Development mobile phase gradients

Figure 7.13 Separation of a test eixture using automated multiple development with a universal mobile phase gradient from acetonitrile through dlchloromethane to carbon disulfide on a silica gel HPTLC plate. The chromatogram was scanned at different wavelengths to enhance the chromatographic information.

Instrumentation requirements for SEC are somewhat simpler than those of other modes of HPLC, since mobile phase gradients are not used however, adequate computer support for data acquisition and processing is essential. Method development involves finding a suitable solvent for the sample and choosing a mixed bed column or, more often, a set of columns in series to match the pore size of the column(s) with the size distribution of the sample. 

Conventioal development procedures of two or more component planar mobile-phase systems form phase gradients. The most polar phase stays near the bottom of the development plate [29]. Besides these mobile-phase gradients, stationary-phase gradients based on having a sorbent of varying composition and medium gradients such as temperature have been studied [27,47],... 

Inject 100 pX of the PTC-amino acid sample and apply the following mobile-phase gradient during development (mark the time of injection, or T = 0, on the chart recorder) ... 

Inject a 100-jul sample of the 20 PTC-amino acid standards and apply the same mobile-phase gradient. The sequence of the PTC-amino acids eluting from the column developed with this gradient is shown in Table 6-1. 

In addition to the stationary and mobile phases, separations obtained in TLC are affected by the vapor phase, which depends on the type, size, and saturation condition of the chamber during development. The interactions of these three phases as well as other factors, such as temperature and relative humidity, must be controlled to obtain reproducible TLC separations. The development process with a single (isocratic) mobile phase is complicated because of progressive equilibration between the layer and mobile phase and separation of the solvent components of the mobile phase as a result of differential interactions with the layer, which leads to the formation of an undefined but reproducible mobile phase gradient. 

The optimization of reversed phase separation by gradient runs is sometimes unavoidable even in process scale. Mobile phase gradients can reduce the development time dramatically as the solvent strength is varied during the chromatographic... 

Gradient development. Gradient developments have been used to achieve separation of complex mixtures such as plant extracts, dyes, etc. However, their application to lipid separation is not very common. Golkiewicz (1996) discusses in detail stationary phase gradients and mobile phase gradients, the theory behind solvent selection, automated techniques and applications of gradient elution in TLC. 

Multicomponent mobile phases often are separated ( demixed ) as they migrate through the layer because different compounds of the solvent mixture are attracted more-or-less strongly to the sorbent. This leads to a mobile-phase gradient effect along the layer and the appearance of extra fronts behind the bulk mobile-phase front. These fronts can aid separations and should not affect the reproducibility of results if TLC conditions are constant from run to run. Results will not be reproducible, however, unless fresh mobile-phase and the same equilibration conditions are used for each development. 

The three types of gradients that have been used the most in TLC are mobile phase, temperature, and stationary phase gradients. Planned mobile phase gradients must be distinguished from the natural, uncontrolled gradients that result from solvent demixing during development. 

Sander and Field (16) used a liquid chromatograph (solvent programmer in conjunction with two pumps) to generate a mobile-phase gradient. The eluent was introduced into the developer trough and distributed across the layer. 

Multiple development may also be performed in the same direction and development distance with different mobile phases gradient multiple development (GMD)). It is also possible to develop preparative plates, especially 0.5 mm layers, with the bivariate multiple development (BMD)... 

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