Ampholytes

Ampholytes are compounds that when dissolved in water (which is itsef an amphoteric compound) can act either as acid or as a base. We illustrate here some properties of ampholytes, taking amino acids as an example. Amino acids are crystalline solids with a melting point higher than those of organic molecules of similar size (amines and carboxylic acids are not crystalline solids). This suggested that amino acids are dipolar ions in which the COOH group occurs in its dissociated form (COO^-) and the NH₂ group is protonated (NH₃⁺). The strong electrostatic forces between positively and negatively charged neighboring molecules can explain their high melting point. Furthermore, their dipolar nature can also explain the good solubility of amino acids in water. For brevity let's indicate a generic amino acid (having a single amino group and a single carboxyl group) as ⁺H₃N-COO^-. When it is dissolved in water, two reactions can take place:

By dividing K_a with K_b and remembering that [OH^-] = Kw/[H₃O⁺], we have

Ka   [H2N-COO-] [H3O+] [+H3N-COO-][H2N-COO-] [H3O+][H2N-COO-] [H3O+]2 ---- = ------------------------------- = ---------------------- = -------------------- Kb   [+H3N-COOH] [OH-] [+H3N-COO-][+H3N-COOH] [OH-][+H3N-COOH] Kw

Solving for [H₃O⁺]² and taking - log we have that:

K_a [⁺H₃N-COOH]
[H₃O⁺]² = ------------------ Kw
K_b [H₂N-COO^-]

pH = 1/2 (pKw + pKa - pKb ) + 1/2 (log [H₂N-COO^-] - log [⁺H₃N-COOH])

For amino acids the values of pK are normally tabulated as pKa (NH₃⁺) and pKa' (COOH), thus it is convenient to express the pH as function of these values. Kb, in the equation above, is the hydrolysis constant of -COO^- then pKw - pKb corresponds to pKa' (see acids and bases) and thus

pH = 1/2 (pKa + pKa') + 1/2 (log [H2N-COO-] - log [+H3N-COOH])

For amino acids, pKa (NH₃⁺) is about 9 and pKa' (COOH) is about 2.

 

Isoelecric point

From

pH = 1/2 (pKa + pKa') + 1/2 (log [H₂N-COO^-] - log [⁺H₃N-COOH])

it is easy to verify that when the concentration of positive charges equals the concentration of negative charges we have

The pH at which negative charges match positive charges is known as isoelectric point and it is generally indicated as pI therefore

pI = 1/2 [pKa(NH₃⁺) + pKa(COOH)]

In other words the pI of an amino acid having a single amino group and a single carboxyl group is the arithmetic average of pKa(NH₃⁺) and pKa(COOH). Remembering that for such amino acids pKa (NH₃⁺) is about 9 and pKa' (COOH) is about 2 we have that pI is 6. For amino acids having additional basic groups (i.e., histidine, lysine and arginine) the pI is higher while for amino acids containing extra acidic groups (i.e., aspartic and glutamic acid) the pI is lower.

Taking in account the definition of pI we can write

pH - pI = 1/2 (log [H₂N-COO^-] - log [⁺H₃N-COOH])

From this equation is easy to verify that:

if pH > pI the ampholyte carries a negative net charge (the larger the difference the larger the amount of negative charge)

if pH < pI the ampholyte carries a positive net charge (the larger the difference the larger the amount of positive charge)

If pH = pI the net charge will be 0

It follows that ampholytes under the action of an electric field (electrophoresis) will move at different speeds and in differents directions (positive charged toward the negative electrode and negative charged toward the positive electrode). Therefore electrophoresis can allow the separation of electrical charged species. More details on electrophoresis will be given later on these pages.

It can be mathematically demonstrated that an ampholyte at its isoelectric point has a minimum of dissociation. In other words, the amount of negative and positive charges is the minimum possible for a given concentration of the ampholyte. This observation accounts for the followings properties of ampholytes at the isoelectric point:

minimum of solubility

minimum of electric conducibility

minimum influence on colligative properties (osmotic pressure, freezing-point depression, boiling-point elevation....).

Ion exchangers are solid materials carrying chemical bound charged groups to which ions of opposite charge can reversibly bound. They are used in a separation technique, known as ion exchange chromatography, of wide spread use in all chemistry fields. The understanding of this technique requires no concepts beyond those discussed so far. Ion exchangers are distinguished into cationic which can exchange cations (1) and anionic which can exchange anions (2):

1. R===SO3- Na+ + M+R===SO3- M+ + Na+

2. R===NH3+ Cl- + M-R===NH3+ M- + Cl-

The binding of a charged specie to the exchanger depends on the amount of net charge it carries and on its ability to compete with the counerion bound to the matrix. Thus, in the case of a cationic exchanger described in 2, Na⁺ (the counterion) should have a lower affinity for the exchanger than M⁺.

Suppose to fill a glass column with a suspension at pH 2 of the cation exchanger (1) and to apply to the column a solution of amino acids at pH 2. Since at this pH all amino acids bring a net positive charge than all the amino acids will bound to the exchanger:

3. R===SO₃^-Na⁺ + AA⁺ R===SO₃^-AA⁺ + Na⁺

If we wash the column with buffers of increasing pH (pH gradient), the increase of pH will lead to a decrease of positive charge on amino acids. The decrease of amino acid net-charge depends on the pI:

4. pH - pI = 1/2 (log [H₂N-COO^-] - log [⁺H₃N-COOH])

and as a consequence, amino acids will move through the column at different rates and will reach the end of the column at different time. Therefore we will be able to collect them separately.

Another mechanism which can allow the displacement of amino acids (or other ampholytes) is the increase of ionic strength. Ionic strength of a salt solution is given by 1/2 S C_iZ_i (where C_i = concentration of a given ion, Z_i = the charge the ion bears). Therefore ionic strength depends on the concentration of the salt and the charge of ions arising from dissociation. Since the ion exchange reaction is reversible, it can be reversed by increasing the concentration of Na⁺ in the eluent. It should be evident that, at given pH, the increase of [Na⁺], will displace first those molecules having a lower positive net charge.

Eventually let's remember that ion exchangers are also distinguished into strong and weak. The strength of a ion exchanger depends on the strength of the basic/acid group. For example,

R===SO₃H is a strong cation exchanger for the acid is completely dissociated.

R===COOH is a weak cation exchanger for the acid is only partially dissociated.

The strength of the exchanger thus influences the number of charge present on the exchangers at a given pH. As an example, the separation of amino acids described above can not be accomplished by using the weak R===COOH. In fact at pH 2 the carboxylic group bears very few charges and practically no amino acids will bind to the column.