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

Choice of Stationary Phase Ideally for a reversed-phase separations, the retention factors (k) for all com- ponents in a sample should lie between 1 and 10 to achieve separation in a reasonable time. For a given stationary phase the k of a particular component can be controlled by changing the solvent composition of the mobile phase. However, the impact of eluent composition will depend on the type of sta- tionary phase and the nature of the components in the mixture. | 368 METHOD DEVELOPMENT Figure 8-13. Optimal wavelength selection for API and related impurities. 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time min Figure 8-14. Determination of peak homogeneity Diode array detection DAD . Reprinted from reference 10 with permission. Also the spectral homogeneity of the peak of interest must be taken into consideration. Diode array spectra at least three points across the peak should be taken to ensure the peak is spectrally homogenous see Figure 8-14. If the peak is not spectrally homogenous the overlay of the spectra will show METHOD DEVELOPMENT CONSIDERATIONS 369 Figure 8-15. Diode array detection for elucidation of coeluting species. Reprinted from reference 10 with permission. distinct differences see Figure 8-15 . However even if the diode array spectra do overlay this does not absolutely ensure that the peak does not contain any coeluting impurities because the impurities could have similar diode array spectra and or if there is a low level of a coeluting species with a different diode array spectrum it may not be determined by this approach. In these cases MS detection needs to be employed to ensure MS spectral homogeneity. MS spectra are taken across the peak and the MS spectra across the peak should not show the presence of any other coeluting species of different masses. This does not absolutely ascertain that the peak is homogeneous since isomers of the same compound will have the same M H and is indistinguishable from the parent compound. Also the impurity that may be coeluting may not have an appreciable ionization efficiency at the particular mobile-phase and mass spectrometric conditions. An example of where using diode array detection may not be helpful is shown in Figure 8-16. Note that for this reaction mixture convergent synthesis the desired product 1 has the same diode array spectra as synthetic precursors 2 and 3. If these two