Lecture 2

Physical organic and bioorganic chemistry

CH-204

Department of Chemistry
IITDH

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Amino
acids and
peptides

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 Acid-base properties of amino acids: Zwitterion
• Every amino acid has a carboxyl group and an amino group, and each group can
exist in an acidic form or a basic form, depending on the pH of the solution
•

• The acidic form (with the proton) predominates if the pH is less than the pKa

of the ionizable group, and the basic form (without proton) predominates if
the pH of the solution is greater than the pK of the ionizable group
a

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 Acid-base properties of amino acids: Zwitterion
• The carboxyl groups of the amino acids have pKa values of approximately 2, and the
protonated amino groups have pKa values near 9. Both groups, are in the acidic form
in a very acidic solution (pH 0). At pH = 7, the pH of the solution is greater than the
pKa of the carboxyl group, but less than the pKa of the protonated amino group;
therefore, the carboxyl group is in the basic form and the amino group is in the acidic
form. In a strongly basic solution (pH 12), both groups are in the basic form.

• At physiological pH (7.4), an amino acid exists as a dipolar ion (zwitterion). A

zwitterion is a compound that has a negative charge on one atom and a positive
charge on a nonadjacent atom. (The name comes from zwitter, German for “hybrid.”)

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Why pKa of amino acid is lower than a normal acid?
•

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 Some side chains have ionizable groups
• A few amino acids have side chains with ionizable hydrogens (Arg, Lys,
Asp, Glu, Cys, Tyr, His ). The protonated imidazole side chain of histidine,
for example, has a pK of 6.04. Histidine, therefore, can exist in four different
a

forms, and the form that predominates depends on the pH of the solution.

pH should be greater than the pKa of the particular functional group for that group to ionize
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 The isoelectric point of an amino acid (pI)
• The isoelectric point (pI) of an amino acid is the pH at which it has no net
charge. In other words, it is the pH at which the amount of positive charge on an
amino acid exactly balances the amount of negative charge:
pI = pH at which there is no net charge

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Determining the pI of an amino acid without
an Ionizable Side Chain: Example: Alanine
• The pI of an amino acid that does not have an ionizable side chain—
such as alanine—is midway between its two pK values.
a

9
•

10
•

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 pI of an amino acid with an Ionizable Side Chain
• The pI of most amino acids that have an ionizable side chain is the average of the
pK values of the similarly ionizing groups (either positively charged groups
a

ionizing to uncharged groups or uncharged groups ionizing to negatively charged

groups). Example: the pI of Lys is the average of the pK values of the two groups
a

that are positively charged in their acidic form and uncharged in their basic form.
The pI of Glu (glutamic acid) is the average of the pK values of the two groups
a

that are uncharged in their acidic form and negatively charged in their basic form.

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 Acid-base equilibria in case of lysine
•

= =
=

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 Separation of amino acids: Electrophoresis
• Electrophoresis separates amino acids on the basis of their pI values. A few drops of
amino acid mixture are applied to the middle of a piece of filter paper (or to a gel).
When the paper is placed between two electrodes and an electric field is applied, an
amino acid with a pI greater than the pH of the solution (such as arginine) has an
overall positive charge and migrates toward the cathode (the negative electrode).

• The farther an amino acid’s pI is from the pH of the solution, the more positive its
overall positive charge and the farther it migrates toward the cathode in a given
amount of time.

• An amino acid with a pI less than the pH of the solution (such as aspartate) has an
overall negative charge and migrates toward the anode (the positive electrode).

• If two molecules have the same overall charge, the larger one will move more slowly
during electrophoresis because the same charge has to move a greater mass.
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 1. Electrophoresis of amino acids
• An amino acid will be overall positively charged if the pH of the solution is
less than the pI of the amino acid, and it will be negatively charged if the pH
of the solution is greater than the pI of the amino acid.

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 2. Reaction with ninhydrin: Forming colored product
• Amino acids form a colored
product when heated with
ninhydrin. The number of amino
acids in the mixture is determined
by the number of colored spots
on the filter paper. The individual
amino acids can be identified by
their location on the paper
compared with a standard.

Spraying a paper with

a solution of
ninhydrin
allows latent
fingerprints (as a
consequence
of amino acids left
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behind by the fingers) Abs maximum of 570 nm
 3. Paper/Thin-Layer Chromatography (TLC)
• Paper chromatography separates amino acids on the basis of their polarity. A few
drops of a solution of an amino acid mixture are applied to the bottom of a strip of
filter paper. The edge of the paper is then placed in a solvent. The solvent moves up
the paper by capillary action, carrying the amino acids with it. Depending on their
polarities, the amino acids have different affinities for the mobile (solvent) and
stationary (paper) phases and, therefore, some travel up the paper farther than others.

• When a solvent is used that is less polar than the paper, the more polar the amino acid,
the more strongly it is adsorbed onto the relatively polar paper. The less polar amino
acids travel farther up the paper because they have a greater affinity for the less polar
mobile phase. Therefore, when the paper is developed with ninhydrin, the colored spot
closest to the origin is the most polar amino acid and the spot farthest away from the
origin is the least polar amino acid

• Less polar amino acids travel father up the paper if the solvent is less polar than the
paper. TLC uses a plate with a coating of solid material instead of filter paper. How
the amino acids separate depends on the solid material and the solvent chosen for the
mobile phase 22
TLC

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 4. Ion-Exchange Chromatography
• A technique called ion-exchange chromatography separates amino acids based on
their overall charge and determines the relative amount of each amino acid in mixture.

• This technique employs a column packed with an insoluble resin. A solution of a

mixture of amino acids is loaded onto the top of the column, and a series of buffer
solutions of increasing pH are poured through the column.

• The resin is a chemically inert material with charged groups.

• Two types: Cation exchange resins and anion exchange resins

• Cations bind most strongly to cation-exchange resins.

• Anions bind most strongly to anion-exchange resins.

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 Cation and anion exchange resins
• A cation-exchange resin exchanges the Na+ counterions
of the SO3- groups for the positively charged species
traveling through the column. In addition, the relatively
nonpolar nature of the column causes it to retain
nonpolar amino acids longer than polar amino acids.
• If a mixture of lysine and glutamate in a solution of pH =
6 is loaded onto the column, glutamate will travel down
the column rapidly because its negatively charged side
chain would be repelled by the negatively charged
sulfonic acid groups of the resin. The positively charged
side chain of lysine, on the other hand, will cause that
amino acid to be retained on the column longer.
• Resins with positively charged groups are called anion-
exchange resins because they impede the flow of anions
by exchanging their negatively charged counterions for Dowex-50 (cation exchange resin)
the negatively charged species traveling through the
column. A common anion-exchange resin, Dowex 1, has
CH2N+(CH3)3Cl groups in place of the SO3-Na+ groups 25
 An Amino Acid Analyzer
• An amino acid analyzer is
an instrument that automates
ion-exchange
chromatography. When a
solution of amino acids
passes through the column of
an amino acid analyzer
containing a cation exchange
resin, the amino acids move
through the column at
different rates, depending on
their overall charge. The
solution that flows out of the
column (the effluent) is
collected in fractions. These
are collected often enough
that each amino acid ends up
in a different fraction (can be
determined by reaction
with ninhydrin and
absorbance at 570nm) 26