Sovent 22 --llilRepacement Method for ) Organc SEC (GPCCoumnsColumn volume of a column can be calculated using the below equation.[Column Volume] = The below table lists the column volumes of different-size columns.< Column Volume (mL) >Column Length(mm) 10 50100150250300 Setting the Pump Limiter's Maximum PressureThe pressure applied to the entire HPLC system (system pressure) and the pressure applied to the column (column pressure) are influenced by eluent type, flow rate, and temperature. Thus, the column pressure may increase during the solvent replacement process. To prevent applying a pressure above the maximum allowable pressure of a column, use a pump limiter's maximum pressure setting.The pressure displayed on the HPLC system is a sum of system pressure and column pressure. Therefore, generally the pump limiter's maximum pressure is set at "the sum of system and column's maximum allowable pressures". However, low flow rate used during the solvent replacement generates a negligible system pressure, thus using the column's maximum allowable pressure as the pump limiter's maximum pressure is a practical choice.If multiple columns are used together, add maximum allowable pressure of all columns when setting the limit.e.g. 1 When using one column with maximum allowable pressure = 3.5 MPa. e.g. 2 When using two columns with both maximum allowable pressures = 3.5 MPa. Set the pump limiter's maximum pressure at 3.5 MPa. Set the pump limiter's maximum pressure at 7.0 MPa. HPLC System's Solvent CompatibilitySome materials used in HPLC system may have low chemical compatibility to organic solvents to be used in SEC analysis. To avoid damaging the HPLC system, please make sure that the chosen solvent is applicable for all HPLC system parts that have contact with the solvent.Please note that there are tubes and fittings made of various materials. Among them, stainless steel types are recommended because of their high durability against various organic solvents. Solvent Replacement Steps1. Reduce the flow rate to the half of the regular flow rate and increase the column oven temperature while keep introducing the current solvent (the temperature required depends on the column and the solvent used).2. Move to next step once the column temperature reaches to the set temperature. Case 1. When replacing the current solvent with a miscible solvent. First introduce about 3 - 5 column volumes of an intermediate solvent (current solvent : new solvent = 1:1 mixture). Then, introduce another 3 - 5 column volumes of 100 % new solvent. e.g. To replace THF to chloroform in KF-803 (8.0 mm I.D., 300 mm L) First introduce 45 - 75 mL of an intermediate solvent (THF : chloroform = 1:1 mixture). Then, introduce 45 - 75 mL of 100 % chloroform. Case 2. When replacing the current solvent with a solvent with low miscibility/solubility to the current solvent. First introduce about 3 - 5 column volumes of a solvent that is miscible/soluble to both solvents. Then, follow the steps in Case 1. e.g. To replace DMF with LiBr to THF in KD-806M (8.0 mm I.D., 300 mm L) First introduce 45 - 75 mL of intermediate solvent 1 (DMF) to wash-out LiBr. Then, introduce 45 - 75 mL of intermediate solvent 2 (DMF : THF = 1: 1 mixture), followed by 45 - 75 mL of 100 % THF.3. Set the flow rate and the temperature to the desired. Start the analysis once a stable baseline is obtained. Additional RecommendationsFrequent eluent replacement may damage the column, and thus not recommended.If analyses using different eluents are frequently expected, it is recommended to have dedicated columns for each solvent.[Inner Diameter (I.D.) ]I.D. (mm)4.60.2--2.54.2-6.0---4.2--π × [Length] 8.0 2.5 5.012.615.167
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