Gradient Elution, Part IV: Dwell-Volume Problems

Jun 01, 2013
Volume 26, Issue 6, pg 330–336

Gradient methods can offer unique solutions to separation problems, but transferring a gradient method from the literature, between laboratories, or even within the same laboratory can be a challenging process.

This is the fourth instalment in a series of "LC Troubleshooting" columns about gradient elution in liquid chromatography (LC). Before we started this series, we considered some techniques for using a gradient scouting run to speed the initial investigations in method development or to quickly obtain a separation under generic conditions (1). This series of column instalments started with a discussion about how we could transfer our intuitive understanding of isocratic separations to gradients (2), and followed this with a way to compare isocratic and gradient methods under "equivalent" conditions (3). Last month (4), we considered some unexpected results, or surprises, that might occur if we inadvertently make changes in one gradient variable without making compensating changes in another. This month, we'll begin looking at some very practical problems related to gradient operation with a discussion of how the gradient dwell volume can impact the results.

What Is Dwell Volume?

Figure 1: Schematices of (a) high-pressure-mixing and (b) low-pressure-mixing LC systems, highlighting differences in dwell volume.
There are two general designs of LC gradient systems, as illustrated in Figure 1. High-pressure-mixing systems [Figure 1(a)] generally comprise two pumps, with mobile-phase blending taking place after the pumps (in the high-pressure region). Such systems are usually limited to two solvents, although switching valves may be included that allow you to switch from one solvent to another. The other design uses low-pressure mixing [Figure 1(b)], in which two to four solvents are blended before they reach a single pump (mixing on the low-pressure side of the pump). The design of both system types results in a measurable volume between the point the solvents are mixed and the inlet to the column — this is the dwell volume, sometimes called the gradient delay volume. From a practical standpoint, the dwell volume is made up of two parts. The first is the physical volume of the various components, including the mixer, any connecting tubing, and usually the injection loop volume. The second is the wash-out volume, which adds to the physical volume the hydraulic characteristics of the various components, especially the mixer, to increase the effective dwell volume of the system. We'll just refer to the combined dwell volume here, although we'll touch on the wash-out volume briefly later.

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