Agilent ZORBAX Eclipse AAA Instructions for Use

Importance of Topic

Amino acid analysis plays a crucial role in protein characterization, nutritional studies, clinical diagnostics, and quality control in biotechnology and pharmaceutical industries. High sensitivity and resolution are essential to detect both primary and secondary amino acids and address complex sample matrices. Automated online derivatization by OPA and FMOC combined with robust HPLC separation streamlines workflows and enhances reproducibility.

Objectives and Study Overview

This work describes a validated protocol using the Agilent ZORBAX Eclipse AAA column in conjunction with Agilent 1100/1200 HPLC systems for high-throughput routine analysis and high-resolution separation of 24 common amino acids. It includes development of low- and high-sensitivity standard preparations, optimization of gradient conditions, detector settings, and strategies for wavelength switching to differentiate OPA- and FMOC-derivatized compounds.

Methodology and Instrumentation

• Derivatization: Online pre-column reaction with OPA for primary amino acids and FMOC for secondary amino acids
• HPLC System: Agilent 1100/1200 with binary/quaternary pump, DAD (338 nm for OPA, 262 nm for FMOC) and FLD (Ex/Em 340/450 nm initial, switching to 266/305 nm)
• Column Configurations: 4.6×75 mm, 3.5 µm for high throughput; 4.6×150 mm, 3.5 µm and 5 µm for high resolution; optional 3.0×150 mm, 3.5 µm for specialized applications
• Mobile Phase: A—40 mM phosphate buffer pH 7.8; B—ACN:MeOH:water (45:45:10); linear gradients specific to column length
• Autosampler Programming: Precise draw and mix sequence in Agilent G1313A or G1367 with conical vial inserts for derivatization reagents and samples

Main Results and Discussion

The protocol achieves complete separation of 24 amino acids in 14 min on a 75 mm column and 26 min on a 150 mm column. The 3.5 µm packing yields higher efficiency than 5 µm but at the expense of backpressure (3.5 µm: 240–300 bar vs. 5 µm: 160–210 bar). Wavelength switching on DAD or FLD enables unambiguous identification of lysine and hydroxyproline by capturing OPA and FMOC signals separately without compromising resolution. Baseline artifacts from derivatization byproducts do not interfere with primary amino acid quantitation.

Benefits and Practical Applications

• High throughput with consistent peak resolution for routine QC and proteomic hydrolysate analysis
• Adaptable sensitivity modes for trace analysis via low- and high-sensitivity standard sets
• Flexible column choices to balance resolution, analysis time, and system pressure
• Automated workflow reduces manual handling, enhances reproducibility, and supports compliance in regulated environments

Future Trends and Potential Applications

• Integration with UHPLC platforms for further reduction in analysis time and solvent consumption
• Expansion to non‐proteinogenic and modified amino acids for advanced metabolomic profiling
• Implementation of real‐time data processing with machine-learning for automated peak assignment and quantitation
• Coupling with high-resolution mass spectrometry to provide complementary structural information

Conclusion

The Agilent ZORBAX Eclipse AAA HPLC protocol offers a robust, flexible, and high‐performance solution for amino acid analysis across diverse applications. Its automated derivatization, optimized gradient schemes, and versatile detection strategies ensure accurate quantitation and high throughput for both routine and research settings.

References

1. Agilent Technologies. Eclipse AAA Technical Note. Publication Number 5980-1193.
2. Agilent Technologies. Agilent ZORBAX Eclipse AAA Protocol. Publication Number 5980-3088EN, June 2008.