Improving Throughput and Analytical Greenness in Pharmaceutical Discovery Using Ultrashort (2.1 x 10 mm) HPLC Columns

Significance of the Topic

High-throughput analytical methods are essential in pharmaceutical discovery to rapidly screen large sample sets for compound identification, purity assessment, and bioanalysis. Traditional HPLC columns can limit workflow speed and increase solvent consumption, highlighting the need for faster, greener separations.

Objectives and Study Overview

This application note evaluates ultrashort 2.1 × 10 mm HPLC columns packed with 2.5 µm XSelect™ HSS T3 stationary phase against 2.1 × 30 mm and 2.1 × 50 mm alternatives. The study compares total analysis time for 96 injections and Analytical Method Greenness Scores (AMGS) to determine improvements in throughput and sustainability.

Methodology and Used Instrumentation

Sample: 250 µg/mL acetaminophen in water loaded into a 96-well QuanRecovery™ plate.

Chromatographic conditions:

• System: ACQUITY™ Premier Binary Solvent Manager with Flow-Through Needle and Column Manager
• Detector: Tunable UV at 254 nm coupled to Xevo™ TQ Mass Spectrometer
• Columns: XSelect HSS T3, 2.1 × 50, 2.1 × 30, and 2.1 × 10 mm, 2.5 µm
• Flow rate: 0.34 mL/min; Column temperature 30 °C; Sample temperature 10 °C
• Injection volumes scaled by column length (1–5 µL); Ballistic gradients optimized per column
• Injection cycle optimized by removing pre/post washes, eliminating air gaps, maximizing syringe draw rate, and enabling load-ahead to minimize inter-injection delay (~29.4 s)

Main Results and Discussion

Analysis of 96 samples showed:

• 50 mm column: 396.1 min total (6.6 h)
• 30 mm column: 252.2 min total (4.2 h), 36.3 % time reduction
• 10 mm column: 160.7 min total (2.7 h), 59.4 % time reduction vs. 50 mm

Shorter columns yielded sufficient performance for single-analyte assays despite lower theoretical efficiency. Peak intensities varied by injection volume adjustments. The ultrashort column reduced solvent use and instrument energy consumption, directly lowering the AMGS metric.

Benefits and Practical Applications

• Up to 60 % faster processing of 96-well plates enables multiple runs per workday
• Lower AMGS values demonstrate improved analytical greenness
• Practical for high-throughput screening where speed outweighs separation resolution

Future Trends and Applications

Continued miniaturization of column formats and integration with advanced mass spectrometry will drive faster, more sustainable analyses. Automation, AI-guided method optimization, and expanded greenness scoring will further enhance throughput and environmental performance.

Conclusion

Ultrashort 2.1 × 10 mm HPLC columns combined with optimized injection cycles and ballistic gradients deliver substantial gains in throughput (up to 60 % faster) and greener operation (lower AMGS) for pharmaceutical discovery screening, with acceptable performance for single-component assays.

Reference

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