Manipulation of Retention of Acids in Ion Exclusion Chromatography

The dominating factor which determines retention is the degree to which the acid is ionized. Separation is based on the electrostatic repulsion between the solute ions and fixed functional groups of the resin. Therefore, ionic species are excluded from the stationary phase while partially ionized or uncharged species partition between the mobile phase and the occluded liquid within the resin pores. Assuming this is the only mechanism, the solute retention time, tR, is given by:

where t0 is the time taken for the interstitial volume of mobile phase (i.e. the volume of mobile phase flowing between the resin beads) to be eluted, ti is the time taken for the volume of occluded liquid inside the pores of the resin to be eluted, and DA is the distribution constant for the solute between the interstitial mobile phase and the occluded liquid. The value of DA is dependent on the degree of ionization of the solute.

If a solute cannot enter the stationary phase because it is fully ionized (ion exclusion), DA = 0. Therefore, the retention time of fully ionized solutes is equal to t0, whilst for an uncharged solute which is free to enter the stationary phase, DA = 1, and its retention time is equal to ti. Thus, in the separation of carboxylic acids, the retention times of the acids depend on their first dissociation constants (pKa). Since the fraction of the ionized solute molecules increases with increasing pH, an increase in the mobile phase pH will reduce the retention time.

The retention times of monocarboxylic acids larger than acetic acid, and dicarboxylic acids larger than succinic acid, show an increase with increasing carbon number, even for solutes with similar pKa values. This increased retention can be attributed to hydro-phobic adsorption of the solutes on to the neutral, unfunctionalized regions of the polymeric resin, in a manner similar to that observed in reversed-phase

HPLC. Hydrophobic adsorption effects can be expected to increase in magnitude as the alkyl chain length of the solute is increased, leading to larger retention times. In the case of aromatic acids, the interaction of re-electrons of the benzene ring of the acid with those of the ion exchanger (such as styrene-divinylbenzene packing materials) leads to much higher retention times than expected from their pKa values. The existence of hydrophobic adsorption effects creates the possibility for manipulation of solute retention by adding typical reversed-phase organic modifiers, such as methanol or acetonitrile, to the mobile phase.

Ion exclusion chromatography is usually carried out on a high-capacity sulfonated PS-DVB resin in the H+ form. However, recently work has also been carried out using a polymethacrylate resin in the H+ form with carboxylate functional groups, bare silica (where the silanol group on the surface of the silica acts as the anionic functional group) and also on silica-based cation exchangers functionalized with alkylsulfonic acid or phenylsulfonic acid groups. Since silica gel is chemically stable and inert to organic solvents, silica-based cation exchangers offer the advantage that high concentrations of organic modifiers can be used. Also, aromatic acids which adsorb strongly on to PS-DVB resin due to re-electron interactions between the aromatic ring and the solid resin network are eluted earlier when using a silica gel column.

Other factors which play a part in the retention process of carboxylic acids in ion exclusion chromatography include the addition of other mobile phase modifiers such as polyols, sugars and inclusion compounds (e.g. ^-cyclodextrin), as well as resin characteristics such as the pore size, the degree of cross-linking, the ion exchange capacity and the ionic form of the resin.

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