When alternative options must be explored

It is normally not too difficult to find a suitable column which will give desirable retention and peak shape for the analyte. One of the reasons why method development is often difficult is that satisfactory retention and peak shape alone are not enough for a method to be suitable. As discussed in Chapter 1, specificity is very important. In pursuing a separation using one particular mode of HPLC, it may be that, despite all the changes in experimental variables that take place in the method optimisation process, it is not possible to separate the analyte from all interferences present in the sample. In considering the initial stages of method development, it is therefore necessary to bear in mind as many approaches to the problem as possible, in case the first option proves not to be successful. 

Another ironic feature of the increasing emphasis on robustness to ensure ease of method transfer is that the quality of HPLC stationary phases is improving. The quality arises from better batch-to-batch reproducibility and more homogeneous surfaces on which only one type of retention mechanism may operate. The irony lies in the fact that in the past it might have been the flaw (e.g. residual silanols on a reversed-phase material) that was responsible for the last bit of selectivity that was needed to achieve a very difficult separation. Now with improved stationary phases, there may be a need for more complex mobile phases thereby compromising robustness once more. 

3 Local considerations. There are many other local factors which dictate the path of method development. An increasingly common example is the development of a method which, as well as serving for quantitation, may be directly transferred to HPLC-mass spectrometry (commonly referred to as LC-MS) to allow the identification of minor components in a sample. This is achievable if the desired separation can be obtained using an ammonium acetate buffer instead of the more usual phosphate buffer. On the other hand, if it was intended that the unknown minor components should be isolated in milligram quantities to allow full structural elucidation using techniques such as H NMR as well as mass spectrometry, then conditions would be developed especially for the isolation unless the conditions for quantitative work coincidentally also lent 

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