Displacement chromatography principles

Additional results from AX purification of DMT-off synthetic oligonucleotides are discussed in the following section, based on the principles of displacement and sample self-displacement chromatography. 

Ampholyte displacement chromatography [43] does not require special packings to be applied. Conventional ion exchangers are sufficient. The main principle is the elution of the column with carrier ampholytes. Leaback and Robinson [43] used CM-cellulose for separation of acetyl hexosamidase isoenzymes (Fig. 4.5.3), Young and Webb [44,50] separated serum proteins on DEAE-cellulose, and Chapuis-Cellier et al. [97] described ampholyte displacement chromatography of hemoglobins on DEAE-cellulose. 

The principle of displacement chromatography for separation is based on the Langmuir isotherm. Only a finite number of sites are on the chromatographic support (stationary phase) for the binding of sample components, and if a site is occupied by one molecule, it is not available to the other sample components. Because the number of binding sites is limited, they are saturated when the concentration of the molecules in the sample is large in comparison to the dissociation constant for the sites. 

A non-equilibrium theory of FFF [14] was elaborated on the principles parallel to the principles of the non-equilibrium theory of chromatography [15]. The solute is displaced in a moving fluid by a combined action of the flow and the fields applied. As long as no flow occurs in the system, a concentration gradient induced by the field will be balanced by diffusion which will lead, after a certain time, to a steady state or to a quasi-equilibrium concentration, c. As soon as the flow is activated, the quasi-equilibrium will be permanently disturbed. The distribution of concentrations across the channel can be described by the relationship... 

In 1940, Tiselius [14] classified chromatography according to the separation principle, namely, elution development, displacement development and frontal analysis. In practice only elution and to a lesser extent, displacement development are commonly used (Figure 1.3, p. 13). 

In principle, the modes and steps in preparative chromatography do not differ from those in analytical chromatography. Four modes are used (Fig. 4) isocratic elution, linear gradient elution, stepwise elution, and displacement development. Frontal analysis is not used for purification of biomolecules, but it is used for... 

The concept of separating sample components in a column was first developed in 1903 by Mikhail Tswett, who introduced the term chromatography in 1906. Unfortunately, his contemporaries showed little interest for the idea and almost 30 years went by before scientists in Germany rediscovered the principle of column liquid chromatography (LC). Then, in 1943 Arne Tiselius (in Sweden) classified chromatography into three modes frontal, elution, and displacement. The elution mode actually became synonymous with almost all chromatography, but in recent years the displacement mode has attracted new interest, particularly in the separation of proteins. 

It should be noted that the displacement of inorganic ions in an exchange adsorption column by addition of dilute acid should proceed according to the principles described above. This has been confirmed 1 some eiqieriments of Brattsten and the author (not yet published). In most cases of ordinary chromatography it may be difficult to say if elution or displacement development is used. If the displacing solution is applied in high concentration or if it is added too fast, there is in-suffident time for a stationary state to be established, and the front of the displacer will overrun the other zones and a sort of mixed elution and displacement will occur. This is also the case if coarse grained adsorbent material is used, in which diffusion into the interior requires too much time to allow the adsorption equilibrium to be established. Nevertheless, it is useful to know the above three ideal types of adsorption analysis even if they cannot always be realized experimentally. 

---