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Amino acid composition analysis

Ninhydrin Color Development Method

Amino acids are organic compounds that make up protein, one of the energy-producing nutrients. Amino acid concentrations in the blood (plasma) are precisely controlled, and variations in the amino acid concentration in the physiological fluid are used as indicators of metabolism and disease in the body.
Many amino acid analyses are performed using automated analysis methods. Among these methods, a post-column method, also known as the ninhydrin method, is often used, in which each amino acid is reacted with the ninhydrin derivatization reagent after column separation and absorbance measurement is carried out. In the ninhydrin method, the sample is pressed into an alkaline-cation exchange resin to elute amino acids by increasing the eluent pH in a stepwise manner. However, the preparation of the buffer used (e.g., pH, concentration) is very complicated. FUJIFILM Wako offers a variety of amino acid analysis reagents, such as color-developing test solutions, analytical standards, and buffer solutions for exclusive use with type LA-8080, L-8900, L-8800 (A), and L-8500 (A) high-speed amino acid analyzers manufactured by Hitachi High-Tech Corporation.

Ninhydrin Color Development Method

Ninhydrin reaction is a method to detect and quantify amino acids via ninhydrin coloration.

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Amino acids reacted with ninhydrin reaction reagent are detected using two wavelengths, i.e., 570 nm and 440 nm.
The primary amine amino acid (α-amino acid) is colored purple at 570 nm, while the secondary amine (imino acid) amino acid is colored yellow at 440 nm.

Protocol

Methods for amino acid analysis include precolumn derivatization and postcolumn derivatization employed by ninhydrin. Postcolumn derivatization is a method in which a sample is reacted with the derivatization reagent after column separation and transferred to the detector.

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Flow diagram of ninhydrin method1)
Measurement Principle
  1. The sample is pressured into an alkaline-cation exchange resin. When the eluent pH is increased in a stepwise manner, a higher amount of acidic amino acid is eluted early.
  2. When this eluate is heated after the addition of Ninhydrin Coloring Solution, a colored substance showing the local maximum absorption is produced at 570 nm for α-amino acid and 440 nm for imino acid.
  3. The peak area of this colored substance is compared with that of the standard solution to determine the concentration of each amino acid in the sample.
  1. Ito, M. et al. : Wako Jun-yaku Jiho, 86 (3), 15 (2018).

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Ninhydrin Coloring Solution Ninhydrin Coloring Solution Kit for HITACHI
Ninhydrin Coloring Solution Kit for JEOL
Amino Acid Standard Solutions Amino Acids Mixture Standard Solution (CRM)
Amino Acid Standard Solution
Buffer Solution for High-Speed Amino Acid Analyzer PH/PF Series
for Amino Acid Automated Analysis

Buffer for High-Speed Amino Acid Analyzer PH/PF Series

There are two types of analytical methods for ninhydrin color development: “physiological fluid analysis method” and “protein hydrolysate analysis method”.
PF series of buffers are used for the physiological fluid analysis method, and PH series are buffers for the protein hydrolysate analysis method.
In the ninhydrin method, the sample is pressed into an alkaline-cation exchange resin to elute amino acids by increasing the eluent pH in a stepwise manner. However, the preparation of the buffer used (pH, concentration, etc.) is complicated. These series are buffer solutions prepared based on the user instruction of the Hitachi High-Tech Corporation type LA-8080, L-890, L-8800 (A), and L-8500 (A) high-speed amino acid analyzer. They do not require fine adjustment of concentration or dilution and can be used by setting them directly on the equipment.
The physiological fluid analysis method is an analytical method for 41 amino acids contained in physiological fluids, such as serum and urine, and it can measure special amino acids such as taurine, urea and, glutathione; amines; oligopeptides; and typical amino acids.
The protein hydrolysate analysis method is an analytical method that targets 17 constituent amino acids for proteins, peptides, etc.
Qualitative evaluation of protein and peptide preparations for drugs and composition analysis of amino acids in proteins for foods can be performed.

Analytical method About Buffer Series for High-speed Amino Acid Analysis
Protein hydrolysate analysis method An analytical method targeting 17 constituent amino acids of proteins, peptides, etc. PH series
Physiological fluid analysis method An analytical method targeting 41 amino acids contained in physiological fluids such as serum and urine. PF series

Analysis Example

Analysis example using Buffer for High-Speed Amino Acid Analyzer PH-SET

Chromatogram

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Analysis conditions

Device Model L-8900 Hitachi Amino Acid Analyzer
Separation column Protein hydrolyzate analysis column #2622 4.6 mm ID×60 mm (Hitachi P/N: 855-3506)
Ammonia filtering column Ammonia filtering column #2650L 4.6 mm ID×40 mm (Hitachi P/N: 855-3523)
Standard sample 2 nmol/20μL Amino Acids Mixture Standard Solution, Type H
2 nmol/20μL L-Tryptophan Reference Material
Detection Ninhydrin reaction (Ninhydrin reagent: Ninhydrin Coloring Solution Kit for HITACHI)
Measurement wavelength 570 nm and 440 nm
Eluent Reaction solution
B1 PH-1 B4 PH-4 R1 Ninhydrin solution
B2 PH-2 B5 Water R2 Buffer
B3 PH-3 B6 PH-RG R3 5 vol% Ethanol solution
Time
(min.)
%B1 %B2 %B3 %B4 %B5 %B6 Flow 1
(mL/min.)
Temp.
(℃)
%R1 %R2 %R3 Flow 2
(mL/min.)
0.0 100 0 0 0 0 0 0.400 57 50 50 0 0.350
2.5 100 0 0 0 0 0
2.6 0 100 0 0 0 0
4.5 0 100 0 0 0 0
4.6 0 0 100 0 0 0
12.8 0 0 100 0 0 0
12.9 0 0 0 100 0 0
27.0 0 0 0 100 0 0
27.1 0 0 0 0 0 100
32.0 50 50 0
32.1 0 0 100
33.0 0 0 0 0 0 100
33.1 0 100 0 0 0 0
34.0 0 100 0 0 0 0
34.1 100 0 0 0 0 0
37.0 0 0 100
37.1 50 50 0
53.0 100 0 0 0 0 0

