Electronic Titration of

Basic, Acidic and

Neutral amino acids
 The pH meter should have been calibrated
to pH 4, 7 and 10 with standard buffers
 Immerse the electrode into 10 mL amino acid
solution at pH 1.0

AA Initial pH
Glycine 3.1
Lysine 6.7
Glutamic Acid 4.3
 After 0.1 M NaOH addition, allow the reaction mixture to come to
equilibrium (20 seconds). Measure the pH.
 Continue titration in this manner until the pH curve has leveled off.
 Plot the corrected meq of NaOH added vs pH.
 Glycine
Volume 12
of NaOH pH
0 1.1 10
0.5 1.4
8
1 2
1.3 2.9
6
1.5 3.4
1.8 4.1 4
2 7.7
2.2 8.8 2
2.5 9.1
2.7 9.9 0
0 0.5 1 1.5 2 2.5 3

X-axis (volume of NaOH added) pK1=2.35

Y-axis (pH) pK2=9.78
pI=6.1
 Glycine
Volume 12
of NaOH pH
0 1.1 10
0.5 1.4
8
1 2
1.3 2.9
6
1.5 3.4
1.8 4.1 4
2 7.7
2.2 8.8 2
2.5 9.1
2.7 9.9 0
0 0.5 1 1.5 2 2.5 3

pK1 error =14.89%

pK2 error =1.27%
pI error=31%
 The –COOH loses its proton at pK1
 Removal of proton is complete and removal of
second will begin at pI
 The –NH3 loses its proton at pK2
 In here, we can say that glycine is a good buffer
at pH near its pK1 and pK2 but not a good buffer
at pH near its pI.
 12
L-Glumatic Acid
Volume of
NaOH pH 10
0 1.1
0.5 1.1 8
1 1.2
1.5 1.3 6

2 1.5
2.5 1.7 4

3 2
3.5 2.9 2

3.8 9
0
4 10.3 0 1 2 3 4 5

pK1=2.19
X-axis (volume of NaOH added) pK2=4.28
Y-axis (pH) pK3=9.66
pI=3.2
 When pH reaches pK1 of glutamic acid,
its –COOH loses its proton. Then, it
reaches its first pI where it completes
the dissociation of –COOH into COO-
and H+ and begins with the next
carbonyl group.
 When titration reaches its pK2, the 2nd
carbonyl group loses its proton. Then it
reaches the 2nd isoelectric point.
 When titration reaches its pK3, the
amino group loses its proton
dissociating NH3 into NH2 and H+
 L-Lysine
12
Volume of
NaOH pH
10
0 1.2
1 1.3
8
2 1.7
3 2.6
6
3.5 3.3
4 8.1 4
4.3 9.1
4.5 9.4 2
4.8 9.7
5 10 0
0 1 2 3 4 5 6

pK1=2.18
X-axis (volume of NaOH added) pK2=8.95
Y-axis (pH) pK3=10.543
pI=9.7
 When the pH reaches pK1, the –COOH
dissociates and loses its proton. Upon arriving
at its pI1, reaction is complete and removal of
next group begins.
 When titration reaches its, pK2, the –NH3 loses
its proton and forwards into reaching the 2nd pI
where removal is completed and removal of the
next amino group begins.
 Upon reaching pK3, the 2nd amino group of the
lysine loses its proton at basic pH.
What is the importance of standardizing the pH
to more than one pH value in this experiment?

 Standardizing/calibrating the pH meter to more than one

pH value makes the reading more accurate compared to
one-point calibration. In this experiment, we calibrated at
three points (pH 4.0, 7.0 and 10.0), so it adjusts the pH
meter up to three different pH values. Meaning, the pH
meter adjusts so that its reading is accurate at three
different points along the linear reading. Thus, being able
to read at wider pH ranges. Also the bulb of the meter is
washed with distilled water before immersing into a
different solutionto avoid or at least minimize
contamination,
Would the use of micro-equipment
improve the results?
 Titration volumes in the experiment are usually relatively
small so using micro-equipments are of great help. This is
useful especially when adding the 0.5 NaOH on the amino
acid solution. Some amino acids require minimal volumes
of NaOH to be able to monitor the pH changes and
appreciate the curve given by the reactions of its
dissociation and equivalence points. If NaOH is added in
excess, there would be a significant increase or jumping
of pH from its previous reading so one should be careful
and mindful in titrating especially when they are near on
the pK of solutions where there is shift of pH
Clinical Correlation
 TITRATION CURVES PREDICT THE ELECTRIC CHARGE OF AMINO ACIDS

 Glycine has a net negative charge at any pH above its pI and will thus
move toward the positive electrode (the anode) when placed in an
electric field. At any pH below its pI, glycine has a net positive charge
and will move toward the negative electrode (the cathode). The
farther the pH of a glycine solution is from its isoelectric point, the
greater the net electric charge of the population of glycine molecules.
 At pH 1.0, for example, glycine
exists almost entirely as the form
H3N-CH2-COOH with a net positive
charge of 1.0. At pH 2.34, where
there is an equal mixture of H3N-
CH2-COOH and H3N-CH2-COO-, the
average or net positive charge is
0.5. The sign and the magnitude of
the net charge of any amino acid at
any pH can be predicted in the same
way