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Amino Acid Analyzer
Nashwa Othman
What are 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
Importance of Amino Acids
• Amino Acids are the building blocks of
proteins
• They are important in many biological
molecules, such as forming parts of
coenzymes
• or as precursors for the biosynthesis of
molecules such as Heme
• They are critical to life, and have many
functions in metabolism
Peptide bond formation
• one amino acid molecule can react with
another and become joined through an amide
linkage. This polymerization of amino acids is
what creates proteins. This condensation
reaction yields the newly formed peptide
bond and a molecule of water.
Peptide bond formation
Classification of Amino Acids
Classification of Amino Acids
Amino Acid Analysis
• Amino acid analysis is essential in the
diagnostic work-up 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
Sample preparation
In physiological fluids, usually, the unbound
amino acids are analyzed. Peptides, proteins
or other highly molecular compounds have to
be removed so as not to obstruct separation
column and this could be done by
• Acid precipitation (by using Sulfosalisilic acid)
• Ultra filtration
• Ultracentrifugation
Sample preparation- 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
Steps of sample analysis
• 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
when required a fresh solution of glutamine
standard is prepared (1.0 µmol / ml ) and injected
then the freshly prepared sample is injected and
glutamine peak is identified from retention time
Autosampler
• 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 ( in comparison with thin layer
chromatography where the slide is the stationary
phase and the buffers are the mobile phase)
• A special pump pumps the buffer to the
separation column and since amino acids are
eluted at different pH medium different pH values
buffers are used
Separation column reaction
• 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
Separation column reaction
• However the
separation are:
two
main
factors
affecting
 Titration, or increasing the pH. As pH increases,
the amino acids go from a positive to a neutral
charge state, and so are released from the fixed
anionic sites in the separation column
 Competitive ion exchange. The positive amino
acids show a higher affinity for the ion-exchange
sites than do the eluting cations and so are
bound strongly to the anionic sites.
Separation column reaction
• Therefore, They are eluted by the continuous
flow of cation or by increasing cation
concentration
developed
by
gradient
formation.
• Once the final amino acid is eluted
regeneration solution is used to regenerate
the separation column by removal of amino
acids remnant on the column
Coil reaction
• After separation the instrument add the
ninhydrin solution that react with the
products at 130° C
• All products give purple color and are
estimated at 570 nm except proline and
hydroxyproline they give yellow color and are
estimated at 440 nm
Photometer
• After the ninhydrin reaction, The resultant
colored species then 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 to those of
reference compounds
Recorder
• 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 those of calibrators by a specific
computer program
proline
Hydroxyproline