This month's installment of "LC Troubleshooting" is prompted by an error that I made in January's installment (1). A reader
had inquired about the source of an observed change in peak width and retention time when moving a gradient liquid chromatography
(LC) method from one LC system to another. I misconstructed the example chromatograms of Figure 1 in (1), which confused the
associated discussion. What concerns me more than the error is the fact that only two readers brought the error to my attention
— usually if I make a mistake, I get dozens of e-mails pointing out my error. This made me decide to return to the topic of
dwell volume for this month's column.
Dwell Volume and Dwell Time
Figure 1
When we refer to "dwell volume," we mean the system volume from the point at which the mobile phase solvents are mixed until
they reach the head of the column. For high-pressure-mixing LC systems, this comprises the mixer, connecting tubing, and autosampler
loop as the primary components (Figure 1). Low-pressure mixing systems combine the solvents upstream from the pump, so additional
tubing plus the volume of the pump head (or heads) is added to the components of the high-pressure mixing system (Figure 2).
Typical dwell volumes for today's LC systems are 1–3 mL for high-pressure mixing and 2–4 mL for low-pressure mixing systems.
These volumes can be reduced to <0.5 mL for systems modified for use with mass spectrometry detection (LC–MS) or can be 5–8
mL, or even larger, for some of the older equipment still in use. Dwell volume (VD) is measured easily, as described in the sidebar.
Figure 2
Dwell volume is of practical importance only for gradient applications. When mobile phase components are mixed on-line for
isocratic separations, the dwell volume still exists, but because the mobile phase concentration is constant, there is no
observed difference between chromatograms run on different dwell-volume systems. Gradient methods, on the other hand, rely
on a change in the concentration of mobile phase over time to facilitate the separation. The delay created by the dwell volume
can make a difference in the appearance of the chromatogram for different gradient systems.
We generally report chromatographic retention in units of time, not volume, so it often is convenient to express the dwell
volume instead as dwell time. Dwell time (tD) is obtained by dividing the dwell volume by the flow rate. So a system with a dwell volume of 3.0 mL run at a flow rate
of 1.5 mL/min would have a dwell time of 2.0 min.
The Chromatogram
Example chromatograms for LC systems with 1.5- and 3.5-mL dwell are shown in Figures 3a and 3b, respectively. Note that this
is identical to the erroneous Figure 1 of reference 1 except the chromatograms are interchanged. If you keep "LC Troubleshooting"
for future reference, I encourage you to photocopy Figure 3 and paste it over Figure 1 of the January 2006 installment. At
a flow rate of 2 mL/min, the gradient reaches the head of the column at (1.5 mL/2 mL/min) = 0.75 min in the run of Figure
3a, as noted by the arrow in the gradient overlay. In a similar manner, the dwell time for Figure 3b is 1.75 min.
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