Method Development Approaches

TABLE 8-7. Equilibration Times for Columns of Different Dimensions 

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 equihbration times. For example, if a 15-cm x 4.6-mm i.d. column and a flow rate of 1 mL/min was used, then the equilibration times for the three methods would be 5 min (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 1 mL/min that has a dwell volume of 1 mL, then you should add an extra minute of equilibration time. This becomes extremely important during method transfers where the instruments in the receiving laboratory may be different. 

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Fekete S. Rudaz S. Fekete J. Guillaume D. Analysis of recombinant monoclonal antibodies by RPLC Toward a generic method development approach. Journal of Pharmaceutical and Biomedical Analysis, 2012, 70, 158-168. 

Chiral CE can be widely applied in release and stability testing, the chiral purity of intermediates, and raw materials. Various generic method development approaches have been developed and published recently. " ... 

This chapter is intended to be a practical overview of the liquid chromatography sorbents, instrumentation, and the various method development approaches used in pharmaceutical laboratories for both relatively small molecules and biomolecules. 

When developing a separation, a strategy of mobile-phase selection is involved. However, when the molecule has a polar and a nonpolar part, the tactics that support the method development strategy differ slightly from the previous discussions. To illustrate the method development approach to be... 

Once a suitable HPLC separation mode has been selected, experimental conditions can be adjusted using either an empirical method or a systematic method development approach. The separation of two sample compounds is conveniently characterized by resolution Rs. ... 

Janiszewski, J.S. Swyden, M.C. Fouda, H.G. High-Throughput Method Development Approaches for Bioanalytical Mass Spectrometry, J. Chromatogr. Sci. 38, 255-258 (2000). 

Method development approaches for biopharmaceutical analysis are not going to be covered in this chapter, but there are some recent pubUcations which may be referred to for a comprehensive overview of this application area [13-15]. 

Other method development approaches used in TLC include unidimensional multiple development [127] and multi-modal separation techniques [127], where TLC, in normal phase mode is used in conjunction with reversed-phase liquid column chromatography [131] or GC [131] to provide additional information in separations. This complementary strategy can prove very inportant even for well... 

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