Gradient separations dwell time

Differences in dwell volumes between different systems are an important reason why conditions for gradient separations on one chromatograph do not necessarily transfer to another. It is helpful to state the dwell volume for your system when you report a gradient separation. One way to compensate for dwell volume is to inject sample at the time fD instead of at t = 0. 

Figure 25-31 Linear gradient separations of the same mixture used in Figure 25-12 in the same column and solvent system [buffer (solvent A) with acetonitrile (solvent B)] at a flow rate of 1.0 mL/min. The dwell time was 5 min.

A mixture of 14 compounds was subjected to a reversed-phase gradient separation going from 5% to 100% acetonitrile with a gradient time of 60 min. The sample was injected at t = dwell time. All peaks were eluted between 22 and 41 min. 

Figure 5.4-2. Set up of a dual-gradient HPLC system. This HPLC-system consist of a normal reversed phase separation system with an additional gradient pump and HPLC-valve. Two complete sets of pre- and separation columns are required. This setup enables the equilibration and sample preconcentration on the one column pair, whereas on the second column pair, the separation is performed. This reduces the dwell time of a connected mass spectrometer and thereby increases the sample throughput per day significantly. The final nano-valve directs the flow of the active separation column to the connected detector.

Gradient profile or solvent composition deviates from the written method —> Check the pump valves for leaks. A common cause are crystallized salts in the valves. The remedy is simple flush with salt-free solvents before the pump is switched off. Caution different HPLC systems have different mixing and dwell volumes. With gradient separations, check the equilibration time between injections and prolong it if necessary. Check the gradient method. 

The upshot of the gradient extension and the dwell time is when %B is reduced from its maximum value % o,max down to zero, then the farther forward that the peaks come in the chromatogram, the greater is the change in the separation. This can lead to a better separation, but also a change for the worse. In the rear section, only a slightly modified separation is to be expected. 

The volume between the solvent mixing point and column inlet is defined as dwell volume, and the corresponding time it takes for the selected composition of mobile phase to reach the column is called dwell time. Although the dwell volume is not the part of the extra-column volume, it is discussed in this section, as it impacts chromatographic performance. Under gradient conditions, the dwell volume for an HPLC system can have a significant effect on separation parameters including retention times (tr) and apparent retention factors fc. ... 

Effect of Dwell Volume on Separation As indicated in Eq. (3.10), the retention time and separation can be impacted by the dwell volume of an HPLC system. The delay time of an analyte due to the dwell volume is inversely proportional to the flow rate used for the separation for a given system, as expressed tdweii = where Vdwell is the dwell volume for a UHPLC or an HPLC system. Figure 3.8 shows gradient separations of phenones using UHPLC and HPLC with a 50 x 2.1 mm column. The first peak elution time is 2.2 min on UHPLC and 4.9 min in HPLC. The... 

A large dwell volume is rather disadvantageous. It delays and smoothes the gradient and requires a long equilibration time before the next separation. Large dwell volumes are not suited for low flow rates. 

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