A Full Introduction to Amino Acid Analysis Methods (Part

One)

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.

1. Chemical analysis

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.

2. Spectral analysis method

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.

3. Electrochemical analysis

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. %.

4. Capillary electrophoresis (CE)

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…