Lab 1 Genetics Lab Medical Club

Titration of amino acids

Welcome to the first genetics lab, we would like to thank doctor Aya Al-Smerat for editing this lab and helping
us to deliver it in the best possible way. Hope you enjoy studying it!
This lecture will be separated into two parts: theoretical and practical. Starting with the theoretical.

Basic Concepts
 Acids and Basis

Defined by Acid Base

Bronsted Lowry Proton (H+) donors Proton (H+) acceptors
Another definition Hydrogen containing molecules which Hydroxyl (OH) containing molecules
“Arrhenius” dissociate in solutions and release H+ which dissociate in solutions and
release OH-

- There are two types of acids:

1) Strong acids (dissociate completely), e.g.  HCl, H2SO4
HCl H+ + Cl-
2) Weak acids (dissociate partially), e.g.  H2CO3, CH3COOH
H2CO3 H+ + HCO3-

- The initial and final concentrations of weak acids and its conjugate base depends on the dissociation
factor Ka.
HA H + + A-
[𝐻 + ][𝐴− ]
𝑘𝑎 = [𝐻𝐴]

- Ka and pKa have an inversed relationship. i.e. the stronger the acid, the higher value of Ka and the
lower value of pKa.
Strong acid
Ka increases Ka decreases
PKa decreases PKa increases
Weak acid
 What is pH?
- pH is related to the actual concentration of H+ by the formula pH = -log[H+], and it is a degree of
measure which describes the acidity or alkalinity of a solution.
pH < 7  acidic pH > 7  alkaline
- Blood concentration of H+ is very low, about 40 nEq/L (0.00000004 Eq/L) and because of this, it is
customary to express this concentration on a logarithm scale using pH unit.
If we calculated pH of blood using the previous equation:
pH = -log[4×10-8]
= 8 - log4
= 8 – 0.6
pHblood = 7.4

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 pH meter
- The most accurate determination of pH is done by pH meter, which is an electric device used to
measure the pH of liquids.
- It is made of 1- Special measuring probe 2- Electronic meter 3- Display screen
- It is used by immersing the probe in the liquid and the reading (result) will be shown on the display
screen.

Probe

Display Screen

 Henderson-Hasselbalch equation
HA H + + A-
[𝐻 + ][𝐴− ]
𝑘𝑎 = [𝐻𝐴]
pH = -log[H+]

- We are already familiar with these equations. Now if we solve the first one for [H+]

- Then we took the log of both sides and multiply by -1, and we substituted -log[H+] with pH and
-log Ka with pKa

- And finally, we rearranged the equation, we will have the Henderson-Hasselbalch.

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 Buffer system
- It is considered as the most important application for Henderson-Hasselbalch equation.
- A buffer system is a solution of weak acid and its conjugate base or weak base and its conjugate
acid, that resist change in pH when another acid or base are added.
- But how pH is not affected by adding acid or base?
It is simple. Let us consider HA H+ + A- , and HA is a weak acid and stays in an equilibrium
with its conjugate base. If we added a strong acid (added H+), they will react with (A-), forming more
HA, which means the reaction shifted to the left (based on Le Chatelier's principle) and we
maintained the equilibrium state (THERE IS NO SIGNIFICANT CHANGE ON THE ORIGINAL [H+]). Now,
we can say that increasing H+ concentration in the right side of the equation will shift the reaction
to the left.
The same applies if we added a strong base (added OH-), they will react with (H+), forming water
H2O. This indicates decreasing in [H +] on the right side of the equation so the reaction will be shifted
to the right (based on Le Chatelier's principle) and more HA will dissociate forming more H + and A-
and maintaining the equilibrium.
- The maximum buffer capacity is when [Acid] = [Conjugate Base]. Remember that log 1 = 0

And this means that the actual maximum capacity is when pH=pKa. But the buffer region (where
the buffer solution can still serve as an effective buffer) is at pH=pKa±1.
- Remember that:
when pH > pKa , [Base] > [acid]
when pH < pKa , [Base < [acid]
when pH = pKa , [Base] = [Acid]

 Amino Acids
- They are the building blocks (units) of
proteins linked by peptide bonds.
- There are about 300 a.a in nature, only 20
of them occur in proteins.
- Each a.a is made of a carboxylic group, amino
group, a hydrogen atom and a side chain (R).
- A.A differ from each other due to the different R groups.

 Chirality
- It is a property that states that an object or a molecule cannot be
superimposed on its mirror image.
- A molecule is considered chiral when it has a chiral atom, i.e.
connected to 4 different groups and has no symmetric plane.
- All amino acids are chiral except for glycine (its R group is hydrogen,
and this makes it connected to 3 different groups and not 4).

