5. Chromatographic Analysis

5.1. High performance cation exchange chromatography

Spackman et al. used a strong acid cation exchange resin to separate amino acids in a cation exchange column by using the characteristics of amino acids forming cations under acidic conditions. The separated amino acids were derivatized with corresponding reagents (such as ninhydrin), and then appropriate detection methods (such as ultraviolet spectrophotometry) were used. Commercially available automatic amino acid analyzers mostly use high performance cation exchange chromatography with one column of ninhydrin derivative photometric detection technology. This method is accurate, reliable, and reproducible. It can determine most kinds of amino acids and their homologs, but the sensitivity is not high and the instrument is expensive. Yang Xuemeng et al. simultaneously measured the contents of L-valine, L-isoleucine and L-leucine in the amino acid preparation "gan ammonia powder" by using an amino acid analyzer

5.2. High-performance anion exchange chromatography integrated pulse amperometric detection

The carboxyl group in the amino acid molecule can form an anion in a strong alkaline medium, and the amino group in the amino acid molecule can undergo an oxidation reaction on the surface of a precious metal (gold, platinum) electrode in a strong alkaline medium at a certain potential. Anion exchange chromatography for amino acids and integrated pulsed amperometric detection. The advantage of this method is that it does not require derivatization of amino acids, can be directly separated and detected, and has a lower detection limit. Cai Yaqi et al. applied high-performance anion exchange chromatography-integrated pulsed amperometric detection to the analysis and determination of amino acid components in fish meal and corn meal hydrolysates. Dionex AminoPacPA10 anion exchange column was used as the analytical column. The column temperature was controlled at 35 ° C. NaOH and NaAc strong alkaline solution is the eluent. Under the proper gradient conditions, it has realized the efficient separation and highly sensitive detection of 17 common amino acids.

5.3. Gas chromatography (GC)

After derivatizing amino acids into substances that are easy to vaporize, multi-component mixed amino acids can be determined by gas chromatography (GC). The advantages of this method are short separation time, high column efficiency, and easy use with mass spectrometry. The disadvantage is that the derivatization reaction is prone to generate interference components. Husek et al. used gas chromatography to analyze 20 amino acids and 30 non-amino organic acids in plasma simultaneously, and the sample preparation time was less than 30 min.

5.4. High Performance Liquid Chromatography (HPLC)

This method includes two types of pre-column derivatization and post-column derivatization. At present, multi-purpose pre-column derivatization and reversed-phase high-performance liquid chromatography(HPLC) are used to first convert amino acids into derivatives suitable for reverse-phase liquid chromatography separation and can be sensitively detected, and then use HPLC to separate the above-mentioned derivatives. This method is sensitive, fast, widely applicable, and easy to automate. Mu Dehai et al. established a method for the determination of amino acid content in samples by reverse phase high performance liquid chromatography with manual pre-column phthalate (OPA) derivatization. The amino acid composition of bovine serum albumin (BSA) and the free content in mouse serum were determined and satisfactory results were obtained; Miao Hong et al. used pre-column derivatization-high performance liquid chromatography to determine the content of 16 amino acids such as aspartic acid, lysine, and leucine in food.

Pre-column derivatization of amino acids

Since the 1980s, research on the determination of amino acids using high-performance liquid chromatography has developed rapidly, especially the research on pre-column-derived reversed-phase high-performance liquid chromatography (RP-HPLC) has made significant progress. Compared to other method, RP-HPLC analysis method is more sensitive (measurable <lpmol level) and faster (to complete the hydrolysis, separation and determination of a protein only 12-30 minutes), the derivatization reaction is also more convenient, easy to combine with the instrument to achieve commercialization .

