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

Significance of the Topic

The separation of chiral amino acids is critical in pharmaceutical development, nutritional analysis, and quality control. Sulfur-containing amino acids such as cysteine and methionine exist as enantiomeric pairs with distinct biological activities. Reliable, high-throughput methods for resolving these enantiomers support drug safety assessments, metabolic studies, and industrial processes.

Objectives and Study Overview

This study evaluates the performance of an Agilent InfinityLab Poroshell 120 Chiral-T column for the enantioselective separation of underivatized cysteine and methionine. The goals were to achieve baseline resolution in a short analysis time using a mass spectrometry-compatible mobile phase and to demonstrate robust, reproducible performance on a superficially porous particle column.

Methodology

Sample Preparation

• Individual D- and L-enantiomers prepared at 2 mg/mL in water and mixed 1:1 to 1 mg/mL each.

Chromatographic Conditions

• Column: 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.

Instrumentation Used

Liquid chromatography system:

• Agilent 1290 Infinity II LC with low-dispersion connections.
• Flexible Pump, Infinity Autosampler, Multicolumn Thermostat.
• Evaporative Light Scattering Detector (ELSD) at 30 °C evaporator and nebulizer, 1 SLM gas flow, 40 Hz signal acquisition.
• OpenLab CDS software for data acquisition.

Main Results and Discussion

Both cysteine and methionine enantiomer pairs were baseline-separated in under four minutes. Key performance metrics:

• Cysteine: k1=0.86, k2=1.50, resolution Rs=4.31, selectivity α=1.75.
• Methionine: k1=0.84, k2=1.76, Rs=5.58, α=2.08.

The superficially porous particles enabled high efficiency at moderate backpressure, yielding sharp peaks and reproducible retention times. The glycopeptide stationary phase demonstrated stability in a reversed-phase-compatible mobile phase, facilitating MS-friendly analysis.

Benefits and Practical Applications

Rapid, robust enantioseparation under common reversed-phase conditions allows integration into existing LC workflows. The method supports:

• Pharmaceutical enantiopurity testing.
• Metabolite profiling in biopharma research.
• Quality control of nutritional supplements.

Future Trends and Potential Applications

Advances in superficially porous chiral phases may extend to other amino acids and small biomolecules. Potential developments include:

• Coupling with high-resolution mass spectrometry for structural elucidation.
• Expansion to supercritical fluid chromatography (SFC) for green analysis.
• Miniaturized or high-throughput platforms for screening applications.

Conclusion

The Agilent InfinityLab Poroshell 120 Chiral-T column delivers fast, high-resolution separation of sulfur-containing amino acid enantiomers using an MS-compatible mobile phase. Its robust performance and compatibility with standard LC infrastructure make it well suited for analytical laboratories in pharma, biopharma, and food science.

References

• Agilent Technologies Application Note 5994-1381EN, Chiral Analysis of Sulfur Amino Acids with Agilent InfinityLab Poroshell 120 Chiral-T Columns, 2019.