Chromatography often goes through the immobile and mobile phases. The immobile phase is provided by a gel matrix, usually an Agarose gel; a linear sugar molecule sourced from algae (Mattiasson et al. 1999). During the process, heterogeneous molecules having specific properties will be separated from other molecules that lack the targeted property. In essence, the target molecules are trapped in the solid medium (Mattiasson et al. 1999). The process then goes into the second phase (mobile) in which the mixture is washed and the target molecule eluted (Melander et al. 1999).
The binding of the molecules to a solid phase can be done using the column chromatography in which the â€œsolid medium is packed into the column and the initial mixture run through the column to allow setting.â€ (Melander et al. 1999) Awash buffer is run through the column, the elution buffer applied, and then collected from the column. Alternatively, this can also be achieved by a newer method that utilizes the advantages of batch setup; this method is referred to as expanded bed adsorption.
In this technique, â€œthe solid phase particles are placed in a column then the liquid phase is pumped from the bottom to exit from the topâ€ (Mattiasson et al. 1999). Gravitational force is exploited in this setup and it ensures that the solid phase remains with the particles as the liquid phase exits from the column (Melander et al. 1999).
Affinity chromatography is utilized in various operations which may include; â€œnucleic acid purification, protein purification from cell-free extracts and purification from bloodâ€ (Melander et al. 1999). The technique is more often used in medical laboratory investigations to purify antibodies from blood serum. In this case, the Agarose gel may contain specific antigens which covalently attach to the antibodies (Melander et al. 1999).
The same can be achieved using theâ€ immobilized metal ion affinity chromatography (IMAC)â€ (Mattiasson et al. 1999). This method is based on the particular covalent bonds that exist between amino acids, specifically histidine and metals (Mattiasson et al. 1999). Separation is usually achieved when the â€œproteins that have an affinity for metal ions are retained in a column containing the metal ionsâ€ (Melander et al. 1999). The metal ions that are commonly used include gallium, cobalt, zinc, nickel, iron, copper among others.
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