The biologics and natural product industries rely heavily on separation technology. Sample analyses are undertaken on the analytical scale, and isolation and purification are undertaken at the preparative scale. Key target components are often isolated to provide standard reference materials for future product quality assurance testing. These products are often very complex mixtures, requiring separation systems to have a high peak capacity for both analytical and preparative scale separations. A technique gaining popularity among companies that require the isolation of pure compounds from complex sample matrices is two-dimensional liquid chromatography, which can be conducted at either the analytical (2D-HPLC) or preparative scale (2D-PHPLC). Operated in a heart-cutting mode, 2D-PHPLC is a technique for which the first dimension essentially serves as an extraction step, simplifying the sample matrix before separation of target analyte(s) in the second dimension. In the comprehensive mode (LC × LC), it is useful for analytical analysis and sample profiling.

PRODUCT FOCUS: BIOLOGICS AND NATURAL PRODUCTS



PROCESS FOCUS: DOWNSTREAM PROCESSING



WHO SHOULD READ: MANUFACTURING, PROCESS DEVELOPMENT, AND ANALYTICAL PERSONNEL



KEYWORDS: COLUMN CHROMATOGRAPHY, SOLVENTS, PACKED BEDS, FLOW PROFILE, TWO-DIMENSIONAL SEPARATIONS



LEVEL: ADVANCED

Multidimensional chromatographic separation techniques use differences in the selectivity of each dimension that are oriented toward specific component attributes within a sample. Hence, different stationary and/or mobile phases are often used in each dimension. For example, common types of multidimensional systems include ion-exchange–reversed-phase, size-exclusion–reversed-phase, and (increasingly more useful) reversed-phase–reversed-phase. As such, a typical operational factor of a two-dimensional separation is that the mobile phase is different in each dimension, nearly always in at least the solvent composition, but often also in solvent type.

Coupled column multidimensional chromatographic separations require that a sample be transported from the first dimension to the second dimension as a dilute solution in a solvent plug. When operated on a preparative scale, this transport (heart-cut) volume fraction may be large to maximize sample recovery. Increasingly, scientists applying combinations of liquid chromatographic modes of separation are becoming aware of the limitations associated with solvent transportation processes, especially those that occur between two different separation dimensions. In particular, the phenomenon known as viscous fingering (VF) has been gaining increasing attention (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17).

Viscous fingering is a flow instability phenomenon that occurs when two fluids with different viscosities come into contact with each other. When one fluid pushes the other, as is the case of a mobile phase and sample in chromatographic elution, the interface between these fluids can become unstable. The lower-viscosity fluid penetrates the higher-viscosity fluid in a complex interaction that resembles fingers. The phenomenon was first noticed by Hill in 1952 (1). But it was Saffman and Taylor who coined the name viscous fingering and investigated it (2). The first important paper detailing the significance of VF in liquid chromatography was published by Moore, who studied the effects of VF in size-exclusion chromatography (3). Numerous researchers have modeled the VF process and some have applied these models specifically toward chromatographic applications (4, 5).

In the mid 1990s, Fernandez and coworkers 6,7,8) were the first to visualize the VF phenomenon in chromatographic columns when they used MRI imaging to view inside a column. These visualization experiments were closely followed by Broyles, Shalliker, and Guiochon, who visualized VF in glass-tube columns and used stationary and mobile phases that had exactly the same refractive indices (9). In this technique, an otherwise opaque resin bed becomes perfectly transparent. Colored solute markers injected into the columns were observed and photographed, recording the VF process. This provided absolute proof that viscous fingering was important in liquid chromatography.

Further studies by Shalliker and coworkers verified that significant changes in band shapes were apparent even when the viscosity contrast between the injection plug and the mobile phase was not significant enough to initiate a fully developed VF phenomenon (10,11,12,13) They coined this effect previscous fingering (11). These were important findings that highlight the need to pay particular attention to the viscosity differences between solute plugs (or heart-cut sections) and mobile phases, even when this difference is moderate and the actual VF process is not initiated — because the result may significantly change how multidimensional HPLC is undertaken, especially at the preparative level (14). How best to improve the performance of your multidimensional preparative HPLC separation (or at least how to minimize solvent-related inefficiencies) given that a viscosity contrast between solvents in each dimension may be a necessary factor in separation problems.