Programmed ternary gradients

Another complication may arise if we choose to vary the selectivity of a gradient program in LC by varying more than one parameter at the same time. For example, the concentration of two organic modifiers may be varied independently in RPLC (so-called ternary gradients) or both the mobile phase composition and the temperature may be programmed. 

In figure 6.9c a ternary gradient is shown in which a small concentration of the second modifier C is present throughout the elution. Figure 6.9d shows a gradient that runs from 100% A to 100% B and subsequently from 100% B to 100% C. This may be a sensible program if C is a considerably stronger solvent than B. 

The selectivity in programmed solvent LC may be varied by varying the solvents used or by the application of ternary or even more complicated gradients. However, most ternary gradients can in fact be reduced to binary ones using mixed (pseudo-) solvents. 

The most useful secondary parameter for the optimization of the selectivity in programmed solvent LC is the nature of the modifter(s) in the mobile phase. The selectivity can be varied by selecting various solvents (pure solvents for binary or ternary gradients mixed solvents for pseudo-binary gradients). Analogous to the situation in isocratic LC, it is possible to use different modifiers (and hence to obtain different selectivity) while optimum retention conditions are maintained for all solutes. This possibility to optimize the selectivity in programmed solvent LC will be discussed below. 

The application of the Simplex procedure for the optimization of the selectivity in programmed solvent LC (e.g. for the application of ternary gradients) has not yet been reported. However, there is no apparent obstacle to the applicability of the Simplex procedure for this purpose. 

Ricci, M.C. Cross, R.F. High performance liquid chromatographic euialyses of sulphonamides and dihydrofolate reductase inhibitors. II. Separations in acetonitrile modified solutions, ternary gradient studies flow programming. J.Liq.Chromatogr. Rel.Technol., 1996,19, 547—564... 

Experimental design methodology was applied for the optimization of the elution program for the RP-HPLC resolution of a mixture of nine phenols, with a ternary solvent system (water-AcOH-MeCN). Important factors were the initial isocratic elution, the gradient running time and the gradient curvature . ... 

Ternary and quaternary systems are also tested with A to C and C to D, respectively, if necessary. The gradient profile conditions (Section 25.3) are programmed, the system is rinsed thoroughly, and the gradient is run and registered. The profile is evaluated as shown in Figure 25.2. 

Examples Include binary, ternary, quaternary, linear and multiple-linear solvent gradients as well as for cases Involving stationary phase programming (coupled column gradient elution experiments). 

Because of the different separation mechanisms employed in interaction chromatography, the solvents used are usually different from the solvents in SEC. Binary or ternary mixtures of organic and aqueous solvents are frequently used as the mobile phase. The ratio of the solvents may be constant during the chromatographic run (isocratic elution) or may be programmed to vary with time (gradient elution). 

Matysik and Jusiak (77) used stepwise gradient development for separation of Chelidonium alkaloids in waste industrial fraction. Binary (toluene, methanol) and ternary (toluene, ethyl acetate, methanol) mobile phases were used and a six-step program was performed. Eight-step stepwise gradient was also used for separation of the glycosides from the Digitalis species (78). 

A gradient former to generate binary, ternary or quaternary mixtures of solvents with a pre-programmed composition profile during a separation (gradient elution). 

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