Chiral Analysis of Aromatic Amino Acids with Agilent InfinityLab Poroshell 120 Chiral-T Columns

Significance of the Topic

Chiral separation of aromatic amino acids such as tryptophan, phenylalanine, and tyrosine is critical for pharmaceutical and biotechnological applications. Enantiomeric purity influences drug efficacy and safety, and reliable analytical methods are essential for quality control and research.

Objectives and Study Overview

This study evaluates the performance of the Agilent InfinityLab Poroshell 120 Chiral-T column for baseline separation of underivatized aromatic amino acid enantiomers using a methanol/ammonium formate buffer mobile phase. Detection was carried out by evaporative light scattering (ELSD) and diode array UV detectors to compare sensitivity and selectivity.

Methodology and Instrumentation

The chromatographic system was based on an Agilent 1290 Infinity II LC configured for low dispersion. Key method parameters included:

• Column: Agilent InfinityLab Poroshell 120 Chiral-T, 2.1 × 100 mm, 2.7 µm
• Mobile phase: 70:30 methanol/25 mM ammonium formate buffer (pH 3.0)
• Flow rate: 0.21 mL/min; column temperature: 30 °C; injection volume: 1 µL
• Sample concentration: 2 mg/mL (1 mg/mL each enantiomer); tyrosine at half concentration due to solubility
• Detection: ELSD (evaporator 30 °C, nebulizer 30 °C, gas flow 1 SLM) and DAD at 220 nm and at each compound’s UV maximum

Main Results and Discussion

All three aromatic amino acid pairs were baseline-separated in under four minutes. Chromatographic metrics (Table 3) showed resolution (Rs) values of 2.58–3.02 and selectivity factors of 1.45–1.50. Superficially porous particles delivered high efficiency at low backpressure, enabling rapid analysis without compromising peak shape. ELSD provided near-universal response, while UV detection at specific wavelengths offered enhanced discrimination.

Benefits and Practical Applications

The described method is robust, fast, and compatible with mass spectrometry-friendly solvents. It integrates seamlessly into laboratories accustomed to reversed-phase LC workflows and supports both universal and selective detection modes. Applications include enantiomeric purity testing in pharmaceutical research, quality control of nutritional supplements, and broader chiral analyses in industrial biochemistry.

Future Trends and Potential Applications

Advances may include extending chiral Poroshell column chemistries to additional amino acids and small molecules, coupling with high-resolution mass spectrometry for structural confirmation, and optimizing mobile phases for ultra-high throughput. Emerging superficially porous particle technologies could further reduce analysis times and expand the range of compatible chiral selectors.

Conclusion

The Agilent InfinityLab Poroshell 120 Chiral-T column provides a reliable and efficient platform for the rapid separation of aromatic amino acid enantiomers. The method balances speed, resolution, and versatility, making it well suited for pharmaceutical, biopharma, and industrial analytical workflows.