Equipment for gradient elution

The quality of an HPLC analysis is often equated with the resolution of the peaks achieved, and as previously discussed this may be optimised by adjusting the solvent composition to obtain appropriate capacity factors for the solutes. For difficult separations, isocratic elution may be inadequate and the desired resolution may only be achieved using gradient elution 

There are two types of system commercially available for gradient forming. 

This system can be used at low compositions (1 %) of A or B (except at low flow-rate). The main disadvantage is cost and inconvenience, as two pumps are required for this set-up and that the system is generally limited to two-solvent gradients. 

The pump consists of two pistons, but solvent is delivered to the column only from one piston, the other piston chamber serves as a reservoir. As illustrated the pistons are 180° out of phase such that when piston A is delivering solvent to the column, the piston reservoir B is being replenished with solvent. The piston volumes are 100 pi and thus at a flow-rate of lmlmin the dehvery fill cycle is 6 s. As the fill cycle takes the same time as the delivery cycle then the proportioning valves on the low pressure side have 6 s to actuate. Mixing of the component solvents occurs inside the pump and connecting tubing dining the transfer of the solvent from the reservoir piston to the delivery piston. 

The routines developed for determining the optimum four-solvent eluant composition are designed to yield a prechosen level of resolution for all pairs of components in a mixture by carrying out no more than seven analyses with various solvent compositions. Evaluation of these chromatograms allows selection of a set of standard conditions for the given application. The technique has a sound basis in theory and appears to have general applicability and has been used with both normal and reverse phase systems. The routines associated with the optimisation process have been reviewed by Glajch et al. [15] and Poile [16] and are discussed in detail in section 6.14. 

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