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The analysis of interactions between biological agents or with surrounding chemicals is important in many areas of modern biochemical, biomedical, and environmental research. Microscale platforms based on affinity chromatography have been shown to be a powerful set of tools for these studies. This approach makes use of an immobilized binding agent as a stationary phase in a microscale platform for either direct examination of the interactions of this agent with an applied target solute or as a secondary capture agent to probe a solution‐phase interaction. This review will examine the various platforms and strategies that have been used in microscale affinity chromatography, or µAC, to characterize and study biointeractions. The general principles of µAC and schemes based on this approach will be examined, along with applications of this technique. Examples of approaches that will be considered will include zonal and frontal analysis methods, as well as a variety of schemes by which µAC can be employed in kinetic studies. In each case, the theory and principles of these methods will be provided along with examples of their use in biointeraction studies. 

The characterization of drug-target interactions is a key component of drug discovery, testing, and development. Affinity chromatography is one approach that can be used for this type of analysis. For instance, this may be done by using an immobilized target as a stationary phase and a drug as the applied solute. This review will discuss the various ways in which affinity chromatographic methods have been used to examine drug-target interactions, with an emphasis on high-performance methods. The general principles of this approach and factors to consider in its use for drug-target interaction analysis will first be examined. Methods based on zonal elution or frontal analysis for binding and competition studies will then be discussed. Various techniques for kinetic studies will next be considered, along with approaches that employ secondary binding agents and hybrid techniques. In each case, the general principles and theory of an approach will be given along with examples of its use in drug-target interaction studies. Advantages or limitations of each approach will be provided as well. This information should make it possible in the future to extend these techniques to other drug-target systems of interest in biomedical research and drug testing or development. 

High‐performance affinity microcolumns with entrapped humic acid were utilized to investigate interactions between this natural carrier agent and several classes of antibiotics that are common emerging environmental contaminants, or micropollutants. Aldrich humic acid was used as a general model for this type of binding agent. Chromatographic studies under various temperature and mobile phase conditions were used to characterize interactions of the humic acid with the antibiotics sulfadiazine and sulfamethoxazole (sulfonamides), clarithromycin (a macrolide), and lincomycin (a lincosamide). It was determined by this approach that sulfadiazine and sulfamethoxazole had moderate affinities for the humic acid at pH 7.0 and 25°C, with distribution equilibrium constants (KD) of ∼2–3 × 10^1 L/kg and global affinities (nK’a) of ∼0.8–1.0 × 10^3 M^−1. Lincomycin and clarithromycin had stronger binding, with KD and nK’a values of 3.8–7.5 × 10^2 L/kg and 1.3–2.6 × 10^4 M^−1. All the antibiotics had a negative for this binding, representing spontaneous reactions, and a negative change in enthalpy; however, the change in free energy due to entropy was positive in some cases but negative in others. The binding strength decreased in each case as the ionic strength increased. A change in pH also affected binding, as was consistent with the presence of significant electrostatic interactions from some of the antibiotics. These experiments demonstrated how affinity microcolumns could be employed to study such interactions quickly and with only small amounts of binding agent. The fundamental information obtained through this analytical technique should be valuable in characterizing the transport and activity of these antibiotics in the environment and in adapting this approach to the study of other binding agents and micropollutants that may be found in water. 

The analysis of biomolecular interactions is important in characterizing and understanding many fundamental processes that occur in the body and biological systems. A variety of methods are available for studying the extent and rate of binding of these interactions. Some of these techniques are homogeneous methods, with all interacting components being present in the solution-phase, while others are heterogeneous, such as involving both solution-phase and solid-phase components. LC and HPLC have often been used to study biomolecular processes. Although these chromatographic methods make use of both a liquid phase (i.e., the mobile phase and applied samples) and a solid phase (the stationary phase and support), they can be used to study solution-phase interactions. This review examines several strategies that have been developed and employed to use LC and HPLC for this purpose. These strategies include the Hummel-Dreyer method, solution-phase frontal analysis, and the use of physical entrapment for a soluble component of a biomolecular interaction. Other strategies that are discussed are those in which the stationary phase of the column is used as a secondary component or capture agent when studying a solution-phase interaction, as occurs in normal-role affinity chromatography and ultrafast affinity extraction. The general principles for each of these strategies will be considered, along with their advantages, potential limitations, and applications. 