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- Because of the chirality of amino acids (except glycine), each one of them has two isomers (called
enantiomers) i.e. L-isomer and D-isomer.
Extra note:

L  Levo which means

left or counterclockwise

D  Dextro which means

right or clockwise

- In amino acids, the position of amine group determines whether its L or D conformation.
- Amino acids that are involved in protein synthesis in our bodies are in L conformation.

 Ionization
- Each ionizable group in an amino acid has its own pKa value. The carboxyl (2-3), amine group (9-10)
and the R group (if ionizable) also has its own pKa.
- These groups are protonated or deprotonated depending on the acidity of the medium.
If pHmedium < pKagroup, the group will be protonated.
If pHmedium > pKagroup, the group will be deprotonated.

 Acidic and Basic properties of Amino Acids

- Amino acids are amphoteric compounds, which means that they have acidic and basic tendencies.
(The carboxyl group can lose a proton and the amino group can accept a proton).
- Free amino acids and the ones in a peptide linkage can act as buffers.
- When amino acids are in an aqueous solution at low pH, all of the ionizable groups are protonated
and they start to lose their protons as pH increases depending on their pKa values (The one with
lowest pKa value, will be deprotonated first).

Form α-COOH α-NH2

Protonated R-COOH R-NH3+
Deprotonated R-COO- R-NH2
- The same is applied to the ionizable side chains. If the pH of the surrounding medium is lower than
its pKa, it will keep its proton and if its higher than its pKa, the proton will be donated.

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 Isoelectric point (pI)

- It is the pH at which the net charge of a
molecule in a solution equals zero.
- Here, the ion is called Zwitterion, which
is a neutral ion with both, positive and negative
charges. (See the following figure).
- For an amino acid with two ionizable groups
pI = (pKa1 + pKa2)/2
- For acidic amino acid pI = (pKa1 + pKa2)/2
- For basic amino acid pI = (pKa2 + pKa3)/2

 Titration of amino acids

- Titration: an experiment in which one solution is added to another
solution using a burette.
- The two solutions contain two compounds that will react with each
other. The solution in burette is added, until there is an evidence that
the reaction is complete.
- Acid-base titration is one of the most common types of titration is
used to determine the concentration of acidic and basic solutions.
- Titration of amino acids is a method used to analyze the acid-base
behavior of amino acids. It is a method to determine 1- buffering
range 2- maximum buffer capacity 3- isoelectric point. And for this
purpose, we use the analysis of the titration curve.
- Titration curve of a weak acid is a plot of the pH of that 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:

- 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 Henderson-Hasselbalch relationship. The titration curve of the neutralization
of acetic acid by NaOH is shown in the figure.
- This curve defines several characteristics of the weak acid
being titrated; (1) the number of ionizable groups of the
weak acid, (2) the pKa of these groups, (3) the buffering
region(s) and (4) the pH where there is maximum buffering
capacity. Based on the number of plateaus on the titration
curve, one can determine the number of ionizable groups in
any chemical species. The one plateau observed when
acetic acid is titrated indicates that it is a mono-protic acid
(i.e., has only one ionizable group). Many organic acids are
poly-protic (have more than one ionizable group). An
example of poly-protic acids are amino acids.
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- Most of the standard amino acids are diprotic molecules since they have only two ionizable groups
that can donate and accept protons; one is the alpha amino group and the other is the alpha carboxyl
group. The side chains of these amino acids have no ionizable groups that can accept and donate
protons. These amino acids are also called “simple amino acid”. A simple amino acid is electrically
neutral under physiological conditions.
- Ionization of a di-protic amino acid will proceed in the following manner:
Dissociation 1:

Dissociation 2:

- As mentioned earlier, the order of proton dissociation depends on the acidity of the ionizable groups;
the group that is most acidic (lower pKa) will dissociate first. Consequently, the H+ on the α-COOH
group (pKa1) will dissociate before the one on the α-NH3 group (pKa2).
- This curve reveals, in addition to the information mentioned above
with a mono-protic 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.
Mathematically, the pI can be determined by taking the average of
the pKa for the ionizable groups.
- 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 an ionizable group that can accept and dissociate
protons in their R group. Under physiological conditions these amino acids will be charged. If the net
charge under physiological conditions is negative, the amino acid is classified as an acidic amino acid
because the R group has a proton that dissociates at a pH significantly below the physiological pH of
7. The remaining tri-protic amino acids are classified as basic amino acids due to having a net positive
charge under physiological conditions and an ionizable R group with a pKa near or greater than pH 7.
Titration curves of tri-protic amino acids generate the same information as those for the diprotic
amino acids.

