Gradient programming composition

Samples were injected as concentrated solutions in methanol. The initial composition of the solvent was 0 methanol/l00 water, but was changed at 5 /min using a linear gradient program, the solvent composition reaching 100 methanol in 20 min. Methanol of HPLC grade (Rathburn Chemical Ltd, Peebleshire,... 

The composition of the mobile phase varies as a function of time at the column inlet. We refer to this as the gradient program. 

We have seen in chapter 3 (table 3.10 b-d) that the composition of the mobile phase is a primary parameter in various forms of LC (LLC, RPLC, LSC). Gradient elution is only relevant for the latter two techniques, because the LLC system is not compatible with mobile phase gradients. Figure 6.6a shows a typical gradient program for LC. The complete program can be divided into a number of segments. 

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. 

A strategy for the optimization of gradient programs based on the actual retention behaviour of some sample components has been described by Jandera and Chura5ek [623, 624]. This approach relies on the possibility to calculate retention and resolution under gradient conditions from known retention vs. composition relationships and plate numbers. Both typical RPLC (eqn.3.45) and LSC (eqn.3.74) relationships can be accommodated in the calculations and linear, convex and concave gradients are all possible because of the use of a flexible equation to describe the gradient function. This equation reads... 

Reservoirs. Most analytical work with HPLC can be accomplished with reservoirs of approximately one-liter capacity. A vessel should be available for each component of the mobile phase if gradient programming (changing mobile phase composition) is employed. Ideally, reservoirs should be unbreakable and... 

Eqs. 5 and 6 could be applied to formulate computer programs that calculate the final values of Rp(j) for a given gradient program and the retention-eluait composition relationships for selected systems. The systems vay often applied in MD contain thin layers of adsorbents with definite groups like diol, cyano, or amine. " The basic equation describing the retention of solutes in such systems has the form of Eq. 2 or the following formula ... 

Gradient programs cause the most problems when viscous solvents are used. Figure 1.11 shows a plot of solvent viscosity versus composition for water/THF, water/methanol, and water/acetonitrile mixtures [19-21]. The resulting curves are nonlinear and always exhibit a maximum value higher than either pure component... 

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