different pH from the mobilephase.• Contamination caused by hydrophobic adsorptionUse solvent containing small percentage of organic solvent.(In case of using buffer as a mobile phase, miscibility of the buffer solution and the organicsolvents need to be checked)*The volume of the cleaning solvent required is 5-10 times the column volume.*Avoid pressure elevation during the cleaning*The cleaning is limited and does not guarantee the full regeneration of the column to its original condition.For your informationOne typical cause of the column pressure elevation is the clogging of solid substances at the inlet filter of the column.In this case, reverse the direction and reduce the flow to 1/3 of the regular flow rate. This may remove the solidsubstance causing the elevated pressure.*Use the solvent specified in the operation manual