Amino Acid Analysis - Application Notebook

Significance of the Topic

Amino acid analysis underpins diverse fields from protein characterization and biopharmaceutical process development to nutritional profiling of foods and feeds. Accurate quantitation of amino acid composition is critical for verifying protein identity, optimizing cell culture media for maximum biopharmaceutical yields, and ensuring the nutritional content of agricultural products.

Objectives and Overview

This web article summarizes the Waters UPLC Amino Acid Analysis Solution and its enhancements. The goals are to review method transfer from HPLC to UPLC for rapid, high‐resolution separations; to compare detector options (TUV, PDA, FLR); and to illustrate applications in protein hydrolysates, cell culture media, foods, and feeds.

Methodology and Instrumentation

The core analytical workflow combines pre‐column derivatization with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (AccQ•Tag Ultra reagent) and separation on a 1.7 µm BEH C18 column using an ACQUITY UPLC H-Class System. Samples include:

• Acid‐hydrolyzed proteins (e.g., BSA)
• Acid or microwave hydrolyzed food/feed samples (with optional performic acid oxidation)
• Cell culture media spiked with free amino acid standards

The system employs quaternary‐gradient eluents, heated separation at 55 °C, and injection volumes of 1 µL. Detector options include tunable UV (TUV, 260 nm), photodiode array (PDA, 260 nm), or fluorescence (FLR, Ex 266/Em 473 nm). Data acquisition and processing are handled in Empower software. Norvaline serves as an internal standard to correct pipetting variability.

Main Results and Discussion

• Method Transfer and Throughput: A reversed‐phase HPLC amino acid method requiring ~60 min per run was migrated to UPLC, reducing analysis time to ~9 min per sample while preserving resolution. This 7× increase in speed enabled in-house processing of >100 samples per month.
• Detector Comparison: TUV and PDA detectors provided equivalent quantitative results (50 fmol to 50 pmol on-column), with TUV exhibiting lower noise. FLR detection offered similar accuracy but varying peak responses due to differential fluorescence yields across amino acids.
• Reproducibility and Sensitivity: Retention times were reproducible to ±0.01 min. Calibration exhibited linearity over three orders of magnitude. Precision for amino acid residues in hydrolyzed BSA and complex feeds was within ±2% RSD across 75 determinations.
• Sample Handling Advances: Microwave‐assisted acid hydrolysis reduced feed sample preparation from 24 h to ~20 min. Prepacked eluents and batch derivatization streamlined routine workflows.

Benefits and Practical Applications

• High Throughput: Rapid separations and automated derivatization allow >5 samples per analyst per day.
• Cost Savings: Elimination of outsourcing and 10× lower solvent usage recouped instrument investment within months.
• Broad Applicability: Single platform supports pure protein, cell culture, food, and feed analyses with consistent, rugged performance.
• Data Integrity: Empower software ensures controlled processing, customizable reporting, and compliance-ready documentation.

Future Trends and Potential Applications

Emerging developments include integration with mass spectrometry for structural characterization, robotic sample preparation for higher automation, multidimensional UPLC separations, real-time process analytical technology (PAT) in biomanufacturing, and miniaturized UPLC systems for field applications in food quality and environmental monitoring.

Conclusion

The Waters UPLC Amino Acid Analysis Solution delivers a robust, high-throughput platform for precise amino acid quantitation across diverse matrices. Detector flexibility (TUV, PDA, FLR), rapid run times, and streamlined sample preparation expand laboratory capacity, reduce costs, and maintain data quality for research, QC, and industrial applications.

Instrumentation

• Waters ACQUITY UPLC H-Class System
• AccQ•Tag Ultra derivatization reagents and borate buffer
• AccQ•Tag Ultra BEH C18, 2.1 × 100 mm, 1.7 µm column
• Tunable UV detector, PDA detector, fluorescence detector
• Empower Chromatography Data Software

References

• Waters ACQUITY UPLC H-Class Amino Acid Analysis System Guide
• Waters UPLC Amino Acid Analysis Application Notebook
• Waters Application Notes: 720001372EN, 720001683EN, 720002913EN