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 Titration curves of some amino acids

I. Glycine: a neutral amino acid with only two ionizable groups (diprotic),
α-COOH (pKa = 2.3) and α-NH3 (pKa = 9.6).
1- At a very low pH (acidic) both groups are fully protonated where the
solution predominantly contains:

2- When the pH is raised, the α-COOH group start to

be deprotonated and the proportion will be:

3- pH= pKa1, where it will act as a buffer and the

solution will contain an equal amount of :

4- Further increase in pH, the solution will

predominantly contain zwitterion and the pH at
this point is equal to pI.

5- As the pH increases, the second group α-NH3+ will be deprotonated.

6- After that, pH=pKa2 where it will work as a buffer and the solution will contain an equal amount of:

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7- More increasing in pH, means more deprotonated form of the α-NH3+, thus:

8- The NH3+ group will dissociate, and this mean that glycine now is fully dissociated.

II. Aspartic acid: an acidic amino acid with three ionizable groups (triprotic) and each one has its own pKa
value.
pKa1 for α-COOH = 2.1
pKa2 for acidic side chain = 3.9
pKa3 for α-NH3+ = 9.7

- α-COOH will be deprotonated first, followed by the side

chain carboxylic group and finally the α-NH3+.
- Note that the net charge of the molecule is zero
between pKa1 + pKa2.

III. Arginine: a basic amino acid with three ionizable groups (triprotic) and
each one has its own pKa value.
pKa1 for α-COOH = 2.2
pKa2 for α-NH3+ = 9
pKa3 for basic side chain = 12.5

- α-COOH will be deprotonated first, followed by the α-NH3+,

and finally the side chain amine group.
- Note that the net charge of the molecule is zero between
pKa2 + pKa3.

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Now let us move to the practical part.

 Titration of alanine with sodium hydroxide.

- Alanine: is a neutral amino acid, with two ionizable groups (diprotic)
and each one has its own pKa value.
pKa1 for α-COOH = 2.34
pKa2 for α-NH3+ = 9.87

 Equipment:
1. Graduated cylinder
- It has a narrow cylindrical shape and has numbered lines on it representing amount of liquid
that has been measured. It is used to measure the volume of liquids.
2. Beaker
- For containing liquids.
3. Erlenmeyer flask
- Used to hold and measure chemical liquid samples. The shape of it allows us to freely shake the
flask without spelling the liquid.

Beaker Graduated cylinder Erlenmeyer Flask

4. Burette
- A long glass tube with volume markings. It is used
to deliver specific amount of liquid.
- As you can see, it has a burette clamp which is
used to hold and secure the burette so that it is
fixed and more convenient for use during the
experiment.
- If you have a closer look at the top of the burette,
you will find that the graduation starts from zero
and as you move down the volume increases.
- At the bottom of the burette we have a stopcock,
used to start or stop the liquid flow. (when its
horizontal it is closed, when vertical its fully
opened.)
- To determine the volume of liquid that has been transferred from the burette, we take the
difference between final and initial volume.
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5. Funnel
- Used to deliver liquids to containers that have small
opening. In our experiment, we will use it to pour
solution in the burette.
6. Ph meter
- Already been discussed.

 Materials:
1. Strong base.
[NaOH] = 0.1 M
2. Amino acid.
[alanine[ = 0.1
3. Distilled water.

 Procedure:
1. Fill the burette with NaOH but do not forget to rinse the burette with NaOH before use.

2. Pour 10 ml of alanine in the graduated cylinder.

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3. Pour the previous 10 ml of alanine to the flask.

4. Add few drops of concentrated HCL to the alanine in flask in order to have low initial pH
reading.

- Now we measure the pH, but before that remember to wash the probe using distilled water
and dry it using a clean tissue.

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5. Immerse the probe in the solution. You can see that initial pH (before adding the base) = 1.61

6. Using the burette, we are going to add 1ml of NaOH to the alanine solution. Shake it well and
measure the pH. We find that pH is 1.86

7. Add another 1ml of NaOH using burette. pH after that is 2.05.

8. We continue to add NaOH until we reach pH = 12. See the following figure:

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9. Here is the titration curve of alanine on an excel sheet based on the information in the previous
table

10. You can see two plateaus, so two ionizable groups for alanine, i.e. α-COOH and α-NH3.
11. These plateaus are the buffer regions in which pH increases slowly (where we have minimal
change in pH).
12. The pKa value for each titratable group can be determined by extrapolating the mid points for
each buffer region (plateaus).
13. pI is the midpoint between pKa1 ( α-COOH) and pka2 (α-NH3+).

Extra note:
Extrapolation : is a type
of estimation, beyond the
original observation range, the
value of a variable on the basis
of its relationship with another
variable.
Like you have three points, and
you connect them by one line,
so you estimated the values
between each adjacent points
(these values were not
calculated but estimated).

If you have any questions, please don’t hesitate to ask us!

٢٠٢٠ ‫مع تحيات فريق عمل النادي الطبي‬

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