The Basic Frontal Method

Frontal analysis (FA) is perhaps the most straightforward of all chromatographic methods. To operate the method, one continuously infuses sample through a stationary phase and monitors breakthrough times. The goal of such analyses is not to separate components of the mixture, but simply to explore the nature of the interaction between column and compound. An example can be found in Fig. 6.1. Here, a large volume sample of caffeine is injected onto a C18 column, sufficient to achieve breakthrough conditions. In this experiment, a single point determination of the breakthrough volume immediately provides a measurement of the amount of compound bound, while a concentration series accurately describes the isotherm governing the compound/stationary phase interaction -different models of interaction behavior can then be applied to rationalize the interaction.

The method has a rich history in the characterization of compound-stationary phase interactions as it supports the determination of thermodynamics and kinetics of interaction between a solute and a stationary phase. What has emerged from these studies is the recognition of FA as the premier chromatographic method for generating interaction data; high precision and accuracy are a direct result of making measurements under undistorted dynamic equilibrium conditions. These advantages offer the opportunity to ''dissect'' the molecular basis for molecular interactions. For example, FA supports the determination of complexity in solute-sorbent interactions as shown in Fig. 6.2, by revealing distinct binding modes. This figure demonstrates that a ''simple'' interaction between a small molecule nortryptiline and a C18 column is better described as a convolution of

Fig. 6.2 An example of a binding isotherm generated from frontal affinity data. This example shows that nortryptiline on a C1g reversed-phase column exhibits complex binding behavior. At least three distinct binding modes exist between the compound and the stationary phase [31]. q* represents the concentration of bound nortryptiline and C the total concentration of applied nortryptiline. Adapted with permission from Elsevier.

Fig. 6.2 An example of a binding isotherm generated from frontal affinity data. This example shows that nortryptiline on a C1g reversed-phase column exhibits complex binding behavior. At least three distinct binding modes exist between the compound and the stationary phase [31]. q* represents the concentration of bound nortryptiline and C the total concentration of applied nortryptiline. Adapted with permission from Elsevier.

at least three distinct types of interaction. This sort of information is useful in the development of advanced materials for high performance chromatography.

Its application to the measurement of biochemical interactions is intuitive -simply replace the conventional analytical stationary phase with ligand, protein, DNA or any relevant biomolecule. A large-scale version of the method was first described in 1975 by Ken-Ichi Kasai [1] and referred to as frontal affinity chromatography (FAC). The method finds application in the process engineering field, where adsorbents are used to study the interaction of proteins on immobilized ligands, for the purpose of optimizing purification schemes [2-4]. The realization of the analytical benefits of FAC was later in coming [5-7]. Through extensive miniaturization of the affinity columns, sensitive FAC assays have been implemented that are comparable to the amounts used in sensitive biosensor applications [8].

Developing a FAC assay for discovering or characterizing molecular interactions involves effort comparable with most bioassay development exercises. Optimal buffer conditions need to be determined, including the use of necessary co-factors (e.g. divalent cations, secondary ligands). Column design requires a valid immobilized form of the protein, ligand or other biomolecule. This is no more problematic than similar requirements found in surface plasmon resonance (SPR) assays and many plate-based assays. It is worth emphasizing that, with the production of recombinant proteins and the ability to selectively insert affinity tags, much of the complexity involved in this stage of assay development has been removed. In addition, new developments in protein entrapment suggest that covalent immobilization can be circumvented in certain situations, as will be discussed in Section 6.3.

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