Affinity adsorbents

The reaction kinetics approximation is mechanistically correct for systems where the reaction step at pore surfaces or other fluid-solid interfaces is controlling. This may occur in the case of chemisorption on porous catalysts and in affinity adsorbents that involve veiy slow binding steps. In these cases, the mass-transfer parameter k is replaced by a second-order reaction rate constant k. The driving force is written for a constant separation fac tor isotherm (column 4 in Table 16-12). When diffusion steps control the process, it is still possible to describe the system hy its apparent second-order kinetic behavior, since it usually provides a good approximation to a more complex exact form for single transition systems (see Fixed Bed Transitions ). 

Dibydropteridine reductase (from human liver) [9074-11-7] Mr52,000 [EC 1.6.99.7]. Purified to homogeneity on a naphthoquinone affinity adsorbent, followed by DEAE-Sephadex and CM-Sephadex... 

Phosphoproteins (various). Purified by adsorbing onto an iminodiacetic acid substituted agarose column to which was bound ferric ions. This chelate complex acted as a selective immobilised metal affinity adsorbent for phosphoproteins. [Muszyfiska et al. Biochemistry 25 6850 1986.]... 

Macromolecules bearing reactive groups in the repeat units along their chains are capable of multiple interaction with the matrix. As early as 1973, Wilchek prepared Sepharose-based supports chemically modified by chemisorbed polylysine and polyvinylamine [41]. The leakage of dyes covalently bonded to these supports was reduced remarkably as compared to non-modified Sepharose activated by cyanogen bromide. Thus, stable and high capacity affinity adsorbents could be prepared by the introduction of macromolecular spacers between a matrix and a biospecific ligand. 

The reaction kinetics approximation is mechanistically correct for systems where the reaction step at pore surfaces or other fluid-solid interfaces is controlling. This may occur in the case of chemisorption on porous catalysts and in affinity adsorbents that involve very slow binding steps. In these cases, the mass-transfer parameter k is replaced... 

Immobilized antibodies may be used as affinity adsorbents for the antigens that stimulated their production (Figure 6.15). Antibodies, like many other biomolecules, may be immobilized on a suitable support matrix by a variety of chemical coupling procedures. 

S. Zhang, X. Huang A microchip electrospray device and column with affinity adsorbents and use of the same, PCT Int. Appl. 2002, 59 pp,... 

An example of a highly specifie affinity adsorbant of this type is where a monoclonal antibody to a particultu compound is immobilised as shown in Figure 15.14. For instance, a gel with a monoclonal antibody to p-interferon attached has been used in industrial scale extraction of the compound from fermentation mixtures. 

As a result of these various requirements, the particle size to pick when designing a new affinity adsorbent will be a compromise between the desired chromatographic performances, properties of the feed stream, and the mechanical strength of the support. Some common selections made in specific cases will be described in the next few sections. 

In the preparation of affinity adsorbents, it is necessary to attach the ligand residues to insoluble supports by chemical bonds or physical interactions. Adsorbents having carbohydrate ligands have been prepared for the most part by chemical reaction of carbohydrate derivatives with the support material. Methods for the preparation of some types of carbohydrate derivatives are described in Section IV. Methods for the preparation of other derivatives of carbohydrates that may be suitable for synthesizing affinity adsorbents have been described in an article on neoglycoproteins.31... 

Affinity adsorbents having carbohydrate ligands have been used for the isolation and purification of many types of macromolecular substances. To illustrate, several types of antibodies, enzymes, lectins, and myeloma proteins have been obtained in highly purified form. All of the substances that have been purified by this procedure exhibit specificity for a particular carbohydrate moiety. It is the purpose of this article to assemble information on the methods for preparing affinity adsorbents having carbohydrate ligands, and to illustrate the use of these adsorbents for the purification of representative, macromolecular substances. 

The interaction between biological macromolecules can be of various degrees of complexity. These interactions may be of high specificity, as in antigen-antibody complexes, or of low specificity, as in protein-lipid associations. The complexes that are formed may be very stable, or highly unstable. These properties of a complex must be considered in selecting an affinity adsorbent to be used for the purification of a specific substance. 

Many types of derivatives of cellulose have been used to prepare affinity adsorbents.51 0-(2-Aminoethyl)cellulose and 0-(carboxy-methyl)cellulose can be coupled to carbohydrates bearing carboxyl or primary amino groups by the carbodiimide method.52,53 Cellulose derivatives bearing amino groups can be coupled to reducing carbohydrates by reductive-amination reactions25 in which the Schiff base produced in the initial reaction is reduced with sodium cyanoborohy-dride.54... 

Cellulose possesses some disadvantages for use as an insoluble support. The microstructure of cellulose and cellulose derivatives interferes with the permeation of substances through affinity adsorbents of these materials. Consequently, long periods are needed for separating and isolating substances on such columns. The linear chains of cellulose mask some ligand residues, and the adsorbent will have a low capacity. The amount of the substance that can be purified on such columns may be significantly diminished. Affinity adsorbents prepared from cellulose may exhibit nonspecific adsorption, and the purification of the desired substance could be difficult to achieve. 

Supports activated with divinyl sulfone, like those activated with bisoxirane, can be coupled to underivatized carbohydrates.21 The supports activated with divinyl sulfone couple to the hemiacetal hydroxyl group of the carbohydrate by the vinyl group. Accordingly affinity adsorbents prepared by this method will contain glycosyl units. The structure of the reaction product of L-fucose with agarose activated with divinyl sulfone is shown in 5. 

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