The Development History of Five Amino Acid Analysis Technologies (Part One)

Amino acid analysis is one of the most important analytical techniques since it plays an important role in research in the fields of life sciences, food science, and clinical medicine. The research and improvement of amino acid analysis methods will continue to be highly valued.

Moore et al. (in 1958) first proposed a method combining cation exchange chromatography with post-column ninhydrin derivatization to analyze the amino acids produced by proteolysis, and realized the automation of amino acid analysis. Since then, traditional amino acid analysis has continued to improve with derivatization methods, column carrier chemistry, sensitivity, and automation.

In recent years, more and more attention has been paid to the research and improvement of amino acid analysis methods. The coexistence and complementarity of multiple current amino acid analysis methods has increased the sensitivity, accuracy, and speed of amino acid analysis to the degree of automation of instruments and equipment to a whole new level, forming a modern amino acid analysis technology with wide adaptability. Here in this article, five methods will be discussed.

First, paper chromatography and thin layer chromatography (TLC)

From the 1940s to the 1950s, the analysis of amino acids by paper chromatography was rapidly developed. Because of its advantages such as simple operation, high separation efficiency, and low cost of required equipment, it was widely used at that time. Paper chromatography uses different chromatographic solutions and color reagents for detection, which can analyze more than 30 amino acids and their derivatives. Thin layer chromatography (TLC) belongs to solid-liquid adsorption chromatography. TLC is an analytical method developed in the 1960s. This method is characterized by convenient operation, simple equipment, fast chromatographic speed, and high sensitivity. It is widely used for the separation and determination of amino acids, and can analyze up to 60 kinds of amino acids and their derivatives. The resolution of thin-layer chromatography is generally 10 to 100 times higher than that of paper chromatography. It can not only separate tens of micrograms of samples, but also can be used to separate and prepare samples larger than 500 mg. For quantitative analysis, it is also possible to develop a color using a corrosive developer such as concentrated sulfuric acid and concentrated hydrochloric acid. Therefore, thin-layer chromatography is more widely used than paper chromatography. With the increasingly active research of amino acid analysis and the rapid development of analytical instruments, and its ability to meet the requirements of complex samples for high sensitivity, short analysis time, and good reproducibility, the application of paper chromatography and thin layer chromatography for amino acid analysis is decreasing.

Second, gas chromatography analysis (GC)

In the 1950s and 1960s, with the development and popularization of gas chromatography (GC), it began to be widely used in the analysis of amino acids. Because gas chromatography is a high-performance, high-selectivity, high-sensitivity, simple-to-operate, and widely used analysis and separation method, and it is easy to use with mass spectrometry to determine the structure of amino acids, it is possible to discover new amino acids. In recent years, amino acid analysis technology has been widely studied in biochemistry, medicine, and food. However, amino acids are not easy to gasify. To analyze amino acids by gas chromatography, the amino acids must first be converted to derivatives that are easily gasified, and then appropriate columns and detectors must be selected for separation and determination. Gas chromatography (GC) has been used for a long time to determine the derivatized amino acids. At present, the better gas chromatographic derivatization method is derivatization of amino acids into N-trifluoroacetyl-n-butyl ester and N-heptafluorobutyryl isobutyl ester or tert-butylsilyl derivative.

HusekN reported that gas chromatography determination of ethyl chloroformate-derived amino acids can be completed within 5 minutes from sample derivation to sample separation.

Gas chromatography–mass spectrometry (GC-MS) is also a powerful method for the analysis of amino acids, especially in the analysis of the enantiomers of amino acids. Although the capillary column of gas chromatography has high separation efficiency and high sensitivity, the conditions for derivatization of amino acids by gas chromatography are relatively harsh and poor in specificity. At the same time, due to the development of other analytical instruments, gas chromatography has been used in the actual analysis and determination of amino acids. It is not widely used in applications.

Third, high-performance liquid chromatography (HPLC) analysis

Since the 1980s, research on the determination of amino acids by high-performance liquid chromatography (HPLC) has become increasingly active, especially in the past 20 years. Pre-column-derived high-performance liquid chromatography analysis of amino acids is a rapidly developing separation and analysis technique. Analytical technology combining high-performance liquid chromatography with various amino acid derivatization methods has developed rapidly and has gradually shown its unique advantages. It has the advantages of fast analysis speed, good accuracy, high sensitivity, short analysis time, and wide applicability. It provides broad prospects for amino acid analysis. HPLC requires the conversion of amino acids before the column into derivatives suitable for reversed-phase chromatography and sensitive detection. The key to pre-column derivatization is the selection of derivatization reagents. The criterion for selecting derivatization reagents is to be able to react quantitatively with each amino acid. Each amino acid generates only one compound and the product has a certain stability. It does not produce or easily eliminate interferences; simple operation. The chromatographic resolution is high, the detection sensitivity is high, and the analysis time is short, which is convenient for automation and enables products to be measured on different types of high-performance liquid chromatography. At present, there have been a large number of reports on the study of amino acid derivatization methods. Representative pre-column derivatization reagents for amino acid analysis are: o-phthalaldehyde (OPA) fl1, phenyl isothiocyanate (PITC), Ethyl methyl chloroformate (FMOC-C1) dimethylaminonaphthalenesulfonyl chloride (Dansyl ～ C1) (tetra), 2,4-dinitrofluorobenzene (DNFB), 6-aminoquinolinyl-N- Pre-column derivatization HPLC analysis of amino acids such as hydroxysuccinimide carbamate (AQC), sulfonyl chloride dimethylamine azobenzene (Dabsyl-C1), etc. It has unique advantages: high sensitivity, short analysis time and good repeatability and so on. In actual work, the specific method should be selected based on indicators such as the speed and simplicity of derivatization, the speed of analysis, the ability to detect secondary amino acids and cystine, interference factors, sensitivity, and repeatability.

To be continued in Part Two…