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.
Imagine yourself driving home but stuck in a traffic jam in which you can only drive at a 10 mph. After the traffic clears
up, you will surely want to increase your driving speed, preferably up to the speed limit, or maybe even a little higher when
no police are around. In any case, you will certainly not continue driving at 10 mph.
However, when running a liquid chromatography (LC) gradient (1–3), maintaining a slow speed is exactly what we all do: The
flow rate we apply is always determined by the pressure bottleneck, occurring when the gradient composition reaches its viscosity
maximum. This is illustrated in Figure 1a, which shows the pressure trace of a 5–95% gradient of acetonitrile–water (red curve)
obtained when a constant flow rate is selected such that the pump does not exceed 1000 bar. In agreement with Darcy's law,
the pressure trace follows the viscosity profile of the varying mobile-phase composition pumped through the column during
the gradient. As can be noted, the pressure maximum of 1000 bar is reached only for a brief moment, when the column is filled
with a 20:80 (v/v) acetonitrile–water mixture (4). Before and after this bottleneck, the pressure drop is considerably lower than the imposed
limit of 1000 bar, leaving a considerable margin to increase the flow rate during these portions of the gradient.
Figure 1: Traces of (a) pressure and (b) flow rate of a 5–95% acetonitrile–water mobile-phase gradient under constant pressure
(black) and constant flow rate (red).
Doing so, and controlling the pump so it delivers the maximum pressure during the entire gradient, the flow rate at the start
and end of the gradient is larger than in the case of a constant flow rate (see the black lines in Figures 1a and 1b). For
the presently considered 5–95% acetonitrile gradient, the average increase in affordable flow rate is about 20%. When running
methanol gradients, a similar situation arises, with a similar percentage gain. The main difference is that in this case the
viscosity maximum occurs at about 50% (vol%) methanol instead of at about 20% acetonitrile.
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.