Method development solvent selectivity

U. Huber, Method development -solvent and column selection with Agilent 1100 Series valve solutions, Agilent Publication No. 5900-8347EN (2002). 

The issue of selectivity is one that is often difficult to address. Initial method development is invariably carried out by using standards made up with pure solvents, i.e. free from any matrix effects. It is often only when real samples are analysed that the true extent of interference becomes apparent and the value of the method can be properly assessed. An added complication is that interferences , by their very nature, are not constant and a number of samples may have a combination of interferences that defy analysis by a method that is otherwise successful on a routine basis (another example of Murphy s law ). 

Regarding development, normal sdica gel layers are tried first if not otherwise indicated. Selection of the best developing solvent has to be performed systematically, and its preparation should be proper. Various modes of development and separation chambers, both of which can influence the separation, have to be considered for development, with focus on the classical method of chromatogram development. Aspects of the chamber climate are helpful as background information and can improve resolution. 

The HcReynolds system of phase constants has become the most widely used systematic approach to solvent selectivity characterisation and virtually all pedlar phases have been characterized by this method. In spite of its popularity the approach is fundamentally flawed and the phase constants are an unreliable indication of i ase properties. The basic approach, however, has influenced the development of other methods of selectivity characterization, and although these methods have inherited many of the deficiencies of their parent, a brief description of the HcReynolds approach is worthwhile to. idicate the general limitations of methods based on retention index differences. 

The selection of a mobile diase for the separation of simple aixtures may not be a particuleurly difficult problem and can be arrived at quite quickly by trial and error. Solvent systems can be screened in parallel using either several development chambers or a device like the Camag Vario KS chamber, which allows the simultaneous evaluation of a number of solvents by allowing each of these to migrate along parallel channels scored on a single TLC plate [8]. However, whenever the number of components in a mixture exceeds all but a small fraction of the spot capacity for the TLC system, a more systematic method of solvent optimization is required. 

Snyder L.R., Dolan J.W., Molnar I., and Djordjevic, N.M., Selectivity control in reversed-phase HPLC methods development varying temperature and solvent strength to optimize separations, LC-GC, 15 (2), 136, 1997. 

The use of the Hildebrand solubility parameter approach to aid solvent selection with a few simple experiments, starting from the liquid solvents used in traditional extraction methods, limits the efforts needed in method development. As for other extraction... 

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

NRTL-SAC has been demonstrated through the case study on Cimetidine as a valuable aid to solubility data assessment and targeted solvent selection for crystallization process design. The average model error is typically 0.5 Ln (x) [1] and is sufficient as a solvent screening tool. Methods that can deliver greater accuracy would increase the value and utility of these techniques. It is impressive in the case of Cimetidine that the NRTL-SAC correlation is capable of reasonable accuracy and predictive capability on the basis of just 2 fitted parameters. Further work to extend the solvent database and optimize the descriptive parameters will be beneficial, and are planned by the developers. 

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