In this lecture

„ Introduction to Amino acids

AMINO ACID COMPOSITION „ Importance of amino acid analysis
ANALYSIS „ Methods of amino acid analysis
Assoc. Prof. PHAM VAN HUNG

SPECTROSCOPY

Introduction Amino Acids

• an amino group (hence "amino"
„ Amino acids acid).
‰ The monomer unit of proteins
• a carboxyl group (-COOH). This
‰ Important constituents of foods both in nutrition gives up a proton and is thus an acid
and quality of foods (hence amino "acid").
• one of 20 different "R" groups. It is
„ Classification the structure of the R group that
„ 20 amino acids determines which of the 20 it is and
„ Different side chain (R groups) its special properties.
„ R groups vary in size, shape and polarity

1
 Amino acids can be classified by R groups

Amine group

SPECTROSCOPY

pKa = 10.5 i id
imidazo
pKa = 3.9 pKa = 4.1

guanidino
pKa = 12.5

Aspartic acid Glutamic acid

Basic amino acids Acidic amino acids

2
 Amino acids can act as acids and bases
Nonionic and zwitterionic forms
Titration of glycine
of amino acids

pH 7

indole
ring
pH 1

Titration curves predict the

electric charge of amino acids
pH 12
Isoelectric point (or isoelectric pH)
Amphoteric
(ampholytes - amphoteric electrolytes) pI = ½ (pk1 + pk2) = ½ (2.34 + 9.60) = 5.97

SPECTROSCOPY

Amino acids differ in their acid-base properties

Amino acids with

pka of the –COOH group: 1.8 – 2.4 Important of analysis
R groups that do not ionize pka of the –NH3+ group: 8.8 – 11.0

Amino acids with ionizable R groups

Amino acid analysis can be used to:
e.g. • to quantify protein and peptides
• to determine the identity of proteins or
peptides
id b d on their
based h i amino
i acid
id
composition
• and to detect atypical amino acids that might
be present in a protein or peptide

three stages (three ionization steps → three pka values)

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 Principle of analysis Procedure for Amino Acid Composition analysis

ƒ A protein/peptide is hydrolyzed into its A. Hydrolysis

individual amino acid constituents and then 1. Overnight in 6 M HCl at 100 oC.
amino acid compositions are analyzed based
2. Enzymes.
on a chromatographic method.
method
• Ion‐exchange chromatography, reversed‐
phase liquid chromatography, and gas–liquid  B. Separation by HPLC.
chromatography are three separation 
techniques used.

SPECTROSCOPY

Protein Hydrolysis Protein Hydrolysis

ƒ Acid hydrolysis is the most common method for • Free amino acids can be obtained from proteins by strong 

acid hydrolysis:
hydrolyzing a protein sample before amino acid
analysis.
6 N HCl
ƒ However, some of the amino acids can be Protein Amino acids
destroyed 100 ºC,, 24 h,,
in vacuo
ƒ A time-course study (i.e., amino acid analysis at
acid hydrolysis times of 24, 48, and 72 hours) is • 3 of the standard aas are lost during acid hydrolysis treatment:
often employed to analyze the starting
concentration of amino acids that are partially Asparagine Aspartic acid Amides go to
destroyed or slow to cleave Glutamine Glutamic acid acids
Tryptophan Decomposed

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 Protein hydrolysis Separation by HPLC
ƒ Hydrolysis Solution:
6 N hydrochloric acid containing 0.1% to 1.0% of phenol. (phenol is
ƒ Ion‐exchange chromatography with postcolumn ninhydrin
to prevent halogenation of tyrosine) detection is one of the most common methods employed
for quantitative amino acid analysis.
ƒ Liquid Phase Hydrolysis
ƒ a Li‐based cation‐exchange system is employed for the
a. Place the protein sample in a hydrolysis tube, and dry.
y of the more complex
analysis p physiological
p y g samples,
p , and
b. Add 200 μL of Hydrolysis Solution per 500 μg of protein. the faster Na‐based cation‐exchange system is used for
c. Freeze the sample tube in a dry ice‐acetone bath, and flame seal the more simplistic amino acid mixtures.
in vacuum.
ƒ Separation of the amino acids on an ion‐exchange column
d. Samples are typically hydrolyzed at 110ºC for 24 hours in is accomplished through a combination of changes in pH
vacuum or inert atmosphere to prevent oxidation. Longer
and cation strength.
hydrolysis times (e.g., 48 and 72 hours) are investigated if there
is a concern that the protein is not completely hydrolyzed. ƒ A temperature gradient is often employed to enhance
separation.

SPECTROSCOPY

Separation by HPLC Detection of peaks
• When the amino acid reacts with ninhydrin, the
reactant has characteristic purple or yellow color. • A detector is usually present as an ultraviolet‐
• Amino acids, except imino acid, give a purple color, visible or fluorescence detector .
and show the maximum absorption at 570 nm.
• The imino acids such as p proline ggive a yyellow color,,
and show the maximum absorption at 440 nm. • A
A recording device (e.g., integrator) is used for 
di d i ( i )i df
• The postcolumn reaction between ninhydrin and transforming the analog signal from the 
amino acid eluted from column is monitored at 440 detector and for quantification. 
and 570 nm.
• and the chromatogram obtained is used for the
determination of amino acid composition.

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1. Amino acids are pre- or
postcolumn derivatized with
ninhydrin, or o-phtalaldehyde
(OPA) form fluorescent
adducts.
2. The amino acids are identified
according to their retention
times on HPLC
3. Amounts of aas present are
The end!
determined fluorescent
intensities.
4. Sensitive: can detect less than 1
pmol of each amino acid.

OPA-amino acid analysis using reverse-phase HPLC

*note OPA does not react with proline so another reagent must be used (FMOC)

SPECTROSCOPY

Mechanism of Ion-Exchange Chromatography of Chromatogram of Amino Acids

Amino Acids
+ pH 2
Na
H3N
+ Moles/Liter VAL
-
SO 3
COOH
+
Na ALA
OH
- +
So 3 H3N
COOH
Exchange Resin
HIS LEU

-
SO 3 H 3N+
ASP
pH3.5 GLU
COOH LYS
-
OH
So 3 H3N+
+ - + -
Na COO H OH = H 2 O

+
Na

SO 3
-
+ pH 2.25 pH 3.25 pH4.25
H3N
- + -
COO H OH = H 2 O
- + pH4.5
So 3 Na