1. Amino Acid Analyzer
Nashwa Othman

3. 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

4. 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

5. 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.

6. Peptide bond formation

7. Classification of Amino Acids

8. Classification of Amino Acids

9. 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

10. 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 Analyzer perform the Quantitative tests

11. The Amino Acid analyzer analysis all known amino acids beside it also analyses the following
Taurine α-Aminoadipicacid
Urea α-Aminobytyricacid
Asparagine Hydroxyproline
Citrulline 1-methyl histidine
Ornithine Ammonia
Homocysteine

12. 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

13. 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 because this pH is less than the PI of all amino acids so that all amino acids will positively charged

14. Steps of sample analysis
The sample go through 4 steps
Autosampler
Separation column reaction
Coil reaction
Photometer

15. 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

16. 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

17. 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

18. 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

19. Separation column reaction
However the two main factors affecting separation are:
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.

20. 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

21. 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

22. 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

23. 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

24. 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

26. Hydroxyproline
proline