Passive modulation is by far the most common form of modulation in two-dimensional liquid chromatography. A 10-port or 8-port 2-position switching valve connects the separation dimensions using two or more sampling loops (see figure below). At any time the first-dimension effluent is sampled by one of the modulation loops (Figure 1: Left). Once the valve switches, the contents of that loop are injected to the second-dimension column, whereas the other loop is connected to the first-dimension separation (Figure 1: Right).
While this modulation strategy is simple and effective, its use imposes a number of constraints on the analytical method. First of all, the volume of the sampling loops cannot be too large in order to prevent overloading the second-dimension column, yet cannot be too small to allow sufficient quantities of the first-dimension effluent to be sampled.
The second constraint is related to this. As a result of the modulation interface, the first-dimension effluent is essentially the injection solvent of the second dimension. Consequently, incompatibility issues may – and often do – arise, regardless whether heart-cut or comprehensive mode is used.
Finally, for comprehensive mode (LC×LC) the static modulation loops impose time constrains to the analytical method. Comprehensive mode requires the entire first-dimension effluent to be subjected to a second-dimension separation. The modulation time, the time at which the valve switches, is therefore determined by the volume of the sampling loops and the first- and second-dimension flow rates. If the first-dimension flow rate is high, the volume of the sampling loops must be large enough to store the fraction until the second-dimension system is ready to receive a new fraction. Second-dimension separations in LC×LC are therefore generally very fast often using ultra-high pressure conditions.