Gradient chromatography methods

An unknown mixture can be screened on a set of orthogonal systems as a first step in the method development procedure. The chromatographic and/or electrophoretic system, on which the best separation was achieved, can then be retained for further method optimization. Sequentially, the pH and the organic modifier composition of the mobile phase can be adjusted to improve the separation on the CS. If necessary, also the temperature can be modified, while for gradient methods the gradient slope can be considered. For CE methods, the optimization steps will be different from RP chromatography methods. Other factors will be optimized depending on the type of CE method, e.g., CZE and MEKC. However, for the development of CE methods, we would like to refer to Chapter 4 of this book. 

K. Stella, M. Stella, A. Pandora, A. Morfis, B. Antonios, K. Maria, New gradient high-performance liquid chromatography method for determination of donepezil hydrochloride assay and impurities content in oral pharmaceutical formulation, J. Chromatogr. A 1189 (2008) 392-397. 

A particularly attractive feature of GC, one that distinguishes it from liquid chromatography methods, is the lack of a sensitive dependence on solvents, modifiers, and gradient elution systems. Prerequisites for the use of GC, however, are volatility, thermostability, and resolvability of the chiral analyte. Obviously, this restricts the utility of the method. 

A reverse-phase chromatography matrix is a hydrophobic surface, such as silica. The protein mixture is loaded in a relatively hydrophilic solvent, so that proteins with hydrophobic patches on their surfaces will bind to the column. The column is then eluted with a solution of increasing hydrophobicity and the proteins are eluted in order of their hydrophobicity as they are displaced by the solvent gradient. This method also separates protein mixtures into many different groups. 

Gradient elution is the norm for the analysis of the MCs, the reason is that the MCs differ significantly in their polarities and therefore in their retention characteristics however, there are a number of limitations inherent to gradient chromatography, namely, long equilibration times between analyses and retention time shift. As a consequence of the difficulties associated with gradient chromatography several isocratic methods have been developed to determine the MCs, many with complimentary, confirmatory mass spectral data (Tables 40.5 and 40.7). 

The determination of lovastatin and its hydroxyacid metabolite in plasma and bile can be accomplished by high performance liquid chromatography (25). Plasma samples are prepared for analysis by solid phase extraction and are analyzed using isocratic elution on a Cl 8 column. Bile samples do not require any sample dean-up prior to HPLC analysis, but do require the use of a gradient elution method to separate the compounds of interest. The HPLC assay has a limit of detection of 25 ng/mL... 

The chromatography columns used are Berger Instruments Amino, Cyano, and Diol in 6-p particle size with a 21.2 x 150mm format. The standard gradient elution methods employed are 5-50% methanol in... 

---