Stationary phase ion exchange

As the solvent mixture also contained 225 mg of tetramethyl ammonium hydroxide pentahydrate per liter at a high water content (75%), the surface of the reverse phase would have been largely covered with the tetramethyl ammonium hydroxide pentahydrate. This would have acted as an adsorbed ion exchange stationary phase. It is clear that the free acids, salicylic acid, acetylsalicylic acid (aspirin) and benzoic acid were retained largely by ionic interactions with adsorbed basic ion exchanger and partly by dispersive interactions with the exposed reversed phase. The acetaminophen and the caffeine, on the other hand, being unionized substances, were retained only by dispersive interactions with the exposed reversed phase. 

We want the acids to be present only as ions, as the two forms of each acid will have different retentions on the ion-exchange stationary phase. As a rough guide, to suppress ionisation completely, we want to buffer at pH = (pKa - 1.5) and to cause complete ionisation we need pH = (p/Ca + 1.5). 

While, displacement chromatography of proteins has, for the most part, been carried out on ion-exchange stationary phases, several investigations have been carried out on stationary phases involving other modes of adsorption. 

Figure 2.18 Effect of radius of hydration on distance between counterion and fixed charge of ion-exchange stationary phase. Cesium, with smaller radius of hydration, is shown with one water molecule (small circle) between it and fixed charge of the bead. Lithium is shown with three water molecules.

GiUespie, E. et al. Evaluation of capillary ion exchange stationary phase coating dis-tiihution and stability using radial capillary column contactless conductivity detection. Analyst 2006, 131, 886-888. 

Polarizable anions include those listed in Group 2 of the table. These anions have a relatively high affinity for the ion-exchange stationary phase and therefore require a stronger eluent for their separation. 

The interaction of solute molecules with the ion-exchange stationary phase can be regarded as a sequential two-step process. Initially the solute must diffuse from the mobile phase (usually aqueous) into the stationary phase (often organic). The distribution between the two phases is largely responsible for the retention of a particular solute. Secondly, the solute must interact with, and diffuse through, the stationary phase. 

The degree to which the ion-exchange stationary phase can interact with the solute molecules is affected by pH. At pH values above the p a of ion-exchange groups, anion-exchangers are neutraUsed by base ... 

In 1967, Horvath developed pellicular ion-exchange stationary phases which were both stable and relatively efficient and enabled the apphcation of higher pressures, thereby facilitating the rapid separation of the mono-, di- and triphosphates of cytidine, uridine, adenosine, thymidine and guanosine. The subsequent development of stationary phases based on porous silica has led to a variety of chromatographic modes which are able to rapidly separate nucleosides and their derivatives. 

HPLC has been used for the analysis and purification of peptides for many years, both in the production of synthetic peptides and in the isolation of naturally occurring peptides. An extensive literature exists on the use of the technique, and the examples quoted here serve only to illustrate either recent developments, or specific and novel applications. In addition, the application of the new ion-exchange stationary phase in the separation of peptides is well documented in literature available from the manufacturers. Application of HPLC to purification, characterisation (peptide mapping), sequencing and purity assessment is now universally recognised. In addition, successful assays based on HPLC have been developed. 

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