EXPERIMENT I
TITRATION CURVES OF AMINO ACIDS AND DETERMINATON
OF ISOELECTRICAL POINT
Amino acids are amphoteric substances and contains two functional groups; carboxylic acid
(-COOH) and amino (-NH2). When an amino acid is dissolved in water, it exists in solution as
the dipolar ion, or zwitterion.
Neutral:
(Zwitterion)
Acidic:
(Cation)
Basic:
(Anion)
A zwitterion can act as either an acid (proton donor) or a base (proton acceptor).
Substances having this dual nature are amphoteric and are often called ampholytes (from
“amphoteric electrolytes”).
Due to their amphoteric nature amino acids can be titrated with both acids and bases. Since
amino acids bear at least two functional groups than can be titrated by they have two
equilibrium constants (K) and two ionization constants (Ki). The characteristic pH at which
the positive and negative electrical charges are equal and net electric charge is zero is called
the isoelectric point or isoelectric pH, designated pI.
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Acid-base titration involves the gradual addition or removal of protons. Figure shows the
titration curve of the diprotic form of glycine. The plot has two distinct stages, corresponding
to deprotonation of two different groups on glycine.
The shaded boxes, centered at about pK1=2.34
and pK2=9.60, indicate the regions of greatest
buffering power. Glisin can be used as a buffer
solution at +/- 1 pH range of these pK values. That
means glycine has two regions of buffering power.
At pH 5.97, the point of inflection between the
two stages in its titration curve, glycine is present
predominantly as its dipolar form, fully ionized but
with no net electric charge, which is the pI of
glycine.
Amino acids with an ionizable R group have more complex titration curves, with three stages
corresponding to the three possible ionization steps
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CHEMICALS
HCl, NaOH, Glycine
METHOD
Prepare 50 mL of 0.1 N Glycine. After calibrating the pH meter, pour 20 mL of glycine solution
to a beaker and determine the pH of the solution. Titrate your solution with 0.1 N HCl. After each
addition of 500 µL of acid mix the content of the beaker vigorously and determine pH. Continue this
process until there is no discernible change of the pH of the solution.
Wash and dry the electrode of the pH meter. Pour 20 mL of glycine solution to another
beaker and determine the pH of the solution. Titrate your solution with 0.1 N NaOH as described
above. Write down the data you obtain in data sheet.
Acid-Base titration data sheet:
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CALCULATION
Draw the graph between added acid or base volume (mL) to pH on a graph paper (milimetric
paper). Using the graph determine pKa, pKb and pI values for glycine. Compare your findings with the
theoretical values for glycine.
QUESTIONS
1- How do you calculate the pI value of glutamic acid and histidine? What is the importance
of different pI values of amino acids?
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