Background: DJ-1 is a protein whose mutation causes rare heritable forms of Parkinson’s disease (PD) and is of interest as a target for treating PD and other disorders. This work used high performance affinity microcolumns to screen and examine the binding of small molecules to DJ-1, as could be used to develop new therapeutics or to study the role of DJ-1 in PD. Non-covalent entrapment was used to place microgram quantities of DJ-1 in an unmodified form within microcolumns, which were then used in multiple studies to analyze binding by model compounds and possible drug candidates to DJ-1. Results: Several factors were examined in optimizing the entrapment method, including the addition of a reducing agent to maintain a reduced active site cysteine residue in DJ-1, the concentration of DJ-1 employed, and the entrapment times. Isatin was used as a known binding agent (dissociation constant, ~2.0 µM) and probe for DJ-1 activity. This compound gave good retention on 2.0 cm × 2.1 mm inner diameter DJ-1 microcolumns made under the final entrapment conditions, with a typical retention factor of 14 and elution in ~8 min at 0.50 mL/min. These DJ-1 microcolumns were used to evaluate the binding of small molecules that were selected in silico to bind or not to bind DJ-1. A compound predicted to have good binding with DJ-1 gave a retention factor of 122, an elution time of ~15 min at 0.50 mL/min, and an estimated dissociation constant for this protein of 0.5 µM. Significance: These chromatographic tools can be used in future work to screen additional possible binding agents for DJ-1 or adapted for examining drug candidates for other proteins. This work represents the first time protein entrapment has been deployed with DJ-1, and it is the first experimental confirmation of binding to DJ-1 by a small lead compound selected in silico. 

The presence of pharmaceuticals as microcontaminants in the environment has become a particular concern given the growing increase in water reuse and recycling to promote global sustainability of this resource. Pharmaceuticals can often undergo reversible interactions with soluble dissolved organic material such as humic acid, which may be an important factor in determining the bioavailability and effects of these compounds in the environment. In this study, high-performance affinity microcolumns containing non-covalently entrapped and immobilized humic acid are used to examine the binding strength and interactions of this agent for tetracycline, carbamazepine, ciprofloxacin, and norfloxacin, all common pharmaceutical microcontaminants known to bind humic acid. The binding constants, as measured with Aldrich humic acid, have good agreement with values reported in the literature. In addition, the effects of temperature, ionic strength, and pH on these interactions are examined with the humic acid microcolumns. This technique made it possible to determine the relative importance of electrostatic interactions vs non-polar interactions or hydrogen bonding on these binding processes. This study illustrates how affinity microcolumns can be used to screen and uniformly quantify binding by pharmaceuticals with humic acid, as well as to study the mechanisms of these interactions, with this information often being acquired in minutes and with small amounts of binding agent (~0.3 mg per microcolumn, which could be used over 200-300 experiments). Use of entrapment and affinity microcolumns can support similar research for a wide range of other microcontaminants with humic acid or alternative binding agents found in water and the environment. 

Chromatography is a robust and reliable separation method that can use various stationary phases to separate complex mixtures commonly seen in metabolomics. This review examines the types of chromatography and stationary phases that have been used in targeted or untargeted metabolomics with methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. General considerations for sample pretreatment and separations in metabolomics are considered, along with the various supports and separation formats for chromatography that have been used in such work. The types of liquid chromatography (LC) that have been most extensively used in metabolomics will be examined, such as reversed-phase liquid chromatography and hydrophilic liquid interaction chromatography. In addition, other forms of LC that have been used in more limited applications for metabolomics (e.g., ion-exchange, size-exclusion, and affinity methods) will be discussed to illustrate how these techniques may be utilized for new and future research in this field. Multidimensional LC methods are also discussed, as well as the use of gas chromatography and supercritical fluid chromatography in metabolomics. In addition, the roles of chromatography in NMR- vs. MS-based metabolomics are considered. Applications are given within the field of metabolomics for each type of chromatography, along with potential advantages or limitations of these separation methods.