A considerable gain in analysis time (10–30%) can be obtained by switching from a constant-flow-rate mode of operation to a constant-pressure gradient-elution mode. This switch will not reduce separation selectivity, because selectivity is volume-based and thus is independent of the flow rate. The peak areas and plate heights are similar in the two modes.
Because the flow rate in the constant flow rate mode is determined by the viscosity bottleneck, the average flow rate in the constant-pressure mode will always be larger than that in the constant-flow-rate mode. As a consequence, the gradient program will always be finished sooner than in the constant-flow-rate mode, thus leading to a noticeable time saving.
Because the possible average increase in the flow rate and the accompanying analysis time savings are determined by the viscosity of the mobile phase, the time savings will heavily depend on the start and end composition of the gradient. When these compositions lie close to the viscosity maximum of the water–organic modifier mixture, the gain will only be on the order of a few percent. This is true, for example, in the case for an acetonitrile–water gradient running between 10 and 30% acetonitrile or for a methanol–water gradient running between 40 and 60% methanol. On the other hand, gains on the order of 16–22% can be realized for gradients running between 5 and 50% or 50 and 95% methanol. For typical scouting gradients — that is, gradients running between 5 and 95% of organic modifier — the time gain is also on the order of approximately 20%, for methanol as well as acetonitrile gradients. For some exotic solvents, or for some complex mobile-phase gradient profiles (segmented gradients), the time gain can even be larger. A more detailed calculation of the time gain that can be achieved is found in the literature (5,6).
The time gain also is dependent on pressure and temperature, because of the temperature and pressure dependency of the viscosity. For the average column pressure of 200 bar typical of high performance liquid chromatography (HPLC) separations (P inlet = 400 bar), the time gain at 30 °C for a linear 20–95% acetonitrile–water gradient is 26% compared to the time gain of 21% for an average column pressure of 500 bar, typical for ultrahigh-pressure LC (UHPLC) separations (P inlet = 1000 bar). With increasing temperature, the time gain further decreases because the viscosity differences between water and an organic modifier such as acetonitrile or methanol become smaller, because the viscosity of water is more sensitive to temperature than that of organic modifier (4). When increasing the column temperature to 60 °C, the time gain is further limited to 16% for P inlet = 1000 bar.
In practice, the time gain that can be achieved is 5–10% greater (leading to an overall gain on the order of 30%) than what would be calculated on the basis of the changing mobile–phase viscosity only. This is because the pressure safety margin that needs to be taken into account in the constant-flow-rate mode is no longer needed in the constant-pressure mode. In the constant-flow-rate mode, this margin is needed to buffer possible pressure fluctuations or a degradation of the column's permeability. In the constant-pressure mode, this margin is not needed because the pressure is controlled to its set point. This robustness advantage of the constant-pressure operation might even be more attractive than the reduction in analysis time, especially in unattended operation or in start-up procedures when a column heats up either by an oven or by frictional heating (because it is a common observation that the pressure drop in the column decreases during the first couple of minutes of high-pressure operation as a result of column heating by frictional heat).
Knowing from the above that a considerable gain in analysis time can be obtained by switching from a constant flow rate to a constant-pressure gradient-elution mode, several questions emerge. How will this switch affect the separation selectivity and the width of the elution window? What will happen to the sensitivity of analysis and the reliability of quantification? And finally, what will happen to the peak broadening? These questions are answered in the following sections.