Biochemical techniques based on
(i) Sedimentation coefficient : The sedimentation coefficient s of a particle is used to characterize its behaviour in sedimentation processes, notably centrifugation. It is defined as the ratio of a particle’s sedimentation velocity to the acceleration that is applied to it (causing the sedimentation).
Sedimentation: In a sedimentation experiment, the applied force accelerates the particles to a terminal velocity Vterm at which the applied force is exactly canceled by an opposing drag force. For small enough particles (low Reynolds number), the drag force varies linearly with the terminal velocity, i.e., Fdrag = fvterm (Stokes flow) where f depends only on the properties of the particle and the surrounding fluid. Similarly, the applied force generally varies linearly with some coupling constant (denoted here as q) that depends only on the properties of the particle, Fapp = qEapp Hence, it is generally possible to define a sedimentation coefficient Sdef = q/f that depends only on the properties of the particle and the surrounding fluid. Thus, measuring s can reveal underlying properties of the particle.
Rate zonal centrifugation : This is a type of density gradient centrifugation. The sample is applied in a thin zone at the top of the centrifuge tube. Under centrifugal force, the particles will sediment through the gradient in separate zones based on their sedimentation coefficient or‘S’value.
(ii) Technique used to separate biomolecules based on polarity : Ion-exchange chromatography: It is defined as the reversible exchange of ions in solution with ions electrostatically bound to some sort of insoluble support medium. Separation is obtained since different molecules have different degree of interaction with the ion- exchanger due to difference in their charges, charge densities and distribution of charge on their surfaces. These interactions can be controlled by varying conditions such as ionic strength and PH.
An ion-exchanger consists of an insoluble matrix to which charged groups have been covalently bound. Ion exchange separations are carried out mainly in columns packed with an ion-exchanger. There are two types of ion-exchanger, namely cation and anion exchangers. Cation exchangers possess negatively charged groups and these will attract positively charged cations. Anion exchangers have positively charged groups that will attract negatively charged anions. After the ion exchange the molecules can be eluted from the matrix by selective desorption. The selective desorption can be achieved by changes in PH and /or ionic concentration or by affinity elution, in which case an ion that has greater affinity for the exchange than has the bound ion is introduced into the system.
