The basic concept to understand AAA

1. Amino Acid
Analyzer
PRESENTED TO: SIR UMAIR

2. Contents
• Introduction
• Principle
• Types
• Working
• Application

3. Introduction
Amino acid Analyzer is specifically configured system
optimized for analysis of free amino acids.
PURPOSE
• Detection of presence of AA in variety of solutions, such
as extracellular and intracellular fluids
• Plant and animal tissues
• Broths and fruits
• Beverage juices
• Detection of presence of hydrolyzed AA, such as found in
protein, collagen, peptides, and processes foods.

5. Key Features
• AAA is fully automated.
• Enables to analyze AA automatically without sample
preparation.
• Reproducible results while minimizing errors between
operators.
• You don't require to prepare reagents and solvents because all
necessities are provided.
• You can easily analyze 40 kinds of primary and secondary
AA with pre-established analysis methods.

6. Principle
The system utilizes ion-exchange chromatography incorporating
post column reaction with Ninhydrin and subsequent detection in
the visible region spectrum.
Improve sensitivity for amino acids, proteins, carbohydrates,
inorganic ions, pesticides, and other samples. In post column
reactions, column effluent is mixed with a reagent before it
enters the detector.
Post column reaction

7. Types of AA Analysis
The following 3 groups of tests are important
• Screening tests This test is done to measure the
level of amino acids in the blood.
• Quantitative tests to monitor treatment or
confirm an initial diagnosis .
• Specific tests that identify an unknown amino
acid.
The Amino Acid Analyzer perform the Quantitative
tests.

8. Working
The sample go through 4 steps
• Auto sampler
• Separation column reaction
• Coil reaction
• Photometer

9. Auto sampler
Before sample analysis commercially available standard solution
(concentration must remain constant all the time) are analyzed to
calibrate instrument.
• Glutamine is not present in standard as it decomposes quickly.
• Fresh glutamine standard solution is prepared.
• Freshly prepared sample is injected.
• Glutamine peak is identified from retention time.
• Following sample preparation the sample is inserted in the specified
place then the auto sampler inject 130 µl of the sample and pass it to
separation column.

10. Separation Column Reaction
• Separation column is the stationary phase.
• Buffers are the mobile phase.
• Special pump pumps the buffer to the separation column.
• As AA are eluted at different pH medium different pH values
buffers are used.
• Ion exchange reaction take place and positively charge AA are
bound to negatively charged sites in the separation column.
• The separation depends on different factors example ion size,
adsorption.

11. • The column temperature is accurately controlled and can
be varied, as necessary, to produce the required
separation.
• The column eluent is mixed with the ninhydrin reagent,
this mixture being passed through the high temperature
reaction coil.

12. Coil Reaction
• After separation instrument add the ninhydrin
solution that react with the products at 130° C.
• In the reaction coil ninhydrin reacts with the
amino acids present in the eluate to form colored
compounds.
• 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.

13. Photometer
• From the reaction coil, the eluate/ninhydrin mixture is fed to the
photometer unit where the amount of each colored compound is
determined by measuring the amount of light absorbed.
• The light absorption is measured at two wavelengths, 570nm and
440nm.
• The amount of colored compound produced is directly proportional
to the quantity of amino acid present in the eluate.

14. Recorder
• The photometer output is connected either to a two channel chart
recorder which plots the amino acid concentrations as a series of
peaks or to an appropriate integration system.
• The retention time of the peak on the chart identifies the amino acid.
• Area under the peak indicating the quantity of amino acid present.
• Amino acid analyser is a comparative instrument, a calibration
analysis must be performed before commencing a series of analyses,
to produce a standard trace for comparison purposes.

16. • Estimate amount of peptide/protein.
• Estimate degree of purity.
• Estimate concentration of protein/peptide solution.
• Determine amino acid composition.
• Estimate amount of unusual amino acids.
Applications