Introduction
In industries such as food science, feed analysis, and biological products, the accurate determination of free amino acid content in protein solutions is of great significance for evaluating the degree of proteolysis, product quality, and process control. Traditional amino acid analysis methods such as HPLC are accurate but expensive and complex to operate. The microtitrameter assay based on chromogenic reaction has become a widely adopted alternative due to its easy operation, low cost, and suitable for routine batch testing. This method is based on the principles of relevant standards at home and abroad, through the specific reaction between specific reagents and free amino acids, and the photometric determination is carried out by microtitrator, so as to achieve rapid and stable analysis of the free amino acid content in protein solution.
Method principle
The core of this method is the indotrione reaction. Under specific pH and heating conditions, the free amino acids in solution react with indotrione to form a violet substance with characteristic absorption at the wavelength of 570 nm (proline and hydroxyproline produce yellow products, which need to be determined at 440 nm). The degree of reaction and the concentration of free amino acids are within a certain range, which is in line with Lambert-Beale's law. The general formula of the reaction can be expressed as:
Amino acids + indotrione → reduced indotrione + ammonia + carbon dioxide
Reduced intrione + ammonia + intrione → purple complex
In this process, the microtitrator precisely controls the reaction temperature and time, and measures the absorbance value of the final reaction solution through the built-in photometer, and calculates the content of free amino acids in the sample through a pre-established standard curve.
Instruments and reagents
The main instruments required are microtitrators with heating modules, magnetic agitation, and photometric detection of 570 nm (and 440 nm) filters. Supporting equipment includes micropipettes, vortex mixers and thermostatic water baths. The key reagents required are shown in the table below.
| Reagent name | Role and requirements |
| Indotrione solution | Main reagent for chromogenic reaction, commonly used concentration 2% (w/v) |
| Reduced intrione | Stabilizes color rendering and enhances sensitivity |
| Acetate buffer (pH 5.5) | Provide a stable reactive acid-base environment |
| Glycine standard solution | Draw standard curves, commonly used concentration gradients |
| Ethanol in water (60%, v/v) | Dissolve and dilute reagents |
| Sample protein solution | Pretreatment is required to remove interfering macromolecules |
Analysis steps
The analysis process mainly includes four parts: sample pretreatment, standard curve preparation, color development reaction and determination, and result calculation.
Sample preparation:Take an appropriate amount of protein solution, add an equal volume of sulfosalicylic acid solution (final concentration 3-5%) to precipitate the protein, vortex mixing, let it stand at 4 °C for 30 minutes, and then centrifuge at 10,000 rpm for 15 minutes. Take the supernatant and dilute it with a buffer of appropriate pH if necessary as the solution to be tested.
Standard curve preparation:Using a glycine standard stock solution, a range of standard working fluids at known concentrations are prepared. Typical concentrations range from 0.05 to 2.0 μmol/mL. Each standard point needs to be set in parallel.
Color development reaction and determination:In the titrator tube, add the exact volume of sample solution or standard, acetate buffer and intrione working solution in sequence. The tube was placed in the titrator heating module that had been preheated to 100°C for 15 minutes. After the reaction is over, it is quickly cooled to room temperature. Add ethanol aqueous solution to each tube to a uniform volume and mix well. Using the photometric module of the microtitrator, the absorbance values of each tube were determined at a wavelength of 570 nm using blank reagents as a reference.
Result calculation:The linear standard curve equation Y = aX + b is fitted by taking the standard solution concentration as the abscissa (X) and the corresponding average absorbance value as the ordinate (Y). The absorbance value measured by the sample was substituted into the equation to calculate the concentration of free amino acids in the sample solution (μmol/mL), and then the free amino acid content in the original protein solution was calculated according to the dilution factor and the initial volume of the sample.
Influencing factors
The accuracy and repeatability of the method are affected by many factors. The pH value of the reaction system needs to be strictly controlled, with an optimal range of 5.0-5.5, and deviations will significantly affect the color development efficiency. Precise control of heating temperature and time is crucial, as fluctuations can lead to inconsistent color development. Adequate protein pre-treatment is necessary for the presence of ammonium salts, peptides, or certain reducing substances in the sample that may cause interference. For samples containing predominantly proline, 440 nm should be used as the detection wavelength. Standard curves should be prepared synchronously for each assay, and reagents should be prepared and used to ensure activity.
Scope of application:
This method is suitable for the routine determination of total free amino acids in various food proteolytes, fermentation broths, feed extracts and biotechnology products. Its advantage is that the operating process is relatively standardized, suitable for batch sample analysis, and requires less instrumentation than chromatographic methods. The limitation is that specific amino acid species cannot be distinguished, the determination results are total free amino acid content, and there may be background interference for dark sample matrices, requiring additional sample blank correction.
References
Moore, S., & Stein, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. Journal of Biological Chemistry.
National Standardization Administration of China. Determination of amino acids in feed. Relevant national standards.
Yemm, E. W., & Cocking, E. C. (1955). The determination of amino-acids with ninhydrin. The Analyst.