Analysis of Amino Acids Using Automatic Pretreatment Function of LC System

Importance of the Topic

The precise quantification of proteinogenic amino acids is essential across fields such as biochemistry, food analysis, clinical diagnostics and pharmaceutical quality control. Automated pre‐column derivatization combined with high‐performance liquid chromatography (HPLC) streamlines sample handling, enhances reproducibility and reduces operator error, facilitating high‐throughput routine analysis.

Objectives and Overview

This application note describes a method for the simultaneous separation and quantification of 20 primary and secondary amino acids using an automated derivatization module and reversed‐phase HPLC on Shimadzu’s Prominence™‐i system. The goal is to demonstrate efficient and reliable amino acid profiling with minimal manual intervention.

Methodology and Instrumentation

Instrumentation Used

• HPLC System: Prominence™‐i (LC‐2030C)
• Column: Shim‐pack™ XR‐ODSII, 100 mm × 3.0 mm, 2.2 μm
• Detector: Fluorescence, channels Ex.350/Em.450 nm and Ex.266/Em.305 nm

Chromatographic Conditions

A low‐pressure gradient elution was employed using three mobile phases: (A) 20 mM sodium acetate buffer pH 6; (B) water/acetonitrile 10/90 (v/v); (C) 20 mM sodium acetate buffer pH 5 with 0.5 mM EDTA-2Na. Flow rate was 1.0 mL/min, column temperature 40 °C and injection volume 1 μL. The 17-minute gradient achieved baseline separation of all 20 amino acids.

Derivatization Protocol

Automated pre‐column derivatization employed:

• Mercaptopropionic acid reagent in borate buffer
• o-Phthalaldehyde (OPA) reagent in ethanol/borate buffer
• FMOC reagent in acetonitrile for secondary amines

Equal volumes of mercaptopropionic acid and OPA reagents were mixed in the autosampler, followed by reaction with sample prior to injection.

Key Results and Discussion

The method resolved all 20 proteinogenic amino acids within a single run with sharp peaks and minimal carryover. Fluorometric detection provided high sensitivity for both primary and secondary amino acids. The automatic pretreatment module ensured consistent derivatization yields and reduced sample‐to‐sample variability.

Benefits and Practical Applications

• High throughput: full panel in under 20 min
• Enhanced reproducibility via automated derivatization
• Sensitive detection suitable for trace analysis
• Reduced manual labor and error risk

Future Trends and Potential Applications

Advances may include integration with mass spectrometric detection for structural confirmation, miniaturized flow cells for lower solvent consumption, and AI-driven data analysis for rapid quantitation in complex matrices such as fermented foods, blood plasma and bioprocess monitoring.

Conclusion

This study demonstrates a robust, fully automated HPLC method for comprehensive amino acid profiling. The combination of automated derivatization and Shimadzu’s XR-ODSII column delivers rapid, reproducible and sensitive analysis, supporting diverse applications from research to routine quality control.

Reference

Application News L529 (JP, ENG), Shimadzu Corporation, First Edition Dec. 2021.