Analysis example using Buffer for High-Speed Amino Acid Analyzer PF-SET

Chromatogram

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Analysis conditions

Device Model L-8900 Hitachi Amino Acid Analyzer
Separation column Body fluid analysis column #2622 4.6 mm ID×60 mm (Hitachi P/N: 855-3507)
Ammonia filtering column Ammonia filtering column #2650L 4.6 mm ID×40 mm (Hitachi P/N: 855-3523)
Standard sample Amino Acids Mixture StandardSolution, Type AN/Type B
L-2-Aminoadipic Acid Reference Material L-Asparagine Standard Solution L-Tryptophan Reference Material
Detection Ninhydrin reaction (Ninhydrin reagent: Ninhydrin Coloring Solution Kit for HITACHI)
Measurement wavelength 570 nm and 440 nm
Eluent Reaction solution
B1 PF-1 B4 PF-4 R1 Ninhydrin solution
B2 PF-2 B5 Water R2 Buffer
B3 PF-3 B6 PF-RG R3 5 vol% Ethanol solution
Time
(min.)
%B1 %B2 %B3 %B4 %B5 %B6 Flow 1
(mL/min.)
Temp.
(℃)
%R1 %R2 %R3 Flow 2
(mL/min.)
0.0 100 0 0 0 0 0 0.350 38 50 50 0 0.300
2.0 30
21.5 100 0 0 0 0 0
21.6 80 20 0 0 0 0 60
33.5 70 30 0 0 0 0
33.6 10 90 0 0 0 0
36.5 40
43.5 10 90 0 0 0 0
43.6 0 100 0 0 0 0
50.5 0 100 0 0 0 0 70
50.6 0 0 100 0 0 0
68.4 45
69.5 0 0 100 0 0 0
69.6 60 0 0 40 0 0
75.0 60 0 0 40 0 0
75.1 0 0 0 100 0 0
82.0 0 0 0 100 0 0
82.1 0 20 0 80 0 0
92.5 70
99.5 0 20 0 80 0 0
99.6 0 0 0 100 0 0
112.5 0 0 0 100 0 0
112.6 0 0 0 0 0 100
116.0 50 50 0
116.1 0 0 100
121.5 0 0 0 0 0 100
121.6 100 0 0 0 0 0
125.0 38
126.0 0 0 100
126.1 50 50 0
148.0 100 0 0 0 0 0

Hitachi High-speed Amino Acid Analyzer: LA8080 AminoSAAYA

Features

  • Focuses on operability and safety
    In addition to the conventional front access design, the reagent-installing unit set at a height for easier operations facilitates routine reagent replacement, sample setting, and maintenance, thereby minimizing manipulations and operations that require a half-sitting posture (the front door can also be removed).
  • The long-term reliability and stability of the analyzer have been proved
    One can utilize the analysis conditions, column (filler), and reagents that have been established so far.
    In addition, unstable ninhydrin reaction reagents usually need to be refrigerated, but for Hitachi amino acid analyzers, refrigeration is not required, since two solutions are mixed immediately before the derivatization reaction.
  • The analyzer is compatible with diverse applications
    Up to six types of buffer solutions and time program for column thermostat can be used.
    Moreover, unique features of the reactor, such as high-temperature proof, enable the development of desired methods.

Product Page: HITACHI High-speed Amino Acid Analyzer LA8080 AminoSAAYA

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Ninhydrin Coloring Solution Kit for Hitachi

Ninhydrin Coloring Solution Kit for JEOL

Amino Acids Mixture Standard Solution (High Range)

These mixture standard solutions are prepared by the highly accurate liquid preparation system cultivated in CRM development, and are mixture of high-concentration amino acids (0.1 to 5.5 μmol/L) which are CRM or TRM; Traceable Reference Material (SI traceable reference material). By using these mixture standard solution, it is possible to obtain accurate quantitative values ​​of amino acids.

Amino Acids Mixture Standard Solution (CRM)

These products are SI traceable, and the reliability of the measured values is guaranteed internationally.

Amino Acid Standard Solution

Since L-asparagine and L-2-aminoadipic acid are unstable in 0.1 mol/L HCl, they are prepared separately from the amino acids mixture standard solution of the HCl solution (Amino Acids Mixture Standard Solution, Type AN).

Buffer Solution for L-8500-PH Series

These products are buffer solutions required for amino acid analysis of protein hydrolyzate. It is adjusted based on the instruction manual of Hitachi High-Speed Amino Acid Analyzer (L-8900, L-8800(A), L-8500(A)). There is no need for fine adjustment and dilution of pH. It can be used immediately.

Buffer Solution for L-8500-PF Series

These products are buffer solutions required for amino acid analysis of protein hydrolyzate. It is adjusted based on the instruction manual of Hitachi High-Speed Amino Acid Analyzer (L-8900, L-8800(A), L-8500(A)). There is no need for fine adjustment and dilution of pH. It can be used immediately.

MCI BUFFER™ PH Series

Manufacturer: Mitsubishi Chemical Corporation

MCI BUFFER™ PF Series

Manufacturer: Mitsubishi Chemical Corporation

Buffer Solution

For research use or further manufacturing use only. Not for use in diagnostic procedures.

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