Affinity adsorption/chromatography

Figure 7-11. Purification ot staphylococcal nuclease by affinity adsorption chromatography on a nuclease-specific agarose column (0.8 x 5 cm). The column was equilibrated with 50mM borate buffer, pH 8.0, containing lOmAf CaClj. Approximately 50 mg of partially purified material containing about 8 mg nuclease was applied in 3.2 ml of the same buffer. After 50 ml of buffer had passed through the column, O.IM acetic acid was added to elute the enzyme. 8.2 mg nuclease and all the original activity was recovered. The flow rate was about 70 ml/hour. [From P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Natl. Acad. Sci. US, 61 636 (1968).]...

Alpha-1-proteinase inhibitor and antithrombin III are used to treat people with hereditary deficiencies of these proteins. Both can be recovered from Cohn Fraction IV (Table 7) using ion-exchange chromatography (52) and affinity chromatography (197), respectively. Some manufacturers recover antithrombin III directiy from the plasma stream by affinity adsorption (56,198,199). 

Porath, J., and Olin, B. (1983) Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22, 1621-1630. 

Occurs in nature in abundance the principal forms are bauxites and lat-erites. The mineral corundum is used to produce precious gems, such as ruhy and sapphire. Activated aluminas are used extensively as adsorbents because of their affinity for water and other polar molecules and as catalysts because of their large surface area and appropriate pore sturcture. As adsorbents, they are used for drying gases and liquids and in adsorption chromatography. Catalytic properties may be attributed to the presence of surface active sites (primarily OFT, 02, and AF+ ions). Such catalytic applications include sulfur recovery from H2S (Clauss catalysis) dehydration of alcohols, isomerization of olefins and as a catalyst support in petroleum refining. 

Adsorption chromatography relies on the different affinity of components of a mixture for a liquid moving phase and a solid stationary phase. The separation mechanism depends upon differences in polarity between the different feed components. The more polar a molecule, the more strongly it will be adsorbed by a polar stationary phase (Varki et al. 1999). Similarly, the more nonpolar a molecule, the more strongly it will be adsorbed by nonpolar stationary phase. It is often employed for relatively nonpolar, hydrophobic materials so that the solvent tends to be nonpolar while the stationary phase is polar. Proteins have a high affinity to polar chromatographic media and the recovery of the sample is usually difficult. Therefore, this method is not commonly used to purify and characterize proteins. 

In high-pressure adsorption chromatography, solutes adsorb with different affinities to binding sites in the solid stationary phase. Separation of solutes in a sample mixture occurs because polar solutes adsorb more strongly than nonpolar solutes. Therefore, the various components in a sample are eluted with different retention times from the column. This form of HPLC is usually called normal phase (polar stationary phase and a nonpolar mobile phase). 

The basic principles of adsorption, ion-exchange, and affinity resins have been explained in the previous section on adsorption. Chromatography is similar to adsorption because both involve the interaction between solute and solid matrix. However, they are different in a sense that chromatography is based on the different rate of movement of the solute in the column, while adsorption is based on the separation of one solute from other constituencies by being captured on the adsorbent. 

At industrial scale, various chromatographic techniques are available adsorption chromatography, which uses physical binding effects which are dependent on pH or salt concentration affinity chromatography, where a specific binding between a molecule and the matrix is achieved and partition chromatography, where product and impurities move through the bed at different rates. 

The formulation of the mass transfer kinetic processes was first presented by Kucera 28] to describe gas adsorption chromatography. This approach was then adapted for modeling in liquid chromatography 129,30) and affinity chromatography 131 ] experiments. The model used to describe the adsorption of the protein on the affinity support is given in Fig. 1 and includes the following steps ... 

Earlier fluorometric methods for analysis of urinary free catecholamines have been replaced by HPLC methods that allow selective quantitation of epinephrine, norepinephrine, and dopamine. Preliminary extraction of urine is stid required and numerous preanalytical cleanup techniques are available. An alumina extraction procedure is typically coupled with ion-exchange or adsorption chromatography. Alumina pretreatment usually involves a batch extraction technique in which catechols are first adsorbed at pH 8.6 and then eluted with boric acid, which forms a complex with cis-diol groups. Purification on boric acid affinity gels provides an alternative procedure for selective adsorption of catecholamines. 

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