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Generally, organic compounds having -COOH (carboxyl group) and -NH 2 (amino group) in the molecular structure are called amino acids.
Fig.1 General structural formula of amino acids
Individual organic groups are bonded to R. Characteristics of each amino acid differ depending on the nature of R.
Fig.2 Structure of Proline (Pro)
Proline has a ring structure. it shows specific optical properties different from other amino acids.
Amino acids are represented by three-letter code almost unified worldwide.
Table 1 Three-letter code of amino acids
(from Instruction manual (main unit) Chapter5 5.5)
In the field of amino acid analysis, Asparagine (Asn) and Glutamine (Gln), may be written as AspNH2 and GluNH2. Similarly, when described as Cys, it may refer to Cysteine.
1) Standard amino acids (Protein hydrolyzate amino acids)
The proteins that make up all animals are composed of amino acids. Amino acids that make up the protein are called standard amino acids (protein hydrolyzate amino acids).
It is said that there are 20 standard amino acids.
Fig.3 Schematic diagram of amino acids, peptides, proteins
2) Physiological fluid amino acids (Free amino acids)
Basics of amino acids are 20 standard amino acids mentioned above, but if you take in them as food, it will be digested and will change to various substances. These amino acids including metabolites and precursors are called physiological fluid amino acids (free amino acids). Generally we analyze about 40 physiological fluid amino acids.
Most of amino acids are difficult to separate and detect because these hydrophilicity are high, UV absorption and fluorescence are low. Hitachi High-speed Amino Acid Analyzer Model L-8900 is based on post-column ninhydrin system.
Fig.4 Flow Diagram of LA8080
Fig.5 Amino acid separation
The cation exchange column has a high chemical strength such as being able to wash with basic solutions, but the physical strength is not so high.
Sudden pressure fluctuations may cause degradation of column, so you should pay attention to pressure of column.
Fig.6 Example of amino acids chromatogram
Table 2 Necessary elements for amino acid analysis buffer
|Ionic strength||NaCl, LiCl|
|buffer capacity||Sodium citrate, Lithiun citrate|
Table 3 Other components of amino acid analysis buffer and purpose of addition
|Ethanol||separation of Thr-Ser|
|Benzyl alcohol||separation of Trp|
|β-thiodiglycol||anti-oxidation of sulfur-containing amino acids|
|Brij-35||Pump pressure reduction|
The buffer solutions are provided as MCI R BUFFER L-8500 SERIES (L-8500 PH-KIT, L-8500 PF-KIT) from Mitsubishi Chemical Corporation.
You can also prepare the buffer solutions according to the "Buffers preparation" section of the instruction manual (main unit). At that time, pH described in the instruction manual is a reference value and adjustment of pH is not necessary.
You have to use special grade reagents or grade for amino acid analysis reagents.
There are two types of derivatization: pre-column and post column. The postcolumn derivatization method adopted for Hitachi High-speed Amino Acid Analyzer L-8900 has advantages such as less influence of contaminants and better reproducibility.
Fig.7 Types of derivatization of amino acids
Fig.8 Chemical reaction of amino acid and ninhydrin
The ninhydrin reaction produces a blue-violet substance (Ruhemann's purple), which is measured the absorbance at 570 nm. Because proline and hydroxyproline produce yellow-red substances which is measured the absorbance at 440 nm.