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27 3 8 2 10 11 1 4,5 6 7 9 12 13 14


the original run and with the analysis time actually reduced by two minutes. Thus, the goal of improving the separation by use of a shallower gradient, without increasing the run time was achieved.


Figure 2. Experiment Run at 30°C.


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


too high, an additional experiment was run where the flow was reduced to 1.5 mL/ min and the gradient time was increased to 35.6 minutes. In this way, the same gradient steepness was obtained, but the shallower gradient was achieved by a combination of increasing the flow rate by a factor of three and increasing the gradient time by a factor of 1.33. The result, shown in Figure 1d, was that the same selectivity was again seen but now with better efficiency. In comparison to the initial steeper-gradient run (Figure 1a) a better separation has been obtained, as the peak capacity increased from 51 to 70 (a 37% improvement), but with essentially no change in run time (in fact the run time was two minutes shorter). Therefore, the goal of improving the separation power without increasing the run time has been achieved.


In order to evaluate the effect of temperature, the chromatogram depicted in Figure 2 was run with conditions similar to Figure 1b, but with the temperature set to 30°C. When using acetonitrile based mobile phases, it has been suggested that the acetonitrile content should be increased by 1% for every 5°C reduction in temperature to maintain equivalent elution strength [11]. Therefore, the high end of the gradient was set to 95% mobile phase B instead of 85%. The gradient time was then adjusted to 120 minutes so that the steepness of the gradient would be equivalent to that of experiment 1b. In this way, an effort was made such that temperature would be the only variable changing between the runs shown in Figure 1b and Figure 2.


While not identical, the chromatograms of Figure 1b and Figure 2 are very similar for the peaks which elute subsequent to the active ingredient. The differences seen in the earlier part of the chromatogram are believed to be due to changes in selectivity and elution order, as a function of the different temperature. The peak capacity was 103, which is only moderately better than what was observed for Figure


1b. The similar quality of the separation at 30°C, compared to 80°C (other than the noted exception), provides support for the contention that the shape selectivity of biphenyl phases are less temperature dependent than what has generally been reported for most columns. It is most likely the ‘slotted’ nature of these stationary phases (i.e. consisting of ligands that are fairly rigid, with room in between the ligands where the analytes can interact) that is responsible for the lesser dependence of shape selectivity on temperature. However, this is speculative and further studies are warranted to better understand the shape selectivity of these phases and the effect of temperature.


Conclusion


Experiments were conducted to evaluate the expectation, from theory, that reducing the steepness of a chromatographic gradient can be accomplished either by increasing the gradient time or by increasing the flow rate, and that the effect on selectivity (or peak spacing) should be essentially equivalent. It was expected that fused core columns, run at elevated temperatures, would be ideal for this purpose as they allow increased flow rates to be used without generating excessive pressures or peak broadening.


Our data generally support the validity of this approach, as similar selectivity was obtained by virtue of elevating the gradient time or the flow rate, but with the run time being faster when using the high-flow-rate alternative. It was found that increasing the flow rate to 2.0 mL/min was too high resulting in a loss of efficiency, which largely cancelled the benefits of the shallower gradient. When the experiment was run with the flow rate reduced to 1.5 mL/min, and the gradient time increased, so as to maintain the same gradient steepness, the peak capacity was 37% larger than that of


Our data suggest that, in addition to the use of a somewhat more moderate flow rate, moderately smaller particle sizes, perhaps in the range of 2.0 to 2.4 µm, would be more optimal. However, particle sizes much smaller than this (i.e. UHPLC columns) are not believed to be ideal as the ability to increase the flow rate significantly becomes limited, unless using very high temperatures. It may also be noted that techniques such as supercritical fluid chromatography, HILIC, and classical normal phase chromatography would lend themselves particularly well to this approach due to the inherently lower pressures that are typically observed; and, in these cases, sub-2-µm particles would be feasible.


Our data also provide support for the hypothesis that biphenyl stationary phases offer a shape selectivity which is less effected by temperature than what has been reported, for other columns, in the literature. Further studies should be run to confirm this and to better understand the nature of the shape selectivity offered by these phases.


Lastly, we note that recent approaches which are used for comparison of chromatographic methods typically evaluate the combination of kinetic factors effecting band broadening, linear velocity, and the maximum column length that can be used given the pressure tolerance of the method. The additional variable of the steepness of the gradient is generally not considered but perhaps should be, given its importance.


References


1. Neue, U.D.; Carmody, J.L.; Cheng, Y.F.; Lu, Z.; Phoebe, C.H.; Wheat, T.E. and Editors, Chapter 3, Design of Rapid Gradient Method for the Analysis of Combinatorial Chemistry Libraries and the Preparation of Pure Compounds, in Advances in Chromatography, Volume 41, CRC Press Taylor and Francis Group, 2001.


2. Neue, U.D.; Alden, B.A.; Iraneta, P.C.; Mendez, A.; Grumbach, E.S.; Tran, K.; Diehl, D.M. and Editors, Chapter 4, HPLC Columns for Pharmaceutical Analysis, in Handbook of Pharmaceutical Analysis by HPLC, Volume 6, Elsevier Academic Press, 2005.


3. Neue, U.D.; Cheng, Y.F.; Lu, Z. and Editors, Chapter 1.2, Fast Gradient


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