Histidine Titration Lab
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
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