l li CoumnCeanngProblems in peak shapes and elution timing changes or elevated column pressure etc. are often caused by insoluble or adsorbing components present in the eluent and reagents being deposited inside the column. These problems may be resolved by cleaning the column.This section describes general signs of column deterioration and column cleaning procedures.Please also read column-specific detailed cleaning procedures included in the product operation manual. Typical signs of column deterioration1. Elevated column pressure2. Abnormal peak shapes (broadening, leading, tailing, and split peaks)3. Change in retention time4. Unstable baseline Standard cleaning procedures1. Insoluble components Insoluble components that block the column inlet may be removed by reversing the flow direction, i.e., introducing the eluent from the column outlet, with flow rate at less than half of the recommended flow rate.2. Adsorbing components For an efficient cleaning, reverse the flow direction and reduce the flow rate at half of the recommended flow rate. Cleaning solvent selection guide Solvents capable of dissolving the adsorbed substances Solvents with high eluting power (depends on separation mode) MethodsReversed phase chromatography columnsSugar analysis columnsAqueous SEC (GFC) chromatography columnsIon exchange chromatography columns*Recommended solvent volume to introduce is 5 to 10 times the column volume.*Pay attention to the column pressure elevation during column cleaning.*Column cleaning is limited and does not guarantee full recovery of the column to its original condition.Use a solvent with higher organic solvent concentration such as methanol, acetonitrile, or THF.(When using a mixture of buffer solution and organic solvent, make sure there is no precipitation of salt.)[Ligand exchange columns (SUGAR series) ]To regenerate the detached counter ions.- Flush or inject aqueous salt solvent which contains the modified counter ion.[Polymer-based amino columns (NH2P series and VG-50 series) ]Adsorption of acidic substances on the amino functional group.- Flush with solvents in the following sequence; water, 0.1 M NaOH (aq.), water, and the eluent.Adsorption of ionic substances.- Use a solvent with higher salt concentration or solvent with different pH from the eluent.Adsorption of hydrophobic substances.- Use a solvent containing organic solvent.(When using a mixture of buffer solution and organic solvent, make sure there is no precipitation of salt.)Adsorption of ionic substances.- Use a solvent with higher salt concentration or solvent with different pH from the eluent.Adsorption of hydrophobic substances.- Use a solvent containing organic solvent.(When using a mixture of buffer solution and organic solvent, make sure there is no precipitation of salt.)Adsorption of protein.- Inject 1 - 2 mL of 0.1 M NaOH (aq.) or 30 % acetic acid (aq.) several times.*use only the solvents allowed in the operation manual68Column Cleaning
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