6
Figure 5. The last sheet is the one with 100% MeOH as eluent B, delivering the best separations and a decent Design Space as we will see it in the following figures.
Figure 9. The separation of the 18 compounds can also be done in acetonitrile with a critical resolution of 2.36 in ca. 50 min at a pressure of ca.1300 psi (back side of the Cube from Figure 7).
DryLab and the QbD movement
In 2002, the FDA instigated the development of the QbD concept which allowed more flexibility in industrial laboratories [14, 15]. DryLab demonstrated as early as 25 years ago, that systematic experimentation in HPLC is required.and has contributed to the development of Quality by Design in the analytical chemistry area. It was the first software demonstrating ’robust resolution maps‘, allowing the estimation of tolerance limits for every important parameter of a separation. DryLab is therefore an important tool to help meet QbD practices.
Figure 6. DryLab 4 Laboratory screen with 2-D and 3-D Resolution space. Red areas with warm colours mean sufficient (baseline) separation of all peaks (Rs,crit > 1.5) whereas cold (blue) colour means peak overlaps. The Cube calculation helps to find robust working points in advance, saving tremendous amount of experimental work supporting green HPLC.
Figure 7. Robust regions in the Cube are shown as irregular geometric forms of the Design Space, in which baseline resolution of all components is possible.
Method transfer
We can see in Figure 8. on the top of the graph the selected method parameter (tG = 46 min, T = 30°C and tC = 100% MeOH as organic eluent) with estimated possible deviations from the nominal value. The temperature is assumed to deviate from the nominal value of 30°C by not more than +- 2°C, i.e. the true temperature is assumed to be in any experiment between 28 and 32°C). On the left graph the ’Frequency Distribution’ shows how often (N) a certain critical resolution (Rs,crit) occurs under any combination of possible, true parameter values. As can be seen from the graph, the failure rate, i.e., the number of experiments that could fall outside the required critical resolution Rs,crit =1.5, is = zero. That means that practically all experiments should fulfil the critical resolution requirement. The position of the “set point” or “working point” is of great importance, as many experiments cost enormous amount of resources. If the point is selected by trial and error, an analyst may have to change it and repeat a large number of experiments to find a new optimum. DryLab can calculate 6 experimental factors at 3 levels, i.e., 36 = 729 experiments in less than 1 minute!
The right graph in Figure 8 (‘Regression Coefficients’) describes the importance of each parameter, related to the selected deviation from the nominal value, for the critical resolution. As can be seen from the graph, temperature has the most important influence; a lower temperature gives a higher global resolution.
Method transfer is a problem in a global economy, where products travel over borders and are used in different location to generate the same analytical result. It is necessary to enhance this process using modelling software to ease the burden using virtual UHPLC models. This method transfer process is often instrument dependent and therefore it is important to understand how to utilise predictive software in method transfer. An example of successful method transfer using this approach is demonstrated in reference [23].
The so called Knowledge Management Protocol, which was discussed above in a short format, is a great help in dealing with regulatory authorities. In this way methods can be developed in an inspection-safe manner.
Economic considerations of modelling in reducing waste
In a steadily growing number of publications, the value and usability of retention modelling for fast and systematic method development has been demonstrated [17-22].
Figure 8. Extended robustness calculation for 3 measured and 3 additional parameters. (a) frequency distribution of critical resolution values and (b) regression coefficients.
INTERNATIONAL LABMATE - APRIL 2013
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