Capillary electrophoresis technology is applied in the chiral separation of L-valine

L-valine, an essential branched-chain amino acid in humans, exhibits significant differences in safety and biological activity from its enantiomer (D-valine) in the food and pharmaceutical fields (e.g., D-valine may interfere with human metabolism). Therefore, achieving efficient chiral separation of the two is of great significance. Capillary electrophoresis (CE) has become an important technique for chiral amino acid separation due to its advantages of high separation efficiency, fast analysis speed, and low reagent consumption. Advances in its application to the chiral separation of L-valine mainly focus on innovations in chiral selectors, optimization of separation modes, and expansion of practical sample analysis, as detailed below:

I. Types and Applications of Chiral Selectors

Chiral selectors are the core of chiral separation in CE. They form diastereomeric complexes with L- and D-valine through hydrogen bonding, hydrophobic interactions, electrostatic attraction, etc., resulting in differences in migration rates. Currently, selectors used for chiral separation of L-valine mainly include the following categories:

Cyclodextrins and their derivatives:

Cyclodextrins (CDs) are the most commonly used chiral selectors. Their hydrophobic cavities can form inclusion interactions with the isopropyl side chain of valine, while the hydroxyl groups at the ports interact with amino and carboxyl groups via hydrogen bonding. β-cyclodextrin (β-CD) exhibits better separation efficiency for valine enantiomers than α- or γ-CD, but its resolution is limited. Modification via methylation or hydroxypropylation (e.g., hydroxypropyl-β-CD, HP-β-CD) can enhance water solubility and enantiomer recognition ability. When the concentration of HP-β-CD is 10–20 mmol/L, the resolution (Rs) of L- and D-valine can reach over 2.5. In addition, sulfobutyl ether-β-CD (SBE-β-CD), which carries a negative charge, can generate electrostatic interactions with positively charged valine (under acidic conditions), further improving separation selectivity, especially suitable for separating valine in complex matrices.

Crown ether compounds:

18-crown-6-tetracarboxylic acid (18C6TCA), as a charged crown ether, can bind to the amino group of valine through its cavity, while carboxyl groups form ion pairs with amino groups, showing excellent chiral recognition ability for aliphatic amino acids. In phosphate buffer at pH 2.5–3.0, when the concentration of 18C6TCA is 3–5 mmol/L, the migration time difference between L-valine and D-valine can exceed 1.5 minutes, with minimal interference from other amino acids (e.g., L-leucine).

Surfactant micelles:

In micellar electrokinetic chromatography (MEKC) mode, sodium dodecyl sulfate (SDS) micelles act as a pseudostationary phase, and separation can be achieved based on differences in hydrophobic interactions with valine enantiomers. Adding chiral additives (e.g., β-CD) to the micellar system to form a "micelle-cyclodextrin" composite selector can enhance chiral recognition through synergistic effects. For example, the combination of SDS (50 mmol/L) and HP-β-CD (15 mmol/L) can increase the resolution of valine enantiomers to over 3.0.

II. Optimization Strategies for Separation Conditions

CE separation efficiency is affected by parameters such as buffer properties, pH, voltage, and temperature. The optimization focus for L-valine includes:

Buffer system:

Phosphate buffer or borate buffer (concentration 20–50 mmol/L) is commonly used. The pH needs to be adjusted according to the charged state of the chiral selector and valine. For example, when using neutral cyclodextrins (e.g., HP-β-CD), a pH of approximately 7.0 allows valine to exist as a zwitterion, achieving separation through hydrogen bonding and hydrophobic interactions with cyclodextrins. If using charged selectors (e.g., SBE-β-CD), the pH should be adjusted to 2.0–4.0 to make valine positively charged, enhancing electrostatic attraction with negatively charged selectors and improving separation efficiency.

Separation voltage and temperature:

Increasing voltage can shorten analysis time, but excessively high voltage causes Joule heating, leading to peak broadening. Typically, 15–20 kV is selected (with a capillary length of 50 cm), and the column temperature is controlled at 25–30°C to reduce the impact of temperature fluctuations on migration time reproducibility (RSD can be controlled within 2%).

Sample pretreatment:

For complex matrices such as food and fermentation broths, valine needs to be enriched via solid-phase extraction (SPE). C18 or ion-exchange solid-phase extraction columns are commonly used to remove interfering substances such as proteins and fats. For samples with low valine concentrations (e.g., plasma samples), field-amplified sample stacking (FASS) can be used to achieve enrichment through conductivity differences between the sample and buffer, with the detection limit reduced to the μmol/L level.

III. Practical Applications and Technical Expansion

The application of CE in chiral separation of L-valine has covered multiple fields:

Food analysis: Used to detect the optical purity of L-valine in fermented foods (e.g., soy sauce, yogurt) to avoid improper accumulation of D-valine. By optimizing HP-β-CD concentration (12 mmol/L) and buffer pH (6.8), separation can be completed within 10 minutes, meeting the demand for rapid detection of batch samples.

Pharmaceutical research and development: In the synthesis of valine derivative drugs (e.g., antiviral drug intermediates), CE can monitor chiral purity to ensure the proportion of L-configuration in the product. Using 18C6TCA as a selector can effectively separate structurally similar chiral impurities with a resolution of 2.8, superior to high-performance liquid chromatography.

Technology coupling: Coupling with mass spectrometry (CE-MS) can improve qualitative capabilities. By selecting volatile buffers (e.g., ammonium formate) and optimizing interfaces, compatibility issues between CE and MS are resolved, enabling high-sensitivity detection of valine enantiomers in complex matrices (with a detection limit as low as 0.1 μmol/L).

Capillary electrophoresis, with its high separation efficiency and flexible chiral selection strategies, has become a key technique for chiral separation of L-valine. Future development will focus on the design of new chiral selectors (e.g., metal-organic frameworks), the development of miniaturized chip CE (shortening analysis time to minutes), and integration with multi-omics technologies, providing more efficient and accurate analytical methods for food quality control and pharmaceutical research.