Liquid Chromatography - LC Mobile Phases

Due to their being stable to extremes of pH, their use for the separation of peptide and proteins at both high and low pH has been well established. An example of a macro-reticulated resin phase used as an exclusion medium in the separation of a crude protein extract is shown in figure 39. The column, 9TSKgel QC-PAK GFC 399GL0, was 15 cm long and 8 mm in diameter and the separation was carried out at a flow rate of 1 ml/min. The mobile phase consisted of 0.5M NaCl in 0.05M sodium phosphate buffer at pH 7.0. The sample was 5ml of a rat liver extract. An excellent separation based on molecular size is obtained from which the molecular weight range of the mixture and even that of the individual components could be estimated.

LC Mobile Phases

The choice of phase system can be very complex, particularly if multicomponent mixtures are to be separated. In the first instance the type of stationary phase needs to be chosen and this choice must be based on the interactive character of the solutes to be separated. If the solutes are predominantly dispersive then the stationary phase must also be dispersive (a reversed phase) to promote dispersive interaction with the solutes and provide adequate retention and selectivity. If the solutes are strongly polar then a polarizable stationary phase (one containing aromatic rings or cyano groups) would be appropriate to separate the solutes by polar and induced polar interactions. If the solutes are weakly polar then a strong polar stationary phase would be required (such as silica gel) to separate the solute by polar interactions.

The mobile phase must be chosen to complement the stationary phase so that the selected interactions are concentrated in the stationary phase. Thus, a reversed phase having strong dispersive interactions would be used with a strongly polar mobile phase (e.g., mixtures of methanol and water acetonitrile and water or tetrahydrofuran and water). In contrast, if the strongly polar silica gel is selected for the stationary phase then a strongly dispersive mobile phase would be appropriate (e.g., n-heptane, n-heptane/methylene chloride or n-heptane with a small quantity of n-propanol or ethanol). In general the mobile phase must be chosen so that the selected interactions strongly dominate in the stationary phase and are minimized in the mobile phase.