A Full Introduction to Amino Acid Analysis Methods (Part One)Posted by beauty33 on March 26th, 2020
Amino acids are the basic units that make up enzymes and proteins in organisms. There are more than 20 kinds of amino acids (also known as basic amino acids) that participate in protein synthesis. They mainly exist in nature in the following two forms. One is in the free state in physiological body fluids (such as plasma, urine, etc.), and food ( (e.gg, meat products, beverages), the other is in the peptide and protein in a bound state. Amino acid analysis is an important research method in the fields of proteomics, biochemistry, food science, clinical medicine, chemical light industry, archeological research, geological inference, and cosmic exploration. Therefore, it is of great significance to research and improve amino acid analysis methods. Introduction to amino acid analysis methods Conventional analytical chemistry methods can be used for amino acid analysis. At present, the commonly used amino acid analysis methods include chemical analysis, spectral analysis, electrochemical analysis, capillary electrophoresis, and chromatography.
Chemical analysis is a traditional amino acid analysis method, which includes formaldehyde titration and Kjeldahl method. 1.1. Formaldehyde titration method The principle of the formaldehyde titration method is to add formaldehyde to a neutral or alkaline amino acid solution. The amino group and formaldehyde of the amino acid can form methylol, titrating the derivative with an alkaline standard solution, and using a phenolphthalein indicator to determine the titration endpoint. This method is simple, easy and inexpensive, but it is difficult to accurately determine the endpoint of the titration in actual operation, so the method is less accurate. 1.2. Kjeldahl method The Kjeldahl method determines the total amount of nitrogen in a sample, and calculates the protein and amino acid content in the sample based on the average nitrogen content of the protein and amino acid in the specific sample. This method was first proposed by Kiel-dahl in 1833. After long-term improvements, it has now evolved into a constant method, a micro method, a semi-micro method, an automatic nitrogen determination method, and an improved Kelvin method. The measurement results of this method have high accuracy, but due to the complicated operation steps, long measurement cycle, and inability to identify the source of nitrogen, it has a limited application range.
Spectroscopy is a relatively mature and easy-to-use method. UV spectrophotometry and fluorescence spectrophotometry are commonly used. 2.1. Ultraviolet spectrophotometry Tyrosine, arginine, and phenylalanine have obvious characteristic absorptions in the ultraviolet region. Because their R groups contain a benzene ring conjugated double bond system, the above amino acid content can be determined by ultraviolet spectrophotometry. This method is simple and easy to implement, but the linear range is slightly smaller, only one order of magnitude. Li Jinming et al. used ultraviolet spectrophotometry to determine the content of arginine in Rana oil capsules. 2.2. Fluorescence Spectrophotometry The amino group, carboxyl group or other reactive groups in the amino acid molecule can chemically react with the derivatization reagent. The content of the derivatized product can be determined by using a fluorescence spectrophotometer, and the amino acid content in the original sample can be calculated. This method may have problems such as long derivatization time, complex derivatization product components, and poor stability. Careful selection of derivatization reagents can partially solve the above problems. Chen Yuejiao et al. used the o-phthalaldehyde fluorescence method to determine the total amount of free amino acids in commercially available ginkgo.
Electrochemical analysis usually uses different types of amino acid selective electrodes to measure various amino acids. Due to its simple, sensitive, non-radiation, and pollution-free characteristics, it has attracted increasing attention. However, in practical applications, the problems of fewer types of amino acids with direct electrochemical activity, poor reversibility of electrochemical reactions, large background interference, and non-chemically active amino acids still need to be derivatized. Appropriate reaction system and reaction conditions have become the key to the research of this method. Amino acid electrochemical analysis can be divided into direct electrochemical analysis and indirect electrochemical analysis. 3.1. Direct electrochemical analysis The above-mentioned amino acids can be directly measured using the electrical activity exhibited when some types of amino acids undergo an oxidation reaction on the electrode. It has been reported that H J can be used to determine the content of tryptophan, tyrosine, cysteine, cysteine and cysteine by direct electrochemical analysis without derivatization. 3.2. Indirect electrochemical analysis (1) Indirect determination of derivatization reaction Most types of amino acids are non-electroactive on metal electrodes and cannot be measured directly. However, the indirect determination can be performed after the above-mentioned amino acid is converted into an electrically active substance by a derivatization reaction. For example, after non-electrically active lysine is derivatized with an aromatic aldehyde, the derivatized product is at a peak potential of 1.12 V at a mercury drop electrode at about 0.3 mol / L in a phosphate buffer solution (pH = 11). A sensitive adsorption reduction wave is generated on the surface, and the derivative wave height has a good linear relationship with the lysine concentration. The detection limit is 3 x 10 mol / LL. (2) Indirect determination of coordination reaction between amino acid and metal ion Amino acid as a ligand can coordinate with a variety of metal ions, and the formed electroactive complex can improve the detection sensitivity of amino acid itself. Gilbert et al. Used Co to catalyze the production of hydrogen waves by cysteine to determine cystine and cysteine. The detection limits were 5 × 10 mol / L and 5 × 10 ～ mol / L. (3) Electrochemiluminescence analysis The principle of electrochemiluminescence analysis is that a substance with electrochemiluminescence activity can interact with a sensitizing reducing substance present in solution under a certain potential condition, and the unstable excited state returned to the ground state can produce a chemical Luminescence, the luminous intensity has a fixed proportional relationship with the content of the test object. Under alkaline conditions, amino acids can significantly enhance the sensitivity of sodium hypochlorite-luminol chemiluminescence system. Lang Huiyun et al. established a new method for the determination of amino acids by reversed-phase flow injection-chemiluminescence based on this principle. This method does not require derivatization or conversion of amino acids, the detection limit is 0.016 g / mL, the sampling frequency is 150 times / h, and the RSD of lg / mL histidine is measured in 12 consecutive parallel runs of 0.9. %.
Capillary electrophoresis uses a high-voltage electric field as the driving force and a capillary as the separation channel to achieve component separation based on differences in mobility and distribution behavior between components in a sample. High speed and low solvent consumption are especially suitable for chiral separation of amino acids. Zhai Haiyun et al. used reversed-phase capillary electrophoresis mode and added a surfactant to the buffer solution to redirect the electroosmotic flow. Without the need for derivatization, rapid separation of mixed amino acids was achieved. To be continued in Part Two… Like it? Share it! More by this author |
