Isocratic method development approach

Lewis, J. A., Snyder, L. R., and Dolan, J. W., Initial experiments in high-performance liquid chromatographic method development. II. Recommended approach and conditions for isocratic separation, ]. Chromatogr. A, 721,15,1996. 

The approach to method development is similar to the one described for HPLC and can be characterized as a rapid stationary phase screen using column and solvent switching with gradient elution followed by development of an isocratic preparative method. SFC has been successfully applied to the analytical and preparative separation of achiral and chiral compounds. 

Another means of controlling eluent strength is the use of ternary or quaternary solvent mixtures instead of the more common binary approach. Each solvent has its own unique properties that can be used to improve the separation of difficult-to-resolve analytes or to shorten the analysis time without sacrificing resolution. Although gradients and more complex solvent matrices are more difficult to model than binary isocratic systems, software exists for such purposes and can assist in method development. 

Two popular approaches are frequently used in method development (1) mobile-phase optimization based on sequential isocratic runs and (2) mobile-phase optimization based on gradient runs. As specified, a primary goal of late-phase method development is to achieve isocratic operating conditions. Accordingly, the following approaches focus initially on the optimization of isocratic conditions. A discussion of gradient optimization follows for mixtures not amenable to isocratic separation. 

The fuU mathematical treatment of gradient elution (Snyder 1979) that led to these relationships (Table 3.2) is too complex to be reproduced here indeed, in practice a gradient elution method is often developed by an essentially trial-and-error approach based on an isocratic method while keeping in mind the restrictions imposed by the qualitative considerations outlined above. However, it is possible to give some flavor of this theory to provide some understanding of the principles, as follows. 

In order to reduce the number of analyses and analysis time standardisation of existing polymer/ additive analysis is important. Obvious advantages are presented by universal methods. For example, Taylor et al. [22] have recently proposed a hybrid SFE/ESE technique as being a general approach to rapid sampling of both polar and non-polar analytes from polymeric matrices. Similarly, the reduction in number of various specific chromatographic methods, all based on the same column, speeds up analysis [23]. Method development needs to keep an analysis as simple as possible, such as isocratic in case of HPLC, in order to facilitate transfer to other laboratories with different equipment. 

Most RP-HPLC separations are done in the isocratic mode (i.e., where the composition of the mobile phase is held constant during the analysis). This approach is suitable when the sample consists of analytes having similar properties or where their hydrophobicities encompass a small or moderate range. Under these conditions, all solutes in the sample will be eluted over a reasonable time span (i.e., not too short to prevent resolution of individual analytes and not too long to result in an inconvenient analysis period). Therefore, proper selection of the mobile-phase composition is essential in the development of any RP separation method. Fortunately, due to the decades of... 

For example, Hermansson et al. [63] employed a diol-modified silica column (250 X1.0 mm, 5-pm particles) and used an isocratic elution with a mobile phase comprising hexane-isopropanol-water-formic acid-triethylamine (628 348 24 2 0.8 v/v). The mass spectrometric detection was in the negative-ion mode, in which either [M-H] or [M-i-HCOO] molecular ions were monitored. The investigators separated most of the common lipid classes and developed a method with MS detection to automatically identify and quantify over 100 lipid species through two-dimensional mapping of elution time and the masses of lipid species (i.e., an SIM approach). 

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