How Viscous Fingering Can Spoil Your Separation And You May Not Even Suspect It

LEVEL: INTERMEDIATE T echniques of both analytical and preparative liquid chromatography have advanced substantially in recent years. Nowadays, samples are often analyzed or purified in systems that incorporate one or more f low-stream changes from one mobile phase to another. It happens, for example, in column switching, in multidimensional separations, and in simulated moving bed (SMB) chromatography, where a feedstock stream enters continuously into a mobile-phase stream. When f low-stream switching is necessary, care must be paid to the compatibility of the different f low solutions. Obviously, the streams must be miscible, but it is less obvious that their viscosities should be similar. An important consideration is the solvent in which a sample is dissolved before injection. Usually that solvent is the same as the mobile phase, but sometimes a solute must be dissolved in a stronger solvent with a different viscosity. In some instances a mismatch between mobile phase and solute plug, or mobile phases in twodimensional (2D) separations, or feedstock and mobile phase in SMB, leads to viscosity differences large enough to cause a phenomenon known as viscous fingering (1–3). Occurrence of viscous fingering (VF) can have a catastrophic effect on separation performance, leading to separation failure (4). In short, viscous fingering is a f low instability phenomenon that occurs at the interface of two f luids of differing viscosities. When a high-viscosity f luid is displaced by a lower viscosity f luid, their interface is unstable. After a time (depending on the viscosity contrast), the lower viscosity f luid penetrates the other in a pattern resembling a set of fingers. Those “fingers” multiply and develop further into a complex network (1–10). VF can be detrimental to chromatographic separations when a low-viscosity mobile phase fingers into a highviscosity solute plug (2), or conversely, when a low-viscosity sample plug fingers into a higher viscosity mobile phase (1). Similar adverse effects take place when a mobile-phase stream is replaced with one of different viscosity in the complex schemes now developed for 2D chromatography (3). Analysis of polymers and isolation of proteins using size-exclusion chromatography (SEC) present ideal environments for development of VF. Solutions of high molecular weight polymers and proteins are more viscous than their mobile phases and do not dilute appreciably during rapid elution (4). When proteins are isolated at the preparative level, sample solutions have a high viscosity for exactly the same reasons as polymer solutions. In a worst-case scenario, a set of multiple peaks instead of a In an extreme case of VF, the band width expands to the column length