What Does a Gradient Profile Look Like?

What Does a Gradient Profile Look Like?

The most frequently used gradient profile is the linear gradient. However, there are a variety of profiles which could be applied, including step, segmented and curved (Figure 2).

Figure 1 Schematic of the programmed gradient, with the corresponding gradient timetable and the actual gradient

Figure 2 Schematics of different gradient profiles; (A) linear, (B) step, (C) segmented and (D) curved profiles

Gradients are controlled in the method via a gradient timetable program. It is regularly written in terms of % of the strong B solvent, as shown in Figure 1. The gradient begins at a lower %B composition, then increases over a period of time, before it either is held isocratically or dropped back to the original starting composition. It is then held isocratically at the original composition in order to re-equilibrate the column for the next injection.

The blue dashed profile follows the timetable described in Figure 1. However, in reality there is a slight deviation in the programmed gradient due to the dwell volume (orange trace). The dwell volume is the volume between the point the two solvents first mix to the head of the column. This value is different for each instrument based on the volume of tubing, mixers, injectors and valves. This dwell causes a delay in when the column experiences the change in mobile phase composition, as illustrated.