In a follow-up study, high-temperature LC, enabling lower organic modifier concentrations, was demonstrated [ 43].
Different parameters, such as substrate (4-HPAA) and enzyme concentrations, reaction time, temperature, additives, and organic modifier concentrations were first optimized for the PAD system used in FIA mode.
The variation of the isotherm parameters with the modifier concentration was accounted for.
The reason for the existence of an optimum modifier concentration is also discussed.
The effect of simultaneously varying the pressure, temperature and modifier concentration was studied to optimize the method in order to obtain excellent chromatographic figures of merit.
The effect of the chiral modifier concentration was also studied and the results revealed a bell-type curve dependence of the enantiomeric excess with the modifier concentration.
One advantage of a higher organic modifier concentration is that higher flow rates can be used in HPLC gradients, which may result in shorter analysis times and higher resolutions.
Here we have illustrated this effect using the PT:MPS system, which has very different binding constants (K′ = 70), and have carried out experiments where the total modifier concentration was fixed at 1 × 10 4 M (equivalent to ca. 1.25 monolayers with the volume of HRSC used).
However, at total modifier concentrations that are closer to those required for monolayer coverage, the equilibrium position resulting from the competitive adsorption will be perturbed because surface binding of the modifier with the highest affinity will deplete its concentration in the bulk phase, reducing its ability to compete for the remaining sites.
By comparing these measurements, the assay window in relation to the different organic modifier concentrations can be estimated.
