Histidine Titration Lab

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4 Titration Curve of an Amino Acid

pH

Simple amino acid

Acidic amino acid

Basic amino acid

7

OH- equivalents

Objectives:

A) To determine the titration curve for an amino acid and B) to use this curve to estimate the pKa values of the ionizable groups of the amino acid and the amino acid’s pI.

Introduction: A titration curve of an amino acid is a plot of the pH of a weak acid against the degree of neutralization of the acid by standard (strong) base. Consider the ionization of a weak organic acid such as acetic acid by NaOH.

CH3COOH(aq) + NaOH

-

+

CH3COO Na + H2O

As more of the strong base (titrant) is added to the aqueous solution, more of the weak acid is converted to its conjugate base. During this process, a buffer system forms and the pH of the system will follow the HendersonHasselbalch relationship. The titration curve of the neutralization of acetic acid by NaOH will look like this:

pH

0.5 equival ent s

pH = pKa

Equivalents of Base

When a weak monoprotic acid is titrated by a base, a buffer system is formed. The pH of this system follows the Henderson-Hasselbalch equation:
This curve empirically defines several characteristics (the precise number of each characteristic depends on the nature of the acid being titrated): 1) the number of ionizing groups, 2) the pKa of the ionizing group(s), 3) the buffer region(s).

100% ApH = pKa - 1;

pH = pKa;
50% HA
50% A-

pH

90% HA
10% A-

Buffer region

90% A- ; pH = pKa + 1
10% HA

100% HA

Eq
Equivalents of Base
Based on the number of plateaus on a titration curve, one can determine the number of dissociable protons in a molecule. The one plateau observed when acetic acid is titrated indicates that it is a monoprotic acid (i.e., has only one dissociable H+). Many organic acids are polyprotic (have > one dissociable H+).

The protein building blocks, amino acids, are polyprotic and have the general structure

H
+

H3N Cα COOH
R

The majority of the standard amino acids are diprotic molecules since they have two dissociable protons: one on the alpha amino group and other on the alpha carboxy group. There is no dissociable proton in the R group. This type of amino acid is called a “simple amino acid”. A simple amino acid is electrically neutral under physiological conditions. NOTE: Under this definition it is possible to have a simple amino acid which is triprotic. Which of the 20 common or standard amino acids are

simple & triprotic? Ionization of a diprotic amino acid will proceed as follows:

Dis s ociation 1:
H

H

+

+

H3N Cα COO- + H+

H3N Cα COOH
R

R

Dis s ociation 2:
H

H
+

H2N Cα COO- + H+

H3N Cα COO-

R

R

The order of proton dissociation depends on the acidity of the proton: that which is most acidic (lower pKa) will dissociate first. Consequently, the H+ on the α-COOH group (pKa1) will dissociate before that on the α-NH3 group (pKa2). The titration curve for this process looks similar to the following:

pH = pKa2

pH

pH = pI

pH = pKa1

Equivalents of Base

This curve reveals, in addition to the same information observed with a monoprotic acid, an additional characteristic of polyprotic acids and that is the pH at which the net charge on the molecule is zero. This pH defines the isoelectric point (pI) of the molecule, a useful constant in characterizing and purifying molecules. Using a titration curve, the pI can be empirically determined as the inflection point between the pKa of the anionic and cationic forms. Mathematically, the pI can be determined by taking the average of the pKa for the anionic and cationic forms. The

ionic form of the molecule having a net charge of zero is called the zwitterion.
A few amino acids are classified as triprotic. This is because, in addition to the ionizable protons of the α-COOH and α-NH3 groups, they also have a dissociable proton in their R group. Although triprotic amino acids can...
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