High-Speed Amino Acid Analysis (AAA) on 1.8 μm Reversed-Phase (RP) Columns

Significance of the Topic

Rapid and accurate amino acid analysis is essential for protein characterization, nutritional profiling of foods, and monitoring cell culture media. Advances in chromatography and derivatization chemistry enable high sensitivity and throughput needed in modern analytical laboratories.

Objectives and Study Overview

This study presents a fast and robust methodology for amino acid analysis (AAA) using subtwo-micron reversedphase columns and automated OPA/FMOC derivatization. The goal was to reduce analysis time, enhance sensitivity for limited samples, and maintain excellent reproducibility.

Methodology and Instrumentation

The method employs sequential precolumn derivatization: primary amino acids react with ophthalaldehyde (OPA), followed by secondary amines labeled with 9fluorenylmethyl chloroformate (FMOC). Separation is achieved on Agilent ZORBAX Rapid Resolution HT Eclipse Plus C18 columns (1.8 µm, 2.1, 3.0 or 4.6 × 50 mm) using an Agilent 1200SL HPLC system comprising a binary pump, degasser, wellplate autosampler, column compartment, diode array detector, and fluorescence detector. Mobile phase A is a borate buffer at pH 8.2, and mobile phase B is acetonitrile/methanol/water (45:45:10).

Main Results and Discussion

The optimized protocol achieved a full AAA cycle in approximately 13.5 minutes, more than halving previous runtimes. Peak shapes for early eluters improved, and reproducibility of primary and secondary amino acids reached average RSDs below 2 %. Linearity across the calibration range yielded r2 values > 0.99. Scalability tests on different column IDs showed consistent retention profiles with adjusted flow rates. Analysis of beer samples demonstrated the method’s practical application in comparing amino acid content across varieties.

Contributions and Practical Applications

• High throughput: allowing double the sample output in the same time
• Enhanced sensitivity: femtomole detection suited for scarce samples
• Robust automation: reduces manual intervention for QA/QC and research
• Flexible configuration: method readily adapts to various column formats

Future Trends and Opportunities

Integration with process analytical technology (PAT) platforms and coupling to mass spectrometry will further expand applications in bioprocess monitoring. Continued column chemistry development may enable even faster separations and broader analyte coverage, including modified peptides and nonproteinogenic amino acids.

Conclusion

The combined use of 1.8 µm reversed-phase columns and automated OPA/FMOC derivatization delivers a fast, sensitive, and reproducible amino acid analysis method. Its flexibility and performance make it a valuable tool in proteomics, food analysis, and biopharmaceutical quality control.

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