Mobile phase parameters, adjusting

Once a selection of the column type has been made, sample resolution is optimized by adjusting the ionic strength, pH, temperature, flow rate, and concentration of buffer or organic modifier in the mobile phase [39a], The influence of these parameters on solute retention is summarized in Table 15.9. 

In supercritical fluid chromatography, fluids above their critical point are used as mobile phases. This chapter discusses the principles of operation, mobile phase considerations, parameters that can be adjusted in method development as well as an overview of instrumentation required and a few pertinent examples from current literature. Not everything can be illustrated, but the advantages of this diverse technology will be highlighted. 

The various separation parameters should be adjusted to provide optimum resolution. These include mobile phase flow rate, stationary phase particle size, gradient elution, and column temperature (using an optional column oven). 

Obtaining data on the effects of these parameters may allow one to judge whether a method needs to be revalidated when one or more parameters are changed. For example, if column performance changes over time, adjusting the mobile-phase strength to compensate for changes in the column may be allowed if such data are included in the validation. 

Among the other factors to be considered in the choice of separative conditions is temperature. Peak resolution and retention time (RT) depend greatly on this parameter an increase in temperature diminishes the viscosity of the mobile phase and of the sample, and a reduction of RT is observed in addition to an increase in the efficiency of the column. However, this effect is not always produced, nor for all supports some sulphonic ion-exchange resins show for analytes such as ethanol, the opposite effect. It is clear that thermostatization of the system must be very thorough so that repeatable results may be obtained. Very commonly used temperatures are those between 25°C and 70°C, values that must be adjusted each time depending on the complex of analytes to be separated. 

In eluent compositions beyond those that are typically part of the routine adjustments carried out by the analyst to meet system suitability parameters. As expected, the use of short columns packed with 3-um particles results in reduced solvent consumption and reduced mobile phase re-equilibration time in gradient elution. 

Dnring the infancy of IPC, retention prediction commonly faced trial-and-error procednres that attempted to make the problem univariate, holding all experimental conditions constant except one. This one-at-a-time changing of parameters, without regard to parameter interactions, is still practiced and may, in a time consuming way, improve performance. The description of the dependence of retention on the mobile phase composition parameters is the focus of interest of model makers becanse an a priori retention prediction is highly desirable. Optimization is finding the nnique combination of values of adjustable parameters that yields the best performance possible for a set of requirements. 

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