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Hitachi High-Technologies GLOBAL

Banner: Amino Acid Analyzer Basic Course

1. Principle and basics of amino acid analysis

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

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)

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)

Asp Aspartic acid Val Valine
Thr Threonine Met Methionine
Ser Serine Ile Isoleucine
Asn Aspargine Leu Leucine
Glu Glutamic acid Tyr Tyrosine
Gln Glutamine Phe Phenylalanine
Pro Proline Lys Lysine
Gly Glycine His Histidine
Ala Alanine Trp Tryptophan
Cys Cystine Arg Arginine

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

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.

Standard amino acids and Physiological fluid amino acid

2. Method for analyzing 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.4 Flow Diagram of LA8080

2-1. Cation exchange column and separation of amino acids

  1. Amino acids are charged to + in acidic solution, and electric attraction is generated to cation exchange resin.
  2. In contrast, amino acids are charged to - in basic solution, so that they repel and pass through cation exchange resin.
    (Utilizing this chemical property, we use sodium hydroxide solution (basic solution) for washing out remaining substances in the column after analysis. It is called regeneration (RG) process.)
  3. In amino acid analysis, separation is carried out by changing the pH of the eluent from acidic to basic.

Fig.5 Amino acid separation

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

Fig.6 Example of amino acids chromatogram

Table 2 Necessary elements for amino acid analysis buffer

Condition Chemicals
pH Citric acid
Ionic strength NaCl, LiCl
buffer capacity Sodium citrate, Lithiun citrate

Table 3 Other components of amino acid analysis buffer and purpose of addition

Reagents Porpose
Ethanol separation of Thr-Ser
Benzyl alcohol separation of Trp
β-thiodiglycol anti-oxidation of sulfur-containing amino acids
Brij-35 Pump pressure reduction
Caprylic acid Anti-corruption

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.

2-2. Derivatization of amino acids

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.7 Types of derivatization of amino acids

Fig.8 Chemical reaction of amino acid and ninhydrin

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.

3. Inside Front Doors

Image: Inside Front Doors