AMINO ACIDS
• Amino acids are organic compounds containing an amine
group, a carboxylic acid group and a side chain that varies
between different amino acids
• Amino Acids are the building blocks of proteins, important in
many biological molecules, such as enzymes , co- enzymes,
precursors for the biosynthesis of molecules, and have many
functions in metabolism
• An amino acid molecule react with other and made linkage to
form polymer of amino acids called proteins. This
condensation reaction yields the newly formed peptide bond
and a molecule of water.
AMINO ACID ANALYSIS
 Amino acid analysis is a technique used to determine
the amino acid composition or content of proteins.
 The sequence of the amino acids in a protein or
peptide determines the properties of the molecule.
• It is used to quantify protein and peptides.
• To determine the identity of proteins or peptides.
• To detect atypical amino acids present in a protein.
• Amino acid analysis is also essential in the diagnostic
of inherited metabolic disorders such as
phenylketonuria (PKU) and maple syrup urine
disease (MSUD) and different other diseases
TYPES OF AMINO ACID ANALYSIS
• The following 3 groups of tests are important
• Screening tests
• Quantitative tests to monitor treatment or confirm
an initial diagnosis
• Specific tests that identify an unknown amino
acid or metabolite
• The Amino Acid
Quantitative tests
Analyzer
perform
the
• The Amino Acid analyzer analysis all known
amino acids beside it also analyses the following
Taurine
Urea
Asparagine
Citrulline
Ornithine
Homocysteine
α-Aminoadipicacid
α-Aminobytyricacid
Hydroxyproline
1-methyl histidine
Ammonia
 It is necessary to hydrolyze a protein/peptide to its
individual amino acid constituents before amino acid
analysis.
 Methods used for amino acid analysis are usually based
on a chromatographic separation of the amino acids
present in the test sample.
 An amino acid analysis instrument is generally a lowpressure or high-pressure liquid chromatography or GC.
 A detector is usually present as an ultraviolet-visible or
fluorescence detector .
• A recording device (e.g., integrator) is used for
transforming the analog signal from the detector and for
quantitation.
SAMPLE PREPARATION
• Accurate results from amino acid analysis require purified
protein and peptide samples.
• Buffer components (e.g., salts, urea, detergents) can interfere
with the analysis so removed from the sample before analysis
1. Precipitating the protein from the buffer using an organic
solvent (e.g., acetone) and drying in a vacuum centrifuge
2. Acid precipitation (by using Sulfosalisilic acid)
3. Dialysis against a volatile buffer or water
4. Ultrafiltration for buffer replacement with a volatile buffer or
water
5. Gel filtration.
pH ADJUSTMENT
• Each amino acid has isoelectric pH (PI) and its
charge will be natural at this pH
• By increasing H ions (decreasing pH) it will be
positively charged and visa versa by increasing
OH ions (increasing pH) it will be negatively
charged
• Therefore, when preparing the sample the pH
should be 1.8-2 because this pH is less than the PI
of all amino acids so that all amino acids will
positively charged
PROTEIN HYDROLYSIS
 Acid hydrolysis is the most common method for
hydrolyzing a protein sample before amino acid
analysis.
 However, some of the amino acids can be
destroyed
 A time-course study (i.e., amino acid analysis at
acid hydrolysis times of 24, 48, and 72 hours) is
often employed to analyze the starting
concentration of amino acids that are partially
destroyed or slow to cleave
METHOD FOR HYDROLYSIS
 Hydrolysis Solution:
6 N hydrochloric acid containing 0.1% to 1.0% of phenol. (
phenol is to prevent halogenation of tyrosine)
 Liquid Phase Hydrolysis
a. Place the protein sample in a hydrolysis tube, and dry.
b. Add 200 μL of Hydrolysis Solution per 500 μg of protein.
c. Freeze the sample tube in a dry ice-acetone bath, and flame
seal in vacuum.
d. Samples are typically hydrolyzed at 110ºC for 24 hours in
vacuum or inert atmosphere to prevent oxidation. Longer
hydrolysis times (e.g., 48 and 72 hours) are investigated if
there is a concern that the protein is not completely
hydrolyzed.
DERIVATIZATION
• Typically, HPLC is used for the analysis of amino acids.
• However, GC also be used and availability of instrument &
operation costs can make it a better choice.
• The polar nature of amino acids requires derivatization prior to
GC analysis.
• The derivatization make an analyte more volatile, less reactive,
and improve its chromatographic behavior.
• Derivatization replaces active hydrogens, OH, NH2, and SH
polar functional groups of AA with a nonpolar moiety.
• Silylation is a very common derivatization technique & useful
for a wide variety of compounds.
• The main disadvantage of this method is its sensitivity to
moisture. The presence of moisture results in poor reaction
yield and instability of the derivatized analytes.
• Other eg are halogination, methylation, glycosylation etc.
SAMPLE ANALYSIS
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The sample go through 4 steps
Autosampler
Separation column reaction
Coil reaction
Photometer
AUTOSAMPLER
• Before samples are analyzed the commercially
available standard solution have to be analyzed to
calibrate the instrument
• The amino acid glutamine is not present in the standard
since it decomposes quickly. Therefore a fresh solution
of glutamine is prepared (1.0 µmol / ml ) and injected
then the freshly prepared sample is injected and
glutamine peak is identified from retention time
• Following sample preparation the sample is inserted in
the specified place then the autosampler inject 130 µl of
the sample and pass it to separation column
SEPARATION COLUMN REACTION
• In this situation the separation column is the
stationary phase and the buffers are the mobile
phase.
• A special pump pumps the buffer to the separation
column.
• In the separation column ion exchange reaction
take place and the positively charged amino acids
are bound to the negatively charged sites in the
separation column
• The separation depends on different factors for
example ion size, Adsorption
METHOD FOR ANALYSIS
 Ion-exchange chromatography with postcolumn ninhydrin
detection is one of the most common methods employed for
quantitative amino acid analysis.
 a Li-based cation-exchange system is employed for the
analysis of the complex physiological samples, and the faster
Na-based cation-exchange system is used for the simple
amino acid mixtures
• Separation of the amino acids on an ion-exchange column is
accomplished through a combination of changes in pH and
cation strength.
• A temperature gradient is often employed to enhance
separation.
COIL REACTION
• When the amino acid reacts at 130oC with ninhydrin,
the reactant has characteristic purple or yellow color.
• Amino acids, except imino acid, give a purple color,
and show the maximum absorption at 570 nm.
• The imino acids such as proline give a yellow color,
and show the maximum absorption at 440 nm
• The postcolumn reaction between ninhydrin and amino
acid eluted from column is monitored at 440 and 570
nm.
• and the chromatogram obtained is used for the
determination of amino acid composition.
PHOTOMETER
• After the ninhydrin reaction, The resultant
colored species are detected with a
spectrophotometer where the absorption of the
colored complex is measured at two
wavelength length 570 and 440 nm
• The quantity of the colored complex produced
is directly proportional to the concentration of
the particular amino acid present in the sample
RECORDER
• Photometer is linked to a two channel recorder where
a series of peaks representing the amino acids are
recorded
• The amino acids are identified by the comparison of
the retention times of the components in the specimen
with reference compounds
• The information is transferred to a specific computer
program where Quantitation of each amino acid could
be done by comparison of specimen peak area or
height with standards.
proline
Hydroxyproline