Solvent optimisation 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... 

When the chromatographic mode, column type, packing and dimensions have been chosen, the final stage of method development involves solvent optimisation and a choice between isocratic or gradient elution. Many separations can be achieved perfectly satisfactorily under isocratic conditions and are preferred to gradient elution techniques, as these are inconvenient due to the time required to re-equilibrate the column. A measure of the quality of separation is given by the resolution factor which can be expressed as follows ... 

Pappa-Louisi, A. Nikitas, P. Papageorgiou, A. Optimisation of multilinear gradient elutions in reversed-phase liquid chromatography using ternary solvent mixtures. J. Chromatogr. A, 2007,1166, 126-134. 

Based on chemical shifts and peak multiplicities, the on-flow HPLC-NMR characterisation of the majority of the components in the mixture of 27 tripeptides was achieved and demonstrated that this approach is likely to be an effective method for compound mixtures. The elution positions of all of the alanyl-containing peptides were determined, with the exception of A-M-M-NH2, which may have co-eluted with another peptide or may have been synthesised in a much smaller quantity. The only other tripeptides for which assignments have not been obtained are the MY2-NH2 isomers and two of the three M2Y-NH2 isomers. These eluted towards the end of the gradient run and are not as well resolved under these HPLC conditions. Additionally, with changes in the relative chemical shifts of the solvent signals, the intensities of the non-TV-terminal a-CH protons and the methionyl [3-methylene signals from these peptides may have been reduced by the effects of the solvent suppression irradiation of the water and acetonitrile resonances, respectively. With further optimisation of the elution conditions, it is possible that all 27 analytes could have been resolved and characterised. 

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