Determination of the column dead volume The extra column volume which is the volume of other equipment (such as connected tubes, HPLC pump, injector, etc.) except the chromatographic column was estimated by the isocratic method. The column was removed and replaced with a short union fitting. Blue dextran was used for the determination of column dead volume. The concentration of blue dextran was 1 g/L and using the mobile phase was a mixture of acetonitrile/water (75:25). The flow rate of mobile phase was varied from 0.3-1.5 mL/min and the UV detector was set to 320 nm. The flow rates and retention times was recoded and calculated to determine the extra column dead volume, which was calculated as the time of the start of blue dextran multiplied by the inverse of flow rate. The extra column volume was determined to be 0.12 mL, which represents 2.92% of the column volume. Therefore, the extra column volume which was very small compared to column volume was neglected.