Rangan Mallik David S. Hage Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA Review Affinity monolith chromatography The combined use of monolithic supports with selective affinity ligands as statio- nary phases has recently given rise to a new method known as affinity monolith chromatography (AMC). This review will discuss the basic principles behind AMC and examine the types of supports and ligands that have been employed in this method. Approaches for placing affinity ligands in monoliths will be considered, including methods based on covalent immobilization, biospecific adsorption, en- trapment, and the formation of coordination complexes. Several reported applica- tions will then be presented, such as the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, and biomimetic chromatography. Other appli- cations that will be discussed are chiral separations and studies of biological interac- tions based on AMC. Keywords: Affinity chromatography / Affinity ligands / Affinity monolith chromatography / Biointeraction studies / Received: April 19, 2006; revised: April 30, 2006; accepted: April 30, 2006 DOI 10.1002/jssc.200600152 1 Introduction The use of monolithic materials in affinity chromatogra- phy is an area which has seen great growth over the last few years. This has given rise to a method that will be referred to in this review as affinity monolith chromato- graphy (AMC). This review will first examine the topics of affinity chromatography and monolithic columns and discuss how monoliths have been used in affinity separa- tions (Sections 1.1 – 1.3). The next items considered will include a summary of the types of supports, ligands, and immobilization methods that have been employed in affinity monoliths (Sections 2 and 3). Finally, the applica- tions of such monoliths will be discussed, along with recent developments in this field (Section 4). 1.1 General principles of affinity chromatography Affinity chromatography is a liquid chromatographic technique that uses a biologically related agent as the sta- tionary phase [1 – 3]. The retention of solutes in this method is based on the specific, reversible interactions found in biological systems, such as binding of an enzyme with a substrate or an antibody with antigen. These interactions are exploited in affinity chromatogra- phy by immobilizing or adsorbing one of a pair of inter- acting substances onto a support and using this as a sta- tionary phase. This immobilized binding agent is referred to as the affinity ligand. Figure 1 shows some common procedures for applying and eluting solutes from affinity columns. In each of these schemes, a sample is applied to the affinity column in a mobile phase known as the application buffer; this solution is generally selected to allow the immobilized ligand to bind its target while other sample components pass through with little or no retention. The retained tar- get is later eluted through one of several routes. If this compound is bound with only a weak or moderate affi- nity (i. e., possessing an association equilibrium constant for the ligand of 10 6 M –1 or less), it is possible to elute the target in the application buffer under isocratic condi- tions. This technique is known as weak affinity chroma- tography or dynamic affinity chromatography [2, 4, 5] and is common in applications of affinity columns invol- ving chiral separations or studies of solute-protein bind- ing [4 – 7]. More strongly retained substances can be eluted by changing the mobile phase or column conditions. For instance, this can be accomplished by using a separate Correspondence: Professor David S. Hage, Department of Chem- istry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA. E-mail: dhage@unlserve.unl.edu. Fax: +1-402-472-9402. Abbreviations: AGP, a1-acid glycoprotein; AMC, affinity mono- lith chromatography; CDI, carbonyldiimidazole; CNBr, cyano- gen bromide; CSP, chiral stationary phase; DHFR, dihydrofolate reductase; DSC, disuccinimidyl carbonate; EDMA, ethylene di- methacrylate; FAC, frontal affinity chromatography; GMA, glyci- dyl methacrylate; IDA, iminodiacetic acid; IMAC, immobilized metal-ion affinity chromatography i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 1686 R. Mallik et al. J. Sep. Sci. 2006, 29, 1686 – 1704