Separation of derivatised D-/L-Amino acids

Significance of the Topic

The precise separation of D- and L-amino acid enantiomers is essential in pharmaceutical quality control, food analysis and biochemical research. Enantiomeric purity influences drug efficacy, safety and metabolic profiling. Robust analytical methods enable accurate quantification of chiral amino acids in complex matrices.

Study Objectives and Overview

This application note describes a reversed-phase HPLC method for baseline separation of 18 D- and L-amino acids derivatized with N-α-(5-fluoro-2,4-dinitrophenyl)-L-valinamide (L-FDVA). The goal is to achieve high resolution, reproducibility and sensitivity for routine enantioselective analyses.

Methodology

A racemic mixture of amino acids was reacted with L-FDVA to form chromophoric diastereomers. Separation was performed on a Eurospher II 100-3 C18 column (250 × 3.0 mm i.d.) under the following conditions:

• Mobile phases: A – acetonitrile/methanol (2:1) with 0.1% TFA; B – water with 0.1% TFA
• Gradient program:
  
  • 0–35 min: 75% B
  • 35–55 min: 75%→60% B
  • 55–70 min: 60%→50% B
  • 70–90 min: 50%→40% B
  • 90–100 min: 40% B
  • 100–105 min: 40%→30% B
  • 105–115 min: 30% B
• Flow rate: 1.0 mL/min
• Column temperature: 25 °C
• Injection volume: 25 µL

Instrumentation Used

The system employed an HPLC configured in reverse-phase mode (Method VBS0028J) equipped with a photodiode array detector. Detection wavelengths were set at 260, 330, 340 and 410 nm (10 Hz) to capture the strong absorbance of the FDVA derivatives.

Main Results and Discussion

The optimized gradient and column selection achieved baseline resolution for all 18 amino acid enantiomer pairs within a 115-minute run. Chromatographic peaks exhibited sharp profiles and resolution values above 1.5. Derivatization with L-FDVA provided enhanced UV response, facilitating sensitive quantitation even at low micromolar concentrations.

Benefits and Practical Applications

This method delivers a reliable platform for enantioselective amino acid analysis in pharmaceutical development, food authenticity testing and metabolomic studies. The use of a robust C18 column and reproducible gradient ensures consistent performance for routine quality control workflows.

Future Trends and Applications

Emerging column technologies, such as sub-2 µm particles and core-shell phases, may shorten run times and further improve resolution. Automation of derivatization and integration with MS detection can expand throughput and sensitivity for high-volume laboratories.

Conclusion

The described reversed-phase HPLC method with FDVA derivatization provides a reproducible and sensitive approach for separating D-/L-amino acids. It fulfills industry requirements for enantiomeric purity assessment and offers a foundation for future method enhancements.