Resin and immobile cations
Figure 3.22 Ion-exchange chromatography. Mobile ions compete for the fixed immobile ions in the resin. The major factors that influence the process are the molar concentration of the ions and the charge that they carry.
conjugate acid) dissociates as the pH increases:
Hence an ion-exchange resin cannot be used at a pH that suppresses the ionization of the group, and weak anion exchange resins, for instance, are only effective over the pH range 2-8. The stronger acids and bases, however, are capable of being used over almost the whole pH range.
The major consideration in selecting a resin is the charge carried by the test ions. In the case of uncomplexed inorganic ions, it is relatively constant but for many molecules, it can be altered considerably by variations in pH.
There are three major factors that determine the binding of ions in such a competitive system. The size of the charge carried will result in divalent ions showing greater affinity for the resin than monovalent ions. The intensity of the charge is also significant and small monovalent ions, e.g. hydrogen, will show greater affinity than large monovalent ions, e.g. potassium. Superimposed on both of these considerations is the effect of the concentration of the ions and this is demonstrated by the fact that a high concentration of a low affinity ion is capable of displacing a low concentration of an ion with a higher affinity for the resin. It is the careful control of these three factors that provides the selectivity in ion-exchange separations (Figure 3.22).
Ion-exchange chromatography may be used for either purification of an individual component or fractionation of a mixture. In order to isolate a particular component
Table 3.10 Ion-exchange media
Effective pH range
Strong acids, e.g. nucleotides Weak acids, e.g. organic acids Weakly polyanionic, e.g. proteins
Strong bases, e.g. amino acids Weak cations, e.g. peptides Weak polycationic, e.g. proteins
Quaternary ammonium Strong 2-11
Tertiary ammonium Intermediate 2-7
Diethylaminoethyl Weak 3-6
Sulphonated Strong 2-11
Carboxylate Intermediate 6-10
Carboxymethyl Weak 7-10
> The form of a resin lefers to the mobile ion associated with It prior to use.
> Gradient separation of amino acids - see Section 10.6.2.
from a mixture, it may be selectively retained on a resin while the unwanted constituents, which under the conditions of the analysis should be uncharged or carry the same charge as the resin, will be eluted. Subsequently the required component can itself be eluted in a small volume of an appropriate buffer. Conversely, the resin may be selected so that the required compound does not bind but unwanted ions do and the sample can be eluted quickly from the column without the contaminating ions. Neutral compounds lend themselves to this technique in which a bed of mixed anion and cation resins can remove unwanted ions and replace them with water (H+ and OH-). The technique of fractionation is used for samples that contain mixtures of similar ions and permits the separation and quantitation of each component, the various components being eluted sequentially as the solvent composition is changed. It is in this form that ion-exchange chromatography is used as an HPLC technique.
The resin should be prepared for use by washing it with a solution containing a high concentration of an ion which has a high affinity for the resin, e.g. hydrochloric acid or sodium hydroxide solution (1.0 mol 1_1). The hydrogen and hydroxyl ions will displace all other ions present from cation- and anion-exchange media respectively. After washing in water or buffer to remove the excess acid or alkali, the resin is said to be in the hydrogen or hydroxyl form respectively. For some applications it may be necessary to convert it to a different ionic form, e.g. the sodium form, that has an affinity for the resin comparable to that of the test ion. The resin is finally equilibrated with the chosen buffer, the pH of which must be carefully selected to ensure the correct ionization of both the test ions and the resin.
A small volume of sample is applied to the column and the components of the mixture eluted using the buffer. A single buffer (isocratic separation) or a gradient technique may be used depending on the complexity of the sample. Gradient elution normally involves either changes in pH (causing alterations in ion affinity for the resin) or changes in the concentration of the buffer (causing displacement of the test ions).
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