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3. Characterization of Vapor Phase
For the characterization of the chamber saturation a marker test dye mixture was proposed by Nyiredy et al. (40). The chromatograms of this dye mixture have to be developed with dichloromethane. The Rf range of the applied dyes depends on the chamber saturation. If the hRf values of the marker dye mixture are the same in different chambers on a given stationary phase, the grade of the chamber saturation is identical. For comparison, the hRf values obtained from different chambers can be depicted in a coordinate system. The h/fy values of any given system are plotted along the y-axis and those from the system being compared along the jt-axis. If the chamber saturation is identical, a linear relationship has to be obtained, and tg a for the line is 1.
Two basic possibilities arise during characterization of chamber saturation. If the hfy values obtained in the second system were smaller, the vapor phase was more saturated and tg à > 1, or a
> 45°. Conversely if tg a < 1, or a < 45° the vapor phase in the second system was Jess saturated. These possibilities are illustrated by results A and Â in Figures 3a and 3b, respectively.
With the help of this approach the chamber type can be characterized for a certain separation (X in Figure 3) without specification of the vapor phase. Obviously, the separation can be influenced by changing the saturation of the vapor phase. The example depicted in Figure 3a, shows that increasing the degree of saturation of the chamber used for separation X would reduce the hRj values of the compounds separated and reduce the resolution obtained. Figure 3b shows that use of a less saturated vapor phase increased the resolution obtained at lower /fy values.
These facts, together with the hfyvalues of the test dye mixture, can be used to characterize the different types of chamber and, simultaneously, indirectly characterize the vapor phase conditions used for a given separation (40). These results can be used for comparison of separations with given stationary and mobile phases, thus enabling prediction of the separation under other vapor phase conditions, e.g., the different forced-flow planar chromatographic techniques. The technique also provides guidelines for the transfer of mobile phases between the different planar chromatographic methods.
As a rule of thumb, if the sample contains less than 7 compounds to be determined quantitatively, saturated chromatographic tanks have to be selected for the development method. If the sample contains more than 7 substances for quantitative determination, or the separation is very difficult, unsaturated chromatographic chambers have to be selected that enable the transfer of the optimized mobile phase for OPLC separation.
C. Suitable Solvent Selection
The basis for the strategy of the solvent selection is the solvent classification by Snyder (25), who classified more than 80 solvents into eight groups for normal phase (NP) chromatography according
tg a < 1----------------------------> a < 45
Figure 3 Calculation of the effect of the chamber saturation on the separation. (Reproduced from Ref. 40, with permission.)
828 Szepesi and Nyi
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to their properties as proton acceptors (xa), proton donors (xd), and their dipole interactions (*„) (see Figure 4, Part I). From these eight groups 29 solvents were chosen and commonly used in planar chromatography (53-56).
For the selection of suitable solvents the first experiments can be carried out on TLC plates with the suggested neat solvents. After these first experiments the solvent strength either has to be reduced or increased so that the substance zones are distributed between fy0.2 and 0.8. If the compounds to be separated migrate in the upper third of the plate the solvent strength has to be reduced by dilution with hexane. If the substances do not migrate with the neat solvents the solvent strength has to be increased by addition of water. In both cases the solvent strength could be varied so that a better distribution of the substance zones is obtained. From the solvents showing good separation their homologs or other solvents of the same group also may be tested. Consequently, the structures and properties of the compounds to be separated do not have to be known for the experiments. After these experiments, the solvents giving adequate separations are chosen for further optimization of the mobile phase (56).
D. Mobile Phase Optimization
Mobile phase optimization is based both on the analyst’s experience and intuition and on modifications of published data. However, as the sample composition becomes more complex, systematic solvent optimization becomes more important (38). The methods used for optimizing isocratic mobile phases in HPLC are generally also applicable, with some modifications, to TLC. Window diagrams have been successfully applied (45-47) for the optimization of TLC mobile phases. Similarly, overlapping resolution maps were used as criteria by Issaq et al. (48) and by Nurok et al. (49), who