Applications of silica supports in affinity chromatography

Abstract

The combined use of silica-based chromatographic supports with immobilized affinity ligands can be used in many preparative and analytical applications. One example is the use of silica-based affinity columns in HPLC, giving rise to a method known as high-performance affinity chromatography (HPAC). This review discusses the role that silica has played in the development of affinity chromatography and HPAC and the applications of silica in these methods. This includes a discussion of the types of ligands that have been employed with silica and the methods by which these ligands have been immobilized. Various formats have also been presented for the use of silica in affinity chromatographic methods, including assays involving direct or indirect analyte detection, on-line or off-line affinity extraction, and chiral separations. The use of silica-based affinity columns in studies of biological systems based on zonal elution and frontal analysis methods will also be considered.

Imobilização de enzimas em suportes cromatográficos: uma ferramenta na busca por substâncias bioativas

“…A natureza específica dessas interações é responsável pela alta seletividade desse tipo de cromatografia, tornando possível o isolamento seletivo de um analito ou grupo de analitos em amostras complexas. 47 Inicialmente, a cromatografia de afinidade foi utilizada quase que exclusivamente para isolamento e purificação de micro ou macromoléculas em misturas biológicas complexas, 61 mas, nos últimos tempos, tem se revelado como uma poderosa ferramenta para monitorar as interações ligante-proteína, substrato-proteína, inibidor-proteína e ligante-receptor 10,13 demonstrando grandes vantagens como uma das técnicas de high throughput screening (HTS). 21,62,63 A cromatografia de afinidade envolve, essencialmente, três passos: a imobilização de um alvo; a avaliação das modificações sofridas pela biomolécula, após a imobilização e, a determinação dos parâmetros de ligação do ligante, depois da inserção da proteína imobilizada no sistema de separação.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…Affinity chromatography (AC) is one of the most versatile and adaptable types of liquid chromatography, since it is the only technique that uses a specific binding agent to purify a biomolecule on the basis of its biological function or individual chemical structure (Schiel et al 2006). Although the early affinity definition was related to the interactions similar to that occurring in many biological systems, such as the binding of an enzyme with a substrate or of an antibody with an antigen (Schiel et al 2006), the meaning of the affinity concept, in the biomolecules separation context, has undergone evolutionary changes over the years, especially as a way to answer to the challenges of purifying new biomolecules with clinical and therapeutic interest.…”

“…Although the early affinity definition was related to the interactions similar to that occurring in many biological systems, such as the binding of an enzyme with a substrate or of an antibody with an antigen (Schiel et al 2006), the meaning of the affinity concept, in the biomolecules separation context, has undergone evolutionary changes over the years, especially as a way to answer to the challenges of purifying new biomolecules with clinical and therapeutic interest.…”

“…Furthermore, a key factor for achieving successful pDNA purification by affinity chromatography is the selection of the ligand used within the column. Since its discovery, affinity chromatography has grown to include a wide variety of ligands for analytical and preparative applications (Schiel et al 2006). However, the biological origin of some affinity ligands is viewed as a limitation (Lowe et al 2001), since these ligands tend to be fragile and associated with low binding capacity.…”

Preparation of an Immunosorbent and its use in the Solid Phase Extraction of Benzodiazepines

“…18,19 One advantage of using silica in HPIAC is the availability of this support in a form that is both reproducible and that gives good chromatographic efficiency. 20 Silica particles with diameters of 3-5µm are now commonly used in HPIAC separations because the relatively small particle diameters result in short diffussional distances for solutes, which helps to increase mass transfer and allows for rapid, efficient separations. Although silica particles with diameters of 5-10 µm and pore sizes of 40-100 Å are commonly used in methods such as reversed chromatography, these pore sizes are not appropriate for many proteins 20 because such pores are comparable in size to the proteins, leading to either the exclusion of proteins from these pores or slow mass transfer as a result of restricted diffusion.…”