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26 May / June 2021


2 3


1 4 5 6 7 9 8 10 11 12 13 14 1,3 2 4,5 7 6 9 8 10 11 12 13 14 1,3 8 2 4,5 10 11 6 7 9 12 13 14


the peaks of interest easy to visualise). It can be seen from the chromatogram that this separation was less than optimal as many of the peaks are not well resolved. The peak capacity was calculated to be 51. Figure 1b shows the result obtained under the same conditions, except that the gradient time was increased by a factor of four, thereby reducing the gradient steepness by the same factor. The resolution obtained is now visibly improved, particularly for the peaks which elute subsequent to the active ingredient (the large peak), such that the peaks are generally baseline resolved. The peak capacity, under these conditions, increased to 89. However, the run time was now longer by a factor of 2.6. The changes observed in the peaks eluting before the active ingredient are believed to be due to changes in selectivity which can occur, in gradient methods, when either the gradient time or fl ow rate are changed [6].


1,3 8 2 4,5 10 11 6 7 9 12 13 14 Figure 1. Experiments Run at 80°C.


The large peak, in the chromatogram, is the active pharmaceutical ingredient, and the other peaks are degradation products of unknown structure.


4. Results and Discussion


The quality of the separations conducted in this study were evaluated with respect to run time and with the metric of peak capacity, values of which were calculated as per equation (3); where tr,last


and tr,fi rst are the


retention times of the last eluting and fi rst eluting peak of interest, respectively, F is the mobile phase fl ow rate, and Waverage


is the


average peak width of the related substance peaks being separated. This relationship has previously been used in the literature [1,3,5,7 72-74] and provides an effective way of expressing the separation power of a gradient method.


just after 17 minutes in Figure 1a). The parameters used for these experiments, as well as the steepness of each gradient, are summarised in Table 1. The peak capacity and the run times are summarised in Table 2.


The starting point, for this study, is the chromatogram obtained with the column at 80°C, with a fl ow rate of 0.5 mL/min, and a gradient time of 26.7 minutes. This is presented in Figure 1a (note that the time axis is expanded in the fi gures so as to make


Figure 1c shows the result obtained when the fl ow rate was increased by a factor of four instead of the gradient time, resulting in a gradient steepness which is equivalent to what was used for the chromatogram of Figure 1b. The selectivity (peak spacing) of the chromatography is very similar to what was observed in Figure 1b; thus, demonstrating that the same gradient steepness has been obtained. The run time in Figure 1c was four times faster than the run of Figure 1b; and, in fact, was actually 6 minutes shorter than the original run (Figure 1a). However, some band broadening has clearly developed in this run which suggests that, despite the use of a fused-core particle at elevated temperature, the fl ow rate of 2.0 mL/min was too high and has resulted in a loss of effi ciency. The peak capacity was determined to be 54. Hence, this run was very similar to Figure 1a, both in terms of separation power and speed. It seems that the benefi t of the improved peak separation due to the shallower gradient was almost exactly cancelled by the loss of effi ciency due to the higher fl ow rate.


Given that the somewhat disappointing separation obtained at 2 mL/min was believed to be due to setting the fl ow rate


Table 2. Peak Capacity and Run Time Corresponding to the Figures


For the purpose of these calculations, the run time was taken as the retention time of the last peak of interest (the peak eluting


Figure 1a Figure 1b Figure 1c Figure 1d Figure 2


Peak Capacity 51 89 54 70


103


Run Time (minutes) 17.1 45.7 11.3 15.1 53.5


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