tailieunhanh - HPLC for Pharmaceutical Scientists 2007 (Part 8C)

Some typical equilibration times for various column dimensions are shown in Table 8-7; however, these should only be used as a guide. If complete equi- libration is not achieved, early eluting components may show differences in retention from run to run. An experiment could be run such that three dif- ferent methods could be run with different equilibration times. | METHOD DEVELOPMENT APPROACHES 385 TABLE 8-7. Equilibration Times for Columns of Different Dimensions Length cm Diameter cm Column Volume CV mL 3 CV mL 5 CV mL 15 15 10 10 10 5 5 the flow rate mL min to determine the time in minutes needed. Therefore the lower the flow rate the longer the equilibration time. Some typical equilibration times for various column dimensions are shown in Table 8-7 however these should only be used as a guide. If complete equilibration is not achieved early eluting components may show differences in retention from run to run. An experiment could be run such that three different methods could be run with different equilibration times. For example if a 15-cm x . column and a flow rate of 1 mL min was used then the equilibration times for the three methods would be 5min 3 CV 9min 5 CV and 11 min 6 CV equilibration times respectively. If the retention of the early eluting components are consistent less than 1 variation in retention time in all three methods then the lowest equilibration time could be used. However if the early eluting components show greater variation in their retention time with the 5-min equilibration time compared to the methods with the 9- and 11-min equilibration time then an equilibration time of greater than 5 min is warranted. Optimization of the optimal equilibration time is required for reproducible methods. Other considerations include differences in dwell volumes from the different HPLC systems. The dwell volume should be determined for all the systems in the laboratory and based on these determinations this should be factored into the calculation of the equilibration time. For example if the maximum dwell volume of all the systems in a particular laboratory to which the method is transferred to is 2mL and you are running on an instrument at 1mL min that has a dwell volume of 1 mL .