Question

The amino acid glycine is often used as a component of buffers in biochemical experiments. The amino group of glycine has a pKa of 9.6, which can exist in the protonated form -NH₃⁺ or the free base -NH₂ as shown in this equilibrium:

        R-NH₃⁺ ⇌ R-NH₂ + H⁺

(a) Using glycine as a buffering agent requires specific pH ranges. Identify the buffer range for glycine’s amine group and describe how this molecule provides buffer capabilities as small amounts of HCl(aq) is added to the solution.

(b) In a 0.1 M solution of glycine at pH = 9, what fraction of glycine has its amino group in the -NH₃⁺ form?

(c) When 90% of glycine is in its -NH₃⁺ form, what is the numerical difference between the pH of the solution and the pKa of the amino group?

Answer 1

(a) Amino acids have both an amine group and a carboxylic group. Glycine is a simple amino acid among all the known 20 amino acids with single hydrogen atom as its side chain. In glycine the COOH group has pKa1 = 2.34 and the H3N+ group has pKa2 = 9.60. The isoelectric point (PI) of glycine is 5.7. At both this pKa glycine forms zwitter ions. The pH range of a buffer is pKa -1 to pKa +1. Thus the buffer regions of glycine are pH 1.3 to pH 3.3 and from pH 8.6 to pH 10.6. When a small amount of HCl (aq) is added to glycine solution, the pH of the solution will decrease i.e there will be more H+ ions in the solution. HCl provides hydronium ions to the amino group of glycine and protonates the amino group of glycine. But when a small amount of HCl is added in the buffer regions of glycine the pH will not change much.

(b) The ratio of base to acid in the solution is

pH = pKa + log ([NH2]/[NH3])

Therefore ([NH2]/[NH3]) = 10pH-pKa

= 109-9.6 = 0.25.

So for every 4 molecules in the NH3+ form there is one molecule in the NH2 form. Thus the fraction of molecules in the NH3+ form is 4/5.

(c) The pKa value for an amino group on any amino acid refers to the equilibrium between the protonated positive and deprotonated neutral nitrogen. The pKA is the pH at which a chemical species will accept or donate proton. So when 90% of glycine is in its NH3+ form, it means that the pH is low with high H+ ion concentration. The isoelectric point (PI) of glycine where it exists in zwitter ionic form (neutral charge) is 5.7. If glycine is kept at lower pH (for eg. 2) than its PI (5.7) where 90% of glycine exists in NH3+ form, then all the NH2 group will be protonated to NH3+ form. So the numerical difference between its pKa and pH of the solution will be 5.7 - 2 = 3.7