Affinity monolith chromatography: a review of principles and recent analytical applications

Abstract

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.

Figure 1

A typical on/off elution scheme used in affinity chromatography.

Figure 2

Comparison of (a) the back pressures of affinity columns containing alpha₁-acid glycoprotein (AGP) that was immobilized to 300 Å pore size, 7 µm HPLC-grade silica particles, a silica monolith or a GMA/EDMA monolith; (b) comparison of the efficiencies, as represented by the total plate height (H_total), for columns based on the same silica particles and silica monoliths as used in (a). All of these columns were prepared using the same type of AGP, the same general type of immobilization method, and the same sample application and elution conditions. The results in (a) are for injections of S-warfarin. The data in (b) have been adjusted to represent the back pressures that would be expected for a 10 cm × 4.6 mm I.D. column. (Adapted from Mallik R, Xuan H, Hage DS (2007) J Chromatogr A 1149:294–304. With permission.)

Figure 3

Typical scheme for the thermal-initiated preparation of a GMA/EDMA monolith. (From Mallik R, Jiang T, Hage DS (2004) Anal Chem 76:7013–7022. With permission. Copyright 2005 American Chemical Society)

Figure 4

Examples of covalent immobilization methods that have been used to attach proteins and other amine-containing agents to GMA/EDMA monoliths: (a) the epoxy method, (b) the Schiff base method, (c) the carbonyldiimidazole (CDI) method, and (d) the disuccinimidyl carbonate (DSC) method.

Figure 5

Example of a silica monolith. (From Vervoort N, Saito H, Nakanishi K, Desmet G (2005) Anal Chem 77:3986–3992. With permission. Copyright 2005 American Chemical Society)

Figure 6

Purification of lactate dehydrogenase using a 6.0 cm × 16 mm I.D. agarose monolith that contained immobilized NAD⁺. These results were obtained for the application of a 50 ml sample to the column at 60 cm/h. The sample was applied and the column was washed using a pH 7 buffer, while the retained target was eluted by using a similar buffer with 1 mM NADH added as a competing agent. (From Gustavsson PE, Larsson PO (1999) J Chromatogr A 832:29–39. With permission.)

Figure 7

Components used to prepare a cryogel comprised of 2-hydroxyethyl methacrylate (HEMA) and containing the immobilized dye Cibacron Blue F3GA (Adopted from Dogan A, Ozkara S, Sari M M, Uzun L, Denizli A (2012) J Chromatogr B 893–894:69–76. With permission.)

Figure 8

An HPLC system for capturing and concentrating glycoproteins using tandem lectin columns based on monolithic supports. (From Selvaraju S, El Rassi Z (2012) J Sep Sci 35:1785–1795. With permission.)

Figure 9

Binding and elution of horseradish peroxidase (HRP) when using an anti-HRP monolith in a small pipette-based column. F1–F5 represent the fractions of the retained HRP that were eluted from the monolith when applying successive 150 µL aliquots of methanol to the column. The gel results were obtained by SDSPAGE without the use of a reducing agent. (From Faye C, Chamieh J, Moreu T, Granier F, Favre K, Dugas V, Demesmay C (2012) Anal Biochem 420:147–154. With permission.)

Figure 10

Use of silica monoliths in small columns containing immobilized HSA or an inert control support to estimate the dissociation rate of warfarin from HSA. The results in (a) show the chromatograms that were obtained for 100 µL injections of racemic warfarin at 4 ml/min onto 1 mm × 4.6 mm I.D. monoliths. The data in (b) show the natural logarithms of the same chromatograms, with the slopes of the tailing edge for the peak on the HSA column being used to estimate the dissociation rate constant for warfarin from this protein. (From Yoo MJ, Hage DS (2011) J Chromatogr A 1218:2072–2078. With permission.)

Figure 11

Typical chiral separation obtained for injections of R/S-propranolol at several flow rates onto a 10 cm × 4.6 mm I.D. silica monolith containing immobilized AGP. The mobile phase was pH 7.4, 0.067 M phosphate buffer containing 3% isopropanol. (From Mallik R, Xuan H, Hage DS (2007) J Chromatogr A 1149:294–304. With permission.)