High-speed analysis of amino acids

Importance of the Topic

Rapid and accurate analysis of proteinogenic amino acids is critical in fields such as biochemistry, food science, clinical diagnostics, and pharmaceutical quality control. High-throughput methods with reliable quantification support metabolic studies, nutritional profiling, and therapeutic monitoring.

Study Objectives and Overview

This application note describes the development of a high-speed HPLC method for the simultaneous separation and quantification of 20 amino acids using Shim-pack XR-ODSII combined with an automated pre-column derivatization system. The aim is to demonstrate efficient separation, sensitive detection, and streamlined sample preparation.

Methodology

• Automated pre-column derivatization to label amino acids prior to injection, improving reproducibility and reducing manual steps
• Low-pressure gradient mode with three mobile phases: (A) 20 mmol/L sodium acetate buffer (pH 6), (B) water/acetonitrile (10/90, v/v), and (C) 20 mmol/L sodium acetate buffer (pH 5) with 0.5 mmol/L EDTA-2Na
• Flow rate of 1.0 mL/min, column temperature at 40 °C, sample cooler maintained at 4 °C, and a 1 µL injection volume
• Dual-channel fluorescence detection: Channel 1 (Ex 350 nm/Em 450 nm) and Channel 2 (Ex 266 nm/Em 305 nm)

Instrumentation Used

• Shim-pack XR-ODSII column (100 mm × 3.0 mm I.D., 2.2 µm particle size)
• HPLC system equipped with an automated pretreatment (derivatization) unit
• Fluorescence detector with temperature-controlled cell and two detection channels

Main Results and Discussion

The method achieved baseline separation of all 20 standard amino acids in a single run with sharp, well-resolved peaks. Automated derivatization enhanced run-to-run reproducibility and significantly reduced sample handling time. Dual fluorescence channels provided complementary sensitivity, ensuring accurate quantification even at low concentrations.

Benefits and Practical Applications

• High throughput and reproducibility suitable for routine laboratory workflows
• Minimal manual intervention through automation, reducing potential for human error
• Applicable to various sectors including biochemical research, food and beverage analysis, clinical diagnostics, and QA/QC in pharmaceuticals

Future Trends and Potential Applications

Further developments in ultra-high-performance columns and automated sample preparation are expected to decrease analysis times even more. Coupling this workflow with mass spectrometry could extend applicability to non-derivatized compounds and broader metabolomic profiling. Integration of AI-based data analysis tools may streamline peak identification and quantification.

Conclusion

The combination of a high-speed Shim-pack XR-ODSII column and automated pre-column derivatization provides a robust and efficient solution for comprehensive amino acid analysis. The method delivers reliable, rapid results suitable for diverse analytical challenges.

References

• Application News an_01-00441, Shimadzu Corporation (2023)