Mobile phase column changes

FIGURE 13.1 Initial Chromatograms were obtained using THF as mobile phase. Each column was then stressed by an abrupt change of mobile phase from THF to methanol. Methanol was run through each column for approximately 16 hours, at which time the mobile phase was changed back to THF. 

The deflection refractometer (Fig. 8.4), which measures the deflection of a beam of monochromatic light by a double prism in which the reference and sample cells are separated by a diagonal glass divide. When both cells contain solvent of the same composition, no deflection of the light beam occurs if, however, the composition of the column mobile phase is changed because of the presence of a solute, then the altered refractive index causes the beam to be deflected. The magnitude of this deflection is dependent on the concentration of the solute in the mobile phase. 

If the separation is still unsuitable after optimizing the experimental conditions and column length, selectivity must be optimized further by changing the stationary phase, the type of column, or the mobile phase by changing it or adding a modifier. These choices were described earlier in this chapter, because the changes needed depend so heavily on the nature of the analytes and/or sample matrix, it is difficult if not impossible to provide concise, general recommendations. 

On the other hand, optimization of the column temperature may significantly improve the overall performance of the chromatographic system. Several studies describe a combination of temperature control with the mobile-phase composition changes and the effect of the combined parameters on reversed-phase separations [10]. 

In liquid chromatography, system selectivity can be modified by a number of techniques, including (1) changing the composition or pH of the mobile phase, (2) changing the column temperature, and (3) changing the type of stationary phase that is employed. Of these, the first method is the easiest to accomplish and is most often the first to be utilized in method development for improving selectivity. 

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