Programmed solvent

Program solvent gradient conditions as described in Table F4.4.1 or Table F4.4.2. 

The program starts and ends at the purge segment (P). The reason for this is related to the typical baseline observed in a gradient elution LC experiment (figure 6.6b). Unlike the situation in GC, the main cause of the blank signal in programmed solvent LC is formed... 

By analogy with the term programmed temperature GC [605] we will use the term programmed solvent LC , although solvent programmed LC is also commonly used. 

A second factor that contributes to the baseline variation is the difference in the background signal (absorption fluorescence) between the two solvents. This effect causes the difference in the baseline level between the left and the centre in figure 6.6b. A more extensive discussion on baseline variations in programmed solvent LC can be found in ref. [607]. 

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. 

In programmed solvent LC the nature of the modifier(s) in the mobile phase is the most common secondary parameter that may be used for the optimization of the selectivity. This is an attractive parameter, because different modifiers may be selected and programmed automatically on various commercial instruments. Therefore, the possibilities for selectivi-... 

The (primary) program parameters may be used to optimize the separation in programmed solvent LC in a non-selective way. Since this involves optimization of the... 

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. 

As with programmed temperature GC, the application of the Simplex optimization procedure to programmed solvent LC is relatively straightforward. The same procedure can be used both for isocratic and for gradient optimization, as long as an appropriate criterion is selected for each case. ... 

An indication of this latter effect can be found in figure 6.11, which shows the result of the Simplex optimization procedure applied to the programmed solvent LC separation of three antioxidants [621]. 

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. 

If larger solute molecules (e.g. proteins) are to be separated by programmed solvent LC, then much higher S values may be expected and consequently (eqn.5.8) a lower B value (shallower gradient) will be required [609]. 

Figure 6.12a shows the resulting optimal chromatogram for the separation of a mixture of seven barbiturates by programmed solvent RPLC This figure was obtained with the following optimization criterion ... 

For the optimization of programmed solvent LC the Sentinel method starts by establishing a suitable binary methanol-water gradient. The approach of Snyder described above may be used for this purpose. For example [627], a gradient from 20 to 100% methanol (in water) in 20 minutes may be the result. 

Figure 6.13 Figure illustrating the 7 linear gradients used in the Sentinel optimization method for programmed solvent LC. Initial and final compositions of the gradients are listed in table 6.4. 

Figure 6.14 Result of a Sentinel optimization of programmed solvent LC. Experimental design according to figure 6.13 and table 6.4. (a) Predicted optimum linear gradient and (b) chromatogram obtained with the optimum linear gradient. Stationary phase Zorbax alkyl silica.

Three methods appear to be available for optimizing the selectivity in programmed solvent LC ... 

The Sentinel method is the outstanding exponent of the group of interpretive methods, as it has already been applied successfully for selectivity optimization in programmed solvent LC. However, other interpretive methods, based either on fixed experimental designs or on iterative procedures, can be applied along the same lines. It was seen in section 6.3.2.3 that the extension of the Sentinel method to incorporate gradient optimization was fairly straightforward. 

FIG. 8 High-performance liquid chromatogram of the pigments extracted from pumpkin seed oil. Elution program solvent A 100% methanol solvent B methanol-hexane (4/1 v/v). Linear gradient solvent B from 0% to 100% in 28 minutes and then isocratic elution for 6 minutes. 

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