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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. 

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. 

Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low‐performance supports such as agarose for off‐line immunoextraction and supports used in high‐performance IAC for on‐line immunoextraction. The use of IAC for protein analysis as a flow‐based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example. 

Antibody‐based therapeutic agents and other biopharmaceuticals are now used in the treatment of many diseases. However, when these biopharmaceuticals are administrated to patients, an immune reaction may occur that can reduce the drug's efficacy and lead to adverse side‐effects. The immunogenicity of biopharmaceuticals can be evaluated by detecting and measuring antibodies that have been produced against these drugs, or antidrug antibodies. Methods for antidrug antibody detection and analysis can be important during the selection of a therapeutic approach based on such drugs and is crucial when developing and testing new biopharmaceuticals. This review examines approaches that have been used for antidrug antibody detection, measurement, and characterization. Many of these approaches are based on immunoassays and antigen binding tests, including homogeneous mobility shift assays. Other techniques that have been used for the analysis of antidrug antibodies are capillary electrophoresis, reporter gene assays, surface plasmon resonance spectroscopy, and liquid chromatography‐mass spectrometry. The general principles of each approach will be discussed, along with their recent applications with regards to antidrug antibody analysis.