Separation methods affinity chromatography

Affinity Absorption A method of separation by affinity chromatography. It may be used, for example, to remove unwanted antibodies from an antibody preparation. The preparation is passed through a column matrix containing antigens against which the unwanted antibodies are directed. Thus, the unwanted antibodies remain bound to the column. The antibody solution leaving the column contains only the desired antibodies, purified by affinity absorption. 

The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). 

Several methods have been used to separate the lanthanides chemically solvent extraction, ion exchange chromatography, HPLC using Q-hydroxyisobutyric acid and, in limited cases, selective reduction of a particular metal cation.40-43 The use of di(2-ethylhexyl)orthophosphoric acid (HDEHP) for the separation of various rare-earth elements via solvent extraction has also been reported.44 16 This separation method is based on the strong tendency of Ln3+ ions to form complexes with various anions (i.e., Cl- or N03 ) and their wide range of affinities for com-plexation to dialkyl orthophosphoric acid. When the HDEHP is attached to a solid phase resin, the lanthanides can be selected with various concentrations of acid in order of size, with the smallest ion being the most highly retained. 

Although affinity chromatography has not been used directly as an analytical method, it may be modified in the future to produce a viable technique. Leucovorin has been used as an effective spacer in obtaining active samples of dihydrofolate reductase.79 If the enzyme could be immobilized without losing its activity, perhaps it could be used to separate folates. 

While the individual components of the mixture may be separated from each other, they will all be contaminated to some extent with the eluting solute. Techniques such as ion-exchange chromatography and various types of adsorption and affinity chromatographies are examples of displacement methods. 

A detailed examination of the affinity of SLPI for the heparinized capillary was next made using a stepwise elution (from 0.1 to 0.9 M NaCl) (Fig. 11). SLPI eluted from the capillary with 0.2 M NaCl. This agreed well with results obtained by traditional affinity chromatography on a heparin-Sepharose matrix. The ACE method has the unique advantages over traditional affinity chromatography in that it requires much smaller quantities of protein and afforded better separation profiles. 

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