EXPERIMENT 1

DETERMINATION OF ISOELECTRIC POINT OF AN AMINO ACID

Amino Acids
A compound that contains an amino group, a carboxyl
group and a side-chain that is specific to each amino
acid.

-Amino acid:

An amino acid in which the amino group is on the

carbon adjacent to the carboxyl group.
Amino Acids
• The building blocks of proteins

• 20 amino acids are naturally incorporated into polypeptides and are called
proteinogenic or standard amino acids. These 20 are encoded by universal genetic code.

• 10 standard amino acids (Lys, Met, His, Leu, Ile, Thr, , Tyr, Phe, Val & Arg) are called
"essential" for humans because they cannot be created from other compounds by the
human body, and so must be taken in as food.
Amino Acids
All amino acids (except glycine) are optically active (chiral).

Two amino acids can react with loss of a water molecule to form a covalent bond.

Amide linkage is planar NH and CO are anti.

Classification of Amino Acids

Classification based on side-chain structure:

– Non-polar amino acids.
– Polar, uncharged amino acids.
– Acidic amino acids.
– Basic amino acids.

Other side chain structural classifications:

– Aromatic, cyclic, hydroxyl, and thiol amino acids.
Isoelectric Point (pI) of an Amino Acid

• The isoelectronic point or isoionic point is the pH at which the amino

acid does not migrate in an electric field.

• This means it is the pH at which the amino acid is neutral, i.e. the
zwitterion form is dominant.
The equations that define acidity and basicity are:

The lower the pKa, the stronger the acid.

From these expressions it is possible to derive the important Henderson-

Hasselbalch equation :

pKa = pH + log [HA] / [A-]

 pKa = pH + log [HA] / [A-]

• It tells us that when the pH = pKa then log [HA] / [A-] = 0 therefore [HA] = [A-], i.e.
equal amounts of the two forms, the acid and the conjugate base.

• If we make the solution more acidic, i.e. lower the pH, so pH < pKa,
then log [HA] / [A-] has to be > 0 so [HA] > [A-].
A stronger acid will cause the formation of HA, the protonated form.

• If instead we make the solution more basic, i.e. raise the pH, so pH > pKa
and log [HA] / [A-] has to be < 0 so [HA] < [A-].
A stronger base will cause the formation of A- , the deprotonated form.
There are 3 cases to consider
• neutral side chains

These amino acids are characterised by two pKas : pKa1 and pKa2 for the carboxylic acid and
the amine respectively.

The isoelectronic point will be halfway between, or the average of, these two pKas,
i.e., pI = 1/2 (pKa1 + pKa2).

At very acidic pH (below pKa1) the amino acid will have an overall +ve charge and at very basic
pH (above pKa2 ) the amino acid will have an overall -ve charge.

For the simplest amino acid, glycine, pKa1= 2.34 and pKa2 = 9.6, pI = 5.97.
• acidic side chains

The pI will be at a lower pH because the acidic side chain introduces an "extra" negative charge.

So the neutral form exists under more acidic conditions when the extra -ve has been neutralised.

For example, for aspartic acid shown below, the neutral form is dominant between pH 1.88 and
3.65, pI is halfway between these two values, i.e. pI = 1/2 (pKa1 + pKa3), so pI = 2.77.
• basic side chains

The pI will be at a higher pH because the basic side chain introduces an 1.82 pKa1
"extra" positive charge.

So the neutral form exists under more basic conditions when the extra +ve
has been neutralised. 6.00 pKa3

For example, for histidine, the neutral form is dominant between pH 6.00 and
9.17, pI is halfway between these two values, i.e. pI = 1/2 (pKa2 + pKa3), so
pI = 7.59. 9.17 pKa2
 The Experiment to Determine Isoelectric Point
1. At the beginning, i.e., before base is added, the pH of the solution is
fairly low because it contains acid.
2. As titration proceeds, acid is neutralized by the added base and pH
rises.
3. Addition of base after all of the acid is neutralized produces a basic
solution with a high pH.
4. The pH of the solution at each interval is monitored by a pH meter.
5. A plot of pH versus the volume of titrant added to the solution gives
Moles of acid in the original
the “S” shaped titration curve.
aliquot is calculated as :

Moles of acid
Successively add small volumes of base, measure pH after each addition, = Vbase at inflection point x Mbase
and plot the titration curve, from which we may find Vbase at the inflection
point (the equivalence point).
A derivative plot needs to be created to determine the pKa
values accurately.
The steps are as follows:
1. Calculate pH/V from the collected pH data for each
addition of the titrant.
2. Plot pH/V against V (volume of titrant added).
3. The plot gives sharp peaks at the equivalence points
corresponding to the sharp jumps in the titration plot.
APPARATUS: pH meter, beaker, burette, pipette, glass rod, spatula.
REAGENT AND MATERIALS: Potassium hydrogen phthalate, glycine, alanine, HCl, NaOH,
phenolphthalein.

EXPERIMENTAL PROCEDURE:

i) Standardisation of NaOH solution

a) Prepare 100 mL 0.1 M KHP (Potassium hydrogen phthalate) solution.
b) Standardize the supplied (~0.1 M) NaOH solution against KHP solution using
phenolphthalein indicator (three results).

Table 1. Preparation of 100 mL standard 0.1 N KHP solution Table 2. Standardization of NaOH solution using standard KHP solution

Weight taken Weight to be Strength of KHP Sl. No. Volume of Burette reading (mL) Average Strength of NaOH
KHP (mL) volume (mL) solution
Initial Final Difference
(g) taken (g) solution
i) Amino acid titration and estimation of equivalence point
a) Transfer exactly 10 mL of the supplied protonated amino acid solution to a clean 100 mL beaker.
b) Add 15 mL of distilled water to the beaker so that the total volume of the amino acid solution is 25 mL.
c) To homogenize the solution, place the beaker on the top plate of a magnetic stirrer and place a 1-inch stir bar in the
beaker. Rinse the pH electrode and submerge it in the solution containing protonated amino acid. Make sure that the
tip of the electrode is clear of the magnetic stir bar in the beaker before starting the stirrer. The rotation rate should be
reasonably fast, but not so vigorous that splashing of the solution occurs.
d) Record the initial pH of the solution. Initiate the pH titration by adding 0.5 mL of NaOH solution from burette.
e) On each addition of base solution, note the pH of the solution. Continue this addition until you find larger gaps
between two subsequent pH values. This indicates approach of the equivalence point. Reduce the volume of addition
of the alkali solution to 0.1 mL until you comfortably cross the sudden jump in pH, indicating the equivalence point.
After the equivalence point is passed, increase each volume of addition to 0.5 mL. Repeat this process if you expect
more than one equivalence points.
f) Discard the solution on completion. Rinse the pH electrode with distilled water till pH meter reading is approximately
equal to that of distilled water. Leave the pH electrode in beaker of distilled water and turn the meter off.
 Table 3. Titration of amino acid solution using standard NaOH solution
Volume of amino acid (mL) =

Sl. No. Volume of NaOH (mL) pH V (mL) pH pH/V

Results: Isoelectric point (pI) of the supplied amino acid = ____________ at _______ oC.