Download Introduction: As the building blocks of proteins, amino acids play a

Introduction:
As the building blocks of proteins, amino acids play a key cellular role in structure and
function. Proteins themselves participate in nearly every physiological event in the cell. In order
to understand acid-base properties of proteins and their behavior as polyionic macromolecules,
we will begin by investigating the properties of their constituent amino acids. Since all amino
acids contain at least one amino and one carboxyl group, they are classified as amphoteric
substances (meaning that they can act as either an acid or as a base). Such a molecule reacts with
acids as follows:
R
+
H3 N C COO
H
R
+
+
H3O
H3 N C COOH
H
Acid
Zwitterio
+
H2 O
+
H2 O
Cationic
and with bases as follows:
R
+
H3 N C COO
H
Zwitterio
R
+
OH
H2 N C CO O
H
Base
Anionic form
Since alpha-carboxyl and alpha-amino groups are weak acids and bases, respectively,
buffering action by these groups will occur.
In this experiment, you will identify an unknown amino acid through acid-base titration.
Titration curves of amino acids are very useful for identification. As you can see in the example
for glycine shown below, a simple amino acid has two dissociation steps corresponding to loss of
H+ from the acidic carboxyl group at low pH followed by loss of H+ from the more basic amino
group at high pH. The pKa value for each dissociable group of an amino acid can be determined
from such a titration curve by extrapolating the midpoint of each buffering region (the plateau) in
the titration curve. The diagram also shows that there is a point in the curve where the amino
Organized by: Badahdah, M. S. & Alghazzawi W. M.
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acid behaves as a "neutral" salt. At this pH, the amino acid is predominantly a zwitterion with a
net charge of zero. This point of the titration curve is the “isoelectric point” (pI) and can be
approximated as halfway between the two points of strongest buffering capacity (the two pKa
values). The isoelectric point (pI) can be estimated by:
PI = (pK1 + pK2)
2
Where K1 and K2 are the dissociation constants of the carboxyl and amino groups, respectively.
Proteins can be separated according to their isoelectric point in a process known as isoelectric
focusing. At a pH below the pI, proteins carry a net positive charge. Above the pI they carry a
net negative charge. The pH of an electrophoresis gel is determined by the buffer used for that
gel. If the pH of the buffer is above the pI of the protein being run, the protein will migrate to the
positive pole (Negative charge is attracted to positive pole). If the pH of the buffer is below the
pI of the protein being run, the protein will migrate to the negative pole of the gel (Positive
charge is attracted to negative pole). If the protein is run with a buffer pH that is equal to the pI,
it will not migrate at all. This also true for individual amino acids.
Due to a preponderance of weakly acid residues in almost all proteins, they are nearly all
negatively charged at neutral pH. At pI the solubility is often minimal and at which mobility in
an electrofocusing system is zero (and therefore the point at which the protein will accumulate
and it will not migrate at all). This also true for individual amino acids.
Organized by: Badahdah, M. S. & Alghazzawi W. M.
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Apparatus & glassware:
For team of two students
 pH meter and standard buffer solutions
 Magnetic stirrer and bar
 Graduated burette and holder
 Beaker (400 ml)
 Graduated cylinder (20 ml)
Chemicals & other material:




Distilled water
NaOH solution (0.5 N)
H2SO4 solution (0.25 N)
Amino acid (Glycine)
Procedure:
1. Use the electronic magnetic stirrer device. Place a
magnetic bar into your 400 ml beaker, and turn on
the stirrer so that the bar turns at a moderate speed.
Turn off the stirrer.
2. Place approximately 20 ml (know and record the
actual volume) of your selected amino acid
solution into the beaker. Assemble the titration
stand, graduated burette, and pH meter so the
electrode is submerged into the amino acid
solution and the burette is positioned to drop
solution into the beaker. Be sure the electrode is
clear of the magnetic stir bar.
3. Record the initial pH of the amino acid solution and the initial reading of your burette (Which
should be 0.0 ml)
4. Turn on the stirrer, check that the magnetic bar does not collide with the electrodes.
Organized by: Badahdah, M. S. & Alghazzawi W. M.
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5. Add enough 0.5 N NaOH solution to the titration burette. It is recommended that you allow
the first portion of NaOH solution to run through the burette to clear the bottom constriction
from residual distilled water (Discard this volume).
6. Record the starting volume reading on the burette, and then carefully titrate the amino acid
solution by dropwise addition of the 0.5 N NaOH.
7. Record the volume addition of NaOH (think!!) and pH of the solution throughout the titration
until a pH > 12 is reached. You do not have to take a reding foreach pH unit shown, but try
not to skip more than two units.
8. Repeat the titration (Step 5 and 6) with 0.25 N.
9. Record the volume of added H2SO4 and pH of the solution throughout the titration until a pH
below 1.5 is reached.
10. Use care in adding the H2SO4 and NaOH, as the pH may change slowly or rapidly depending
on the pH and buffering range of the ionizable group(s). Have patience!!!
Near the equivalence point, a single drop may change the pH dramatically. It is strongly
recommended that you proceed drop by drop.
Note:
 Each student will clean their benches and dispose of the waste chemicals.
 Each student will submit a lab report the following week to report their findings.
Organized by: Badahdah, M. S. & Alghazzawi W. M.
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Result sheet
Data Collection:
Initial pH reading _______
H2SO4
ml added
pH
0.2
0.4
0.6
1.0
2.0
3.0
Volume added _0.0 ml_
NaOH
ml added
pH
0.2
0.4
0.6
0.8
1.0
2.0
3.0
4.0
End point
(1.5)
End point
(12.00)
Answer the following questions.
1. Plot pH versus ml of NaOH and H2SO4 solutions added.
2. Use pH as the dependent variable (Y-axis) and ml of NaOH or H2SO4 as the independent
variable (X-axis)
3. Determine the pI of your amino acid.
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References:
1.
2.
3.
4.
http://www.colby.edu/chemistry/CH367/laboratory/
http://www.unm.edu/~rrobergs/titration.htm
http://en.wikipedia.org/wiki/Isoelectric_point
http://www.daviddarling.info/encyclopedia/G/glycine.html
Animation links:
Amino acid titration curve and pKa values
Organized by: Badahdah, M. S. & Alghazzawi W. M.
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