Separation selectivity, displacement

Thus, a 2-D separation can be seen as 1-D displacement operating in two dimensions. The 2-D TLC separation is of no interest if selection of the two mobile phases is not appropriate. With this in mind, displacement in either direction can be either selective or non-selective. A combination of two selective displacements in 2-D TLC will lead to the application of different separating mechanisms in each direction. As an extreme, if the solvent combinations are the same (5ti = 5t2 5vi = va) or very similar (5ti 5vi 5ya), the compounds to be separated will be poorly resolved or even unresolved, and as a result a diagonal pattern will be obtained. In such circumstances, a slight increase in resolution might occur, because of an increase by a factor of V2 in the distance of migration of the zone (4). 

The Effects of Eluent Temperature. m agreement with previous studies (67), both solute retention and separation selectivity were found to increase significantly as the eluent temperature was reduced from 30 to 0 C, as shown in Fig. 6 for the enantiomers of tryptophan. The change is much larger than what is observed on an octadecylsilica reversed-phase column and the improved separation selectivity is attributed to more pronounced inclusion complex formation. Solute retention decreases precipitously when the temperature of the eluent is raised to or above 60 C, an important fact that can be utilized advantageously for the rapid removed, of strongly adsorbed components (such as displacers) from the cyclodextrin-silica column. 

Thus, in conclusion, it can be stated that retention studies such as the ones outlined above permit the selection of operation conditions (type and concentration of the organic modifier, concentration and pH of the buffer, temperature of the eluent) which lead to sufficient initial solute retention (k >10) and maximum separation selectivity necessary for a successful displacement chromatographic separation. 

In some cases, natural convective flow plays an integral role in separations. For example, thermogravitational (TG) columns rely on a combination of thermal convective flow and relative (selective) displacement by thermal diffusion. 

FIGURE 8 Displacement histogram and UV detector trace for a selective displacement process. (A) Displacement separation of a three-component protein mixture using streptomycin sulfate A as a displacer. Column 100 X 5 mm i.d. strong cation exchange (8 m) carrier 30 mM sodium phosphate buffer, pH 6.0 feed 1.6 mL of 0.392 mAI ribonudease A, 0.42 mM horse cytochrome c and 0.34 mM lysozyme in the carrier. Total column loading 12.7 mg/mL column displacer 25 mM streptomycin sulfate A flow rate 0.2 mL/min fraction size 200 /iL. (Kundu et al.43) (B) UV detector trace monitored at 280 nm for the displacement separation shown below. 

Fki. 20. Contribution of solvent-solute hydrogen bonding to separation selectivity (see text). The data are for MTBE (O) and ACN ( ) as basic solvent. The ACN curve is displaced vertically by +0.2 log units. Reprinted from Snyder (19). 

Active ester formation by the mixed anhydride method is accompanied by the side reaction of esterification at the carbonate moiety of mixed anhydride 51 which generates mixed carbonate 52 (Scheme 12).This decreases the yields, but is more of a nuisance than an obstacle as the side products do not interfere with crystallization of the esters as the former are soluble in the crystallizing solvent. More mixed carbonate is formed from derivatives of the hindered amino acids and proline none is formed from a-unsubstituted acids. A-Hy-droxysuccinimide gives rise to much less byproduct than 4-nitrophenol other phenols generate intermediate amounts. Less byproduct is generated when the reagent is isopropyl chloroformate. The impurity can be readily removed from a solution of the ester by adsorption of the compounds on reverse-phase chromatography beads followed by separation by selective displacement. ... 

When the solute isotherms cross one another, the situation becomes more complex. It then becomes possible to experience selectivity reversal that is, at one displacer concentration, the solutes elute in the order A first, then B, whereas at another displacer concentration, the order is B first, then A. In a study of this problem, Antia and Horvath showed the existence of the separation gap. This is a region in the isotherm plane, the position of which depends on the ratio of the saturation capacities of the solutes in question. If the operating line is outside the separation gap, displacement occurs in the normal fashion. The elution order of the solutes then depends on the position of the operating line relative to the separation gap. However, if the operating line is within the separation gap, displacement operation does not separate the displaced solutes, but results in the elution of a mixture of the solutes. 

As discussed earlier in this chapter, one of the characteristic features of the competitive Langmuir isotherm is that the selectivity between any pair of components is constant, independent of their concentrations. As a consequence, the requirements for their successful separation by displacement chromatography would merely be that the selected displacer is retained more strongly than either feed... 

Note that actual chromatograms will look like those shown in Figures 12.1 to 12.4 only if the concentrations of each of the feed components are plotted versus the volume of eluent passed. This requires either the use of a selective detector e.g., LC/MS), or of fraction collection and analysis. The chromatogram in Figure 12.17 illustrates the difficulties encountered in developing a separation by displacement chromatography. Unless the isotachic train is fully formed and the response factors of all feed components are similar, the recording of the response of a nonselective detector is very difficult to accoxmt for. 

The initial mixture, as well as the final separated state, may consist of either a single phase or a collection of immiscible phases. If separation is desired in a feed consisting of two immiscible phases, then each component has to be selectively displaced toward its designated phase. Such component-specific displacement and the separation achieved thereby may or may not lead to pure phases. It is, however, a prerequisite to any separation. 

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