Amino acids play a crucial role as essential building blocks in the composition of living organisms. They actively participate in and regulate metabolic processes within the body, significantly influencing the growth and development of all living organisms. Therefore, the detection of amino acids holds paramount significance in nutritional food research and assessing the nutritional status of organisms.
Various methods, such as spectrophotometry, liquid chromatography, gas chromatography, and amino acid analyzers, can be applied for amino acid detection. However, each of these methods has its limitations, including low sensitivity, complexity in operation, and high operational costs, which, to some extent, restrict their application in amino acid detection.
What is Capillary Electrophoresis (CE) Technology?
Capillary electrophoresis (CE) has emerged as an efficient separation technique driven by an electric field, developed in the 1980s. This method offers advantages such as low cost, minimal environmental impact, high efficiency, and simplicity in operation. As a result, capillary electrophoresis has become a hot topic in amino acid analysis, particularly in fields such as food safety, biotechnology, and medical diagnostics.
Capillary electrophoresis in conjunction with various detectors, facilitates qualitative and quantitative analysis of each amino acid. Commonly used detectors in capillary electrophoresis include ultraviolet detectors (UV), laser-induced fluorescence detectors (LIF), mass spectrometric detectors (MS), and electrochemical detectors (EC).
Electropherogram showing separation of the PTC-amino acid (Ren et al., 2012)
Capillary Electrophoresis with Ultraviolet Detection (CE-UV)
CE-UV is a technique used for the qualitative and quantitative analysis of amino acids based on their ultraviolet absorption properties. Some amino acids exhibit weak intrinsic UV absorption. To enhance analysis sensitivity, it is necessary to add background electrolyte (buffer solution) and derivatization agents to the sample solution. Therefore, capillary electrophoresis with ultraviolet amino acid analysis methods includes both direct UV detection and derivatization-based UV detection.
Direct UV Detection Method:
The direct UV detection method requires the addition of a buffer solution to the sample solution to create a stable electroosmotic flow system. According to research, the movement direction and stability of particles on the inner surface of the capillary column, as well as the movement direction and stability of the amino acids to be separated, are influenced by the pH value. Therefore, by controlling the pH of the buffer solution, effective separation of amino acids in different samples can be achieved.
Derivatization-Based Ultraviolet Detection Method:
The derivatization-based ultraviolet detection method aims to enhance the sensitivity of amino acid detection by introducing a chromophore into the solution. This chromophore intensifies the ultraviolet absorption of amino acids, thereby improving the performance of the ultraviolet detector. The selection of a suitable derivatization agent is crucial, emphasizing minimal by-product generation and high selectivity. Commonly employed derivatization agents for this method include ortho-phthaldialdehyde (OPA), chloroformate-9-fluorenylmethyl ester (FMOC-Cl), fluorescein isothiocyanate (FITC), and naphthalene-2,3-dicarboxaldehyde (NDA).
Capillary Electrophoresis-Mass Spectrometry (CE-MS)
Capillary Electrophoresis-Mass Spectrometry (CE-MS) combines capillary electrophoresis separation with mass spectrometry to achieve qualitative and quantitative analysis of amino acids. This technique offers high sensitivity, accuracy in quantitative analysis, and minimal interference. Researchers have successfully applied CE-MS to analyze amino acids in the urine of gastric cancer patients, providing accurate insights into health conditions. It has also been utilized to detect non-protein amino acids in olive oil, enabling the analysis of adulteration. In forensic investigations, CE-MS enhances the accuracy of amino acid analysis in fingerprint residues and improves case resolution. To address sample dilution issues at the interface between capillary electrophoresis and mass spectrometry, an electrospray ionization device (CE-ESI-MS) has been introduced. Experimental evidence indicates that CE-ESI-MS is cost-effective, detecting a broader range of amino acids compared to CE-MS.
Capillary Electrophoresis-Fluorescence Detection (CE-LIF)
Capillary Electrophoresis-Fluorescence Detection (CE-LIF) requires fluorescence-absorbing amino acid samples, necessitating derivatization of the samples. CE-LIF offers extremely high sensitivity and requires minimal sample injection, making it particularly valuable in disease diagnosis, assessment, and monitoring response to drug therapy. The success of CE-LIF relies on careful selection of derivatization agents, separation conditions, and detection conditions.
Capillary Electrophoresis-Electrochemical Detection (CE-EC)
Capillary Electrophoresis-Electrochemical Detection (CE-EC) directly detects non-derivatized amino acids, demonstrating excellent selectivity and cost-effectiveness. This method is commonly employed in the analysis of clinical samples like physiological fluids. Experimenters often optimize CE-EC by modifying working electrodes, choosing materials such as carbon, copper, gold, and platinum. The selection of working electrodes should consider having a specific response to the analyte, resistance to liquid contamination, and cost-effectiveness.
Application of Capillary Electrophoresis in Chiral Amino Acid Separation
In recent years, the separation and detection of chiral amino acids have emerged as both a research focus and a challenging endeavor. This is attributed to the distinct metabolic roles of L- and D-amino acids (chiral amino acids) in biological systems and their divergent implications in pharmaceutical applications. Capillary electrophoresis technology, known for its advantages of minimal sample requirement, high efficiency, and time efficiency, has found extensive use in the separation and detection of chiral amino acids.
The separation of chiral amino acids is achieved by altering their optical rotation, primarily explored in pharmaceutical preparation and clinical sample analysis. Commonly employed chiral separation agents include cyclodextrins, metal complexes, macrocyclic antibiotics, and crown ethers.
Researchers have adopted various chiral separation agents to enhance the chemical selectivity of the method. Contemporary research often involves the addition of derivatizing agents and appropriate background electrolytes to the sample solution. This approach prioritizes chemical reactions between the chiral agent and the functional groups of amino acids, thereby improving the chemical selectivity of the chiral agent.
In a notable experiment, β-cyclodextrin was selected as the chiral reagent, economically viable 9-fluorenylmethyl chloroformate (FMOC) as the derivatizing agent, and sodium dodecyl sulfate (SDS) with isopropanol and boric acid as background electrolytes. This combination successfully separated L- and D-amino acids, demonstrating the desired outcomes. The experiment underscored the substantial assistance provided by SDS in enhancing the chemical selectivity of the chiral agent.
In the realm of capillary electrophoresis chiral separation techniques, the careful selection of background electrolytes, derivatizing agents, and separation conditions is paramount. This is particularly crucial when opting for cyclodextrins as chiral separation agents, where temperature control plays a pivotal role; typically, higher temperatures can weaken the binding forces between the chiral separation agent and amino acids.
Reference
- Ren, Yanli, et al. "Analysis of free amino acids during fermentation by Bacillus subtilis using capillary electrophoresis." Biotechnology and Bioprocess Engineering 17 (2012): 1244-1251.