RP-HPLC requires that amino acids be converted into derivatives suitable for reversed-phase chromatography and sensitively detected before the column. The key lies in the choice of derivatization reagents. Criteria for the selection of derivatization reagents include: the reagent can quantitatively react with each amino acid; each amino acid generates only one compound; the target product has a certain stability, does not produce or easily exclude interferences; facilitates automation; products can be used in Determination of u on different models of high performance liquid chromatography. At present, the widely used pre-column derivatization reagents are listed in Table 1. The most commonly used pre-column derivatization reagents are o-phthalaldehyde (OPA), phenyl isothiocyanate (PITC), and methyl chloroformate (FMOC— C1), dansyl chloride (Dansyl-C1) and dinitrofluorobenzene (FDNB).

1. Orthophthalaldehyde (OPA) Derivation

The o-phthalaldehyde (OPA) derivatization method was first proposed by Jone B N et al. 201 3. It has the characteristics of simple derivatization steps, fast reaction speed, and the remaining reagents do not interfere with the determination. It is most widely used in pre-column derivatization reagents.

Under the action of the reducing agent p-mercaptoethanol, OPA reacts rapidly with primary amino acids to form thio-2-alkylisoindole. The derivatization reaction can be completed within 13 minutes. Derivatives can be detected by fluorescence after separation by RP-HPLC with sensitivity up to fmol. Chromatography of aspartic acid / serine and valine / methionine derivatives when D-mercaptoethanol is used as reducing agent resolution is poor. It is now replaced with 3-mercaptopropionic acid (3-MPA). By introducing a carboxyl group, the hydrophobicity of the above two pairs of amino acid derivatives is relatively weakened, and acetonitrile is used instead of methanol as the mobile phase, and the gradient is optimized. The program makes it completely separate.

There are still some shortcomings in the pre-column derivatization reagent itself. First, the OPA reagent itself is easily oxidized and degraded by oxygen in the air (from colorless and transparent to yellowish), and the reagent is difficult to store. Combining OPA with 0-mercaptoethanol as a derivatization reagent can partially solve this problem. The above reagents can be stored stably for 1 week under dark conditions at 40 ° C. Second, the derivatized products of OPA and amino acids are also unstable, and injection analysis is required immediately after derivatization. To solve this problem, the method adopted in practice is to add borate buffer and reduce and fix the time from the derivatization operation to the completion of the manual injection.

2. Derivative method of phenyl isothiocyanate (PITC)

PITC can react with primary and secondary amino acids to form phenylaminothioformyl derivatives (PTC). The reaction (Figure 2) only needs to be performed at room temperature for 20 min, and then completed by two-step rapid evaporation.

The derivative of this reaction is single and stable. After being separated by RP-HPLC, it is detected at 254nm with a UV detector. At present, this method has been extended to the analysis of modified amino acids such as phosphorous amino acids and sulfur amino acids.

However, this method also has the following disadvantages: vacuum drying to remove excess derivatization reagents during amino acid derivatization makes it difficult to automate the operation of the PIT C method; PITC is highly toxic and requires a special derivatization device to be performed under anhydrous conditions; PITC It will reduce the service life of the analytical column. The ordinary C column has poor durability. After a period of use, the separation effect decreases rapidly, and it is difficult to improve by adjusting the mobile phase gradient.

3. 9 Derivatives of fluorene methyl monochloroformate (FMOC-C1)

9 Methyl monochloroformate is an amino protective agent for peptide synthesis. It can form stable derivatives with primary and secondary amino acids at a pH of 7.7, and its fluorescence detection sensitivity can reach the level of fmo1. FMOC-C1 reacts quickly with amino acids and can be completed in about 30s at room temperature.

The biggest advantage of this method is that the emission wavelength of the derivative is fixed and there is no endogenous interference. In addition, in the presence of piperidine and DMF, FMOC-C1 can easily fall off the amino group and obtain the original amino acid, which is beneficial to the further analysis of amino acids.

The disadvantage of this method is that FMOC-Cl and its hydrolyzed product, FMOC-OH, have similar fluorescence phenomena as FMOC-Cl amino acid derivatives. Although interference can be removed with pentane extraction, the extraction efficiency is only about 70% per step, which can only partially solve the problem.

To be continued in Part Three…