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analogous basic exchanger may be prepared by reacting cross-linked polystyrene with
chloromethyl ether, and then reacting the chloro groups with tertiary amines. These 
CH2N+ (CH3)3Cl- groups are ionized at all but very alkaline pH values.
Chemically modified celluloses have proved to be a particularly useful alternative to
the polystyrene- based exchangers. Cellulose is a high molecular weight compound which
can be obtained in a highly pure state (arboxymethyl cellulose (CM- CH2 OCH2 COOH
cellulose), and DEAE- cellulose (-CH2OCH2CH2N(CH2CH3)2) are examples of the main
derivates of practical value.
Ionizable groups:
Ionizable groups charged groups are attached to the matrix and the type of group
defines the nature and strength of the ion exchanger. These groups may be either anionic or
cationic, according to the nature of their affinity for either negative or positive ions. For
example, the cation exchanger materials exchange positive ions, so it is the charge carried by
the exchangeable ion which decides whether a material is anionic or cationic and not the
charge carried on the matrix. (figure 27.3) these two types can be further divided into
materials that contain strongly ionized groups, such as  SO3H and  NR3, and the weakly
ionized groups, such as  COOH, -OH and  NH2. The strong ion exchange resins are
completely ionized and exist in the charged form except at extreme pH values:
- SO3H SO-3 + H+
+
-NR3OH NR3 + OH-
The weak ion exchange materials, on the other hand, contain groups whose ionization is
dependent on the pH, and they can only be used at maximum capacity over a narrow pH
range.
-COOH -COO- + H+
-NH3+ -NH2 + H+
As a rough guide, resins containing carboxyl groups have a maximum capacity above pH 6,
while those with amino groups are effective below pH6.
The number of ionizable group determines the capacity of the ion
exchanger. The total capacity is the number of ionizable groups per gram of material,
whereas the available capacity is the amount of a given molecule that can bind under defined
experimental conditions. In the case of some materials, large molecules may be unable to
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penetrate the matrix and can only react with charged groups on the surface. In this case, the
available capacity will be considerably less than the total capacity.
The ionizable groups commonly met are given in table 27.3 together with their
abbreviations.
Ion exchange equilibria. The typical way that an ion exchange material function is illustrated
in the following sample, where an anion exchange resin containing amino groups is used to
separate two negatively charged ions X- and Y- (figure 27.4).
Although ion exchange materials are claimed to be monofunctional, in that only the
ion exchange process is used in separation, in practice some molecular sieving and
adsorption can occur. The adsorption is small, but it can sometimes be used to separate
closely related compounds.
Elution of bound ions. The bound ions can be removed by changing the pH of the buffer.
For example, as the pH of a protein moves towards its isoeletric point, the net charge
decreases and the macro-molecule is no longer bound. Separation is achieved as other
charged proteins remain on the column alternatively, ions can be removed by increasing the
ionic strength, when high concentrations of ions in the solvent displace the bound ions by
increasing the competition for the charged groups of the ion exchange material. The pH or
ionic strength can be altered sharply, by changing the eluting buffer, or, gradually, by means
of a gradient.
Preparation of material. Ion exchange materials are first allowed to swell in the buffer and
the fines removed.
The ion exchange material is then obtained in the required ionic form by washing with the
appropriate solution. For example, the H+ form of a cation exchange resin is obtained by
washing the material with hydrochloric acid then water until the washing are neutral.
Similarly the Na+ form is prepared by washing the resin with sodium chloride or sodium
hydroxide then water as above.
The final stage before preparing the column is to equilibrate the material by stirring
with the eluting buffer.
The separation of amino acids by ion exchange chromatography:
MATERIALS
1. Chromatography column (20 cm × 1.5 cm) 5
2. Strongly acidic resin 30 g.
3. Hydrochloric acid (4 ml/ litre) 1 litre.
4. Hydrochloric acid (0.1 ml/ litre) 4 litres
5. Glass wool 
6. Amino acid mixture 10 mg
(Dissolve aspartic acid, histidine of each and lysine in 0.1 mol/ litre HCl to a final
concentration of 2mg/ ml)
7. tris-HCl buffer (0.2 mol/ litre, pH 8.5) 3 litres
8. Sodium hydroxide (0.1 mol/litre) 2 litres
9. Separating funnels (500 ml) 10
10. Acetate buffer (4 mol/ litre, pH 5.5) 250 ml
11. Ninhydrin reagent. (Dissolve 20 g of ninhydrin and 3 g of 1 litre hydrindation in 750 ml
of methyl cello solve and add 250 ml of acetate buffer). Prepare fresh and store in a brown
bottle.
12. Methyl cello solve (ethylene glycol monomethyl ether) 1 litre.
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13. Ethanol (50 percent v/v) 1 litre.
14. Ninhyrin (2 g/litre in acetone). (Care: carcinogenic!) 100 ml
15. Oven at 105Úc 1.
Method:
PREPARATION OF THE COLUMN- gently stirs the resin with 4 mol/ litre HCl until
fully swollen (15- 30 ml/ g dry resin). Allow the resin to settle, and then decant the acid.
Repeat the washing with 0.1 mol/ litre HCl, resuspend in this solution, and prepare the
column as previously described.
Elutions of amino acids carefully apply 0.2 ml of the amino acid mixture to the top of the
column, open the tap and allow the sample to flow into the resin. Add 0.2 ml of 0.1 mol/
litre HCl, allow to flow into the column as before, and repeat the process twice. Finally, [ Pobierz całość w formacie PDF ]